Hypoxic cancer-associated fibroblast exosomal circSTAT3 drives triple negative breast cancer stemness via miR-671-5p/NOTCH1 signaling.

[BACKGROUND] The hypoxic tumor microenvironment, particularly hypoxia-conditioned cancer-associated fibroblasts (CAFs), drives breast cancer (BC) progression and therapy resistance. However, the molecular mechanisms linking hypoxic CAFs to BC plasticity and chemoresistance remain elusive.

[METHODS] Primary CAFs were isolated from high-grade BC tissues (Grade III) and characterized (α-SMA⁺/CD34⁻/pan-CK⁻), with normal fibroblasts (NFs) from reduction mammoplasty as controls. Hypoxic CAF-derived exosomal circSTAT3 stability was validated using RNase R resistance and actinomycin D assays. Exosomes were characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and marker profiling (CD63⁺/TSG101⁺/Alix⁺, calnexin⁻). Functional effects of hypoxic CAF exosomes on TNBC cells (MDA-MB-231, SUM159) were assessed through proliferation/migration assays, stemness/epithelial-mesenchymal transition (EMT) marker analysis, and siRNA-mediated circSTAT3 knockdown. Mechanistic studies employed luciferase assays and RNA immunoprecipitation (RIP). Chemoresistance was evaluated by cisplatin half-maximal inhibitory concentration (IC₅₀). In vivo tumor growth and stemness enrichment were analyzed in xenografts. Clinical validation used BC tissues (n = 60) and plasma exosomes from BC patients (n = 40) versus healthy controls (n = 25).

[RESULTS] Hypoxic CAF-derived exosomes efficiently transferred circSTAT3 to TNBC cells, promoting proliferation, migration, EMT, and stemness marker expression. SiRNA-mediated circSTAT3 knockdown reversed these effects. Mechanistically, circSTAT3 acted as a competitive endogenous RNA (ceRNA), sponging miR-671-5p to derepress NOTCH1. Hypoxic CAF exosomes increased cisplatin IC₅₀ in TNBC cells, while circSTAT3 depletion restored chemosensitivity. In vivo, hypoxic CAF exosomes accelerated tumor growth, enriched CD44⁺/NOTCH1⁺ populations, and elevated circulating exosomal circSTAT3. Clinically, circSTAT3 was significantly upregulated in advanced BC tissues (p < 0.01) and patient plasma exosomes (p < 0.01), correlating with lymph node metastasis.

[CONCLUSION] This study identifies a hypoxia-driven feedforward loop wherein CAF-derived exosomal circSTAT3 promotes TNBC stemness and chemoresistance via miR-671-5p/NOTCH1 signaling. CircSTAT3 redefines stromal-tumor crosstalk as a circRNA-driven process and serves as both a circulating non-invasive biomarker and a promising therapeutic target to disrupt stromal-mediated resistance in aggressive TNBC.