abstract: Breast cancer is the second leading cause of disease related death in women, contributing over 40,000 fatalities annually. The severe impact of breast cancer can be attributed to a poor understanding of the mechanisms underlying cancer metastasis. A primary aspect of cancer metastasis includes the invasion and intravasation that results in cancer cells disseminating from the primary tumor and colonizing distant organs. However, the integrated study of invasion and intravasation has proven to be challenging due to the difficulties in establishing a combined tumor and vascular microenvironments. Compared to traditional in vitro assays, microfluidic models enable spatial organization of 3D cell-laden and/or acellular matrices to better mimic human physiology. Thus, microfluidics can be leveraged to model complex steps of metastasis. The fundamental aim of this thesis was to develop a three-dimensional microfluidic model to study the mechanism through which breast cancer cells invade the surrounding stroma and intravasate into outerlying blood vessels, with a primary focus on evaluating cancer cell motility and vascular function in response to biochemical cues. A novel concentric three-layer microfluidic device was developed, which allowed for simultaneous observation of tumor formation, vascular network maturation, and cancer cell invasion/intravasation. Initially, MDA-MB-231 disseminated from the primary tumor and invaded the acellular collagen present in the adjacent second layer. The presence of an endothelial network in the third layer of the device drastically increased cancer cell invasion. Furthermore, by day 6 of culture, cancer cells could be visually observed intravasating into the vascular network. Additionally, the effect of tumor cells on the formation of the surrounding microvascular network within the vascular layer was evaluated. Results indicated that the presence of the tumor significantly reduced vessel diameter and increased permeability, which correlates with prior in vivo data. The novel three-layer platform mimicked the in vivo spatial organization of the tumor and its surrounding vasculature, which enabled investigations of cell-cell interactions during cancer invasion and intravasation. This approach will provide insight into the cascade of events leading up to intravasation, which could provide a basis for developing more effective therapeutics. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2017

Cigarette smoke's influence on cancer has primarily been a subject of epidemilogic and tumorigenic studies. There have been no proper investigations with interests focused on how cigarette smoke affects the cellular mechanics of metastasis. Gathering an understanding of how smoke influences metastatic invasion could be vital in regulating or possibly eliminatings cancer's ability to initiate new tumor growth sites. This project focuses on cigarette smoke's influence on cellular mechanics of endothelial cells, and the invasive potential of cancer against a fully active endothelium. It is already known that cigarette smoke has a carcinogenic effect, but it is hypothesized that the cigarette smoke causes the endothelium to exhibit pro-invasive characteristics. Cancer cells are often ignorant to extra-cellular stimuli. It is suspected that there will be a less pronounced degradation of cellular mechanics of cancerous cells than endothelial cells when exposed to similar concentrations of cigarette smoke.

The investigations conducted within this dissertation centers around the use of electric cell-substrate impedance sensing (ECIS). This system is able to characterize in real-time analysis, the adhesion of cells to their substrate and neighboring cells. With this, valuable information can be gathered with in-vitro experiments regarding a tissue culture's response to physiological stimulation. This dissertation has taken advantage of ECIS' ability to analyze toxicology, barrier function, and cancer invasion on a tissue culture. With proper analysis modifications, trans-epethelial resistance (TER) can be used as a cytotoxicity assay with higher sensitivity than previously thought. In vitro assessment of cytotoxicity based on TER needs more quantitative methods to analyze the alteration of cell morphology and motility. Here, we applied ECIS to evaluate dose-dependent responses of human umbilical vein endothelial cells (HUVEC) and mouse embryonic fibroblasts (NIH 3T3) exposed to cytochalasin B and protein kinase inhibitor H7. To detect subtle changes in cell morphology, the frequency-dependent impedance data of the cell monolayer were measured and analyzed with a theoretical cell-electrode model. To detect the alternation of cell micromotion in response to cytochalasin B and H7 challenge, time-series impedance fluctuations of cell-covered electrodes were monitored and the values of power spectrum, variance, and variance of the increment were calculated to verify the difference. While a dose-dependent relationship was generally observed from the overall resistance of the cell monolayer, the analysis of frequency-dependent impedance and impedance fluctuations distinguished cytochalasin B levels as low as 0.1µM and H7 levels as low as 10 µM for HUVEC and 3T3 layers. Even though overall resistance values are relatively small for 3T3 layers, and frequency scan measurements are negligible, impedance fluctuation analysis reveals significant micromotion for cytotoxic detection. Our results show that cytochalasin B and H7 causes a decrease of junctional resistance between cells and an increase of membrane capacitance. Cigarette smoke is cytotoxic and tumorigenic. Initial studies were conducted to evaluate the cytotoxicity of cigarette smoke condensate (CSC) on HUVEC layers. The focus was then turned to investigations involving in vitro cancer invasion assays with CSC on HUVEC layers. ECIS is an excellent investigative device that can be utilized to observe cancer invasion on normal tissue cultures due to the significantly higher impedance signature of cancer cells. The investigation in this dissertation focused on cigarette smoke's influence on cellular mechanics of endothelial cells and the invasive potential of two ovarian cancer cell lines (ALST and OVCA429) against a fully active endothelium. The HUVEC cultures responded to CSC with an increase in junctional binding, where as ALST and OVCA429 relieved adhesion thereby providing an improved motility when evaluated in wound healing assays. Transmigration of the HUVEC layer by ALST cells exhibit a pre-CSC exposure time-dependence affecting the effectiveness of ALST transmigration. The HUVEC layer's decreased tight junction binding that resulted from CSC exposure, allowed for a more aggressive ALST layer formation that occurred during simulated intravasation. Increased HUVEC layer tight junction binding that occurred in the first five hours in response to CSC during extravasation contributes to impeding ALST transmigration at high concentrations of CSC. Overall, CSC has an impeding effect on ALST transmigration during extravasation while causing aggressive transmigration during intravasation.