Final Report Summary - ENCITE (European Network for Cell Imaging and Tracking Expertise)
Executive Summary:
The European Network for Cell Imaging and Tracking Expertise (ENCITE) has been a strong collaboration of 29 project partners from eleven countries, working together within a multi-disciplinary project of nine disciplines with the task of seeking solutions to cell imaging and tracking. ENCITE had the long-term mission to improve and test existing and new MR and optical imaging methods and biomarkers to get a more comprehensive picture of cell fate and to ultimately improve and further develop cell therapy for the benefit of the European patient. Over the past four and a half years, the scientists have achieved ground-breaking results on the preclinical validation of techniques towards the application into the clinic.
Novel chemical probes, reporter genes and mouse models were properly developed and designed for the set-up of innovative imaging procedures in the field of cell tracking and cell therapy.
To label cells in a more efficient way, non invasive imaging methods to monitor specific cellular decisions affecting cell fate were developed. These included tools for the detection of processes, such as cell migration, differentiation, proliferation and death.
With respect to a pre-clinical validation, existing generic and specific imaging tools were further developed and validated for the application of cell-based therapies for neurological disease and stroke, cardiovascular disease, musculoskeletal disorders, diabetes and cancer. The tools included: novel probes for cell labelling, novel imaging sequences and protocols for visualisation and quantification of cell fate in vivo, and novel imaging procedures for assessing tissue response to treatment.
A key aspect of the research work focussing on the translation towards clinical application was dedicated to the improvement of the translation and implementation of novel techniques into the clinic, for the benefit of patients. Major breakthroughs were realised in establishing the infrastructure for the production of tracers for in patient use and testing novel; tools for patient monitoring in patients with cancer, diabetes and ischemic heart disease.
Training and dissemination were important activities in the project. One of the highlights was the ENCITE Multi Centre Cluster for Training to provide flexible access to face-to-face trainings, e-courses and teaching files on ENCITE’s new technologies and to offer a virtual database serving as a repository of probes and procedures in the fields of chemistry and biology. In the long term, it is planned to further establish this cluster as a key node for trainings to the entire research community and hence, to also support the implementation of innovative tools across Europe as a standard.
ENCITE’s novel MR and multimodal imaging methods should serve to increase the rate of progress from preclinical studies to early clinical trials and their ultimate patient application. Cell therapy perhaps holds the future promise of personalised medicine with the potential to cure chronic diseases. The ultimate successful implementation of cell therapy as a novel treatment modality in the clinic requires a means to follow the fate of transplanted cells once inside a patient by widely applicable non-invasive monitoring tools. A promotional video is showcasing how in vivo image-guided cell therapy is revolutionising medicine in the field of cancer and diabetes. To this end, ENCITE is intending to further exploit the project results towards realistic clinical applications within follow-up joint actions in the future.
ENCITE’s scientists are confident that these technologies will help to speed up developments in cell therapies and that their entry into wider routine clinical practice could offer potential cures for many different types of diseases, including stroke, musculoskeletal and cardiovascular disease, diabetes and cancer.
Project Context and Objectives:
1. ENCITE background and context
The mission of the European Network for Cell Imaging and Tracking Expertise (ENCITE) project was to develop and test new MR and optical imaging methods and biomarkers to get a more comprehensive picture of cell fate and the reaction of the immune system and to ultimately improve and further develop cell therapy for the benefit of the European patient.
Cell therapy can be defined as the transplantation of living cells for the treatment of medical disorders. Although the concept of cell therapy has existed for centuries, and has been part of medical practice for decades in terms of blood transfusion and bone marrow transplantations, more recently cell therapy has received increased attention and has become a main focus of (bio)medical research.
Different principles underlie this increased interest in cell therapy: The transplanted cells can be used as an “active drug”; the transplanted cells can be used to replace damaged and degenerated tissue; or the transplanted cells can be used as a drug delivery vehicle. For all these applications promising results have been obtained in pre-clinical and clinical studies, however their success rates have been variable and this limits the clinical benefits associated with stem cell therapy.
One major challenge is that the mechanisms underlying how cell therapy works are still poorly understood, and currently there is no single imaging modality that meets the requirements of stem cell therapy. Yet the ability to non-invasively monitor the fate and action modes of transplanted cells over time is crucial for a better understanding of how cell therapy works in various disease states.
ENCITE has aimed to address these issues by developing and testing MRI imaging methods and biomarkers in order to get detailed information on the in vivo fate of transplanted cells and the hosts reaction to the transplanted cells. Ultimately, the project objectives were to translate improved techniques from the laboratory to the clinic, and apply them in the treatment of stroke and neurological, cardiovascular and musculoskeletal diseases as well as diabetes and cancer.
Magnetic Resonance Imaging (MRI) is one of the most powerful tools in clinical diagnosis and biological research due to its exceptional spatial and temporal resolution in the visualisation of soft tissue, including brain, heart and liver. However, the main disadvantage of this visualisation method is its limited sensitivity: target cells must be labelled with a large amount of MRI probe (commonly magnetic nanoparticles) to achieve high definition visualisation. Thus one of the key objectives addressed by the ENCITE scientists was to improve MRI sensitivity by increasing probe stability within biological systems, by enhancing cellular uptake as well as strengthening individual probe efficiency.
2. ENCITE’s main objectives
2.1 Generic and disease-specific objectives
In order to be able to address the extremely wide variety of cell therapies, the overall project objectives were divided into generic and disease-specific categories as follows:
The generic objectives included the development of new imaging methods to improve the spatio-temporal tracking of labelled cells, the generation of dual- and multimodality imaging procedures to cross-validate each individual approach, and the generation of new contrast agents and procedures that would improve the sensitivity and specificity of cellular labelling. For the generation of molecular and cellular imaging reporters, the potential of combining molecular biology with multimodal imaging techniques was explored, and novel cell and animal reporter systems were generated that enable detection of the location and function of individual cells and small cell subsets within the target organ. Appropriate cellular labelling techniques, that do not interfere with cellular functions and therapeutic efficacy, were established and new methods for quantitative assessment were developed to generate reliable biomarkers of cell fate and therapeutic effects. The overall aim was to establish and validate cell homing for therapeutic delivery to target organs. The legal and ethical issues associated with the use of new agents and methodologies were also properly addressed.
The disease-specific project objectives were to establish the clinical potential of novel imaging approaches in preclinical studies related to neurological, cardiovascular and musculoskeletal diseases as well as to cancer and diabetes, and also to improve existing cell-based therapy regimens in cancer.
2.2 ENCITE’s main objectives of its individual research fields
The ENCITE project was divided into five areas in research entitled novel imaging technologies, novel imaging reporter probes, novel tools for cell labelling, pre-clinical validation and translation towards clinical application as well as one area dedicated to the development and long-lasting establishment of training activities. These work programmes addressed the following main objectives:
2.2.1 Novel Imaging Technologies
This research field focused on the development and improvement of MR imaging techniques used in cell tracking, the evaluation of cell fate and the validation of cell therapy: on one hand, it aimed to provide new imaging methods to improve the spatio-temporal resolution of the imaging techniques, which in turn allows for the more detailed tracking of labelled cells. In addition, new methods for the quantitative assessment of transplanted cells and generated new and reliable biomarkers of cell fate and therapeutic effects were developed. On the other, the research aimed to develop novel image post-processing techniques to allow the stable and reproducible evaluation of experimental results. To achieve these goals, new hardware and advanced concepts were developed and utilised to develop new labels and to discover a more effective use for existing ones. This included advanced imaging sequences for cells labelled with iron oxide nanoparticles, novel fluorine containing compounds, imaging techniques for musculoskeletal imaging and multispectral biomarkers for the imaging of neo-angiogenesis and brain diseases. These post-processing techniques included tools for visualisation, co-registration as well as algorithms for quantitative evaluation. A major goal was the integration and co-registration of different imaging modalities to allow a more comprehensive assessment of the different modalities that have been developed.
2.2.2 Novel Imaging Reporter Probes
By addressing this research the overall aim was to develop chemical probes, reporter genes and mouse models designed for the establishment of innovative procedures in the field of cell tracking and cell therapy. Additional objectives were to increase the number of reporter genes available for MRI by the evaluation of novel candidate reporters as well as to increase the utilisation of MRI reporter genes by applying them to monitor the constitutive expression of genes and the detection of changes in gene expression as well as imaging of cell differentiation. To date, no imaging approach has been reported as being suitable for the direct detection of cell proliferation, and bioluminescence imaging has been used instead to determine increases in cell number. The objective was to combine strategies for the simultaneous imaging of cell proliferation, differentiation and death, and therefore multi-parametric information on the state and fate of the transplanted cells would be retrieved. The further development and validation of such strategies focussed on cell labelling is described in the next work programme of novel tools for cell labelling.
2.2.3 Novel Tools for Cell Labelling
In this research area the aim was to develop non-invasive imaging methods to monitor specific cellular decisions that affect cell fate. These methods included the development of tools for the detection of processes such as cell migration, differentiation, proliferation and death. Apoptosis, or programmed cell death, is a process which plays an important role in the development of multi-cellular organisms and in the regulation and maintenance of cell populations in tissues under certain physiological and pathological conditions. The ultimate objective was to establish a technique that would enable the visualisation of intra-cellular protein interactions known to occur during apoptosis. Such a method would be valuable for in-vivo imaging and for identifying new proteins involved in the apoptotic process using small interfering RNA (siRNA) screens. Analogous approaches have been taken to report in the MRI images on specific enzymatic activities.
2.2.4 Preclinical Validation
In the research area of preclinical validation, the overall objectives were to develop and validate generic and specific imaging tools for five disease-specific application fields of cell-based therapy, entitled neurological disease and stroke, cardiovascular disease, musculoskeletal disorders, diabetes and cancer.
In neurological disease and stroke, the main objective was to increase the capability of non-invasive imaging to monitor the migration, integration, functional status, and differentiation status of transplanted stem cells in addition to their interactions with other cell types. Another objective was to develop an imaging method to monitor the improvement in the functional outcome following cell therapy, such that not only the fate of the transplanted cells, but also the functional fate of the receiving organ, is monitored.
Similarly, in relation to cardiovascular disease, the aim was to develop tools that allow for an integrated assessment of cell fate, cell function and their therapeutic effect in cellular cardiomyoplasty taking into account, that from an imaging perspective, the heart represents a significant challenge. Its complex anatomical structure, continuously changing form (due to the heart beat), the large volumes of blood flowing through it (blood circulation), and its location in an environment filled with air (chest cavity and lungs) that is also incessantly moving (respiration), all contribute towards the imaging challenge.
For musculoskeletal disorders three main objectives were pursued: to develop methods for in vivo observation of the regeneration of injured tissues, the tissues being tendons, particular cartilage and the nucleus and the annulus components of invertebrate disks; to design methods using mesenchymal stem cells to induce regeneration of injured tissue; as well as to follow by MRI the fate of stem cells that were introduced to the injured tissue.
In diabetes, attention has been devoted to the development of new in vivo image methods suitable for the guidance of insulin islets transplantation into the liver.
One of ENCITE’s major objectives for preclinical validation was related to the improvement of existing cell-based therapies in the treatment of cancer. High importance was attached to the development of imaging tools facilitating the use of cell therapy in cancer by antagonising the mechanisms by which cells promote tumour progression, and additionally to the development of approaches for utilising cell homing ability for therapy delivery to the tumour. Furthermore, work has been devoted to overcome the factors in the tumour microenvironment impairing cell trafficking, cell delivery and immune effector function. In the oncological application of cells for targeted drug delivery, it was aimed to develop tools for assessing bystander effects in inducing cytotoxicity to tumour cells.
2.2.5 Translation towards Clinical Application
The key objectives focused on the translation and implementation of the novel techniques and probes developed in the previously mentioned work programmes into the clinic for the benefit of patients.
Progress made in the development of new imaging techniques and evaluation procedures during the ENCITE project demonstrates that results of preclinical studies can be successfully applied in clinical studies. An example of labelling of pancreatic islets (PI) by superparamagnetic iron oxide nanoparticles and following MR studies on an animal model proved that these imaging techniques can also be used for description of a position and fate of human pancreatic islets transplanted to patients with type 1 diabetes. In addition, the results of preclinical studies also show that MR imaging of labelled PI can be used for prediction of functional failure of PIs before clinical symptoms occur. This is an important message for future clinical applications of transplantation procedures.
Research has shown that the relationship between the immune system and human cancer is complex, highly dynamic and variable between individuals. Considering the complexity, enormous effort and costs involved in optimising immunotherapeutic approaches, clinically applicable tools to monitor therapy-induced immune responses in vivo are most warranted. However, the development of such tools is complicated by the fact that a developing immune response encompasses several body compartments, for example peripheral tissues, lymph nodes, lymphatic and vascular systems as well as the tumour site itself. Moreover, the cells that comprise the immune system are not static but constantly circulate through the vascular and lymphatic system. Molecular imaging is considered the favourite candidate to fulfil this task. The progress in imaging technologies and modalities during the ENCITE project life time has provided a versatile toolbox to address these issues.
2.3 ENCITE’s objective on dissemination and training actions
Particular attention on dissemination and training was paid to the development of a multi-disciplinary training platform providing flexible access to individual face-to-face trainings, e-courses and teaching files on ENCITE’s new technologies as well as a virtual database serving as a repository of probes and procedures in the fields of chemistry and biology. This training platform aimed to strengthen the exchange and interactions between project partners on improving their research work and further developing specific trainings, such as: 19F MRI, diffusion tensor imaging (DTI), diffusion weighted imaging (DWI), dynamic contrast enhanced MRI, vessel size imaging (VSI), imaging with ultra short and variable TE and high resolution anatomical imaging. Further emphasis was also given to optical imaging aspects, reporter probes and pre-clinical investigations, etc.
The training concept shall ensure that ENCITE’s technologies will be available to researchers and clinicians throughout Europe. A training platform, namely ENCITE Multi Centre Cluster for Training established in Belgium, France, Germany, Israel, Italy and The Netherlands, will continue to offer training activities in the long-term future in order to increase mutual benefits for the European molecular imaging community and for those groups interested in cell imaging and tracking.
To widely communicate the project advancements, the promising techniques for (pre-)clinical validation and application, and the multidisciplinary training opportunities to peers and experts, the ENCITE consortium partners focussed on carrying out all the actions described in the ENCITE Dissemination Plan. To ensure intense promotion of its advancements and trainings an effective communication network has been established by all communication channels and means available within and outside the consortium, such as: project and consortium partners’ websites, websites of co-operational partners, online event calendars and portals, magazines and publications, e-mailings and e-newsletters, (booth) presentations and individual conversations at public events, publications, individual meetings and printing materials (flyers, posters and brochures, booth presentations, etc.).
Project Results:
Along the four and a half years of activities ENCITE has achieved a number of important results ranging from very innovative proofs of concepts to the set up of protocols already suitable for clinical applications. In the following section the main results and foregrounds are summarised.
1. Novel Imaging Technologies
The research field of “Novel Pulse Sequences” was mainly focused on the development of dedicated acquisition protocols for new imaging biomarkers and new MR imaging reporter probes. The utilisation of new techniques and hardware was used to generate novel types of sequences that feature the higher sensitivity and signal strength that is the crucial requirement for the future of MR imaging technologies.
The studies included the accurate in vivo detection and segmentation of cells, or clusters of cells, and imaging methods for specific contrast agents, such as fluorine magnetic resonance imaging (19F-MRI) and ultra-short TE (UTE) imaging for bright contrast. Special attention was devoted on the translation of the newly developed sequences to the clinic. Thus, an alternative positive contrast method was developed based on a double contrast 3D spoiled gradient echo (SPGR) sequence with a variable first TE (vTE) and highly asymmetric readout for visualisation of SPIO islets on an animal model and on a patient. In addition, this novel vTE technique was used for musculoskeletal imaging.
MR imaging sequences for multiple biomarkers with clinically approved contrast agents that are used in the monitoring of cell fate were further improved and tested in vivo to facilitate the evaluation and image post-processing of these markers for a potential translation into the clinical routine. This topic received major attention since it is expected to provide more valuable information for the monitoring of patient treatments out of each single examination without the need to sacrifice patient comfort and time.
As the tracking of bacteria in vivo could enlighten multiple disease mechanisms, SPIO-mediated contrast with highly sensitive susceptibility weighted contrast imaging of bacteria for the visualisation of bacterial leakage in vivo were utilised. The potential of MR for detecting iron oxide-labelled T cells has been explored and investigated.
As the development of alternative labels in MR remains an attractive topic, especially with the development of new fluorine-compounds with enhanced sensitivity, this topic has been further investigated. A new complex has been used together with an improved UTE sequence to monitor labelled tumour cells in an animal model over a period of eight days and macrophages for more than one week. This method combined the specificity of fluorine MRI with the ease of proton MRI and it can be therefore performed on any standard MRI scanner.
Within the domain of “Image Post-processing”, molecular, functional and anatomical imaging data were integrated into a comprehensive dataset that could be investigated as a whole rather than looking into individual examinations. This was a particularly important topic since the development of new imaging methods and advanced imaging techniques yields a great number of new modalities that needed to be correlated to each other to deploy their full potential. New algorithms were investigated to be used to facilitate registration, matching and correction of motion at the anatomical level and detection and tracking of functional events at the cellular level.
The main task was the integration of molecular, functional and anatomical imaging data. 3D and 4D datasets from the brain and heart were generated and these were recorded under variable conditions using different MRI contrast and spatial resolution. The usefulness of the chosen imaging acquisition procedure and image analysis methods was evaluated, and an articulated atlas for image registration of follow-up studies was successfully created.
A number of the image analysis challenges which emerged from longitudinal pre-clinical molecular imaging studies were addressed. New algorithms were investigated that can be used to facilitate registration, matching and correction of motion at the anatomical level, and detection and tracking of functional events at the cellular level. The newly developed methodologies will enable quantified monitoring of disease progression and treatment response that is not possible with current visual interpretation, and will be playing a key role in the quantitative evaluation of future therapies.
2. Novel Imaging Reporter Probes
In the field of “Probe Chemistry”, research activities were devoted to the development of new labelling probes and their subsequent efficacy testing in vitro and in vivo. Several new MRI agents were synthesised, including paramagnetic metal complexes and superparamagnetic iron oxide particles (SPIO). The use of these MRI agents in cellular labelling has led to the achievement of enhanced labelling sensitivity and specificity. In the field of SPIO, much work has been carried out in the rational synthesis of particles of defined size and improved magnetic properties.
To fully exploit the full potential of the high spatial and temporal resolution of MRI in molecular imaging applications, it was deemed necessary to improve the sensitivity and specificity of the currently available probes. Regarding sensitivity, a new high relaxivity tetrameric gadolinium (Gd)-based agent was shown to provide an impressive seven-fold sensitivity enhancement in respect to the commercially available agents, while also demonstrating an analogous safety profile. Regarding specificity, an enzyme responsive Gd probe which reports on the activity of beta-galactosidase, an enzyme commonly used as reporter of gene expression, was synthesised and tested. Other Gd-containing probes responsive to tyrosinase and glutammato decarboxylase (GAD) have been prepared and tested on cellular and animal models.
Concerning the use of Gd containing chelates in cellular labelling, particular attention was devoted to monitoring the effect of compartmentalisation on the observed relaxivity, the fate of the internalised complexes over time and the establishment of efficient cellular uptake routes, such as endosome escape and hyposmotic shock.
The ability of Magneto-Liposomes (MLs) to act as a targeted contrast agent was also evaluated, and the successful targeting of ASGP1 receptors of hepatocytes has been reported. In comparison with the non-functionalised anionic MLs, specific uptake of the functionalised MLs by hepatocytes was observed in cultures of hepatocyte-like cell lines, primary hepatocytes, progenitor cells differentiated into hepatocyte-like cells, and in vivo after systemic administration. Furthermore, the labelling efficacy of these iron-based nanoparticles was further assessed in four different types of cells (Panc, Capan, HeLa and Jurkat).
