Enabling Technology to Study Mechanosensitive and Mechanoresistant Cancer Cells in Flow

肿瘤细胞在固体肿瘤环境中的机械转导是一个活跃的研究领域，但远远不够 人们已经研究了癌细胞在血液流动环境中的生物学行为。最近， 机械刺激，如剪切力，因其对癌症进展的影响而受到关注。为 例如，研究表明，剪切力与增强转移和癌细胞有关 死亡。在申请人的实验室中，剪切力对肿瘤坏死因子相关的协同作用 细胞凋亡诱导配体(TRAIL)诱导循环肿瘤细胞(CTCs)凋亡。 以及癌细胞在极高的剪切力脉冲下存活的独特能力，可与血液相媲美 细胞。这些机械信号可以通过以下过程转化为细胞中的生化反应 机械转导。建议在多井中使细胞悬浮液受到反复的剪应力脉冲 平板格式用于研究剪应力响应并开发将被 表型和基因特征的目标是识别使癌细胞产生的驱动因素 才能在循环中生存。此外，鉴于血液中CTC聚集体的存在发出了更多的信号 侵袭性和转移性疾病，多细胞聚集体模拟分离和 前列腺癌患者的血液样本将在体外进行机械反应测试， 并用于指导实验小鼠注射模型细胞和球体的发育 血行转移的模型。本研究围绕三个具体目标展开：具体目标1： 开发一种新的高通量设备来研究流体剪应力对癌细胞反应的影响。一个 基于BioJet打印机的多孔板配置将实现对多孔板的直接分析 流式细胞仪和分光光度计。钙内流、膜和线粒体损伤及细胞凋亡 将检查癌细胞对剪应力信号的反应，以及“机械阻力”前列腺癌 细胞的发育和特征。具体目标2：研制剪切流装置及培养条件 研究与基质细胞相互作用所调制的剪应力响应。循环中的肿瘤细胞聚集体 将从前列腺癌患者分离的血液样本进行表征，并用于建立模型 供进一步研究的集合体。剪切流中异质肿瘤细胞聚集体的稳定性和存活率 然后才会被研究。具体目标3：研究癌细胞的机械增敏作用和 体内转移性肿瘤负荷的机械抵抗。应用细胞原位转移研究 将执行调制剪切灵敏度。机械抗药性癌细胞与亲本癌细胞将是 在实验小鼠转移模型中进行比较，并将注射的细胞聚集体的命运研究为 井。

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.