A 3-D in vitro platform of tumor metastasis (PQ24)

描述（由申请人提供）：当肿瘤获得转移到远处器官的能力时，生存率急剧下降。迄今为止，转移过程的复杂性已经排除了专门靶向转移的批准治疗剂。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。该申请回应了挑衅性问题#24：鉴于研究转移的困难，我们能否开发新的方法，如工程组织移植物，以研究肿瘤扩散的生物学？肿瘤的血管供应是转移的主要途径，因为已经经历上皮间充质转化（EMT）的能动癌细胞可以内渗到血管中并通过循环运输到远处部位，例如肿瘤。 就像肝脏。肿瘤的血管供应是极其渗漏的，并且由曲折和无组织的血管组成，其对肿瘤微环境具有深远的影响，包括生长因子（例如，转化生长因子（TGF）、间质压和氧含量。目前还没有体外模型来概括肿瘤微环境的这些突出特征，从而概括早期转移事件。采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体外高通量的人体微组织模型（~ 1 mm 3），该模型由活的动态人体微血管灌注。该模型是准备纳入肿瘤球状体，并创建一个新的肿瘤微环境，其中包括通过人体微血管流动的基本特征的体外模型。我们的应用程序的中心目标是创建一个高通量的3-D人类肿瘤球体灌注的人类微血管，模仿EMT和内渗平台。我们假设腔内流体切应力影响内渗和肿瘤细胞进入体循环的效率。这一假设可以通过我们的体外平台得到独特的解决。具体目标集中在平台的开发、验证和调查：1）通过整合上皮来源的肿瘤球体，开发和优化灌注的人类肿瘤的3-D模型（人结直肠癌）到3-D人微组织的灌注网络中; 2）通过刺激肿瘤转移的体外平台来验证肿瘤转移的体外平台。（与TGF β和/或缺氧）和表征EMT和3-D灌注微组织内的人肿瘤球体的内渗;和3）检验管腔内流体剪切力通过影响细胞间连接完整性而影响内皮渗透性的假设，从而提高了内渗和进入“体”循环的效率。完成特定目标将创建并验证一种新的肿瘤转移体外模型，该模型将显著提高该过程的时间和空间分辨率。最后，该模型是灵活的，虽然我们的应用将集中在内渗， 也可用于检查转移中的其他步骤，例如外渗和远端部位的存活。 公共卫生相关性：我们的应用程序的中心目标是创建一个高通量的3-D人肿瘤球体平台灌注的人微血管，模仿转移的早期事件，包括上皮间质转化（EMT）和内渗。灵活的平台将为研究转移的潜在机制提供新的机会，从而提供新的治疗选择。

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. PUBLIC HEALTH RELEVANCE: 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 the early events of metastasis including epithelial mesenchymal transition (EMT) and intravasation. The flexible platform will provide new opportunities to study underlying mechanisms of metastasis, and thus new therapeutic options.