PROJECT SUMMARY Primary tumors shed circulating tumor cells (CTCs) into the bloodstream that metastasize preferentially to distant organs, resulting in 90% of cancer related fatalities. For example, estrogen receptor positive (ER+) breast cancers exhibit high rates of metastasis to bone, with decreased rates to liver and lung. CTCs exhibit heterogeneous gene expression programs and functional phenotypes, which are selected by soluble and mechanical interactions within each metastatic “niche.” A critical challenge is to predict how patient-specific CTCs disseminate throughout the body and respond to therapeutic treatments. An exciting strategy is to culture CTCs ex vivo for drug screening informed by genomic and transcriptional profiling. We seek to elucidate how CTCs respond to different features of the metastatic niche by engineering controlled interactions with tissue-specific extracellular matrix (ECM) and with human primary stromal cells, which may recapitulate disease progression and therapeutic resistance in these microenvironmental contexts. Our long-term goal is to establish preclinical assays for patient CTCs to predict metastatic disease progression and screen targeted inhibitors. Realization of this goal involves several technical challenges, including: 1) Tissue-mimetic matrix with tunable biochemistry and mechanics, 2) Multicellular tissue constructs with controlled size and cellular composition, 3) Gene expression profiling of CTCs in response to soluble and mechanical stimuli, 4) Spatiotemporal analysis of phenotypic heterogeneity, including the epithelial- mesenchymal transition (EMT), and 5) Validation with human patient samples. Thus, our overall objective is to understand how CTC gene expression and mechanophenotype is regulated by matrix or stromal interactions in tissue-mimetic microenvironments. This discovery-based approach can deconstruct patterns of gene expression driven by decellularized extracellular matrix or the secretome of human stromal cells. Such bioinformatics analyses can identify possible therapeutic strategies based on existing patient, xenograft, and high-throughput screens. PI: Wong is a New Investigator with expertise in cancer cell migration, biomaterials, and microfluidics. Mentor: Reichner is an expert on directed cell migration and mechanobiology and Mentor: Bertone is an expert on single cell analyses in cancer. We investigate the relative contributions of microenvironmental stimuli using three aims: AIM 1 will elucidate how individual breast cancer cells interact with tissue-mimetic matrix and AIM 2 will engineer co-cultured multicellular spheroids with stromal cells.