Spatial mapping of fibroblast subtypes in primary colorectal cancer.

e15665 Background: Colorectal cancer (CRC) is the third leading cause of cancer related death. CRC arises from colon stem cells, the equilibrium of colon stem cell maintenance and differentiation is heavily dependent on fibroblast signals along the crypt-villous axis. Such fibroblast cues may be important determinants of CRC cell states and progression as well. Hence, in this study, we phenotyped fibroblast subtypes and analyzed their spatial mapping in primary CRC. Methods: We performed Xenium spatial transcriptomics of 38 primary CRC samples from 35 CRC cases (8 with metastatic disease at diagnosis), with varying depths of tumor invasion. Data was analyzed using R Seurat package. Results: There were 5 major fibroblast subtypes with distinct spatial arrangement – WNT5A+BMP+, WNT5A+hypoxic, ADAMDEC1+stromal, matrix-cancer associated fibroblasts (mCAFs) and Inflammatory-CAFs (iCAFs). The key markers of the WNT5A+ BMP+ fibroblasts were WNT5A, WNT5B, NRG1, BMP5 and corresponded to normal epithelial crypt niche fibroblasts. mCAFs showed higher expression of POSTN, SULF1, and FN1 and iCAFs had higher expression of C3. The WNT5A+hypoxic fibroblasts had overlapping gene expression with WNT5A+BMP+ and mCAFs and over-expressed hypoxia markers ( VEGFA, NDRG1 ). They may represent a transitional state between WNT5A+BMP+ and mCAFs based on trajectory analysis. Spatially, the WNT5A+BMP+ and ADAMDEC1+stromal fibroblasts were predominantly located in the adjacent normal appearing colon tissue. The WNT5A+BMP+ fibroblasts, which lines normal intestinal epithelium, was also detected along the lining of tumor regions in mucosa and submucosa including at deep stromal invaginations into tumor nests. The presence of normal intestinal WNT5A+BMP+ fibroblasts lining tumor nests in submucosa is interesting, temporal data is necessary to understand if tumor cells induce this transcriptional state in neighboring fibroblasts or if fibroblasts migrate with tumor cells as they invade. Several markers of the WNT5A+BMP+ fibroblasts (NRG1, WNT5A, BMPs) are involved in epithelial differentiation and intestinal injury repair and may maintain epithelial identity in CRC tumor cells. Notably, higher NRG1 and WNT5A expression in primary CRC bulk RNA sequencing data was associated with better overall survival (log rank p = 0.04 and p = 0.01 respectively) in The Cancer Genome Atlas (TCGA). The WNT5A+hypoxic was the most abundant fibroblast subset lining tumor nests throughout the colon, they were absent farther away from the tumor nests. mCAFs are the predominant population throughout the tumor stroma and their proportion at the lining of tumor nests increased in deeper layers of the colon. Conclusions: Taken together, we describe key fibroblast populations and their spatial distributions in primary CRC, their impact on tumor cell phenotype through paracrine signaling needs further investigation.