Spontaneous migration of cancer cells under conditions of mechanical confinement

Abstract

When cancer cells spread away from the primary tumor, they often follow the trajectories of lymphatic vessels, nerves, white matter tracts, or other heterogeneous structures in tissues. To better understand this form of guided cell migration we designed a series of microfluidic devices that mechanically constrain migrating cancer cells inside microchannels with cross-section comparable to cell size. We observed unexpectedly fast and persistent movement in one direction for several hours of cancer cells of different types. The persistent motility occurs spontaneously, in the absence of external gradients, suggesting the presence of intrinsic mechanisms driving cancer cell motility that are induced in conditions of mechanical confinement. To probe the mechanisms responsible for this behavior, we exposed cancer cells inside channels to drugs targeting the microtubules, and measured a significant reduction in the average migration speed. Surprisingly, a small number of cells appeared not to be affected by the treatment and displayed fast and persistent migration, comparable to the untreated cells. The new matrix-free, 3D-confined motility assay replicates critical interactions that cancer cells would normally have inside tissues, is compatible with high-content, high-throughput analysis of cellular motility at single cell level, and could provide useful insights into the biology of cancer cell migratory phenotype.

Fig. 1. Single cell motility analysis and schematics of the microfluidic device

(a) Cancer cell migration is observed inside channels formed between a PDMS piece and a glass coverslip. (b) Multiple devices are fixed in the wells of a 96-well plate for high-throughput motility screening. One such device is presented in more detail in the inset.

Fig. 2. Persistent migration of cancer cells under mechanical confinement inside the channels

(a) MDA-MB 231 breast cancer cells move persistently away from the seeding chamber. Many cells that reverse their direction maintain the same speed of migration as before. (b) Sequential frames over 4 h show the displacement of two cells, one with amoeboid and one with mesenchymal morphology, inside the same channel.

Fig. 3. Average motility of MDA-MB 231 breast cancer in channels with different cross-section area

(a) Inside 3 μm tall channels (filled circles), the velocity of migration increases with the increasing cross-section of the channels, reaching an optimum at 25 μm width, and then decreases progressively inside wider channels. In 12 μm tall channels (empty squares), migration appears not to be altered by the width of the channel for the range of dimensions tested. While the cells completely fill the smaller channels, they are preferentially distributed towards the sides inside the wider channels. (b) Confocal imaging of MDA-MB 231 loaded with cell tracker dye orange (CMRA, Invitrogen) shows that the majority of moving cells are in contact with all four walls of the channel, and that a smaller number of cells will only touch three or two of the walls.

Fig. 4. Average motility of cells from different cell lines

The migration of seven cell lines was tested in channels with rectangular, 12 ± 15 μm cross-section. Cell lines known to be more aggressive display highest motility. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles.

Fig. 5. The effects of Taxol and nocodazole on MDA-MB 231 breast cancer line

(a) Taxol inhibited persistent motility at concentrations above 1.6 μM and nocodazole at concentrations above 120 nM. Only nocodazole at concentrations above 1.2 μM and Taxol above 16 μM appear to inhibit the migration of the fastest moving cells. (b) Individual tracks of cell displacement in the presence of nocodazole (1.2 μM) and Taxol (16 μM) show slower and less persistent movement of the treated cells that enter the channels.