CXCL12 ⁺ pericytes as drivers of lymphatic dissemination in pancreatic cancer: Integrated single-cell and spatial analysis.

Abstract

4126 Background: Pancreatic ductal adenocarcinoma (PDAC) exhibits poor prognosis largely due to early lymph node metastasis, yet the cellular and molecular mechanisms governing lymphatic dissemination remain incompletely understood. Methods: We integrated multi-omics approaches including single-cell RNA sequencing (scRNA-seq) of 19 PDAC samples, spatial transcriptomics of 6 specimens, bulk RNA sequencing of 174 cases, and multiplex immunofluorescence of 166 clinical samples stratified by lymph node metastasis status. Cell-cell communication analysis, trajectory inference, and functional validation were performed to elucidate pro-metastatic mechanisms. Results: Single-cell profiling of 145,283 cells revealed substantial cellular heterogeneity, with fibroblasts and pericytes serving as dominant signaling hubs within the tumor microenvironment. Lymph node metastasis-positive (pLNM) tumors exhibited enhanced stromal-epithelial crosstalk mediated by PTN and ANGPT pathways. We identified invasive epithelial subpopulations (Epi_MUC5AC_CD24 and Epi_NEAT1_MALAT1) significantly enriched in pLNM samples, characterized by activation of EMT and invasion-related programs. Critically, high-resolution subclustering uncovered a CXCL12-expressing pericyte subset (PCs_CXCL12) preferentially expanded in metastatic tumors. Multiplex immunofluorescence validation in 166 specimens demonstrated that CXCL12+ pericyte infiltration correlated with perineural invasion, lymph node metastasis, advanced TNM stage, and inferior survival outcomes. Multivariate analysis established CXCL12+ pericyte score as an independent prognostic factor for both disease-free and overall survival. Mechanistically, we identified MYC as the master transcriptional regulator driving CXCL12 secretion in pericytes. Functional studies revealed that MYC-driven pericyte-derived CXCL12 promoted PDAC cell proliferation, invasion, and EMT. Spatial transcriptomics demonstrated co-localization of MYC, CXCL12, and its receptor CXCR4 in perineural pericyte niches. Orthotopic xenograft models confirmed that pericyte-specific Myc depletion significantly reduced tumor growth, CXCL12+ pericyte density, and tumor-associated lymphangiogenesis. Conclusions: This study establishes the pericyte MYC-CXCL12 axis as a critical driver of PDAC lymphatic metastasis through promotion of tumor cell invasion, EMT, and lymphangiogenesis. CXCL12+ pericytes represent a novel prognostic biomarker and potential therapeutic target for preventing lymph node metastasis in PDAC patients.