978-1-4244-6714-3/10/$26.00 ©2010 IEEE Abstract—Metastasis is the process by which cancer spreads to form secondary tumors at downstream locations throughout the body. This uncontrolled spreading is the leading cause of death in patients with epithelial cancers and is the main reason that suppressing and targeting cancer has proven to be so challenging. Tumor cell extravasation is one of the key steps in cancer’s progression towards a metastatic state. This occurs when circulating tumor cells found within the blood stream are able to transmigrate through the endothelium lining and basement membrane of the vasculature to form metastatic tumors at secondary sites within the body. Predicting the likelihood of this occurrence in patients, or being able to determine specific markers involved in this process could lead to preventative measures targeting these types of cancer; moreover, this may lead to the discovery of novel anti-metastatic drugs. We have developed a microfluidic device that has shown the extravasation of fluorescently labeled tumor cells across an endothelial cell lined membrane coated with matrigel followed by the formation of colonies. This device provides the advantages of combining a controlled environment, mimicking that found within the body, with real-time monitoring capabilities allowing for the study of these biomarkers and cellular interactions along with other potential mechanisms involved in the process of extravasation. I. INTRODUCTION UMOR cell extravasation is the last step before circulating tumor cells (CTCs) invade downstream locations and initiate the formation of metastatic tumors. Previous steps that cancer must first progress through leading up to this include tumor growth along with the recruitment of blood supply by neoangiogenesis, tumor cell migration from the primary tumor into the blood stream or lymphatics (intravasation), and the survival and transport of viable cells within the vasculature to secondary sites of invasion [1]. This cascade of events including extravasation is collectively defined as cancer metastasis which terminates with the formation of metastatic masses at target organs or tissues within the body. Benjamin E. Reese is a student in the Department of Mechanical, Aerospace, and Biomedical Engineering at the University of Tennessee, Knoxville, TN. Siyang Zheng is with the Department of Bioengineering at Pennsylvania State University. Boyd Evans is with Monolithic Systems Development in the Measurement Science & Systems Engineering Division at Oak Ridge National Laboratory. Ram H. Datar, Thomas Thundat, and Henry K. Lin are affiliated with the Nanoscale Science & Devices department of the Biosciences Division at Oak Ridge National Laboratory. It has been established that only a small population of CTCs within the blood stream undergo extravasation making this key step in metastasis of great clinical importance to understanding the underlying factors contributing to this process [2]. Recently, many groups have made progress towards developing assays allowing for the capture and study of CTCs [3-5]. A modified Boyden chamber/Transwell assay can be employed for the study of tumor cell invasiveness by seeding the cells of interest on one side of a chamber while a chemoattractant is introduced across the interlaying membrane favoring transmigration [6]. While this device is able to diagnose tumor cell invasiveness, it is unable to distinguish the underlying interactions occurring between adhesion molecules influenced by the biophysical factors such as shear stress and fluid flow that could also be contributing to transmigration [7,8]. It is therefore critical to further explore a system capable of controlling the numerous parameters associated with these dynamic interactions. In this study, we have developed a microfluidic device mimicking the endothelial cell and basement membrane lining of the vasculature coupled with real-time monitoring capabilities and have demonstrated its ability to reproduce tumor cell extravasation in an in-vitro model system. II. MATERIALS AND METHODS A. Fabrication of microfluidic device The design of this microfluidic device is made up of three layers as shown in figure 1. The first and third layers are constructed using the polymer PDMS (polydimethyl siloxane) that was chosen for both its biocompatible and optical properties [9,10]. The top layer forms the main channel through which the tumor cells will flow mimicking the blood. The size of blood vessels are known to range from 25mm down to capillaries as small as 8 µm in diameter. Following these guidelines to model capillaries, the top channel ranges in diameter as it tapers from 150 µm on one end to 25 µm on the other end, and has a common height of 150 µm across the entire 5 mm length of the channel. The bottom layer is similar in its tapered design as the bottom channel ranges in diameter from 300 µm on one end to 50 µm on the other end and maintains a height of 150 µm along the 12 mm length of the channel. The middle layer is an etched parylene membrane consisting of pores 9 µm in diameter to mimic the supporting structures found within the vasculature. These pores will allow enough space for the adherent tumor cells to pass through into the bottom channel. The PDMS channels for both the upper and lower layers were fabricated using replicate molding on a master created by patterning. The master was prepared by applying SU8 2100 photoresist onto a silicon wafer and patterned using standard Microfluidic Device for Studying Tumor Cell Extravasation in Cancer Metastasis Benjamin E. Reese 1 , Siyang Zheng 2 , Boyd Evans 3 , Ram H. Datar 4 , Thomas Thundat 4 , Henry K. Lin 4,* T