Abstract 122: Highly multiplexed, spatially-resolved tissue imaging of genetically engineered mouse models of cancer to discover and characterize immune regulators of tumorigenesis
Authors: Gaglia Giorgio , Burger Megan , Ritch Claire , Argyropoulou Danae , Dai Yang , Coy Shannon , Lin Jia-Ren , Sorger Peter , Jacks Tyler , Santagata Sandro 
Published: July 2021

Abstract
Despite the encouraging success of immunotherapy for certain malignancies, most patients with solid tumors do not yet benefit from such therapies. It is therefore critical that we identify factors dictating responsiveness to immunotherapy by more completely characterizing tumor-immune interactions. Even though immunotherapies are used in humans, in-depth analysis of murine models is essential for a mechanistic understanding of the crosstalk among tumor, immune, and stromal components of the tumor microenvironment (TME): only animal models have the necessary manipulability and reproducibility for causal, mechanistic studies. However, murine modelling must be combined with spatially resolved analytical methods such as highly multiplexed tissue imaging that enable accurate characterization of the TME at a single-cell level. Here we use tissue cyclic immunofluorescence (t-CyCIF) multiplexed imaging to characterize the immune microenvironment of a mouse lung adenocarcinoma model initiated via lentiviral delivery of Cre recombinase into the lungs of KrasLSL-G12D/+;p53fl/fl (KP) mice. Using this new platform, we identify tumor cells and immune cell types (dendritic cells, NK cells, macrophages, B cells, helper T cells, regulator T cells and cytotoxic T cells). We characterize the effects of expressing tumor antigen (i.e., LucOS), of CRISPRa based upregulation of the chemokines (e.g., CXCL10), and of combination immune check point inhibitor treatments (e.g., one-week treatment with anti-PD-1 and anti-CTLA-4 (PC)). Remarkably the total immune composition of the mouse lungs remains relatively unchanged following these perturbations, but there are marked changes in the immune cell localization in tumor nodules, in the number and size of immune cell networks and in the functional activation states of cytotoxic T cells. For example, one week of PC treatment did not affect tumor burden and did not change the extent of immune cell infiltration however, it did drastically change cytotoxic T cell phenotypes with increased effector phenotypes (i.e., GzmB and Perforin expression and proliferation) and decreased exhaustion-like phenotypes (i.e., PD-1 and Tim-3 expression). Lymphocyte networks decreased in number but were closer to tumors. Using high-dimensional protein expression data to characterize GEMM models following in situ genetic or therapeutic perturbation is a powerful new platform to investigate tumor-immune interactions.
Citation Format: Giorgio Gaglia, Megan Burger, Claire Ritch, Danae Argyropoulou, Yang Dai, Shannon Coy, Jia-Ren Lin, Peter Sorger, Tyler Jacks, Sandro Santagata. Highly multiplexed, spatially-resolved tissue imaging of genetically engineered mouse models of cancer to discover and characterize immune regulators of tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 122.