Exosomal circ_0005397 promotes TNBC progression by inducing M2 macrophage polarization via the miR-204-5p/STAT6 axis.

OBJECTIVE

To elucidate the functional role and mechanistic basis of TNBC-derived exosomal circ_0005397 in driving the malignant progression of triple-negative breast cancer (TNBC).

METHODS

Exosomes were harvested from MDA-MB-231 cells by differential ultracentrifugation and validated. Circ_0005397 abundance in exosomes from cultured cell lines and patient-derived specimens was measured by RT-qPCR (comprising 20 TNBC patients and 20 healthy controls). A co-culture system was established by incubating THP-1-derived M0 macrophages with circ_0005397-overexpressing exosomes, followed by assessment of macrophage polarization markers. The circ_0005397/miR-204-5p/STAT6 regulatory axis was validated using FISH, RIP, RNA pull-down, and dual-luciferase reporter assays, in conjunction with miR-204-5p overexpression rescue experiments. The impact of M2-polarized macrophages on TNBC cell behavior was evaluated through EdU proliferation, colony formation, and Transwell migration/invasion assays. A subcutaneous xenograft model in nude mice was further used to confirm these results at the in vivo level.

RESULTS

Circ_0005397 was markedly overexpressed in TNBC cell lines as well as in serum exosomes from TNBC patients relative to healthy controls. Circ_0005397-overexpressing exosomes were efficiently internalized by macrophages, leading to elevated M2 polarization markers with concomitant reduction of M1 markers. Mechanistically, circ_0005397 operated as a ceRNA to sequester miR-204-5p, thereby relieving its post-transcriptional suppression of STAT6 and triggering STAT6 pathway activation; notably, these effects were abrogated upon miR-204-5p overexpression. The resulting M2 macrophages markedly potentiated TNBC cell proliferation, migration, and invasion in vitro. In vivo experiments further demonstrated that circ_0005397-overexpressing exosomes significantly promoted tumor growth, accompanied by elevated CD206 and phosphorylated STAT6 (p-STAT6) levels; dual-immunofluorescence staining confirmed that this M2 polarization and STAT6 activation were localized to the CD68+ tumor-associated macrophage compartment.

CONCLUSION

TNBC cell-derived exosomal circ_0005397 drives M2 macrophage polarization via the miR-204-5p/STAT6 ceRNA network, consequently reshaping the tumor immune microenvironment and accelerating TNBC progression. This axis may serve as a promising candidate for TNBC early diagnosis and a potential target for immunotherapy-based interventions.