Proteomics-driven analysis and robotics-enabled drug testing of microdissected tumors and their microenvironment

Technologies that combine the microscale manipulation of tissues and fluids offer the exciting possibility of miniaturizing disease models and drug testing workflows. Such technologies enable inexpensive, more efficient tests of high clinical biomimicry, maximizing the use of scarce human biopsies while minimizing animal testing. Notable examples are patient-derived organoids (PDOs) and organs-on-chips (OOCs). Traditional approaches using tissue culture and animal models offer a nearly-infinite supply of tumor tissue, but, unfortunately, they are extremely poor predictors of disease outcomes and lack key anatomical and pathophysiological features of the real patient. PDOs and OOCs can only test effects on the tumor cells or on reconstituted tumor microenvironments (TMEs) because the rest of the TME, including the immune TME, is compromised during the process of tissue growth in culture. In contrast, microdissected tumor tissues (µDTs), e.g., minced tumor “spheroids” or “ex vivo tumor fragments” tested directly in culture without passaging, preserve key features of the native TME. Though lack of expansion in culture leads to lower throughput, maintaining an intact TME critically enables more clinically-relevant cancer disease models, empowers drug evaluation for the next generation of combination therapies and immunotherapies, and helps deploy more effective personalized oncology approaches. The Folch and Gujral labs have developed a high-throughput µDT approach in which thousands of regularly- sized µDTs are rapidly dissected from a single biopsy. The tumor (or slices) are mechanically cut into ~400 µm- wide, cuboidal-shaped µDTs (or “cuboids”). The cuboids retain key features of the TME, including vascular structures and immune cells, as revealed by immunostaining, proteomics, and cytokine profiling. Using a custom- made robotic platform, we hydrodynamically “lift”-and-place individual cuboids into 384-well plates to obtain high-dimensional molecular readouts of drug effects and TME composition with proteomics (Villen lab), complemented by standard viability readouts, multi-immunohistochemistry, and cytokine secretion assays. Using cuboids, we have demonstrated TME-dependent cancer treatments, such as immunotherapy by checkpoint inhibition, and proteomics analysis of cuboids that provides a readout of cell type, immune processes, and drug responses. Here we will validate the robotic platform combined with proteomics to test TME-dependent combination therapies on individual cuboids from mouse and human tumors at scale and high depth and will integrate proteomics and functional responses to model therapeutic efficacy and resistance. Project Number: 1R01CA299650-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: ALBERT FOLCH (+2 co-PIs) | Institution: UNIVERSITY OF WASHINGTON, SEATTLE, WA | Award Amount: $686,271 | Activity Code: R01 | Study Section: Cellular and Molecular Technologies Study Section[CMT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11295115