The high affinity of Yeast Cell Wall Particles for immune system cells was exploited to label macrophages with the aim of tracking them by MRI experiments performed on J774 murine macrophages. These experiments demonstrated a high and very fast uptake of the paramagnetic particles with good temporal persistence of the contrast and an extremely low cytotoxicity.
Important advances have also been made in the field of Chemical Exchange Saturation Transfer (CEST) agents and their use in cellular labelling. The use of these MRI probes has significant advantages over the use of the more classical relaxation agents. Being frequency-encoding systems, it is possible to visualise (using different colours) more probes in a single image as every CEST agent is responsive to a specific irradiation frequency. The frequency-encoding property of this class of agents has been exploited for pursuing the visualisation of different types of cells in the same anatomical region. Two approaches have been followed: entrapping paramagnetic CEST agents into cells or, by exploiting the intracellular water molecules as a source of exchangeable protons once their absorption frequency has been suitably shifted by the entrapment of a proper Lanthanide Shift Reagent (Cell-CEST).
In addition to develop fluorescent probes to detect the differentiation or functional states of cells and tissues, novel non-linear imaging modalities which enable the detection of tissue structure and function have also been developed, including second and third harmonic generation which can be excited and detected by multiphoton microscopy. Second harmonic generation is the frequency doubling of light, which is excited by collagen fibres of healthy and diseased tissue and by actomyosin bundles present in myofibres. Third harmonic generation is the frequency tripling of light, which can be exploited by infrared excitation to detect tissue interfaces, including adipocytes, nerve tracks, erythrocytes, and surfaces of collagen bundles. The ENCITE scientists were the first group to exploit the intravital use of combined second and third harmonic generation microscopy to map the invasion routes of cancer cells during the metastatic cascade.
In conclusion, highly efficient probes for cellular labelling have been developed and tested either in cell or in vivo. A number of promising systems were developed ranging from molecular paramagnetic Gd-based reporters to nanosized iron based probes and to micron sized Gd-based probes (YCWPs).
Moreover in the field of MRI very important results have been obtained with CEST agents showing that multiplex visualisation is possible also with this modality. Finally, fluorescent probes and innovative non linear imaging modalities have been investigated to enhance the imaging information on cellular differentiation and assessment of tissue functional states.
In the field of “Molecular Biology Probes”, the objective was to develop two reporter genes for MRI which could be used to non-invasively monitor gene expression in vivo. This could be of use for example in developmental studies, tracking of implanted cells, or monitoring therapeutic transgene expression. Following the addition of Mn-apoferritin or ferritin to the growth medium of cultured cells, TIM2 (T-cell Immunoglobulin and Mucin domain 2) expression has been shown to shorten T1 and T2 relaxation times of cells in vitro. Preliminary results suggest that TIM2 expression can produce negative contrast in T2-weighted images in vivo. Since TIM2 expression does not compromise cell growth or viability, and ferritin and Mn-apoferritin uptake are well tolerated, ENCITE scientists suggest that TIM2 is a suitable protein to be used as a reporter gene.
OATP (human organic anion transporting polypeptide) expression has been shown to shorten T1 relaxation times of cells incubated with gadolinium chelates in vitro, in comparison to control cells. Additionally, OATP expression does not seem to prevent luciferase expression suggesting that the transduced cells remain viable.
3. Novel Tools for Cell Labelling
Within ENCITE a number of cell labelling strategies have been evaluated and developed. Regarding imaging probes, the work carried out focused on both generic strategies, using exogenous non-specific probes such as iron oxide particles, Gd-chelates and Fluorine (19F) compounds, and also on more tailored strategies involving smart or responsive probes and reporter genes. While it was found that some general principles regarding cell labelling protocols could be defined, it was recognised that for each specific combination of cell type and contrast agent/imaging probe, tailored approaches are required, including the technique to be used for optimal probe incorporation into the cell and assessment of retention of cell functionality.
A number of experiments were successfully carried out regarding novel tools for cell labelling and cell fate imaging, including the monitoring of cell recruitment, differentiation, and cell death. Optimal labelling conditions were defined for a number of different contrast media, for example, cellular incorporation of a responsive contrast agent into murine neural progenitor cells and labelling of mesenchymal stem cells with Gd-DTPA. For probes based on Gd-chelates it was found that the MRI signature (relaxivity) is dependent on the intra-cellular compartment the probe is contained in. Depending on probe characteristics and/or the labelling techniques used, probes either mainly accumulate in endosomes or in the cytosol. In many cases however, probes enter the cell via endocytosis and consequently accumulate in endosomes. For Gd-based probes this has a profound effect on the MRI signature, referred to as quenching of the relaxivity. Within ENCITE techniques were developed and evaluated to “rescue” such probes from endosomal entrapment. One such strategy encompassed the use of photochemical internalisation (PCI) techniques. Initial cell labelling experiments indicated the ability of porphyrins incorporated in endosomal membranes to induce the endosomal escape of contrast media by UV irradiation of labelled cells. Endosomal escape techniques based on PCI technology have been further developed for application in vivo to enhance the delivery of all molecules taken into the cell by endocytosis. Clinical Phase I/II studies in patients with cutaneous and subcutaneous tumours are currently being carried out at the University College Hospital of London. These trials are using the commercial photosensitiser AmphinexTM (TPCS2a) and the cytotoxic agent bleomycin.
As a means to potentially achieve in vivo targeted labelling of cells, the use of local sonoporation was studied. An in vitro model was established in which it was shown that cellular uptake of iron oxide nanopartcles from the surrounding medium is enhanced by local sonoporation. Local sonoporation was achieved through sonication of targeted ultrasound microbubbles with tailored pulse sequences from an ultrasound transducer. With optimised pulse sequences an up to 7 times increase of SPIO uptake compared to the control with 90% cell viability could be achieved. A ~10% labelling should be sufficient for in vivo MRI tracking. This technique might therefore provide a safe in vivo labelling strategy for endothelial cells with SPIO, where other cell labelling techniques may not be applicable.
Regarding responsive contrast agents a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions, was developed. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase, expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may extend the usefulness of MRI for functional imaging on a cellular level.
In contrast to labelling cells with a probe, use of a reporter gene to label cells avoids the serial dilution of the label associated with cell division. Use of ferritin as a reporter gene for MRI has been demonstrated by a large number of laboratories and in multiple model systems. The ENCITE scientists built on this research by introducing the use of heavy chain ferritin (h-ferritin) as a reporter gene, which allowed expression activation detection in cancer cells as well as in transgenic mice under tetracycline regulation. Such over-expression was found to elevate the level of intracellular iron with no adverse effect on animal wellbeing, even when expressed throughout the lifetime of the mice. Ferritin over expression was also used to detect systemic recruitment of tumour associated fibroblasts to the angiogenic rim of ovarian carcinoma xenografts. Bi-exponential relaxometry mapping provided information not only on the distribution of the cells, but also quantitative information on the fraction of labelled cells within a mixture of exogenously administered cells, native tumour and tumour stroma cells. Such information has not been derived using any other type of cell tracking method.
The main limitation of the use of ferritin in MRI is its relatively low relaxivity. The ENCITE consortium sought to improve the magnetic properties of ferritin’s iron core by introducing a peptide derived from an iron binding protein of the magnetosome machinery of magnetic bacteria. The chimeric magneto-ferritin gene affected iron sedimentation, and cells expressing the new magnetoferritin showed enhanced iron uptake, higher relaxivity and increased MRI contrast.
4. Preclinical validation
In regenerative medicine, the goal is to repair damaged/degenerated tissue with an intrinsic low capacity for self healing by transplantation of stem cells. In order to achieve this goal, and to ultimately achieve restoration of function of the damaged tissue, it is crucial to know what happens to the cells after they have been translated into the patient (or animal model).
Within the ENCITE consortium, several imaging tools have been developed and validated in pre-clinical models. These tools encompass novel techniques by which cell survival, cell differentiation and therapeutic effects mediated by the transplanted cells can be monitored in vivo. They focus on five different application fields, namely neurological disease (stroke), cardiovascular disease (myocardial infarction), musculoskeletal disease, diabetes and cancer.
4.1 Neurological disease
Within the application field of neurological disease/stroke some major advances were made regarding the ability to monitor neuronal stem cell engraftment, cell differentiation and neurological tissue repair using sophisticated high-field MR imaging and spectroscopy techniques, as well as with novel 19F- and Gd-based probes and reporter gene technology.
Through double labelling of neuronal progenitor cells with 19F probe and a luciferase reporter gene distribution and survival of these cells upon transplantation could be monitored. Cells remained visible for several weeks by 19F-MRI, but rapid loss of cell vitality, as assessed by bioluminescence imaging, was noted within the first two weeks after grafting. Immunohistochemical analysis showed that this loss of cell vitality was due to a massive inflammatory reaction to the graft. Studies directed at optimisation of the bioluminescent signal revealed that the timing and route of administration of the luciferase substrate can significantly influence signal levels. Furthermore, the sensitivity of various different luciferases for the specific purpose of neuro-imaging has been compared. Luc2 was found to be by far the most intense, leading to highest sensitivity of small grafted cell groups. Putting these two studies together, an in vivo bioluminescence protocol specifically optimised for the requirements of neuro-imaging has been developed and validated for the first time.
Through MR spectroscopy approaches and responsive contrast agents, changes in cell function were monitored based on shifts in the expression of specific molecules associated with a certain cell function stage. The visualisation and assessment of the functional state of transplanted stem cells in brain lesions was achieved by 19F cell labelling techniques and the use of a GABAergic cell specific responsive contrast agent, respectively. In a transgenic model based on doublecortin (DCX) promoter activity neurogenesis was monitored non-invasively using bioluminescence. The differentiation of human neural stem cells was achieved by metabolite profiling using magnetic resonance spectroscopy (MRS). In response to transplanted cells in stroke lesions, angiogenesis was monitored using reporter genes that were associated with the formation of novel blood vessels. These approaches have significantly improved our ability to monitor the distribution of transplanted cells, as well as determining their functional status and effect on host tissue.
4.2 Cardiovascular disease
From an imaging perspective the heart represents a significant challenge. Its complex anatomical structure, continuously changing shape (due to the heart beat), the large volumes of blood flowing through it (blood circulation), and its location in an environment filled with air (chest cavity and lungs) that is also incessantly moving (respiration), all contribute towards the imaging challenge. These anatomical and physiological conditions lead to the requirement of tailored MR imaging approaches which are able to compensate for the imaging artefacts that can be caused by motion and blood flow. Specific advances made by the ENCITE scientists include the ability to perform parametric imaging techniques on the heart which allow for more sensitive and specific detection of labelled stem cells. In addition, they also developed and/or validated new methods for the monitoring of transplanted stem cell fate in the myocardium and for the assessment of myocardial function.
While mapping techniques and the use of a post-processing pipeline were shown to be robust in small animal models and have advantages regarding sensitive detection of cells labelled with either iron oxide or gadolinium-based probes, some limitations of these techniques were also highlighted especially for the use of cells labelled with iron oxides. While various in vitro studies have demonstrated that specific MRI parameters, i.e. the R2 and R2* values, are differently affected depending on the compartmentalisation of iron oxide particles, and that the difference in the signatures of these parameters can be used as an indicative measure of cell survival, the ENCITE scientists have demonstrated that this principle can not be applied in vivo. Potential differences in R1, R2 or R2* relaxation rate, as a measure of overall cell viability for mesenchymal stem cells labelled with Gd-liposomes (Gd-MSCs) or iron oxide nanoparticles (SPIO-MSCs), were investigated. Cells were also transduced with a luciferase vector, facilitating a correlation between MRI findings and cell viability using bioluminescence imaging (BLI). Viable Gd-MSCs were clearly distinguishable from nonviable Gd-MSCs under both in vitro and in vivo conditions, clearly differing quantitatively (?R1 and ?R2) as well as by visual appearance (hypo- or hyperintense contrast). Immediately post-injection, viable Gd-MSCs caused a substantially larger ?R2 and lower ?R1 effect compared with nonviable MSCs. With time, the ?R1 and ?R2 relaxation rate showed a good negative correlation with increasing cell number following proliferation. Upon injection, no substantial quantitative or visual differences between viable and nonviable SPIO-MSCs were detected. Moreover, nonviable SPIO-MSCs caused a persisting signal void in vivo, which compromises the specificity of this contrast agent. In vivo persistence of SPIO particles was confirmed by histological staining. These findings indicated Gd to be the favourable contrast agent in qualitative and quantitative evaluation of labelled cell fate in cell therapy experiments.
For the novel approach of using adipose tissue derived stem cells ENCITE succeeded in the isolation, characterisation and transduction with a GFP reporter probe of pig adipose tissue derived mesenchymal stem cells (pASC) and modelling of ischaemic damage in a pig model. This allowed the study of the fate of pASC upon intra-coronary infusion, by performing PCR analysis of the GFP gene on various myocardial tissue regions. This showed that GFP+ was only detected in the coronary arteries and ischemic damaged tissue (left ventricle) and was absent in the remote myocardium, atriums, right ventricle and all the aorta different portions. Confocal microscopy analysis also revealed the presence of GFP+-cells in the ischemic cardiac tissue. Cardiac magnetic resonance (CMR) was performed with a 3T clinical magnet at baseline (post-MI) and 7days post-MI/ASC infusion in a pig model. Biventricular function, myocardial edema, reperfusion hemorrhage, necrosis and no-reflow phenomena were assessed. Interestingly, histopathological analysis revealed a histopathologic correlation with the 3T-MRI findings at T2* sequence (myocardial hemorrhage, infarct zone and microvascular obstruction). However, CMR analysis showed no differences in cardiac performance, the degree of cardiac damage, and no-reflow phenomena between those animals treated with ASC-GFP+ and the placebo controls. However, histopathological analysis of vascular density (lectin staining) revealed a significant increase in neovascularisation within the ischemic cardiac tissue of those animals administered ASC as compared to controls.
A new approach to analyse tagged cardiac MRI data was developed. The proposed method is based on a Bayesian estimation framework, implemented by means of reversible jump Markov chain Monte Carlo (MCMC) methods, and combines information about the heart dynamics, the imaging process, and tag appearance. In phantom studies this method was shown to perform superior to other commonly used techniques. This new method was validated in rat models, pig models, and on human tagged cardiac MRI (see also section on clinical translation).
4.3 Musculoskeletal disease
Regarding the application field of musculoskeletal disease, new MR imaging parameters were defined as markers for intervertebral disc (IVD) and cartilage degeneration using advanced MR imaging techniques such as double quantum filtered MRI and multinuclear NMR. For the first time scientists were able to characterise the changes occurring following the ablation of the nucleus pulposus part on the intervertebral disc, mimicking the effect of disc degeneration, and the differences in NMR parameters in the cartilage of osteoporosis patients.
One major breakthrough was the use of CEST to assess glycosaminoglycan concentration in the intervertebral disc. The CEST method for hydroxyl protons of GAG is shown to be sensitive to changes in GAG concentrations in the nucleus pulposus of the intervertebral disc, both in spectroscopy and in ex vivo imaging. It could potentially be used for monitoring GAG depletion in the nucleus pulposus, which plays a central role in the study and diagnostics of disc degeneration. As a consequence, gagCEST may hold potential not only for the assessment of disc pathophysiology in vivo, but also for medical intervention and therapeutic monitoring.
By tracking mesenchymal stem cells (MSCs) in vivo it was shown that they can be successfully implanted into, and survive in, a degenerated mouse IVD. This may facilitate the early diagnosis of disc degeneration and represent a method to monitor repair mechanisms by stem cells.
4.4 Diabetes
In diabetes the application of five new MR imaging techniques for sensitive detection of islets labelled with SPIO particles that were developed within ENCITE were evaluated. The first one, an echo-dephased SSFP method, can successfully visualise SPIO-labelled human and rat pancreatic islets yielding a positive contrast in MR images, as opposed to signal loss when using classical MR imaging techniques, This so-called “positive contrast with echo dephased SSFP sequence” method allows for the unambiguous and sensitive visualisation of transplanted pancreatic islets in a physiological tissue environment. This technique is sensitive enough to detect single pancreatic islets.
The second technique comprised Ultra Short Echo (UTE) and was successfully tested on an experimental MR system. A similar approach was used on a 3T system where a double contrast selective 3D spoiled gradient echo sequence (SPGR) was used for animal studies as well as for human applications.
The third technique involved an approach of double contrast, which uses the combination of T2 imaging (for cell labelling) and T1 imaging following intravenous application of contrast agents to enhance contrast between labelled cells and surrounding tissue. It was shown that this approach could suppress image artifacts and the segmentation of pancreatic islets was improved.
The fourth technique assesses vascularisation of artificial transplant sites. From the techniques available on the MR systems, the Dynamic Contrast Angiography (DCA) technique was chosen to study vascularisation around pancreatic islets deposits.
The fifth technique using the balanced SSFP (bSSFP) sequence with phase cycling was successfully implemented for SPIO-labelled human and rat pancreatic islets yielding a negative contrast. The bSSFP sequence is sensitive enough to detect single pancreatic islets and was routinely used in a clinical protocol.
For several of these techniques, semiautomatic and automatic methods for the calculation of the number of pancreatic islets were tested and are now ready for routine application. In addition, new bimodal probes were synthesised and tested for labelling of pancreatic islets (and also STEM cells). Bimodal contrast agents are agents that can be used by two independent imaging modalities. This study focussed on the synthesis and evaluation of two different types of novel probes for MR and optical imaging: “positive” T1 contrast agent based on cyclodextrin Gd complexes and “negative” T2 contrast agent based on perovskite manganite nanoparticles, both with a fluorescein marker for optical imaging.
For the bimodal fluorescein /MRI cyclodextrin-based contrast agent cell toxicity was found using second generation dendrimers. Efforts were therefore focused further on analogous aggregates with a cyclodextrine core. In ?-cyclodextrine the -OH groups of the upper ring were replaced with amines and thereby, one molecule of fluorescein and six molecules of the Gd(III) complex with DO3A-CH2-PO2H-Bz-NH2 acid were attached to the molecule of cyclodextrine. The T1 relaxivity of A-CD-F is high, about 25 s–1mM–1 per one Gd(III) ion at 20 MHz and temperature of 25 ºC. The labelling of stem cells was verified by fluorescent microscopy and in vivo tests on rat models were also carried out. The potential of magnetic nanoparticles based on the La0.75Sr0.25MnO3 perovskite manganite for magnetic resonance imaging (MRI) was studied. Dual imaging probes where the magnetic cores are combined with a fluorescent moiety were synthesised and tested. Viability tests show that the particles are suitable for labelling of PIs. No fatal interference with the vitality and insulin releasing ability of labelled pancreatic islets was observed. Relaxometric measurements confirm high T2 relaxivities at magnetic fields of B0 = 0.5–3 T.
Experimental models were also studied to investigate whether MR imaging can predict failure of pancreatic islets transplanted into hepatic portal vein. It is known, that labelled PIs are visible in liver or in other organs for a relatively long time. Syngeneic transplantation and allogeneic transplantations with two different immunosuppressive regimes were studied for 12 weeks. The result shows that the failure of PI can be seen after 3-4 week after the transplantation by MR imaging of labelled PI. This result could be useful for clinical applications.
