ARTICLES PUBLISHED ONLINE: 15 JUNE 2014 | DOI: 10.1038/NMAT4009

Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium Ovijit Chaudhuri1,2,3, Sandeep T. Koshy1,2,4, Cristiana Branco da Cunha1,2,5, Jae-Won Shin1,2, Catia S. Verbeke1,2, Kimberly H. Allison6 and David J. Mooney1,2* In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.

T

he microenvironment in tumours often differs from that in normal tissue, exhibiting altered composition and density of ECM proteins, stromal and immune cells, and growth factors. This altered microenvironment has been implicated as contributing to cancer development and progression1 . Indeed, in the context of breast cancer, the tumour microenvironment exhibits higher stiffness2,3 , and the ability of mammography and palpation to identify regions of dense tissue is typically used as an initial screen4 . The connection between altered ECM and tumour progression has been elegantly probed through numerous studies using cultures of non-malignant mammary epithelial cells within a reconstituted basement membrane (rBM) matrix, which has become a standard in vitro model of the mammary epithelium5,6 . Under basal conditions, mammary epithelial cells growing within a three-dimensional (3D) rBM matrix form growth-arrested acinar structures. However, alteration of the ECM composition or cell–ECM interactions can induce changes representative of tumorigenesis, such as invasion of the basement membrane, uncontrolled proliferation, and loss of apicobasal polarity, which are commonly referred to as a malignant phenotype2,5–7 . It has long been hypothesized that increased ECM stiffness leads to such malignant phenotypes. Previous studies have found that a malignant phenotype results when the stiffness of 3D rBM– collagen matrices increases as the concentration of type I collagen added to the matrix is increased2,8 . However, increased binding of type I collagen to integrins is known to activate distinct signalling pathways and alter cell behaviour independently of changes in mechanics9–12 . Indeed, in another approach in which both collagen concentration was held constant and the stiffness of rBM–collagen matrices was increased through enhanced collagen crosslinking, external oncogene activation was found to be necessary for

invasion3 . However, the range of ECM stiffness accessible by this approach is limited (elastic moduli range from 100 to 150 Pa), and the availability and presentation of ligands is altered as collagen is bundled. A third approach has been to encapsulate cells in floating or attached collagen gels, and then examine differences in cell behaviour as the matrices are contracted to different extents by celltraction forces13 . However, the altered mechanics and composition (for example, matrix density) of the cellular microenvironment that result from gel contraction are unknown, and are likely to independently cause differences in behaviour. More generally, type I collagen is not found in the basement membrane of the mammary epithelium, and rBM–collagen matrices may not be physiologically relevant for epithelial cells that have not breached the basement membrane and are unable to interact with the type I collagenrich stroma14 . There is clearly a need for an in vitro model of the mammary epithelium in which matrix stiffness alone, independent of ECM composition and architecture, can be altered. Polyacrylamide gels coated with collagen are typically used to modulate stiffness independent of ligand density for 2D cell culture15–19 . However, a 3D microenvironment has been shown to be necessary for the formation of mammary acinar structures in vitro, and is also probably more relevant to the process of invasion5,6 . Polyethylene glycol20 , alginate21 , collagen–agarose22 and hyaluronic acid23 matrices have previously been used to better control matrix stiffness independent of ligand density in 3D cell culture, but can physically inhibit migration and proliferation. Further, these do not contain the basement membrane components, such as laminin, that are critical for acini formation. Stiffness of the rBM matrix itself has been altered directly by adding transglutaminase to crosslink the matrix24 , but this method may alter matrix pore size25 , the structure

1 School

of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA, 2 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA, 3 Department of Mechanical Engineering, Stanford University, Stanford, California 94305, USA, 4 Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA, 5 Institute of Molecular Pathology and Immunology, Instituto de Engenharia Biomédica, and Faculty of Medicine of the University of Porto, Porto 4150-180, Portugal, 6 Department of Pathology, Stanford University Medical Center, Stanford, California 94305, USA. *e-mail: [email protected] 970

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NATURE MATERIALS DOI: 10.1038/NMAT4009

