Project Summary Tumor stroma, encompassing both extracellular matrix (ECM) and cells, regulates essentially all aspects of tumor growth and metastasis. Signaling among cancer cells, stromal cells, and ECM in tumors promotes proliferation of cancer cells and drug resistance among other key outcomes. Therefore, disrupting stroma- cancer cells signaling is essential to restoring drug sensitivity of cancer cells and improving outcomes for patients. Despite this recognition, the lack of physiologic, high throughput human tumors models significantly impedes drug development and discovery efforts targeting tumor-stromal interactions. We will address this need by developing a high throughput tumor microtissue technology to recreate the complexity of native tumors and enable drug testing against tumor-stromal signaling. This facile technology is based on two-step robotic micropatterning of user-defined cancer cells, stromal cells, and ECM using a polymeric aqueous two-phase system in microwell plates. A 3D mass of cancer cells is formed in an aqueous nanodrop settled at the bottom of a microwell and immiscible from the immersion aqueous phase. A second aqueous drop containing the stromal components is then dispensed to merge with the nanodrop and surround the cancer cell mass to spontaneously generate a microtissue upon incubation. This approach uniquely offers the flexibility of incorporating tissue-specific matrix proteins and different stromal cells to reproduce physicochemical properties of tumors in vivo. We will validate this technology using triple negative breast cancer (TNBC) as a disease model, demonstrating effects of carcinoma-associated fibroblasts (CAFs) and ECM on proliferation and matrix invasion of cancer cells. These studies will use engineered tumor models of both TNBC cell lines and patient-derived CAFs to establish the feasibility of tailoring our tumor model to patient-specific cells. Through this research, we expect to establish our tumor model as a transformative advance that will be implemented broadly for drug discovery and mechanistic studies of breast cancer.