Interrogation of glioma immune microenvironment identifies a noncanonical role for microglial Galectin-9 in tumor cell adhesion and phagocytosis

Pravesh Gupta^1*Silvana Valdebenito-Silva²Gayatri Kumar¹Prashanth Chakrapani³Shivangi Oberai⁴Minghao Dang⁵Hiroshi Katyama³Linghua Wang⁵Eliseo A Eugenin²Krishna Bhat^1*

• ¹Cancer Biology, Mayo Clinic Arizona, Phoenix, United States
• ²Neurobiology, The University of Texas Medical Branch John Sealy School of Medicine, Galveston, United States
• ³Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, United States
• ⁴Mayo Clinic Minnesota, Rochester, United States
• ⁵Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, United States

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 exists. 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 (FcgR, phospholipase D, Rab family proteins etc.) pathways that were significantly upregulated in Galectin-9+ compared to Galectin-9lo/- MG and MACs. Using ex vivo primary human microglia (pMGs) and patient derived glioma stem cells (GSCs) co-culture system, we evaluated the functional role of Galectin-9. Confocal imaging analyses of co-cultured pMGs 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 pMGs. Altogether, our study This is a provisional file, not the final typeset article underscores 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.