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Figure 1 | The stiffness of interpenetrating networks of alginate and basement-membrane matrix can be modulated independently of cell-adhesion-ligand density for 3D cell culture. a, Schematic of a cell encapsulated in an interpenetrating network (IPN) of alginate (blue) and reconstituted basement membrane (rBM) matrix (green), and binding of cellular integrin receptors to ligands. Deformation of the rBM matrix due to cellular traction or protrusion forces is restricted by the crosslinked alginate network. b, Schematic of calcium ion crosslinking of the alginate network in the IPNs. Increasing Ca2+ (red) leads to a greater number of crosslinks within G blocks (indicated by the blue ridged region along the alginate polymer). As a result of this zonal crosslinking, gel architecture and pore size are not altered as crosslinking increases. c, Scanning electron micrograph of an IPN. No microscale phase separations are observed. Scale bar, 10 µm. d, Image of fluorescent alginate in IPNs. Scale bar, 50 µm. e, Histogram of fluorescent-alginate intensity per pixel taken from ten images. f, Histogram of fluorescently labelled laminin antibody per pixel taken from 18 images. The presence of alginate and laminin staining in each pixel of e and f demonstrates that these two networks are interpenetrating at this scale. g, Storage modulus at 1 Hz of IPNs with different concentrations of calcium ions. h, Storage modulus at 1 Hz of rBM matrix at different concentrations. Error bars are smaller than data symbols. i, Diffusion coefficient of bovine serum albumin (BSA) as a function of matrix stiffness for IPNs and rBM matrix normalized by the diffusion coefficient in an IPN with a storage modulus of 30 Pa. The differences in the coefficients for diffusion in the rBM matrix are statistically significant (Student’s t-test, ∗ p < 0.05, ∗∗ p < 0.01). j, Time evolution of the fluorescence intensity of laminin-antibody binding to the cross-section of IPNs. All data are shown as mean ± s.d.

of ligand binding sites, and also may nonspecifically crosslink membrane proteins directly to the matrix. Interpenetrating polymer networks are combinations of polymer networks in which one network is formed in the presence of another, and have long been used in industrial and biomedical applications, and for cell culture, to provide an overall material exhibiting a combination of useful biochemical and/or physical properties of the different networks26–32 . Here we introduce the use of interpenetrating networks of alginate and rBM matrix to investigate the effect of matrix stiffness on inducing malignant phenotypes in nonmalignant mammary epithelial cells in 3D culture.

Interpenetrating networks of alginate and rBM matrix Interpenetrating networks of alginate and rBM matrix can be used to modulate ECM stiffness independent of ligand density and polymer concentration (Fig. 1a,b). Alginate is a polysaccharide derived from seaweed, composed of mannuronic and guluronic acid, and is a flexible polymer that presents no intrinsic celladhesion ligands33 . Blocks along the polymer, consisting of sequential guluronic acid residues (‘G blocks’), can be crosslinked by divalent cations such as calcium (Ca2+ ). For rBM matrix, Matrigel—a mixture of ECM proteins including laminin 111 and collagen IV extracted from the EHS mouse tumour—was used. A

mixture of the two components formed interpenetrating networks (IPNs), as no microscale phase separations are observed with scanning electron microscopy, and histograms of pixel-fluorescence intensity from confocal microscopy images revealed single-peak distributions of both alginate and laminin with values above the background throughout (Fig. 1c–e and Supplementary Fig. 1). By modulating the initial concentration of Ca2+ used to crosslink the gels alone, the storage modulus at 1 Hz can be tuned from 30 to 310 Pa while maintaining a constant polymer composition (Fig. 1g and Supplementary Fig. 2). For a Poisson’s ratio of 0.5, this corresponds to Young’s moduli of 90–945 Pa, spanning the range from normal breast tissue to the lower end of malignant-breasttissue stiffness3 . Atomic force microscopy measurements confirmed that this range of Young’s moduli holds for stiff IPNs at the nanoscale (Supplementary Fig. 3). The moduli of matrices formed by different concentrations of rBM also span the same range, and rBM matrices and IPNs exhibit similar frequency-dependent rheology (Fig. 1h and Supplementary Fig. 2). Importantly, we find that the architecture of the IPNs—that is, pore structure and ligand accessibility—is not altered as the stiffness of the IPNs is increased through enhanced calcium crosslinking. Diffusion of bovine serum albumin (BSA, 67 kDa) and dextran (10 kDa or 70 kDa) was not altered as the modulus of the IPNs

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Figure 2 | Enhanced stiffness alone leads to the malignant phenotype in MCF10As. a, Schematic of the experimental design: MCF10A proliferation and development into growth-arrested acinar structures or invasive clusters as a function of IPN stiffness is assessed. b,c, Bright-field images of MCF10A clusters in IPNs at the indicated stiffness at day 11 and day 19, respectively. Cells were seeded in all gels in this study at a density of 25,000 cells ml−1 . d, Confocal immunofluorescence imaging of stainings of the indicated components from cryosections of MCF10A clusters in IPNs with moduli of 30 or 310 Pa at day 19. DAPI staining is in blue. e, Quantification of cluster area and percentage of invasive clusters as a function of day and stiffness. f, Cluster area and percentage of invasive clusters shown as a function of storage modulus at day 19. Both quantities show a statistically significant dependence on storage modulus, with a Spearman’s rank correlation coefficient of 0.8 and 0.9 (∗∗∗∗ p < 10−9 ) for cluster area and invasiveness, respectively. g, Time series of a cluster invading an IPN with a modulus of 310 Pa from day 11 to 13. h, The activity of the PI3K pathway is indicated by a quantification of AkT phosphorylated at Serine 473 in cells within IPNs with moduli of 30 or 310 Pa using an ELISA assay. Values normalized by the value for IPNs with a modulus of 30 Pa (Student’s t test, ∗ p < 0.05). i, mRNA expression of ESR1, the gene for the oestrogen receptor, in IPNs with moduli of 30 or 310 Pa, normalized by the value in IPNs with a modulus of 30 Pa (Student’s t test, ∗ p < 0.05). j, H&E stains from grade-1 ER+ invasive-ductal-carcinoma human-breast-cancer tissue samples. Benign and invasive glands are indicated. All data are shown as mean ± s.d. Scale bars in all images, 50 µm.

