Novel device to study tumor metastasis in vitro

描述（由申请人提供）：血管系统和循环通过其的肿瘤细胞之间的相互作用在肿瘤转移中起着核心作用。由于介导循环肿瘤细胞和内皮细胞之间相互作用的分子具有物种特异性，因此使用啮齿动物体内模型研究人类肿瘤细胞的转移可能会产生误导性结果。我们开发了一种新的体外技术，一种三维（3D）流动室装置，它将允许研究人员1）定量测量循环肿瘤细胞外渗级联的每一步; 2）评估局部微环境对循环肿瘤细胞外渗的影响。该提案的目的是完善3D设备原型的设计，目标是制造一次性和负担得起的3D腔室。在具体目标1中，我们将完成3D设备上隔室的设计。在第一组实验中，我们将优化用于生长单层内皮细胞的上部插入物的孔径。最佳孔径将确保转移性与非转移性肿瘤测试细胞的最有效的迁移，所述测试细胞已经循环并随后在剪切应力下粘附到内皮单层。第二组实验被设计为优化每个腔室的威尔斯孔的数量。需要这些实验以确保可以定量测量迁移细胞的数量以产生统计学显著的结果。作为实验模型，我们建议使用人肿瘤细胞系MDA-MB-435和MDA-MB-468。在特定目标2中，我们将测试3D流动室装置是否可用于评估局部微环境对循环肿瘤细胞和内皮单层之间粘附相互作用的影响。本Aim的目标是优化3D器械下隔室的设计。需要调整两个参数以最终确定将用于生长局部微环境的细胞的下部插入物。在第一组实验中，我们将优化内皮细胞和微环境细胞之间的距离。为了实现这一目标，我们将制造和测试具有不同高度边界的下刀片。在第二组实验中，我们将确定下部插入件的最佳孔径。需要进行该试验，因为由于肿瘤细胞迁移的不同条件，上部和下部衬垫的最佳孔径可能彼此不同：上部衬垫的剪切应力与下部衬垫的静态条件。此外，不同类型的贴壁细胞将在上部和下部插入物上培养。我们建议使用人MDA-MB- 435肿瘤细胞系作为模型，因为这些细胞在体内和体外表现出高的粘附内皮细胞的能力。原代人肺源性成纤维细胞将用于模拟局部微环境。本1期申请中提出的研究将允许最终确定3D器械的设计，并提供原理证明证据，证明该器械可充分代表循环细胞与体内血管系统相互作用的条件。拟议的研究将导致开发一种新的商用体外技术，以研究介导人类肿瘤细胞迁移的分子机制，发现新的靶点，并测试靶向肿瘤细胞迁移和转移形成的候选药物。 公共卫生相关性：我们开发了一种新的体外技术-三维流动室设备-填补了肿瘤转移的基础和临床前研究的关键利基。该装置允许定量评估在生理剪切应力条件下人肿瘤细胞外渗级联的每个步骤。拟议的研究一旦成功完成，将产生一种新的商业化体外技术，用于研究肿瘤细胞转移，发现新的靶点，并测试干扰肿瘤细胞转移过程的候选药物。

DESCRIPTION (provided by applicant): The interactions between the vascular system and the tumor cells circulating through it play a central role in tumor metastasis. Because the molecules mediating the interactions between the circulating tumor cells and endothelium are species-specific, the use of in vivo models in rodents to study metastasis of human tumor cells can generate misleading results. We developed a novel in vitro technology, a 3-dimensional (3D) flow chamber device, which will allow researchers 1) to quantitatively measure each step of extravasation cascade of circulating tumor cells; and 2) to assess the effect of the local microenvironment on extravasation of circulating tumor cells. The objective of this proposal is to refine a design of the prototype of the 3D device with the goal to manufacture disposable and affordable 3D chambers. In Specific Aim 1, we will finalize the design of the upper compartment of the 3D device. In the first set of experiments we will optimize the pore size of the upper insert used for growing the monolayer of the endothelial cells. The optimal pore size will assure the most efficient transmigration of metastatic versus non- metastatic tumor test-cells that have circulated and subsequently adhered to the endothelial monolayer under shear stress. The second set of experiments is designed to optimize the number of wells per chamber. These experiments are required to assure that the number of transmigrated cells can be quantitatively measured to generate statistically significant results. As experimental model we propose to use the human tumor cell lines MDA-MB-435 and MDA-MB-468. In Specific Aim 2, we will test whether the 3D flow chamber device can be used to assess the effect of the local microenvironment on the adhesive interactions between circulating tumor cells and the endothelial monolayer. The goal of this Aim is to optimize the design of the lower compartment of the 3D device. Two parameters need to be adjusted to finalize the lower insert that will be used for growing cells of the local microenvironment. In the first set of experiments, we will optimize the distance between the endothelial cells and cells of microenvironment. To achieve this goal, we will manufacture and test the lower inserts with different heights of the enclosure-boundary. In the second set of experiments, we will determine the optimal pore size for the lower insert. This test is required since the optimal pore size for the upper and lower inserts is likely to be distinct from each other due to different conditions for transmigration of the tumor cells: shear stress for the upper inserts versus static conditions for the lower inserts. Furthermore, the different types of adherent cells will be cultured on the upper and lower inserts. We propose to use human MDA-MB- 435 tumor cell line as the model since these cells demonstrated high ability to adhere to endothelial cells in vivo and in vitro. Primary human lung-derived fibroblasts will be used to model local microenvironment. The studies proposed in this Phase 1 application will allow to finalize the design of the 3D device and provide proof-of-principle evidence that this device fairly represents conditions under which the circulating cells interact with vasculature in vivo. The proposed studies will lead to the development of a new commercially available in vitro technology to study molecular mechanisms that mediate migration of human tumor cells, to discover novel targets and to test drug candidates that target tumor cell migration and metastasis formation. PUBLIC HEALTH RELEVANCE: We developed a novel in vitro technology - a 3-dimensional flow chamber device - that fills a critical niche in the basic and preclinical study of tumor metastasis. This device allows to quantitatively evaluate each step of the extravasation cascade of human tumor cells under conditions of physiological shear stress. The proposed studies, when successfully completed, will lead to a new commercially available in vitro technology to study tumor cell metastasis, to discover novel targets and to test drug candidates that interfere with the process of tumor cell metastasis.