TY - JOUR
AU - Suggs, Laura, J
AB - Abstract Soft tissue tumors, including breast cancer, become stiffer throughout disease progression. This increase in stiffness has been shown to correlate to malignant phenotype and epithelial-to-mesenchymal transition (EMT) in vitro. Unlike current models, utilizing static increases in matrix stiffness, our group has previously created a system that allows for dynamic stiffening of an alginate–matrigel composite hydrogel to mirror the native dynamic process. Here, we utilize this system to evaluate the role of matrix stiffness on EMT and metastasis both in vitro and in vivo. Epithelial cells were seen to lose normal morphology and become protrusive and migratory after stiffening. This shift corresponded to a loss of epithelial markers and gain of mesenchymal markers in both the cell clusters and migrated cells. Furthermore, stiffening in a murine model reduced tumor burden and increased migratory behavior prior to tumor formation. Inhibition of FAK and PI3K in vitro abrogated the morphologic and migratory transformation of epithelial cell clusters. This work demonstrates the key role extracellular matrix stiffening has in tumor progression through integrin signaling and, in particular, its ability to drive EMT-related changes and metastasis. tumor microenvironment, extracellular matrix, migration, mechanotransduction, matrix stiffening, epithelial-to-mesenchymal transition Insight box/paragraph Extracellular matrix stiffness plays a key role in tumor progression; however, its role in the invasive transformation of cancer cells is still unclear. Here, we utilize a dynamic 3D hydrogel capable of dynamic stiffening to interrogate the role of matrix stiffening on the invasive transformation of mammary epithelial cells. We demonstrate that dynamic stiffening induces an invasive and migratory transformation in epithelial cells, both in vitro and in vivo, and that it is conducted utilizing mechanical signaling pathways. Through analyzing the early steps of the metastatic cascade with a dynamic hydrogel model of ECM stiffening, we establish the role of matrix stiffening on this stage of tumor progression. INTRODUCTION Cancer, and specifically breast cancer, is the second leading cause of death in the USA and has been one of the most prevalent pathologies of the previous few years [1]. An increase in local extracellular matrix (ECM) stiffness is a hallmark characteristic of soft tissue tumors, including breast cancer, where ECM stiffness is used to detect tumor formation via palpation [2]. In fact, current preclinical treatments for breast cancer act by targeting this increase in matrix stiffness with the most prominent being lysyl oxidase (LOX) inhibitors that act in part by reducing LOX-dependent collagen crosslinking and downregulating focal adhesion kinase (FAK) [3–5]. Research groups have elucidated the link between increased matrix stiffness and tumor progression and have found that increased collagen density, alignment and matrix stiffness can regulate an invasive phenotype in mammary epithelial cells [6, 7]. Further models of the ECM stiffening in cancer have also corroborated the link between the modulus of the underlying substrate and tumorigenic shift [8–12]. However, many of these studies have been conducted in hydrogel models with static mechanical properties, which do not accurately model the dynamic tumor formation process [13]. A dynamically stiffening culture system is therefore needed to investigate the role of physiologically relevant increases in matrix stiffness on tumor progression. Previous studies to examine the role of static increases of matrix modulus on cell morphology have used alginate–matrigel interpenetrating networks (IPNs), as alginate is a bio-inert and increasing alginate cross-linking increases overall modulus without altering the availability of adhesive ligands [14]. This property of alginate–matrigel IPNs makes them an ideal candidate as a base system to investigate the role of ECM modulus on cell phenotype. Our group has developed an ECM model using an alginate–matrigel IPN loaded with liposomes, which contain gold nanorods (AuNR) and soluble calcium [15]. Upon near-infrared irradiation, the soluble calcium is released, cross-linking the alginate and dynamically increasing matrix modulus. This model has been used to investigate the development of an invasive phenotype in normal MCF10A mammary epithelial cells following an increase in matrix stiffness after irradiation [16]. While the development and progression of primary tumors is extensively studied, it is thought that up to 90% of cancer deaths result from metastasis [17]. Additionally, in breast cancer, the extent of tumor cell invasion in the breast dictates the surgical decision-making as lumps are excised until a clear margin exists [18, 19]. Thus, it is crucial to not only interrogate the ability of a primary tumor to metastasize, but invade the surrounding tissue as well. Epithelial-to-mesenchymal transition (EMT) has been identified as a process critical to the metastatic cascade in which cells from the primary tumor detach, migrate in to the vasculature and extravasate to a secondary site [20]. EMT is a phenotypic transformation by which cells lose epithelial traits, such as strong cell–cell adhesions and gain mesenchymal traits, including increased migratory capacity and strong cell–ECM adhesions [21]. In cancer, migration due to EMT is critical early in the metastatic process as cells must break strong epithelial cell–cell adhesions, form new cell–ECM adhesions and then proceed to migrate away from the primary tumor [22]. The molecular mechanism through which EMT acts is complex, and a variety of signaling cascades must converge, though transforming growth factor-β (TGF-β) has been found to be a key regulator [21, 23]. Additionally, previous work has shown increased matrix stiffness to promote many transcription factors most notably Yap/Taz nuclear localization associated with EMT potentially linking cell mechanosensing with metastasis [9, 24]. Here, we utilized a dynamic stiffening hydrogel to investigate how increases in matrix stiffness contribute to EMT both in vitro and in vivo. To our knowledge, this is the first time dynamic stiffening has been used to probe EMT-driven migratory transformation. The invasive and migratory transformation