Enabling Technology to Study Mechanosensitive and Mechanoresistant Cancer Cells in Flow

在流动中研究机械敏感和机械抗性癌细胞的技术

• 批准号: 10306077
• 负责人: Michael R. King
• 金额: $ 29.79万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10306077
• 关键词: Address; Apoptosis; Area; Attention; Behavior; Biochemical; Biological; Blood; Blood Cells; Blood Circulation; Blood flow; Blood specimen; CRISPR/Cas technology; Calcium; Cancer Patient; Cardiac; Cell Death; Cell Line; Cell Survival; Cell model; Cells; Chemoresistance; Clinical; Coculture Techniques; Cues; Development; Devices; Disease; Environment; Epithelial; Fibroblasts; Flow Cytometry; Gene Expression; Genes; Genotype; Goals; Injections; Laboratories; Ligands; Liquid substance; Malignant Neoplasms; Malignant neoplasm of prostate; Measurement; Measures; Mechanics; Membrane; Mesenchymal; Metastatic Prostate Cancer; Mitochondria; Modeling; Morphology; Necrosis; Neoplasm Circulating Cells; Neoplasm Metastasis; Peripheral; Phenotype; Physiologic pulse; Piezo 1 ion channel; Play; Population; Primary Neoplasm; Process; Production; Protocols documentation; Research; Role; Seeds; Signal Transduction; Site; Solid Neoplasm; Spectrophotometry; Stimulus; Stromal Cells; Suspensions; TNF gene; Technology; Translating; Tumor Burden; Tumor-associated macrophages; Work; base; biological adaptation to stress; cancer cell; cancer type; demographics; experimental study; fluid flow; in vitro testing; in vivo; knock-down; macrophage; mechanotransduction; model development; mouse model; neoplastic cell; new technology; prostate cancer cell; prostate cancer cell line; response; shear stress; trait; transcriptome sequencing; tumor progression

Mechanotransduction of cancer cells in the solid tumor environment is an active area of research, yet far less work has been done to examine the biological behavior of cancer cells in the blood flow environment. Recently, mechanical stimuli such as shear stress have received attention for their effects on cancer progression. For instance, studies have shown that shear stress has been associated with enhanced metastasis and cancer cell death. In the applicant’s laboratory, the synergistic effect of shear stress on tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis of circulating tumor cells (CTCs) was demonstrated, as well as the unique ability of cancer cells to survive extremely high pulses of shear stress, comparable to blood cells. These mechanical cues can be translated into biochemical responses in cells through the process of mechanotransduction. It is proposed to subject cell suspensions to repeated shear stress pulses in a multiwell plate format to study shear stress response and to develop “mechanoresistant” cell lines that will be phenotypically and genotypically characterized with the goal of identifying the drivers that enable cancer cells to survive in circulation. Moreover, given that the presence of CTC aggregates in the blood signal more aggressive and metastatic disease, multicellular aggregates modeled after aggregates isolated and characterized from prostate cancer patient blood samples will be tested in vitro for their mechanical responses, and also used to guide the development of model cells and spheroids to be injected into experimental mouse models of bloodborne metastasis. This research is organized around three specific aims: Specific Aim 1: To develop a new high throughput device to study the effect of fluid shear stress on cancer cell responses. A multiwell plate configuration based on a BioJet printer will enable direct analysis with multiwell plate-capable flow cytometers and spectrophotometers. Calcium influx, membrane and mitochondrial damage, and apoptosis of cancer cells in response to shear stress signals will be examined, and “mechanoresistant” prostate cancer cells developed and characterized. Specific Aim 2: To develop the shear flow device and culture conditions to study shear stress responses modulated by interactions with stromal cells. Circulating tumor cell aggregates isolated from prostate cancer patient blood samples will be characterized, and used to develop model aggregates for further study. The stability and survival of heterogeneous tumor cell aggregates in shear flow will then be studied. Specific Aim 3: To examine the roles of cancer cell mechanosensitization and mechanoresistance on metastatic tumor burden in vivo. Orthotopic metastasis studies using cells with modulated shear sensitivity will be performed. Mechanoresistant cancer cells vs. parental cancer cells will be compared in an experimental mouse model of metastasis, and the fate of injected cell aggregates studied as well.

实体瘤环境中癌细胞的机械转导是一个活跃的研究领域，但远低于 已经进行了研究癌细胞在血流环境中的生物学行为的工作。最近， 诸如剪切应力的机械刺激因其对癌症进展的影响而受到关注。为 例如，研究表明，剪切应力与增强转移和癌细胞 死亡在申请人的实验室中，剪切应力对肿瘤坏死因子相关的细胞的协同作用， 细胞凋亡诱导配体（TRAIL）诱导的循环肿瘤细胞（CTC）的细胞凋亡被证实， 以及癌细胞在极高的剪切应力脉冲下存活的独特能力， 细胞这些机械提示可以通过以下过程转化为细胞中的生化反应： 机械传导提出了在多孔介质中使细胞悬浮液经受反复的剪切应力脉冲， 板格式研究剪切应力反应，并开发“机械抗性”细胞系， 表型和基因型特征，目的是鉴定使癌细胞 才能在流通中存活此外，鉴于血液信号中存在更多的CTC聚集体， 侵袭性和转移性疾病，在分离的聚集体后建模的多细胞聚集体， 将在体外测试其机械响应， 并用于指导模型细胞和球体的发育，以注射到实验小鼠体内 血源性转移的模型。本研究围绕三个具体目标组织：具体目标1： 开发一种新的高通量装置来研究流体剪切力对癌细胞反应的影响。一 基于BioJet打印机的多孔板配置将使能够使用多孔板进行直接分析 流式细胞仪和流式细胞仪。钙内流、膜和线粒体损伤与细胞凋亡 将检查癌细胞对剪切应力信号的响应，并且“机械抗性”前列腺癌 细胞发育并鉴定。具体目标2：开发剪切流装置和培养条件， 研究与基质细胞相互作用调节的剪切应力反应。循环肿瘤细胞聚集体 将表征从前列腺癌患者血液样品中分离的前列腺癌细胞，并用于开发模型 聚合物以供进一步研究。剪切流中异质性肿瘤细胞聚集体的稳定性和存活性 将被研究。具体目标3：检查癌细胞机械敏化作用和 机械抗性对体内转移性肿瘤负荷的影响。原位转移研究，使用具有 将执行调制剪切灵敏度。机械抗性癌细胞与亲本癌细胞将是 在转移的实验小鼠模型中进行比较，并且作为对照研究注射的细胞聚集体的命运。 好.