Immobilized Transforming Growth Factor-Beta 1 in a Stiffness

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Immobilized Transforming Growth Factor-Beta 1 in a StiffnessTunable Artificial Extracellular Matrix Enhances Mechanotransduction in the Epithelial Mesenchymal Transition of Hepatocellular Carcinoma Rui-Zhi Tang,†,‡ Sai-Sai Gu,†,§ Xin-Ting Chen,§ Li-Jie He,⊥ Kai-Ping Wang,§ and Xi-Qiu Liu*,§

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‡

School of Basic Medicine and §School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, P.R. China ⊥ Graphitene Ltd., Flixborough, North Lincolnshire DN15 8SJ, United Kingdom S Supporting Information *

ABSTRACT: Cancer progression is regulated by multiple factors of extracellular matrix (ECM). Understanding how cancer cells integrate multiple signaling pathways to achieve specific behaviors remains a challenge because of the lack of appropriate models to copresent and modulate ECM properties. Here we proposed a strategy to build a thin biomaterial matrix by poly(L-lysine) and hyaluronan as an artificial stiffness-tunable ECM. Transforming growth factor-beta 1 (TGF-β1) was used as a biochemical cue to present in an immobilized and spatially controlled manner, with a high loading efficiency of 90%. Either soft matrix with immobilized TGF-β1 (i-TGF) or bare stiff matrix could only promote HCC cells to form the epithelial phenotype, whereas stiff matrix with i-TGF was the only condition to induce the mesenchymal phenotype. Further investigation revealed that i-TGF increased the specific TGF-β1 receptor (TβRI) expression to activate PI3K pathway. i-TGF-TβRI interactions also promoted HCC cell adhesion to enlarge contact area for stiffness sensing, resulting in the raising expression of the mechano-sensor (β1 integrin). Mechanotransduction would then be enhanced by the β1 integrin/vinculin/p-FAK pathway, leading to a noble PI3K activation. Using our model, a novel mechanism was discovered to elucidate regulation of cell fates by coupling mechanotransduction and biochemical signaling. KEYWORDS: ECM mimicking, mechanotransduction, growth factor presentation, epithelial−mesenchymal transition, hepatocellular carcinoma

1. INTRODUCTION

presentation to cells are spatially regulated by the physical properties of the ECM.9−11 However, it is still a matter of debate if GFs are capable to initiate a mechanical response during the cancer development, and the signaling pathways in response to these integrated signals are not well understood. Transforming growth factor-β1 (TGF-β1) is one of the most important members of the transforming growth factor family. This cytokine contributes to tumor progression and metastasis through the induction of epithelial-mesenchymal transition (EMT), a hallmark of the dissemination of malignant hepatocytes during HCC progression.12 However, how the ECM properties influence the responses of HCC cells to TGFβ1 remains unclear, due to the lack of appropriate cell culture models. For in vitro cell culture, soluble GFs are usually added into the culture medium, but they are rapidly degraded and

The local microenvironment (or niche) of cancer cells plays important roles in cancer development. Recently, increasing attention has been paid to the physical factors of the local cellular microenvironment in cancer studies.1 Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers in the world, and it is the third leading cause of cancerrelated death in adults.2 Approximately 80% of HCCs develop in the context of advanced liver fibrosis or cirrhosis,3 accompanying with stereotypical changes in the cellular microenvironment. Among them, liver stiffness (a noninvasive, indirect measure of liver fibrosis) has been found to be positively correlated with HCC risk.4−6 Malignant HCC cell behaviors are also highly influenced by biochemical and biomechanical cues in the surrounding microenvironment. Studies have shown that the extracellular matrix (ECM) plays a significant role in cancer progression including tumor growth, invasion, and metastasis.7,8 Growth factors (GFs) are chemical cues incorporated into the ECM, whose activation and © XXXX American Chemical Society

Received: February 26, 2019 Accepted: April 1, 2019

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DOI: 10.1021/acsami.9b03572 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Surface characteristics of multilayer films after polydopamine (pDA) and TGF-β1 loading. (A) Nitrogen/carbon (N/C) molar ratio and the photo (inset) of films without and with pDA loading. (B) Surface Young’s modulus of films without and with pDA loading. (C) Immobilization efficiency of TGF-β1 on pDA-coated films. (D) Atomic force microscopy images (5 μm × 5 μm) of films without pDA coating, with pDA coating, and after TGF-β1 immobilization on pDA-coated films.

