SUMMARY Tumor microenvironment (TME) is a key determinant of tumor growth, disease progression and resistance to chemotherapy. There is a diverse repertoire of distinct cell type that are found in the tumor microenvironment, with many that have not been fully enumerated. In addition, little is known about the “geography” of the tumors and the spatial organization of the distinct cell types. There is therefore, an immediate need of transformative tools to enable in-depth understanding of the TME in many forms of malignancies. Spatial Genomics Inc is commercializing seqFISH+ (Eng et al, Nature 2019) which can achieves multiplexing of 10,000 genes at the mRNA level in single cells in tissues with spatial information preserved. Using the seqFISH+ transcriptome profiles, novel cells types in situ and spatial organizations can be directly identified and visualized in tumor tissue slices. These capabilities make seqFISH+ a powerful tool for examining the different cell types and their interactions in the tumor microenvironment. In this SBIR proposal, we will develop an instrument and the associated software and reagents that will allow researchers to perform seqFISH+ in tumor slices. In collaboration with Amgen and Children’s Hospital at LA (CHLA), we will apply seqFISH+ to image the tumor microenvironment in both mouse model systems and human pediatric neuroblastoma samples.