Project Summary Patient-derived tumor organoids (PDO), involving the ex vivo culture of fresh tumor fragments, have emerged as promising models for predicting patient drug response for personalized cancer therapy. PDOs recapitulate the tumor micro-environment (TME), resemble the source tumor phenotypically and genomically, and are compatible with high-throughput drug screening. However, the lack of preservation of immune cells in PDOs has been a major roadblock to modeling immunotherapy. Our team recently demonstrated a new type of PDO that cultures tumor fragments as a cohesive unit, allowing the in situ preservation of diverse immune cell types alongside tumor cells without artificial reconstitution. This approach has enabled the modeling of patient-specific responses to immune checkpoint inhibitors. One of the first steps in the generation of PDOs is the dissection of patient tumor specimen into small fragments. Mechanical dissection, instead of enzymatic digestion, is critical in preserving the in vivo association between tumor cells and endogenous immune and non-immune elements. The ability to preserve endogenous immune cells, including tumor-infiltrating lymphocytes (TIL), is particularly important for personalized immunotherapy testing. However, current mechanical dissection relies primarily on manual mincing of tumor specimen into small fragments. It results in fragments with a broad size range, and is imprecise and irreproducible. Fragments that are too large suffer from inadequate nutrient supply, suboptimal oxygenation and viability, and poor drug penetration. Fragments that are too small are unlikely to preserve sufficient stromal cells to support PDO growth, and/or endogenous immune cells which may be present at low concentrations. As such, there is an unmet need for a better way to generate tumor fragments of controllable and uniform size, and identify optimal size(s) to increase the reproducibility and yield of viable PDOs that can preserve the cellular contexture and tumor architecture. This project aims to address this need by developing a new method to mechanically dissect tumor specimen into uniform fragments. Performance measures include fragment size uniformity, PDO viability, preservation of immune cells, and tumor cytotoxicity in response to immunotherapy. Other cutting methods including manual mincing will be used as benchmarks.