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 1

CLEC5A is preferentially expressed by hypoxic TAM-IMs within the perinecrotic niche and correlates with a poor prognosis.

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CLEC5A is preferentially expressed by hypoxic TAM-IMs within the perinec...
(A) scRNA-seq Seurat and UMAP analysis clustered samples into 30 distinct gene expression profiles. Four distinct cell subpopulations were identified as follows: cancer cells (GAP43+, GPM6B+, PTN+, GFAP+, SOX2+, OLIG2+, CD44+, CCND2+, and PARP1+); TAM-IMs (AIF+, CD68+, CD163+, and MSR1+); TAM-INFs (AIF+, CD68+, CD86+, and CD163–); and T cells (CD3G+). (B) UMAP visualization of a large-scale human GBM scRNA-seq dataset (>338,000 cells), showing major cell populations including neoplastic cells, myeloid cells, lymphoid cells, and vascular and glial cell lineages. (C) Spearman correlation reveals 49 genes highly associated with CD163 among 3 independent GBM datasets. (D) Forest plot shows the top CD163-related genes, together with their association with survival. CLEC5A had the highest association with survival in TCGA RNA microarray dataset. (E) Kaplan-Meier overall survival for patients with GBM with high versus low CLEC5A expression in TCGA RNA microarray dataset. (F) Time-dependent ROC curves display a CLEC5A expression predictive capacity for 1- and 2-year survival of patients with GBM in TCGA database. (G) Representative flow cytometric plots of CLEC5A+CD163+, CD206+CD86–, and CD206–CD86+ THP-1 cell populations under normoxia versus hypoxia. (H) Representative flow cytometric plots and quantification of CLEC5A+CD163+, CD206+CD86–, and CD206–CD86+ MDM populations under normoxia versus hypoxia. (I and J) THP-1 protein expression levels of CLEC5A in normoxic (Norm) and hypoxic (Hyp) conditions detected by Simple Western (Jess) (unpaired, 2-tailed Student’s t test). (K) IF staining for CAIX, IBA1, CLEC5A, and CD163 performed on perinecrotic and distal necrotic regions in necrotic GBM, as well as non-necrotic regions in non-necrotic tumor. Scale bars: 100 μm. Original magnification, ×20. (L) Histogram depicting the proportion of CLEC5A+CD163+ cells relative to IBA1+ cells across 3 distinct areas: perinecrotic areas, distal necrotic zones in necrotic tumors, and in non-necrotic gliomas, by IF staining (1-way ANOVA with Tukey’s multiple-comparison test). ****P < 0.0001. All quantitative data are presented as mean ± SD.