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Using microfluidic single cell culture to characterize cancer cell asymmetric division

使用微流控单细胞培养来表征癌细胞不对称分裂

基本信息

批准号：
9237393
负责人：
Ronald J Buckanovich
金额：
$ 43.31万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2017-08-25
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9237393
关键词：
Acidosis Address Alpha Cell Biologic Characteristic Biological Models Cancer Model Cell Culture Techniques Cell Differentiation process Cell Separation Cell division Cells Clinical Computer software Data Data Analyses Devices Disease Environmental Impact Event Gene Expression Genes Genomic Instability Growth Growth Factor Human Hypoxia In Vitro Link Malignant Neoplasms Malignant neoplasm of ovary Metals Microfluidic Microchips Microfluidics Modeling Molecular Molecular Analysis Molecular Profiling Monitor Mothers Multipotent Stem Cells Pathway interactions Patients Pattern Phenotype Population Procedures Quantitative Reverse Transcriptase PCR Recurrent disease Resistance Retrieval Solid Neoplasm Specimen Stem cells Stress System Technology Testing Therapeutic Tumor Initiators actionable mutation cancer cell cancer cell differentiation cancer stem cell cancer therapy cell growth cell type chemotherapy daughter cell design differential expression high throughput analysis in vivo interpatient variability neoplastic cell new technology new therapeutic target novel novel therapeutics progenitor prototype stem cell biology stem cell fate stem cell niche stem-like cell targeted treatment therapeutic target tool transcriptome sequencing tumor initiation

项目摘要

ABSTRACT Whether cancer follows a hierarchical (linear) or stochastic (random) model of differentiation has critical clinical implications, yet remains highly controversial. The hierarchical model mirrors normal stem cell biology with cancer stem-like cells (CSC) following an established pattern of asymmetric divisions to give rise to distinct progeny. In this model only CSC can initiate recurrent disease. In the stochastic model, any given tumor cell can asymmetrically divide to give rise to the other cells of the tumor and initiate recurrent disease. The key to determining the pattern of differentiation is characterizing asymmetric division potential. However the asymmetric division potential of primary human cancer cells has not been directly interrogated due to a lack of technology. We hypothesize that cancer cells will follow a hierarchical differentiation pathway, but rare stochastic dedifferentiation events occur allowing some cells to obtain a CSC state. We further hypothesize that factors regulating the asymmetric division of cancer cells, both hierarchical and stochastic, will significantly impact cancer growth and represent therapeutic targets. To address the technology deficiency, we propose SA1: To develop a high-throughput single-cell microfluidic culture device with selective live-cell retrieval. A highly parallel, compact, 1024 microwell design will allow efficient single cell capture, growth, and characterization of asymmetric division of primary human cancer cells. Automated data analysis and selective single-cell retrieval capacity will allow for high-throughput functional and molecular analysis of common and rare events. In order to validate this important new technology we will use ovarian cancer as a model system. We propose: SA2: To directly interrogate the asymmetric division potential of primary ovarian cancer cells. Our preliminary data indicates that ovarian cancer follows a hierarchical differentiation model with rare (~1/3000) putative stochastic ‘dedifferentiation’ events wherein a progenitor cell gives rise to putative CSC. We will expand our studies of the asymmetric division potential to assess inter-patient variability and determine the impact of environmental stresses on dedifferentiation rates. Critically, using selective cell retrieval we can evaluate the biologic characteristics (chemotherapy resistance, tumor initiation capacity) of single cells derived following an asymmetric division to confirm the biologic implications of differentiation and dedifferentiation events. Finally, we propose SA3: To identify regulators of cancer cell differentiation and drivers of dedifferentiation. In parallel to the functional studies in SA2, mother and daughter cells from asymmetric divisions will be isolated and expression of distinct stem cell related genes assessed via single cell qRT-PCR. Factors differentially expressed between mother and daughter cells will then be evaluated for their impact on CSC growth and differentiation. To identify molecular drivers of the rare dedifferentiation events, we will perform RNASeq comparing clones derived from hierarchical differentiation vs. stochastic dedifferentiation events. Putative molecular drivers of dedifferentiation will then be confirmed both in vitro and in vivo.

摘要