4.5 Cancer
In cell-based therapy approaches for cancer, the main results are directed towards monitoring the fate and function of adoptively transferred DC and T cells. Good progress regarding multi-modal imaging techniques and imaging probes was achieved. A new 19F-based imaging probe was developed to quantitatively monitor the fate of DC by MRI. A major achievement in DC therapy studies was the observation of DC migrating to the draining lymph nodes in patients enrolled in a Phase I Clinical Trial using Indium-111 scintigraphy (further discussed in the translation towards clinical applications below). Only a small proportion of injected DCs migrated from the injection site. MR imaging allowed assessment of both accurate DC delivery and inter-/intranodal migration patterns. In addition, the mechanism by which DCs activate Natural Killer (NK) cells in the presence of stimuli of bacterial origin was identified, showing a crucial role of DCs in NK cell activation.
Detection of CFSE-labelled Cytotoxic T-cells in dual-colour tumours was achieved. Through the introduction of third harmonic generation in multiphoton microscopy, otherwise undetected structures of the tumour stroma could be visualised. With regards to monitoring the dynamics of tumour and tumour microenvironment, apoptosis was detected using a histone H2B/GFP construct, and a quantitative nuclear condensation score to discriminate apoptosis from necrosis was developed, which is instrumental in the real-time monitoring of preclinical immunotherapy.
Using multiphoton and intra-vital imaging approaches, dynamic interactions between various immune cells and between immune cells and tumour cells were monitored in vivo. This study included techniques for the detailed analysis of T-cell effector functions, based on the assessment of tumour cell viability and growth, and techniques to monitor tumour cell–T cell interactions and cell apoptosis. Pre-clinical T-cell therapy studies resulted in the successful generation and expansion of cytotoxic T lymphocytes (CTL) which kill different OVA-peptide positive target cells, including B16F10 melanoma cells.
Detection of CFSE-labelled Cytotoxic T-cells in dual-colour tumours was achieved. Through the introduction of third harmonic generation in multiphoton microscopy, otherwise undetected structures of the tumour stroma could be visualised. With regards to monitoring the dynamics of tumour and tumour microenvironment, apoptosis was detected using a histone H2B/GFP construct, and a quantitative nuclear condensation score to discriminate apoptosis from necrosis was developed. This approach will be valuable as a third colour to monitor the tumour stroma in the projected dual-colour reporter mouse. In addition, a significant impact of p53 expression in fibroblasts on tumour progression was detected.
A tumour rejection model based on antibody-dependent cellular cytotoxicity was successfully established. Tumour cell death in response to chemotherapy and tumour stroma activation, using two photon intravital microscopy, was successfully monitored.
Besides tracking what happens to the transplanted cells (cell fate) another question to be addressed in the various disease states is “what do the transplanted cells do?”. To answer this question, tools were developed that are highly specific for the targeted disease, such as in neuronal differentiation as well as new methods which have been developed to quantify glycosaminoglycan levels in cartilage. Other tools, although developed and validated within a specific disease model, have a more general applicability, such as the assessment of cell survival.
The developed tools are important in helping to elucidate the mechanisms of disease and repair of tissue, and to further our understanding of how stem cell therapy works, to make its use more effective and broadly applicable Some of the developed tools are suitable for direct translation for clinical studies, or have already been tested in a clinical setting. For example, tools for monitoring the survival of transplanted pancreatic islets are already being tested in in-patient studies, or in the patient DC study described in more detail below.
5. Translation towards Clinical Applications
The main results in the translation of research findings towards clinical application are specific to cancer, diabetes and cardiovascular disease.
5. 1 Cancer
Beyond these in vitro and in vivo mouse models, in ENCITE it has been also possible to monitor the migration and anti-tumour function of immune cells in cancer patients during therapy. One focus was on specialised antigen-presenting cells (DC), which were generated from patients’ monocytes by a standardised in vitro culture method and then loaded with melanoma-specific tumour antigens. In ethically approved phase I-II trials, melanoma patients with advanced disease repeatedly received their antigen loaded-DC as “tumour vaccines”. After therapeutic application, DC migrate to various lymphoid organs and instruct the CTL to identify and combat the melanoma antigen. If successful, CTL and other immune effector cells are activated by the DC, invade the metastases and destroy the tumour cells.
Cancer patients were recruited to a clinical trial and were monitored with a tracer in order to detect an antigen-specific immune response in vivo shortly after vaccination. This tracer offers a sensitive tool to study the kinetics, localisation and involvement of proliferating lymphocyte subsets. On the trial several patients demonstrated a broad response to numerous peptides prior to vaccination. Furthermore, tetramer-based 8-colour flow cytometry has become a routine monitoring assay, allowing extended phenotypic and functional characterisation of T-cell subpopulations. The identification of polyfunctional T-cells was of particular interest, since these have been shown to elicit more effective immune responses in HIV vaccination trials.
In summary, DC were exported to vaccinate melanoma patients and it was demonstrated there is a statistically significant correlation between a favourable clinical outcome and the presence of vaccine-related tumour antigen specific T cells in delayed type hypersensitivity (DTH) skin biopsies. However, this favourable clinical outcome was only observed in a minority of the treated patients. It was obvious that the DC-based vaccination protocols need to be improved, and the fate, interactions and effectiveness of the injected DC became the subject of some proof of principle trials. Our results confirm that DC immunotherapy is well feasible non toxic and effective in some cancer patients. However, many questions still remain. One of the concerns related to ex-vivo generated DC is how to ensure effective migration to the T cell areas in the lymph node.
5.2 Diabetes
With respect to diabetes, a protocol for measurement of high resolution MR images at 3T was developed. The purpose of the studies was to optimise the set of MR sequences and evaluate techniques suitable for routine examination of patients before and after PI transplantation, which allow quantitative data post-processing including quantification of transplanted PIs. The MR imaging and spectroscopy was performed on a 3T Siemens Trio scanner equipped with a body resonator surface coil. Different sequences and protocols were tested.
SSFP sequences were modified and two other types of sequences were developed. The echo dephase SSFP sequence was successfully applied on an animal model, however unfortunately its application on patients was not successful. Therefore a sequence, based on an ultra short echo protocol, was tuned and tested on a Siemens Trio system and used later as a part of the examination protocol to prove the presence of SPIO nanoparticles in human liver.
Different evaluations methods of liver MR images with labelled pancreatic islets were tested. Manual, semi-automatic calculation, dual contrast technique and texture analysis described precisely the position and number of labelled pancreatic islets in the liver and the data can be used for description of therapeutic function of transplanted pancreatic islets.
In total, 17 patients with transplanted PI according to a standard clinical procedure were examined, 11 of them were transplanted repeatedly. As a result, there were a total of 24 transplantations and 92 MR repeated examinations. Labelled PI were visible in human liver as hypo-intense spots on the MR image 6 months following transplantation.
The increasing concentration of fat in the liver and around the transplanted PI provides interesting information about the fate of islets and the surrounding tissue. A conventional spoiled gradient echo (SPGR) sequence for detection of the tissue was modified with very short T2 components and quantification was performed, e.g measured T2* maps and water/fat distribution. The protocol was tested on volunteers and patients with transplanted liver who underwent liver biopsy. Obtained data served as a reference to the MRI and MRS data of patients with transplanted PI into the liver.
An excellent agreement between MR results of preclinical experiments and clinical tests was found which confirms that MRI and MRS represents a suitable tool for observation of labelled pancreatic islets, changes in the tissue around the grafted islets and monitoring of cell transplantations in humans in general.
5.3 Cardiovascular disease
In validating novel imaging tools for clinical application, studies were performed in a clinically relevant pig model in which cardiac magnetic resonance techniques for evaluation of cardiac damage were validated by histopathological analysis. A distinct correlation between the histopathological findings and the 3T-MRI reading at T2 sequence (myocardial hemorrhage, infarct zone and microvascular obstruction) was observed. These techniques may therefore provide a non-invasive approach for assessing various types of cells, such as adipose derived MSCs, and routes of administration over time and evaluate both short term effects and long term effects on cardiac performance.
The newly developed tool for myocardial strain analysis based on a Bayesian estimation framework, implemented by means of reversible jump Markov chain Monte Carlo (MCMC) methods, in combination with information about the heart dynamics, the imaging process, and tag appearance has been tested in rat models, pig models and on human tagged cardiac MRI. The new method was shown to improve the performance of even the best of four frequently used methods. The new method yields higher consistency, accuracy, and intrinsic tag reliability assessment; the proposed method allows for improved analysis of cardiac motion
6. ENCITE’s ethical statement on the use of animals
The ENCITE consortium has explicitly stated in written how they apply the Directive on the Protection of Animals used for Experimental and Other Scientific Purposes and described how they have considered and implemented the three principles replacement, refinement and reduction as follows:
a. Replacement
Wherever possible, animal studies were replaced by experiments in silico or in cell cultures in vitro. This principle has been employed exten¬sively, for example in the sections dealing with cancer.
b. Refinement
Refinement was applied in breeding, accommodation and care and in animal experiments, where methods were applied eliminating or reducing to the minimum any possible pain, suffering, distress or lasting harm to the animals.
c. Reduction
The replacement and refinement principles were both contributed to the overall goal of reducing the use of animal experiments. Animal studies were only be used at advanced stages of investigations when few, specific and highly relevant questions were addressed by a limited number of experiments. In addition, minimum number of animals was used in all experiments to achieve statistically significant data. Experimental methods reducing significantly the number of animals were preferred, e.g. MR imaging, allowing for repetitive and safe measure of status of animals during long time periods, was used. To avoid potential overlaps and unnecessary duplication of animal experiments they were controlled by ENCITE’s senior scientists in their organisations’ research work.
A minimum number of animals were used in all experiments to achieve statistically significant data. All animals were treated humanely according to Institutional Animal Care and Use Committee guidelines.
Furthermore, the care and welfare of laboratory animals was ensured in line with the following conditions:
a.
Animals were kept under standardised environmental conditions of temperature and humidity and a day/night cycle; animals were kept in appropriate cages. Any restrictions on the extent to which an animal can satisfy its physiological and ethological needs were kept to a minimum. Animals were provided an environment, food, water and care appropriate to their health and well-being.
b.
Surgical procedures were always performed under local or general anaesthesia. Analgesia or another appropriate method was used to ensure that pain, suffering and distress are kept to a minimum.
c.
At the end of experiments animals were killed using a painless, quick and appropriate method (as specified in the Directive).
d.
Methods alternative to the use of animals, such as cell cultures, were employed where possible and applicable, and the assessments of biological actions of novel probes for imaging were always first carried out and validated in vitro, prior to animal studies.
Potential Impact:
1. Potential Impact on European Medicine and Healthcare (clinical application)
The ENCITE project has set-up and will continue to maintain an interdisciplinary platform integrating groups working on translational imaging across Europe. The project has been able to integrate skills in physics, chemistry, cell biology, immunology and medicine, all centred on imaging that is unique. The research work carried out in this consortium has led to the achievement of a number of relevant results.. ENCITE brought together skills and facilities from two distinct imaging communities: the more clinically oriented MRI community (radiologists, probes’ and sequences’ developers) and the more biomedical oriented basic research community (biologists and immunologists) MRI and optical imaging have been the modalities most used in the project. This collaboration is of major importance for the further integration of the imaging field as the key-tool for the development of cell based therapies.
Looking towards the future and critically reflecting on the statement “cell imaging and tracking plays a central role for tomorrow’s medicine”, ENCITE scientists highlight how their core research work has potential key impact on European medicine and future healthcare along the following direction:
1.1 Novel Imaging Technologies
The achievements reached are well suited to enable more efficient and sensitive diagnostic and clinical tools in the field of cell therapy. A number of new imaging biomarkers, MR probes and new post processing algorithms were developed and are on the brink of translation into the clinical environment, greatly facilitated through the interdisciplinary collaboration within the ENCITE consortium. Given the broad range of applications, the difference in the species observed (from mouse to man), the range of field strengths (3-11.7T) and the different platforms, it is anticipated that the developed toolboxes and techniques will be sufficiently flexible such that they can be adapted to a range of different specific studies. These new techniques will help in the development and quantitative evaluation of new therapeutic concepts. The development of advanced techniques for new hardware will also facilitate the use and distribution of these newly available techniques and machines. The main benefits for European medicine are the increasing availability of non invasive in vivo imaging tools that are also more efficient and allow a more comprehensive look on new therapeutical questions. One of the major limitations of MR imaging was the high cost and the long examination times that are not well tolerated by the patients and limit the use of MR imaging technologies in clinical studies. The collaborating partners enabled the development of advanced techniques that are more efficient and sensitive and will therefore promote the use of these techniques for more patients.
1.2 Novel Imaging Reporter Probes
The results obtained are extremely encouraging with respect to the final goal of providing highly sensitive agents and procedures for an efficient labelling of cells. The design of efficient labels is crucial for pursuing "in vivo" cell tracking. The partners’ achievements have allowed the set up of a large library of powerful imaging reporter probes. The availability of such a repository of chemical and biological reporter probes will facilitate tackling novel advanced applications in the field of cell therapies. Furthermore, the novel cellular labelling procedures, which have been developed as part of the ENCITE project, have been shown to be more effective than the standard methods currently being used, and they are expected to have a high impact on future clinical practice.
1.3 Novel Tools for Cell Labelling
The outcome of the research work is a portfolio of tools for non-invasive monitoring of cells within living organisms. It is anticipated that these tools will help improve the understanding of disease processes, and thus could aid in development and monitoring of cell-based therapeutic interventions.
1.4 Pre-clinical Validation
The various tools developed for the different application fields are expected to provide a better understanding of the fate of transplanted cells and of how cell-based therapies provide a therapeutic benefit. Thanks to the multi-disciplinary nature of this consortium and the strong collaboration, ENCITE members were able to leverage each other’s expertise in order to successfully achieve the pre-clinical validation of the newly developed probes and methodologies and their translation towards a clinical application. Although various tools were developed and validated within studies focused on a specific application field, a large number of these developed tools will have a broader impact, such as novel contrast agents (for example bimodal agents as developed in studies focused on neurological disorders and diabetes) or Gd-liposomes (as developed in studies focused on cardiovascular disease) and reporter genes for neo-angiogenesis. Next to these innovations, a number of field specific advancements were linked to specific pathologies associated with specific disorders. These include tools for monitoring lineage specific differentiation, assessment of tissue function e.g. analysis of tagged cardiac images, assessment of GAG content and assessment of T-helper function. The complete portfolio will have a significant impact on research in the specific application fields addressed but also in general on cell imaging studies. A number of the developed and validated tools are only suitable for preclinical studies but various tools have potential for (routine) clinical application. Most of the techniques for assessing tissue response have this potential, but also various cell imaging techniques show promising results as specified below.
1.5 Translation towards Clinical Application
ENCITE expects that the newly developed imaging tools will significantly improve the translation of cell-based therapies towards the clinic. By leveraging the knowledge obtained in the other ENCITE research work programmes, cell therapy treatment strategies can be optimised to reap the full therapeutic potential of cell-based therapies.
The ENCITE consortium partners used the opportunity to perform clinical studies on the transplantation of SPIO labelled PI to patients. Significant conclusions are as follows: the clinical outcome of a patient group with labelled islets does not differ from the patients with native islets; the islet visualisation was successful but less efficient during a labelling period below 16 hours; a significant decrease of islet spots occurred in the first week suggesting early islet destruction or impaired engraftment; afterwards, islet spot numbers decrease slowly, and the islets are visible for at least 24 weeks; significant C-peptide production, glycosylated hemoglobin (HbA1c) were found in all patients, near to normal values; insulin dose reduction were achieved following one or two islet transplants, but only one patient became fully insulin independent; an excellent agreement between animal models and clinical experiments was found.
ENCITE’s novel MR and multimodal imaging methods should serve to increase the translation from preclinical studies to early clinical trials and their ultimate patient application. Several developments are currently under evaluation for approval, including the anti-angiogenesis treatment for Glioblastoma (approved and currently under clinical evaluation), the first fluorine compound for clinical DC vaccines in a patient (approved by the Food and Drug Administration) and the process using new generation fluorinated polylactic-co-glycolic acid (PLGA) particles (currently under evaluation). In 2013 clinical trials can hopefully be started using these particles within in vivo settings for cancer patients.
ENCITE’s scientists are confident that these technologies will help to speed up developments in cell therapies and that their entry into wider routine clinical practice could offer potential cures for many different types of diseases, including cancer, cardiovascular disease and diabetes, as well as in stroke and musculoskeletal diseases.
Cell therapy appears to holds the future promise of personalised medicine with the potential to cure chronic diseases. The ultimate successful implementation of cell therapy as a treatment modality in the clinic requires a means to follow the fate of transplanted cells once inside a patient by widely applicable non-invasive monitoring tools. To this end, ENCITE has developed robust tools that make realistic their application in the treatment of major diseases.
In the long-term, ENCITE’s ultimate goal is to see cellular therapy and image guidance enter widespread clinical practice allowing for early detection and progression of disease or effectiveness of therapy.
2. Potential impact on socio-economic and wider societal implications
Up to now cell therapy and imaging techniques for the monitoring of cell therapy are not well established for many patients and diseases. As cell therapy itself is a highly dynamic field with a broad spectrum of inventions and applications it holds the potential to change the outcome of diseases that cannot be treated adequately. Since the new techniques developed by the members of the ENCITE consortium feature higher sensitivity and efficiency they will be available for more patients and provide more insights in novel therapeutic concepts and therefore facilitate the invention of new therapies for a number of diseases.
Cell-based therapy offers unique treatment options for severely debilitating or fatal disorders and diseases. The potential socio-economic impact of ENCITE’s achievements is therefore immense. In both regenerative medicine approaches and adoptive treatment strategies for cancer the advancements of ENCITE will significantly contribute on three different levels:
a.
Development of novel therapeutics due to a better understanding of the disease processes and the mechanism of action of such novel therapeutics
b.
Assessment of the efficacy of existing and novel therapeutics and thus patient management
c.
Optimisation of treatment protocols in terms of route and timing of administration of therapeutics
3. Potential impact on collaborations with relevant initiatives and EU-funded projects
The long-lasting and strong interactive way of working together has led to an extensive collaboration on the development of novel imaging tools and the implementation at the level of translational medicine across Europe with a global impact on future joint actions.
3.1 Close collaboration among ENCITE scientists
The close collaboration among the scientists has gone beyond pure institutional cooperation to achieve the work programme during the project life time and also beyond project end:
A number of project partners, mainly the experts from King’s College London (UK), Radboud University Medical Center (NL), Catholic University Leuven (BE), University of Münster (DE), University Hospital Freiburg (DE) and Max Planck Institute for Neurological Research (DE) will continue their collaboration on the use of 19F MRI. They are confident that, in a joint multi-disciplinary effort, the challenges associated with the use of 19F MRI in a pre-clinical and clinical setting can be overcome, providing biomedical researchers with potential new and exciting tools for in vivo imaging.
In addition to this interaction, the Institute for Clinical and Experimental Medicine (CZ) and the University Hospital Basel (CH) will continue their research in joint projects with respect to the development of a new modification of special sequences for the evaluation of fat in human liver.
3.2 Collaboration between ENCITE scientists and other projects
Further collaborations are intended to start with a group of experts outside the ENCITE consortium:
The FP7-funded project Euro-BioImaging (www.eurobioimaging.eu) has revealed that there is significant demand among its community for advanced training in the field of cell imaging. Such requirements can be met by the ENCITE Multi Centre Cluster for Training. Following this collaboration work the exchange of knowledge between both groups represents an excellent potential to showcase the cutting-edge research findings and training resources.
Within the framework of the EU-funded Marie Curie project “BetaTrain” (http://www.betatrain.eu) the Catholic University Leuven (BE) and the Institute for Clinical and Experimental Medicine (CZ) will continue their intense relationship for additional science activities on the evaluation of new contrast agents based on F19 where several joint experiments are prepared. The exchange between a number of students from both project partners will start in spring 2013.
