Abstract: In this SBIR grant, AtlasXomics Inc. proposes to develop a novel spatial immunophenotyping device that maps immune infiltrates in the mouse brain tumor microenvironment (TME) by spatially co-profiling ~20 immune surface protein biomarkers and the whole transcriptome (WT) with cellular resolution. In collaboration with researchers at the National Cancer Institute (NCI), we will demonstrate the ability of this device to richly characterize their potentially transformative immunotherapy for glioblastoma (GBM). Company founder Professor Rong Fan developed the basis for the device, Deterministic Barcoding in Tissue for spatial omics sequencing (DBiT-seq), to enable cellular-resolution mapping of the whole transcriptome, high- plex proteome, and genome-wide epigenome. AtlasXomics proposes in Phase I to develop a next-generation immunophenotyping device to map immune infiltrates in the TME by combining same-section WT and proteomic data. DBiT- seq’s unique microfluidics approach enables co-profiling of WT and proteins. The next-generation immunophenotyping device will build on the proven ability of protein biomarkers to characterize immune phenotypes by incorporating both cellular-resolution mapping and the discovery potential imparted by same-section WT coverage. Aim 1: Develop a spatial multi-omics immunophenotyping device (proteome and whole transcriptome) to identify and map immune infiltrates in the GBM microenvironment. Aim 2: Demonstrate application of the next generation immunophenotyping device by mapping and identifying immune infiltrates in NCI’s GBM mouse model. After Phase I, we will have demonstrated that the next generation immunophenotyping assay achieves cellular resolution co-profiling of whole transcriptome and a tailored protein panel, thereby enabling mapping and characterization of immune infiltrates in the TME. This device will help improve understanding of the mechanisms underlying various immunotherapy treatments. Upon demonstrating the value of the novel spatial immunophenotyping device to NCI’s vaccine development program, AtlasXomics will scale the product's throughput, robustness through semi-automation and advanced chip designs. An optimized mouse brain-specific multi-omics immunophenotyping device would enable cancer researchers to better understand how immune infiltration underpins cancer immunotherapy. A next generation immunophenotyping device combining cellular resolution proteomics and WT will become a standard tool to promote solid tumor cancer immunotherapy treatment development.