Supplemental data

Tensional homeostasis and the malignant phenotype Matthew J. Paszek, Nastaran Zahir, Kandice R. Johnson, Johnathon Lakins, Gabriela I. Rozenberg, Amit Gefen, Cynthia A. Reinhart-King, Susan S. Margulies, Micah Dembo, David Boettiger, Daniel A. Hammer, and Valerie M. Weaver Supplemental experimental procedures Elastic moduli measurements The elastic moduli of the polyacrylamide gels and reconstituted extracellular matrices were measured using an RFS II fluids spectrometer (Rheometrics, Inc.). The dynamic shear modulus was first determined using the instrument with parallel plates (25 mm) in oscillatory mode (deformation frequency of 1 radians/second) and variable maximal strain amplitudes from 1 to 100%. The shear storage modulus G’ was determined from the dynamic shear stress in phase with a shear strain of 2% maximum amplitude by standard techniques (Yeung et al., 2004; Georges et al., 2004). G’ was then related to the elastic modulus (E) with the following expression:

)1('2 υ+= GE where υ is the Poisson’s ratio of the material. The elastic moduli of the polyacrylamide gels were additionally measured with the steel ball indentor method (Munevear et al., 2001). A small steel ball (0.025 in diameter, Hoover Precision Instruments) was placed on a polyacrylamide gel in a phosphate buffer filled chamber at 37 oC, and gel deformation due to the weight of the ball was measured using microscopy. The deformation is related to the elastic modulus of the material by the following equation:

E = 3(1 – υ 2)2f 2/4d 3/2r1/2 where f is the buoyancy-corrected weight of the steel ball, d is the depth of the indentation created by the ball, and r is the radius of the ball. Shear force manipulations Shear force manipulations were performed using a spinning disk device (Garcia et al., 1998). For shear rescue and adhesion strength studies, log-phase NIH-3T3 fibroblasts were serum starved overnight and seeded uniformly at 10 cells/mm2 (shear rescue) or 400 cells/mm2 (adhesion strength) on fibronectin coated polyacrylamide gels in Dulbecco’s modified Eagle’s medium (DMEM) plus calf serum (10%). After cell attachment (30 min), gels were attached to the stage of a motorized device designed to spin at a controlled rotational velocity, and the device stage was submerged in a fluid filled chamber containing Dulbecco’s phosphate buffered saline supplemented with 2 mM glucose (see schematic below). Spinning Disk Device Schematic For shear rescue experiments, cells on the gels were spun (5 min; 2000 RPM) and either immediately fixed (2% paraformaldehyde) or first incubated in DMEM plus serum (30 min) and then fixed. Cell morphology and immunofluorescence analysis were performed as described in Experimental Procedures (Indirect Immunofluorescence and Morphometric Analysis) except that cell images and radial position were simultaneously recorded manually or

Gel

Motor

Stage

Fluid filled chamber

Cells

automatically using a mechanical microscope stage, LabVIEW software, and custom programs. Applied shear stress is linearly related to radial position and was calculated using the following equation:

3800.0 ρµωτ r= where τ, r, ρ, µ, ω are the applied shear stress, radial position, density of the buffer, viscosity of the buffer, and rotational velocity of the gel respectively. Using this relation we calculated cell area, adhesion size, and FAKpY397 activity as a function of shear stress. For adhesion strength measurements, cells on the gels were spun (10 min; 5000 RPM), fixed and permeabilized (95% ethanol; 1% Triton X-100; 10 min), and stained with ethidium homodimer (Molecular Probes) nuclear dye. Using a mechanical microscope stage and ImagePro software, nuclei per microscope field (60 fields; 0.5 mm2/field) and radial position were simultaneously measured automatically. To calculate the strength of adhesion (τ50) cell detachment profiles were constructed by plotting the fraction of adherent cells (f) versus applied shear stress (τ). τ50 was calculated by nonlinear fitting (SigmaPlot) of the detachment profiles to sigmoidal curves of the form:

( )[ ]50exp11

ττ −+=

bf

where b is the slope of the inflection point (Garcia et al., 1998). References Garcia, A.J., Huber, F., and Boettiger, D. (1998). Force required to break alpha5beta1 integrin-fibronectin bonds in intact adherent cells is sensitive to integrin activation state. J Biol Chem 273, 10988-10993. Georges, P.C. and Janmey P.A. (2005). Cell type-specific response to growth on soft materials. J Appl Physiol 98, 1547-1553. Munevar, S., Wang, Y., and Dembo, M., (2001). Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts. Biophys J, 80, 1744-1757. Yeung T., Georges, P.C., Flanagan, L.A., Marg, B., Ortiz, M., Funaki, M., Zahir, N., Ming, W., Weaver, V., and Janmey, P.A. (2005). Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 60, 24-34.

