Project Summary Personalized immunotherapy using cancer vaccines made of dendritic-tumor (DC/Tumor) hybrid cells is being developed for a range of cancers, starting with acute myeloid leukemia (AML) and now being adapted to other blood cancers (lymphoma, multiple myeloma) and solid tumors (renal cell carcinoma, breast cancer). These cancer vaccines have demonstrated durable remissions, long-lasting protection, and minimal toxicity. During processing, DC-based vaccines use fusion of millions of patient-derived tumor cells and autologous DCs to create hybrid cells that present a broad array of tumor antigens, including neoantigens generated by the unique mutational profile of an individual's tumor, in the context of DC-mediated co-stimulation. Despite their promise, several technical challenges exist during vaccine production stemming from the low and variable DC-tumor fusion efficiency. One challenge involves obtaining adequate tumor tissue, which can restrict vaccine applicability in early-stage tumors and solid cancers. More significantly, low fusion efficiency means that many tumor cells do not contribute to the vaccine, which may limit the neoantigen repertoire and the ultimate efficacy of the vaccine. To attain high fusion efficiency, we propose to adapt a microfluidic method for cell fusion to a centrifugal format that uses a microfluidic spinning disc to fuse hundreds of thousands of cells. In this R21 we propose to address two high-risk questions necessary before further development can take place: 1) can we fuse clinically relevant quantities of cells at high efficiency, and 2) does increased fusion efficiency provide any biological (and, thus, potentially clinical) benefits. We thus propose two specific aims: Aim 1: Development of a centrifugal microfluidic disc platform for high-efficiency fusion of DC/AML cells. We will adapt a microfluidic cell fusion platform to a spinning disc centrifugal format, which processes cells over a large area. Aim 2: Ex vivo and in vivo evaluation of disc-made DC/AML fusion cancer vaccines. We will utilize ex vivo human patient-derived and murine AML models to evaluate disc-made DC/AML fusion cells in comparison to conventionally processed fusion cells.