exhibit high turnover.13,14 Tethering GFs on biomaterial substrates can extend their half-life and improve the persistence of GFs presented to the cells.15,16 Moreover, presentation of GFs in an immobilized manner can mimic their physiological conditions because most GFs are bound to proteins and glycosaminoglycans (GAG) in the ECM.10 Therefore, use of ECM-mimicking bioactive scaffolds is highly required to recapitulate a correct molecular milieu in vitro cell culture for better understanding of HCC development. As the ECM is a dynamic network of various signaling molecules with distinctive physical and biochemical properties,17 one possibility to imitate an ECM environment would be to create a sophisticated scaffold to present signaling molecules in combination with surrounding mechanics (ex. stiffness). The layer-by-layer technique is a facile method for depositing polyelectrolytes alternatively to fabricate a thin biomimetic matrix. It is possible to engineer multilayer films that mimic the thin matrix and contain bioactive molecules such as peptides and proteins by choosing appropriate film components or physicochemical conditions.18,19 However, effective presentation of GFs and preservation of their bioactivities remain as a challenge in the field. Conventionally, physical entrapment has been used to present GFs but the possibility to control orientation and distribution of such entrapped GFs is still limited.20 In the present study, we built multilayer films made of poly(L-lysine) (PLL) and hyaluronan (HA) as potential in vitro platforms for modeling the ECM of HCC. The films with elastic modulus of 180 kpa and 400 kpa were particularly focused, to represent ECM stiffness of liver cirrhosis with HCC21 as well as metastasis in the spinal cord.22 Then a polydopamine (pDA) deposition process was performed for anchoring TGF-β1 on the surface of films with a high efficiency. Immobilized TGF-β1 revealed its specific functions to promote HCC cell cytoskeleton rearrangement, migration, EMT progression, particularly to enhance signaling activation in mechanotransduction. Our results highlight the ability of controlling GF presentation manner in a stiffness-tunable artificial ECM allows extended investigation of HCC cell behaviors during tumorigenesis.

2. RESULTS 2.1. Characterization of ECM-Mimicking Thin Matrix. The polyelectrolyte multilayer films are hydrophilic polymeric networks with tunable physical and mechanical properties, which are commonly designed to mimic ECM composition and to study cell−microenvironment interactions.23 Therefore, applications as coatings are requested to modulate the surface properties and bind bioactive molecules without any negative effect on their bioactivity. In our study, the bioinspired coating by pDA was carried out on the surface of films under simple and solvent-free conditions with minimal change in chemical structure of synthetic biomaterials, as a starting point for further applications. The films developed a brown color due to the oxidation of dopamine and corresponding polymerization to pDA. The nitrogen/carbon (N/C) molar ratios were also calculated by measuring relative atomic compositions (Figure 1A). After 1 h of pDA coating, the N/C molar ratios on the surfaces increased from 0.085 ± 0.006 to 0.120 ± 0.004, which was similar to the theoretical value of dopamine molecule (N/ C molar ratio: 0.125). Thus, we could conclude that the homogeneous pDA coating may have been achieved. Similar results were obtained by measuring peak intensity from other materials, such as poly(l-lactide-co-ε-caprolactone) films.24 Next, we established the experimental conditions for TGFβ1 immobilization in pDA-coating cross-linked (PLL/HA) films. Two cross-linking concentrations were selected based on the previous physicochemical characterization of such films,25 and marked as soft and stiff matrix, respectively. pDA-based new surface modification method allowed development of a compatible biomaterial without affecting the mechanical properties of the material, and the Young’s modulus was similar without and with pDA coating (Figure 1B). The soft film showed 187.2 ± 31.4 kpa of stiffness, which was maintained as 190.8 ± 49.6 kpa after pDA coating. The stiff film itself exhibited 422.0 ± 83.2 kpa of stiffness, whereas Young’s modulus was slightly changed to 456.4 ± 76.6 kpa without significant differences. We found no evidence of change in the stiffness properties of films, suggesting that the process of pDA coating was not detrimental to the substrates, in accordance with the previous publication.24 B


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Figure 2. Adhesion and morphology of HCC cells cultured in different matrix. (A) Fluorescence images, scale bar: 20 μm. Quantitative analysis of the cell spreading area (B), circularity (C), and aspect ratio (D) **: p < 0.01, ***: p < 0.001. (E) Cell density after 24 h incubation. (F) Phasecontrast images (upper) exhibiting three major phenotypes of cell morphology, scale bar: 20 μm; Percentage analysis of each phenotype (bottom). (No TGF, in the bare matrix; i-TGF, in the matrix with immobilized TGF-β1; s-TGF, in the matrix with soluble TGF-β1).

