Xuqing Shi, Hangqi Liu, Jianru Sun, Xiaoding Liu, Xinping Jv, Longyun Chen, Yuhan Zhang, Hui Zhang, Xudong Xing, Ruiyu Li, Xinyi Ke, Jun Wang, Xianglin Yin, Bohan Liu, Qixian Liu, Yuan Wang, Junliang Lu, Shiyi Liu, Junyi Pang, Yumeng Cai, Menghua Dai, Fan Bai, Huanwen Wu, Zhiyong Liang
ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly immunosuppressive and desmoplastic tumor microenvironment (TME) that limits the efficacy of immunotherapy. However, the evolution of this immunosuppressive TME and the underlying mechanisms remain incompletely understood. Here, we construct a dynamic single‐cell atlas spanning uninvolved adjacent pancreatic tissue (UNIN), intraductal papillary mucinous neoplasm (IPMN), and PDAC. We confirm the stepwise establishment of an immunosuppressive milieu, accompanied by the emergence of LRRC15 + fibroblasts as determinants. Functional assays further identify tumor‐derived LAMB3 as a regulator of LRRC15 + fibroblast differentiation. Mechanistically, LAMB3 promotes FOSL2‐dependent transcriptional activation of LRRC15 through the ITGB1/FAK/MAPK signaling axis, ultimately suppressing T cell cytotoxicity. Orthotopic models reveal that LAMB3 overexpression increases the LRRC15 positive area and impairs T cell cytotoxicity, whereas FAK inhibition partially reverses these effects. In parallel, LAMB3 knockdown reduces the LRRC15 positive area and improves the efficacy of PD‐1 blockade. Moreover, glycolytic reprogramming in PDAC ductal cells upregulates LAMB3 expression and correlates with increased LRRC15 + fibroblast enrichment. Clinically, co‐enrichment of LAMB3 + PDAC ductal cells and LRRC15 + fibroblasts is associated with inferior overall survival. Collectively, our findings define a dynamic ductal‐fibroblast‐immune multicellular axis underlying PDAC pathogenesis and provide insights into potential therapeutic strategies.