Abstract
In New Zealand, the incidence and mortality rates of colorectal cancer (CRC) are among the highest worldwide. Despite drastic advances in our understanding of this disease, current treatments for metastatic CRC remain largely ineffective, highlighting the urgent need for more effective therapies. Recently, therapeutic strategies that harness anti-tumour immune responses – called immunotherapies – have achieved unsurpassed clinical efficacy in a subset of cancers. Given the crucial role of the immune system in controlling CRC progression and metastasis, immunotherapies represent a promising approach for the treatment of this disease. However, despite their tremendous potential, technical constraints currently limit the clinical application of cancer immunotherapies for solid tumours such as CRC. Most notably, the identification of targetable tumour antigens remains a highly expensive, difficult, and laborious process. Consequently, simplified approaches to immunotherapy that circumvent the need to identify specific tumour antigens are attractive alternatives, offering both clinical efficacy and broad applicability.
Tumour lysates provide a source of both undefined tumour antigens and endogenous adjuvants – called danger-associated molecular patterns (DAMPs) – to induce anti-tumour immune responses. Tumour lysates can therefore serve clinical applications for immunotherapy – including vaccination and adoptive cell therapy – without having to identify specific tumour antigens. Utilising the MC38 murine CRC model, this thesis investigated the feasibility of using tumour lysates as a simplified but broadly applicable immunotherapy for CRC.
In the first study, longitudinal immune phenotyping revealed dynamic changes in T cell infiltration during progressive MC38 tumour outgrowth. Important immune-suppressive mechanisms identified in this model include the upregulation of inhibitory immune molecules, the presence of deleterious or dysfunctional immune populations, and the physical exclusion of T cells from the tumour parenchyma. Importantly, these mechanisms reflect those identified in human CRC patients that contribute to immunotherapy resistance. Consequently, MC38 represents a clinically relevant model for investigating immunotherapies – especially human CRC subtypes that exhibit similar biological mechanism(s) of immune evasion.
In the second study, methodology to improve the therapeutic efficacy of tumour lysates was investigated. Specifically, the exposure of tumour cells to either oxidation (using hypochlorous acid) or heat shock (at 42°C and 56°C) were evaluated as strategies to enhance tumour lysate immunogenicity. The immune-stimulatory properties of different tumour lysate preparations were then compared in vitro and in vivo. Despite poor correlates of immunogenicity in vitro, prophylactic vaccination with tumour lysates could elicit protective anti-tumour immunity in vivo. However, the results of this study provided little evidence that the induction of cell stress could enhance the immune-stimulatory properties of tumour lysate preparations. Importantly, findings from this chapter advocate for the careful characterisation and optimisation of methodology for tumour lysate preparation, as well as careful selection of in vitro and in vivo models to assess their immunogenicity.
In the third study, tumour lysate-loaded antigen-presenting cells were investigated as a platform for the expansion of tumour-specific T cells. In a model antigen system, lysates prepared from tumour cells exposed to severe heat shock could facilitate the ex vivo expansion of antigen-specific CD8+ T cells. These T cells retained desirable phenotypic and functional attributes for use in adoptive immunotherapy but failed to mediate robust tumour regression when transferred into tumour-bearing hosts. This failure was attributable to mechanisms of immune-suppression intrinsic to the MC38 model, highlighting the need to target active immune-resistance mechanisms to potentiate the efficacy of immunotherapies.
In the fourth and final study, a potential mechanism underlying the immunogenicity of lysates prepared from severe heat-shocked tumour cells was investigated. Results from this chapter offer evidence that the degradation of antigen by stress-induced proteases can enhance subsequent cross-presentation in vitro. Important mediators implicated in this phenomenon include tumour-derived calpains and the association of antigenic peptides with necrotic tumour cell debris. Together, these findings suggest that stress-induced proteases can increase the availability of antigenic substrates for cross-presentation, thereby enhancing the immunogenic potential of tumour cell death. Importantly, this data extends the links between cell death and immunity and may have profound implications in the contexts of infectious disease and autoimmunity, in addition to cancer immunotherapy.
In conclusion, this thesis demonstrates that immune-suppressive mechanisms active in CRC tumours represent a prominent barrier to effective immunotherapy and highlights inherent limitations to the use of tumour lysates as a therapeutic approach. Thus, while simplified approaches to immunotherapy may offer the advantage of widespread applicability, more sophisticated approaches are needed to realise the therapeutic potential of immunotherapies in the context of CRC. In this regard, personalised combination strategies that are tailored to individual patients’ specific tumour microenvironments hold immense promise for the next generation of cancer immunotherapies. Finally, findings contained within this thesis may also broaden the current concept of what determines immunological outcomes in response to cell death and open new avenues for investigation.