of epithelial cells was demonstrated through higher number of cells migrating both from the initial hydrogel in vitro and injection site in vivo. Furthermore, it is shown that FAK and PI3K signaling are required for this process, cementing the role of integrin signaling in tumor progression and their role as targets of anti-metastatic therapy. EXPERIMENTAL Cell culture Epithelial Py2T cells were derived from murine MMTV-PyMT tumors and serve as a model of TGF-β-induced EMT; they were provided generously by Dr. Gerhard Christofori [25]. Py2T cells were cultured in 4.5 mg/ml glucose DMEM (Caisson Labs) supplemented with 10% fetal bovine serum (Thermo Scientific) and 1% penicillin-streptomycin (Life Technologies). Py2T cells were transformed to a mesenchymal Py2T-LT phenotype through supplementing culture media with 2 ng/ml TGF-β (R&D Systems). TGF-β supplement was maintained in cultures containing Py2T-LT cells. NMuMG cells were cultured in the same media and conditions with 10 μg/ml supplement of insulin. Hydrogel formulation and temporal stiffening The alginate–matrigel utilized here has been described in detail in previous publications [15, 16]. Briefly, Pronova UP MVG sodium alginate (FMC Biopolymer) was dissolved in nanopure water at 4% w/v. Crosslinked alginate gels were generated utilizing insoluble CaCO3 and glucono-δ-lactone (GDL), which hydrolyzes upon dissolution in water to release calcium ions [26]. Gels were formulated at 1.6% w/v alginate with 5 mM CaCO3 and 10 mM GDL, liposomes were included at 20% gel volume and growth factor reduced Matrigel (Corning) was included at 25% gel volume. Gels were mixed thoroughly with cells and pipetted in tissue-culture-treated well plates and allowed to gel for 60 minutes at 37°C. Culture media were then carefully pipetted over the gels and changed every other day. Gels were initially formed at 150 Pa and stiffened through irradiation to 1200 Pa. Temporal stiffening was accomplished through incorporation of CaCl2-loaded liposomes within the gel as described previously [15]. Briefly, 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Avanti Polar Lipids) liposomes containing gold nanorods (AuNR, NanoHybrids) and CaCl2 were synthesized through interdigitation fusion [27]. Irradiation of the AuNR induces surface plasmon resonance and local heating. This heating induces a phase transition at 41°C in the lipid bilayer, allowing calcium ions to permeate the bilayer and crosslink the gels [28, 29]. Irradiation was conducted 3 days after gelation with a 2 W 808-nm diode laser (Lasermate Group). Brightfield and confocal microscopy Brightfield images were acquired utilizing a Leica EC3 camera on a Leica DM IRB microscope. Quantification of cell cluster size and circularity were conducted through tracing and utilizing built-in ImageJ measurements. A minimum of 25 clusters were measured per experimental condition. Confocal microscopy images were acquired utilizing a Fluoview FV10i (Olympus) with either a 10× or a 60× objective. Images of each fluorescent channel were acquired separately and merged utilizing ImageJ. Immunocytochemistry Anti-E-cadherin (Cell Signaling Technology, 3195S), anti-N-cadherin (Novus Biologicals, NBP1–51612) and YapTaz (Cell Signaling Technology, 8418S) were purchased. Gels were fixed with 4% paraformaldehyde for 60 minutes on day 14. To fully allow antibody permeation, gels were broken through gentle pipetting. Gels were incubated in blocking solution of 10% goat serum and 0.1% Triton-X-100 in DPBS. Primary antibodies were diluted 1:100 in 10% goat serum and 0.1% Triton-X-100 in DPBS and incubated overnight at 4°C. Following this, gels were washed in DPBS with 10% and 0.1% Triton-X-100 once. The secondary antibody was diluted 1:1000 in 10% goat serum and 0.1% Triton-X-100 in DPBS and incubated for 90 minutes. DAPI was added at 5 μg/ml and incubated for 5 minutes. Gels were washed three times in DPBS and pipetted on to glass slides, coverslipped and sealed. Mean fluorescence intensity of E-cadherin staining was quantified over a maximum z-projection of a three-dimensional cell cluster. RNA extraction and RT-qPCR To differentially isolate RNA from cells contained within the gel and at the bottom of the well plate, the gels were chemically dissolved in a mixture of 4 U/ml dispase (Sigma-Aldrich) and 0.5% sodium citrate (Sigma). Media were gently aspirated from the top of the gel, and the dispase–citrate buffer was added; after 5 minutes of gentile agitation, the partially dissolved gel was pipetted in to a separate tube and rotated at 37°C to fully dissolve the gel. After this, the solution was spun at 500 × g for 5 minutes to pellet cells and Trizol (Life Technologies) was added to lyse the cells. Cells that remained at the bottom of the well plate were washed three times with DPBS and Trizol was added to lyse the cells. RNA isolation was conducted utilizing the RNeasy Mini Kit (Qiagen) following the manufacturer’s specifications. GAPDH, CDH1, CDH2, VIM, SNAI1, TWIST, ZEB1 and ZEB2 for RT-qPCR were obtained from RealTimePrimers (RealTimePrimers.com). RT-qPCR was conducted on a Viia7 system utilizing Sybr Green (Fisher Scientific). In vivo tumor model All gel components were mixed as described previously and placed in a 1-ml insulin syringe quickly and on ice to prevent gelation. The gel mixture was injected orthotopically in the fourth inguinal mammary fat pad of matched female FBV/N mice aged 3–6 months. To incorporate temporal stiffening, 3 days postinjection, mice were anesthetized and hair around the gel site was removed with Nair. After hair removal, the gel site was irradiated with a 2 W 808-nm diode laser. Tumor size was measured with calipers and recorded daily and mice were sacrificed when tumor volume exceeded 750 mm3. To evaluate downstream metastatic burden, axillary lymph nodes were extracted and frozen in Tissue Tek OCT compound (Fisher Scientific). To evaluate the early timepoint migration, a subset of mice were injected with cells labeled with CM-DiI (Thermo Fisher) according to manufacturer’s specifications. Mice were sacrificed 2 days after irradiation and the fourth inguinal fat pad was extracted and frozen in Tissue Tek OCT compound (Fisher Scientific) in an isopropanol-dry ice bath. Blocks were stored at −80°C until sectioning. Histology and image quantification Frozen tissue blocks were sectioned