Periodic Reporting for period 1 - TxImmuneOrganoids (Divide and conquer: using patient-derived tumour organoids to dissect intra-tumour immune heterogeneity of non-small cell lung cancer)
Periodo di rendicontazione: 2021-08-01 al 2023-07-31
Context
The immune system has the capacity to control cancer growth, as illustrated by the recent successes in therapies aimed at reinvigorating anti-tumour immunity. It is becoming increasingly clear that there is heterogeneity within individual tumours with respect to their interaction with the immune system. For example, regions heavily infiltrated with immune cells (‘hot’ regions) and immune-deserted regions (‘cold’ regions) can coexist in the same tumour. At the same time, driven by work from the host (Swanton) lab and other labs, we now know that cancers are subject to the force of genetic evolution, resulting in multiple cancer subclones that harbour distinct genetic mutations.
The extent to which separate subclones differ in their capacity for immune evasion, the tumour-intrinsic mechanisms underlying any such heterogeneity, and its impact on cancer immunosurveillance remain largely unexplored. This question has both biological and clinical relevance, as resistance to cancer immunotherapy could be driven by (minor) resistant subclones. Identifying the mechanisms that drive subclonal immune evasion could therefore ultimately lead to the development of novel immunotherapies that will result in deeper responses compared to current practice. Determining the ability of individual cancer subclones to elicit an immune cell response has been challenging so far because of the lack of patient-derived model systems that can recapitulate the interaction between cancer cells and immune cells at the level of single clones.
Overall objectives
Patient-derived tumour organoids (PDTOs) are three-dimensional cultures of cancer cells that can be established directly from patient tumours. PDTOs can be co-cultured with patient-matched immune cells to create a miniaturised test system for anti-tumour immunity at the level of an individual patient. Here, we leverage the multi-region TRACERx lung cancer evolution study to generate a patient-derived study platform that allows the evaluation of T-cell responses to individual cancer subclones. Our overall objective is to evaluate whether distinct subclones differ in their ability to elicit a T-cell response, and the mechanistic basis underlying any such heterogeneity (Fig. 1)
The immune system has the capacity to control cancer growth, as illustrated by the recent successes in therapies aimed at reinvigorating anti-tumour immunity. It is becoming increasingly clear that there is heterogeneity within individual tumours with respect to their interaction with the immune system. For example, regions heavily infiltrated with immune cells (‘hot’ regions) and immune-deserted regions (‘cold’ regions) can coexist in the same tumour. At the same time, driven by work from the host (Swanton) lab and other labs, we now know that cancers are subject to the force of genetic evolution, resulting in multiple cancer subclones that harbour distinct genetic mutations.
The extent to which separate subclones differ in their capacity for immune evasion, the tumour-intrinsic mechanisms underlying any such heterogeneity, and its impact on cancer immunosurveillance remain largely unexplored. This question has both biological and clinical relevance, as resistance to cancer immunotherapy could be driven by (minor) resistant subclones. Identifying the mechanisms that drive subclonal immune evasion could therefore ultimately lead to the development of novel immunotherapies that will result in deeper responses compared to current practice. Determining the ability of individual cancer subclones to elicit an immune cell response has been challenging so far because of the lack of patient-derived model systems that can recapitulate the interaction between cancer cells and immune cells at the level of single clones.
Overall objectives
Patient-derived tumour organoids (PDTOs) are three-dimensional cultures of cancer cells that can be established directly from patient tumours. PDTOs can be co-cultured with patient-matched immune cells to create a miniaturised test system for anti-tumour immunity at the level of an individual patient. Here, we leverage the multi-region TRACERx lung cancer evolution study to generate a patient-derived study platform that allows the evaluation of T-cell responses to individual cancer subclones. Our overall objective is to evaluate whether distinct subclones differ in their ability to elicit a T-cell response, and the mechanistic basis underlying any such heterogeneity (Fig. 1)
Main results
We generated libraries of >20 separate non-small cell lung cancer (NSCLC) organoid lines, based on isolating individual (clonal) organoids established from multiple spatially separated tumour regions. Each organoid subline was co-cultured with autologous tumour infiltrating lymphocytes (TIL) to evaluate how they differ in their capacity to elicit a T-cell response. We combined functional assays with DNA, RNA and T-cell receptor (TCR) sequencing to perform an in-depth characterisation of 44 individual organoid lines derived from 6 separate tumour regions from two patients with NSCLC to identify the mechanistic basis driving subclonal immune evasion.
We focused our analyses on organoid libraries from two different patients. For patient 1, T-cell recognition strongly differed between separate clones established from one individual tumour region. This was not due to differences in antigen expression, as the same heterogeneity between organoid sublines was seen when tested for their ability to elicit T-cell responses against a defined (and exogenously provided) model antigen. DNA sequencing showed that immune-escaping clones (‘cold’ clones) shared distinct copy number alterations that set them apart from hot clones. RNA sequencing of hot and cold clones revealed that immune evasion was associated with a transcriptional profile linked to quiescence. Current experiments are aimed at determining a potential causal link between quiescence and immune evasion.
For patient 2, organoids were derived from four different tumour regions that differed strongly in histomorphology, with two regions featuring a papillary growth pattern, and two other regions are solid growth pattern. Organoids derived from papillary tumour regions escaped T-cell detection in vitro, in contrast to organoids from the solid tumour compartment. Tumour regions that gave rise to hot or cold organoids diverged greatly at the genetic level. Immune escape was antigen-dependent, suggesting that regional heterogeneity in antigen expression drives subclonal immune escape in this patient’s tumour. Current work is focused at experimental identification of the target antigen and validation of differences in expression that drive subclonal immune escape.