was increased (Fig. 1i and Supplementary Fig. 4 and Supplementary Table 1). This indicates that the average pore size is similar for all IPNs. This is expected, as the polymer concentration is constant for all IPNs. Also, it has been previously reported that in alginate networks an increase in Ca2+ crosslinks serves only to further bridge the G blocks already crosslinked and alters neither the pore size of the matrix21 nor the polymer conformation34 , consistent with our findings here. In contrast, increasing the concentration of rBM matrix does reduce diffusion of BSA, as would be expected with a reduced pore size (Fig. 1i). Even with a similar pore structure, it is possible that the presentation of ligand-binding sites could be altered as the stiffness 972

of the IPNs is increased. To exclude this as a possibility, the availability of ligand-binding sites was assessed using laminin-111 as a representative ligand, as laminin-111 is a major component of rBM (ref. 35). Laminin-111-antibody binding to cross-sections of IPNs with different stiffness was measured using quantitative fluorescence microscopy. Both the kinetics and concentration of laminin-111-antibody binding to the IPNs of different stiffness are similar, demonstrating that ligand accessibility is not altered as the stiffness is increased (Fig. 1j). Together, these show that the stiffness of the IPNs can be modulated independent of polymer concentration, cell-adhesion-ligand density and ECM architecture. NATURE MATERIALS | VOL 13 | OCTOBER 2014 | www.nature.com/naturematerials

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NATURE MATERIALS DOI: 10.1038/NMAT4009

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Figure 3 | Altered ECM composition can enhance or completely abrogate the effect of increased stiffness on the phenotype of MCF10As. a, Bright-field images of MCF10A clusters in pure rBM matrix of different stiffness at day 19. Cells were seeded in all gels at a density of 25,000 cells ml−1 . Scale bar, 50 µm. b, Quantification of MCF10A cluster area and % invasiveness as a function of stiffness and day in rBM matrix. c,d, Percentage invasiveness and cluster area, respectively, for MCF10As in IPNs and rBM matrix of different stiffness. Statistically significant differences are noted (Student’s t test, ∗∗ p < 0.01, ∗ p < 0.05). e, MCF10A cluster area and % invasiveness in rBM matrix, IPNs and RGD–IPNs with an RGD concentration of 400 µM, all at 80 Pa. f, MCF10A cluster area and % invasiveness in IPNs with a modulus of 80 Pa, with the indicated concentration of RGD coupled to alginate in the matrix. g, MCF10A cluster area and % invasiveness in rBM matrix, IPNs and RGD–IPNs with an RGD concentration of 400 µM, all at 30 Pa. All data are shown as mean ± s.d.

Increased ECM stiffness leads to malignant phenotype With this new method to tune ECM stiffness independent of composition and architecture, the effect of altered stiffness on the phenotype of the MCF10A non-malignant mammary epithelial cell line was examined. The underlying idea for this approach is that the alginate network restricts deformation of the rBM matrix due to tensional or protrusive forces by cells encapsulated within the IPNs, thereby regulating local ECM stiffness around the cells. When encapsulated within IPNs of the lowest modulus, single MCF10A cells formed organotypic mammary acini—characterized by growth arrest, lumen formation, apicobasal polarization and basement membrane formation, as represented by laminin 332 deposition— as in pure rBM matrix (Fig. 2a–e). These clusters also exhibit a rotational movement that is characteristic of acinar structures36

(Supplementary Movie 1). This demonstrates that alginate in the IPNs does not inhibit acinar formation. As stiffness is increased by enhanced crosslinking of the alginate network, clusters are not growth-arrested, as cluster area increases significantly (Spearman’s rank correlation, p < 10−9 ), apicobasal polarization is altered, and clusters increasingly invade the matrix (Fig. 2b–e, Spearman’s rank correlation, p < 10−9 ). Cell–cell contacts are typically maintained during invasion as β-catenin localizes to cell–cell junctions, and collective cell invasion is observed by timelapse microscopy, although single cells do bud off in some cases (Fig. 2d,g and Supplementary Movies 2–4). These effects are due to the altered stiffness alone, as increasing the concentration of Ca2+ in rBM matrix alone, or in the cell culture media, over the range used to crosslink the IPNs does not inhibit acinar formation

