The cyto-linker and scaffolding protein ‘’Plectin’’ mis-localization leads to softening of cancer cells

Discovering tools to prevent cancer progression requires understanding the fundamental differences between normal and cancer cells. More than a decade ago, atomic force microscopy (AFM) revealed cancer cells' softer body compared to their healthy counterparts. Here, we investigated the mechanism underlying the softening of cancerous cells in comparison with their healthy counterparts based on AFM high resolution stiffness tomography and 3D confocal microscopy. We showed microtubules (MTs) network in invasive ductal carcinoma cell cytoskeleton is basally located and segmented for around 400 nm from the cell periphery. Additionally, the cytoskeleton scaffolding protein plectin exhibits a mis-localization from the cytoplasm to the surface of cells in the carcinoma which justifies the dissociation of the MT network from the cell's cortex. Furthermore, the assessment of MTs' persistence length using a worm-like-chain (WLC) model in high resolution AFM images showed lower persistence length of the single MTs in ductal carcinoma compared to that in the normal state. Overall, these tuned mechanics support the invasive cells to ascertain more flexibility under compressive forces in small deformations. These data provide new insights into the structural origins of cancer aids in progression.

c) 20 single isolated nuclei from MCF10a and BT20 cells aquired from force-volume maps, using BEC contact mechanics model with fitting range of 1500 nm.Cancer cells' nuclei are significantly softer than their healthy counterparts.d) Statistical analysis was performed using Mann-Whitney U-Test for the nuclei measurements.
Data showed that the difference between MCF10a-nucleus and BT20-nucleus with respect to the dependent variable was statistically significant, U = 216287179.5,***P < 0.001, r = 0.21.Studying cell's cytoskeleton support in small deformations using high-resolution imaging on clusters of cells.High resolution imaging using quantitative nanomechanical mapping-PeakForce Tpping mode (PFT-QNM) revealed the distribution of the well-resisting cytoskeleton components of the cells against the applied maximum force of 1nN (Fig. S6).Fig. S6a-c illustrate the height, PeakForce error, and dissipation images of a single normal mammary cell (MCF10a), respectively.The cell shows packed and high cytoplasmic filaments integrity to support the cell's inner components, specifically in sheltering the nucleus, analogous to the results of the fluorescence microscopy images of these cells.Fig. S6d-f illustrate the height, PeakForce error, and dissipation images of single ductal carcinoma (BT20), respectively.The ductal carcinoma cell contrary to normal mammary cells illustrates a few cytoskeleton filaments in the cell's periphery while they show up the scarce presence of filaments on top of the nucleus and its rim (Fig. S6e).
Stiffness distribution histogram on a carcinoma cell (BT20) acquired from PFT-QNM DMTimages shows a mode value of 2.0 -2.1 kPa with an average value of 13.2 kPa (a mode value of 3.2 -3.3 kPa for 5 cells) and that of a single normal mammary cell was 3.3 -3.4 kPa with an average value of 13.3 kPa (mode value of 6.6 -6.7 kPa for 7 cells), (stiffness distribution histograms and corresponding cells images are provided in supplementary figure S8).Given that the microtubules network is located far lower than the cortex of the cell in cancerous cells (~ 400 nm) shown in the force-volume mapping and fluorescence microscopy experiments, it is justified that in the small indentation range of ~ 300 nm in PFT QNM (supplementary figure S7) the tip does not sense the tubules in the peripheral region of cancer cells.
Accordingly, the ductal carcinomas are measured to be generically softer than the healthy mammary cells.Figure S6g, and S6h show the distribution of the DMT modulus (Derjagin, Muller, Toropov -1975) [1] of the normal mammary cell and the ductal carcinoma, respectively, derived from the locations indicated by the lines on the dissipation images (Fig. S6c, and S6f, accordingly).
The DMT values of cancer cells show high elastic modulus in the center of its nuclear region (Fig. S6h).The validity of this observation is being questioned specifically for the cancerous cell's nucleus domain.This doubt is discussed in the manuscript by the results from cytolinker protein scrutiny.plectin deficient cells appear with a drastic decrease in the elastic moduli compared to the intact MECs (7.07 kPa, 7.56 kPa, 12.04, respectively).This shows either with F-actin collapse or cyto-linker absence the cortex of the cell becomes highly flexible.When microtubule (MT) support is absent beneath the cell cortex (in colchicine-treated cells), the mechanical integrity of the cell cortex is significantly compromised, as F-actin alone cannot adequately maintain it (a decrease from 12.04 kPa to 9.80 kPa).Cancer cells show an overall lower stiffness median value of 8.30 kPa and 9.59 kPa for invasive and non-invasive ductal carcinoma, respectively, compared to that of normal cell with the median value of 12.04 kPa, in the small deformation range that is justified by majorly basally positioning of MTs in invasive cells.For a) and b) data the c) Kruskal-Wallis test revealed a significant difference among the groups.To identify the specific pairs with significant differences, a Dunn-Bonferroni test was conducted, comparing the groups pairwise.Dunn-Bonferroni post-hoc test; ns P = 0.113 and ***P0.001.d) Violin plots of kernel smoothed density estimates of the stiffness of MCF10a, cytoskeleton controls, and BT20 cells aquired from force-volume maps of 20 single cells from each cell lines and controls, using DMT contact mechanics model with a fitting range of 1500 nm (full indentation range) for the full cells body.The control measurements on MEC with disrupted cytoskeleton filaments or silencing plectin proteins in the cells show higher elastic values compared to intact MEC cells which signifies the bottom-effectartifacts.

