Deciphering MFAP5 + Fibroblasts in Pancreatic Cancer Progression via Multi‐Regional Single‐Cell RNA Sequencing With Experimental Validation

Recent studies highlight the critical role of cancer-associated fibroblasts (CAFs) in tumor invasion, but research on the regulation of MFAP5+ fibroblasts by the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) remains limited. Therefore, understanding the function, spatial distribution, and communication dynamics of MFAP5+ fibroblasts within the PDAC tumor microenvironment is essential. We used multi-regional single-cell RNA sequencing to examine the biological characteristics of MFAP5+ fibroblasts in PDAC. A pseudo-time analysis technique was then applied to infer the evolution of CAF subtypes. To investigate this functional role and spatial relationship, we employed multiplex immunofluorescence to observe the spatial distribution of MFAP5+ fibroblasts and endothelial cells. Our study investigated PDAC tumor heterogeneity through a comprehensive analysis of 59,829 cells, which integrated our own multi-regional sampling (GSE285264) with publicly available datasets (GSE277782, GSE155698, GSE212966). We found that MFAP5+ fibroblasts were associated with FABP4+ endothelial cells and VWF+ endothelial cells via key tumor-promoting pathways (e.g., TGF-β, VEGF, FGF). Multiplex immunofluorescence and semi-quantitative analysis confirmed increased prevalence of FABP4+ and VWF+ endothelial cells in areas with high MFAP5+ fibroblast expression, along with elevated VEGF and FGF signaling. Our study reveals a potential pro-tumorigenic mechanism of MFAP5+ fibroblasts in PDAC, suggesting that the MFAP5+ fibroblast-endothelial cell axis may represent a potential target for future therapeutic strategies.