LAUSR

In the last decades, several major breakthroughs in cancer treatment were achieved through a better understanding and therapeutic targeting of the immune system. Multiparametric flow cytometry is a robust and powerful tool to evaluate the frequency and activation status of a large variety of immune cells. However, for flow cytometric analysis tumor, tissue must be digested into a single cell suspension, and thereby the information on the tumor architecture as well as the spatial distribution of immune cells is lost. The distribution of immune cells in the tumor microenvironment is often heterogeneous and a favorable immune cell distribution is crucial for an efficacious anti-tumor immunotherapy. The recent advances in multiplex immunofluorescence (mIF) allows the analysis of multiple parameters simultaneously so that the frequency as well as the spatial distribution of multiple immune cells can be analyzed. In this study, immunotherapy-responsive tumor cell lines (colon carcinoma cell lines MC38-CEA and CT26wt as well as melanoma cell line Clone M3) were implanted in the mammary fat pad (subQperiorTM), which leads to reduce ulceration rate and homogenous tumor growth compared to subcutaneous implantation. Anti-PD-1 immunotherapy reduced the tumor growth in all three tumor models significantly. Formalin-fixed paraffin-embedded tumor samples were collected from control and anti-PD-1 treated animals. Murine specific InSituPlex® (ISP) technology (Ultivue) was used to perform multiplex immune profiling of the markers CD3, CD4, CD8 and FOXP3 on mouse whole slide FFPE tumor serial sections. Slides were then imaged for high quality images of the four targets and downstream analysis performed. The evaluated T cell frequency and spatial distribution was compared to flow cytometric data of anti-PD-1 treatment in these models. In summary, flow cytometry and multiplex immunofluorescence (mIF) are valuable tools in the evaluation of immune responses in pre-clinical tumor models. Both technologies enable an improved understanding of treatment responses and facilitate the development of novel treatment modalities as well as the identification of potential predictive biomarkers. Citation Format: Philipp Metzger, Carla Castro, Cynthia Obodozie, Holger Weber. Immunophenotyping of responses to immunotherapy in syngeneic tumor models using multiplex immunofluorescence compared to multiparametric flow cytometry. [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 4148.