Fig. S2
Fig. S2 Representative cross-sections from the MTs networks of invasive ductal cells (BT20) and normal mammary epithelial cells (MCF10a).Each graph corresponds to the fluorescence intensity profile along the corresponding line color indicated on Fig. 2c for MCF10a, and Fig. 2g for BT20 cells.

Fig. S3
Fig. S3 Live cell force-volume mapping (trigger point F = 1 nN) combined with fluorescence imaging of MCF10a cell; a) F-actin and b) MTs.Cross-sectional profiles (x-z) of the local elastic modulus as function of the z-position at the location indicated by the blue lines 1 and 2 in MTs image are shown in e), and f), respectively.The red arrow bars in f) are 390 nm.BT20 cell; e) F-actin and f) MTs.Cross-sectional profiles (x-z) of the local elastic modulus as function of the z-position at the location indicated by the blue lines 1, 2, and 3 in the MTs image are shown in g), h), and i), respectively.Images a), b), c), and d) have the same scale bar of 5 µm in xplane.Images e), f), g), h), and i) have the same scale bar of 5 µm in the x-plane and 0.5 µm in the z-plane.

Fig. S4
Fig. S4 Ridgeline plots of kernel smoothed density estimates of the stiffness of MCF10a and BT20 cells aquired from force-volume maps of 10 representative single cells from each cell lines, using BEC contact mechanics model, with a fitting range of 1500 nm (full indentation range) for the full cells body.a) 10 representatives single

Fig. S5
Fig. S5 Violin plots of kernel smoothed density estimates of the stiffness of MCF10a and BT20 cells and their isolated nuclei aquired from force-volume maps of 20 single cells from each cell lines, using BEC contact mechanics model, a) with a fitting range of 1500 nm (full indentation range) for the full cells body.Cancer cells show an overall higher stiffness median value of 14.58 kPa and 14.89 for invasive and non-invasive ductal carcinoma, respectively, compared to that of normal cell with the median value of 13.93 kPa, in the large deformation range.Dunn-Bonferroni post-hoc test; ns P = 0.113 and ***P0.001.A comprehensive Kruskal-Wallis test values are given in figure S13.b) Fitting range of 300 nm for the full cells body.Cancer cells show an overall lower stiffness median value of 9.22 kPa and 8.04 kPa for invasive and non-invasive ductal carcinoma, respectively, compared to that of normal cell with the median value of 11.39 kPa, in the small deformation range.Dunn-Bonferroni post-hoc test; ***P0.001.Invasive ductal carcinomas are around 1 kPa softer than noninvasive ductal carcinomas only in small deformation range.

