A 3D microfluidic platform for quantitative assessments of tumor cell migration

描述(由申请人提供)：癌细胞的运动性、趋化性及其通过内皮层迁移的能力在癌细胞的转移级联中起着重要作用。肿瘤转移是一个动态而复杂的过程，它涉及癌细胞离开原发肿瘤，进入血液循环，滞留血管，侵袭远处器官，生长新的肿瘤。转移瘤不是原发肿瘤，而是大多数癌症死亡的原因。尽管癌症转移在临床上很重要，但人们对它们的了解仍然很少。目前的基因/蛋白质表达谱研究揭示了许多与癌症转移有关的分子因素。在动物模型中的活体细胞成像首次将癌细胞的转移行为与体内的分子机制直接联系起来，并提供了对癌细胞转移途径的洞察。尽管我们对肿瘤转移过程的了解有了很大的进步，但从这些研究中衍生出来的抑制剂要么缺乏特异性，要么在临床上无效。这在一定程度上是因为我们缺乏对癌细胞永远不会单独行动的理解。它们通过分泌趋化因子、生长因子以及细胞外基质(ECM)的重塑，积极地与微环境相互作用。了解不同细胞类型和细胞外基质之间复杂的相互作用已成为最终了解癌症转移的关键组成部分。我们建议汇集微化学、微机械工程和成像(吴博士)、血管和癌细胞生物学(Swartz博士)和癌细胞生物学(Yen博士)方面的专业知识，共同应对基础癌细胞生物学及其在癌症转移临床化疗中的潜在应用方面的挑战。我们的短期目标是建立一个生理相关(3D)、机械和化学可调的体外模型，并将肿瘤转移步骤中的两个重要步骤--迁移和血管内转移--置于灯光下。我们的长期目标是找到肿瘤细胞微环境中的关键分子，这些微环境是癌细胞转移行为的基础。为了实现这一目标，我们提出了以下具体目标Aim1：开发一种3D高通量、基于水凝胶的微流体体外模型，具有可调节的微化学和微机械环境，用于模拟癌细胞转移中的两个重要步骤--肿瘤细胞在ECM内的迁移和从3D ECM通过血管的血管内转移。目的：开发一种计算算法，结合新开发的4D成像技术，对肿瘤细胞的侵袭性(以细胞运动性、趋化性和细胞移行率为特征)进行自动评分。这一目标对于实现真正的高吞吐量系统至关重要。目的：定量评价多种趋化因子、生长因子、细胞外基质成分以及有无免疫细胞和基质细胞对肿瘤细胞体外侵袭力的影响。 公共卫生相关性：转移是大多数癌症死亡的原因。这项拟议的工作将结合新兴的微流控技术和一种新的4D活细胞跟踪成像技术来研究微环境在癌细胞侵袭中的作用。实验结果将推动基础癌细胞生物学的发展；它还将产生微流控体外工具，将直接应用于高通量癌症抑制物筛选。

DESCRIPTION (provided by applicant): Cancer cell motility, chemotaxis as well as its ability to transmigrate through an endothelium layer play important roles in cancer cells' metastatic cascade. Cancer metastasis is a dynamic and complex process, it involves cancer cells leaving the primary tumor, entering blood circulation, arresting in blood vessel, invading a distant organ, and growing a new tumor. Rather than primary tumors, metastases are responsible for most cancer deaths. Despite their clinical importance, cancer metastases remain poorly understood. Current gene/protein expression profiling work has revealed many molecular factors that are responsible for cancer metastases. Intra-vital cell imaging in animal models has, for the first time, connected the cancer cell metastatic behavior directly with the molecular mechanism in vivo and provided insights into the cancer cell metastatic pathways. Despite of all the advances in our understanding of the cancer metastasis processes, inhibitors derived from these studies have either lacked specificity and/or been ineffective clinically. This is, in part, due to our lack of understanding that cancer cells never act alone. They are actively interacting with the microenvironment via the secretion of chemokines, growth factors, as well as the remodeling of the extracellular matrices (ECM). The understanding of the intricate interactions among different cell types and the extracellular matrices has becoming a critical component towards the eventual understanding of cancer metastases. We propose to bring together expertise on micro-chemical, micro-mechanical engineering and imaging (Dr. Wu), vascular vessel and cancer cell biology (Dr. Swartz) and cancer cell biology (Dr. Yen) to the challenges in both fundamental cancer cell biology and its potential applications in clinical chemotherapy for cancer metastases. Our short term goal is to build a physiologically relevant (3D), mechanically and chemically tunable in vitro model, and to bring the two important steps in cancer metastasis steps, migration and intravasation, under the light. Our long term goal is to find the key molecular players in the tumor cell microenvironment that underlie the cancer cell's metastatic behavior. To achieve this, we propose the following Specific Aims Aim1: To develop a 3D high throughput, hydrogel based, microfluidic in vitro model, with tunable micro- chemical and micro-mechanical environments, for mimicking two important steps in cancer cell metastasis - tumor cell migration within a ECM and intravasation from a 3D ECM through a vascular vessel. Aim2: To develop a computation algorithm, in conjunction with a newly developed 4D imaging technique, to automatically score the tumor cell invasiveness (characterized by cell motility, chemotaxis and cell transmigration rate). This Aim is critical in the realization of a truly high throughput system. Aim3: To assess quantitatively the tumor cell invasiveness in vitro under the influences of various chemokines, growth factors, ECM compositions, as well as the presence/absence of immune cells and stromal cells. PUBLIC HEALTH RELEVANCE: Metastases are responsible for most cancer deaths. The proposed work will use the emerging microfluidic technology in conjunction with a novel 4D alive cell tracking imaging technique to investigate roles of microenvironments in cancer cell invasiveness. Experimental results will advance the basic cancer cell biology; and it will also generate microfluidic in vitro tools that will find direct applications for high throughput cancer inhibitor screening.