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

利用单细胞蛋白质组学对融合癌蛋白驱动的儿科癌症转移进行深度表型分析

基本信息

批准号：
10687394
负责人：
Julea Vlassakis
金额：
$ 138.97万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-11 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687394
关键词：
3-Dimensional Address Award Biochemical Biological Biological Assay Cell Communication Cells Cellular Structures Coculture Techniques Confocal Microscopy Cytoplasmic Protein Cytoskeleton Disease Engineering Ewings sarcoma Focal Adhesions Fractionation Fusion Oncogene Proteins Heterogeneity Image Immune Immune Evasion Immunocompetent Integrins Invaded Malignant Childhood Neoplasm Malignant Neoplasms Mediating Metastatic Ewing&apos s Sarcoma Methods Microscopy Neoplasm Metastasis Nuclear Proteins Patients Pattern Performance Phenotype Population Proteins Proteomics Research Structure Testing Therapeutic Tumor-infiltrating immune cells United States National Institutes of Health biophysical properties cancer cell cancer invasiveness cell motility design fluorescence imaging high throughput screening instrumentation migration monomer mouse model novel protein complex protein expression single cell proteins single molecule superresolution microscopy tumor microenvironment

项目摘要

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.

SOW/摘要： NIH新创新者奖的拟议研究旨在生物化学和生物病理学表征转移性融合癌蛋白的完整细胞骨架表型- 通过设计新颖的高通量单细胞仪器来驱动儿科癌细胞。我们将我们的生物学假设集中在转移性尤文肉瘤细胞的免疫逃避上，死于转移性疾病的患者比例几乎不变（约20-30%），几十年）。为了提供无与伦比的蛋白质复合物定量评估表达和结构（例如，的细胞骨架，整合素，局灶性粘连），我们将开发用于高通量（1000个单细胞）的高度多重荧光成像的测定，细胞结构或细胞侵袭迁移模式沿着蛋白质表达定量。在目的一中，我们将设计一种微尺度的电清洗方法来去除蛋白质免疫染色前单体细胞质和核蛋白的背景尤文肉瘤细胞与免疫细胞的3D细胞共培养。我们会评估成像电清除方法的性能，预期用于宽视场成像 (for更快速的高通量屏幕代替共聚焦显微镜）和超分辨率显微术（即，以减少单分子定位显微术的背景）。为目的二，我们将开发与下游相容的集成3D细胞侵袭测定，单细胞蛋白质复合物分级分离。因此，我们将确定蛋白质复合物的模式表达存在于持续侵袭性癌细胞中，以鉴定治疗性融合癌蛋白介导的细胞转移的脆弱性。