Microfluidic 3D Assays for Metastatic Cancer

描述（由申请人提供）：从原发性肿瘤迁移并通过细胞外基质（ECM）、穿过细胞屏障的内渗以及远端部位的外渗和再浸润包括癌症转移的关键步骤。模拟体内肿瘤微环境的生理学相关和良好控制的模型将能够更好地理解转移的步骤和评估潜在的治疗效果。体内模型具有生理相关性，但本质上缺乏高水平的控制。体外癌症模型提供了控制，但缺乏肿瘤微环境的关键成分。我们提出了一种新技术，即微流控转移测定（μMA），它复制了体内肿瘤微环境的基本组成部分，包括3D ECM和血管系统，同时提供了对生化和生物物理参数的严格控制。 本论文的目标是扩展我们在R21 IMAT项目下的工作，以进一步开发和评估我们的μMA。提出了几种扩展，包括：（i）创建受控的低氧环境，（ii）在血管隔室中引入真实水平的剪切应力， (iii)使用肿瘤球状体模拟EMT，和（iv）扩大目前使用的ECM材料的范围（目标1）。另一个新的方向是开发一种类似的测定方法来研究外渗和药物化（目的2）。最后，为了促进其他研究人员使用μMA并进行高通量研究，该平台被复用，并开发了塑料制造方法（目标3）。如所开发的，μMA具有单独可寻址的通信区域，用于癌细胞和其他肿瘤相关细胞接种在3D胶原凝胶中，以及用于内皮细胞（EC），其排列在第二通道中以模拟脉管系统。该配置允许癌细胞从凝胶内的肿瘤球状体向EC内衬通道迁移。EC层模拟体内血管屏障，允许观察癌细胞的内渗。类似的装置允许接种在通道中的癌细胞穿过EC层外渗到ECM中。良好的光学通路将允许对癌细胞迁移、内渗和外渗进行真实的实时观察。结合图像处理技术的光学访问将量化癌细胞的形态和迁移参数，从而识别新的入侵指标，量化癌细胞的转移潜力。最后，我们将利用μMA的功能筛选抗转移药物。 这些目标将使μMA成为定量研究癌细胞转移生物学机制的有用模型。解决转移过程多个步骤的治疗显然将受益于使用μMA作为开发平台，因为该系统在严格控制的微环境条件下提供了良好表征的EC层。未来的发展将使μMA成为癌细胞诊断设备和高通量药物开发工具。

DESCRIPTION (provided by applicant): Migration from the primary tumor and through extra-cellular matrix (ECM), intravasation across a cellular barrier, and extravasation and recolonization in a remote site comprise the critical steps of cancer metastasis. Physiologically relevant and well-controlled models that mimic the in vivo tumor microenvironment will enable better understanding of the steps of metastasis and evaluation of potential therapy efficacy. In vivo models have physiological relevancy, yet inherently lack a high level of control. In vitro cancer models provide control, yet lack critical components of the tumor microenvironment. We propose a new technology, a microfluidic metastasis assay (μMA) that replicates essential components of the in vivo tumor microenvironment, including a 3D ECM and vasculature, while providing tight control of biochemical and biophysical parameters. The objective of the proposed work is to extend our previous work under the R21 IMAT program to further develop and evaluate our μMA. Several extensions are proposed including: (i) creating a controlled hypoxic environment, (ii) introducing realistic levels of shear stress in the vascular compartment, (iii) use of tumor spheroids to simulate EMT, and (iv) expanding the range of ECM materials currently being used (Aim 1). Another novel direction is to develop a similar assay to investigate extravasation and recolonization (Aim 2). Finally, to promote use of the μMA by other researchers and for high throughput studies, the platform is multiplexed and methods are developed for manufacturing in plastic (Aim 3). As developed, the μMA has separately addressable communicating regions for cancer cells and other tumor-associated cells seeded in a 3D collagen gel, and for endothelial cells (EC) that line a second channel to simulate the vasculature. The configuration permits migration of cancer cells from tumor spheroids within the gel toward the EC-lined channel. The EC layer mimics the in vivo vascular barrier allowing observation of cancer cell intravasation. A similar device allows cancer cells seeded in the channel to extravasate across an EC layer into ECM. Excellent optical access will permit real time observation of cancer cell migration, intravasation and extravasation. The optical access combined with image processing techniques will quantify cancer cell morphological and migratory parameters, leading to identification of novel invasion metrics that will quantify the metastatic potential of cancer cells. Finally, we will leverage the capability of the μMA for use a a functional screen for anti-metastatic drugs. These aims will establish the μMA as a useful model for quantitative research of biological mechanisms governing cancer cell metastasis. Therapies that address multiple steps of the metastatic process would clearly benefit from using the μMA as a development platform, as the system provides a well-characterized EC layer under tightly controlled microenvironmental conditions. Future development will enable the μMA to serve as a cancer cell diagnostic device and a high throughput drug development tool.