Deep phenotyping of fusion oncoprotein-driven pediatric cancer metastasis with single-cell proteomics

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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.