Microfluidic 3D Assays for Metastatic Cancer

描述（由申请人提供）：从原发肿瘤通过细胞外基质（ECM）迁移，穿过细胞屏障内渗，外渗并在远处重新定殖，这些都是癌症转移的关键步骤。模拟体内肿瘤微环境的生理相关和良好控制的模型将有助于更好地了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，微流控转移试验（uMA），它复制了体内肿瘤微环境的基本组成部分，包括3D ECM和脉管系统，同时提供了严格的生化和生物物理参数控制。拟议工作的目标是扩展我们之前在R21 IMAT计划下的工作，以进一步开发和评估我们的uMA。提出的几个扩展包括：(i)创造一个可控的缺氧环境；（ii）在血管室中引入实际的剪切应力水平；

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 (uMA) 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 uMA. 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 uMA 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 uMA 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 uMA for use a a functional screen for anti-metastatic drugs. These aims will establish the uMA 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 uMA as a development platform, as the system provides a well-characterized EC layer under tightly controlled microenvironmental conditions. Future development will enable the uMA to serve as a cancer cell diagnostic device and a high throughput drug development tool.