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NATURE MATERIALS DOI: 10.1038/NMAT4009 α6 integrin

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Figure 4 | Mechanotransduction and malignant phenotype of MCF10As in IPNs is mediated through β4 integrin signalling. a, Staining of the different hemidesmosome components in MCF10A clusters at the indicated stiffness. DAPI co-stain is shown in blue. Scale bar, 100 µm. b, Transmission-electron-micrograph images of the cell/ECM interface of MCF10A cells after 19 days of culture in soft or stiff IPNs. Electron-dense regions that exhibit the characteristic features of hemidesmosomes are observed at the membrane/ECM interface as indicated by arrows. 1, Sub-basal dense plate/outer plaque; 2, inner plaque; 3, keratin filament; 4, anchoring filaments in the basement membrane. Scale bar, 500 nm. c, Quantification of the number of hemidesmosomes formed per 4 µm of plasma membrane for MCF10A clusters in the two conditions (Student’s t test, ∗∗∗∗ p < 0.0001). d, Control β4 integrin (β4-GFP; top) or a dominant negative β4-integrin mutant without a cytoplasmic tail (β4-1tail-GFP; bottom) were expressed in MCF10As to assess the role of the cytoplasmic tail of β4 integrin in mediating the malignant phenotype. e, Representative images of MCF10A clusters for cells transduced with β4-GFP and β4-1tail-GFP constructs. Scale bar, 100 µm. Inset image is a magnification of the region in the larger panel indicated by a red box. f, Quantification of cluster area and invasiveness of β4-GFP and β4-1tail-GFP mutants (Student’s t test, ∗ p < 0.05, ∗∗ p < 0.01) in IPNs of 310 Pa. g, Quantification of cluster area of β4-1tail-GFP mutants in IPNs of different stiffness. Data are shown as mean ± s.d.

of MCF10As (Supplementary Fig. 5). In contrast to mammary epithelial cells patterned in 2D culture under compression37 , the invasion observed here is not mediated through ECM deposition, as deposition of fibronectin, type-1 collagen and laminin 332 was not localized to the periphery of clusters, as would be expected if these were necessary for invasion; however, lack of basement membrane deposition may facilitate invasion (Fig. 2d and Supplementary Fig. 6). Furthermore, the activity of the PI3K pathway, which is known to drive proliferation and migration and is often activated in breast cancer38 , is significantly enhanced in stiff IPNs, as demonstrated by increased phosphorylation of AkT, a downstream effector of the PI3K pathway (Fig. 2h). A RT-PCR screen of 67 genes associated with breast cancer revealed significantly altered expression in 14 of these genes for MCF10As in stiff IPNs, relative to these cells in soft IPNs (Supplementary Table 2). Most notably, a fivefold enhancement of expression was measured in ESR1, the gene encoding for oestrogen receptor α, known to play an important role in the initiation and growth of a subclass of breast cancers39 (Fig. 2i). Interestingly, the phenotypes observed here resemble welldifferentiated oestrogen receptor α positive (ER+) invasive ductal carcinomas, as both show invasion while maintaining cell–cell contacts, sustained proliferation and lack of a basement membrane (Fig. 2j and Supplementary Fig. 7). This subtype of breast cancer also typically shows elevated PI3K activity40 . Taken together, these results show that enhanced stiffness alone, given a constant ECM composition and architecture, leads to a phenotype representative of 974

tumorogenesis in non-malignant MCF10A cells (see Supplementary Note 1).

ECM composition mediates the effect of stiffness Next, the effect of altered cell-adhesion-ligand type and density on mediating the response of MCF10A cells to altered ECM stiffness was probed. Interestingly, MCF10A cells formed growth-arrested acini, independent of moduli tested, in ECM composed solely of rBM matrix (Fig. 3a–d). This demonstrates that stiffness alone does not determine the phenotype of these cells. To determine if this result was simply due to the increased cell-adhesion-ligand density presented by the increased rBM matrix concentration generally, or alternatively due to increased concentration of specific components of the rBM matrix, MCF10A cells were encapsulated in IPNs with the RGD cell-adhesion motif coupled to alginate. The RGD motif binds primarily to αv - and β1-containing integrins whereas components of rBM matrix bind to α6β4 integrin, β1containing integrins, and a variety of other non-integrin receptors41 . In this case, the malignant phenotype was again observed for all concentrations of RGD tested, with invasiveness enhanced as the adhesion-ligand density increased at an intermediate stiffness of 80 Pa (Fig. 3e,f). The enhanced malignant phenotype observed in the RGD–IPNs at higher stiffness indicates that the suppression of the malignant phenotype in pure rBM matrix with a high stiffness is probably due to an increase in specific receptor–ligand interactions. Interestingly, at a modulus of 30 Pa, the phenotype NATURE MATERIALS | VOL 13 | OCTOBER 2014 | www.nature.com/naturematerials

