The perinecrotic niche of glioblastoma drives tumor-associated macrophage polarization and immunosuppression via podoplanin-mediated CLEC5A activation
Jiabo Li, Xuya Wang, Luqing Tong, Bo Feng, Ling-kai Shih, Steven M. Markwell, Hannah Nuszen, Tomasz Gruchala, Nicholas G. Lam, Petros Basakis, Erika Ruiz-Yamamoto, Deyu Fang, Roger Stupp, Xuejun Yang, Daniel J. Brat
Jiabo Li, Xuya Wang, Luqing Tong, Bo Feng, Ling-kai Shih, Steven M. Markwell, Hannah Nuszen, Tomasz Gruchala, Nicholas G. Lam, Petros Basakis, Erika Ruiz-Yamamoto, Deyu Fang, Roger Stupp, Xuejun Yang, Daniel J. Brat
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Research Article Neuroscience Oncology

The perinecrotic niche of glioblastoma drives tumor-associated macrophage polarization and immunosuppression via podoplanin-mediated CLEC5A activation

Abstract

Glioblastoma (GBM), isocitrate dehydrogenase-WT (IDH-WT) (WHO grade 4) is the most common malignant glioma in adults and is characterized by a hypoxic and immunosuppressive tumor microenvironment (TME). Bone marrow–derived tumor-associated macrophages (TAMs) dominate the immune landscape in GBM and are recruited to the perinecrotic niche following the onset of necrosis. C-type lectin domain–containing 5A (CLEC5A) has the strongest association with poor clinical outcomes among immune-related genes in GBM and is preferentially expressed in hypoxic, perinecrotic TAMs. CLEC5A overexpression promotes TAM polarization toward an immunosuppressive phenotype and secretion of immunoregulatory cytokines. Using the replication-competent avian sarcoma-leukosis virus long terminal repeat with a splice acceptor (RCAS)/tumor virus A (tv-a) system GBM model with bone marrow transplantation from Clec5a–/– donor mice, we demonstrated that CLEC5A loss prolonged survival, delayed tumor progression, and attenuated TME immunosuppression. Mechanistically, podoplanin (PDPN) expressed on glioma cells directly engaged CLEC5A and triggered downstream Syk/JAK/STAT3 signaling in TAMs. Pharmacologic Syk inhibition suppressed glioma growth, diminished TAM infiltration and polarization, reversed the immunosuppressive TME, and prolonged survival in vivo. Collectively, our findings indicate that the PDPN/CLEC5A/Syk/STAT3 axis orchestrates TAM polarization and TME immunosuppression in the perinecrotic niche of GBM, highlighting CLEC5A/Syk as a promising therapeutic target for reversing the immunosuppressive TME and improving outcomes.

Authors

Jiabo Li, Xuya Wang, Luqing Tong, Bo Feng, Ling-kai Shih, Steven M. Markwell, Hannah Nuszen, Tomasz Gruchala, Nicholas G. Lam, Petros Basakis, Erika Ruiz-Yamamoto, Deyu Fang, Roger Stupp, Xuejun Yang, Daniel J. Brat

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Figure 5

Tumor-derived PDPN promotes TAM-IM polarization through CLEC5A in vivo.

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Tumor-derived PDPN promotes TAM-IM polarization through CLEC5A in vivo. ...
(A) Schematic diagram of in vivo experimental design using the RCAS/tv-a mouse model to evaluate the effects of sh-PDPN in glioma cells. (B) Kaplan-Meier survival curves for RCAS/tv-a mice treated with sh-PDPNs (n = 12 per group). (C) Histogram showing tumor volumes in mice in control, sh-PDPN-1, and sh–PDPN-2 groups 28 days after injection of DF-1 cells (1-way ANOVA, Dunnett’s multiple-comparisons test). (D) Kaplan-Meier survival analysis of tumor-bearing mice in the RCAS/tv-a, PDGFB-driven glioma model with control, tumor cell PDPN knockdown (sh-PDPN), BMT-Clec5a–/–, or combined sh-PDPN plus BMT-Clec5a–/– (n = 10 per group). (E) Representative 2D MRI images (top) and corresponding 3D tumor reconstructions (bottom) from each group at day 21 and day 35 time points. Scale bar: 1 mm. (F) Quantification of tumor volume across the groups shown in D and E (1-way ANOVA with Tukey’s multiple-comparison test). (G–I) Representative flow cytometry images showing the proportion of CLEC5A (G), CD206 (H), and CD86 (I) in tumor-infiltrating myeloid cells across the indicated groups. (J) Quantification of CLEC5A, CD206, and CD86 expression within CD45+ myeloid cells across groups (2-way ANOVA with Tukey’s multiple-comparison test). (K) IF staining for HIF-1α, IBA1, CD163, and CLEC5A in mouse brain sections. Scale bar: 50 μm. Original magnification, ×20. (L) Representative flow cytometry images of CD8+ cells in CD3+ T cells across groups. (M and N) Representative flow cytometry plots and quantification of IFN-γ+granzyme B+ expression within CD8+ T cells across groups (1-way ANOVA, Tukey’s multiple-comparisons test). (O–Q) Representative images of PD-1 (O), TOX (P) (terminal exhausted), and TCF-7 (Q) (precursor) expression in CD8+ T cells across groups. (R) Quantification of PD-1, TOX, and TCF-7 expression in CD8+ T cells (2-way ANOVA with Tukey’s multiple-comparison test). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.