on a CryoStar NX50 cryostat (Thermo Fisher). About, 8-μm sections were taken at a depth greater than 50 μm from injected gel or serially from extracted lymph nodes and fixed in 4% paraformaldehyde for 10 minutes. Slides were washed three times and stained with anti-Ki67 (Abcam, ab16667) and goat anti-rabbit IgG-AF488 (Abcam, ab150077) at 1 μg/ml as described previously or with anti-wide spectrum Cytokerain (Abcam, ab9377) and an HRP/DAB chromagen IHC detection kit (Abcam, ab64261). Migratory or cytokeratin-positive cells were quantified through an in-house Matlab script to quantify the area of positively stained regions. CM-DiI- and Ki67-positive regions were normalized to the total area of DAPI to control for fat pad area. Small molecule inhibition Small molecule inhibitors were added to the gels 3 days after gelation at the same time as temporal stiffening. The inhibitors (all from Sigma) consisted of PF573228 (FAK, 5 μM), LY294002 (PI3K, 20 μM) and NSC23766 (Rac1, 70 μM) and were added to culture media, concentration was based on previous publication [7, 16, 30]. Inhibitors were maintained in culture media for 11 additional days. Gels were then assayed for morphology, migratory capacity or gene expression as previously described. Statistical analysis All data in bar graphs are presented as mean ± standard deviation and in box and whisker plots as median with quartile ranges. Statistical significance was determined utilizing Student’s t-test for comparisons between two groups, a two-way ANVOA with post hoc Tukey test for multiple comparisons for multiple groups, an extra sum of squares F test on fitted exponential curves for tumor burden data, or a Mantel-Cox log-rank test with post hoc Bonferroni tests for multiple comparisons for survival data. P-values of less than 0.05 were considered significant. RESULTS Py2T cells are a model of TGF-β-induced EMT in vitro To determine the role of matrix stiffness on EMT, a cell line derived from murine MMTV-PyMT tumors as a model of TGF-β-induced EMT was utilized. These cells, denoted Py2T (Polyoma-middle-T tumor), are defined as epithelial, through expression of both luminal and basal cytokeratins [25]. Upon exposure to TGF-β, these cells undergo EMT and lose epithelial markers. These resulting cells are denoted Py2T-LT for long-term culture. These traits were confirmed through examination of cell monolayer morphology, where the epithelial Py2T cells displayed classic cobblestone morphology, and mesenchymal Py2T-LT became more spindle like (Fig. 1A). Additionally, transformation to Py2T-LT resulted in reduced expression of epithelial marker E-cadherin and increased expression of mesenchymal markers N-cadherin, vimentin and fibronectin (Fig. 1B). Importantly, the morphologic and genotypic changes upon TGF-β stimulation match earlier characterization of the Py2T cell line [25]. Thus, the Py2T cell line serves as a specific model of TGF-β-induced EMT and will be utilized in our further studies. TGF-β has been shown to play an important role in EMT and is utilized to induce a mesenchymal transformation in many experimental studies [31]. Figure 1 Open in new tabDownload slide Model Py2T cells are induced towards a mesenchymal phenotype after culture with TGF-β. Epithelial Py2T cells were treated with 2 ng/ml TGF-β and were transformed to mesenchymal Py2T-LT cells. (A) Mesenchymal Py2T-LT cells exhibit a more elongated, less-cobblestone phenotype as compared to epithelial Py2T. (B) Transformation corresponds to loss of epithelial marker E-cadherin and increase in mesenchymal markers N-cadherin, vimentin and fibronectin (*P < 0.05). (C) These cells were utilized in a dynamic stiffening hydrogel that was irradiated at 808 nm 3 days after gelation, to allow for small cluster formation, to increase the elastic modulus from 150 to 1200 Pa. Figure 1 Open in new tabDownload slide Model Py2T cells are induced towards a mesenchymal phenotype after culture with TGF-β. Epithelial Py2T cells were treated with 2 ng/ml TGF-β and were transformed to mesenchymal Py2T-LT cells. (A) Mesenchymal Py2T-LT cells exhibit a more elongated, less-cobblestone phenotype as compared to epithelial Py2T. (B) Transformation corresponds to loss of epithelial marker E-cadherin and increase in mesenchymal markers N-cadherin, vimentin and fibronectin (*P < 0.05). (C) These cells were utilized in a dynamic stiffening hydrogel that was irradiated at 808 nm 3 days after gelation, to allow for small cluster formation, to increase the elastic modulus from 150 to 1200 Pa. Dynamic increase in matrix stiffness results in an invasive, phenotypic shift in epithelial cells Our group has previously reported on the role of dynamic stiffening on human mammary epithelial cells and found that MCF10A cell clusters became more invasive and lost polarity upon stiffening [16]. However, these data did not interrogate possible mesenchymal transformation on cell clusters and the use of human MCF10A cells renders advancement towards a murine in vivo model difficult. To remedy this, murine Py2T or Py2T-LT cells were mixed with gel components to form soft (150 Pa) hydrogels. Hydrogels underwent irradiation after 3 days to externally increase the modulus to a final stiffness of 1200 Pa (Fig. 1C). Though this timeline does not exactly mimic native in vivo tumor stiffening, it provides a dynamic increase in modulus on the same scale seen in tumor development [32]. Furthermore, the formation of small cell clusters prior to external stiffening allows for the development of additional cell–cell and cell–ECM interactions, which have been shown to influence epithelial cell behavior [21, 33]. Cell clusters in either soft 150 Pa gels or stiffened 1200 Pa gels were evaluated with transformed, mesenchymal Py2T-LT cells serving as a control. Though Py2T cells cultured in gels that were stiffened formed larger cell clusters with an invasive, protrusive morphology reminiscent of mesenchymal Py2T-LT clusters in either soft or stiffened gels (Fig. 2A). Additionally, these Py2T clusters formed larger clusters upon stiffening relative to those that remained soft, though significance was not established (Fig. 2B). However, clusters in stiffened gels became less circular due to the extension of protrusions from the main cluster body, indicative of invasive shift (Fig. 2C). EMT is classically determined through a shift