Cancer arises when a group of malfunctioning cells undergoes abnormal growth, invades and destroys adjacent tissue organization, and spreads to other parts of the body via the bloodstream and/or lymphatic system, a process known as metastasis. Despite more than 2 million cancer related papers published to date, cancer malignancy remains a complex disease with low survival rates. Understanding the underlying principles of the cellular and molecular mechanisms of malignancy is crucial in guiding the successful design of anti-cancer drugs and new point-of-care diagnostics.
The process of cancer metastasis consists of a series of highly coordinated, sequential, interrelated steps. Each of these steps can be rate limiting, as a failure of any one of the steps can abort the entire process. The metastatic cascade is initiated when cells from the primary tumor detach and infiltrate the extracellular matrix. A fraction of the invading cells penetrates into the systemic circulation (intravasation). The tumor cells in the blood form emboli with platelets and leukocytes, adhere to the endothelium of certain organs, extravasate and colonize the secondary sites to form site-specific metastases.
Metastasis accounts for the vast majority of cancer-associated mortality. However much remains to be learned about the biology of this process. Many of the remaining questions arise from the fact that metastasis is a "hidden process" which occurs inside the body and is inherently difficult to observe. Despite significant improvements in diagnostic and surgical techniques to combat cancer progression in recent years, our understanding of cancer biology remains incomplete. The laboratory investigation of each step of the metastatic cascade is vital to improve this fundamental understanding. This thesis describes efforts to develop and improve biological assays via the use of microfabrication techniques, which can be used to probe the fundamental biophysical and biomechanical properties of metastasizing cancer cells.
In this work, we first demonstrate a novel method for creating multifunctional surfaces which present proteins (e.g. antibody) in well-defined geometries. The method relies on the serial application of microcontact printing and microfluidics, thus allowing control over the shape, size, and chemical functionality of the discrete functionalized patches. We utilized such surfaces to selectively immobilize and sort cancer cells from a mixture of tumor cells and healthy blood cells. This assay showed potential in the sensing and diagnostics of disease states, and the study of the biophysics of cellular adhesion.
To study the cell-protein or cell-cell adhesive interactions under physiological shear flow conditions, we developed a microfluidics (MF)-based adhesion assay as an alternative to the parallel plate flow chamber. The dimensions of the MF environment are comparable to the intrinsic dimensions of cells and blood vessels, which not only better mimics physiological conditions but also greatly reduces operational reagent volumes. Coupling this MF device with micropatterned adhesive protein (i.e. selectin) surface, we examined the ability of cancer cells to tether and roll on well-defined selectin coated patches using the MF device. We were able to identify the critical size of selectin patches required for initiating cell binding and to extract cell binding kinetic parameters.
We further extended the MF device to study the interaction of dendritic cells (DCs) with selectin-coated surfaces under shear flow. DCs are potent antigen presenting cells, which warrant their potential as candidates for cancer immunotherapy. Characterizing the adhesive molecules expressed by DCs may help to generate DCs with high immunostimulatory capacity that will maximize vaccine delivery. DCs are extremely difficult to isolate in the laboratory, so the small volume requirement of the MF device is especially important in these assays. Using this novel system, we characterized the role of tetrasaccharide carbohydrate determinant, sialyl Lewis X, on DC surfaces binding to selectin molecules.
Lastly, to simulate the physiological microenvironment for cancer cell migration or transmigration, we generated a MF-based cell migration assay containing micropores. In this device we utilized a self-sustaining chemotactic gradient to induce cell locomotion through the well-defined micropores, a process akin to intravasation and extravasation. Dynamic real-time analysis of both population-scale and single-cell movement were achieved at high resolution. Interior surfaces of the device were functionalized through adsorption of extracellular matrix components, and pharmacological agents were administered to cells through the chemotactic reservoir. We demonstrate the capability of direct comparison of migratory potential of multiple cell types in a single enclosed system.