Single-cell analysis explores GJB2-mediated interactions in cancer-associated pericyte subgroups that promote lymph node metastasis in lung adenocarcinoma.

e20012 Background: Lymph node metastasis remains a critical determinant of recurrence and adverse outcomes in invasive lung adenocarcinoma (IAC). Methods: To elucidate microenvironmental drivers of metastatic progression, we conducted single-cell RNA sequencing analysis of the IAC niche (12 samples from 4 patients), revealing a previously unrecognized pericyte subpopulation with tumor-promoting properties, designated cancer-associated pericytes (CAPs). Results: Mechanistic investigations identified connexin-encoding GJB2 as a pivotal regulator initiating CAP differentiation, alongside a paracrine signaling axis mediated by the CCL11-ACKR2 ligand-receptor complex between CAPs and tumor cells. Conditional ablation of Gjb2 inhibits tumor progression and invasion in Cre-loxP-based orthotopic lung cancer mice model. Functional validation demonstrated that pericytes suppress tumor proliferation while paradoxically enhancing invasive capacity in vitro , with reciprocal tumor-mediated inhibition of pericyte tubulogenesis. GJB2 knockdown experiments revealed its dual role in constraining pericyte motility. Conclusions: Our findings establish CAPs as central architects of the metastatic microenvironment and implicate GJB2-dependent intercellular crosstalk as a critical mediator of vascular co-option. This work advances the conceptual framework for vascular normalization strategies and unveils potential therapeutic targets for disrupting metastatic signaling cascades in IAC. Top 10 marker genes had significant upregulation of GJB2 in cancer-associated pericytes (CAPs) cell type. Gene p_val avg_log2FC pct.1 pct.2 p_val_adj cluster(0,CAP) CTHRC1 9.91E-305 4.066613028 0.986 0.141 2.88E-300 0 MMP2 1.82E-277 3.565836058 0.96 0.246 5.29E-273 0 LUM 2.93E-270 3.332542613 0.999 0.439 8.52E-266 0 SFRP2 4.03E-269 4.106443715 0.917 0.053 1.17E-264 0 THBS2 1.31E-263 2.619653362 0.967 0.233 3.81E-259 0 FBLN1 1.79E-263 2.916227506 0.971 0.336 5.21E-259 0 DCN 8.92E-261 2.870365695 1 0.631 2.59E-256 0 VCAN 1.77E-250 2.752535218 0.991 0.324 5.16E-246 0 COL10A1 4.41E-248 2.736812508 0.85 0.028 1.28E-243 0 FAP 5.42E-244 1.711320514 0.915 0.133 1.57E-239 0