Abstract Introduction: Subsets of cancer-associated fibroblasts (CAFs) in colorectal cancer remain poorly characterized preventing their use as biomarkers and drug targets. Results: Integrated scRNA seq profiling and multiplex staining identified four human CAF subsets These were A1 (PDGFRA+/FAP-/TF), PDGFRAhighA2 (PDGFRA+/FAP+/TF-), PDGFRAlowA2 (PDGFRA-/FAP+/TF-) and A3 (PDGFRA+/FAP-/TF+) (TF: Tissue Factor).Spatial analyses identified spatial associations indicative of specific functional interactions of fibroblast subsets with cancer cells or immune cells. Explorative analyses in six CRC cases suggested, in general, favorable effects of FAP-negative subsets (A1 and A3) on immune features and cancer cell proliferation, whereas FAP-positive subsets PDGFRAhighA2 and PDGFRAlowA2 overall showed pro-tumoral associations. To extend findings, niches were quantitated in U-CAN cohort of approximately 200 stage I-IV cases with around 80% stage II/III. Spatial screening generated more than 1600 different multi-marker-defined tissue metrics. Analyses identified a higher average of A1 density in areas surrounding proliferating vs non-proliferating T-cells, and a higher average PDGFRAlowA2 peri-T-cell density in the low proliferating T-cell group. Regarding cancer cell proliferation, high proliferating cancer islands showed lower peri-epithelial density of A3 and PDGFRAlowA2 cells as compared to low proliferating cancer islands. As for T-cell cancer island infiltration, high peri-cancer island density of A1 and PDGFRAhighA2 was associated with high T-cell cancer island infiltration. Additional analyses related CAF metrics to stage and survival. Density of PDGFRAlow A2 cells increased with stage (I-III), whereas density of A1 showed an opposite pattern with an inverse association with stage (I-III). Regarding survival associations, these were in general stronger for metrics collected in peripheral regions of tumors. Among prognosis signals related to niches referred to above, it was noted that A1 density in peri-cancer regions was significantly associated with good prognosis. Interestingly, this association was not detected when A1 density was scored in total stroma. Among all metrics, the strongest metrics were A1 density in 20μm A1 expansion areas (good prognosis) and T-reg density in the 10μm A3 expansion areas (bad prognosis) (P<0.001; univariate Cox-regression); both in peripheral regions. Conclusion: Four novel CRC CAF subsets, defined by PDGFRalpha, FAP and TF, was identified by integrated single cell RNA seq analyses and multiplex staining with associated digital image analyses. Non-random spatial distribution of these subsets and compartment-specific prognosis associations are compatible with distinct and different functions of these subsets. Citation Format: Linglong Huang, Mercedes Herrera, Jonas Sjölund, Vladimir Chocoloff, Simon Joost, Rasul M. Tabiev, Lina Wik Leiss, Carina Strell, Luis Nunes, Artur Mezheyeuski, David Edler, Anna Martling, Fredrik Pontén, Bengt Glimelius, Tobias Sjöblom, Maria Kasper, Kristian Pietras, Arne Östman. identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6970.