Abstract B11: A microfluidic platform to evaluate soluble signaling in the metastasis microenvironment
Authors: Blaha Laura , Zhang Chentian , Ryu Byungwoo , Alani Rhoda , Cabodi Mario , Wong Joyce 
Published: May 2015

Cancer mortality varies widely across different cancer types, but over 90% of cancer deaths, regardless of tumor origin, are caused by metastasis. Recent work to elucidate the mechanisms of metastasis has focused on the early stages of metastasis, specifically migration within a primary tumor and intravasation into vessels of the circulatory system. What is still relatively unknown is how these circulating cancer cells extravasate from the vessels to form micrometastases in distant organs. Chemokine gradients have long been associated with cell migration in embryogenesis and have also been implicated in promoting migration of cancer cells in vitro. This study was designed to investigate how competing gradients of chemotactic cytokines produced by cells in the metastatic niche communicate with cancer cells trapped in the vasculature to promote extravasation and colonization of new tumors.

A microfluidic channel having five inlets and outlets was fabricated from polydimethylsiloxane (PDMS) bound to glass, and metastatic breast cancer cells (MDA-MB-231-4175 cell line, ATCC) were seeded into the device. Following cell attachment, a syringe pump supplied cells with a constant gradient of either epidermal growth factor (EGF) or stromal cell-derived factor 1 (SDF-1)-two known breast cancer chemoattractants-in serum-free media. Cells in the device were imaged regularly for 8 hours, and the timelapse data was used to quantify velocity, directionality, and chemotactic index of the cells. Following single-chemokine gradient studies, EGF and SDF-1 gradients of equal strength but opposite direction were introduced to the chip simultaneously. Cells in the device were again imaged for 8 hours, and velocity, directionality, and chemotactic index of cells in the competing gradients were quantified.

To study chemokine gradients involved specifically in hematogenous metastasis, an extravasation microenvironment was built inside a microfluidic channel similar to the device previously described. Evenly spaced PDMS posts extending the height of the channel separated the channel into 5 lanes. A collagen gel was polymerized in two lanes to simulate niche extracellular matrix and to provide structural support for endothelial cells grown to confluence in the shape of a vessel. The shape of the EGF diffusion gradient formed across the endothelial monolayer was characterized using fluorescein isothiocyanate (FITC)–dextran as a diffusion marker.

These experiments test the ability of both EGF and SDF-1 to promote directional migration of MDA-MB-231-4175 both individually and when applied in opposition, allowing us to test the hypotheses that chemotaxis of metastatic breast cancer cells can be controlled by chemokine gradients and that cells can sense and respond to multiple gradients simultaneously. In regard to hematogenous metastasis, characterization of an EGF gradient across an endothelial monolayer in both the absence and presence of cancer cells will assess the function of endothelial cell tight junctions in establishing chemokine gradients in the metastasis microenvironment.

Combined, these studies will evaluate the hypotheses that: (1) Metastatic cancer cells trapped in the vasculature must initiate communication by modifying tight junctions between endothelial cells and (2) Once chemokines are free to diffuse into the vessel, extravasation and colonization are influenced by communication via a variety of competing chemokines that the cancer cells can sense and respond to simultaneously. Which chemokines ultimately “win” the competition and succeed in attracting cancer cells to colonize a target organ is the driving question of this research and will be the focus of future work.

Citation Format: Laura Blaha, Chentian Zhang, Byungwoo Ryu, Rhoda Alani, Mario Cabodi, Joyce Wong. A microfluidic platform to evaluate soluble signaling in the metastasis microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B11. doi:10.1158/1538-7445.CHTME14-B11