277317 A Microfluidic Device to Measure Traction Forces During Confined Cancer Cell Migration towards Chemoattractant
Cell migration represents a key facet of cancer metastasis, since cells depend on motility to navigate through interstitial tissues, intravasate to the circulatory or lymph systems, and seed new metastatic foci. Cell motility mechanisms are influenced by the physical and chemical cues of the microenvironment. Mechanical forces are constantly being exerted by migrating cells on the surrounding cells as well as the extracellular matrix. Simultaneously, forces are being continuously exerted on the cells themselves from the extracellular environment. To fully comprehend the combinatorial effects of mechanical forces and chemotaxis, we engineered a novel two-layer microfluidic-based migration device to measure traction forces exerted by cells migrating in microchannels in response to a chemotactic stimulus.
The fully formed migration device consists of confining microchannels with a “floor” consisting of deflectable micropillars. Cells are induced to migrate through these channels by a chemotactic stimulus. Molds for the top and bottom layers are fabricated using multi-step photolithography. The device layers themselves are formed by replica molding with PDMS. The top layer of the device is composed of parallel microchannels (separated by 50 µm) of height 4 µm and length 200 µm. Channels vary in width from 10 to 50 µm. The microchannels are aligned in a ladder-like configuration orthogonal to and connecting two larger main channels which serve as a cell seeding source and a chemokine reservoir, respectively. The bottom portion of the microfluidic migration chamber consists of micropillars (diameter - 3 µm; height - 9 µm; center-to-center spacing - 6 µm) that are located within channels of different widths, mirroring the top portion of the device. Using a micromanipulator and a stereoscopic microscope, the top portion of the microfluidic device is aligned precisely over the bottom portion, thereby defining the channels.
To demonstrate the general utility of our migration device for traction force measurement, we performed experiments with metastatic human breast adenocarcinoma cells (MDA-MB-231). MDA-MB-231 cells were seeded and allowed to migrate within the microchannels over micropillars coated with type I collagen towards the chemoattractrant. Once the cells were on top of the micropillars, time lapse video microscopy was performed to record the real-time pillar deflections due to cellular traction forces. The pillar deflections were converted into forces by multiplying by the spring constant of the micropillar, which was calculated using beam bending theory. Our preliminary data revealed that colchicine treatment leading to the disruption of microtubules results in higher pillar deflections relative to untreated controls for cells migrating through a 50 µm-wide channel. This increase can be explained by an increase in traction that can be attributed to the loss of compression-supporting capacity of microtubules, resulting in a shift of load and energy from the microtubules to the substrate. We wish to further characterize the cell traction forces in response to increasing confinement, manifested in our design by decreasing channel width. Thus, our novel microfluidic design, which incorporates micropillars within microchannels to quantify cellular tractions forces during directed migration under confinement, is a powerful tool for the study of cellular chemotaxis and is suitable for a wide range of biological and biomedical applications.
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