from epithelial E-cadherin to mesenchymal N-cadherin, over which process E-cadherin is trafficked from the cell membrane to the cytosol [34]. To investigate possible EMT, cell clusters were immunostained for E- and N-cadherin. Analysis of cadherin expression within cell clusters demonstrates a reduction in epithelial E-cadherin localization upon stiffening, particularly for Py2T clusters (Fig. 2D). Furthermore, the mean fluorescence intensity of the E-cadherin stain was reduced in epithelial Py2T cells upon stiffening (Fig. 2E). This reduction in E-cadherin is matched with a corresponding increase in mesenchymal N-cadherin expression; however, localization to cell–cell contacts is not yet apparent (Fig. 2F). The shift in cadherin protein expression is corroborated though reduction in E-cadherin and increase in N-cadherin mRNA transcripts upon stiffening in Py2T clusters (Fig. 2G). Stiffening also results in an increase in vimentin expression (Fig. 2G), which has been shown to regulate EMT in breast cancer [35]. The alteration in Py2T cluster morphology and cadherin expression is indicative of mesenchymal transformation and EMT induced via the dynamic stiffening of the hydrogel. To investigate the pathway responsible for the mesenchymal transition observed, canonical and non-canonical EMT pathways were investigated. Increases in mRNA levels were seen in canonical EMT transcription factors Twist, Zeb1 and Zeb2 upon stiffening in both the Py2T cells and Twist and Zeb1 in Py2T-LT cells, though no significant increases in Snai1 were seen (Supplementary Fig. 1). Interestingly, no change was seen in Yap/Taz nuclear localization in Py2T cells (Supplementary Fig. 2). Yap/Taz nuclear localization is traditionally thought to be associated with early stages of EMT as a result of matrix binding; however, a variety of mechanisms are proposed to induce EMT shift [24, 36–41]. Interestingly, matrix-dependent breast cancer progression in three-dimensional culture has been shown to be independent of YAP nuclear localization in MCF10A cells [42]. Figure 2 Open in new tabDownload slide Dynamic stiffening induces EMT in epithelial Py2T cell clusters. Dynamic stiffening transforms Py2T cells from an epithelial-to-mesenchymal phenotype. (A) Py2T cell clusters in gels that have been dynamically stiffened exhibit a more invasive morphology similar to those of mesenchymal Py2T-LT cells. (B) Quantification of cell cluster area. Py2T cells in stiffened gels form larger clusters than in 150 Pa gels. (C) Quantification of cell cluster circularity. Py2T cells in stiffened gels form less circular clusters than those in soft gels (#P < 0.05, Students t-test). (D) Representative images of E-cadherin staining shows stiffening reduces E-cadherin expression and organization in stiffened gels. (E) Quantification of E-cadherin immunocytochemical stain mean fluorescence intensity averaged over cell clusters. (F) Representative images of N-cadherin staining in gels demonstrates mesenchymal shift in stiffened gels and those containing Py2T-LT clusters. (G) Epithelial-to-mesenchymal transformation in Py2T cells is corroborated with reduction in E-cadherin and increase in N-cadherin and vimentin in stiffened gels (*P < 0.05, two-way ANOVA, post hoc Tukey test). Figure 2 Open in new tabDownload slide Dynamic stiffening induces EMT in epithelial Py2T cell clusters. Dynamic stiffening transforms Py2T cells from an epithelial-to-mesenchymal phenotype. (A) Py2T cell clusters in gels that have been dynamically stiffened exhibit a more invasive morphology similar to those of mesenchymal Py2T-LT cells. (B) Quantification of cell cluster area. Py2T cells in stiffened gels form larger clusters than in 150 Pa gels. (C) Quantification of cell cluster circularity. Py2T cells in stiffened gels form less circular clusters than those in soft gels (#P < 0.05, Students t-test). (D) Representative images of E-cadherin staining shows stiffening reduces E-cadherin expression and organization in stiffened gels. (E) Quantification of E-cadherin immunocytochemical stain mean fluorescence intensity averaged over cell clusters. (F) Representative images of N-cadherin staining in gels demonstrates mesenchymal shift in stiffened gels and those containing Py2T-LT clusters. (G) Epithelial-to-mesenchymal transformation in Py2T cells is corroborated with reduction in E-cadherin and increase in N-cadherin and vimentin in stiffened gels (*P < 0.05, two-way ANOVA, post hoc Tukey test). Stiffening induces migratory transformation The key consequences of such a mesenchymal shift include increased migratory capacity, adoption of stem-like traits and resistance to apoptosis [43]. EMT-induced migration is a crucial step in the metastatic cascade and therefore cancer mortality [44]. By forming gels within well plates, migratory cells are able to collect on the treated well-plate bottom and quantification of these cells can serve as a measure of migration (Fig. 3A). Figure 3 Open in new tabDownload slide Dynamic matrix stiffening induces migratory shift in epithelial Py2T cells. (A) Schema of stiffening gel system with migrant cells highlighted. Gels can be removed and migrant cells isolated for analysis. (B) Brightfield images of cells that have migrated to well bottom after 14 days of culture. Both Py2T and Py2T-LT cells migrate and cells that migrate maintain morphology typically seen in 2D culture. (C) Immunocytochemical staining of E-Cadherin of cells in migratory compartment show minimal cell deposition after 24 hours. (D) However, significant numbers of cells have migrated after 14 days and those Py2T cells that migrated in stiffened gels reduced E-cadherin expression and localization. (E) Analysis of mRNA corroborates the reduction in E-Cadherin upon stiffening and in Py2T-LT cells (*P < 0.05, two-way ANOVA, post hoc Tukey test). (F) Py2T cells migrated in stiffened gels at a rate comparable to that of Py2T-LT cells in all conditions (*P < 0.05, compared to soft Py2T, two-way ANOVA, post hoc Tukey test). Figure 3 Open in new tabDownload slide Dynamic matrix stiffening induces migratory shift in epithelial Py2T cells. (A) Schema of stiffening gel system with migrant cells highlighted. Gels can be removed and migrant cells isolated for analysis. (B) Brightfield images of cells that have migrated to well bottom after 14 days of culture. Both Py2T and