Abstract P38: Spatial Profiling of Colorectal Cancer Extracellular Milieu Reveals Novel Axes of Immunosuppression and Inflammation

Abstract Colorectal cancer (CRC) progression is driven by a complex interplay of immunosuppressive and inflammatory mechanisms involving various cell types, functional states, and extracellular milieu. To unravel this intricate relationship, we took a systems biology approach using high resolution spatial distributions of multiple key markers in clinically diverse CRC tumors. We employed Multiplexed Ion Beam Imaging (MIBI-TOF) to capture 65 protein markers across 3, 700 fields of view from 250 CRC tumors. This allowed us to deeply characterize the phenotypes and functional states of 5. 7 million cells. To incorporate extracellular geography information, we developed a computational approach named CellExt. This quantified and modeled the gradated abundance of key structural and functional proteins found extracellularly, such as fibronectin, granzymeB and vimentin, from each cell boundary to a 40-micron distance in pixelwise steps. CellExt detected a distinct immunosuppressive modality characterized by extracellular PDL1, TNF-related apoptosis-inducing ligand (TRAIL), and matrix metalloproteinase-9 (MMP9). Investigating the cell types involved led us to map the potential receivers and emitters by linking extracellular trends to cellular enrichment and gene expression. We found that Pankeratin-high epithelial cells were acting as TRAIL receivers with downstream activation of non-canonical pro-tumorigenic signaling through S6 phosphorylation. In parallel, we found that T cell suppressive CD40+ antigen-presenting B cells with PD1 expression were acting as MMP9 emitters. Additionally, CellExt was able to identify two spatially segregated inflammatory modalities: IFNgamma cytokine with mucin protein, MUC5AC and prostaglandin synthase, cyclooxygenase-2 with extracellular matrix component, Collagen1A1. This suggests a strong inflammatory role for Collagen1A1 in CRC. We then contextualized our identified modalities by genetic background, tumor stage and CMS subtype of CRC tumors. Taken together, our approach revealed novel axes of immunosuppression and inflammation and puts forward potential extracellular targets for therapeutic intervention in specific CRC patient subsets. Citation Format: Reema Baskar, Vairavan Lakshmanan, Audrey Lee, Wilfred Wong, Dane Bagaoisan, Yuhan Peng, Marc Bosse, Ferda Filiz, Christine Fullaway, Sean Bendall, Iain Beehuat Tan, Shyam Prabhakar. Spatial Profiling of Colorectal Cancer Extracellular Milieu Reveals Novel Axes of Immunosuppression and Inflammation abstract. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85 (15Suppl): Abstract nr P38.