High-Dimensional Flow Cytometry of Tumor and Immune Cell Marker in High Grade Glioma

Abstract

High grade glioma and particular glioblastoma are malignant brain tumors with a poor prognosis. Median overall survival with current standard therapy is only 14 months. Although immunotherapy revolutionized the treatment of other types of cancer with many ongoing trials, major advances have not yet been made in the treatment of malignant brain tumors. Recent studies have shown that the tumor microenvironment is dominated by myeloid cells with an immune suppressive phenotype, programmed by the tumor, which promotes tumor progression. T cells on the other hand are often not efficiently primed, are terminally exhausted, or inactivated by immune checkpoints. The aim of this study was to identify targetable surface markers on tumor cells and macrophages in high grade glioma. We first performed a screen using spectral flow cytometry to identify surface markers expressed on four human glioblastoma cell lines. Initially, 361 surface markers expressed on tumor cell lines were identified. Using various databases and survival data from the Cancer Genome Atlas, this list was narrowed down to markers that correlated with a negative impact on survival in glioblastoma patients. Further analysis using single-cell RNA sequencing datasets helped to determine which markers were specifically expressed on tumor cells and macrophages, while excluding those highly expressed on normal brain cells or other normal tissues. After creating and testing panels of brain tumor and immune cell surface markers, samples from 46 patients with high grade glioma were analyzed by high dimensional spectral flow cytometry. By comparing surface marker expression between IDH mutant and IDH wild-type high grade gliomas, we identified specific tumor cell and macrophage subpopulations that are more abundant in IDH wildtype tumors. The signature on tumor cells included the expression of GPR56, CD271, CD71, CD112, CD323, CD146, CD276, PDL1, CD56, CD151, CD51, CD304, CD63 and PDL2. Correlation of our signature with survival revealed a significant association with shorter survival comparing IDH mutant and IDH wildtype patients in our cohort. Various immunosuppressive macrophage subsets were more abundant in IDH-wildtype tumors. Higher frequency of exhausted CD8 T cells were associated with longer survival, whereas immunosuppressive macrophage subsets including FOLR2+CD39+CD163+MoMacs, cycling CD169+MERTK+CD163+CD206+MoMacs, and CD39+CX3CR1+ microglia were associated with shorter survival. These MoMacs co-expressed markers that were also present on our tumor samples, such as CD39, CD112, CD31, CD276, PDL1, HLADR, CD116, CD63, CD51, CD304 and PDL2. Endothelial cells in our samples distinguished themselves by the expression of CD146, CD138 and CD151. Overall, our study was able to generate a specific surface marker atlas with signatures for brain tumor cells, microglia, and monocyte derived macrophages as well as endothelial cells in high grade glioma. Our study reveals potential new targets for development of immunotherapies, such as for example antibody cytokine fusions or CAR-macrophages, particularly for the more aggressive IDH wildtype glioblastoma