Use of heparin to rescue immunosuppressive monocyte reprogramming by glioblastoma-derived extracellular vesicles

Abstract

OBJECTIVE The profound immunosuppression found in glioblastoma (GBM) patients is a critical barrier to effective immunotherapy. Multiple mechanisms of tumor-mediated immune suppression exist, and the induction of immunosuppressive monocytes such as myeloid-derived suppressor cells (MDSCs) is increasingly appreciated as a key part of this pathology. GBM-derived extracellular vesicles (EVs) can induce the formation of MDSCs. The authors sought to identify the molecular consequences of these interactions in myeloid cells in order to identify potential targets that could pharmacologically disrupt GBM EV–monocyte interaction as a means to ameliorate tumor-mediated immune suppression. Heparin-sulfate proteoglycans (HSPGs) are a general mechanism by which EVs come into association with their target cells, and soluble heparin has been shown to interfere with EV-HSPG interactions. The authors sought to assess the efficacy of heparin treatment for mitigating the effects of GBM EVs on the formation of MDSCs. METHODS GBM EVs were collected from patient-derived cell line cultures via staged ultracentrifugation and cocultured with monocytes collected from apheresis cones from healthy blood donors. RNA was isolated from EV-conditioned and unconditioned monocytes after 72 hours of coculture, and RNA-sequencing analysis performed. For the heparin treatment studies, soluble heparin was added at the time of EV-monocyte coculture and flow cytometry analysis was performed 72 hours later. After the initial EV-monocyte coculture period, donor-matched T-cell coculture studies were performed by adding fluorescently labeled and stimulated T cells for 5 days of coculture. RESULTS Transcriptomic analysis of GBM EV–treated monocytes demonstrated downregulation of several important immunological and metabolic pathways, with upregulation of the pathways associated with synthesis of cholesterol and HSPG. Heparin treatment inhibited association between GBM EVs and monocytes in a dose-dependent fashion, which resulted in a concomitant reduction in MDSC formation (p < 0.01). The authors further demonstrated that reduced MDSC formation resulted in a partial rescue of immune suppression, as measured by effects on activated donor-matched T cells (p < 0.05). CONCLUSIONS The authors demonstrated that GBM EVs induce broad but reproducible reprogramming in monocytes, with enrichment of pathways that may portend an immunosuppressive phenotype. The authors further demonstrated that GBM EV–monocyte interactions are potentially druggable targets for overcoming tumor-mediated immune suppression, with heparin inhibition of EV-monocyte interactions demonstrating proof of principle.

Effect of homotypicvs. heterotypic interactions on the cellular uptake of extracellular vesicles

“…HSPGs are abundantly present in the brain endothelium [55–57] and are known to be involved in the receptor-mediated endocytosis of HIV-1 particles [57, 58] and HIV-TAT protein [59]. Many reports have demonstrated that EVs from cancer cells [60–62] interact with HSPGs and the role of the HSPG inhibition on the effects of EV-HSPG interactions. Atai et al demonstrated involvement of HSPG receptors in the attachment and internalization of EVs derived from U87/GBM 11/5 and 293T/HUVEC into the respective recipient cells [21].…”

“…HSPGs are abundantly present in the brain endothelium [53][54][55] and are known to be involved in the receptor-mediated endocytosis of HIV-1 particles [55,56] and HIV-TAT protein [57]. Many reports have demonstrated that EVs from cancer cells [58][59][60] Assuming that heparin blockade of HSPG receptors in recipient cells would inhibit dynamindependent endocytosis [53] and macropinocytosis [61] pathways, our goal was to determine if HSPG blockade may have an effect on the uptake of EVs into the recipient BECs. The internalization/adherence of EVs have been observed as early as 30-minutes post EV exposure to the recipient cells [62,63].…”

Understanding the immunosuppressive microenvironment of glioma: mechanistic insights and clinical perspectives

“…In GBM, the secretion of EVs involves a unique mechanism in which these vesicles interact with heparan sulfate proteoglycans (HSPGs) and MDSCs, inducing the transformation of MDSCs [441]. This process can be inhibited by heparin, leading to a reduction in the number of MDSCs in GBM [449]. EVs derived from GBM cells can reprogram normal MONs, promoting their differentiation into MDSCs and subsequent suppression of T cell function [449].…”

“…This process can be inhibited by heparin, leading to a reduction in the number of MDSCs in GBM [449]. EVs derived from GBM cells can reprogram normal MONs, promoting their differentiation into MDSCs and subsequent suppression of T cell function [449]. Again, heparin can inhibit this reprogramming process and restore T cell function.…”

Effects of glioblastoma-derived extracellular vesicles on the functions of immune cells

“…Another mechanism of heparin action is that heparin causes aggregation of vesicles ( Atai et al, 2013 ). Finally, heparin treatment helped to avoid monocyte reprogramming during incubation with patient-derived glioblastoma vesicles ( Himes et al, 2022 ). Unfortunately, studies on heparin in glioblastoma model in vivo were not found in literature.…”