4. Impact created by training activities
The high-valued impact created by ENCITE’s training developments is immense:
4.1 Exchange of knowledge among ENCITE scientists
Presenting the status of methodological developments on their current pulse sequence developments and fostering interactions between all experts involved in this research field the University Hospital Freiburg (DE) conducted an internal workshop to discuss the needs on new methods for cell tracking and imaging biomarkers and to identify action items to successfully finish the relevant work within a limited number of months following this meeting, held in 2009.
After a research phase of two years the ENCITE consortium decided to arrange a consortium-internal workshop to foster interactions and collaboration between the partners themselves but also between the expert groups of individual work packages and/or between different key subjects for the development of specific techniques. This first consortium-internal workshop was held at the University of Mons (BE) in 2010.
After another successful year of research, the ENCITE experts have met each other again to internally present the status quo of their current developments on imaging technologies, reporter probes, cell labelling and pre-clinical tools and to discuss their relevance towards the pre-clinical validation and possible clinical application. They focussed on the real clinical needs and the most appropriate tools for translation into the clinic as well as on rules and regulations with respect to clinical trials. In conclusion, ideas were presented for specific further developments and improvements and with respect to future research strategies for optimal detection sensitivity, quantification and imaging cell differentiation. This workshop was held at the Weizmann Institute of Science (IL) in spring 2011.
Following the discussions during this workshop, more than 20 scientists from ten ENCITE research organisations have met each other at the Max Planck Institute in late 2011 to discuss pitfalls and challenges towards a practical use in clinical studies. According to a questionnaire prior to the workshop, the participants compared 19F MRI protocols, set up and achieved sensitivity for cell tracking. In this respect, the workshop found answers to the issues as how to improve the sensitivity by new 19F compounds, strategies to increase 19F uptake by cells, the design of designated 19F MR hardware and the optimisation of MRI methods. The expert group showed the high potential of 19F MRI to monitor the fate of chemotherapeutics, to image dendritic cells implanted for the treatment of cancer, and to better image neural stem cells donated for tissue regeneration in the damaged brain. In conclusion, the workshop provided an excellent overview of the highly developing field of 19F MRI for cellular and molecular imaging. The pitfalls and challenges towards a practical use in preclinical and clinical studies were discussed in detail.
Consecutively, a 2-day symposium was organised at the Radboud University in autumn 2012. The first day was open to all European scientists, and the second one was reserved for detailed discussions among the ENCITE experts only. A wide range of topics were presented and discussed for further improvements, such as novel probes, design and optimisation of imaging sequences, cell labelling and hardware development. In total, an audience of more than 50 participants and 15 abstract submissions were received. Both workshops supported substantial interactions between researchers working on the new 19F MRI hot topic. It is planned to continue these meetings on a regular basis to allow further improvements and a better visibility among the imaging community.
4.2 Establishment of a knowledge dissemination platform
Looking towards a future training concept in an effective and sustainable way, the ENCITE Multi Centre Cluster for Training represents an efficient knowledge dissemination platform which will play a crucial role in the success of the post-project impact. The consensus at the end of this project is that training, especially advanced on-site courses, is highly valued by the research community throughout Europe.
Seven centres at the Universities of Torino, Mons, Nijmegen and Paris-Descartes (FR) as well as Max Planck and Weizmann have developed a programme to ensure a co-ordinated training approach for the wider scientific community with a significant impact on trained staff members in molecular and cellular imaging (research and clinics). The training programmes address senior researchers, graduate students and young researchers using the new technologies and demonstrating their benefits for cell imaging and tracking, imaging probes, nanoparticles, in vivo molecular imaging, multi-modal imaging, intravital microscopy, imaging tools and magnetic labelling. The centre established by the University of Paris-Descartes has been mainly focussing on radiology, image processing and the clinical application, thus ideally complementing the other centres.
Within the Cluster, more than 20 teaching files (one designed with an audiovisual video explaining the contents) and training documents as well as one e-course dedicated to the project’s novel and improved techniques have been prepared, in addition to the face-to-face trainings.
The centres have addressed and will continue on further developing different training modules offering hands-on courses and additional educational programmes on their specific technologies in the fields of chemistry and biology, developed within ENCITE. All centres’ activities will be complementary to each other to make use of synergies and mutual benefits.
Such an efficient knowledge dissemination platform will play a crucial role in the success of the post-project impact, leading to the implementation of the newly developed innovative tools and methodologies across Europe.
5. Main dissemination activities
Led by the European Institute for Biomedical Imaging Research (EIBIR) and in close collaboration with a majority of the project partners, a huge number of promotion activities and materials have been carried out to ensure widespread information on ENCITE, its promising results as well as on its dedicated training programme, based on the detailed ENCITE Dissemination Plan.
These activities ensured the information flow to different target groups of molecular and cellular imaging, of other imaging-relates disciplines and the general public at large to raise the awareness of ENCITE’s collaboration for further research and technologies, regulations and collaboration activities to strengthen the future work in this research sector.
5.1 Key carriers of ENCITE messages
The most important visual carriers of ENCITE’s messages were the development of a Project CI and logo specified on the project’s key subject and the website from the beginning onwards and a promotional video about the key outcome to be used also after project end:
Project CI and logo
The project CI and logo have proved very efficient throughout the project, allowing easy recognition of any project communication by the relevant scientific community. Information flyers on the project have been distributed widely at relevant scientific meetings and among other stakeholders (including industry) in order to inform about the advances achieved within the project.
ENCITE Website
The ENCITE website was updated regularly, in particular the Quick Links, News and the Press sections which were used to advertise research findings, upcoming (training) events, publications and references, annual reports and press releases on ENCITE workshops’ outcome and interim results to summarise key research findings. In addition, the ENCITE Multi Centre Cluster for Training webpage including the Interdisciplinary Training Platform was regularly updated. Each centre’s training activities, teaching files and additional materials as well as documentation for novel probes and associated information were uploaded onto the cluster’s website and the online repository for making the information available for download to the public.
http://www.encite.org
http://www.encite.org/cms/website.php?id=/en/index/mcc_training.htm
ENCITE Blog
The partners contributing to the Cluster for Training created an Internet Blog to ensure additional communication on ENCITE’s news and trainings to the scientific community during the project life time and particularly beyond the project’s end. This is intended to provide a resource on a long-term where posting of news and training updates can be continued on a long-term basis.
http://www.encite.org/blog/
ENCITE video “living cells as an active drug”
Launched at the final workshop, a promotional video within the framework of ENCITE’s translation of technologies into the clinics was produced. The video is showcasing how in vivo image-guided cell therapy is revolutionising medicine and describing ENCITE’s strong contribution to this revolution. ENCITE has made a real impact on the treatment of healthcare problems that affect our every day lives. To better understand how cell therapy works, prominent researchers have been working together to develop imaging tools for novel cellular therapies. The project partner RUNMC took over the scientific responsibility of the video contents and format; EIBIR promoted the video to all its contacts in the research and academia communities as well as to the EIBIR shareholders and the interested public. The video is also seen as kind of an educational kit to raise the awareness of ENCITE’s training cluster. The video has been provided to the World Wide Web users on different websites, such as: ENCITE, EIBIR, ENCITE Blog, Facebook, Twitter, several project partners’ websites, YouTube, etc. A dedicated press release and advertisements for this video was also published in the EIBIR Scientific Annual Report, the European Radiology, ECR Today, MAGMA, Auntminnie.com and several Internet channels.
http://youtu.be/xnVNKwEFZyE
5.2 ENCITE final workshop on the future of cell imaging and tracking
In addressing the questions “What are the clinical needs for imaging and improved disease diagnosis?” and “What are the best tools for translation to the clinics?” the ENCITE opened their workshop by presenting the most advancing technologies in the morning sessions. In the afternoon session, international key note lectures presented topic-related developments over the years. Based on this input, the ENCITE experts and workshop participants enjoyed a lively Round Table Discussion addressing the questions “What will cell imaging look like over the next 10 years?”. Within this discussion round, special emphasis was given on the idea of an entirely new system for funding major multinational clinical research on diagnostic products that are needed to be created by industry, academics, and regulatory bodies. The workshop was held at the Leiden University Medical Centre in Leiden/NL in November 2012.
5.3 ENCITE presence at public events, conferences and workshops
Extensive dissemination activities and trainings took place in collaboration with the European Society for Magnetic Resonance in Medicine and Biology (ESMRMB Congress 2009-2012 including the ENCITE Mini-Categorical courses 2011-2012 and the ENCITE Lectures on MR 2011-2012), the European Society of Molecular Imaging (EMIM Congress 2011-2012, TOPIM 2011-2012) and the World Molecular Imaging Congress in 2009, 2010 and 2012.
An educational session was held at the World Molecular Imaging Congress (FR) in 2008. There were an estimated 100-120 people in the audience for this educational session. Although, MRI plays a junior role at this International Molecular Imaging conference participation by the audience was very good. Further and more intense discussions went on directly following the session in personal discussions between individuals and the speakers. The high interest was also reflected in the lively discussions in the hall after the session.
In 2011, the University Hospital Freiburg hosted a European symposium on the future prospects of preclinical MR imaging where a number of promising advancements and future prospects were presented and discussed, together with internationally renowned experts.
In addition, dissemination activities were carried out and presentations were held at other related congresses, including Deutscher Röntgen Kongress 2011, the International Society for Magnetic Resonance in Medicine 2011-2012, the Radiological Society of North America Congress, the European Congress of Radiology 2010-2012, the European Association of Nuclear Medicine Congress 2011-2012 and the Journées Francaises de Radiologie 2011-2012.
Under the lead of EIBIR, the Institute for Clinical and Experimental Medicine, the University Hospital of Basel, the University Hospital Freiburg, Max Planck, the University of Torino and the Leiden University Medical Centre were actively involved in these congress actions.
5.4 ENCITE promotion materials
Various dissemination materials were developed and produced for different target sectors of experts in sciences, relevant industry sectors and the general public at large, such as: fact sheets, flyers, posters, booth presentations at congresses, ENCITE event calendars, print newsletter ENCITE inside and the brochure about the proceedings and outlook, etc.
They were distributed at a huge number of relevant workshops, congresses and public events, which captured ENCITE’s progress and highlighted its key successes in science and training, and to the ENCITE scientists’ partners at individual meetings of ENCITE scientists.
5.5 Media work and press campaign
At the beginning of the project, the Austrian National Contact Point published a “success story” about the project and its ambitious vision in suitable diffusion quality targeted to the German-speaking general public at large.
Press mailings with interim results were distributed to EIBIR’s media list. As a result, some (e-)newspapers published some spotlights about ENCITE, such as: Der Standard, The Times UK, Le Soir, Die Zeit, medical physics web, Life Science Vienna region, WorldPharmaNews, Universum Magazin and EC Research Information Centre, etc.
On an annual basis, a summary on the project progress and interim achievements was published in the EIBIR Scientific Annual Reports between 2008 and 2012. The report was distributed to all EIBIR Network Members (ca. 280), project and other partners (ca. 130) and at public events and conferences.
Over the years, major intermediate results including regular updates and public ENCITE sessions and lectures were drafted and communicated by EIBIR, in close collaboration with individual partners.
In addition, interim results, programmes and event and training announcements were published in a variety of scientific-related magazines several times per year (e.g. ECR Today, European Radiology, MAGMA, different congress programmes (ESMRMB, TOPIM, EMIM, European Congress of Radiology (ECR), WMIC), ESR/EIBIR/ENCITE e-newsletters and ENCITE dedicated mailings, etc.).
A European-wide press campaign was set up in 2011 dedicated to regularly communicate on ENCITE’s promising findings and its ENCITE’s training courses and its outcome to all scientists and stakeholders interested in the field molecular and cellular imaging and tracking and imaging-related disciplines.
At the end of the project, a media briefing was held with the journalist from Auntminnie.com (Auntminnie Molecular Imaging Insider! Europe) who published a press release about the Round Table Discussion at the ENCITE final workshop, held in Leiden/NL in November 2012. The experts focussed on the discussion of hundreds of promising target molecules identified in preclinical studies for possible use as novel cell imaging agents and tried to find answers on the questions “How many are ever likely to be used in real patients? And who will pay the costs of the clinical trials needed to prove their value?”.
5.5 ENCITE communication channels and target groups
The dissemination of ENCITE’s scientific advances, newly developed technologies and trainings was aimed at distributing it to the peers and experts of the scientific community in molecular imaging and all other disciplines related to cell imaging and tracking, including researchers and clinicians, the medical imaging industry as well as the media and the general public at large.
In addition to the project and partners’ websites, the websites of ESMRMB, European Hospital, Auntminnie, eHealth news and CORDIS have become the external online key communication channels for ENCITE. In addition, a high number of websites from co-operating partners, online platforms and event calendars as well as social media platforms (such as Facebook and Twitter) have been used to communicate regular updates, project results and scientific advancements as well as public events and trainings.
In total, a mailing list of more than 9.000 addresses was used for regular updates as well as event and training announcements. This list includes EIBIR Network Members (ca. 250), EIBIR stakeholder organisations (8) and industry partners (8), ESR Research Committee members, ESR members interested in molecular imaging (a. 8.000) participants of ENCITE events and trainings between 2009-2012 (ca. 400), the media mailing list (ca. 60), ESMRMB members (ca. 1000), ESMI mailings, and other stakeholders throughout Europe, etc.
For the entire dissemination activities including participation in conferences and workshops, publications, printing materials, web applications, press releases, presentations, posters, thesis and interviews, etc. please see the list on dissemination activities.
6. Exploitation of results
One of ENCITE’s major objectives has been to develop and test new MR and optical imaging methods and biomarkers to get a more comprehensive picture of cell fate and the reaction of the immune system and to ultimately improve and further develop cell therapy for the benefit of the European patient.
6.1 Scientific publications and presentations
To demonstrate these benefits for the European healthcare and the citizens of this multidisciplinary collaboration in the pre-clinical and clinical sectors on a European level and to raise the awareness of ENCITE’s impact on treatment, a huge number of publications, posters, presentations, dissemination activities and discussions were produced and held. The consortium partners published more than 170 (peer-reviewed) publications in scientific journals, highlighting their research results and presenting the achievements of the implementation of the project work programme. More publications are in preparation.
One of the publication’s highlights is a special issue of Contrast Media and Molecular Imaging dedicated to the major achievements of the ENCITE project, is currently in preparation. This will be a joint publication of mini-reviews and research articles. This publication is an opportunity to underline ENCITE’s strong collaboration and its significant achievements. Key topics, among other, are reporter genes, optimised multimodal neuro-imaging strategy for cell tracking, islet/beta cell imaging, cell imaging, clinical translation and toxicity, imaging biomarkers and sequences, nano systems for cell labelling, multimodal in vivo cell tracking and strategies for cellular labelling with paramagnetic metal complexes. The contributing scientists are from the Universities of Torino, Leuven, Paris-Descartes, Mons, Freiburg, Basel, Nijmegen and Erlangen as well as from Erasmus MC, Weizmann, Max Planck, Leiden University Medical Centre, Institute for Clinical and Experimental Medicine, King’s College London and the Foundation for Applied Medical Research. The publication of this special issue including this joint paper of approximately 100 pages is expected in summer/autumn 2013.
6.2 Exploitable foreground
In order to bring the expected results into pre-clinical and clinical practice, some of the ENCITE consortium members have started with specific exploitation paths. Several ENCITE consortium partners have been actively seeking protection of intellectual property:
With respect to exploitable foreground within ENCITE, the Hebrew University of Jerusalem has investigated in general advancement of knowledge with respect to both the methods for cell labelling and imaging in vivo and for cell injection to intervertebral discs in the research and medicine sectors. They have investigated on the following to be further worked on in future: first, labelling and imaging methods aimed to monitor stem cell survival and biodistribution. Hence, these methods could be exploited for further research of stem cell therapies. Second, do cell injection to the intervertebral disc. These results could be exploited for further research in cell therapies for intervertebral disc degeneration. Further research will be required in order to develop efficient stem cell therapies for degenerate discs. The expected impact of these findings would be of greatest value in the biomedical research field.
Four patents on ENCITE foreground have been filed by the Agencia Estatal Consejo Superior de Investigaciones Cientificas (CSIC) (ES), Erasmus MC, University Hospital Erlangen (DE) and University of Torino (IT). The University of Torino has applied for a patent on CEST agents allowing the visualisation of more cell typologies in the same MR Image. If approved, CEST agents may be then exploited for tracking different cell typologies. The University Hospital of Erlangen has a patent filed for dendritic cell therapy in cancer.
Particular importance is placed on the fact that two partners are currently preparing the establishment of spin-off companies:
CSIC is planning to create a spin-off company entitled BIOIMAG for the commercialisation of collagen and other extracellular matrix components functionalised with magnetic nanoparticles to be used as an “intelligent” contrast agent capable of reporting enzymatic activity. Cage Chemicals has introduced in its catalogue the most effective Gd-based agents developed in ENCITE.
The University of Mons has been working on the development of simultaneously reproducible batches of negatively charged or neutral PEG coated particles. These nanoparticles are available to all partners of the ENCITE consortium through a spin-off company entitled AGECO, which is currently under development.
The detailed list of scientific publications as well as the list of patents and exploitable foreground can be found below.
6.3 Collaboration with SMEs
During the project life time, all small and medium sized companies (SMEs) have obtained great advantages from the collaborative work carried out during the project life time. Several ENCITE research organisations have intensified their collaboration with SMEs in order to establish a joint strategy with respect to new approaches for technologies and exploitation to support specific research activities which might not bring immediate commercial benefits but could be significant in the longer term. Their positioning in their respective market appears definitively stronger when compared with the relevant numbers at the beginning of the project.
Cage Chemicals has doubled its staff and settled its activities in a larger space (from 66m2 to ca. 180m2). Thanks to the intensive collaboration work within ENCITE between Cage Chemicals, the University of Torino and Weizmann an important cooperation agreement has been signed between CAGE Chemicals and the Israelian company ASPECT Imaging. Based on the agreement, CAGE Chemicals will provide ASPECT costumers with a dedicated line of Gd-based products, in part generated by ENCITE. Furthermore, CAGE Chemicals has added few new products to its new catalogue and has definitely improved its visibility in the market of products for Imaging.
Max Planck and the SME Medres medical research GmbH (Medres) (DE), have developed new anesthesia gas monitor systems to improve the accurate determination of the inhaled gas mixture during longitudinal small animal experiments. These systems have successfully been installed in high-field MRI systems at the laboratories of MP. Within this collaboration, an integrated animal bed/monitoring system has also been developed and continuously improved for a separate version for MRI application of mouse and rat, respectively.
The close co-operation between the experts of Max Planck and the SME Biospace Lab. has led to the improvement of animal pedestals that have been designed and applied for allowing simultaneous optical imaging recording of three viewing angles. This set-up is now in routine application at the optical imager of Max Planck.
Finally, prototype designs have been tested between Max Planck and Medres concerning an innovative, non-invasive stimulation system for fMRI in anesthetised rodents. This new design has clear potential for improved performance while being at the same time, of reduced stress for the experimental animal. This device is not yet in application routine.
List of Websites:
The address of the project public website is: www.encite.org
Contact details:
European Institute for Biomedical Imaging Research (EIBIR), Project Coordination
Neutorgasse 9, 1010 Vienna (AT)
Phone: +43-1-5334064-29
office@eibir.org
www.eibir.org www.encite.org
Scientific Coordinator: Prof. Gabriel P. Krestin, ERASMUS MC, Rotterdam/NL, g.p.krestin@erasmusmc.nl
The European Network for Cell Imaging and Tracking Expertise (ENCITE) has been a strong collaboration of 29 project partners from eleven countries, working together within a multi-disciplinary project of nine disciplines with the task of seeking solutions to cell imaging and tracking. ENCITE had the long-term mission to improve and test existing and new MR and optical imaging methods and biomarkers to get a more comprehensive picture of cell fate and to ultimately improve and further develop cell therapy for the benefit of the European patient. Over the past four and a half years, the scientists have achieved ground-breaking results on the preclinical validation of techniques towards the application into the clinic.