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NATURE MATERIALS DOI: 10.1038/NMAT4009

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Figure 5 | Mechanotransduction and malignant phenotype of MCF10As in stiff IPNs is mediated through the PI3K signalling pathway and Rac1 activation. a, Representative images of MCF10A clusters with the indicated inhibitor added to the media. Inhibitors, and the concentrations they were used at, were: 70 µM of NSC23766 to inhibit Rac1, 20 µM of LY294002 to inhibit PI3K, 5 µM for PF573228 to inhibit FAK, 10 µM of PD98059 to inhibit MAPK, and 10 µM of Y27632 to inhibit ROCK. A control sample was treated with the same amount of dimethyl sulphoxide as in the drug-inhibition conditions. Scale bar, 200 µm. b, Area and invasiveness of MCF10A clusters in IPNs with a stiffness of 310 Pa, with inhibitors to the indicated pathways added to the media at day 17 (Student’s t test, ∗∗ p < 0.01). c,d, MCF10A cluster area in IPNs with a modulus of 30 Pa or 310 Pa, with inhibitors to the indicated pathways added to the media. e, Representative images and quantification of cluster area and invasiveness for MCF10As expressing the indicated shRNA in stiff IPNs at 310 Pa (∗ p < 0.05, ∗∗ p < 0.01). Scale bar, 100 µm. f, Representative images and quantifications of cluster area and invasiveness for MCF10As overexpressing constitutively active Rac1 or PI3K, or the control vectors for MCF10As, in rBM matrix at 80 Pa (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.0001). Scale bar, 100 µm. Data are shown as mean ± s.d.

was the same in all IPNs, demonstrating the robustness of the acinar phenotype at low stiffness (Fig. 3g). Laminin-111, a major component of rBM, can bind to α6β4 integrin42–44 . The increase in α6β4 integrin ligands in rBM at high stiffness, but not in the RGD–IPNs at high stiffness, suggests the possibility that an increase in α6β4 integrin binding of laminin-111 may play a role in maintaining a normal phenotype at higher stiffness in pure rBM matrix. More broadly, these findings demonstrate that ECM stiffness acts together with ECM composition to regulate the phenotype of MCF10As.

Mechanotransduction through β4 integrin, PI3K and Rac1 We next sought to elucidate the molecular components mediating the response of the MCF10As to the combination of ECM stiffness and composition presented by IPNs. First, we investigated the role of α6β4 integrin in mediating the MCF10A response to IPN stiffness, as increases in ligands for α6β4 integrin (that is, laminin) were associated with maintenance of the non-malignant phenotype. α6 integrin, β4 integrin, plectin and keratin were all found to localize at the periphery of the cell clusters at lower IPN stiffness, indicating hemidesmosome formation (Fig. 4a).

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NATURE MATERIALS DOI: 10.1038/NMAT4009

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Acini

Figure 6 | Proposed mechanism for the impact of ECM stiffness and composition on malignant phenotype. In ‘normal’ ECM (left panel), fluctuations of α6β4 integrins–laminin units are large enough such that they overlap, and can cluster. Increased ECM stiffness (middle panel) reduces the fluctuations and mobility of bound α6β4 integrins, leading to reduced clustering when laminin density is held constant. α6β4 integrins that are not associated with hemidesmosomes, such that the cytoplasmic tail of β4 integrin is not bound to plectin, have multiple sites that are available for phosphorylation by receptor tyrosine kinases (RTKs). Phosphorylation of distinct sites on the cytoplasmic tail of β4 integrin can lead to activation of the PI3K pathway and Rac1 signalling. However, when an increase in ECM stiffness is accompanied by an increase in laminin density (right panel), clustering is restored because the average distance between the α6β4 integrins–laminin units is smaller, reducing the barrier to clustering.