Py2T-LT cells migrate and cells that migrate maintain morphology typically seen in 2D culture. (C) Immunocytochemical staining of E-Cadherin of cells in migratory compartment show minimal cell deposition after 24 hours. (D) However, significant numbers of cells have migrated after 14 days and those Py2T cells that migrated in stiffened gels reduced E-cadherin expression and localization. (E) Analysis of mRNA corroborates the reduction in E-Cadherin upon stiffening and in Py2T-LT cells (*P < 0.05, two-way ANOVA, post hoc Tukey test). (F) Py2T cells migrated in stiffened gels at a rate comparable to that of Py2T-LT cells in all conditions (*P < 0.05, compared to soft Py2T, two-way ANOVA, post hoc Tukey test). Both epithelial Py2T and mesenchymal Py2T-LT cells migrate in all conditions (Fig. 3B). Interestingly, migrant cells generally resume 2D morphology: Py2T migrants form cobblestone sheets and Py2T-LT migrants maintain a spindle shape. Cells migrate progressively from the gel to the well plate as minimal numbers of cells are seen on the plate 24 hours after gelation (Fig. 3C). After 14 days, however, significant migrant Py2T and Py2T-LT cells can be observed (Fig. 3D). Further Py2T cells that migrated from stiffened gels showed reduced E-cadherin expression and loss of localization, similar to Py2T-LT cells in both soft and stiffened gels, which exhibited little to no E-cadherin expression. This result was confirmed through gene analysis, which showed that E-cadherin expression was reduced upon stiffening among cells that migrated to the bottom of the well plate (Fig. 3E). Interestingly, this relative E-cadherin reduction in migrant cells was less than that observed for cell clusters remaining in the gel. Differences in cadherin expression in 2D versus 3D culture have been investigated in other cell types [45] and would suggest that our 3D culture model affects in vitro EMT to some extent. Total migratory capacity was also determined through quantification of the total migrated population at the well surface. We found that Py2T cells in stiffened gels migrated at a far higher quantity, almost four times as frequently, than those in soft gels. This migration rate is comparable to mesenchymal Py2T-LTs in each gel condition (Fig. 3F). Furthermore, the increase in the number of migrant cells is independent of total cell metabolic activity, indicating a change in overall migratory capacity rather than in proliferative capacity (Supplementary Fig. 3). These data suggest that dynamic increases in matrix modulus not only induce a phenotypic shift in cells within stiffened gels but also induce a phenotypic shift to a more migratory state. Although EMT and migration are typically closely associated, they are not coincident and may be decoupled in certain cancers [46]. The similar migratory capacity of Py2T from stiffened gels and all Py2T-LT suggests that stiffness-induced migration operates through TGF-β, which correlates strongly with cancer metastasis [47]. In vivo dynamic stiffening reduces tumor burden and increases cell migratory capacity Py2T or Py2T-LT cells were pooled with gel components, as described previously, and orthotopically injected in the fourth inguinal fat pad of FVB/N mice. Gels for each cell type were irradiated in situ 3 days postinjection to dynamically stiffen the gels from 150 to 1200 Pa (Fig. 4A). Larger tumors formed more quickly from soft, unstiffened gels containing Py2T cells compared to all other conditions, which formed large tumors at roughly the same rate (Fig. 4B). Upon fitting exponential growth curves to the tumor burden curves, the rate constant of the tumors formed from soft gels containing Py2T cells was significantly different than all other conditions, with no additional significant differences between them. These data are corroborated through Kaplan–Meier survival analysis (Fig. 4C) where mice implanted with soft gels containing Py2T cells had a reduced time to survival condition as compared to all other groups. Median survival for mice implanted with Py2T gels that were not stiffened was 22 days postinjection with all other groups ranging from 27 to 29 days postinjection. It is important to note that all mice were sacrificed when primary tumor burden exceeded 750 mm3 as determined by IACUC protocol. Figure 4 Open in new tabDownload slide Dynamic stiffening reduces tumor burden and increases migration from injection site in mice injected with Py2T cells. (A) Gel components were injected in to the fourth inguinal mammary fat pad of mice, allowed to gel and form small clusters for 3 days then transdermally irradiated to increase the modulus from 150 to 1200 Pa. (B) Gel injection with mesenchymal Py2T-LT cells reduces tumor burden as compared to epithelial Py2T cells. Upon stiffening Py2T cell tumors tumor burden is reduced as well. Tumor burden data was fit with exponential growth curves and tumors formed significantly quicker from Py2T cells in 150 Pa gels (extra sum of squares F test of rate constant, P < 0.05). (C) Survival analysis of tumor size further demonstrates the reduced tumor burden from stiffened gels containing Py2T cells. Mice implanted with 150 Pa gels containing Py2T cells had a reduced survival time as compared to all other groups (Mantel-Cox log-rank test, post hoc Bonferroni multiple comparisons, P < 0.05). Figure 4 Open in new tabDownload slide Dynamic stiffening reduces tumor burden and increases migration from injection site in mice injected with Py2T cells. (A) Gel components were injected in to the fourth inguinal mammary fat pad of mice, allowed to gel and form small clusters for 3 days then transdermally irradiated to increase the modulus from 150 to 1200 Pa. (B) Gel injection with mesenchymal Py2T-LT cells reduces tumor burden as compared to epithelial Py2T cells. Upon stiffening Py2T cell tumors tumor burden is reduced as well. Tumor burden data was fit with exponential growth curves and tumors formed significantly quicker from Py2T cells in 150 Pa gels (extra sum of squares F test of rate constant, P < 0.05). (C) Survival analysis of tumor size further demonstrates the reduced tumor burden from stiffened gels containing Py2T cells. Mice implanted with 150 Pa gels containing Py2T cells had a reduced survival time as compared to all other groups (Mantel-Cox log-rank test, post hoc Bonferroni multiple comparisons, P < 