TMIC-27. PDPN ACTIVATION OF CLEC5A IN THE PERI-NECROTIC NICHE DRIVES TAM POLARIZATION AND TME IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract

Abstract Glioblastoma, IDH-wildtype (GBM, WHO grade 4) is the most common malignant glioma of adults and is characterized by a severely hypoxic and immunosuppressive tumor microenvironment (TME). CLEC5A regulates immune responses in inflammatory and infectious diseases. However, its role in GBM immune regulation remain unclear. Here, we found that CLEC5A has the strongest association with poor clinical outcomes among all immune-related genes in GBM. Tumor-associated macrophages (TAMs) account for the majority of immune cells in the GBM and CLEC5A expression in this population is highest in hypoxic, peri-necrotic zones. Both hypoxia and GBM conditioned media cause increased CLEC5A expression by THP-1 cells, and overexpression of CLEC5A enhanced TAM polarization, migration toward glioma cells, and increased secretion of cytokines that mediate immunosuppression. We show that CLEC5A is a cell surface receptor activated by podoplanin (PDPN) and induces TAM polarization through Syk-JAK-STAT3 signaling. Co-IP assay showed that CLEC5A and PDPN could be binded together using antibodies for CLEC5A, Flag-CLEC5A or PDPN when we cocultured control THP-1 cells and THP-1 cells overexpressing Flag-CLEC5A with primary GBM cells (NU02068). PDPN blocking antibody (α-PDPN) reduced the specific binding between PDPN and CLEC5A, as well as inhibited the TAM-IM polarization. Overexpression of CLEC5A significantly upregulated the expression of p-Syk, Jak2, p-Jak2, STAT3, p-STAT3, CD163, CD206 and PD-L1. Moreover, Bay 61-3606, a selective Syk inhibitor, rescued CLEC5A overexpression-mediated TAM-IM polarization and Syk-JAK-STAT3 activation in THP-1 cells. In vivo, both silencing CLEC5A in TAMs and silencing PDPN in GBM cells, as well as pharmacologically targeting Syk delayed glioma growth, prolonged survival and was associated with diminished immunosuppressive TAMs. Collectively, we found that PDPN-CLEC5A-Syk-JAK-STAT3 signaling causes TAM polarization and enhances TME immunosuppression in GBM and can be targeted to suppress growth.