Novel chemical probes, reporter genes and mouse models were properly developed and designed for the set-up of innovative imaging procedures in the field of cell tracking and cell therapy.
To label cells in a more efficient way, non invasive imaging methods to monitor specific cellular decisions affecting cell fate were developed. These included tools for the detection of processes, such as cell migration, differentiation, proliferation and death.
With respect to a pre-clinical validation, existing generic and specific imaging tools were further developed and validated for the application of cell-based therapies for neurological disease and stroke, cardiovascular disease, musculoskeletal disorders, diabetes and cancer. The tools included: novel probes for cell labelling, novel imaging sequences and protocols for visualisation and quantification of cell fate in vivo, and novel imaging procedures for assessing tissue response to treatment.
A key aspect of the research work focussing on the translation towards clinical application was dedicated to the improvement of the translation and implementation of novel techniques into the clinic, for the benefit of patients. Major breakthroughs were realised in establishing the infrastructure for the production of tracers for in patient use and testing novel; tools for patient monitoring in patients with cancer, diabetes and ischemic heart disease.
Training and dissemination were important activities in the project. One of the highlights was the ENCITE Multi Centre Cluster for Training to provide flexible access to face-to-face trainings, e-courses and teaching files on ENCITE’s new technologies and to offer a virtual database serving as a repository of probes and procedures in the fields of chemistry and biology. In the long term, it is planned to further establish this cluster as a key node for trainings to the entire research community and hence, to also support the implementation of innovative tools across Europe as a standard.
ENCITE’s novel MR and multimodal imaging methods should serve to increase the rate of progress from preclinical studies to early clinical trials and their ultimate patient application. Cell therapy perhaps holds the future promise of personalised medicine with the potential to cure chronic diseases. The ultimate successful implementation of cell therapy as a novel treatment modality in the clinic requires a means to follow the fate of transplanted cells once inside a patient by widely applicable non-invasive monitoring tools. A promotional video is showcasing how in vivo image-guided cell therapy is revolutionising medicine in the field of cancer and diabetes. To this end, ENCITE is intending to further exploit the project results towards realistic clinical applications within follow-up joint actions in the future.
ENCITE’s scientists are confident that these technologies will help to speed up developments in cell therapies and that their entry into wider routine clinical practice could offer potential cures for many different types of diseases, including stroke, musculoskeletal and cardiovascular disease, diabetes and cancer.
Project Context and Objectives:
1. ENCITE background and context
The mission of the European Network for Cell Imaging and Tracking Expertise (ENCITE) project was to develop and test new MR and optical imaging methods and biomarkers to get a more comprehensive picture of cell fate and the reaction of the immune system and to ultimately improve and further develop cell therapy for the benefit of the European patient.
Cell therapy can be defined as the transplantation of living cells for the treatment of medical disorders. Although the concept of cell therapy has existed for centuries, and has been part of medical practice for decades in terms of blood transfusion and bone marrow transplantations, more recently cell therapy has received increased attention and has become a main focus of (bio)medical research.
Different principles underlie this increased interest in cell therapy: The transplanted cells can be used as an “active drug”; the transplanted cells can be used to replace damaged and degenerated tissue; or the transplanted cells can be used as a drug delivery vehicle. For all these applications promising results have been obtained in pre-clinical and clinical studies, however their success rates have been variable and this limits the clinical benefits associated with stem cell therapy.
One major challenge is that the mechanisms underlying how cell therapy works are still poorly understood, and currently there is no single imaging modality that meets the requirements of stem cell therapy. Yet the ability to non-invasively monitor the fate and action modes of transplanted cells over time is crucial for a better understanding of how cell therapy works in various disease states.
ENCITE has aimed to address these issues by developing and testing MRI imaging methods and biomarkers in order to get detailed information on the in vivo fate of transplanted cells and the hosts reaction to the transplanted cells. Ultimately, the project objectives were to translate improved techniques from the laboratory to the clinic, and apply them in the treatment of stroke and neurological, cardiovascular and musculoskeletal diseases as well as diabetes and cancer.
Magnetic Resonance Imaging (MRI) is one of the most powerful tools in clinical diagnosis and biological research due to its exceptional spatial and temporal resolution in the visualisation of soft tissue, including brain, heart and liver. However, the main disadvantage of this visualisation method is its limited sensitivity: target cells must be labelled with a large amount of MRI probe (commonly magnetic nanoparticles) to achieve high definition visualisation. Thus one of the key objectives addressed by the ENCITE scientists was to improve MRI sensitivity by increasing probe stability within biological systems, by enhancing cellular uptake as well as strengthening individual probe efficiency.
2. ENCITE’s main objectives
2.1 Generic and disease-specific objectives
In order to be able to address the extremely wide variety of cell therapies, the overall project objectives were divided into generic and disease-specific categories as follows:
The generic objectives included the development of new imaging methods to improve the spatio-temporal tracking of labelled cells, the generation of dual- and multimodality imaging procedures to cross-validate each individual approach, and the generation of new contrast agents and procedures that would improve the sensitivity and specificity of cellular labelling. For the generation of molecular and cellular imaging reporters, the potential of combining molecular biology with multimodal imaging techniques was explored, and novel cell and animal reporter systems were generated that enable detection of the location and function of individual cells and small cell subsets within the target organ. Appropriate cellular labelling techniques, that do not interfere with cellular functions and therapeutic efficacy, were established and new methods for quantitative assessment were developed to generate reliable biomarkers of cell fate and therapeutic effects. The overall aim was to establish and validate cell homing for therapeutic delivery to target organs. The legal and ethical issues associated with the use of new agents and methodologies were also properly addressed.
The disease-specific project objectives were to establish the clinical potential of novel imaging approaches in preclinical studies related to neurological, cardiovascular and musculoskeletal diseases as well as to cancer and diabetes, and also to improve existing cell-based therapy regimens in cancer.
2.2 ENCITE’s main objectives of its individual research fields
The ENCITE project was divided into five areas in research entitled novel imaging technologies, novel imaging reporter probes, novel tools for cell labelling, pre-clinical validation and translation towards clinical application as well as one area dedicated to the development and long-lasting establishment of training activities. These work programmes addressed the following main objectives:
2.2.1 Novel Imaging Technologies
This research field focused on the development and improvement of MR imaging techniques used in cell tracking, the evaluation of cell fate and the validation of cell therapy: on one hand, it aimed to provide new imaging methods to improve the spatio-temporal resolution of the imaging techniques, which in turn allows for the more detailed tracking of labelled cells. In addition, new methods for the quantitative assessment of transplanted cells and generated new and reliable biomarkers of cell fate and therapeutic effects were developed. On the other, the research aimed to develop novel image post-processing techniques to allow the stable and reproducible evaluation of experimental results. To achieve these goals, new hardware and advanced concepts were developed and utilised to develop new labels and to discover a more effective use for existing ones. This included advanced imaging sequences for cells labelled with iron oxide nanoparticles, novel fluorine containing compounds, imaging techniques for musculoskeletal imaging and multispectral biomarkers for the imaging of neo-angiogenesis and brain diseases. These post-processing techniques included tools for visualisation, co-registration as well as algorithms for quantitative evaluation. A major goal was the integration and co-registration of different imaging modalities to allow a more comprehensive assessment of the different modalities that have been developed.
2.2.2 Novel Imaging Reporter Probes
By addressing this research the overall aim was to develop chemical probes, reporter genes and mouse models designed for the establishment of innovative procedures in the field of cell tracking and cell therapy. Additional objectives were to increase the number of reporter genes available for MRI by the evaluation of novel candidate reporters as well as to increase the utilisation of MRI reporter genes by applying them to monitor the constitutive expression of genes and the detection of changes in gene expression as well as imaging of cell differentiation. To date, no imaging approach has been reported as being suitable for the direct detection of cell proliferation, and bioluminescence imaging has been used instead to determine increases in cell number. The objective was to combine strategies for the simultaneous imaging of cell proliferation, differentiation and death, and therefore multi-parametric information on the state and fate of the transplanted cells would be retrieved. The further development and validation of such strategies focussed on cell labelling is described in the next work programme of novel tools for cell labelling.
2.2.3 Novel Tools for Cell Labelling
In this research area the aim was to develop non-invasive imaging methods to monitor specific cellular decisions that affect cell fate. These methods included the development of tools for the detection of processes such as cell migration, differentiation, proliferation and death. Apoptosis, or programmed cell death, is a process which plays an important role in the development of multi-cellular organisms and in the regulation and maintenance of cell populations in tissues under certain physiological and pathological conditions. The ultimate objective was to establish a technique that would enable the visualisation of intra-cellular protein interactions known to occur during apoptosis. Such a method would be valuable for in-vivo imaging and for identifying new proteins involved in the apoptotic process using small interfering RNA (siRNA) screens. Analogous approaches have been taken to report in the MRI images on specific enzymatic activities.
2.2.4 Preclinical Validation
In the research area of preclinical validation, the overall objectives were to develop and validate generic and specific imaging tools for five disease-specific application fields of cell-based therapy, entitled neurological disease and stroke, cardiovascular disease, musculoskeletal disorders, diabetes and cancer.
In neurological disease and stroke, the main objective was to increase the capability of non-invasive imaging to monitor the migration, integration, functional status, and differentiation status of transplanted stem cells in addition to their interactions with other cell types. Another objective was to develop an imaging method to monitor the improvement in the functional outcome following cell therapy, such that not only the fate of the transplanted cells, but also the functional fate of the receiving organ, is monitored.
Similarly, in relation to cardiovascular disease, the aim was to develop tools that allow for an integrated assessment of cell fate, cell function and their therapeutic effect in cellular cardiomyoplasty taking into account, that from an imaging perspective, the heart represents a significant challenge. Its complex anatomical structure, continuously changing form (due to the heart beat), the large volumes of blood flowing through it (blood circulation), and its location in an environment filled with air (chest cavity and lungs) that is also incessantly moving (respiration), all contribute towards the imaging challenge.
For musculoskeletal disorders three main objectives were pursued: to develop methods for in vivo observation of the regeneration of injured tissues, the tissues being tendons, particular cartilage and the nucleus and the annulus components of invertebrate disks; to design methods using mesenchymal stem cells to induce regeneration of injured tissue; as well as to follow by MRI the fate of stem cells that were introduced to the injured tissue.
In diabetes, attention has been devoted to the development of new in vivo image methods suitable for the guidance of insulin islets transplantation into the liver.
One of ENCITE’s major objectives for preclinical validation was related to the improvement of existing cell-based therapies in the treatment of cancer. High importance was attached to the development of imaging tools facilitating the use of cell therapy in cancer by antagonising the mechanisms by which cells promote tumour progression, and additionally to the development of approaches for utilising cell homing ability for therapy delivery to the tumour. Furthermore, work has been devoted to overcome the factors in the tumour microenvironment impairing cell trafficking, cell delivery and immune effector function. In the oncological application of cells for targeted drug delivery, it was aimed to develop tools for assessing bystander effects in inducing cytotoxicity to tumour cells.
2.2.5 Translation towards Clinical Application
The key objectives focused on the translation and implementation of the novel techniques and probes developed in the previously mentioned work programmes into the clinic for the benefit of patients.
Progress made in the development of new imaging techniques and evaluation procedures during the ENCITE project demonstrates that results of preclinical studies can be successfully applied in clinical studies. An example of labelling of pancreatic islets (PI) by superparamagnetic iron oxide nanoparticles and following MR studies on an animal model proved that these imaging techniques can also be used for description of a position and fate of human pancreatic islets transplanted to patients with type 1 diabetes. In addition, the results of preclinical studies also show that MR imaging of labelled PI can be used for prediction of functional failure of PIs before clinical symptoms occur. This is an important message for future clinical applications of transplantation procedures.
Research has shown that the relationship between the immune system and human cancer is complex, highly dynamic and variable between individuals. Considering the complexity, enormous effort and costs involved in optimising immunotherapeutic approaches, clinically applicable tools to monitor therapy-induced immune responses in vivo are most warranted. However, the development of such tools is complicated by the fact that a developing immune response encompasses several body compartments, for example peripheral tissues, lymph nodes, lymphatic and vascular systems as well as the tumour site itself. Moreover, the cells that comprise the immune system are not static but constantly circulate through the vascular and lymphatic system. Molecular imaging is considered the favourite candidate to fulfil this task. The progress in imaging technologies and modalities during the ENCITE project life time has provided a versatile toolbox to address these issues.
2.3 ENCITE’s objective on dissemination and training actions
Particular attention on dissemination and training was paid to the development of a multi-disciplinary training platform providing flexible access to individual face-to-face trainings, e-courses and teaching files on ENCITE’s new technologies as well as a virtual database serving as a repository of probes and procedures in the fields of chemistry and biology. This training platform aimed to strengthen the exchange and interactions between project partners on improving their research work and further developing specific trainings, such as: 19F MRI, diffusion tensor imaging (DTI), diffusion weighted imaging (DWI), dynamic contrast enhanced MRI, vessel size imaging (VSI), imaging with ultra short and variable TE and high resolution anatomical imaging. Further emphasis was also given to optical imaging aspects, reporter probes and pre-clinical investigations, etc.
The training concept shall ensure that ENCITE’s technologies will be available to researchers and clinicians throughout Europe. A training platform, namely ENCITE Multi Centre Cluster for Training established in Belgium, France, Germany, Israel, Italy and The Netherlands, will continue to offer training activities in the long-term future in order to increase mutual benefits for the European molecular imaging community and for those groups interested in cell imaging and tracking.
To widely communicate the project advancements, the promising techniques for (pre-)clinical validation and application, and the multidisciplinary training opportunities to peers and experts, the ENCITE consortium partners focussed on carrying out all the actions described in the ENCITE Dissemination Plan. To ensure intense promotion of its advancements and trainings an effective communication network has been established by all communication channels and means available within and outside the consortium, such as: project and consortium partners’ websites, websites of co-operational partners, online event calendars and portals, magazines and publications, e-mailings and e-newsletters, (booth) presentations and individual conversations at public events, publications, individual meetings and printing materials (flyers, posters and brochures, booth presentations, etc.).
Project Results:
Along the four and a half years of activities ENCITE has achieved a number of important results ranging from very innovative proofs of concepts to the set up of protocols already suitable for clinical applications. In the following section the main results and foregrounds are summarised.
1. Novel Imaging Technologies
The research field of “Novel Pulse Sequences” was mainly focused on the development of dedicated acquisition protocols for new imaging biomarkers and new MR imaging reporter probes. The utilisation of new techniques and hardware was used to generate novel types of sequences that feature the higher sensitivity and signal strength that is the crucial requirement for the future of MR imaging technologies.
The studies included the accurate in vivo detection and segmentation of cells, or clusters of cells, and imaging methods for specific contrast agents, such as fluorine magnetic resonance imaging (19F-MRI) and ultra-short TE (UTE) imaging for bright contrast. Special attention was devoted on the translation of the newly developed sequences to the clinic. Thus, an alternative positive contrast method was developed based on a double contrast 3D spoiled gradient echo (SPGR) sequence with a variable first TE (vTE) and highly asymmetric readout for visualisation of SPIO islets on an animal model and on a patient. In addition, this novel vTE technique was used for musculoskeletal imaging.
MR imaging sequences for multiple biomarkers with clinically approved contrast agents that are used in the monitoring of cell fate were further improved and tested in vivo to facilitate the evaluation and image post-processing of these markers for a potential translation into the clinical routine. This topic received major attention since it is expected to provide more valuable information for the monitoring of patient treatments out of each single examination without the need to sacrifice patient comfort and time.
As the tracking of bacteria in vivo could enlighten multiple disease mechanisms, SPIO-mediated contrast with highly sensitive susceptibility weighted contrast imaging of bacteria for the visualisation of bacterial leakage in vivo were utilised. The potential of MR for detecting iron oxide-labelled T cells has been explored and investigated.
As the development of alternative labels in MR remains an attractive topic, especially with the development of new fluorine-compounds with enhanced sensitivity, this topic has been further investigated. A new complex has been used together with an improved UTE sequence to monitor labelled tumour cells in an animal model over a period of eight days and macrophages for more than one week. This method combined the specificity of fluorine MRI with the ease of proton MRI and it can be therefore performed on any standard MRI scanner.
Within the domain of “Image Post-processing”, molecular, functional and anatomical imaging data were integrated into a comprehensive dataset that could be investigated as a whole rather than looking into individual examinations. This was a particularly important topic since the development of new imaging methods and advanced imaging techniques yields a great number of new modalities that needed to be correlated to each other to deploy their full potential. New algorithms were investigated to be used to facilitate registration, matching and correction of motion at the anatomical level and detection and tracking of functional events at the cellular level.
The main task was the integration of molecular, functional and anatomical imaging data. 3D and 4D datasets from the brain and heart were generated and these were recorded under variable conditions using different MRI contrast and spatial resolution. The usefulness of the chosen imaging acquisition procedure and image analysis methods was evaluated, and an articulated atlas for image registration of follow-up studies was successfully created.
A number of the image analysis challenges which emerged from longitudinal pre-clinical molecular imaging studies were addressed. New algorithms were investigated that can be used to facilitate registration, matching and correction of motion at the anatomical level, and detection and tracking of functional events at the cellular level. The newly developed methodologies will enable quantified monitoring of disease progression and treatment response that is not possible with current visual interpretation, and will be playing a key role in the quantitative evaluation of future therapies.
2. Novel Imaging Reporter Probes
In the field of “Probe Chemistry”, research activities were devoted to the development of new labelling probes and their subsequent efficacy testing in vitro and in vivo. Several new MRI agents were synthesised, including paramagnetic metal complexes and superparamagnetic iron oxide particles (SPIO). The use of these MRI agents in cellular labelling has led to the achievement of enhanced labelling sensitivity and specificity. In the field of SPIO, much work has been carried out in the rational synthesis of particles of defined size and improved magnetic properties.
To fully exploit the full potential of the high spatial and temporal resolution of MRI in molecular imaging applications, it was deemed necessary to improve the sensitivity and specificity of the currently available probes. Regarding sensitivity, a new high relaxivity tetrameric gadolinium (Gd)-based agent was shown to provide an impressive seven-fold sensitivity enhancement in respect to the commercially available agents, while also demonstrating an analogous safety profile. Regarding specificity, an enzyme responsive Gd probe which reports on the activity of beta-galactosidase, an enzyme commonly used as reporter of gene expression, was synthesised and tested. Other Gd-containing probes responsive to tyrosinase and glutammato decarboxylase (GAD) have been prepared and tested on cellular and animal models.
Concerning the use of Gd containing chelates in cellular labelling, particular attention was devoted to monitoring the effect of compartmentalisation on the observed relaxivity, the fate of the internalised complexes over time and the establishment of efficient cellular uptake routes, such as endosome escape and hyposmotic shock.
The ability of Magneto-Liposomes (MLs) to act as a targeted contrast agent was also evaluated, and the successful targeting of ASGP1 receptors of hepatocytes has been reported. In comparison with the non-functionalised anionic MLs, specific uptake of the functionalised MLs by hepatocytes was observed in cultures of hepatocyte-like cell lines, primary hepatocytes, progenitor cells differentiated into hepatocyte-like cells, and in vivo after systemic administration. Furthermore, the labelling efficacy of these iron-based nanoparticles was further assessed in four different types of cells (Panc, Capan, HeLa and Jurkat).
The high affinity of Yeast Cell Wall Particles for immune system cells was exploited to label macrophages with the aim of tracking them by MRI experiments performed on J774 murine macrophages. These experiments demonstrated a high and very fast uptake of the paramagnetic particles with good temporal persistence of the contrast and an extremely low cytotoxicity.
