Abstract Background Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. Methods Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression. Results GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. Conclusion These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

Glioblastoma is one of the most common malignant brain tumors. It has poor prognosis: the survival rate is 14-15 months, even with treatment by surgery, radiation, and chemotherapy. To develop more efficacious therapies, it is essential to generate preclinical mouse models that enable mechanistic studies. Multiple murine glioblastoma models have been generated, each with distinct advantages and disadvantages. The traditional Cre-LoxP system specifically targets glioblastoma-related genes but requires extended experimental timelines. CRISPR-Cas9 methods require less time to generate mouse models, yet the offtarget effects lead to variable glioblastoma phenotypes. Transposon-based insertional mutagenesis models can intercept and promote transcription but has strict limitation of insertional transgene size. Allograft cell line injection into immunocompetent mice prevents immune rejection but fails to recapitulate various features of human glioblastoma. Intracranial injection of patient-derived xenograft cell lines into immunocompromised mice preserves features of human glioblastoma but does not allow the study of immune cell function in preclinical immunotherapeutic approaches. Finally, humanized mouse models offer the potential to analyze the human adaptive immune response but not the innate

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.