Abstract P04: Extracellular Cartography Reveals Immunomodulatory Axes in Colorectal Cancer Tumor Microenvironment

Abstract The tumor microenvironment (TME) orchestrates immune evasion and therapeutic resistance in colorectal cancer (CRC), particularly in microsatellite stable (MSS) tumors that exhibit poor responses to immunotherapy. While the cellular component of the TME has been extensively characterized, the contribution of the extracellular component (ECM and other proteins) to intercellular signaling and immune modulation remains poorly understood. In this study, we employed Multiplexed Ion Beam Imaging to profile 71 cellular and 18 extracellular protein markers from 250 CRC tumors in and around 5.7 million single cells. To enable high-resolution quantification of extracellular protein deposition, we developed CellExt, a computational framework that measures protein abundance from the cell membrane outward to 40 microns in pixelwise increments. This enables the systematic analysis of extracellular geography and its impact on cell signaling. CellExt revealed that distinct extracellular protein deposition patterns serve as predictors of CRC subtypes and tumor location. We identified prognostic signaling modalities, including the deposition of TRAIL ligand and interferon-gamma around pankeratin-high, poorly differentiated epithelial regions, which were associated with downstream activation of phosphorylated S6 and mucin proteins, respectively. Furthermore, strong co-localization between cyclooxygenase-2 (COX2) and Collagen1A1 implicated extracellular matrix remodeling as a driver of inflammation within the TME. Key predictive proteins included proteases MMP11 and HTRA1, while the presence of mucin proteins TFF3 and MUC5AC distinguished immunosuppressive and inflammatory microenvironments. Notably, elevated extracellular levels of IL-33 correlated with improved patient survival, suggesting an anti-tumorigenic function. Together, these findings reveal previously unrecognized immunomodulatory pathways mediated by the TME’s extracellular component and identify putative therapeutic targets for CRC, with relevance to refractory MSS tumors. Our spatial proteomics approach, enabled by the CellExt framework, provides a generalizable strategy for dissecting extracellular-mediated communication in the TME with further applications for precision immunotherapy. Citation Format: Reema Baskar, Vairavan Lakshmanan, Dane Bagaoisan, Wilfred Wong, Marc Bosse, Ferda Filiz, Christine Fullaway, Sean Bendall, Iain Tan, Shyam Prabhakar. Extracellular Cartography Reveals Immunomodulatory Axes in Colorectal Cancer Tumor Microenvironment [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr P04.