Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix

Abstract

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.

FIGURE 1:

Tumorigenic cancer cells exhibit a basal-like phenotype that is fostered by ECM stiffness and EMT. (a) Schematic of the 3D collagen bioreactor. Collagen I hydrogels are made in a media reservoir as described previously (Cassereau et al., 2015), and the application of 10% strain leads to an increase in effective gel stiffness from 0.5 to 2.5 kPa. (b) SEM images of representative 2.5 mg/ml collagen hydrogels in strained and unstrained conditions. Porosity and fiber orientation are unaltered at 10% strain; increased stiffness may reflect fiber bundling. Scale bar, 2 µm. (c) A 3D collagen invasion assay of multicellular spheroids composed of tumorigenic PyMT cells in 2.5 mg/ml 3D collagen gels that are unstrained (soft, 0.5 kPa) or strained (stiffened, 2.5 kPa) for 72 h. Tumorigenic PyMT cells recapitulate invasive behavior when induced to undergo EMT via treatment with TGFβ. Scale bar, 100 µm. (d) Quantification of invasion parameters, total cross-sectional area of spheroid and protrusions, number of protrusions, and number of migrating cells normalized to the unstrained, untreated condition. Mean ± SEM (*p < 0.05; ***p < 0.001; >20 cells). (e) Immunofluorescence imaging of PyMT cell spheroids in 3D collagen gel stained for β-catenin (green) and DNA (DAPI, blue). β-Catenin strongly localizes to cell–cell junctions in cells cultured in soft 3D gels, whereas it is found more diffusely within the cytoplasm and nucleus in strain-stiffened gels and TGFβ-treated cells. (f) PyMT cell spheroids in a 3D collagen bioreactor express basal epithelial marker K14 at the leader cells when cultured in strain-stiffened gels but maintain luminal epithelial K8/18 in the noninvasive core. Induction of EMT via TGFβ treatment also leads to expression of K14 in leader cells. (g) Snail transcription visualized by Snail-YFP is up-regulated in leader cells in strain-stiffened gels and TGFβ-treated cells. Scale bar, 50 µm (e–g), 10 µm (inset).

FIGURE 2:

Cells with a basal phenotype display enhanced migration through microconstrictions. (a) Representative time-lapse image sequences of PyMT cells, PyMT cells treated with TGFβ, and Met1 cells migrating through a 1 × 5 µm² constriction inside a microfluidic device. Cells express NLS-GFP to visualize the nucleus. Time displayed as hours:minutes. Scale bar, 20 µm. (b) Transit times for cell passage through constrictions ≤10 µm² in cross-sectional area, demonstrating that both Met1 cells and PyMT cells treated with TGFβ migrate faster through the small constrictions. Mean ± SEM (*p < 0.05, ***p < 0.001, by Kruskal–Wallis test with Dunn’s multiple comparisons; >70 cells from three independent experiments). (c) Representative images of cells inside the microfluidic devices after 48 h of confined migration. Fixed cells were stained for F-actin (phalloidin) and DNA (Hoechst 33342). Scale bar, 50 µm. (d) Incidence of cells successfully migrating through the first row of constrictions within 36 h relative to the total number of cells inside the microfluidic channels. Error bars represent SE of the binomial distribution (*p < 0.05; ***p <0.001; >500 cells by Fisher’s exact test with Bonferroni correction).

FIGURE 3:

Cells with a basal phenotype are mechanically more compliant. (a) Schematic of AFM used to measure effective cell stiffness. Cells are seeded on a 2.7-kPa PA gel conjugated with fibronectin and indented using a 2.5-µm beaded tip on the AFM cantilever. (b) Hertz model and parameters used to estimate elasticity from a bead contacting a flat plane. (c) Representative example surface plots of elasticity for PyMT, TGFβ-treated PyMT, and Met1 cells, showing elasticity as function of cell topology. (d) Representative force–indentation curves for a given pixel in the surface map, showing the extension and retraction of the cantilever. The Hertz model is fit to these data to extract an effective cell elasticity. (e) Quantification of cell indentation stiffness for tumorigenic PyMT cells, with and without TGFβ treatment, and metastatic Met1 cells. Met1 cells are modestly softer on average than PyMT cells, with TGFβ treatment not eliciting a statistically significant response. Mean ± SEM (*p < 0.05; >15 cells). (f) Micropipette aspiration of PyMT and TGFβ-treated PyMT cells showing the deformation of the nucleus over time. Cell nuclei were visualized by staining DNA with Hoechst 33342. (g) Compiled nuclear protrusion length (ΔL) as a function of time, indicating some nuclear softening in response to TGFβ treatment. Mean ± SEM (>120 cells from three independent experiments). Scale bar, 20 µm.

