ABSTRACT Tumor cell heterogeneity and dynamic changes to tumor cell states are major contributors to tumor initiation, growth, metastasis, and treatment resistance. The Snyder lab and the Yanai lab are each developing their own technologies in separate NCI funded studies for respectively visualizing tumor heterogeneity in mouse models with hyperspectral lineage tracing (NCI IMAT, Snyder, MousePaint) or reconstructing spatial relationships and cancer cell states using single cell genomics technology (NCI ITCR, Yanai, SNAP). Our hypothesis is that application of the MousePaint and SNAP technologies on the same dataset will benchmark these technologies for (1) viewing thousands of colors (i.e. clones) in vivo and for (2) inferring tumor neighborhoods from scRNAseq data. Two aims are proposed. Aim 1. To generate MousePaint cell lines for assessing color diversity and clonal neighborhoods using hyperspectral imaging. Aim 2. Benchmarking SNAP and MousePaint by coregistering single cell genomics data with MousePaint imaging data. The collaborative supplement will have substantial positive impact on each parent award by providing new models and algorithms for benchmarking Dr. Snyder’s hyperplex lineage tracing strategies as well as providing experimental models for Dr. Yanai’s SNAP single cell genomics approaches. We expect these studies to 1) validate resources for the research community to study tumor heterogeneity and 2) provide new insight into the early changes that occur in the tumor microenvironment during cancer initiation and growth.