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. 2020 Apr 10;9(4):935.
doi: 10.3390/cells9040935.

Single-Cell Probe Force Studies to Identify Sox2 Overexpression-Promoted Cell Adhesion in MCF7 Breast Cancer Cells

Jagoba Iturri  1 , Andreas Weber  1 , María dM Vivanco  2 , José L Toca-Herrera  1
Affiliations

Single-Cell Probe Force Studies to Identify Sox2 Overexpression-Promoted Cell Adhesion in MCF7 Breast Cancer Cells

Jagoba Iturri et al. Cells. .

Abstract

The replacement of the cantilever tip by a living cell in Atomic Force Microscopy (AFM) experiments permits the direct quantification of cell-substrate and cell-cell adhesion forces. This single-cell probe force measurement technique, when complemented by microscopy, allows controlled manipulation of the cell with defined location at the area of interest. In this work, a setup based on two glass half-slides, a non-fouling one with bacterial S-layer protein SbpA from L. sphaericus CMM 2177 and the second with a fibronectin layer, has been employed to measure the adhesion of MCF7 breast cancer cells to fibronectin films (using SbpA as control) and to other cells (symmetric vs. asymmetric systems). The measurements aimed to characterize and compare the adhesion capacities of parental cells and cells overexpressing the embryonic transcription factor Sox2, which have a higher capacity for invasion and are more resistant to endocrine therapy in vivo. Together with the use of fluorescence techniques (epifluorescence, Total Internal Fluorescence Microscopy (TIRF)), the visualization of vinculin and actin distribution in cells in contact with fibronectin surfaces is enabled, facilitating the monitoring and quantification of the formation of adhesion complexes. These findings demonstrate the strength of this combined approach to assess and compare the adhesion properties of cell lines and to illustrate the heterogeneity of adhesive strength found in breast cancer cells.

Keywords: AFM; MCF7 cells; Sox2 overexpression; TIRF; cell adhesion; epifluorescence; single-cell probe.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(a) Schematic drawing of a cell positioned onto a poly-L-lysine (PLL)-fibronectin functionalized cantilever, and a description of a usual Force experiment. The black arrows point at different adhesion events as the cell is detached from the surface. The inset on the right-hand side (b) shows a real retraction force–distance curve derived from such an experiment, following the conditions described at the materials and methods section after 90 s of contact. The analyzed parameters are highlighted graphically: the maximum adhesive force represents the minimum in the force curve, the work of adhesion the area under the curve, and the steps the gradual zero-force recovery pattern.
Figure 2
Figure 2
Cell–substrate interactions. (a) The resulting force–distance plots for the two cell types when placed in contact with the corresponding substrate (SbpA vs. fibronectin). (b) The column plots obtained for adhesion force (in nN) and the work of adhesion (in J, 1E-15) for the respective cases under study.
Figure 3
Figure 3
Contact time dependence on the tether formation for cells in contact with fibronectin (20 µg/mL) surfaces. (a) Representative tether recording at three different contact times for MCF7 cells, and (b) the corresponding magnified view from the area highlighted by a dashed rectangle. The plateau length and rupture force factors are indicated in the plot. The vertical and tilted black lines highlight the position of single-step and intermediate rupture events, respectively. (c) Rupture force distributions. The horizontal line represents the median and the values range from the 5th to the 95th quantile. (d) Mean rupture force calculation for both control (MCF7), and Sox2 overexpressing cells. The significance of the variations in the p < 0.05 and p < 0.005 level is indicated by * and *** accordingly.
Figure 4
Figure 4
Influence of soluble fibronectin. (a) Representative force–distance plots for Sox2 cells interacting with Fibronectin substrates in L15 medium either with or without fibronectin addition, and increasing contact times (30, 60 and 120 s). (b) Calculated values of maximum adhesion force and adhesion work for both MCF7 and Sox2 cells before (filled columns, values from Figure 2) and after (empty columns) the injection of soluble fibronectin.
Figure 5
Figure 5
Contact time-dependent cell–cell interactions. (a) Contact time-dependent averaged force–distance plots for symmetric (MCF7–MCF7 and Sox2–Sox2), and the asymmetric (MCF7–Sox2) interactions for cells on top of fibronectin. (b) Adhesion force and work of adhesion values for the three systems under analysis.
Figure 6
Figure 6
(a–f) Epifluorescence images of both MCF7 (a–c) and Sox2 (d–f) cell lines on top of Fibronectin (20 µg/mL) film upon actin (in green) and vinculin (in red) staining. The scale bars correspond to 50 µm. (g) Boxplot showing the distribution of the cell area values. N = 50. The square (□) shows the mean value, the horizontal line represents the median, and the values range from the 5th to the 95th quantile. (h) Magnification over the cell–cell connection in Sox2 cells, showing the localized agglomeration of vinculin along the contact line.
Figure 7
Figure 7
Total Internal Fluorescence Microscopy (TIRF) images of MCF7 and Sox2-overexpressing cells for individual actin (in red) and vinculin (in green) channels, and the merged image. An evanescent field of 90 nm was used while keeping the illumination time constant. The bottom image shows a wide (220 nm) field applied on the same area of interest.
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