TGF, indicating that differences existed in cell morphology during incubation in two distinct mechanical microenvironments (Figure 2C). The data of aspect ratio was also collected and it released the similar information. Although the aspect ratio was notably increased in the group of i-TGF, there were still differences between soft and stiff matrix, which suggested that stiffness could also play an important role in determining cell morphologies (Figure 2D). Therefore, it is worth further analyzing cell morphology in the different conditions. We first confirmed that the number of adhered cells remained similar among all the groups (Figure 2E), then three types of cell morphology were found apparent: (1) round cells that were attached but had not begun to spread; (2) cells with a cobblestone-like shape, implicating the possibility of starting to spread and form protrusions; and (3) cells with a fibroblast-like morphology displaying a distinct leading edge and retracting tail, indicating a strong motile behavior. We quantified the percent of cells in each category (Figure 2F). In the soft matrix, the majority (>95%) of cells grown are round in the conditions of No TGF and s-TGF. All three types of morphology were seen in the presence of i-TGF (53.4 ± 1.0% for cobblestone-like, 26.9 ± 2.7% for fibroblastlike, and 19.7 ± 2.3% for round), which explained why a wide range existed in circularity. Higher stiffness induced an obvious decline in the proportion of round cells, and only approximately 10% of cells were round in all the conditions of stiff matrix. Of note, cells had mainly cobblestone-like shapes (>75%) for No TGF and s-TGF, indicating the formation of actin-driven protrusions. A significant increase in elongated cells occurred on the stiff i-TGF, and the fibroblastlike phenotype already became the dominant morphology (84.8 ± 3.3%) revealing the obvious status of mesenchymal cells. Taken together, the results suggested that stiffness and iTGF were two key factors in the matrix to determine HCC cell adhesion and morphology. PDA coating did not exhibit obvious promotion on cell area and elongation (Figure SI2),

The pDA strategy has been investigated to link peptides and proteins on the surface of other synthetic and natural scaffolds. To create a tumor progressive coating, the films presenting a pDA layer was treated with TGF-β1. To quantify the amount of TGF-β1 immobilized on the surfaces, we soaked samples in a solution of TGF-β1 and measured the amounts of unbound TGF-β1 before and after the adsorption. The binding efficiency was high and similar in soft matrix (92.9 ± 6.9%) and stiff matrix (93.1 ± 1.8%) (Figure 1C), which meant the amount of immobilized TGF-β1 was 6.19 ± 0.46 and 6.21 ± 0.12 ng/cm2, respectively. The amount of released TGF-β1 was negligible over 48 h (Figure SI1). Furthermore, we confirmed that neither pDA coating nor TGF-β1 binding would change surface topography (Figure 1D), and the following cell behaviors were caused by different stiffness and presentation modes of TGF-β1. 2.2. Cell Adhesion and Morphology. Adhesion and morphology of HCC cells were investigated by fluorescence microscopy after actin staining (Figure 2A). HCC cells responded to surrounding microenvironment and transform into different morphologies that could direct their future behaviors. On the bare soft matrix (No TGF), cells were round and poorly spread (506.5 ± 97.7 μm2), whereas cells were significantly more spread (954.5 ± 248.8 μm2) in the matrix with immobilized TGF-β1 (i-TGF) (p < 0.001). However, this effect did not happen when TGF-β1 was presented in a soluble manner (s-TGF). Cells of No TGF in the stiff matrix exhibited a larger area (623.3 ± 114.1 μm2) compared to those on the soft one (p < 0.01). s-TGF did not show any effect on cell adhesion and spreading on stiff matrix, whereas cells progressively elongated in a spindle and well spread morphology (1155.1 ± 252.3 μm2) in the presence of i-TGF (Figure 2B). Meanwhile, cell circularity was significantly lower for both soft and stiff matrix with i-TGF (p < 0.001), where cells were otherwise less spread in other conditions. The values of circularity were generally higher for cells cultured on the soft matrix and there was a wide range (0.2−0.8) in the group of iC


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Figure 3. EMT phenotypes of HCC cells cultured for 48 h in different matrix. (A) Immunostaining of E-cadherin (red) and nuclei (blue), scale bar 20 μm. (B) Immunostaining of Vimentin (green) and nuclei (blue), scale bar 20 μm. (C) Western blot analysis of E-cadherin, N-cadherin, and Vimentin expression. The ratios of EMT marker/β-actin bands were calculated (*: p < 0.05 and **: p < 0.01).