FIGURE 4:

Cells with a basal phenotype exert higher traction forces. (a) PyMT and Met1 cells were seeded on 2.7-kPa PA gels conjugated with fibronectin and embedded with fluorescent beads for TFM. The displacement fields reflect the relative positions of beads before and after cell lysis. At a substrate stiffness comparable to that of tumor ECM (∼2.7 kPa), Met1 cells are more spread and exert more traction forces on the substrate. Induction of EMT in PyMT cells by treatment with TGFβ recapitulates the increase in traction forces observed with Met1 cells, with total traction force even greater than for the Met1 cells. (b) Quantification of cell traction parameters. Metastatic cells have significantly increased total cellular and maximum traction forces. Mean ± SEM (*p < 0.05; >5 cells). (c) TFM of MCF10ATs. Cells that undergo TGFβ-induced EMT exert higher traction forces than untreated cells. (d) Quantification of cell traction parameters. TGFβ-treated MCF10ATs have significantly higher traction forces. Mean ± SEM (*p < 0.05; >5 cells). Scale bar, 20 µm (a, c).

FIGURE 5:

Motor-clutch model of cellular traction predicts higher force and spread area for cells with a basal phenotype. (a) Motor-clutch model of cell traction, including myosin contractility, actin assembly/treadmilling/disassembly, clutches (linear elastic springs) with constant k_on and variable k_off rates (dependent on clutch strain, x_clutch), and a linear elastic compliant substrate. (b) Parameter descriptions and values used in motor-clutch simulations. (c) Model predictions of traction force calculated from the results of the simulation. Increasing number of motors and clutches leads to an increase in traction force with substrate stiffness and shifts the peak traction to a higher stiffness. (d) Model predictions of cell adhesion area that is extrapolated from the length of assembled filaments, which in turn depends on the number of motors and clutches. (e) TFM of MCF10A cells with and without TGFβ treatment seeded on PA gels of varying stiffness. Cell spread area was quantified from bright-field images, average stress was calculated from the displacement fields, and total force was quantified by multiplying average traction stress by the spread area of the cell (>10 cells). The total force values represent the sum of individual pixel forces underneath the cell body as a whole (average stress times spread area). Scale bar, 20 µm.

FIGURE 6:

Cells with a basal phenotype exert higher integrin-dependent force. (a) Schematic of integrin MTSs. (b) FRET efficiency is converted into force using a calibration curve previously determined (Grashoff et al., 2010) and modified for the fluorescent dyes used here. (c) When seeded on a coverslip densely coated with MTSs, Met1 cells and TGFβ-treated PyMT cells spread more readily and generate higher integrin-mediated forces than untreated PyMT cells. (d) Quantification of the total force and average force per integrin MTS, which are both higher for Met1 than with PyMT cells. TGFβ treatment of PyMT cells resulted in an increase in cell spread area (unpublished data), total integrin-mediated force per cell, and average force per MTS, similar to the values measured for Met1 cells (***p < 0.001; >15 cells by the Wilcoxon rank sum test). Box-and-whisker plots display the median (red), 25th and 75th percentiles (bottom and top edges of the box, respectively), and the most extreme data points not considered outliers (edge of whiskers). (e) Integrin MTS measurements for MCF10A cells treated with TGFβ. (f) There is an increase in total force and average force for the TGFβ-treated MCF10As compared with the untreated ones (***p < 0.001; >15 cells by the Wilcoxon rank sum test). In d and f, the total force values represent the sum of individual pixel forces specifically within segmented adhesions. Scale bar, 10 µm (c, e).

FIGURE 7:

TGFβ-induced EMT alters adhesion organization and stability. (a) Live-cell imaging of paxillin-mEmerald and tensin-mCherry to visualize focal adhesion turnover in MCF10AT cells with and without TGFβ treatment. Top row: control, bottom row: TGFβ-treated. Scale bar, 10 µm (main image), 1 µm (inset). (b) Cumulative distributions of adhesion lifetimes. Inset, quantification of mean adhesion lifetime; mean ± SEM (***p < 0.001; >150 adhesions from five cells total). (c) Focal adhesion microstructure of MCF10AT seeded on MTSs overexpressing paxillin-eGFP and imaged with TIRF and 3B superresolution microscopy. Scale bar, 3 µm (main image), 1 µm (zoom). (d) Quantification of mean adhesion length of MCF10AT cells. TGFβ-treated cells have significantly longer adhesions; mean ± SEM (***p < 0.001; >100 adhesions from five cells). (e) Tensin-positive fibrillar adhesion formation in MCF10AT overexpressing paxillin-mEmerald and tensin-mCherry. Scale bar, 3 µm. (f) Quantification of the spatial colocalization between tensin and paxillin in adhesions. TGFβ-treated cells have significantly more paxillin-tensin colocalization (*p < 0.05; >10 cells). Pearson’s correlation was calculated based on pixels within the focal adhesion areas. (g) Paxillin-mEmerald (N-terminal) axial position in focal adhesions in MCF10As measured with SAIM. Paxillin position is plotted as the relative axial distance from the silicon wafer coated with fibronectin. Scale bar, 5 µm. (h) SAIM axial height measurements of adhesion proteins tagged with fluorescent proteins in MCF10As with and without TGFβ treatment. Paxillin is the only protein to significantly change axial position >10 nm, suggesting that the position of paxillin moves relative to that of the adhesion as a whole. (i) Quantification of paxillin-GFP axial position in MCF10As with and without TGFβ treatment. Paxillin in treated cells is located at a significantly higher axial nanoscale position (*p < 0.05; >10 cells). (j) Representative images of the axial position of paxillin-mEmerald (N-terminal) in focal adhesions in PyMT and Met1 cells. Scale bar, 5 µm.

