Targeting the tumour microenvironment to enhance immunotherapy against cancer

Abstract

Cancer immunotherapies have shown astounding clinical results within the last decade, with complete eradication of advanced malignancies in certain cancer types, particularly melanoma and non-small cell lung cancer (NSCLC). These successes lead to clinical approval in multiple countries for checkpoint blockade and chimeric antigen receptor (CAR) T cell therapies, and the award of the 2018 Nobel Prize in Physiology or Medicine to James P. Allison and Tasuku Honjo for their research on checkpoint blockade molecules. Despite this, many patients receive minimal benefit and focus has shifted to understanding how to predict and enhance immunotherapy responses. It is now well established that the tumour microenvironment (TME) is a major limiting factor for immunotherapy efficacy. Studies in mice using genetically identical tumour lines implanted in different tissues have demonstrated that the location of tumour growth can directly impact the composition of the TME and response to anti-cancer therapies. Retrospective analysis of checkpoint blockade treated patients’ revealed tissue-specific patterns of response, where metastases in certain anatomical sites were more responsive than others. To date studies investigating the tissue-specific influence on immunotherapy responses in vivo have limited clinical relevance, and studies in patients are minimal. In this thesis, we investigated the influence of the tissue-specific TME on immunotherapy responses in vivo and assessed tissue-specific patterns in the TME of breast cancer metastases from patient samples.

First, we investigated a murine breast cancer model comparing responses of primary tumours to tumours in the liver and lungs as common metastatic sites to two immunotherapies, anti-PD-1/anti-CTLA4 and trimAb (anti-4-1BB, anti-CD40, anti-DR5). We reported that the 67NR tumour line growing in the lungs was resistant to immunotherapy, whereas the same tumour line growing in the mammary fat pad (MFP, primary tumour site) or liver could be completely eradicated in a portion of mice. Our analysis revealed that the resistance of lung tumours was independent of the tumour cells, vasculature or drug delivery and that the immune TMEs of lung and MFP tumours were distinct. Specifically, we demonstrated that lung tumours had a more immunosuppressive TME, with increased myeloid derived suppressor cells (MDSCs), decreased T cells and decreased activation of T cells and natural killer (NK) cells. Furthermore, upon depletion of various immune subsets alongside therapeutic intervention we found that NK cell depletion had a significant impact on lung tumours, but not MFP tumours. Taken together our data suggests that tumours grown in different tissues sculpt different TMEs with varied levels of immunosuppression and require different immune cell subsets, and perhaps different immune stimulants, for optimal anti-tumour responses. Following on from this study, we next wanted to assess responses to immunotherapy in vivo in models where multiple tumours in different anatomical sites were present. The rationale of this model was to investigate a more accurate representation of advanced cancer, where tumours have metastasised to multiple locations throughout the body. We hypothesised that co-existing tumours in different sites with disparate TMEs could influence immunotherapy responses compared to tumours existing alone. Our results indicated that the presence of a concomitant MFP tumour enhanced responses of lung tumours to trimAb or anti-PD-1/anti-CTLA4 therapies compared with mice bearing only lung tumours. We observed a decrease in lung metastasis burden in mice with simultaneous MFP tumour growth even before therapy commencement, which likely contributed to enhanced therapy responses. Upon interrogation we found that CD8+ T cells were responsible for the decrease in lung tumour burden and that the lungs of mice with co-existing MPF tumours had more tumour reactive CD8+ T cells. From our results, we hypothesised that the presence of a tumour in a more immunogenic location, such as the MFP, promoted T cell priming within the tumour draining lymph node (TdLN) at this site and led to a systemic response against distal tumours, such as tumours within the lungs. Lastly, we aimed to identify tissue-specific patterns within metastases from human tumours. Herein, we utilised metastatic tumour samples collected as part of the cancer tissue collection after death (CASCADE) rapid autopsy program from three estrogen receptor positive (ER+) breast cancer patients and one triple negative breast cancer (TNBC) patient. We analysed the immune profiles of these samples by transcriptomic and immunohistochemical (IHC) analyses. Our data demonstrated that, although there were potential tissue-specific differences within the TME, the most significant trend delineated immunological differences between ER+ and TNBC patients. These results confirmed previous research describing a higher immune infiltrate in TNBC samples compared with ER+ samples. Our research highlights the potential of investigating metastatic tumour samples however, future studies with separation of disease subtypes and increased sample sizes are needed to truly investigate tissue-specific patterns within the TME.

In summary, the data presented in this thesis highlights the importance in further defining tissue-specific response patterns and mechanisms in patients to optimise current and future immunotherapies. Our results indicate that an in depth understanding of the tissue-specific TME could reveal novel treatment options in tumours that are non-responsive to current immunotherapies.