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Development of 3D Microfluidic Gradient Model for Metastasis
Tumor Cell Biology I
Background : Recent revolution in microfabrication technology has enabled the fabrication of devices in which structures of micron dimensions can be produced. Previous works by the PI have successfully addressed the development of microfabricated devices capable of generating well-defined chemical gradients on micron scales in 2D. This proposal will develop and test a novel design that will enable stable chemoattractant gradient in 3D gels. Metastatic cancer cells will be embedded and allowed to migrate in 3D microenvironment, better simulating the complex tissue microenvironment than ECM coated hard substrates with 2D gradients. Objective/Hypothesis: Complex interactions between tumor cells and its environment (extracellular matrix and soluble factors) that result in cell migration play an important role in cancer metastasis. By exposing the tumor cells (MDA MB-231) to different chemoattractants (chemokines CXCL12 and growth factors EGF), we will gain better understanding on the influence of the chemicals on the migratory behavior of cancer cells in3D environment. We will compare the results of migration in 3D gels with results from 2D migration. This understanding may lead to new means of therapeutic intervention that target protease and/or integrins. Specific Aims: To design, fabricate, and characterize microfluidics-based chemotaxis chamber that can actively control spatial and temporal gradients of chemoattractants in 3D collagen gel. To perform quantitative measurements of breast cancer cell migration in various gradients of chemokines and growth factors. Study Design: Based on our extensive experience on generating 2D gradients, we will design and fabricate a microfluidic device capable of generating gradients in 3D gel. Metastatic breast cancer cells (MDA MB-231) will be embedded in (or allowed to invade) 3D collagen gel and exposed to precise, well-defined concentration gradient of chemokines and growth factors. The chemokine gradients will be generated using a custom microfludics device fabricated by soft lithography and microfabrication. First, the effects of different chemokine and growth factor gradients (CXCL12 and EGF) on migration of MDA MB-231 will be systematically quantified for different conditions. These results will then be compared to migration on 2D substrates.
Metastasis, spreading of tumor cells into other parts of the body, is the main cause of treatment failure in cancer but its process is poorly understood. Detailed understanding of the steps in metastasis such as cell invasion and migration could lead to therapies that block the successful establishment of secondary tumors. Currently, molecular level investigation of cell migration is limited by the availability of techniques that can provide information about how cancer cells navigate and migrate in complex 3 dimensional tissues. Conventional assays such as Boyden chamber and Matrigel invasion assays can not produce stable, well-defined chemoattractant gradients that are responsible for directed migration and spreading of cancer cells to other organs by metastasis. In addition to being a simple end-point assay that is not compatible with advanced imaging techniques, these assays use cells migrating on artificial 2D substrates that poorly resemble the in-vivo environment. Efforts to investigate the roles of ECM composition and property as well as different soluble factor gradients on cancer cell invasion would benefit from a technique that could generate and maintain gradients of biologically active materials. Although cancer cells’ native environment in tissue is composed of complex 3D architecture of ECM proteins and other stromal and immune cells, there is no existing method to generate stable gradients in 3D tissues or gels. Building on our pioneering work on microfluidic devices for 2D soluble gradients, the new device proposed in this work will provide stable gradient in 3D collagen gels by taking advantage of the microfabrication and soft lithography processes. These techniques have been developed in the microelectronics industry for manufacturing integrated circuits (i.e. Intel Pentium Microchips) and can produce fluidic “chips” with extremely small channels. This microfluidic device will be able to deliver and maintain precise gradients of soluble factors (chemicals that attract and guide their migration) to the cancer cells embedded in 3D collagen gel. This experimental platform will closely mimic the tumor microenvironment and allow real-time observation of the migrating cells. The ability to expose cells to spatial gradients of chemokines, growth factors and other biologically important compounds while in 3D environment will be broadly useful in cancer cell migration research. The use of microfluidic device will help provide better understanding of how cells navigate in complex 3D environments and will find applications in development and testing of new drugs for breast cancer metastasis.