Transcriptional regulators in tumor cells drive spatial heterogeneity and control T cell infiltration: Development & application of methods for single cell and spatial omics data

Abstract

T cell exclusion correlates with resistance in solid malignancies. Consequently, an outstanding question is to understand the mechanisms of T cell exclusion. Spatial analyses of the tumor microenvironment are revealing distinct immune hubs that partially explain T cell location. However, the “ultimate orchestrators” that recruit suppressive immune cells or, conversely, create “hot” immune hubs, remain understudied. Cancer cells are plastic, with diverse phenotypes inside a tumor mass: single cell RNAseq (scRNAseq) has enabled deep and granular profiling of individual cells, revealing the full spectrum of states that they assume. In Chapter 1, we propose correspondence analysis, a count-based alternative to principal component analysis (PCA), as an alternative approach to dimension reduction in the “standard” scRNAseq pipeline to better preserve structure in the data. Shifting focus to exploratory data analysis human breast cancer, in Chapter 2, we investigated the cancer cell phenotypes that correlated with T cell exclusion in patients and mice in highly multiplexed immunofluorescence data. We profiled the tumor cells that exclude T cells and compared them with those that allow T cell infiltration to identify the features most strongly associated with T cell exclusion. To look for associations between tumor cells and how infiltrated they are, we proposed a robust negative binomial generalized linear model approach with bootstrapping to model T cell neighbors as a function of tumor markers and field effects of nearby tumor cells. In Chapter 3, we delve deeper into mechanisms of immune exclusion and evasion. By applying computational methods to infer gene regulatory networks, we identified transcriptional regulators (TRs) that enable these cancer cells to create immunosuppressive hubs. To screen these candidate TRs for targets that specifically modulate the tumor cell’s sensitivity to cytotoxic T cells, we developed an experimental framework for conducting in vitro competitive killing assay. Knock-out cancer cells implanted in mice further validated our predictions, with some showing increased T cell infiltration, and demonstrating that we have identified cancer-intrinsic regulators that shape the tumor immune microenvironment. We combine these approaches into an iterative experimental platform bringing together in silico, in vitro, and in vivo profiling to characterize these TRs of interest in tumor cells, how they interact with the immune system, and whether chemically perturbing them in T cells affects cytotoxic function. This opens the door to investigation of small-molecule inhibitors with the potential to both repurpose existing compounds and to uncover new drug targets that would synergize with immunotherapy.