Summary Breast cancer occurs in one in eight women in the United States and leads to 40,000 deaths annually. The leading cause of breast cancer mortality is metastasis — cancer cells spreading and growing in distant organs throughout the body. It is speculated that only a small subset of tumor cells, considered cancer stem cells (CSCs), can initiate metastases. The development of CSC-targeted therapies is crucial in treating metastasis. However, the intricate metastatic process, complex and diverse genetic backgrounds, responses to various treatments and tumor microenvironmental stimuli all lead to the functional heterogeneity of metastatic CSCs, making the identification and treatment of these cells extremely challenging. A potential approach to overcoming this challenge is the development of individualized treatments that rely on the characterization of the unique molecular properties of CSCs from a particular patient, enabling the targeting of specific cancer- promoting signals. The success of this individualized treatment strategy depends heavily on collecting the biopsies that reveal the current tumor biology in a patient. Circulating tumor cells (CTCs), shed from primary or metastatic tumors into the systemic circulation, contain potential metastatic CSCs, and venipuncture offers a feasible, minimally invasive liquid biopsy source for obtaining small quantities of these rare CTCs. Our recently pioneered ex vivo culture method for expanding these rare CTCs has enabled a proof-of-concept study to identify metastatic CSCs from the CTCs and perform drug sensitivity testing. The recent success of using a large panel of tumor cell lines to perform drug sensitivity testing and identify subcategories that predict effective treatments has also validated the concept of individualized medicine. However, a systemic approach using patient-derived metastatic CSCs to establish such biomarkers and therapy predictions has never been done before. To fill this crucial gap, in this proposal, we plan to expand CTCs obtained from metastatic breast cancer patient blood samples in vitro and evaluate their tumorigenesis in immunodeficient mice. This will allow us to functionally identify metastatic CSCs, analyze their unique molecular properties, investigate their interactions with their microenvironments, and examine their susceptibility to clinical-grade drug panels. Results from these analyses will be combined to match metastatic CSC-specific genetic and transcriptomic biomarkers with effective treatment regimens, which will be applied in the future to prescribe therapies targeting metastatic CSCs in breast cancer patients. Our success will advance the field of individualized medicine by identifying effective targeted therapies based upon the unique molecular biomarkers of metastatic CSCs from liquid biopsies. My training in genetics, CTC biology, xenografted animal studies, and drug susceptibility screens uniquely prepared me to face the challenges of this innovative research proposal and move toward the era of personalized medicine.

Project Narrative Majority of breast cancer related deaths are due to metastasis, initiated by rare metastatic cancer stem cells (CSCs) shed into systemic circulation. This proposal aims to identify metastatic breast CSCs from liquid biopsied circulating tumor cells from patient's peripheral blood, analyze their unique molecular properties, and perform clinical-grade drug sensitivity screens in order to deduce metastatic CSC-specific biomarkers and corresponding effective therapeutic strategies. Completion of this proposed research will potentially lead to a significant advancement towards personalized metastatic cancer treatment.