GENO-14SINGLE-CELL TRANSCIPTOMICS AND GENOMICS REVEALS A DIVERSITY OF TUMOR AND IMMUNE CELL POLARIZATION SIGNALS IN GBM

Abstract

Tumor associated macrophages (TAM) are a critical component of the tumor microenvironment, and have functions in tumor invasion, proliferation, angiogenesis and immunosuppression. To assess the heterogeneity of macrophage polarization states, and associated signaling from tumor cells, we single-cell sequenced a cohort of 5 primary GBM biopsies. Using high-throughput microfluidics, followed by RNA-sequencing we analyzed the transcriptomes of 768 single cells. For each case, approximately 96 cells were isolated from disassociated, fresh tumor tissue. For 3 of the 5 cases a second sample was taken and (prior to single-cell isolation) enriched for inflammatory infiltrate via a density gradient, followed by an LS column selecting for CD11b. For all cases, DNA from whole tumor and matched blood was subjected to exome sequencing. Somatic mutations were identified from deeply-sequenced exome data, and the heterogeneity of expressed mutations was tabulated from the single-cell data. Comparing the transcriptional profiles of GBM cells, to transcriptomes obtained from single-cell sequencing human fetal brain tissue, we identified fractions of cells with a low frequency of expressed mutations that have genetic signatures similar to progenitor cell populations found in the developing brain. These sub-populations express high levels of periostin, a potent TAM attractant. We identified a large fraction of cells with a TAM signature. In each sample, the putative TAM population demonstrated gradients of markers for polarization, including CD163, CD11c and MHC class II. The results of differential analysis between these polarized fractions, co-expression analysis of tumor-TAM signaling genes and the ongoing co-culture experiments will be presented.