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. 2012;7(11):e50443.
doi: 10.1371/journal.pone.0050443. Epub 2012 Nov 30.

Attenuation of Cell Mechanosensitivity in Colon Cancer Cells During in Vitro Metastasis

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Free PMC article

Attenuation of Cell Mechanosensitivity in Colon Cancer Cells During in Vitro Metastasis

Xin Tang et al. PLoS One. .
Free PMC article

Abstract

Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks [1]. Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HCT-8 E and R cells and MKF cells cultured on stiffness-gradient PA substrates with stiffness varying continuously from 1 to 20 kPa (left to right).
(a–c) Phase contrast images of the harvested HCT-8 R cells, HCT-8 E cells, and normal MKF cells on the gradient-stiffness PA gel substrates. The respective 3 square panels (enclosed by yellow dash boxes) show the representative magnified views on 1–5 kPa, 8–12 kPa, and 15–20 kPa stiffness domains. The white arrows in magnified views indicate the single, non-contact cells, while the yellow arrows indicate the contacting cells in colonies. Scale bars in magnified view panels are 100 μm. (d) The single cells' projected area of 3 cell types across the stiffness range are shown. Here they do not have any contact with their neighboring cells on different stiffness substrates. (e) The spread area of single cells in contact with neighboring cells on different stiffness substrates. (f) The apparent cell colony area of 3 cell types on different stiffness substrates. (g) The cell shape factor of 3 cell types, which are not in contact with their neighboring cells on different stiffness substrates. (h) The cell shape factor of single cells, which are in contact with neighboring cells on different stiffness substrates.
Figure 2
Figure 2. The coulter counter assay is used to measure specific homotypic cell-cell adhesion rates for HCT-8 E and R cells.
(a) Comparison of cell-cell adhesion rates of original HCT-8 E cells (never exposed to 21 kPa PA gels), disassociated HCT-8 R cells harvested from 21 kPa PA gels, and normal non-cancerous epithelial Ma104 cells. HCT8 R cells have the lowest cell-cell adhesion. Each data point consists of 3 duplicates, and each duplicate consists of 5×105 cells of respective cell types.
Figure 3
Figure 3. Stiffness and morphology of HCT-8 E cells correlate with substrate rigidity.
Using Atomic Force Microscopy, the stiffness of HCT-8 E cells cultured on the gradient substrate is determined. The HCT-8 E cells increase their cell stiffness as the substrates become more rigid. To facilitate the comparison between different cells on same substrate stiffness, five equal-spaced regions across the entire stiffness range are designated: region 1 covers 1–4 kPa, region 2 covers 5–8 kPa, region 3 covers 9–12 kPa, region 4 covers 13–16 kPa, and region 5 covers 17–20 kPa. (a) From region 1 to region 5, the E cell stiffness progressively increases with values 1.42±0.85 kPa to 1.90±0.77 kPa, 2.06±1.39 kPa, 2.15±1.28 kPa, and 3.82±1.98 kPa, respectively. In contrast, on gel substrates with same stiffness gradient, the post-metastatic R cells show almost invariant cell stiffness. (b) Phase-contrast pictures of HCT-8 E cells on gradient PA substrates.
Figure 4
Figure 4. Surface non-specific adhesion of E cell islands measured using a micro-fabricated bio-MEMS force sensor.
(a) The non-functionalized micro-fabricated Si force sensor with a flat probe and with known force-deflection relation is manipulated by a high-resolution x-y-z Piezo-stage to contact cell islands' lateral convex surface (on x-y plane). (b) Confocal microscopy of cell islands show the height of islands is on the order of 30∼50 μm. The vertical height of bio-MEMS probe is 5∼10 μm. (c) After a 2-minute contact, force sensor is horizontally pulled away at a constant speed of 2.1±0.4 μm/s. While the cell adhesion between the probe and cell surface hinders retraction of the sensor, the sensor beams deform by δ, giving the force F. Note that the probe is not functionalized. The 2-minute contact between the probe and cells prevents the activation of cell integrins and the formation of any cell focal adhesion, which takes >30 minutes to form.
Figure 5
Figure 5. Measurement of E cell island intercellular adhesion by determining detachment force using a Bio-MEMS probe.
(a) Intercellular adhesive detachment force of a cell island on the MEMS probe. The force increases monotonically with stretch until detachment. (b–e) Phase contrast images of one typical adhesion experiment. Force is calculated from the deformation of the sensor beam D and the force-deformation calibration curve. The critical detachment force, Fc, is the maximum force on the force-displacement curves. During stretching, the contact angle θ between the probe and the cell island increases, but the contact zone size between the probe and the cell island keeps reducing. Scale bar  = 40 um.
Figure 6
Figure 6. Surface non-specific adhesion of R cells measured by micro-fabricated bio-MEMS force sensor.
(a) Adhesive force of R cells on MEMS probe as the probe is moved away from the cells after 2 min contact (n = 25). (b–e). Phase-contrast images of R cells and MEMS probe when non-specific adhesion between them is measured. The maximum detachment force measured is <2.5 nN, while the cell deformation is barely noticeable. Scale bar: 40 μm.

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