Figure S1. Matrix rigidity destabilizes adherens junctions Confocal immunofluorescence images of MEC colonies on soft and stiff BM gels (140 versus 5000 Pa) co-stained for β-catenin (red), E-cadherin (green) and nuclei (blue) before and after triton X-100 extraction. Co-localization of β-catenin and E-cadherin is shown in the far right panels (Merge; yellow). White arrows indicate diffuse staining patterns of β-catenin and E-cadherin. β-catenin could be extracted from sites of cell-cell interaction in MEC colonies formed on a stiff but not on a soft BM gel (compare 140 Pa, Extracted to >5000 Pa, Extracted), indicating that adherens junctions are likely less stable in MEC structures formed on stiff gels.

Figure S2. Effect of polymerized ligand-dependent integrin activation on FAKpY861 and β1 integrin co-localization Confocal immunofluorescence images of MECs in suspension with (bottom panel) and without (top panel) polymerized BM co-stained for FAKpY861 (red), β1 integrin (green) and nuclei (blue). Top panel: White arrow indicates little to no co-localization of FAKpY861 and β1 integrin at the plasma membrane in suspension cultures of cells in the absence of BM ligand (see far right; Merge). Bottom panel: White arrows indicate regions of co-localized FAKpY861 and β1 integrin at the plasma membrane in suspension cultures of cells ligated with polymerized BM (Merge; yellow)

Figure S3. Forced integrin clustering does not affect amount of ligand bound integrin Left: Representative immunoblot of ligand bound β1 integrin (HUTS-21 binding) in MECs in 3D BM. Bottom panel shows the loading control, E-cadherin. Right: Quantification of E-cadherin normalized HUTS-21 immunoblot results shown on the left.

Figure S4. Inhibiting cytoskeletal-tension normalizes cell phenotype Confocal immunofluorescence images of β-catenin (red; top), FAK pY397 (red; bottom) and nuclei (blue) in MEC colonies in a compliant 3D BM for 21 days, expressing active Rho (V14Rho) treated with vehicle or the myosin inhibitor Blebbistatin (V14Rho+Blebbistatin).

Figure S5. Enhanced ERK activity in tumor cells correlates with altered ROCK expression Left: Representative immunoblot of endogenous ROCK, phospho-ERK, and ERK (top, middle and bottom panels, respectively) in S-1 (normal) and T4-2 (malignant) 3D MEC acini. Right: Quantification ROCK (top) normalized to E-cadherin (not shown) and specific ERK activity (bottom – quotient of phospho and total ERK, both normalized to E-cadherin).

Figure 1D enlargement. Confocal IF images of β1 integrin adhesions (green) in MECs in BM/COL I gels of 175 and 1200 Pa for 16 days (3D BM; soft versus stiff), or 70% confluent monolayers on BM-coated polystyrene (2D stiff), costained for FAKpY397, FAKpY861, vinculin (red), or actin (red) and nuclei (blue).

Figure 2D enlargement. Confocal IF images of β1 integrin adhesions (green) in MECs on soft and stiff BM gels (400 versus 5000 Pa), costained for FAKpY397, FAKpY861, talin, vinculin (red), actin (red), and nuclei (blue). Inset: 3× magnification.

Figure 3B enlargement. Confocal IF images of α5(β1) integrin adhesions (green) in fibroblasts on soft and stiff FN gels (400 versus 5000 Pa) costained for FAKpY397, FAKpY861, talin, vinculin (red), actin (red), and nuclei (blue).

Figure 4C enlargement. Confocal IF images showing time course of changes in α5β1 integrin (green), FAKpY397 (red), and nuclei (blue) in fibroblasts on soft FN gels before (Soft; Control) and after (Soft; 5 and 35 min) 140 dynes shear force.