Abstract 5263: The spatial landscape of tumor-infiltrating lymphocytes in stage III colorectal cancers by single-cell spatial analysis
Authors: HoJoon Lee, Andrew Su, Minh Tran, Richard Delacruz, Anuja Sathe, Xiangqi Bai, Ignacio Wichmann, Lance Pflieger, Bryce Moulton, Tyler Barker, Derrick Haslem, David Jones, Lincoln Nadauld, Quan Nguyen, Hanlee P. Ji, Terence Rhodes
Journal: Cancer Research (2025)

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related deaths. An important contributor to CRC development, maintenance and metastasis is the tumor microenvironment (TME). This cellular milieu contains neighboring tumor epithelial cells, normal epithelium, fibroblasts, endothelial cells, immune cells and many others. The characterization of the TME is critical for understanding tumor biology as well as determining specific features with clinical implications. Representing a subset of the TME, the tumor immune microenvironment (TIME) has specific cellular characteristics that are important predictors for the response to treatments such as immunotherapy. However, the accurate interpretation of complex spatial cell patterns poses significant challenges. To address these issues, we developed a novel computational framework that systematically analyzes spatial data. We conducted a spatial analysis using imaging mass cytometry applied to stage III colorectal adenocarcinomas. This study used multiplexed markers to distinguish individual cells and their spatial organization from 52 colorectal cancers. We determined the landscape features of cellular spatial features in the CRC tumor microenvironment. This spatial single-cell analysis identified 10 unique cell phenotypes in the tumor microenvironment that included stromal and immune cells with a subset which had a proliferative phenotype. These special features included spatial neighborhood interactions between single cells as well as different tissue niches, especially the tumor infiltrating lymphocyte regions. We applied a robust statistical analysis to identify significant correlations of cell features with phenotypes such as microsatellite instability or recurrence. We determined that microsatellite stable (MSS) colorectal cancers had an increased risk of recurrence if they had the following features: 1) a low level of stromal tumor-infiltrating lymphocytes, and 2) low interactions between CD4+ T cells and stromal cells. Our computational framework precisely defined two types of tumor-infiltrating lymphocytes (TILs) based on their spatial properties in the local tumor microenvironment: 1) intra-tumoral TILs within cancer CNs and 2) sTILs within stromal CNs. These spatial characterizations of TILs provide deeper insights into their role, beyond simple lymphocyte abundance. Moreover, our results point to the utility of spatial single-cell interaction analysis in defining novel features of the tumor immune microenvironments and providing useful clinical cell-related spatial biomarkers. Citation Format: HoJoon Lee, Andrew Su, Minh Tran, Richard Delacruz, Anuja Sathe, Xiangqi Bai, Ignacio Wichmann, Lance Pflieger, Bryce Moulton, Tyler Barker, Derrick Haslem, David Jones, Lincoln Nadauld, Quan Nguyen, Hanlee P. Ji, Terence Rhodes. The spatial landscape of tumor-infiltrating lymphocytes in stage III colorectal cancers by single-cell spatial analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5263.