Single-cell heterogeneity of EGFR and CDK4 co-amplification is linked to immune infiltration in glioblastoma

Glioblastoma (GBM) is the most aggressive brain tumor, with a median survival of -15 months. Targeted ap-proaches have not been successful in this tumor type due to the large extent of intratumor heterogeneity. Mosaic amplification of oncogenes suggests that multiple genetically distinct clones are present in each tu-mor. To uncover the relationships between genetically diverse subpopulations of GBM cells and their native tumor microenvironment, we employ highly multiplexed spatial protein profiling coupled with single-cell spatial mapping of fluorescence in situ hybridization (FISH) for EGFR, CDK4, and PDGFRA. Single-cell FISH analysis of a total of 35,843 single nuclei reveals that tumors in which amplifications of EGFR and CDK4 more frequently co-occur in the same cell exhibit higher infiltration of CD163+ immunosuppressive macrophages. Our results suggest that high-throughput assessment of genomic alterations at the single -cell level could provide a measure for predicting the immune state of GBM.

Automated summary

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. The heterogeneity of GBM has hindered the success of targeted therapies. By using spatial protein profiling and single-cell fluorescence in situ hybridization (FISH), we investigated the relationship between genetically diverse subpopulations of GBM cells and the tumor microenvironment. Our findings suggest that the co-occurrence of amplifications in EGFR and CDK4 in the same cell is associated with higher infiltration of immunosuppressive macrophages. Assessing genomic alterations at the single-cell level could provide insights into the immune state of GBM.