Project Summary/Abstract Adoptive cell therapy has the potential to circumvent cancer immune evasion by employing patient-derived, tumor-specific cytotoxic T-cells to attack cancer. While promising responses of advanced cases to cancer immunotherapy bolster the optimism surrounding these therapeutic strategies, interpatient variations in treatment responses demand a new method to select highly effective tumor-specific cytotoxic T-cells for better and safe immunotherapy. We propose a high-throughput, single-cell level method for identifying and recovering highly functional T cells that secrete tumor-killing signals in the presence of tumors. Our proposed technology builds on transformational advances in the generation of droplet emulsions, secretomics, and new hydrogel-based sensing techniques we recently developed. In this assay, tumor-killing T cells and cancer cells will be encapsulated in pairs in a uniformly sized, secreted factor-sensitive hydrogel droplet using a microfluidic droplet generator. The resulting isolated reaction compartments for each cell pair will prevent the mixing of T cells' multiple secreted factors and will concentrate them to allow sensitive detection. The hydrogel will respond to a factor secreted by functional T cells via a dramatic size change, allowing hydrogels with active T cells to be reliably isolated from the heterogeneous mixture using a hydrodynamic size-based particle sorting mechanism. The sorted pairs will be individually recovered using an aspiration pipette and the hydrogel shell dissolved without cell damage, allowing for the recovery of functional individual T cells. We propose to use this high-throughput assay, capable of screening millions of T cells, to identify tumor-responsive T cells and to recover them for downstream analysis or proliferation. This assay is simple, sensitive, and versatile enough to be used by a wide research community or eventually within clinical settings for isolating and profiling rare cells with target secretory phenotypes.

Project Narrative (3 sentences) The objective of the proposed study is to develop a high-throughput, cost-effective but analytical and quantitative method for identifying and recovering highly functional T cells that kill tumor cells from a large background population based on their secretory phenotype. This assay could accelerate the identification of tumor-specific T cells from a patient’s blood to more rapidly create personalized immunotherapy and enable more rapid research into the development of engineered cell immunotherapy. It could also serve as a research tool to precisely identify mechanisms of immune cell activation to help usher in better therapeutic strategies.