ANXA2⁺/TNR^- glioblastoma subpopulation drives tumor progression through via Src-STAT3 axis and neutrophil remodeling

Glioblastoma (GBM) exhibits remarkable cellular heterogeneity, but the functional roles of distinct tumor subpopulations remain incompletely understood.

We employed single-cell RNA sequencing, flow cytometry, and in vitro functional assays to identify and characterize an ANXA2⁺/TNR^- subpopulation in GBM. The stem-like phenotype, key signaling pathways, and interactions with neutrophils were evaluated using limiting dilution assays, 3D culture, co-culture systems, and transcriptomic profiling. Functional relevance was validated by pharmacological inhibition and xenograft models.

ANXA2⁺/TNR^- GBM subpopulation with prominent stem-like features characterized by enhanced self-renewal and tumor-initiating capacity. Functional assays revealed that ANXA2 enhanced GBM cell proliferation, migration, and 3D growth while repressing TNR expression, correlating with aggressive clinicopathological features. Mechanistically, ANXA2 activated the Src-STAT3 signaling cascade, which reinforced stemness-associated transcriptional programs and sustained malignant progression. Additionally, ANXA2 activated the Src-STAT3 pathway, inducing a STAT3-dependent secretory program characterized by robust IL-8 release. In concert with tumor-derived TGF-β, ANXA2-driven IL-8 polarized neutrophils toward an ELANE⁺/N2 immunosuppressive phenotype, which further secreted MMP2/9 to amplify tumor invasion and growth. Pharmacological inhibition of Src or STAT3 effectively impaired stemness maintenance, suppressed neutrophil N2 polarization, and attenuated GBM growth in xenograft models.

Our findings identify ANXA2⁺/TNR^- cells as a critical driver of GBM stemness and immune evasion through the Src-STAT3 axis and neutrophil N2 polarization. Targeting this subpopulation and its signaling network may provide new therapeutic strategies for overcoming GBM aggressiveness.