Dissecting the Microenvironment of Urothelial Bladder Cancer : Therapy, Modelling and Biomarkers

Abstract: The complexity of the tumor microenvironment (TME) impacts therapy responses and the survival of cancer patients. The scope of this thesis is to study the effects of immune modulation on tumor-infiltrating leukocytes, and to explore the TME of urothelial cancer to provide the research society with new knowledge and potential therapeutic targets.In paper I, a small molecule MALT1 inhibitor demonstrated in vitro selective inhibition of immune suppressive T regulatory cells (Tregs). However, the inhibitor failed to demonstrate anti-tumor therapeutic effects in vivo. The effect of the MALT1 inhibitor was assessed on immune cell subsets in tumors, as well as in secondary lymphoid organs. Ultimately, the inhibitor was found to have a depleting effect on antigen-specific T cells.Paper II describes the development and characterization of a novel murine urothelial cancer model in both sexes by using a low-dose carcinogen exposure via drinking water combined with a unique transgenic strain. The model recapitulates the profile of basal histology subtype urothelial cancer with a progression outcome, presents transcriptomic tumor heterogeneity, and displays early immune activation signatures despite its failure to respond to checkpoint blockade immunotherapy. Moreover, the study demonstrates that the female sex displayed accelerated tumor development, but also benefited from therapeutic TLR9 stimulation in contrast to males.Therefore, in paper III, an in-depth transcriptomic analysis of the immune cell compartment of the TME was performed in both sexes by using this novel model of urothelial cancer. Infiltrating immune cell subsets varied between stages, while cell communication analysis pinpointed distinct interactions among immune and tumor cells and pathways of immune regulation suitable for therapeutic targeting. Also, differentially expressed genes in immune cell subsets of the TME between sexes revealed higher immune responsiveness in some female immune cell subsets and higher metabolic activity in several male immune cell subsets.In paper IV, proximity extension assay (PEA) analysis of urine and plasma was assessed for liquid biopsy biomarker discovery. Samples from patients at first diagnosis revealed that high MMP12 plasma levels were associated with poor survival. This finding was reproduced in serum PEA analysis of an independent urothelial cancer patient cohort. Single cell transcriptomic analysis of public datasets demonstrated that the production of MMP12 in urothelial cancer could be attributed to tumor-infiltrating macrophages.In conclusion, this thesis illustrates the multi-faceted opportunities and challenges of targeting and studying the microenvironment of urothelial cancer in murine models and human patients.