Functional optical imaging for rapid, label-free predictions of treatment response and clonal evolution in patient-derived cancer organoids - PROJECT SUMMARY / ABSTRACT Improved treatment strategies for patients with cancer require enhanced tools to better predict patient response, model clonal heterogeneity, and identify novel treatment options for individual patients. Patient-derived cancer organoids (PDCOs) are a major advance providing more representative models of the human disease, including the maintenance of molecular alterations, cell-cell communication, and the 3D architecture found in cancers. Our groups have significant experience using PDCOs to predict treatment response for patients across cancer types. These models, however, have unique challenges when used for translational studies, including (1) heterogeneity between organoids from the same patient, (2) assessment techniques that require sample fixation or reagents that prevent time-course studies of clonal evolution, and (3) lack of single organoid assessment and low- throughput culture techniques that limit screens for new drugs. We have developed optical metabolic imaging (OMI) using two-photon (2P) microscopy to measure treatment response without a need for reagents (e.g., dyes, labels) or fixation. Our prior studies demonstrated that 2P OMI can predict treatment response for patients with cancer. However, 2P microscopy is high cost, low throughput, and complex to operate. To expand the use of OMI across multi-center translational studies of PDCOs, more readily accessible imaging and analysis methods are needed for high/moderate throughput drug screening and assessments of organoid metabolic heterogeneity over time. To enhance the accessibility of this technology to more laboratories and facilitate the expanded use of PDCOs, we have developed a one-photon wide-field (WF) OMI technique with single-organoid tracking and leading-edge segmentation methods for significantly reduced cost, reduced complexity, and increased throughput compared to 2P microscopy. The goal of this proposal is to validate WF OMI techniques for PDCOs that can be widely used for patient treatment planning, heterogeneity analyses, and new drug development. OMI non-invasively images response in a 3D sample using the intrinsic fluorescence of the metabolic co-enzymes NAD(P)H and FAD. OMI can dynamically quantify heterogeneous drug response over a treatment time-course. We have and will continue to develop hundreds of PDCO lines from metastatic colorectal cancer (CRC) patients. These cultures will be used to validate new methods for more widely accessible OMI tools to predict patient response, identify metabolic/genetic heterogeneity that underlies resistance to targeted therapy, and perform screens of new drug candidates. The completion of this work will create technologies to perform high-sensitivity patient-matched drug screens in a clinical setting, predict the evolution of drug resistance for individual patients, and perform new drug development in samples that reflect the diversity of human cancers.