Important advances have also been made in the field of Chemical Exchange Saturation Transfer (CEST) agents and their use in cellular labelling. The use of these MRI probes has significant advantages over the use of the more classical relaxation agents. Being frequency-encoding systems, it is possible to visualise (using different colours) more probes in a single image as every CEST agent is responsive to a specific irradiation frequency. The frequency-encoding property of this class of agents has been exploited for pursuing the visualisation of different types of cells in the same anatomical region. Two approaches have been followed: entrapping paramagnetic CEST agents into cells or, by exploiting the intracellular water molecules as a source of exchangeable protons once their absorption frequency has been suitably shifted by the entrapment of a proper Lanthanide Shift Reagent (Cell-CEST).
In addition to develop fluorescent probes to detect the differentiation or functional states of cells and tissues, novel non-linear imaging modalities which enable the detection of tissue structure and function have also been developed, including second and third harmonic generation which can be excited and detected by multiphoton microscopy. Second harmonic generation is the frequency doubling of light, which is excited by collagen fibres of healthy and diseased tissue and by actomyosin bundles present in myofibres. Third harmonic generation is the frequency tripling of light, which can be exploited by infrared excitation to detect tissue interfaces, including adipocytes, nerve tracks, erythrocytes, and surfaces of collagen bundles. The ENCITE scientists were the first group to exploit the intravital use of combined second and third harmonic generation microscopy to map the invasion routes of cancer cells during the metastatic cascade.
In conclusion, highly efficient probes for cellular labelling have been developed and tested either in cell or in vivo. A number of promising systems were developed ranging from molecular paramagnetic Gd-based reporters to nanosized iron based probes and to micron sized Gd-based probes (YCWPs).
Moreover in the field of MRI very important results have been obtained with CEST agents showing that multiplex visualisation is possible also with this modality. Finally, fluorescent probes and innovative non linear imaging modalities have been investigated to enhance the imaging information on cellular differentiation and assessment of tissue functional states.
In the field of “Molecular Biology Probes”, the objective was to develop two reporter genes for MRI which could be used to non-invasively monitor gene expression in vivo. This could be of use for example in developmental studies, tracking of implanted cells, or monitoring therapeutic transgene expression. Following the addition of Mn-apoferritin or ferritin to the growth medium of cultured cells, TIM2 (T-cell Immunoglobulin and Mucin domain 2) expression has been shown to shorten T1 and T2 relaxation times of cells in vitro. Preliminary results suggest that TIM2 expression can produce negative contrast in T2-weighted images in vivo. Since TIM2 expression does not compromise cell growth or viability, and ferritin and Mn-apoferritin uptake are well tolerated, ENCITE scientists suggest that TIM2 is a suitable protein to be used as a reporter gene.
OATP (human organic anion transporting polypeptide) expression has been shown to shorten T1 relaxation times of cells incubated with gadolinium chelates in vitro, in comparison to control cells. Additionally, OATP expression does not seem to prevent luciferase expression suggesting that the transduced cells remain viable.
3. Novel Tools for Cell Labelling
Within ENCITE a number of cell labelling strategies have been evaluated and developed. Regarding imaging probes, the work carried out focused on both generic strategies, using exogenous non-specific probes such as iron oxide particles, Gd-chelates and Fluorine (19F) compounds, and also on more tailored strategies involving smart or responsive probes and reporter genes. While it was found that some general principles regarding cell labelling protocols could be defined, it was recognised that for each specific combination of cell type and contrast agent/imaging probe, tailored approaches are required, including the technique to be used for optimal probe incorporation into the cell and assessment of retention of cell functionality.
A number of experiments were successfully carried out regarding novel tools for cell labelling and cell fate imaging, including the monitoring of cell recruitment, differentiation, and cell death. Optimal labelling conditions were defined for a number of different contrast media, for example, cellular incorporation of a responsive contrast agent into murine neural progenitor cells and labelling of mesenchymal stem cells with Gd-DTPA. For probes based on Gd-chelates it was found that the MRI signature (relaxivity) is dependent on the intra-cellular compartment the probe is contained in. Depending on probe characteristics and/or the labelling techniques used, probes either mainly accumulate in endosomes or in the cytosol. In many cases however, probes enter the cell via endocytosis and consequently accumulate in endosomes. For Gd-based probes this has a profound effect on the MRI signature, referred to as quenching of the relaxivity. Within ENCITE techniques were developed and evaluated to “rescue” such probes from endosomal entrapment. One such strategy encompassed the use of photochemical internalisation (PCI) techniques. Initial cell labelling experiments indicated the ability of porphyrins incorporated in endosomal membranes to induce the endosomal escape of contrast media by UV irradiation of labelled cells. Endosomal escape techniques based on PCI technology have been further developed for application in vivo to enhance the delivery of all molecules taken into the cell by endocytosis. Clinical Phase I/II studies in patients with cutaneous and subcutaneous tumours are currently being carried out at the University College Hospital of London. These trials are using the commercial photosensitiser AmphinexTM (TPCS2a) and the cytotoxic agent bleomycin.
As a means to potentially achieve in vivo targeted labelling of cells, the use of local sonoporation was studied. An in vitro model was established in which it was shown that cellular uptake of iron oxide nanopartcles from the surrounding medium is enhanced by local sonoporation. Local sonoporation was achieved through sonication of targeted ultrasound microbubbles with tailored pulse sequences from an ultrasound transducer. With optimised pulse sequences an up to 7 times increase of SPIO uptake compared to the control with 90% cell viability could be achieved. A ~10% labelling should be sufficient for in vivo MRI tracking. This technique might therefore provide a safe in vivo labelling strategy for endothelial cells with SPIO, where other cell labelling techniques may not be applicable.
Regarding responsive contrast agents a new tool for neuroimaging with MRI, enabling the selective detection of GABAergic neurons under in vivo conditions, was developed. The specific imaging contrast is achieved by a novel paramagnetic contrast agent, which responds to the activity of the enzyme glutamic acid decarboxylase, expressed solely by inhibitory neurons. The relaxivity of the complex is increased upon decarboxylation of two glutamic acid moieties, thus allowing increased water access to the inner and outer coordination spheres of the paramagnetic ion. The mechanism and specificity of activation were proven with tissue lysates and further applied to a differentiation protocol for murine embryonic stem cells. The relaxation enhancement was studied quantitatively and revealed decreased longitudinal relaxation times in the inhibitory neuron samples compared to the naïve stem cells in vitro and in vivo. Furthermore, this approach offers not only the discrimination of inhibitory, GABAergic neurons in the brain but also may extend the usefulness of MRI for functional imaging on a cellular level.
In contrast to labelling cells with a probe, use of a reporter gene to label cells avoids the serial dilution of the label associated with cell division. Use of ferritin as a reporter gene for MRI has been demonstrated by a large number of laboratories and in multiple model systems. The ENCITE scientists built on this research by introducing the use of heavy chain ferritin (h-ferritin) as a reporter gene, which allowed expression activation detection in cancer cells as well as in transgenic mice under tetracycline regulation. Such over-expression was found to elevate the level of intracellular iron with no adverse effect on animal wellbeing, even when expressed throughout the lifetime of the mice. Ferritin over expression was also used to detect systemic recruitment of tumour associated fibroblasts to the angiogenic rim of ovarian carcinoma xenografts. Bi-exponential relaxometry mapping provided information not only on the distribution of the cells, but also quantitative information on the fraction of labelled cells within a mixture of exogenously administered cells, native tumour and tumour stroma cells. Such information has not been derived using any other type of cell tracking method.
The main limitation of the use of ferritin in MRI is its relatively low relaxivity. The ENCITE consortium sought to improve the magnetic properties of ferritin’s iron core by introducing a peptide derived from an iron binding protein of the magnetosome machinery of magnetic bacteria. The chimeric magneto-ferritin gene affected iron sedimentation, and cells expressing the new magnetoferritin showed enhanced iron uptake, higher relaxivity and increased MRI contrast.
4. Preclinical validation
In regenerative medicine, the goal is to repair damaged/degenerated tissue with an intrinsic low capacity for self healing by transplantation of stem cells. In order to achieve this goal, and to ultimately achieve restoration of function of the damaged tissue, it is crucial to know what happens to the cells after they have been translated into the patient (or animal model).
Within the ENCITE consortium, several imaging tools have been developed and validated in pre-clinical models. These tools encompass novel techniques by which cell survival, cell differentiation and therapeutic effects mediated by the transplanted cells can be monitored in vivo. They focus on five different application fields, namely neurological disease (stroke), cardiovascular disease (myocardial infarction), musculoskeletal disease, diabetes and cancer.
4.1 Neurological disease
Within the application field of neurological disease/stroke some major advances were made regarding the ability to monitor neuronal stem cell engraftment, cell differentiation and neurological tissue repair using sophisticated high-field MR imaging and spectroscopy techniques, as well as with novel 19F- and Gd-based probes and reporter gene technology.
Through double labelling of neuronal progenitor cells with 19F probe and a luciferase reporter gene distribution and survival of these cells upon transplantation could be monitored. Cells remained visible for several weeks by 19F-MRI, but rapid loss of cell vitality, as assessed by bioluminescence imaging, was noted within the first two weeks after grafting. Immunohistochemical analysis showed that this loss of cell vitality was due to a massive inflammatory reaction to the graft. Studies directed at optimisation of the bioluminescent signal revealed that the timing and route of administration of the luciferase substrate can significantly influence signal levels. Furthermore, the sensitivity of various different luciferases for the specific purpose of neuro-imaging has been compared. Luc2 was found to be by far the most intense, leading to highest sensitivity of small grafted cell groups. Putting these two studies together, an in vivo bioluminescence protocol specifically optimised for the requirements of neuro-imaging has been developed and validated for the first time.
Through MR spectroscopy approaches and responsive contrast agents, changes in cell function were monitored based on shifts in the expression of specific molecules associated with a certain cell function stage. The visualisation and assessment of the functional state of transplanted stem cells in brain lesions was achieved by 19F cell labelling techniques and the use of a GABAergic cell specific responsive contrast agent, respectively. In a transgenic model based on doublecortin (DCX) promoter activity neurogenesis was monitored non-invasively using bioluminescence. The differentiation of human neural stem cells was achieved by metabolite profiling using magnetic resonance spectroscopy (MRS). In response to transplanted cells in stroke lesions, angiogenesis was monitored using reporter genes that were associated with the formation of novel blood vessels. These approaches have significantly improved our ability to monitor the distribution of transplanted cells, as well as determining their functional status and effect on host tissue.
4.2 Cardiovascular disease
From an imaging perspective the heart represents a significant challenge. Its complex anatomical structure, continuously changing shape (due to the heart beat), the large volumes of blood flowing through it (blood circulation), and its location in an environment filled with air (chest cavity and lungs) that is also incessantly moving (respiration), all contribute towards the imaging challenge. These anatomical and physiological conditions lead to the requirement of tailored MR imaging approaches which are able to compensate for the imaging artefacts that can be caused by motion and blood flow. Specific advances made by the ENCITE scientists include the ability to perform parametric imaging techniques on the heart which allow for more sensitive and specific detection of labelled stem cells. In addition, they also developed and/or validated new methods for the monitoring of transplanted stem cell fate in the myocardium and for the assessment of myocardial function.
While mapping techniques and the use of a post-processing pipeline were shown to be robust in small animal models and have advantages regarding sensitive detection of cells labelled with either iron oxide or gadolinium-based probes, some limitations of these techniques were also highlighted especially for the use of cells labelled with iron oxides. While various in vitro studies have demonstrated that specific MRI parameters, i.e. the R2 and R2* values, are differently affected depending on the compartmentalisation of iron oxide particles, and that the difference in the signatures of these parameters can be used as an indicative measure of cell survival, the ENCITE scientists have demonstrated that this principle can not be applied in vivo. Potential differences in R1, R2 or R2* relaxation rate, as a measure of overall cell viability for mesenchymal stem cells labelled with Gd-liposomes (Gd-MSCs) or iron oxide nanoparticles (SPIO-MSCs), were investigated. Cells were also transduced with a luciferase vector, facilitating a correlation between MRI findings and cell viability using bioluminescence imaging (BLI). Viable Gd-MSCs were clearly distinguishable from nonviable Gd-MSCs under both in vitro and in vivo conditions, clearly differing quantitatively (?R1 and ?R2) as well as by visual appearance (hypo- or hyperintense contrast). Immediately post-injection, viable Gd-MSCs caused a substantially larger ?R2 and lower ?R1 effect compared with nonviable MSCs. With time, the ?R1 and ?R2 relaxation rate showed a good negative correlation with increasing cell number following proliferation. Upon injection, no substantial quantitative or visual differences between viable and nonviable SPIO-MSCs were detected. Moreover, nonviable SPIO-MSCs caused a persisting signal void in vivo, which compromises the specificity of this contrast agent. In vivo persistence of SPIO particles was confirmed by histological staining. These findings indicated Gd to be the favourable contrast agent in qualitative and quantitative evaluation of labelled cell fate in cell therapy experiments.
For the novel approach of using adipose tissue derived stem cells ENCITE succeeded in the isolation, characterisation and transduction with a GFP reporter probe of pig adipose tissue derived mesenchymal stem cells (pASC) and modelling of ischaemic damage in a pig model. This allowed the study of the fate of pASC upon intra-coronary infusion, by performing PCR analysis of the GFP gene on various myocardial tissue regions. This showed that GFP+ was only detected in the coronary arteries and ischemic damaged tissue (left ventricle) and was absent in the remote myocardium, atriums, right ventricle and all the aorta different portions. Confocal microscopy analysis also revealed the presence of GFP+-cells in the ischemic cardiac tissue. Cardiac magnetic resonance (CMR) was performed with a 3T clinical magnet at baseline (post-MI) and 7days post-MI/ASC infusion in a pig model. Biventricular function, myocardial edema, reperfusion hemorrhage, necrosis and no-reflow phenomena were assessed. Interestingly, histopathological analysis revealed a histopathologic correlation with the 3T-MRI findings at T2* sequence (myocardial hemorrhage, infarct zone and microvascular obstruction). However, CMR analysis showed no differences in cardiac performance, the degree of cardiac damage, and no-reflow phenomena between those animals treated with ASC-GFP+ and the placebo controls. However, histopathological analysis of vascular density (lectin staining) revealed a significant increase in neovascularisation within the ischemic cardiac tissue of those animals administered ASC as compared to controls.
A new approach to analyse tagged cardiac MRI data was developed. The proposed method is based on a Bayesian estimation framework, implemented by means of reversible jump Markov chain Monte Carlo (MCMC) methods, and combines information about the heart dynamics, the imaging process, and tag appearance. In phantom studies this method was shown to perform superior to other commonly used techniques. This new method was validated in rat models, pig models, and on human tagged cardiac MRI (see also section on clinical translation).
4.3 Musculoskeletal disease
Regarding the application field of musculoskeletal disease, new MR imaging parameters were defined as markers for intervertebral disc (IVD) and cartilage degeneration using advanced MR imaging techniques such as double quantum filtered MRI and multinuclear NMR. For the first time scientists were able to characterise the changes occurring following the ablation of the nucleus pulposus part on the intervertebral disc, mimicking the effect of disc degeneration, and the differences in NMR parameters in the cartilage of osteoporosis patients.
One major breakthrough was the use of CEST to assess glycosaminoglycan concentration in the intervertebral disc. The CEST method for hydroxyl protons of GAG is shown to be sensitive to changes in GAG concentrations in the nucleus pulposus of the intervertebral disc, both in spectroscopy and in ex vivo imaging. It could potentially be used for monitoring GAG depletion in the nucleus pulposus, which plays a central role in the study and diagnostics of disc degeneration. As a consequence, gagCEST may hold potential not only for the assessment of disc pathophysiology in vivo, but also for medical intervention and therapeutic monitoring.
By tracking mesenchymal stem cells (MSCs) in vivo it was shown that they can be successfully implanted into, and survive in, a degenerated mouse IVD. This may facilitate the early diagnosis of disc degeneration and represent a method to monitor repair mechanisms by stem cells.
4.4 Diabetes
In diabetes the application of five new MR imaging techniques for sensitive detection of islets labelled with SPIO particles that were developed within ENCITE were evaluated. The first one, an echo-dephased SSFP method, can successfully visualise SPIO-labelled human and rat pancreatic islets yielding a positive contrast in MR images, as opposed to signal loss when using classical MR imaging techniques, This so-called “positive contrast with echo dephased SSFP sequence” method allows for the unambiguous and sensitive visualisation of transplanted pancreatic islets in a physiological tissue environment. This technique is sensitive enough to detect single pancreatic islets.
The second technique comprised Ultra Short Echo (UTE) and was successfully tested on an experimental MR system. A similar approach was used on a 3T system where a double contrast selective 3D spoiled gradient echo sequence (SPGR) was used for animal studies as well as for human applications.
The third technique involved an approach of double contrast, which uses the combination of T2 imaging (for cell labelling) and T1 imaging following intravenous application of contrast agents to enhance contrast between labelled cells and surrounding tissue. It was shown that this approach could suppress image artifacts and the segmentation of pancreatic islets was improved.
The fourth technique assesses vascularisation of artificial transplant sites. From the techniques available on the MR systems, the Dynamic Contrast Angiography (DCA) technique was chosen to study vascularisation around pancreatic islets deposits.
The fifth technique using the balanced SSFP (bSSFP) sequence with phase cycling was successfully implemented for SPIO-labelled human and rat pancreatic islets yielding a negative contrast. The bSSFP sequence is sensitive enough to detect single pancreatic islets and was routinely used in a clinical protocol.
For several of these techniques, semiautomatic and automatic methods for the calculation of the number of pancreatic islets were tested and are now ready for routine application. In addition, new bimodal probes were synthesised and tested for labelling of pancreatic islets (and also STEM cells). Bimodal contrast agents are agents that can be used by two independent imaging modalities. This study focussed on the synthesis and evaluation of two different types of novel probes for MR and optical imaging: “positive” T1 contrast agent based on cyclodextrin Gd complexes and “negative” T2 contrast agent based on perovskite manganite nanoparticles, both with a fluorescein marker for optical imaging.
For the bimodal fluorescein /MRI cyclodextrin-based contrast agent cell toxicity was found using second generation dendrimers. Efforts were therefore focused further on analogous aggregates with a cyclodextrine core. In ?-cyclodextrine the -OH groups of the upper ring were replaced with amines and thereby, one molecule of fluorescein and six molecules of the Gd(III) complex with DO3A-CH2-PO2H-Bz-NH2 acid were attached to the molecule of cyclodextrine. The T1 relaxivity of A-CD-F is high, about 25 s–1mM–1 per one Gd(III) ion at 20 MHz and temperature of 25 ºC. The labelling of stem cells was verified by fluorescent microscopy and in vivo tests on rat models were also carried out. The potential of magnetic nanoparticles based on the La0.75Sr0.25MnO3 perovskite manganite for magnetic resonance imaging (MRI) was studied. Dual imaging probes where the magnetic cores are combined with a fluorescent moiety were synthesised and tested. Viability tests show that the particles are suitable for labelling of PIs. No fatal interference with the vitality and insulin releasing ability of labelled pancreatic islets was observed. Relaxometric measurements confirm high T2 relaxivities at magnetic fields of B0 = 0.5–3 T.
Experimental models were also studied to investigate whether MR imaging can predict failure of pancreatic islets transplanted into hepatic portal vein. It is known, that labelled PIs are visible in liver or in other organs for a relatively long time. Syngeneic transplantation and allogeneic transplantations with two different immunosuppressive regimes were studied for 12 weeks. The result shows that the failure of PI can be seen after 3-4 week after the transplantation by MR imaging of labelled PI. This result could be useful for clinical applications.
