Ultrahigh-plex Spatial Phenotyping of the Glioblastoma Multiforme Immune Microenvironment

Abstract The glioblastoma multiforme (GBM) microenvironment is highly immunosuppressive, leading to poor clinical outcomes and median survival of less than 15 months. Information about the nature of immunosuppressive and immunostimulatory cell types in GBM is fragmented, and a better understanding is urgently needed. We have developed a spatial phenotyping application that permits comprehensive characterization of the human GBM immune microenvironment. The Phenocycler-Fusion is a fast spatial biology solution that affords ultrahigh-plex single-cell spatial readouts. We used this solution for deep immune phenotyping of >50 proteins, comprising immune cell lineages, activation states and checkpoints, as well as markers for tumor, vascular and neuronal landscapes of GBM. Via single cell spatial phenotyping, we then isolated spatial signatures within the GBM tissue immune microenvironment. We focused on large numbers of glioma-associated macrophages that were identified via the combinatorial expression of key markers CD68, CD163, STAT1, STAT6, Iba-1 and SIRPα. The macrophages and their biomarker expression profiles displayed significant inter- and intra-tumoral heterogeneity, indicative of the complex biology of GBM. In addition, we profiled other immunosuppressive elements, including Tregs and exhausted T cells. Our data provide a comprehensive account of diverse immune cell populations in GBM and they may uncover systematic differences among GBM patient samples. Our application has enormous potential to further our understanding of the GBM immune microenvironment and is ready to be deployed by other laboratories.