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: 10458022
• 负责人: Michael R. King
• 金额: $ 36.37万
• 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/10458022
• 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; 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; 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; mechanical signal; mechanical stimulus; 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，但要少得多 已经完成了检查癌细胞在血流环境中的生物学行为的工作。最近， 机械刺激（例如剪切应力）因其对癌症进展的影响而受到关注。为了 实例，研究表明，剪切应力与转移和癌细胞的增强有关 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协同作用 凋亡诱导的配体（TRAIL）诱导的循环肿瘤细胞凋亡（CTC）被证明，如 以及癌细胞在极高的剪切应力脉冲中生存的独特能力，可与血液相当 细胞。这些机械提示可以通过通过 机械转导。提议主体细胞悬浮液，以重复的剪切应力脉冲 板格式研究剪切应力反应并开发“机械抗性”细胞系 以表观和基因型为特征，其目的是识别使癌细胞的驱动因素 在循环中生存。此外，鉴于血液信号中CTC聚集物的存在更多 侵略性和转移性疾病，以分离骨料为单位的多细胞聚集体， 从前列腺癌患者血液样本中的特征将在体外测试其机械反应， 还用来指导模型细胞和球体的开发，以注入实验小鼠 血源转移的模型。这项研究围绕三个特定目的组织：特定目的1： 开发一种新的高吞吐装置，以研究液体剪切应力对癌细胞反应的影响。一个 基于生物释放打印机的多井板配置将通过具有多韦板的能力进行直接分析 流式细胞仪和分光光度计。钙影响，膜和线粒体损伤以及凋亡 响应于剪切应力信号的癌细胞将被检查，“机械抗性”前列腺癌 细胞开发和表征。特定目的2：开发剪切流量和培养条件 研究剪切应力反应通过与基质细胞相互作用调节。循环肿瘤细胞聚集体 将从前列腺癌患者血液样本中分离出来，并用于开发模型 骨料进行进一步研究。剪切流中异质肿瘤细胞聚集物的稳定性和存活 然后将研究。特定目的3：检查癌细胞机理的作用和 体内转移性肿瘤伯嫩的机械固定。原位转移研究使用细胞 将执行调制剪切灵敏度。机械癌细胞与亲本癌细胞将是 在转移的实验小鼠模型中，注射细胞骨料的命运作为 出色地。