Project Summary An important question in cancer immunology is how malignant cells and T cells communicate and impact the efficacy of anti-tumor immunity. To generate hypotheses for how these cells interact, we have applied single cell RNA sequencing to profile the transcriptomes of >700,000 malignant, immune, and stromal cells in 2 cohorts: (1) 65 patients with two types of primary untreated CRC (31 MSS and 34 MSI tumors) which are characterized by vastly different immunotherapy response rates; (2) pre- and post-treatment specimens from 20 BRAF-mutant metastatic CRC patients treated with BRAFi/MEKi/anti-PD1. These data led us to formulate the central hypothesis for this proposal: malignant cells that express interferon-stimulated genes (ISGs) acutely promote anti-tumor immunity, while chronic interferon responses in malignant cells impair anti-tumor immunity and therapeutic responses. The proposed work is anticipated to reveal fundamental insights into human tumor immunology and provide a novel perspective for the design of robust biomarkers and new therapeutic strategies. We will address three major questions. First, is the MSI/responder-associated ISG program in malignant cells part of an intra-tumoral feed-forward loop that is driving anti-tumor immunity? This will be answered by spatially mapping the intratumoral cellular interaction network between ISG+ malignant cells and T cells in untreated primary CRC specimens, and in post-treatment specimens of patients treated with BRAFi/MEKi/anti-PD-1, which will reveal association of this spatial network with tumor regression. Second, what are the drivers of malignant ISGs in human CRC, and can interferons impact immune responses and responses to targeted therapy by epigenetically reprogramming human CRC cells? To address this, we will use interferons and innate immune stimuli to induce ISGs in CRC organoids, map the transcriptional and epigenetic signatures to the signatures found in freshly isolated CRC cells, and nominate ISG-inducing upstream regulators. IFN-stimulated and subsequently rested organoids will be assessed for epigenetic memory and for modulated secondary immune and drug responses. Third, how do pre-existing ISG signatures in malignant cells impact CTL-mediated anti-tumor responses? Using peptide-cognate T cell lines, we will test the in vitro killing of ISG+ and ISG- peptide-loaded CRC organoids. We will furthermore test how ISGs modulate in vivo tumor killing by temporally controlling malignant ISG expression in transplantable tumor models. In summary, we use scRNAseq to predict malignant cell – T cell interactions, and then test these hypotheses with cutting-edge technologies such as spatial profiling, single cell epigenomics, and human organoid T cell co-cultures. The results should elucidate how malignant cells that respond to interferons impact anti-tumor immunity and resistance, generate foundational results and model systems for an R01 proposal, and prepare the candidate for...