Abstract 3863: Development of specific multiplexed immunofluorescence immune assays to study mouse models of tumorigenesis
Authors: Cajigas Yvette , Hamer Peter , Ainbinder Alina , Chatterjee Gourab , Vasaturo Angela , Manesse Mael , Maclean Kirsteen 
Published: June 2022

Abstract
Immunotherapy has transformed the treatment of metastatic and recurrent solid tumors. Advances in technology in the past few years have created unprecedented opportunities to identify biomarkers of disease processes, especially by using multi-omics technologies and datasets to derive valid and useful signatures of disease. Mouse tumor models are widely used tools to demonstrate activity of novel immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. The use of specific mouse tissue phenotyping and multiplex immunofluorescence (mIF) assays offer the unique advantage of preserving the architectural features of the tumor and revealing the spatial relationships between tumor cells and immune cells. The urgency to discover and implement new biomarkers lays bare the need to integrate a variety of advanced pre-clinical tools to probe the dynamic nature of events happening in the tumor immune microenvironment (TiME). Here, we present an established mIF assay, InSituPlex® technology, now enabled for rapid staining and visualization of dedicated 4-plex murine panels for deep, spatial phenotyping in the tumor microenvironment. Murine specific InSituPlex technology was used to perform singleplex and multiplex immune profiling on mouse FFPE tumor serial sections. Alternating serial sections were stained with a cocktail of four primary antibodies or a single primary antibody, in parallel, using Leica Biosystems BOND RX autostainer. Slides were imaged on the Zeiss Axio Scan Z1 providing high-quality images of the four targets (CD3, CD4, CD8, and FOXP3) in a single workday. The images were exported for downstream analysis with Indica Labs HALOv3.1 software. Concordance of the singleplex to the 4-plex mouse specific InSituPlex assay was assessed by quantifying the percent difference in cellular density of immune cell subtypes from 1-plex to 4-plex. Qualitative assessment and quantitative analysis revealed a high level of reproducibility of the assay, with each single markers’ coefficients of variation falling within an acceptable range. Each single marker tested showed an expected level of expression and expression pattern. The multiplex assay was highly concordant to the singleplex assay. Unique phenotypes from whole slide analysis followed the expected pattern, as indicated by the tumor type. Our protocol of mIF staining on mouse tissue provides an improved workflow to investigate the immune system, including the analysis of the tumor immune microenvironment and mechanisms of action of immune-related drugs in preclinical models.
Citation Format: Yvette Cajigas, Peter Hamer, Alina Ainbinder, Gourab Chatterjee, Angela Vasaturo, Mael Manesse, Kirsteen Maclean. Development of specific multiplexed immunofluorescence immune assays to study mouse models of tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3863.