FIGURE 8:

Proteomics identifies unique proteins recruited to paxillin in the integrin adhesions during EMT. (a) BioID technique. A control BirA*-eGFP construct and a BirA*-paxillin-eGFP construct are stably expressed in MCF10A cells, which are then treated with TGFβ to induce EMT. BirA*-paxillin-GFP localizes to FAs and biotinylates proteins adjacent to paxillin when biotin is added to the medium. Biotinylated proteins are isolated and identified by mass spectrometry. (b) Normalized spectral counts for the consensus adhesome were clustered on the basis of an uncentered Pearson correlation using Cluster 3.0 and visualized using Java TreeView. From a subgroup of known paxillin interactors, we identify GIT2, LIMS1 (PINCH), and vinculin as enriched in TGFβ-treated cells. Black indicates no protein detected in the +TGFβ:–TGFβ scale bar, and gray indicates no enrichment relative to the control. (c) Model for potential paxillin associations altered in response to TGFβ treatment. PINCH, GIT2, and vinculin localize adjacent to paxillin, and these associations are enriched in TGFβ-treated cells. (d–f) Immunofluorescence images of paxillin (green) and ILK (magenta). ILK is enriched in paxillin-rich adhesions in (d) MCF10A cells treated with TGFβ and (e) PyMT cells treated with TGFβ. Scale bar, 10 µm (main image), 5 µm (inset). (f) Immunofluorescence images of paxillin and ILK in PyMT cells on stiff (6 kPa) and soft (0.4 kPa) PA gels coated with fibronectin, showing that stiffness and TGFβ treatment modulate the recruitment of ILK to paxillin-rich plaques. Scale bar, 10 µm (main image), 2 µm (inset).

FIGURE 9:

Enhanced paxillin phosphorylation permits invasion in cells with a basal phenotype. (a) Paxillin localization to the basal surface in MCF10A cells with and without TGFβ treatment visualized with immunofluorescence staining and TIRF imaging of paxillin and phosphorylated paxillin, pPax[31]. Scale bar, 10 µm (main image), 1 µm (inset). (b) Paxillin expression in PyMT, Met1, and PyMT + TGFβ visualized with immunofluorescence staining and confocal imaging of paxillin and phosphorylated paxillin pPax[31]. Scale bar, 10 µm (main image), 1 µm (inset). (c) Paxillin expression of PyMT cell spheroids visualized in soft and stiffened 3D collagen gel. Leader cells in stiffened gel have higher levels of phosphorylated paxillin indicated by pPax[31] immunostaining. Scale bar, 20 µm (main image), 10 µm (inset). (d) A 3D collagen invasion assay of multicellular spheroids composed of Met1 cells overexpressing either paxillin^WT or paxillin^Y31/118F in 2.5 mg/ml 3D collagen gels for 72 h. Scale bar, 100 µm. (e) Quantification of invasion. Paxillin^WT overexpression significantly increased invasion compared with control or paxillin^Y31/118F overexpression, as determined by cross-sectional area of spheroid and protrusions and single-cell invasion (*p < 0.05; >20 spheroids). (f) A 3D collagen invasion assay of multicellular spheroids composed of metastatic Met1 cells treated with dimethyl sulfoxide or 20 nM dasatinib, a Src/Abl inhibitor, in 2.5 mg/ml 3D collagen gels for 72 h. Dasatinib addition significantly decreased cell invasion, as determined by cross-sectional area and single-cell invasion (*p < 0.05; >15 spheroids). Scale bar, 100 µm. Dasatinib addition decreased levels of paxillin phosphorylation visualized by pPax[31] immunofluorescence. Scale bar, 5 µm. (g) Kaplan–Meier survival curve of invasive breast cancer patients expressing either high or low levels of paxillin. High paxillin levels are associated with a significantly worse chance of survival (p < 0.001; hazard ratio 1.46 at 10 yr). (h) Src phosphorylation levels via RPPA in TCGA breast cancer patients separated into control and paxillin–up-regulated groups. Paxillin up-regulation is correlated with a significant increase in Src phosphorylation at tyrosine 416 (*p < 0.05; >4200 patients).

FIGURE 10:

Mechanistic model. (a) ECM stiffness and induction of EMT enhance metastatic behavior, evidenced by increased invasion in 3D collagen matrices, expression of basal epithelial markers and Snail transcription in leader cells, and a higher propensity to migrate through small constrictions. (b) Adoption of a mesenchymal phenotype results in biophysical properties similar to those of basal-like cells, including higher integrin-mediated traction forces and more stable, elongated focal adhesions. (c) Axially scaled model of adhesion architecture changes in basal-like cells (TGFβ treated), showing the average increase in paxillin height of 15 nm. (d) EMT promotes the association of the ILK-PINCH-parvin (IPP) complex, vinculin, and Git2 directly to or in the vicinity of paxillin. Paxillin phosphorylation, mediated through kinases such as Src, regulate adhesion turnover and cell invasion.