PIGU modulates CCL5-associated tumor cell–macrophage interactions in esophageal squamous cell carcinoma

Although tumor-associated macrophages (TAMs) are well-recognized drivers of esophageal squamous cell carcinoma (ESCC), the intrinsic signals within tumor cells that control macrophage recruitment and functional reprogramming remain incompletely defined. Publicly available datasets were analyzed and integrated with data from 50 paired ESCC and adjacent non-cancerous specimens. THP-1-derived macrophages were used for conditioned-medium and indirect co-culture assays. Recombinant human C-C motif chemokine ligand 5 (rhCCL5) supplementation and CCL5 neutralization were used to examine macrophage responses. CHIR-99021 treatment included both control and PIGU-knockdown cells, with assessment of CCL5 mRNA, whole-cell and nuclear-fraction β-catenin, and related proteins. In vivo effects were assessed in male BALB/c nude mice bearing xenografts and treated with clodronate liposomes. The signal-overlap area fraction of phosphatidylinositol glycan anchor biosynthesis class U (PIGU) and CD206 was elevated in tumor tissues and was associated with advanced TNM and T stage. Silencing PIGU impaired macrophage migration and reduced CD163/CD206-associated M2-like traits, while also weakening the ability of conditioned macrophages to support ESCC cell proliferation, colony formation, migration, and invasion. Loss of PIGU lowered CCL5 expression and secretion. rhCCL5 supplementation partially restored macrophage responses, whereas CCL5 neutralization reduced migration and CD163/CD206 expression induced by control-cell medium, with no significant further reduction in the PIGU-knockdown condition. Whole-cell β-catenin, c-Myc, and Cyclin D1 levels, together with nuclear-fraction β-catenin abundance, decreased after PIGU knockdown and increased following CHIR-99021 treatment in PIGU-silenced cells. CHIR-99​021 also increased CCL5 mRNA levels in PIGU-silenced cells, whereas no significant change was observed in control cells. In vivo, both PIGU knockdown and clodronate-liposome treatment reduced xenograft burden. PIGU knockdown in ESCC cells reduces macrophage migration and M2-like features, with endogenous CCL5 contributing to these responses. Macrophages conditioned by PIGU-silenced ESCC cells subsequently exert weaker tumor-promoting effects. These changes are accompanied by reduced levels of β-catenin and related proteins, although β-catenin-dependent regulation of CCL5 remains unproven.