0.05). Interestingly, dynamic increases in stiffening did not lead to differences in tumor burden or survival in mice injected with gels containing Py2T-LT cells, which corresponds well to the previous in vitro data collected where stiffening did not alter Py2T-LT genotype or migratory behavior within hydrogels. The role of matrix rigidity on mesenchymal stem cells has been extensively studied and has been shown to regulate mesenchymal cell morphology, phenotype and differentiation [48–52]. Thus, while mesenchymal cells are clearly able to sense the mechanical properties of their environment, ECM stiffness does not appear to be crucial to the growth dynamics of a primary tumor of specific Py2T-LT mesenchymal origin. Differential tumor burden, however, did not correlate to differential metastatic burden as determined via analysis of draining axillary lymph nodes. Small micrometastases were seen in both Py2T and Py2T-LT tumors visualized through positive cytokeratin staining (Supplementary Fig. 4). However, the proportion of each node that stained positive was not different. Furthermore, the metastatic lesions were of approximately the same size in each group. It is unexpected that stiffness-induced EMT does not appear to influence downstream metastatic burden. Current understanding of the role of EMT in tumor progression is that it plays a critical role in the metastatic cascade by promoting cancer cell migration from the primary tumor and extravasation to the vasculature [44, 53–55]. However, the metastatic cascade is incredibly complex and the transformation required at the metastatic site for a circulating tumor cell to intravasate and form a micrometastasis cannot be controlled for here. This is particularly pertinent to our system as EMT plasticity has shown to influence metastasis [56]. Further interrogation of the early in vivo migratory behavior of injected cells was conducted via labeling with CM-DiI prior to injection of the gel mixture. Migratory cells positive for CM-DiI were found in the fat pad of all conditions away from the injection site 2 days after stiffening (Fig. 5A). Cells that migrated from Py2T-LT-derived tumors were found in greater quantity than those that migrated from Py2T-derived tumors (Fig. 5B). Further in mice with tumors formed from epithelial Py2T cells, CM-DiI signal trended higher in those that had been stiffened. Importantly, this increase in migratory cells is not due to increased proliferative behavior as no increase in Ki67 signal was seen in CM-DiI-positive cells (Fig. 5C). The increase in early timepoint migratory behavior suggests a stiffness-dependent EMT shift driving cell migration from the injection site leading to a smaller initial tumor forming population and decreased tumor burden as seen previously. Furthermore, cell migratory behavior has been closely tied to metastasis as migration from the primary tumor site to the surrounding stroma is a critical step in the metastatic cascade [57, 58]. Figure 5 Open in new tabDownload slide Stiffening increases Py2T-LT migration from injection gel to the surrounding stroma independent of proliferation. (A) Representative images of excised fat pad tissue containing CM-DiI-positive migratory cells and further stained with Ki67 to determine proliferation. (B) Quantification CM-DiI positive signal in fat pad relative to DAPI signal indicates more migratory cells from mesenchymal Py2T-LT injection sites. (C) Quantification of Ki67 positive signal in cells positive for CM-DiI indicates increased signal in the fat pad is due to increased migration rather than proliferation. Figure 5 Open in new tabDownload slide Stiffening increases Py2T-LT migration from injection gel to the surrounding stroma independent of proliferation. (A) Representative images of excised fat pad tissue containing CM-DiI-positive migratory cells and further stained with Ki67 to determine proliferation. (B) Quantification CM-DiI positive signal in fat pad relative to DAPI signal indicates more migratory cells from mesenchymal Py2T-LT injection sites. (C) Quantification of Ki67 positive signal in cells positive for CM-DiI indicates increased signal in the fat pad is due to increased migration rather than proliferation. Inhibition of FAK and PI3K abrogates morphologic and migratory transformation The role of the important signaling molecules focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K) and Rac1 as possible mediators of stiffness-dependent EMT was investigated. These pathways were chosen as they feature prominently in integrin-dependent signaling and have been established as possible targets of therapeutics in cancers [40, 59–62]. Py2T cells in stiffened gels treated with FAK or PI3K inhibitors produced cell clusters of similar area to cell clusters in soft gels (Fig. 6A). These clusters were also significantly smaller than Py2T clusters in stiffened gels without inhibitors. Py2T cells in stiffened gels without inhibitors were additionally the only group to show reduced circularity, due to protrusions from the cluster body (as compared to cell clusters in soft gels, Fig. 6B). These protrusions are clearly identified through brightfield illumination. Smaller clusters in those groups treated with either FAK, PI3K or Rac1 inhibitor is also evident (Fig. 6C). Figure 6 Open in new tabDownload slide Small molecule inhibition of mechanosensing pathways abrogates stiffness induced invasive and migratory phenotype. (A) Small molecule inhibition of FAK and PI3K resulted in no stiffness-dependent increase in cluster size. (B) Stiffening reduced cell cluster circularity only in Py2T clusters cultured without small molecule. (C) Representative brightfield images of cell clusters in stiffened gels, arrows were used to highlight cell clusters. Gels cultured with small molecule inhibitor of FAK, PI3K or Rac1 formed smaller, more circular clusters. (D) Stiffening further reduced relative migration of cells to the bottom of the well plate to that of 150 Pa gels alone. (E) Inhibition of FAK or PI3K abrogates the overall trend of decreasing E-cadherin expression in gels stiffened from 150 to 1200 Pa (&P < 0.05, compared to 150 Pa, no inhibitor, #P < 0.05, compared to stiffened control, two-way ANOVA, post hoc Tukey test). Figure 6 Open in new tabDownload slide Small molecule inhibition of mechanosensing pathways abrogates stiffness