Abstract Glioblastoma (GBM) remains one of the biggest challenges in oncology, with median survival of ~15 months. Extreme heterogeneity of the GBM cells within each tumor results in resistance to most of the available therapies. Understanding the relationships between the heterogenous populations of GBM cells and the diverse niches they rely on is key to developing more effective treatment for this aggressive brain tumor. Analysis of the spatial localization of cells with different properties can shed light on their microenvironmental preferences, interactions and potential vulnerabilities. Therefore, we set out to profile 20 GBM cases using methods allowing for single-cell assessment of select genomic features and protein marker expression in intact tissue samples from each tumor. We constructed a tissue microarray (TMA), containing four distant areas from each tumor block to represent the intratumor heterogeneity within each tumor fragment. Based on multicolor immunofluorescent staining with markers of glial cells, immune cells and vasculature, we selected 96 regions of interest, which were then subject to digital spatial profiling for 90 protein markers associated with glioblastoma biology, neurobiology and immunology. Pearson’s correlation and Shannon index calculation based on our profiling results showed that majority of the regions cluster by their tumor of origin, however several highly diverse tumors were also identified. To link the diversity of the protein expression found with the digital spatial profiling with underlying genomic alterations, using the same TMA slide, we performed multiplexed fluorescence in situ hybridization for genes most frequently amplified in GBM. Automated quantification allowed us to collect information from 38,000 individual cells. Based on the presence or absence of the amplifications each cell was assigned a genotype. Since mutation in hTERT promoter has been reported as one of the drivers of GBM, we used STAR-FISH, an in situ single-cell mutation detection assay, to record the frequency and spatial localization of cells harboring this mutation on our TMA. Our datasets provide a unique opportunity to link single-cell genetic diversity to the phenotypic heterogeneity of the tumor cells and their microenvironment. Spatial localization of cells with distinct genotypes allows us to further dissect the interactions between the distinct subclones of GBM cells and uncover their novel dependencies. Citation Format: Kacper A. Walentynowicz, Shreya Yadav, Cristina Vincentelli, Michalina Janiszewska. Uncovering the ecology of glioblastoma with spatial digital profiling and single cell genotyping [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA10.