ROBO3 Drives Endometriosis Progression <i>via</i> Dual Wnt/<i>β</i>-Catenin Activation and M2 Macrophage Polarization

Endometriosis, a prevalent gynecological disorder marked by ectopic growth of endometrial-like tissue, demonstrates malignant tumor-like properties including aggressive adhesion and invasiveness. Emerging evidence implicates roundabout guidance receptor 3 (ROBO3) in cellular pathophysiology, yet its role in endometriosis remains unexplored. In this study, we first found abnormally high ROBO3 expression in endometriosis by bioinformatics analysis. Next, functional assays revealed that ROBO3 is a key regulator promoting the invasion and migration of endometriotic stromal cells (ESCs) in vitro. Mechanistically, ROBO3 activates the Wnt/β-catenin signaling pathway, evidenced by increased phosphorylation of glycogen synthase kinase 3β, nuclear β-catenin accumulation, and upregulated c-myc expression. Pharmacological inhibition of Wnt/β-catenin with MSAB (5 μM) reversed ROBO3-mediated pro-invasive and pro-migratory effects. Furthermore, we discovered that ROBO3 enhances the secretion of chemokines CCL2 and CCL5 in ESCs, which subsequently promote macrophage polarization toward the M2 phenotype, as indicated through elevated expression of interleukin-10 and Arg-1. Collectively, our findings elucidate a dual mechanism whereby ROBO3 drives endometriosis progression through both intrinsic activation of Wnt/β-catenin signaling and extrinsic modulation of tumor-associated macrophages, underscoring ROBO3 as a promising therapeutic target for endometriosis.