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. Graphical abstract In recurrent glioblastoma, CA9 is preferentially enriched in hypoxia-associated MES-like glioma stem cells. Elevated CA9 enhances ANXA1 expression and secretion under hypoxic conditions. Secreted ANXA1 engages FPR1-associated signaling in SPP1 + tumor-associated macrophages, promoting an M2-like immunosuppressive phenotype. This tumor-to-myeloid paracrine circuit contributes to neuroimmune remodeling and supports recurrent GBM progression.

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

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2026年6月29日

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Hangqi Gao,
Guiting You,
Jianhuang Huang,
Yijing Lin,
Jiexin Huang,
Risheng Liang,
Caihou Lin

摘要

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.

Graphical abstract

In recurrent glioblastoma, CA9 is preferentially enriched in hypoxia-associated MES-like glioma stem cells. Elevated CA9 enhances ANXA1 expression and secretion under hypoxic conditions. Secreted ANXA1 engages FPR1-associated signaling in SPP1 + tumor-associated macrophages, promoting an M2-like immunosuppressive phenotype. This tumor-to-myeloid paracrine circuit contributes to neuroimmune remodeling and supports recurrent GBM progression.