4.5 Cancer
In cell-based therapy approaches for cancer, the main results are directed towards monitoring the fate and function of adoptively transferred DC and T cells. Good progress regarding multi-modal imaging techniques and imaging probes was achieved. A new 19F-based imaging probe was developed to quantitatively monitor the fate of DC by MRI. A major achievement in DC therapy studies was the observation of DC migrating to the draining lymph nodes in patients enrolled in a Phase I Clinical Trial using Indium-111 scintigraphy (further discussed in the translation towards clinical applications below). Only a small proportion of injected DCs migrated from the injection site. MR imaging allowed assessment of both accurate DC delivery and inter-/intranodal migration patterns. In addition, the mechanism by which DCs activate Natural Killer (NK) cells in the presence of stimuli of bacterial origin was identified, showing a crucial role of DCs in NK cell activation.
Detection of CFSE-labelled Cytotoxic T-cells in dual-colour tumours was achieved. Through the introduction of third harmonic generation in multiphoton microscopy, otherwise undetected structures of the tumour stroma could be visualised. With regards to monitoring the dynamics of tumour and tumour microenvironment, apoptosis was detected using a histone H2B/GFP construct, and a quantitative nuclear condensation score to discriminate apoptosis from necrosis was developed, which is instrumental in the real-time monitoring of preclinical immunotherapy.
Using multiphoton and intra-vital imaging approaches, dynamic interactions between various immune cells and between immune cells and tumour cells were monitored in vivo. This study included techniques for the detailed analysis of T-cell effector functions, based on the assessment of tumour cell viability and growth, and techniques to monitor tumour cell–T cell interactions and cell apoptosis. Pre-clinical T-cell therapy studies resulted in the successful generation and expansion of cytotoxic T lymphocytes (CTL) which kill different OVA-peptide positive target cells, including B16F10 melanoma cells.
Detection of CFSE-labelled Cytotoxic T-cells in dual-colour tumours was achieved. Through the introduction of third harmonic generation in multiphoton microscopy, otherwise undetected structures of the tumour stroma could be visualised. With regards to monitoring the dynamics of tumour and tumour microenvironment, apoptosis was detected using a histone H2B/GFP construct, and a quantitative nuclear condensation score to discriminate apoptosis from necrosis was developed. This approach will be valuable as a third colour to monitor the tumour stroma in the projected dual-colour reporter mouse. In addition, a significant impact of p53 expression in fibroblasts on tumour progression was detected.
A tumour rejection model based on antibody-dependent cellular cytotoxicity was successfully established. Tumour cell death in response to chemotherapy and tumour stroma activation, using two photon intravital microscopy, was successfully monitored.
Besides tracking what happens to the transplanted cells (cell fate) another question to be addressed in the various disease states is “what do the transplanted cells do?”. To answer this question, tools were developed that are highly specific for the targeted disease, such as in neuronal differentiation as well as new methods which have been developed to quantify glycosaminoglycan levels in cartilage. Other tools, although developed and validated within a specific disease model, have a more general applicability, such as the assessment of cell survival.
The developed tools are important in helping to elucidate the mechanisms of disease and repair of tissue, and to further our understanding of how stem cell therapy works, to make its use more effective and broadly applicable Some of the developed tools are suitable for direct translation for clinical studies, or have already been tested in a clinical setting. For example, tools for monitoring the survival of transplanted pancreatic islets are already being tested in in-patient studies, or in the patient DC study described in more detail below.
5. Translation towards Clinical Applications
The main results in the translation of research findings towards clinical application are specific to cancer, diabetes and cardiovascular disease.
5. 1 Cancer
Beyond these in vitro and in vivo mouse models, in ENCITE it has been also possible to monitor the migration and anti-tumour function of immune cells in cancer patients during therapy. One focus was on specialised antigen-presenting cells (DC), which were generated from patients’ monocytes by a standardised in vitro culture method and then loaded with melanoma-specific tumour antigens. In ethically approved phase I-II trials, melanoma patients with advanced disease repeatedly received their antigen loaded-DC as “tumour vaccines”. After therapeutic application, DC migrate to various lymphoid organs and instruct the CTL to identify and combat the melanoma antigen. If successful, CTL and other immune effector cells are activated by the DC, invade the metastases and destroy the tumour cells.
Cancer patients were recruited to a clinical trial and were monitored with a tracer in order to detect an antigen-specific immune response in vivo shortly after vaccination. This tracer offers a sensitive tool to study the kinetics, localisation and involvement of proliferating lymphocyte subsets. On the trial several patients demonstrated a broad response to numerous peptides prior to vaccination. Furthermore, tetramer-based 8-colour flow cytometry has become a routine monitoring assay, allowing extended phenotypic and functional characterisation of T-cell subpopulations. The identification of polyfunctional T-cells was of particular interest, since these have been shown to elicit more effective immune responses in HIV vaccination trials.
In summary, DC were exported to vaccinate melanoma patients and it was demonstrated there is a statistically significant correlation between a favourable clinical outcome and the presence of vaccine-related tumour antigen specific T cells in delayed type hypersensitivity (DTH) skin biopsies. However, this favourable clinical outcome was only observed in a minority of the treated patients. It was obvious that the DC-based vaccination protocols need to be improved, and the fate, interactions and effectiveness of the injected DC became the subject of some proof of principle trials. Our results confirm that DC immunotherapy is well feasible non toxic and effective in some cancer patients. However, many questions still remain. One of the concerns related to ex-vivo generated DC is how to ensure effective migration to the T cell areas in the lymph node.
5.2 Diabetes
With respect to diabetes, a protocol for measurement of high resolution MR images at 3T was developed. The purpose of the studies was to optimise the set of MR sequences and evaluate techniques suitable for routine examination of patients before and after PI transplantation, which allow quantitative data post-processing including quantification of transplanted PIs. The MR imaging and spectroscopy was performed on a 3T Siemens Trio scanner equipped with a body resonator surface coil. Different sequences and protocols were tested.
SSFP sequences were modified and two other types of sequences were developed. The echo dephase SSFP sequence was successfully applied on an animal model, however unfortunately its application on patients was not successful. Therefore a sequence, based on an ultra short echo protocol, was tuned and tested on a Siemens Trio system and used later as a part of the examination protocol to prove the presence of SPIO nanoparticles in human liver.
Different evaluations methods of liver MR images with labelled pancreatic islets were tested. Manual, semi-automatic calculation, dual contrast technique and texture analysis described precisely the position and number of labelled pancreatic islets in the liver and the data can be used for description of therapeutic function of transplanted pancreatic islets.
In total, 17 patients with transplanted PI according to a standard clinical procedure were examined, 11 of them were transplanted repeatedly. As a result, there were a total of 24 transplantations and 92 MR repeated examinations. Labelled PI were visible in human liver as hypo-intense spots on the MR image 6 months following transplantation.
The increasing concentration of fat in the liver and around the transplanted PI provides interesting information about the fate of islets and the surrounding tissue. A conventional spoiled gradient echo (SPGR) sequence for detection of the tissue was modified with very short T2 components and quantification was performed, e.g measured T2* maps and water/fat distribution. The protocol was tested on volunteers and patients with transplanted liver who underwent liver biopsy. Obtained data served as a reference to the MRI and MRS data of patients with transplanted PI into the liver.
An excellent agreement between MR results of preclinical experiments and clinical tests was found which confirms that MRI and MRS represents a suitable tool for observation of labelled pancreatic islets, changes in the tissue around the grafted islets and monitoring of cell transplantations in humans in general.
5.3 Cardiovascular disease
In validating novel imaging tools for clinical application, studies were performed in a clinically relevant pig model in which cardiac magnetic resonance techniques for evaluation of cardiac damage were validated by histopathological analysis. A distinct correlation between the histopathological findings and the 3T-MRI reading at T2 sequence (myocardial hemorrhage, infarct zone and microvascular obstruction) was observed. These techniques may therefore provide a non-invasive approach for assessing various types of cells, such as adipose derived MSCs, and routes of administration over time and evaluate both short term effects and long term effects on cardiac performance.
The newly developed tool for myocardial strain analysis based on a Bayesian estimation framework, implemented by means of reversible jump Markov chain Monte Carlo (MCMC) methods, in combination with information about the heart dynamics, the imaging process, and tag appearance has been tested in rat models, pig models and on human tagged cardiac MRI. The new method was shown to improve the performance of even the best of four frequently used methods. The new method yields higher consistency, accuracy, and intrinsic tag reliability assessment; the proposed method allows for improved analysis of cardiac motion
6. ENCITE’s ethical statement on the use of animals
The ENCITE consortium has explicitly stated in written how they apply the Directive on the Protection of Animals used for Experimental and Other Scientific Purposes and described how they have considered and implemented the three principles replacement, refinement and reduction as follows:
a. Replacement
Wherever possible, animal studies were replaced by experiments in silico or in cell cultures in vitro. This principle has been employed exten¬sively, for example in the sections dealing with cancer.
b. Refinement
Refinement was applied in breeding, accommodation and care and in animal experiments, where methods were applied eliminating or reducing to the minimum any possible pain, suffering, distress or lasting harm to the animals.
c. Reduction
The replacement and refinement principles were both contributed to the overall goal of reducing the use of animal experiments. Animal studies were only be used at advanced stages of investigations when few, specific and highly relevant questions were addressed by a limited number of experiments. In addition, minimum number of animals was used in all experiments to achieve statistically significant data. Experimental methods reducing significantly the number of animals were preferred, e.g. MR imaging, allowing for repetitive and safe measure of status of animals during long time periods, was used. To avoid potential overlaps and unnecessary duplication of animal experiments they were controlled by ENCITE’s senior scientists in their organisations’ research work.
A minimum number of animals were used in all experiments to achieve statistically significant data. All animals were treated humanely according to Institutional Animal Care and Use Committee guidelines.
Furthermore, the care and welfare of laboratory animals was ensured in line with the following conditions:
a.
Animals were kept under standardised environmental conditions of temperature and humidity and a day/night cycle; animals were kept in appropriate cages. Any restrictions on the extent to which an animal can satisfy its physiological and ethological needs were kept to a minimum. Animals were provided an environment, food, water and care appropriate to their health and well-being.
b.
Surgical procedures were always performed under local or general anaesthesia. Analgesia or another appropriate method was used to ensure that pain, suffering and distress are kept to a minimum.
c.
At the end of experiments animals were killed using a painless, quick and appropriate method (as specified in the Directive).
d.
Methods alternative to the use of animals, such as cell cultures, were employed where possible and applicable, and the assessments of biological actions of novel probes for imaging were always first carried out and validated in vitro, prior to animal studies.
Potential Impact:
1. Potential Impact on European Medicine and Healthcare (clinical application)
The ENCITE project has set-up and will continue to maintain an interdisciplinary platform integrating groups working on translational imaging across Europe. The project has been able to integrate skills in physics, chemistry, cell biology, immunology and medicine, all centred on imaging that is unique. The research work carried out in this consortium has led to the achievement of a number of relevant results.. ENCITE brought together skills and facilities from two distinct imaging communities: the more clinically oriented MRI community (radiologists, probes’ and sequences’ developers) and the more biomedical oriented basic research community (biologists and immunologists) MRI and optical imaging have been the modalities most used in the project. This collaboration is of major importance for the further integration of the imaging field as the key-tool for the development of cell based therapies.
Looking towards the future and critically reflecting on the statement “cell imaging and tracking plays a central role for tomorrow’s medicine”, ENCITE scientists highlight how their core research work has potential key impact on European medicine and future healthcare along the following direction:
1.1 Novel Imaging Technologies
The achievements reached are well suited to enable more efficient and sensitive diagnostic and clinical tools in the field of cell therapy. A number of new imaging biomarkers, MR probes and new post processing algorithms were developed and are on the brink of translation into the clinical environment, greatly facilitated through the interdisciplinary collaboration within the ENCITE consortium. Given the broad range of applications, the difference in the species observed (from mouse to man), the range of field strengths (3-11.7T) and the different platforms, it is anticipated that the developed toolboxes and techniques will be sufficiently flexible such that they can be adapted to a range of different specific studies. These new techniques will help in the development and quantitative evaluation of new therapeutic concepts. The development of advanced techniques for new hardware will also facilitate the use and distribution of these newly available techniques and machines. The main benefits for European medicine are the increasing availability of non invasive in vivo imaging tools that are also more efficient and allow a more comprehensive look on new therapeutical questions. One of the major limitations of MR imaging was the high cost and the long examination times that are not well tolerated by the patients and limit the use of MR imaging technologies in clinical studies. The collaborating partners enabled the development of advanced techniques that are more efficient and sensitive and will therefore promote the use of these techniques for more patients.
1.2 Novel Imaging Reporter Probes
The results obtained are extremely encouraging with respect to the final goal of providing highly sensitive agents and procedures for an efficient labelling of cells. The design of efficient labels is crucial for pursuing "in vivo" cell tracking. The partners’ achievements have allowed the set up of a large library of powerful imaging reporter probes. The availability of such a repository of chemical and biological reporter probes will facilitate tackling novel advanced applications in the field of cell therapies. Furthermore, the novel cellular labelling procedures, which have been developed as part of the ENCITE project, have been shown to be more effective than the standard methods currently being used, and they are expected to have a high impact on future clinical practice.
1.3 Novel Tools for Cell Labelling
The outcome of the research work is a portfolio of tools for non-invasive monitoring of cells within living organisms. It is anticipated that these tools will help improve the understanding of disease processes, and thus could aid in development and monitoring of cell-based therapeutic interventions.
1.4 Pre-clinical Validation
The various tools developed for the different application fields are expected to provide a better understanding of the fate of transplanted cells and of how cell-based therapies provide a therapeutic benefit. Thanks to the multi-disciplinary nature of this consortium and the strong collaboration, ENCITE members were able to leverage each other’s expertise in order to successfully achieve the pre-clinical validation of the newly developed probes and methodologies and their translation towards a clinical application. Although various tools were developed and validated within studies focused on a specific application field, a large number of these developed tools will have a broader impact, such as novel contrast agents (for example bimodal agents as developed in studies focused on neurological disorders and diabetes) or Gd-liposomes (as developed in studies focused on cardiovascular disease) and reporter genes for neo-angiogenesis. Next to these innovations, a number of field specific advancements were linked to specific pathologies associated with specific disorders. These include tools for monitoring lineage specific differentiation, assessment of tissue function e.g. analysis of tagged cardiac images, assessment of GAG content and assessment of T-helper function. The complete portfolio will have a significant impact on research in the specific application fields addressed but also in general on cell imaging studies. A number of the developed and validated tools are only suitable for preclinical studies but various tools have potential for (routine) clinical application. Most of the techniques for assessing tissue response have this potential, but also various cell imaging techniques show promising results as specified below.
1.5 Translation towards Clinical Application
ENCITE expects that the newly developed imaging tools will significantly improve the translation of cell-based therapies towards the clinic. By leveraging the knowledge obtained in the other ENCITE research work programmes, cell therapy treatment strategies can be optimised to reap the full therapeutic potential of cell-based therapies.
The ENCITE consortium partners used the opportunity to perform clinical studies on the transplantation of SPIO labelled PI to patients. Significant conclusions are as follows: the clinical outcome of a patient group with labelled islets does not differ from the patients with native islets; the islet visualisation was successful but less efficient during a labelling period below 16 hours; a significant decrease of islet spots occurred in the first week suggesting early islet destruction or impaired engraftment; afterwards, islet spot numbers decrease slowly, and the islets are visible for at least 24 weeks; significant C-peptide production, glycosylated hemoglobin (HbA1c) were found in all patients, near to normal values; insulin dose reduction were achieved following one or two islet transplants, but only one patient became fully insulin independent; an excellent agreement between animal models and clinical experiments was found.
ENCITE’s novel MR and multimodal imaging methods should serve to increase the translation from preclinical studies to early clinical trials and their ultimate patient application. Several developments are currently under evaluation for approval, including the anti-angiogenesis treatment for Glioblastoma (approved and currently under clinical evaluation), the first fluorine compound for clinical DC vaccines in a patient (approved by the Food and Drug Administration) and the process using new generation fluorinated polylactic-co-glycolic acid (PLGA) particles (currently under evaluation). In 2013 clinical trials can hopefully be started using these particles within in vivo settings for cancer patients.
ENCITE’s scientists are confident that these technologies will help to speed up developments in cell therapies and that their entry into wider routine clinical practice could offer potential cures for many different types of diseases, including cancer, cardiovascular disease and diabetes, as well as in stroke and musculoskeletal diseases.
Cell therapy appears to holds the future promise of personalised medicine with the potential to cure chronic diseases. The ultimate successful implementation of cell therapy as a treatment modality in the clinic requires a means to follow the fate of transplanted cells once inside a patient by widely applicable non-invasive monitoring tools. To this end, ENCITE has developed robust tools that make realistic their application in the treatment of major diseases.
In the long-term, ENCITE’s ultimate goal is to see cellular therapy and image guidance enter widespread clinical practice allowing for early detection and progression of disease or effectiveness of therapy.
2. Potential impact on socio-economic and wider societal implications
Up to now cell therapy and imaging techniques for the monitoring of cell therapy are not well established for many patients and diseases. As cell therapy itself is a highly dynamic field with a broad spectrum of inventions and applications it holds the potential to change the outcome of diseases that cannot be treated adequately. Since the new techniques developed by the members of the ENCITE consortium feature higher sensitivity and efficiency they will be available for more patients and provide more insights in novel therapeutic concepts and therefore facilitate the invention of new therapies for a number of diseases.
Cell-based therapy offers unique treatment options for severely debilitating or fatal disorders and diseases. The potential socio-economic impact of ENCITE’s achievements is therefore immense. In both regenerative medicine approaches and adoptive treatment strategies for cancer the advancements of ENCITE will significantly contribute on three different levels:
a.
Development of novel therapeutics due to a better understanding of the disease processes and the mechanism of action of such novel therapeutics
b.
Assessment of the efficacy of existing and novel therapeutics and thus patient management
c.
Optimisation of treatment protocols in terms of route and timing of administration of therapeutics
3. Potential impact on collaborations with relevant initiatives and EU-funded projects
The long-lasting and strong interactive way of working together has led to an extensive collaboration on the development of novel imaging tools and the implementation at the level of translational medicine across Europe with a global impact on future joint actions.
3.1 Close collaboration among ENCITE scientists
The close collaboration among the scientists has gone beyond pure institutional cooperation to achieve the work programme during the project life time and also beyond project end:
A number of project partners, mainly the experts from King’s College London (UK), Radboud University Medical Center (NL), Catholic University Leuven (BE), University of Münster (DE), University Hospital Freiburg (DE) and Max Planck Institute for Neurological Research (DE) will continue their collaboration on the use of 19F MRI. They are confident that, in a joint multi-disciplinary effort, the challenges associated with the use of 19F MRI in a pre-clinical and clinical setting can be overcome, providing biomedical researchers with potential new and exciting tools for in vivo imaging.
In addition to this interaction, the Institute for Clinical and Experimental Medicine (CZ) and the University Hospital Basel (CH) will continue their research in joint projects with respect to the development of a new modification of special sequences for the evaluation of fat in human liver.
3.2 Collaboration between ENCITE scientists and other projects
Further collaborations are intended to start with a group of experts outside the ENCITE consortium:
The FP7-funded project Euro-BioImaging (www.eurobioimaging.eu) has revealed that there is significant demand among its community for advanced training in the field of cell imaging. Such requirements can be met by the ENCITE Multi Centre Cluster for Training. Following this collaboration work the exchange of knowledge between both groups represents an excellent potential to showcase the cutting-edge research findings and training resources.