Fig. S6
Fig. S6 PFT QNM-AFM a) Height, b) PeakForce error, c) dissipation images acquired from a healthy mammary cell (MCF10a), as well as d) height, e) PeakForce error, f) dissipation images acquired from a ductal carcinoma cell (BT20).Images were taken with a setpoint of 0.5-1 nN, with oscillation frequency of 250 Hz and oscillation amplitude of 300 nm.Scan rate was set to 0.1 Hz.The images are cropped out from a larger size image of 91  91 µm 2 with high resolution of around 20 nm per pixel.DMT values distribution of g) healthy mammary cell (MCF10a), and h) ductal carcinoma (BT20) derived from the locations indicated in c, and f, respectively.Dashed vertical lines show the domain of the cells' nuclei each corresponding to the same cross-section color.

Fig. S7
Fig. S7Indentation range in PFT measurements on a) MCF10a, and b) BT20 cells.Yellow and red curves illustrate the trace trajectories, and green and blue curves depict the retrace trajectories.The indentation into both cells in full scans was not more that 300 nm due to PFT-oscillation-amplitude-limit (max.300 nm).

Fig. S8
Fig. S8 PFT QNM-AFM a) Height, b) PeakForce error, c) DMT images acquired from a normal mammary cell (MCF10a), as well as d) height, e) PeakForce error, f) DMT images acquired from a ductal carcinoma cell (BT20).Images were taken with a setpoint of 1 nN, with oscillation frequency of 250 Hz and oscillation amplitude of 300 nm.Scan rate was set to 0.1 Hz. g) Stiffness histogram of MCF10a cells (green) with the mode value of 6.6 -6.7 kPa for 7 cells, and stiffness histogram of MCF10a cells (red) with the mode value of 3.2 -3.3 kPa for 5 cells.

Fig. S9
Fig. S9 3D confocal microscopy of MCF10a and BT20 cells' cytoskeleton.Cells were stained for filamentous actin (CellMask Actin-Tracking) and -tubulin and DNA were counterstained with DAPI.MCF10a; a) -f) Sum slices projection of an image stack consisting of 25 slices each.The squares illustrate the cells selected for assessing their MTs network.g) Integrated intensity of MTs network in z-direction.All MCF10a cells show a homogenous distribution of MTs in the full cell body.BT20; h) -m) Sum slices projection of image stack consisting of 27 slices each.The squares illustrate the selected cells for assessing their MTs network.n) Intensity of MTs network in z-direction.The distribution of MTs throughout the cell body is highly uneven.The cancer cells show higher intensity of MTs in the slices close to the substrate compared to the normal cells, and the curves show a sudden decrease after 8-10 slices (6 -6.5 µm) which is in mid of the cells' volume.This shows a disruption in the MTs network and majorly basal location of the network in cancer cells.This confirms the results from cells tomography.The scale bar is 20 µm.

Fig. S11
Fig. S11 3D confocal microscopy of invasive ductal carcinoma cells (BT20).Cells were stained for filamentous actin (CellMask Actin-Tracking) (in green), -tubulin (in red), plectin proteins (in magenta), and DNA were counterstained with DAPI (in blue).a) -d), f) -i), and k -n) Max intensity projection of an image stack consisting of 18 slices each.The arrows on d), i), and n) illustrate the accumulated plectin protein on top of the cells' nuclei in invasive cells.The green vertical and horizontal lines in e) and o) are the locations at which orthogonal views are derived (Fig. 8, invasive ductal carcinoma).The scale bar is 20 µm.