induced invasive and migratory phenotype. (A) Small molecule inhibition of FAK and PI3K resulted in no stiffness-dependent increase in cluster size. (B) Stiffening reduced cell cluster circularity only in Py2T clusters cultured without small molecule. (C) Representative brightfield images of cell clusters in stiffened gels, arrows were used to highlight cell clusters. Gels cultured with small molecule inhibitor of FAK, PI3K or Rac1 formed smaller, more circular clusters. (D) Stiffening further reduced relative migration of cells to the bottom of the well plate to that of 150 Pa gels alone. (E) Inhibition of FAK or PI3K abrogates the overall trend of decreasing E-cadherin expression in gels stiffened from 150 to 1200 Pa (&P < 0.05, compared to 150 Pa, no inhibitor, #P < 0.05, compared to stiffened control, two-way ANOVA, post hoc Tukey test). Furthermore, inhibition of FAK or PI3K reduced the number of migratory cells from stiffened gels to the rate of soft gels (Fig. 6D). This change was not seen in those cells treated with Rac1 inhibitor. Correspondingly, E-cadherin expression was not reduced in stiffened gels treated with FAK or PI3K inhibitors (Fig. 6F). Collectively, these changes show the complete abrogation of the morphologic and migratory transformation induced through stiffening and suggest several important mechanotransduction pathways, particularly FAK and PI3K, are important to stiffness induced EMT. NMuMG cells were encapsulated within soft gels and stiffened as described previously to interrogate the conservation in the stiffening-induced migratory transformation in other mammary cell lines. NMuMG cells undergo EMT upon treatment with TGF-β and form tumors in vivo and thus serve as a good control [63, 64]. In conjunction with data collected previously from epithelial Py2T cells, over 4-fold more NMuMG cells migrate from stiffened gels after 14 days of culture (Fig. 7A). However, this migratory transformation is not associated with differences in E-cadherin protein expression or localization (Fig. 7B). Furthermore, dynamic matrix stiffening does not increase traditional EMT markers N-cadherin or vimentin (Fig. 7C). Thus, migratory but not EMT transformation was conserved in the NMuMG cell line, though EMT leads to a range of migratory phenotypes not completely captured by downstream marker expression [65]. Figure 7 Open in new tabDownload slide Migratory transformation but not EMT is conserved in NMuMG cells upon dynamic matrix stiffening. NMuMG mammary gland cells were encapsulated in soft gels and stiffened as described previously. (A) After 14 days over 4-fold more cells had migrated to the bottom of the well plate in the stiffened gels as compared to the soft gels. Confirming the transformation seen in Py2T cells. (B) However, E-cadherin protein expression and (C) mRNA expression of E-cadherin, N-cadherin and vimentin were unchanged due to stiffening. This indicates a non-canonical EMT transformation in these cell lines. Figure 7 Open in new tabDownload slide Migratory transformation but not EMT is conserved in NMuMG cells upon dynamic matrix stiffening. NMuMG mammary gland cells were encapsulated in soft gels and stiffened as described previously. (A) After 14 days over 4-fold more cells had migrated to the bottom of the well plate in the stiffened gels as compared to the soft gels. Confirming the transformation seen in Py2T cells. (B) However, E-cadherin protein expression and (C) mRNA expression of E-cadherin, N-cadherin and vimentin were unchanged due to stiffening. This indicates a non-canonical EMT transformation in these cell lines. DISCUSSION Increased matrix stiffness has been shown to promote EMT within cell clusters and associated EMT signaling with metastasis [9, 66]. This work has extended the role of increasing matrix stiffness on driving EMT through defining a migratory transformation both in vitro and in vivo. Upon dynamic stiffening, epithelial cells clusters lose typical rounded morphology and develop invasive protrusions. This morphologic shift corresponded with a shift from epithelial E-cadherin expression to mesenchymal N-cadherin. The genotypic change corresponded to an increase in migration and further loss of E-cadherin expression in migrated cells in vitro. Importantly, the observed transformation was confirmed in vivo where dynamic stiffening increased migration from the injection site prior to tumor formation resulting in a decreased tumor burden at later timepoints. This corresponds to the increase in migratory capacity seen in cells driven undergoing EMT [56]. Furthermore, the morphologic transformation was seen to be dependent on FAK and PI3K signaling, though E-cadherin expression was not as affected. EMT is a complex process that can be regulated by multiple pathways, including canonical drivers Snail, Twist, ZEB1 and ZEB2. Upon stiffening, both Py2T and Py2T-LT cells upregulated Twist and downstream transcription factors Zeb1 and Zeb2 [67–69]. No significant increase in Snai1 was observed, although Snai1 is known to cooperate with Twist to upregulate expression of the Zeb family, indicating Snai1 mRNA expression may have been upregulated at an earlier timepoint [25, 67–69]. Notably, many of the relative expression changes reported herein are consistent with what is reported in the literature, particularly for the Py2T and Py2T-LT cells and in other stiffening systems [9, 25, 67]. To investigate the potential for other mechanisms to contribute to the partial EMT observed, Yap/Taz nuclear localization was interrogated. Interestingly, no change in Yap/Taz nuclear localization, which is associated with the early stages of EMT due to matrix binding and the formation of focal adhesions, was seen in the Py2T cells [9, 70, 71]. Together, these data suggest that the cadherin shift and cellular migration upon stiffening is at least partially induced by Snail and Twist cooperation. Zeb1, in particular, is associated with invasion, which is consistent with the migratory increase seen in vitro and in vivo [68, 69]. However, full characterization of EMT is difficult due to the ability of cells to undergo transient, partial EMT and express a mixture of both epithelial and mesenchymal traits [72, 73]. Furthermore, complete EMT programming does not appear to be required for increased breast cancer cell migratory behavior indicating future studies