Abstract 6: Comprehensive single-cell transcriptomic analysis of a mouse model of bladder cancer identifies endothelial cells as a principal target of interferon gamma during response to combined PD-1, CTLA-4 immune checkpoint inhibitor treatment
Authors: Freshour Sharon L. , Fisk Bryan , Chen Timothy Hung-Po , Shen Haolin , Mosior Matthew , Fronick Catrina , Bolzenius Jennifer , Griffith Obi L. , Arora Vivek , Griffith Malachi 
Published: April 2023

Abstract
Bladder cancer is the tenth most common cancer worldwide, accounting for over 500,000 new cancer cases and 200,000 cancer-related deaths per year. Depending on the degree of muscle invasiveness and disease spread at the time of diagnosis, 5-year survival rates can range from as high as 96% to as low as 6%. In recent years, use of checkpoint inhibitor treatments for bladder cancer has become more common. Currently, there are five PD-1/PD-L1 checkpoint inhibitors approved by the FDA for treatment of bladder cancer and these treatments have been used across the spectrum of disease, from non-muscle invasive to metastatic disease. However, many patients still do not receive benefit from checkpoint inhibitor treatments. For example, clinical trials have shown response rates of only 20-30% in advanced disease. To improve treatment responses and identify patients who will benefit most from checkpoint inhibitor treatment, a deeper understanding of mechanisms of treatment response is needed. In an effort to explore mechanisms of response to combined PD-1/CTLA-4 checkpoint inhibitor treatment, we generated single cell RNA expression sequencing from a carcinogen-induced mouse model of muscle invasive urothelial carcinoma that included three conditions: untreated tumor, tumor treated with combination treatment, and tumor treated with combination treatment after Cd4+ T-cell depletion. After classifying tumor cells based on per cell detection of somatic variants identified using exome sequencing and assigning non-tumor cell types using SingleR, we performed differential expression, overrepresentation, and gene set enrichment analysis (GSEA) within each cell type, comparing each pairwise set of conditions. This analysis revealed that MSigDB’s hallmark interferon gamma response gene set was overrepresented across multiple cell types. Furthermore, GSEA indicated that with combined checkpoint blockade treatment, endothelial cells showed significant enrichment of upregulated interferon gamma response genes. Functional analysis demonstrated that knocking out Ifngr1 in endothelial cells inhibited the treatment response seen in wild type treated mice. Together, these results indicate that interferon gamma response in endothelial cells is a key mediator of combined PD-1/CTLA-4 checkpoint inhibitor treatment response.
Citation Format: Sharon L. Freshour, Bryan Fisk, Timothy Hung-Po Chen, Haolin Shen, Matthew Mosior, Catrina Fronick, Jennifer Bolzenius, Obi L. Griffith, Vivek Arora, Malachi Griffith. Comprehensive single-cell transcriptomic analysis of a mouse model of bladder cancer identifies endothelial cells as a principal target of interferon gamma during response to combined PD-1, CTLA-4 immune checkpoint inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6.