Intra-tumor heterogeneity has fundamental implications for our understanding of cancer biology and therapeutic responsiveness, but is masked by averaging signals over million of cells in bulk tissue assays currently used in cancer research and diagnostics. To address this problem, single-cell data is instrumental to advance the diagnosis, prognosis, and treatment of human cancer, and implement effective personalized oncology strategies. Here, we propose to use single-cell sequencing and single-cell in situ assays to delineate intra-tumor heterogeneity in Breast Cancer (BC) in response to chemotherapy. In project year 1 (proof-of-principle), we will integrate single-cell, genome-wide DNA/RNA sequencing with targeted single-cell in situ analyses of nuclei acids in specimens obtained from BC patients treated with neoadjuvant (NA) chemotherapy. In project year 2 (prospective trial), we will start applying the established platform to investigate the clinical validity of spatial and temporal intra-tumor heterogeneity measurements for predicting the response to NA therapy.