SOW/Abstract: The proposed research for the NIH New Innovator Award seeks to biochemically and biophysically characterize complete cytoskeletal phenotypes of metastatic fusion oncoprotein- driven pediatric cancer cells by designing novel high-throughput single-cell instrumentation. We focus our biological hypotheses on immune evasion of metastatic Ewing sarcoma cells given the practically unwavering percentage of patients who succumb to metastatic disease (~20-30% for decades). In order to provide unparalleled quantitative assessment of protein complex expression and structure (e.g., of the cytoskeleton, integrins, focal adhesions) we will develop assays for high-throughput (1000s of single-cells) highly multiplexed fluorescence imaging of cellular structure or cell invasion migration patterns along with protein expression quantitation. In Objective I, we will design a microscale electro-clearing method to remove protein background from monomeric cytoplasmic and nuclear proteins prior to immunostaining single Ewing sarcoma cells in 3D cell co-culture with immune cells. We will evaluate the imaging performance of the electro-clearing approach anticipating use cases for both widefield imaging (for more rapid high-throughput screens in place of confocal microscopy) and super resolution microscopy (i.e., to reduce background for single-molecule localization microscopy). For Objective II, we will develop integrated 3D cell invasion assays compatible with downstream single-cell protein complex fractionation. Thus, we will identify the patterns of protein complex expression present in persistently invasive cancer cells towards identifying therapeutic vulnerabilities in fusion-oncoprotein mediated cell metastasis.

Project Narrative: When cancers lack immunocompetent mouse models, we have limited access to evaluate the effect of tumor-immune cell interactions on metastatic capacity. To address this gap, we propose introduction of integrated engineered tumor microenvironments with multiplexed single-cell electrophoretic analysis capable of quantifying protein complex expression, cell structure and motility. We will test specific hypotheses on the metastatic phenotypic heterogeneity of Ewing sarcoma cells in the presence of distinct immune cell populations in the tumor microenvironment.