A 3-D In Vitro Platform of Tumor Metastasis (PQ24)

描述(申请人提供)：当肿瘤获得转移到远处器官的能力时，存活率急剧下降。到目前为止，转移过程的复杂性排除了专门针对转移的批准疗法。随着我们对转移机制理解的提高，新的治疗选择将变得可用。这一应用程序回答了挑衅性的问题#24：鉴于研究转移的困难，我们能否开发新的方法，例如工程组织移植，来研究肿瘤扩散的生物学？肿瘤的血管供应是肿瘤转移的主要途径，因为经过上皮间充质转化(Emt)的可移动的癌细胞可以进入血管，并通过循环输送到远处，如 作为肝脏。肿瘤的血管供应极其渗漏，由弯曲和无组织的血管组成，这些血管对肿瘤微环境有深刻的影响，包括生长因子(如转化生长因子、转化生长因子)的浓度、间质压力和含氧量。目前还没有体外模型可以概括肿瘤微环境的这些显著特征，从而也就是肿瘤转移的早期事件。利用微流体和组织工程技术，我们的研究团队最近开发了一种人体微组织的体外高通量模型(~1mm3)，该模型由活的动态人体微血管灌流。该模型旨在整合肿瘤球体，并创建一种新的肿瘤微环境体外模型，其中包括通过人体微血管流动的基本特征。我们应用程序的中心目标是创建一个高通量的3-D人体肿瘤球体平台，该平台由人体微血管灌流，模拟EMT和血管内注射。我们假设腔内液体剪切力影响血管内和肿瘤细胞进入体循环的效率。这一假说可以通过我们的体外平台独一无二地解决。具体目标集中在平台开发、验证和研究：1)通过将上皮源性肿瘤球体(人结直肠癌)整合到三维人体微组织灌流网络中，建立和优化三维人体肿瘤灌流模型；2)通过刺激(用转化生长因子和/或低氧)和表征肿瘤球体在三维灌流微组织内的EMT和血管内侵入，验证肿瘤转移的体外平台；以及3)验证腔内液体剪切通过影响细胞间连接的完整性，从而影响血管内和退出到“体循环”的效率来影响内皮通透性的假设。完成特定的目标将创建和验证一个新的肿瘤转移体外模型，该模型将显著提高这一过程的时间和空间分辨率。最后，该模型是灵活的，尽管我们的应用程序将重点放在血管内注射上，但该模型 也可以用来检查转移的其他步骤，如外渗和远处的存活。

DESCRIPTION (provided by applicant): Survival rates drop precipitously when a tumor acquires the ability to metastasize to distant organs. The complexity of the metastatic process has, to date, precluded the approval therapeutics that specifically target metastasis. New options for treatment will become available as our mechanistic understanding of metastasis improves. This application responds to the Provocative Question #24: Given the difficulty of studying metastasis, can we develop new approaches, such as engineered tissue grafts, to investigate the biology of tumor spread? The vascular supply to a tumor is a major route for metastasis as motile cancer cells that have undergone epithelial mesenchymal transition (EMT) can intravasate into the vessels and be transported through the circulation to a distant site, such as the liver. The vascular supply to the tumor is extremely leaky and comprised of tortuous and unorganized vessels that have a profound impact on the tumor microenvironment including the concentration of growth factors (e.g., transforming growth factor ¿, TGF¿), interstitial pressure, and oxygen content. There are currently no in vitro models that recapitulate these prominent features of the tumor microenvironment, and thus the early events of metastasis. Employing microfluidic and tissue engineering technologies, our team of investigators has recently developed an in vitro high-throughput model of human microtissues (~ 1 mm3) that is perfused by living dynamic human microvessels. The model is primed to incorporate tumor spheroids and create a novel in vitro model of the tumor microenvironment that includes the essential feature of flow through human microvessels. The central objective of our application is to create a high-throughput platform of 3-D human tumor spheroids perfused by human microvessels that mimics EMT and intravasation. We hypothesize that intraluminal fluid shear stress impacts the efficiency of intravasation and tumor cell exit into the systemic circulation. This hypothesis can be uniquely addressed by our in vitro platform. The specific aims focus on platform development, validation, and investigation: 1) develop and optimize a 3-D model of perfused human tumors by incorporating epithelial- derived tumor spheroids (human colorectal cancer) into a perfused network of 3-D human microtissues; 2) validate an in vitro platform of tumor metastasis by stimulating (with TGF¿, and/or hypoxia) and characterizing EMT and intravasation of human tumor spheroids within 3-D perfused microtissues; and 3) test the hypothesis that intraluminal fluid shear impacts endothelial permeability by affecting intercellula junctional integrity, and thus the efficiency of intravasation and exit into the "systemic" circulation. Completing the specific aims will create and validate a new in vitro model of tumor metastasis that will significantly enhance the temporal and spatial resolution of the process. Finally, the model is flexible and, although our application will focus on intravasation, the model could also be used to examine other steps in metastasis such as extravasation and survival at a distant site.