Fig. S12
Fig. S12 3D confocal microscopy of nonnoninvasive ductal carcinoma cells (BT20).Cells were stained for filamentous actin (CellMask Actin-Tracking) (in green), -tubulin (in red), plectin proteins (in magenta), and DNA were counterstained with DAPI (in blue).a) -d), f) -i), and k -n) Max intensity projection of an image stack consisting of 18 slices each.d), i), and n) illustrate evenly distribution of plectin proteins all over the cells 'body in noninvasive cells.The green vertical line in e) and vertical and horizontal lines in o) are the locations at which orthogonal views are derived (Fig. 8, noninvasive ductal carcinoma).The scale bar is 20 µm.

Fig. S13
Fig. S13 Violin plots of kernel smoothed density estimates of the stiffness of MCF10a, cytoskeleton controls, and BT20 cells aquired from force-volume maps of 20 single cells from each cell lines and controls, using BEC contact mechanics model, a) with a fitting range of 1500 nm (full indentation range) for the full cells body.MEC cells show the softest value among all cases when MTs are disrupted which signifies the major role of MTs in supporting cell's stiffness.b) Fitting range of 300 nm for the full cells body.F-actin disrupted and cyto-linker

Fig. S14
Fig. S14 fluorescence microscopy images of MTs in healthy mammary epithelial cells (MCF10a) and invasive ductal carcinoma cells (BT20).Cells were stained for -tubulin (in red).MTs in MCF10a cells a-b represent approximately a planar/straight conformation, while the tubes in BT20 cells e-h depict compacted and coiled conformation.

Fig. S15
Fig. S15 PFT QNM-AFM a-d) height images of MCF10a cells cytoskeleton in scan size of 10  10 µm 2 with high resolution of 2 nm per pixel.e) force, f) logDMT and g-h) height images of BT20 cells cytoskeleton in scan size of 10  10 µm 2 with high resolution of 2 nm per pixel.White dashed lines in images are representatives of tangent-tangent correlation, the yellow line with the corresponding blue normal lines in images are representatives of beam deflection model.Images were taken with a setpoint of 10 nN, with oscillation frequency of 2 kHz and oscillation amplitude of 100 nm.Scan rate was set to 0.1 Hz.

Fig. S16
Fig. S16 Hight profiles of single MT and MT-bundles derived from location marked with same colored circle in Fig. 9b and 9e PFT QNM images.a) height profile of single MTs, b) MTs bundles in MCF10a cells.c) height profile of single MTs, d) MTs bundles in BT20 cells.Single tubes in both cell lines have full-width-at-halfmaximum of 25-32 nm and MT-bundles have FWHM of 100-120 nm.

Fig. S17
Fig. S17 Actin depolymerization experiments.Cells were loaded with live-staining agents CellMask Actin-Tracking green and Tubulin-Tracker deep red at ratio of 1:1000 in FluoroBrite DMEM for 40 min in 37 °C / 5% CO2 incubator.a) F-actin, b) MTs, and c) merged image of MCF10a cells before drug loading.d) F-actin, e) MTs, and f) merged image of MCF10a cells after cytochalasin-D loading for 40 min.asterisks in d) depict the cells that were measured by AFM.

Fig. S18
Fig. S18 MTs disruption experiments.Cells were loaded with live-staining agents CellMask Actin-Tracking green and Tubulin-Tracker deep red at ratio of 1:1000 in FluoroBrite DMEM for 40 min in 37 °C / 5% CO2 incubator.a) F-actin, b) MTs, and c) merged image of MCF10a cells before drug loading.d) F-actin, e) MTs, and f) merged image of MCF10a cells after colchicine loading for 1.5 h.asterisks in e) depict the cells that were measured by AFM.

Fig. S19
Fig. S19 Plectin knockdown western blot experiments.Based on the results the best timepoints to measure the effect of plectrum was 48 hours post transfection.

Fig. S20 A
Fig. S20 A representative force-indentation curve trace trajectory acquired on MCF10a cell.Each 5 nm intervals of the curve is fit to Herz-Sneddon model for obtaining cell's stiffness tomography.