Hemidesmosomes are adhesive structures, consisting of clustered α6β4 integrins connecting laminin in the basement membrane to the keratin intermediate-filament network within the cell through the cytoplasmic tail of β4 integrin and plectin45 . Hemidesmosomes are critical to normal signalling and acini formation46 . However, at higher stiffness, these components did not localize to the periphery of clusters, suggesting failure of hemidesmosome formation (Fig. 4a). This was confirmed by transmission electron microscopy, which revealed formation of hemidesmosomes for clusters at low stiffness, but the failure of hemidesmosome formation for clusters at higher stiffness (Fig. 4b,c and Supplementary Figs 8,9). Flowcytometry measurements demonstrated that β4-integrin-receptor expression, as well as the expression of other surface receptors relevant to MCF10A–ECM interactions, were similar for MCF10As extracted from IPNs of different stiffness (Supplementary Fig. 10). This indicates that a change in β4-integrin-receptor expression did not underlie the change in hemidesmosome formation as stiffness was increased. Non-hemidesmosomal β4 integrins can play a potent role in carcinogenesis, with phosphorylation of the cytoplasmic tail of β4 integrin by receptor tyrosine kinases leading to PI3K and Rac1 activation and malignant phenotypes45,47,48 . The role of β4 integrin in mediating the malignant phenotype of MCF10As in IPNs was directly tested by expressing a dominant negative mutant form of β4 integrin lacking a cytoplasmic tail (β4 1tail)44 in these cells to reduce the number of signalling-competent α6β4 integrins (Fig. 4d). Cluster area and invasiveness were both drastically diminished in MCF10As expressing the β4 1tail construct relative to a control β4-integrin construct in stiff IPNs (Fig. 4e,f). In addition, the area of clusters formed by the tail-less mutant cells in low stiffness IPNs was similar to that in high stiffness IPNs, indicating that a lack of β4 signalling tail leads to a loss in mechanosensitivity (Fig. 4g). It is not cell growth that is inhibited in these β4 1tail mutants, as 976

the cluster area of the mutants is still greater than that of wild type MCF10A acini in soft IPNs. Together, these findings demonstrate that the cytoplasmic tail of β4 integrin mediates the malignant phenotype and mechanotransduction of MCF10As in the IPNs. With the cytoplasmic tail of β4 integrin having been previously found to be associated with PI3K activation and Rac1 signalling47,49 , we examined the role of these and other signalling pathways in mediating the malignant phenotype in the stiff IPNs. MCF10As in stiff IPNs were treated with a panel of pathway and protein inhibitors (Fig. 5a–d and Supplementary Fig. 11). This screen revealed that the phenotype, and mechanotransduction specifically, was mediated through Rac1 and the PI3K pathway, with PI3K activity already noted to be enhanced by increased stiffness in the IPNs (Fig. 2h). In contrast, a malignant phenotype was still observed when Rho adhesion kinase (ROCK), focal adhesion kinase (FAK) or the MAPK signalling pathway was inhibited. To more rigorously test the involvement of Rac1 and PI3K in mediating the malignant phenotype, shRNAs for Rac1 and P110α, a catalytically active subdomain of PI3K, were expressed in MCF10As to inhibit Rac1 activation and PI3K signalling (Supplementary Fig. 12). A significant decrease in both cluster area and invasiveness were observed in MCF10A mutants encapsulated in stiff IPNs relative to MCF10As expressing scrambled shRNA (Fig. 5c). Although some basal level of both PI3K and Rac1 signalling is thought to be required for survival and basic cell growth6,50 , the finding that the cluster size of MCF10As with decreased Rac1 or PI3K activity (due to inhibition or shRNA) in soft IPNs is on the same order as, or greater than, that of wild type MCF10As that form acinar structures in soft IPNs, indicates that basic cell survival and growth is not being negatively impacted here (Fig. 5c–e). Together, these confirm the role of Rac1 and PI3K in mediating the malignant phenotype. Further, overexpression of constitutively active forms of Rac1 and PI3K in MCF10As led to the malignant phenotype in soft rBM matrices, demonstrating that overactivation of these pathways is sufficient for a malignant phenotype (Fig. 5d and Supplementary Fig. 12). Altogether, these results suggest a mechanism in which the interplay of stiffness and ligand composition and density controls hemidesmosome formation and the malignant phenotype. Under this explanation, an increased ratio of ECM stiffness per bound α6β4 integrin reduces α6β4 integrin clustering and therefore hemidesmosome assembly (Fig. 6). Previous results suggest that signalling from the cytoplasmic tail of β4 integrins that are not associated in hemidesmosomes can lead to an increase in Rac1 and PI3K signalling. Binding of α6β4 integrin to laminin reduces α6β4 integrin mobility and clustering51 . Increased ECM stiffness in turn could further reduce the mobility or lateral fluctuations of α6β4 integrins bound to laminin by providing higher resistance to contractile forces, increasing the barrier for α6β4 integrin clustering into hemidesmosomes. A variety of other factors, including alterations in binding kinetics, stress relaxation in the ECM, active contractile forces and the mechanical remodelling of the matrix and associated feedback between cells and the ECM, may also play important roles, but this is the simplest model that explains the observed results.