should incorporate understanding of the plasticity of EMT states [42, 74–76]. In fact, partial EMT may impart the ability for these cells to become mesenchymal and disseminate within the body and then undergo MET to form stable metastases [77]. Matrix stiffness has been shown to influence a variety of cellular functions, including stem cell differentiation, cell morphology and cytoskeletal dynamics [14, 78–80]. In cancer, local increases in ECM stiffness have been shown to correlate with tumor progression and chemotherapeutic resistance [81–83]. Many groups have consequently investigated the interplay between matrix stiffness and tumor progression in vitro, finding that cells cultured on stiffer substrates develop a more invasive, malignant phenotype [6, 16, 84]. However, these findings have not fully characterized the stiffness-dependent mesenchymal transformation, though studies have interrogated Snail, Twist, Zeb1 and Zeb2 mRNA expression; YAP/TAZ nuclear localization and loss of E-cadherin expression, which correspond to the early transition towards a mesenchymal phenotype [9]. Here, we evaluated the downstream migratory transformation both in vitro and in vivo showing increased cell migratory capacity to the surrounding environment through stiffness-induced EMT. Cell migration from the primary tumor site to the surrounding stroma and vasculature is a key early stage in the metastatic cascade [85]. As the majority of cancer mortalities are due to metastatic spread, understanding this early stage of metastatic transformation is important for both tumor progression and patient prognosis [86]. However, it was unexpected that tumor burden derived from epithelial Py2T in stiffened gels would be significantly less than that of the soft gels. However, mesenchymal cells are quite migratory and cells migrating from the injection site sooner and at a higher frequency would likely reduce the initial population of cells that form the tumor and thus reduce the early primary tumor burden. Further supporting this is the increased migration of more invasive, migratory Py2T-LT cells from the injection site to the fat pad prior to tumor formation. Furthermore, this is consistent with the in vitro increase in Zeb1 mRNA expression of the tumor cells in stiff gels, and further increased in the Py2T-LT cells in comparison with the Py2T cells, as Zeb1 is regarded as essential for tumor invasion [68, 69]. Tumor cell invasion to the surrounding stroma is critical to clinical decision-making, as positive margins can lead to additional surgical excision [87]. Additionally, tumor stroma mesenchymal cells have been found to be indicative of metastatic burden [88, 89]. Several groups have also found primary tumor and metastatic burden to be uncoupled, corroborating the profile of tumors of Py2T and Py2T-LT origin described here [90–92]. The previously described in vitro phenotypic shift and in vivo migratory transformation were expected to result in an increase in the metastatic burden of tumors in mice with stiffened gels. The lack of differential metastatic burden, however, can be attributed to the complexity of the metastatic cascade. As noted previously, metastasis is a dynamic process of which EMT is only a single component [20]. The ability of cells to colonize the secondary site through the reverse MET process or survival the circulation as circulating cancer cells are important for metastatic nodule formation and not interrogated here [93, 94]. Furthermore, EMT is a dynamic process and full transformation to a mesenchymal phenotype may not develop cells with the highest metastatic potential [56]. Further interrogation in to the extent of partial EMT that has been induced by matrix stiffening will inform the degree of invasive and metastatic transformation of these cells [72, 73]. Additionally, other studies have suggested that EMT may not be critical to the metastatic cascade at all [95, 96]. This, along with the relationship between stiffening-induced EMT described here highlights the complex nature of the metastatic process and demonstrates the need for further investigation. Focal adhesion kinase plays an important role in cell signaling and has been particularly associated with integrin signaling [60]. Additionally, both FAK and PI3K have been tied closely to cell migratory behavior [97–99]. As such, the reduction in invasive morphology and migratory behavior seen in epithelial cells in stiffened gels is expected. It is interesting that a corresponding significant reduction in E-cadherin expression was not seen; however, groups treated with FAK or PI3K inhibitor did not show a decreasing trend in expression, which is consistent with findings in other carcinoma models [100]. Furthermore, it has been shown that FAK signaling in particular is critical to EMT and metastasis in breast cancer cell lines [3, 101]. Collectively, this reinforces role of FAK and integrin signaling as a potential therapeutic target for anti-metastatic therapies. CONCLUSIONS Dynamic increases in matrix stiffness through the use of a light-triggered stiffening hydrogel drove EMT in mammary epithelial cells. This resulted in invasive cell clusters that downregulated E-cadherin and upregulated N-cadherin and vimentin in vitro and increased migration of cells in vitro and in vivo. The in vitro changes were shown to be reliant on FAK and PI3K signaling. In vivo, the early, local invasion of tumor cells into the surrounding tissue likely led to a reduction of primary tumor burden in stiffened gels. Collectively, this reinforces the role of matrix stiffness in tumor progression and integrin signaling as a potential target for anti-tumor therapies. Acknowledgements We would like to gratefully acknowledge Dr Gerhard Christofori for providing the Py2T cell line and Dr Collene Jeter for providing the NMuMG cell line. Funding This work was supported by the Cancer Prevention and Research Institute of Texas (RP130372). 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TI - Dynamic extracellular matrix stiffening induces a phenotypic transformation and a migratory shift in epithelial cells
JF - Integrative Biology
DO - 10.1093/intbio/zyaa012
DA - 2020-06-19
UR - https://www.deepdyve.com/lp/oxford-university-press/dynamic-extracellular-matrix-stiffening-induces-a-phenotypic-680inQPC04
SP - 161
VL - 12
IS - 6
DP - DeepDyve
ER -