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
View: Text | PDF
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

×

Figure 3

CLEC5A deletion in TAMs delays tumor progression by reprogramming the immunosuppressive TME.

Options: View larger image (or click on image) Download as PowerPoint
CLEC5A deletion in TAMs delays tumor progression by reprogramming the im...
(A) Schematic diagram of the RCAS/tv-a mouse model showing procedures for bone marrow collection from donor mice, irradiation, BMT, and intracranial injection of DF-1 cells. (B) Kaplan-Meier survival curves for RCAS/tv-a mice in control, BMT-WT, and BMT-Clec5a–/– groups (n = 12 per group). (C) Representative 2D MRI monitoring and 3D reconstruction mapping depicting neoplastic growth of tumor-bearing mice 28 and 35 days following injection of DF-1 cells. Scale bars: 1 mm. (D) Histogram quantification showing the proportion of CLEC5A+, CD206+, and CD86+ cells among CD45+ cells (2-way ANOVA with Tukey’s multiple-comparison test). (E) Flow cytometry of digested mouse brain tissue from control, BMT-WT, and BMT-Clec5a–/– groups, showing the proportion of CLEC5A+ cells among CD45+ cells. (F and G) Representative flow cytometry plots and quantification of CD45+CD11b+ myeloid cells in mouse brain tumors across control, BMT-WT, and BMT-Clec5a–/– groups (1-way ANOVA with Tukey’s multiple-comparison test). (H and I) Flow cytometry of digested mouse brain tissue showing the proportion of CD206+ (H) and CD86+ (I) cells among CD45+ cells across control, BMT-WT, and BMT-Clec5a–/– groups. (J and K) Representative flow cytometry images and quantification of CD8+ cells in CD3+ T cells across the indicated groups (1-way ANOVA with Tukey’s multiple-comparison test). (L and M) Representative flow cytometry images and quantification of PD-1+ cells in CD8+ T cells across the indicated groups (1-way ANOVA with Tukey’s multiple-comparison test). (N) IF staining of mouse brain sections for IBA1, CD8, and PD-1 expression across control, BMT-WT, and BMT-Clec5a–/– groups. Scale bar: 50 μm. Original magnification, ×20. (O and P) Quantification of the proportion of CD8+ T cells within total cells (O) and PD-1+ cells among CD8+ T cells (P) by IF staining (1-way ANOVA with Tukey’s multiple-comparison test). (Q and R) Flow cytometry plots and histogram of digested mouse brain tissue showing the proportion of IFN-γ +/granzyme B+ cells among CD8+ cells across the indicated groups (1-way ANOVA with Tukey’s multiple-comparison test). **P < 0.01, ***P < 0.001, and ****P < 0.0001.