Conclusions
Taken together, these results show (i) that tumour evolution can give rise to distinct cancer clones with intrinsic differences in immune evasion capacity and (ii) provide an approach to prospectively identify and isolate immune evading subclones from a patient with cancer.
We generated libraries of >20 separate non-small cell lung cancer (NSCLC) organoid lines, based on isolating individual (clonal) organoids established from multiple spatially separated tumour regions. Each organoid subline was co-cultured with autologous tumour infiltrating lymphocytes (TIL) to evaluate how they differ in their capacity to elicit a T-cell response. We combined functional assays with DNA, RNA and T-cell receptor (TCR) sequencing to perform an in-depth characterisation of 44 individual organoid lines derived from 6 separate tumour regions from two patients with NSCLC to identify the mechanistic basis driving subclonal immune evasion.
We focused our analyses on organoid libraries from two different patients. For patient 1, T-cell recognition strongly differed between separate clones established from one individual tumour region. This was not due to differences in antigen expression, as the same heterogeneity between organoid sublines was seen when tested for their ability to elicit T-cell responses against a defined (and exogenously provided) model antigen. DNA sequencing showed that immune-escaping clones (‘cold’ clones) shared distinct copy number alterations that set them apart from hot clones. RNA sequencing of hot and cold clones revealed that immune evasion was associated with a transcriptional profile linked to quiescence. Current experiments are aimed at determining a potential causal link between quiescence and immune evasion.
For patient 2, organoids were derived from four different tumour regions that differed strongly in histomorphology, with two regions featuring a papillary growth pattern, and two other regions are solid growth pattern. Organoids derived from papillary tumour regions escaped T-cell detection in vitro, in contrast to organoids from the solid tumour compartment. Tumour regions that gave rise to hot or cold organoids diverged greatly at the genetic level. Immune escape was antigen-dependent, suggesting that regional heterogeneity in antigen expression drives subclonal immune escape in this patient’s tumour. Current work is focused at experimental identification of the target antigen and validation of differences in expression that drive subclonal immune escape.
Conclusions
Taken together, these results show (i) that tumour evolution can give rise to distinct cancer clones with intrinsic differences in immune evasion capacity and (ii) provide an approach to prospectively identify and isolate immune evading subclones from a patient with cancer.
Progress beyond state-of-the-art and expected impact
A direct assessment of the heterogeneity in the ability of distinct cancer subclones to escape immune predation has, to the best of our knowledge, not been performed previously. We were able to generate patient-derived functional models from individual cancer clones, that can be co-cultured with autologous immune cells. This allowed moving beyond descriptive data and provided direct evidence that cancer subclones show intrinsic differences in their ability to elicit a productive T-cell response. Current work is aimed at identifying the specific mechanism underlying subclonal immune escape, and the generalisability in a wider patient cohort, to determine the potential for therapeutic targeting. Moreover, we anticipate that a better understanding of how clonal heterogeneity contributes to immune escape will drive the design of more rational (combination) immunotherapies aimed at targeting subclonal resistance.
Exploitation and dissemination
Insights driven in part by work from this project have led to the publication of two review articles:
- Hughes T, Dijkstra KK, Rawlins EL & Hynds R. Open questions in human lung organoid research. Front Pharmacol. 2022;13:1083017.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9880211/
- Dijkstra KK, Wu Y & Swanton C. The impact of clonal heterogeneity on cancer immunosurveillance. Ann Rev Cancer Biol 2023;7:131-47.
Final publication date April 7 2023.
A primary research paper is in preparation and will be submitted to an open-access peer-reviewed scientific journal.
This work has been presented at the Cancer Research UK Cancer Evolution symposium, the Crick postdoc symposium, and the annual meeting of the American Association of Cancer Resarch. Abstracts have been submitted to the annual conference of the European Association of Cancer Resarch (EACR) and the EACR’s immune-oncology conference in May and June 2023, respectively.
During this project, talks for lay audiences have been given at the Crick’s ‘Outwitting Cancer’ exhibition and ‘Pint of Science’.
A direct assessment of the heterogeneity in the ability of distinct cancer subclones to escape immune predation has, to the best of our knowledge, not been performed previously. We were able to generate patient-derived functional models from individual cancer clones, that can be co-cultured with autologous immune cells. This allowed moving beyond descriptive data and provided direct evidence that cancer subclones show intrinsic differences in their ability to elicit a productive T-cell response. Current work is aimed at identifying the specific mechanism underlying subclonal immune escape, and the generalisability in a wider patient cohort, to determine the potential for therapeutic targeting. Moreover, we anticipate that a better understanding of how clonal heterogeneity contributes to immune escape will drive the design of more rational (combination) immunotherapies aimed at targeting subclonal resistance.
Exploitation and dissemination
Insights driven in part by work from this project have led to the publication of two review articles:
- Hughes T, Dijkstra KK, Rawlins EL & Hynds R. Open questions in human lung organoid research. Front Pharmacol. 2022;13:1083017.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9880211/
- Dijkstra KK, Wu Y & Swanton C. The impact of clonal heterogeneity on cancer immunosurveillance. Ann Rev Cancer Biol 2023;7:131-47.
Final publication date April 7 2023.
A primary research paper is in preparation and will be submitted to an open-access peer-reviewed scientific journal.
This work has been presented at the Cancer Research UK Cancer Evolution symposium, the Crick postdoc symposium, and the annual meeting of the American Association of Cancer Resarch. Abstracts have been submitted to the annual conference of the European Association of Cancer Resarch (EACR) and the EACR’s immune-oncology conference in May and June 2023, respectively.
During this project, talks for lay audiences have been given at the Crick’s ‘Outwitting Cancer’ exhibition and ‘Pint of Science’.