Chakrapani, Prashanth

Glioma-associated microglia/macrophages (GAMs) have traditionally been described as immunosuppressive. However, within their highly heterogeneous repertoire, pro-phagocytic and cytotoxic subsets with anti-tumoral properties exist. Although macrophages (MACs) can exhibit tumor-suppressive functions, their anti-glioma properties largely remain elusive. To identify anti-glioma myeloid cell effectors, we performed directionally concatenated ligand-receptor (L-R) interactome analyses from dendritic cells (DCs) and microglia (MG) to lymphoid (CD4 + T, Tregs, CD8 + T, NK, and NKT) cells identified by our recent single-cell transcriptomics interrogation of tumor-associated CD45 + leukocytes from tumor brains of eighteen isocitrate dehydrogenase (IDH)–stratified glioma patients. Within DC-specific and MG-specific interactomes, we identified LGALS9 , which encodes Galectin-9, as a key mediator of cell–cell interactions in IDH-wild type (IDH-wt) gliomas. Spectral cytometry, immunohistochemistry, and Western blotting analyses confirmed the abundant expression of Galectin-9 in glioma-associated MG, but not in tumor cells. Furthermore, differential gene enrichment analyses revealed transcripts associated with cellular adhesion (coronin 1A, integrin) and phagocytosis (FcγR, phospholipase D, Rab family proteins, etc.) pathways that were significantly upregulated in Galectin-9 + compared to Galectin-9 − MG and MACs. Using an ex-vivo primary human microglia (pMG) and patient-derived glioma stem cells (GSCs) co-culture system, we evaluated the functional role of Galectin-9. Confocal imaging analyses of co-cultured pMG with GSCs revealed that siRNA-mediated knockdown or antibody-based neutralization of Galectin-9 significantly impaired pMG–GSC adhesion. In addition to reduced adhesion, phagocytosis of GSCs was dramatically attenuated across all Galectin-9 silenced or neutralized pMG. Altogether, our study underscores the unappreciated non-canonical role of Galectin-9 in MG as a regulator of glioma cell adhesion and phagocytosis that can be harnessed for glioblastoma immunotherapy.

Abstract Innate immune cells of macrophage family predominate tumor immune microenvironment of CNS malignancies. Although macrophages exhibit myriads of protective functions such as clearance of malignant cells, immune surveillance, inflammation resolution and tissue repair, they are generally linked to immunosuppression in gliomas. To identify anti-glioma myeloid cell effectors, we performed directionally concatenated ligand-receptor (L-R) interactome analyses from dendritic cells (DC) and microglia (MG) to lymphoid (CD4+T, Tregs, CD8+T, NK and NKT) cells identified by single cell transcriptomics analyses of ~145,000 tumor-associated CD45+ leukocytes from tumor brains of eighteen high-grade and low-grade glioma patients. Amongst DC-specific (e.g. adhesion molecules) and MG-specific (e.g. chemokine family) interactomes, we identified LGALS9 encoding Galectin-9 as the most pertinent interactions (e.g. SORL1 encoding sortilin-related receptor 1) in MG of IDH-wild type gliomas. Spectral cytometry, immunohistochemistry and western blotting analyses of MG, macrophage, DC, lymphoid cells and tumor cells confirmed abundant expression of Galectin-9 in glioma-associated MG and not in Nestin+ tumor cells. Furthermore, differential gene enrichment analyses revealed transcripts associated with cellular adhesion (coronin 1A, integrin) and phagocytosis (FcγR, phospholipase D, Rab family proteins etc.) was significantly upregulated in Galectin-9hi compared to Galectin-9lo/- MG and macrophages predicting putative role of Galectin-9 in tumor cell adhesion and phagocytosis. Using glioblastoma relevant patient derived ex vivo primary microglia (pMGs; 707, 2103, 1805) and glioma stem cell (GSC) co-culture model system, we evaluated the functional role of Galectin-9. Confocal imaging analyses of co-cultured pMGs with GSC revealed that siRNA or neutralizing antibody mediated downregulation of Galectin-9 led to significantly impaired adhesion (p<0.001) between pMGs and GSC. Not only adhesion but even phagocytosis of GSC was dramatically attenuated (p<0.0001) across all Galectin-9 silenced or neutralized pMGs. Altogether, our study underscores unappreciated role of Galectin-9 in MG as regulator of glioma cell adhesion and phagocytosis that can be harnessed for glioblastoma immunotherapy.