Poster H4 - Incorporating Viscosity into In Vitro Metastasis Models

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Primary Investigator(s)

SS

Sarah Shelton

Assistant Professor
North Carolina State University & University of North Carolina at Chapel Hill
Cary, North Carolina, United States

Presenting Author(s)

Karthik Ramakrishnan

Undergraduate Student
Biomedical Engineering/UNC & NC State, United States

Introduction: Cancer is a heterogeneous class of diseases characterized by the uncontrolled growth of cells within the human body. Solid tumors may invade nearby tissues and eventually enter the bloodstream and travel throughout the body. Cancer is divided into four stages that describe the degree to which cancer cells have invaded tissue and spread throughout the body. Stage IV, metastatic disease, is the most invasive stage in which cancer cells have spread from the primary tumor to distant organs in a process known as metastasis. One major way in which cancer cells are able to do this is by transport through the circulatory system. As such, defining how the fluid dynamics of blood flow influence cancer cells is a key component of studying this process.

An essential property of all fluids, including blood, is their viscosity. Viscosity is a measurement of how resistant a fluid is to deformation under stress and for blood, this value is heavily impacted by blood flow velocity, hematocrit (the volume fraction of red blood cells), and the composition of plasma proteins. The goal for this project is to explore the impact of viscosity on the process of metastasis. Currently, most in vitro models neglect viscosity, despite evidence that viscosity is a crucial factor in the metastasis with higher blood viscosity leading to a faster rate of metastasis. Quantitative measurements of the metastatic potential of cell lines in vitro will enable a better understanding of exactly what factors are impacting metastasis, and to what degree.

Materials and

Methods: For this project, we use microfluidic devices to replicate blood vessels in vitro. The devices were designed in Fusion 360 (a Computer-Aided-Design software), milled in polyoxymethylene (Delrin®) plastic, and fabricated from polydimethylsiloxane (PDMS) due to its biocompatibility and ease of manufacturing. These devices have a single channel connected to two fluidic inlets/outlets. Cylindrical channels within an extracellular matrix are formed by first filling the channel with collagen and then applying a small pressure gradient with a low-viscosity fluid. This process, known as viscous channel patterning, creates an open channel with a circular cross section as the collagen polymerizes. We then line the inner surface of the channel with HUVECs in order to replicate a blood vessel. After the endothelial cells form a consistent monolayer within the channel, we then perfuse cancer cells through the device to mimic tumor cell circulation. Rather than perfusing cancer cells in whole blood, we instead generated a blood-analog fluid to replicate average blood viscosity. A solution of high-molecular weight dextran (average molecular weight 1,500,000-2,800,000) was used to increase the viscosity of cell culture medium. The in vitro set-up enables imaging of fluorescently labeled live cells using confocal microscopy to quantify the number of cells adhered during flow and the fraction of adherent cells that transmigrate from the luminal side of the vessel to the basolateral side, also known as the process of extravasation.

Results, Conclusions, and Discussions: While developing the viscous patterning protocol, we tested different volume differentials ranging from 10 to 35 microliters to determine the diameters of the channels that resulted . We found that lower volume differentials led to a larger collagen wall thickness, and determined that the 30-microliter volume differential generated optimal devices. We successfully coated this channel with GFP-HUVEC to replicate a blood vessel, as seen in Figure 1a.

After this, we perfused MDA-MB-231 cancer cells to mimic tumor cell circulation. These breast cancer cells are known as “triple negative” (lacking hormonal receptors for estrogen, progesterone, and HER2), which make many treatments for other breast cancer cell lines ineffective. MDA-MB-231 cells are known for their enhanced metastatic capabilities in comparison to other cell lines, with elevated shear stress (and therefore viscosity) increasing metastasis within these cells. As such, MDA-MB-231 cells make a useful model when studying fluid parameters’ influence on metastasis.

A solution of 2.5% (wt./vol.) high-molecular weight dextran yielded a viscosity of 3.08 cP across shear rates ranging from 10 s-1 to 8940 s-1, measured using a cone-plate rheometer at 37 degrees Celsius, as seen in Figure 1b. This compares well to the average blood viscosity of ~3-4 cP, and will allow us to mimic the average range of shear stress values experienced in capillaries.

In next steps, metastasis will be quantified in the microfluidic devices by counting the number of cancer cells that extravasate (escape the endothelial layer within the device) and migrate into the collagen layer. This will be done over specified time intervals (e.g. 1 hour, 24 hours, 48 hours) in order to determine the relationships between fluid flow and the extravasation step of metastasis. In future experiments, we plan to investigate the role of a glycoprotein known as von Willebrand Factor (vWF) and how shear and viscosity affects its expression as well as the subsequent rate of extravasation and metastasis.

Acknowledgements (Optional):