Background Glioblastomas make up more than 60% of adult primary brain tumors and carry a median survival of less than 15 months despite aggressive therapy. Immunotherapy, now standard of care for many peripheral solid tumors, offers an appealing alternative platform that may improve survival outcomes for patients with glioblastoma; however, predictive features that could inform responsiveness to different immunotherapeutic modalities remains to be elucidated. Recent studies have demonstrated that patients whose tumors show radiographic contact with the lateral ventricle have diminished survival outcomes compared to patients whose tumors do not contact the lateral ventricle. While greater immune infiltrate correlates with more favorable outcomes and more effectual responses to immunotherapy, the anti-tumor immune response in the ventricle is unknown. We hypothesized that ventricle contact may provide a uniquely immunosuppressive microenvironment within the brain that promotes tumor growth by suppressing anti-tumor immunity, that may be overcome with appropriate targeting strategies.

Methods Primary glioblastoma tumors obtained in accordance with the Declaration of Helsinki and with institutional IRB approval (#131870) were disaggregated into single-cell suspensions. Radiographic contact with the LV was identified by MRI imaging and confirmed by a trained neurosurgeon. Multi-dimensional single-cell mass cytometry (CyTOF) then measured >30 immune parameters in thirteen immune subpopulations infiltrating human glioblastomas, including T cells, natural killer cells, B cells, microglia, peripheral macrophages, and myeloid-derived suppressors cells (MDSC). Computational machine-learning pipelines including Citrus, t-SNE, FlowSOM, and MEM identified key differences in the abundance and phenotypes of immune infiltrates.

Results On the basis of glioblastoma contact with the ventricle, we computationally identified consequential distinctions in the abundance of T cell, macrophage, and microglia subsets constituting five immunotype signatures among glioblastoma patients. Immunotypes associated with CD69+CD32+CD44+ peripheral macrophages and PD-1+TIGIT+ CD8 T cells correlated with ventricle contact, whereas immunotypes associated with enriched γδ T cells, B, NK cell, and tissue-resident microglial cells correlated with tumors distal to the ventricle. Further, immune infiltration in the tumor microenvironment correlated with patient outcome, with higher lymphocyte infiltrates correlating with more favorable outcomes, and immune exhaustion correlating with less favorable outcomes.

Conclusions Single-cell mass cytometry in conjunction with the machine learning tools identified key differences in immune cell abundance between lateral ventricle contacting and non-contacting glioblastomas. These results provide key insights into the immune microenvironment of glioblastomas and elucidate several clinically actionable immunotherapeutic targets that may be used to optimize treatment strategies for glioblastomas based on ventricle contact status.

Ethics Approval This study was approved by Vanderbilt University’s Institutional Ethics Board, approval number 131870