Abstract 6381: Quantitative multiplex analysis of soluble and cellular immune checkpoint proteins in response to interferon gamma across multiple murine tumor models

Abstract Immune checkpoint proteins are key regulators of the immune system and drug targets of cancer immunotherapy. Immune checkpoint molecules have been found on the cell surface of various tumor cells and immune cells as immune activating or inhibitory receptors and ligands. Recent studies have shown these immune checkpoint proteins can undergo alternative splicing or proteolytic cleavage leading to soluble isoforms. IFNγ is a pleiotropic and multifunctional cytokine commonly released by cytotoxic immune cells. IFNγ production can be enhanced in response to immunotherapy and further modulates the expression of immune checkpoints in the tumor microenvironment. To better understand the immune checkpoint signatures in tumors, we developed a 28-plex mouse checkpoint protein multiplex immunoassay for quantitative analysis of soluble (secreted) and cellular immune checkpoint proteins. Using this immunoassay, we tested 16 mouse cell lines of tumor (n=13) and normal tissues as control (n=3): 2 melanomas (B16F10, YUMM1.7), 3 mammary carcinoma (4T1, NT5, AT-3), 1 lung carcinoma (LLC), 1 prostate cancer (MycCaP), 1 fibrosarcoma (MCA205), 1 ovarian cancer (ID8), 1 bladder cancer (MB49), 1 glioma (CT-2A), 2 blood cancer (RAW, EL4), 1 dendritic cells (DC2.4), 1 myoblast cells (C2C12), and 1 endothelial cells (bEnd.3). We analyzed changes in immune checkpoint protein expression and secretion in these cell lines cultured in normal conditions or upon exposure to IFNγ to reflect an immune inflamed microenvironment. Cells were cultured in 96-well plate, 100 mm, or 150 mm Petri dishes, and treated with either 10 ng/ml or 100 ng/mL mouse IFNγ. Cell culture supernatants and cell lysates were harvested after 6h, 12h, or 24h incubation with IFNγ and processed for multiplex Luminex bead-based immunoassay. The soluble and cellular checkpoint proteins most substantially modulated by IFNγ treatment in these cell lines included: CD137, BTLA, HVEM, PD-L1. The tumor-specific signature of cellular and soluble checkpoint proteins and its response to IFNγ may have implications in the tumor microenvironment and in the outcome to immunotherapy. Citation Format: Wen-Rong Lie, Ryan Bucktrout, Sanjukta Chakraborty, Andrea Orlando, Inna Serganova, Christine Kornmeier, James Hoberg, Roberta Zappasodi. Quantitative multiplex analysis of soluble and cellular immune checkpoint proteins in response to interferon gamma across multiple murine tumor models [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 6381.