Abstract A066: MMR-stratified spatial programs of normal and cancer-associated fibroblasts and their association with lymphocyte pervasiveness in CRC

Abstract Background: Fibroblast-enriched transcriptional signatures in colorectal cancer (CRC) associate with poor prognosis and with mismatch repair–proficient (MMRp) disease, which derives limited benefit from immune checkpoint inhibition (ICI). We hypothesized that fibroblast composition and spatial organization differ between MMRp and mismatch repair–deficient (MMRd) tumors and define microenvironments relevant to ICI resistance. Methods: Fresh primary tumors from 41 CRC patients (MMRp n=34, MMRd n=7) underwent scRNA-seq. Matched FFPE sections and an independent archival FFPE cohort (MMRp n=50, MMRd n=30) were profiled by multiplex whole-slide imaging (up to 1.2×106 cells/image). Fibroblast subtypes were defined by scRNA-seq analysis and mapped to images using HALO classifiers on eight-marker panels. Spatial profiling quantified cell densities at regular intervals from the tumor surface to the invasive edge and modeled focal aggregates using a density-based clustering algorithm. Results: scRNA-seq data resolved 5 fibroblast subtypes conserved across MMRp/MMRd: myofibroblastic cancer-associated fibroblasts (myCAFs; fibrillar ECM genes), inflammatory CAFs (iCAFs; cytokine genes), and 3 normal fibroblast (NF) subtypes—subepithelial myofibroblasts (SEMFs; contractility genes), lamina propria fibroblasts (LPFs; growth factor activity genes), and crypt fibroblasts (CFs; fibrillar and non-fibrillar ECM genes). CAF densities were enriched at the invasive margin directly bordering the tumor core versus distal margin: 4.6-fold in MMRp (p<0.01) and 3.5-fold in MMRd (p<0.01), defining a CAF-enriched niche. In this niche, CAFs formed focal aggregates, within which CAF composition diverged by MMR status. In MMRp tumors, myCAFs were 16-fold more prevalent than iCAFs (p<0.01); in MMRd tumors, iCAFs were 3-fold more prevalent than myCAFs (p<0.03). Higher myCAF:iCAF ratios were correlated with lower CD8+ tumor-infiltrating lymphocyte (TIL) density in the tumor core (p<0.05), but not CD4+ TILs. NFs were collectively as abundant as CAFs in the tumor stroma, with MMR-dependent spatial patterns. In MMRp tumors, LPFs were 3-fold more prevalent within the CAF-niche versus the deeper invasive margin (p<0.01), whereas CF peak densities were observed in deeper invasive margin. In both MMR settings, SEMFs localized closest to tumor cells and in MMRd, SEMFs were 2-fold more prevalent in the niche (p<0.05). CF prevalence was inversely correlated with CD4+ TIL density in the tumor core (p<0.02), but not CD8+ TILs. Conclusions: CRC harbors MMR-dependent fibroblast niches characterized by focal CAF aggregates and distinct NF localization. MMRp tumors exhibit myCAF-dominant aggregates at the tumor core border, whereas MMRd tumors show iCAF-dominant aggregates and SEMF enrichment. Our data are consistent with previous reports of immunosuppressive microenvironments in MMRp CRC, and correlate CAF and NF spatial programs to lymphocyte infiltration. Citation Format: Debanjan Barua, Tze Guan Tan, Shu Wen Samantha Ho, Michael T. Wong, Iain B. Tan, Jennifer H. Yearley, Su-Yang Liu. MMR-stratified spatial programs of normal and cancer-associated fibroblasts and their association with lymphocyte pervasiveness in CRC [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A066.