Within the framework of the EU-funded Marie Curie project “BetaTrain” (http://www.betatrain.eu) the Catholic University Leuven (BE) and the Institute for Clinical and Experimental Medicine (CZ) will continue their intense relationship for additional science activities on the evaluation of new contrast agents based on F19 where several joint experiments are prepared. The exchange between a number of students from both project partners will start in spring 2013.
4. Impact created by training activities
The high-valued impact created by ENCITE’s training developments is immense:
4.1 Exchange of knowledge among ENCITE scientists
Presenting the status of methodological developments on their current pulse sequence developments and fostering interactions between all experts involved in this research field the University Hospital Freiburg (DE) conducted an internal workshop to discuss the needs on new methods for cell tracking and imaging biomarkers and to identify action items to successfully finish the relevant work within a limited number of months following this meeting, held in 2009.
After a research phase of two years the ENCITE consortium decided to arrange a consortium-internal workshop to foster interactions and collaboration between the partners themselves but also between the expert groups of individual work packages and/or between different key subjects for the development of specific techniques. This first consortium-internal workshop was held at the University of Mons (BE) in 2010.
After another successful year of research, the ENCITE experts have met each other again to internally present the status quo of their current developments on imaging technologies, reporter probes, cell labelling and pre-clinical tools and to discuss their relevance towards the pre-clinical validation and possible clinical application. They focussed on the real clinical needs and the most appropriate tools for translation into the clinic as well as on rules and regulations with respect to clinical trials. In conclusion, ideas were presented for specific further developments and improvements and with respect to future research strategies for optimal detection sensitivity, quantification and imaging cell differentiation. This workshop was held at the Weizmann Institute of Science (IL) in spring 2011.
Following the discussions during this workshop, more than 20 scientists from ten ENCITE research organisations have met each other at the Max Planck Institute in late 2011 to discuss pitfalls and challenges towards a practical use in clinical studies. According to a questionnaire prior to the workshop, the participants compared 19F MRI protocols, set up and achieved sensitivity for cell tracking. In this respect, the workshop found answers to the issues as how to improve the sensitivity by new 19F compounds, strategies to increase 19F uptake by cells, the design of designated 19F MR hardware and the optimisation of MRI methods. The expert group showed the high potential of 19F MRI to monitor the fate of chemotherapeutics, to image dendritic cells implanted for the treatment of cancer, and to better image neural stem cells donated for tissue regeneration in the damaged brain. In conclusion, the workshop provided an excellent overview of the highly developing field of 19F MRI for cellular and molecular imaging. The pitfalls and challenges towards a practical use in preclinical and clinical studies were discussed in detail.
Consecutively, a 2-day symposium was organised at the Radboud University in autumn 2012. The first day was open to all European scientists, and the second one was reserved for detailed discussions among the ENCITE experts only. A wide range of topics were presented and discussed for further improvements, such as novel probes, design and optimisation of imaging sequences, cell labelling and hardware development. In total, an audience of more than 50 participants and 15 abstract submissions were received. Both workshops supported substantial interactions between researchers working on the new 19F MRI hot topic. It is planned to continue these meetings on a regular basis to allow further improvements and a better visibility among the imaging community.
4.2 Establishment of a knowledge dissemination platform
Looking towards a future training concept in an effective and sustainable way, the ENCITE Multi Centre Cluster for Training represents an efficient knowledge dissemination platform which will play a crucial role in the success of the post-project impact. The consensus at the end of this project is that training, especially advanced on-site courses, is highly valued by the research community throughout Europe.
Seven centres at the Universities of Torino, Mons, Nijmegen and Paris-Descartes (FR) as well as Max Planck and Weizmann have developed a programme to ensure a co-ordinated training approach for the wider scientific community with a significant impact on trained staff members in molecular and cellular imaging (research and clinics). The training programmes address senior researchers, graduate students and young researchers using the new technologies and demonstrating their benefits for cell imaging and tracking, imaging probes, nanoparticles, in vivo molecular imaging, multi-modal imaging, intravital microscopy, imaging tools and magnetic labelling. The centre established by the University of Paris-Descartes has been mainly focussing on radiology, image processing and the clinical application, thus ideally complementing the other centres.
Within the Cluster, more than 20 teaching files (one designed with an audiovisual video explaining the contents) and training documents as well as one e-course dedicated to the project’s novel and improved techniques have been prepared, in addition to the face-to-face trainings.
The centres have addressed and will continue on further developing different training modules offering hands-on courses and additional educational programmes on their specific technologies in the fields of chemistry and biology, developed within ENCITE. All centres’ activities will be complementary to each other to make use of synergies and mutual benefits.
Such an efficient knowledge dissemination platform will play a crucial role in the success of the post-project impact, leading to the implementation of the newly developed innovative tools and methodologies across Europe.
5. Main dissemination activities
Led by the European Institute for Biomedical Imaging Research (EIBIR) and in close collaboration with a majority of the project partners, a huge number of promotion activities and materials have been carried out to ensure widespread information on ENCITE, its promising results as well as on its dedicated training programme, based on the detailed ENCITE Dissemination Plan.
These activities ensured the information flow to different target groups of molecular and cellular imaging, of other imaging-relates disciplines and the general public at large to raise the awareness of ENCITE’s collaboration for further research and technologies, regulations and collaboration activities to strengthen the future work in this research sector.
5.1 Key carriers of ENCITE messages
The most important visual carriers of ENCITE’s messages were the development of a Project CI and logo specified on the project’s key subject and the website from the beginning onwards and a promotional video about the key outcome to be used also after project end:
Project CI and logo
The project CI and logo have proved very efficient throughout the project, allowing easy recognition of any project communication by the relevant scientific community. Information flyers on the project have been distributed widely at relevant scientific meetings and among other stakeholders (including industry) in order to inform about the advances achieved within the project.
ENCITE Website
The ENCITE website was updated regularly, in particular the Quick Links, News and the Press sections which were used to advertise research findings, upcoming (training) events, publications and references, annual reports and press releases on ENCITE workshops’ outcome and interim results to summarise key research findings. In addition, the ENCITE Multi Centre Cluster for Training webpage including the Interdisciplinary Training Platform was regularly updated. Each centre’s training activities, teaching files and additional materials as well as documentation for novel probes and associated information were uploaded onto the cluster’s website and the online repository for making the information available for download to the public.
http://www.encite.org
http://www.encite.org/cms/website.php?id=/en/index/mcc_training.htm
ENCITE Blog
The partners contributing to the Cluster for Training created an Internet Blog to ensure additional communication on ENCITE’s news and trainings to the scientific community during the project life time and particularly beyond the project’s end. This is intended to provide a resource on a long-term where posting of news and training updates can be continued on a long-term basis.
http://www.encite.org/blog/
ENCITE video “living cells as an active drug”
Launched at the final workshop, a promotional video within the framework of ENCITE’s translation of technologies into the clinics was produced. The video is showcasing how in vivo image-guided cell therapy is revolutionising medicine and describing ENCITE’s strong contribution to this revolution. ENCITE has made a real impact on the treatment of healthcare problems that affect our every day lives. To better understand how cell therapy works, prominent researchers have been working together to develop imaging tools for novel cellular therapies. The project partner RUNMC took over the scientific responsibility of the video contents and format; EIBIR promoted the video to all its contacts in the research and academia communities as well as to the EIBIR shareholders and the interested public. The video is also seen as kind of an educational kit to raise the awareness of ENCITE’s training cluster. The video has been provided to the World Wide Web users on different websites, such as: ENCITE, EIBIR, ENCITE Blog, Facebook, Twitter, several project partners’ websites, YouTube, etc. A dedicated press release and advertisements for this video was also published in the EIBIR Scientific Annual Report, the European Radiology, ECR Today, MAGMA, Auntminnie.com and several Internet channels.
http://youtu.be/xnVNKwEFZyE
5.2 ENCITE final workshop on the future of cell imaging and tracking
In addressing the questions “What are the clinical needs for imaging and improved disease diagnosis?” and “What are the best tools for translation to the clinics?” the ENCITE opened their workshop by presenting the most advancing technologies in the morning sessions. In the afternoon session, international key note lectures presented topic-related developments over the years. Based on this input, the ENCITE experts and workshop participants enjoyed a lively Round Table Discussion addressing the questions “What will cell imaging look like over the next 10 years?”. Within this discussion round, special emphasis was given on the idea of an entirely new system for funding major multinational clinical research on diagnostic products that are needed to be created by industry, academics, and regulatory bodies. The workshop was held at the Leiden University Medical Centre in Leiden/NL in November 2012.
5.3 ENCITE presence at public events, conferences and workshops
Extensive dissemination activities and trainings took place in collaboration with the European Society for Magnetic Resonance in Medicine and Biology (ESMRMB Congress 2009-2012 including the ENCITE Mini-Categorical courses 2011-2012 and the ENCITE Lectures on MR 2011-2012), the European Society of Molecular Imaging (EMIM Congress 2011-2012, TOPIM 2011-2012) and the World Molecular Imaging Congress in 2009, 2010 and 2012.
An educational session was held at the World Molecular Imaging Congress (FR) in 2008. There were an estimated 100-120 people in the audience for this educational session. Although, MRI plays a junior role at this International Molecular Imaging conference participation by the audience was very good. Further and more intense discussions went on directly following the session in personal discussions between individuals and the speakers. The high interest was also reflected in the lively discussions in the hall after the session.
In 2011, the University Hospital Freiburg hosted a European symposium on the future prospects of preclinical MR imaging where a number of promising advancements and future prospects were presented and discussed, together with internationally renowned experts.
In addition, dissemination activities were carried out and presentations were held at other related congresses, including Deutscher Röntgen Kongress 2011, the International Society for Magnetic Resonance in Medicine 2011-2012, the Radiological Society of North America Congress, the European Congress of Radiology 2010-2012, the European Association of Nuclear Medicine Congress 2011-2012 and the Journées Francaises de Radiologie 2011-2012.
Under the lead of EIBIR, the Institute for Clinical and Experimental Medicine, the University Hospital of Basel, the University Hospital Freiburg, Max Planck, the University of Torino and the Leiden University Medical Centre were actively involved in these congress actions.
5.4 ENCITE promotion materials
Various dissemination materials were developed and produced for different target sectors of experts in sciences, relevant industry sectors and the general public at large, such as: fact sheets, flyers, posters, booth presentations at congresses, ENCITE event calendars, print newsletter ENCITE inside and the brochure about the proceedings and outlook, etc.
They were distributed at a huge number of relevant workshops, congresses and public events, which captured ENCITE’s progress and highlighted its key successes in science and training, and to the ENCITE scientists’ partners at individual meetings of ENCITE scientists.
5.5 Media work and press campaign
At the beginning of the project, the Austrian National Contact Point published a “success story” about the project and its ambitious vision in suitable diffusion quality targeted to the German-speaking general public at large.
Press mailings with interim results were distributed to EIBIR’s media list. As a result, some (e-)newspapers published some spotlights about ENCITE, such as: Der Standard, The Times UK, Le Soir, Die Zeit, medical physics web, Life Science Vienna region, WorldPharmaNews, Universum Magazin and EC Research Information Centre, etc.
On an annual basis, a summary on the project progress and interim achievements was published in the EIBIR Scientific Annual Reports between 2008 and 2012. The report was distributed to all EIBIR Network Members (ca. 280), project and other partners (ca. 130) and at public events and conferences.
Over the years, major intermediate results including regular updates and public ENCITE sessions and lectures were drafted and communicated by EIBIR, in close collaboration with individual partners.
In addition, interim results, programmes and event and training announcements were published in a variety of scientific-related magazines several times per year (e.g. ECR Today, European Radiology, MAGMA, different congress programmes (ESMRMB, TOPIM, EMIM, European Congress of Radiology (ECR), WMIC), ESR/EIBIR/ENCITE e-newsletters and ENCITE dedicated mailings, etc.).
A European-wide press campaign was set up in 2011 dedicated to regularly communicate on ENCITE’s promising findings and its ENCITE’s training courses and its outcome to all scientists and stakeholders interested in the field molecular and cellular imaging and tracking and imaging-related disciplines.
At the end of the project, a media briefing was held with the journalist from Auntminnie.com (Auntminnie Molecular Imaging Insider! Europe) who published a press release about the Round Table Discussion at the ENCITE final workshop, held in Leiden/NL in November 2012. The experts focussed on the discussion of hundreds of promising target molecules identified in preclinical studies for possible use as novel cell imaging agents and tried to find answers on the questions “How many are ever likely to be used in real patients? And who will pay the costs of the clinical trials needed to prove their value?”.
5.5 ENCITE communication channels and target groups
The dissemination of ENCITE’s scientific advances, newly developed technologies and trainings was aimed at distributing it to the peers and experts of the scientific community in molecular imaging and all other disciplines related to cell imaging and tracking, including researchers and clinicians, the medical imaging industry as well as the media and the general public at large.
In addition to the project and partners’ websites, the websites of ESMRMB, European Hospital, Auntminnie, eHealth news and CORDIS have become the external online key communication channels for ENCITE. In addition, a high number of websites from co-operating partners, online platforms and event calendars as well as social media platforms (such as Facebook and Twitter) have been used to communicate regular updates, project results and scientific advancements as well as public events and trainings.
In total, a mailing list of more than 9.000 addresses was used for regular updates as well as event and training announcements. This list includes EIBIR Network Members (ca. 250), EIBIR stakeholder organisations (8) and industry partners (8), ESR Research Committee members, ESR members interested in molecular imaging (a. 8.000) participants of ENCITE events and trainings between 2009-2012 (ca. 400), the media mailing list (ca. 60), ESMRMB members (ca. 1000), ESMI mailings, and other stakeholders throughout Europe, etc.
For the entire dissemination activities including participation in conferences and workshops, publications, printing materials, web applications, press releases, presentations, posters, thesis and interviews, etc. please see the list on dissemination activities.
6. Exploitation of results
One of ENCITE’s major objectives has been to develop and test new MR and optical imaging methods and biomarkers to get a more comprehensive picture of cell fate and the reaction of the immune system and to ultimately improve and further develop cell therapy for the benefit of the European patient.
6.1 Scientific publications and presentations
To demonstrate these benefits for the European healthcare and the citizens of this multidisciplinary collaboration in the pre-clinical and clinical sectors on a European level and to raise the awareness of ENCITE’s impact on treatment, a huge number of publications, posters, presentations, dissemination activities and discussions were produced and held. The consortium partners published more than 170 (peer-reviewed) publications in scientific journals, highlighting their research results and presenting the achievements of the implementation of the project work programme. More publications are in preparation.
One of the publication’s highlights is a special issue of Contrast Media and Molecular Imaging dedicated to the major achievements of the ENCITE project, is currently in preparation. This will be a joint publication of mini-reviews and research articles. This publication is an opportunity to underline ENCITE’s strong collaboration and its significant achievements. Key topics, among other, are reporter genes, optimised multimodal neuro-imaging strategy for cell tracking, islet/beta cell imaging, cell imaging, clinical translation and toxicity, imaging biomarkers and sequences, nano systems for cell labelling, multimodal in vivo cell tracking and strategies for cellular labelling with paramagnetic metal complexes. The contributing scientists are from the Universities of Torino, Leuven, Paris-Descartes, Mons, Freiburg, Basel, Nijmegen and Erlangen as well as from Erasmus MC, Weizmann, Max Planck, Leiden University Medical Centre, Institute for Clinical and Experimental Medicine, King’s College London and the Foundation for Applied Medical Research. The publication of this special issue including this joint paper of approximately 100 pages is expected in summer/autumn 2013.
6.2 Exploitable foreground
In order to bring the expected results into pre-clinical and clinical practice, some of the ENCITE consortium members have started with specific exploitation paths. Several ENCITE consortium partners have been actively seeking protection of intellectual property:
With respect to exploitable foreground within ENCITE, the Hebrew University of Jerusalem has investigated in general advancement of knowledge with respect to both the methods for cell labelling and imaging in vivo and for cell injection to intervertebral discs in the research and medicine sectors. They have investigated on the following to be further worked on in future: first, labelling and imaging methods aimed to monitor stem cell survival and biodistribution. Hence, these methods could be exploited for further research of stem cell therapies. Second, do cell injection to the intervertebral disc. These results could be exploited for further research in cell therapies for intervertebral disc degeneration. Further research will be required in order to develop efficient stem cell therapies for degenerate discs. The expected impact of these findings would be of greatest value in the biomedical research field.
Four patents on ENCITE foreground have been filed by the Agencia Estatal Consejo Superior de Investigaciones Cientificas (CSIC) (ES), Erasmus MC, University Hospital Erlangen (DE) and University of Torino (IT). The University of Torino has applied for a patent on CEST agents allowing the visualisation of more cell typologies in the same MR Image. If approved, CEST agents may be then exploited for tracking different cell typologies. The University Hospital of Erlangen has a patent filed for dendritic cell therapy in cancer.
Particular importance is placed on the fact that two partners are currently preparing the establishment of spin-off companies:
CSIC is planning to create a spin-off company entitled BIOIMAG for the commercialisation of collagen and other extracellular matrix components functionalised with magnetic nanoparticles to be used as an “intelligent” contrast agent capable of reporting enzymatic activity. Cage Chemicals has introduced in its catalogue the most effective Gd-based agents developed in ENCITE.
The University of Mons has been working on the development of simultaneously reproducible batches of negatively charged or neutral PEG coated particles. These nanoparticles are available to all partners of the ENCITE consortium through a spin-off company entitled AGECO, which is currently under development.
The detailed list of scientific publications as well as the list of patents and exploitable foreground can be found below.
6.3 Collaboration with SMEs
During the project life time, all small and medium sized companies (SMEs) have obtained great advantages from the collaborative work carried out during the project life time. Several ENCITE research organisations have intensified their collaboration with SMEs in order to establish a joint strategy with respect to new approaches for technologies and exploitation to support specific research activities which might not bring immediate commercial benefits but could be significant in the longer term. Their positioning in their respective market appears definitively stronger when compared with the relevant numbers at the beginning of the project.
Cage Chemicals has doubled its staff and settled its activities in a larger space (from 66m2 to ca. 180m2). Thanks to the intensive collaboration work within ENCITE between Cage Chemicals, the University of Torino and Weizmann an important cooperation agreement has been signed between CAGE Chemicals and the Israelian company ASPECT Imaging. Based on the agreement, CAGE Chemicals will provide ASPECT costumers with a dedicated line of Gd-based products, in part generated by ENCITE. Furthermore, CAGE Chemicals has added few new products to its new catalogue and has definitely improved its visibility in the market of products for Imaging.
Max Planck and the SME Medres medical research GmbH (Medres) (DE), have developed new anesthesia gas monitor systems to improve the accurate determination of the inhaled gas mixture during longitudinal small animal experiments. These systems have successfully been installed in high-field MRI systems at the laboratories of MP. Within this collaboration, an integrated animal bed/monitoring system has also been developed and continuously improved for a separate version for MRI application of mouse and rat, respectively.
The close co-operation between the experts of Max Planck and the SME Biospace Lab. has led to the improvement of animal pedestals that have been designed and applied for allowing simultaneous optical imaging recording of three viewing angles. This set-up is now in routine application at the optical imager of Max Planck.
Finally, prototype designs have been tested between Max Planck and Medres concerning an innovative, non-invasive stimulation system for fMRI in anesthetised rodents. This new design has clear potential for improved performance while being at the same time, of reduced stress for the experimental animal. This device is not yet in application routine.
List of Websites:
The address of the project public website is: www.encite.org
Contact details:
European Institute for Biomedical Imaging Research (EIBIR), Project Coordination
Neutorgasse 9, 1010 Vienna (AT)
Phone: +43-1-5334064-29
office@eibir.org
www.eibir.org www.encite.org
Scientific Coordinator: Prof. Gabriel P. Krestin, ERASMUS MC, Rotterdam/NL, g.p.krestin@erasmusmc.nl