Discussion We have demonstrated a new biomaterial system that allows one to alter matrix stiffness independently of ligand density and architecture for 3D cell culture, and have used this system to reveal that ECM stiffness and composition act together in the induction of malignant phenotypes in a normal mammary epithelium. It has been a long-standing challenge to modulate matrix stiffness independently of composition and architecture for 3D cell culture of mammary epithelial cells while capturing their complex biological behaviours. The IPNs described here address this need, enabling the investigation of the role of mechanical cues in other pathological or developmental contexts that involve basement membrane. This NATURE MATERIALS | VOL 13 | OCTOBER 2014 | www.nature.com/naturematerials

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NATURE MATERIALS DOI: 10.1038/NMAT4009 approach can be used directly to address numerous questions about how mechanical cues crosstalk with other biochemical or microenvironmental cues to induce or suppress invasion by the mammary epithelium. The collective cell invasion of the matrix that is observed in the stiffer IPNs could also serve in the future as a model system to study collective cell migration in 3D. Further, the type and density of small adhesion peptides coupled to the alginate can be modulated, and this approach of using alginate-based IPNs to control stiffness is likely to be useful with a variety of ECM molecules and integrin-binding peptides. Our finding that enhanced stiffness alone, given a constant ECM composition, leads to malignant phenotype contrasts with previous work using rBM–collagen matrices, in which it was found that external oncogene activation was necessary for invasion3 . This difference might result from the absence of type I collagen in the IPNs, as type I collagen binding to integrins is associated with activation of distinct signalling pathways and cell morphologies. Indeed, single cells exhibit spread morphologies in matrices consisting of pure type I collagen, but maintain a rounded morphology in rBM and at early timepoints in the IPNs, highlighting the potent impact of type I collagen on cell behaviour52 . Taken together, these indicate that there are multiple mechanisms by which the mammary epithelium senses and responds to ECM cues, and that the mechanism reported here might be more relevant to initial invasion of an intact basement membrane that does not contain type I collagen. Although ECM deposition does not seem to mediate invasion of the matrix, mechanical remodelling of the matrix by cells and mechanical feedback between cell and local ECM may play a role in the development of invasion. The potential importance of ECM cues on initial invasion of the basement membrane is suggested by a recent study in which it was found that single oncogene activation does not lead to invasion of the basement membrane53 . The finding that stiffness and composition act together to regulate malignant phenotypes in the mammary epithelium highlights the importance of considering specific receptor–ligand interactions in in vitro studies of mechanotransduction. Our finding that an increase in basement membrane ligands can abrogate the effect of increased ECM stiffness indicates the possibility that mechanical cues may have completely different effects on cell behaviour, depending on which integrin receptors are engaged. To our knowledge, this work presents the first evidence of β4 integrin playing a key role in mechanotransduction, although this receptor may have played an unrecognized role in previous studies investigating the effect of ECM cues in rBM on different processes. In the context of breast cancer, the similarity of the phenotypes observed here to ER+ invasive ductal carcinoma is compelling, and suggests that mechanical cues may be particularly relevant to the progression of this subtype of breast cancer. More generally, these results indicate that although increased ECM density is considered a key indicator, the relationship between the altered density and composition may be more directly predictive of risk for breast cancer.

Methods IPN matrix formation. All IPNs in this study consisted of a concentration of 4.4 mg ml−1 Matrigel (BD Biosciences) for the rBM matrix component, and 5 mg ml−1 alginate. For each gel, alginate reconstituted at 2.5% in Dulbecco’s modified Eagle’s medium (DMEM) was first delivered into a 1.7 ml centrifuge tube and put on ice. After the alginate had cooled, Matrigel, also on ice, was added to the alginate, and mixed ∼30–60 times with a pipette, being careful not to generate bubbles. As the initial Matrigel concentration varied from batch to batch (7.6 mg ml−1 –11.3 mg ml−1 ), different amounts of serum-free DMEM were added to the mixture, as necessary, to achieve the final concentration of 4.4 mg ml−1 Matrigel in the final IPN. The mixture was kept on ice before IPN formation. Next, a solution containing calcium sulphate, used to crosslink the alginate, was prepared. We note that the use of calcium sulphate as the crosslinking agent is critical to the formation of uniform IPNs because calcium sulphate is partially insoluble and thus the delivery of calcium to crosslink the alginate is not instantaneous. Calcium sulphate was first reconstituted in water at

