Hypoxic CA9-high mesenchymal-like glioma stem cells promote ANXA1-FPR1-dependent macrophage polarization in recurrent glioblastoma

BACKGROUND: Recurrent glioblastoma (rGBM) is characterized by marked myeloid remodeling and a profoundly immunosuppressive microenvironment. Although carbonic anhydrase 9 (CA9) is a canonical hypoxia-inducible molecule linked to aggressive glioblastoma (GBM) behavior, its cell-state-specific distribution in rGBM and its role in glioma stem cell (GSC)-macrophage crosstalk remain incompletely understood. METHODS: We integrated public transcriptomic cohorts, single-cell RNA sequencing, quantitative proteomics, paired clinical specimens, and in vitro/in vivo functional assays to define the role of CA9 in rGBM. Malignant-cell states and tumor-associated macrophage (TAM) subpopulations were resolved at single-cell resolution, and intercellular communication was inferred computationally. CA9 gain- and loss-of-function models were established in GBM cell lines and patient-derived GSCs. ANXA1 expression and secretion were assessed by qRT-PCR, Western blotting, ELISA, and promoter-reporter assays. Macrophage polarization was evaluated using conditioned-medium transfer, recombinant ANXA1 rescue, and pharmacological FPR1 blockade with cyclosporin H. RESULTS: CA9 was upregulated in GBM, further enriched in mesenchymal and recurrent tumors, and associated with inferior survival. Single-cell analysis localized CA9 predominantly to a hypoxia-associated MES-like GSC subpopulation that expanded in recurrent samples and exhibited elevated stemness. Recurrent tumors also displayed increased SPP1 + immunosuppressive TAMs. Proteomic, transcriptomic, and cell-cell communication analyses prioritized ANXA1 as a CA9-associated downstream mediator with secreted immunomodulatory potential. Hypoxia induced both CA9 and ANXA1, and mutation of a hypoxia-response element attenuated ANXA1 promoter activation. CA9 depletion reduced ANXA1 expression and secretion, impaired GBM growth phenotypes, and attenuated hypoxia-driven ANXA1 induction. CellChat highlighted enhanced signaling between hypoxic MES-like GSCs and SPP1 + TAMs in recurrent GBM, nominating ANXA1-FPR1 as a leading ligand-receptor axis. Functionally, conditioned medium from CA9-high GBM cells promoted M2-like macrophage polarization, which was weakened by CA9 knockdown, partially restored by recombinant ANXA1, and markedly suppressed by FPR1 antagonism, supporting ANXA1 as a contributory paracrine mediator, and markedly suppressed by FPR1 antagonism. In vivo, CA9 depletion, and in CA9-intact tumors FPR1 blockade, reduced M2-like macrophage-associated signals. CONCLUSIONS: CA9 is enriched in hypoxic MES-like GSCs in rGBM and promotes an ANXA1-dependent paracrine program that supports immunosuppressive macrophage polarization. These findings identify a CA9-ANXA1-FPR1-associated neuroimmune crosstalk axis linking hypoxic stem-like tumor states to myeloid remodeling in recurrent GBM, with ANXA1 functioning as a contributory paracrine mediator rather than a sole effector of CA9-driven immunosuppression.