ARTICLES 1.22 M and then autoclaved. This solution was then diluted tenfold in serum-free DMEM just before the experiment. For each gel, 100 µl of serum-free DMEM containing the appropriate amount of the calcium sulphate slurry was added to a 1 ml Luer lock syringe (Cole-Parmer) and kept on ice. For experiments in which cells were encapsulated in the IPNs, the cells were then trypsinized, washed, filtered through a 40 µm cell strainer, counted, diluted to the appropriate concentration, and then placed on ice. Once all three components had been individually prepared, the gels were formed one by one. First, cells were pipetted into the Matrigel/alginate tube and mixed ∼60 times with a pipette. Then the cell/Matrigel/alginate solution was transferred with a pipette into a cooled 1 ml Luer lock syringe. Bubbles often formed during this step, so excess volume of this solution was always prepared to compensate for the loss of volume when bubbles were purged from the syringe. The syringe with the calcium sulphate solution was then agitated to mix the calcium sulphate uniformly, and then the two syringes were connected together with a female–female Luer lock coupler (ValuePlastics), taking care not to introduce bubbles or air into the mixture. Finally, the two solutions were mixed rapidly together with four pumps of the syringe handles and immediately deposited into a well in a plate that had been pre-coated with Matrigel (to prevent cells sensing and spreading onto a tissue-culture-plastic surface). A transwell insert (Millipore) was placed on top of the gel immediately following deposition in most experiments to prevent the gels from floating, and to form a disk-shaped gel in all cases. The plate was then placed in an incubator, and the IPNs were allowed to gel for 30–50 min before media was added to the wells. rBM matrix formation. rBM matrices with a concentration of 4.4 mg ml−1 were formed exactly the same way as IPNs were formed, except that DMEM was used instead of alginate. For rBM of concentration of 8.0 mg ml−1 or 17 mg ml−1 , cells and DMEM were mixed together in a centrifuge tube and then pipetted directly into the well to form gels. rBMs with a concentration of 17 mg ml−1 were made from a high-concentration matrigel product (BD Biosciences) to achieve high modulus rBM matrices. Mechanical properties of IPNs and pure rBM matrix. The mechanical properties of the IPNs and pure rBM matrix were characterized with an AR-G2 stress-controlled rheometer (TA Instruments). IPNs without cells were formed as described above, except that they were deposited directly onto the surface plate of the rheometer after mixing. A 20 mm plate was immediately brought down before the IPN started to gel, forming a 20 mm disk of IPN. The plate was warmed to 37 ◦ C, and the mechanical properties were then measured over time. The storage modulus at 0.5% strain and at 1 Hz was recorded periodically until the storage modulus reached its equilibrium value (∼20–40 min, Supplementary Fig. 1). Then, a strain sweep was performed to confirm this value was within the linear elastic regime, followed by a frequency sweep (Supplementary Fig. 2). No prestress was applied to the IPNs for these measurements. The storage modulus at 1 Hz of the IPNs was measured to range from 30 to 310 Pa. For Poisson’s ratio of 0.5, this corresponds to Young’s moduli of 90–945 Pa using the equations: √ E = 2G (1 + ν) and G = G02 + G002 where E is Young’s modulus, ν is the Poisson ratio of the material, G is the shear modulus, G0 is the shear storage modulus, and G00 is the shear loss modulus. AFM measurements of Young’s modulus of IPNs were performed with an MFP-3D system (Asylum Research) using silicon nitride cantilevers (MLCT, Bruker AFM Probes). The stiffness was calibrated from the thermal fluctuations of the cantilever in air, and cantilevers with a stiffness of ∼13 pN nm−1 were used. The cantilever was moved towards the stage at a rate of 1 µm s−1 for indentations, and force–indentation curves were fit using the Hertzian model with a pyramid indenter54 .

Received 24 July 2013; accepted 13 May 2014; published online 15 June 2014

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Acknowledgements The authors acknowledge the help of A. Li, D. Klumpers, A. Mao and other members of the Mooney lab. The authors also thank J. Brugge (Harvard Medical School) for providing the β4-integrin and Rac1 mutant plasmids, L. Lichten (Qiagen) for help with RNA arrays, Louise Jawerth/Weitz lab for help/use of the rheometer, M. Ericsson and L. Trakimas of the Harvard Medical School EM facility for help with transmission electron microscopy, P. Mali (Harvard Medical School) for discussions, and the Bauer Core for flow sorting. This work was supported by an NIH F32 grant to O.C. (CA153802), fellowships from NSERC and HHMI for S.T.K., fellowships from FCT, FCG and FLAD for C.B.d.C., and NIH (R01EB015498) and MRSEC (DMR-0820484) grants to D.J.M. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN).

Author contributions O.C. and D.J.M. designed the IPNs. O.C., S.T.K. and D.J.M. designed the experiments. O.C., S.T.K., C.B.d.C., J-W.S. and C.S.V. conducted experiments and analysed data. C.B.d.C. designed and conducted the RNA expression arrays. K.H.A. conducted the comparison of in vitro results to human-breast-cancer samples. O.C., S.T.K., C.B.d.C. and D.J.M. wrote the manuscript.

Additional information Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to D.J.M.

Competing financial interests The authors declare no competing financial interests. NATURE MATERIALS | VOL 13 | OCTOBER 2014 | www.nature.com/naturematerials

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