and it could be considered as the anchorage layer for bioactive molecules.26,27 In addition, the increasing i-TGF (∼30 ng/ cm2) did not significantly promote cell adhesion and spreading (Figure SI3), which indicated the current i-TGF amount was sufficient to perform its biological functions to HCC cells. 2.3. Distinct Inducing Effects on EMT Phenotypes. As known that EMT is a complex change from an epithelial phenotype to a narrow, fibroblast-like phenotype, which is considered as a critical step in tumor metastasis and invasion.28 The different phenotypes in cell morphology have been found in different stiffness and TGF-β1 presentation modes. Next, we analyzed the expression of relevant markers for monitoring EMT progression after 48 h incubation. The regular growth and proliferation of HCC cells in all conditions were confirmed (Figure SI4) by comparison of cell viability at 12 and 48 h incubation (p < 0.001). The loss of epithelial E-cadherin and the gain of mesenchymal N-cadherin expression is a major hallmark of EMT.29 A mesenchymal phenotype accompanies by the expression of intermediate filament protein, such as vimentin.30 Immunostaining of E-cadherin showed cells having strong cell−cell junctions in the soft matrix with i-TGF, as well as the stiff one with No TGF and s-TGF (Figure 3A). The cells in the stiff i-TGF condition displayed minimal cell−cell junctions with a negligible E-cadherin staining. Immunostaining of the mesenchymal marker vimentin showed negligible or weak expression in all the groups, except for the stiff i-TGF condition (Figure 3B). Cells were disseminated with staining in the cytoplasm, accompanied by the spindle-shaped morphology, confirming that the cells transitioned from the epithelial to mesenchymal type. Protein levels of E-cadherin, N-cadherin, and vimentin were also studied to confirm their roles in revealing EMT (Figure 3C). The expression of Ecadherin for soft i-TGF, as well as the stiff matrix with No TGF and s-TGF was almost 2-fold and statistically significant as compared to other conditions (p < 0.01). Meanwhile, the highest expression of N-cadherin and vimentin both occurred in the stiff i-TGF condition, and a much weaker expression was observed in other groups with a round or cobblestone-like cell

morphology, which was in accordance with the results of immunostaining. 2.4. Individual and Crowed Cell Migration. TGF-β1 in the tumor microenvironment is known to trigger cell migration in the process of metastasis.31 We addressed the question whether matrix stiffness and presentation manner of TGF-β1 could also influence cell migration and investigated in two different modes of cell migration: individual and crowed cultures. The individual cell migration behaviors were studied by time-lapse microscopy. Representative migration tracks of cells and quantification of cell migration speeds were shown (Figure 4A). In the absence of TGF-β1, HCC cells migrated randomly at 0.41 ± 0.10 μm/min and 0.73 ± 0.10 μm/min on soft and stiff matrix, respectively. s-TGF slightly increased cell migration speed to 0.48 ± 0.08 μm/min on the soft matrix and to 0.79 ± 0.13 μm/min on the stiff one. However, i-TGF significantly increased cell migration to 0.99 ± 0.13 μm/min on the soft matrix and to 1.52 ± 0.27 μm/min on the stiff one (p < 0.001). To analyze the crowded cell migration behavior, we performed a wound healing assay in a high-density cell culture. Figure 4B showed the representative images of magnified cell adhesion on the margin at different time points after the culture inserts were removed. On the soft matrix, HCC cells inclined to form clusters at the conditions of No TGF and sTGF, which was very unfavorable to cover wound gaps. Otherwise, wound width decreased from 595.6 ± 33.4 μm to 338.3 ± 24.6 μm in the condition of i-TGF after 24 h incubation (p < 0.01). The crowded cell migration acted differently on the stiff matrix, and HCC cells held an average potential of wound healing at the conditions of No TGF (445.6 ± 49.5 μm) and s-TGF (405.6 ± 16.4 μm). Meanwhile, the most obvious wound healing effect existed in the condition of i-TGF and 60% of wound gap could be covered after 24 h incubation (p < 0.05). Therefore, the individual and crowded migration behaviors possessed a similar tendency with stiffness and i-TGF being two determined factors to stimulate HCC cell D


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Figure 4. Individual and crowed HCC cell migration cultured in different matrix. (A) Representative trajectories and quantitative analysis of the individual cell migration speeds (***: p < 0.001). (B) Wound healing assay for crowded cell migration. The migration was assessed by the microscope images at beginning and end of cell migration (left panel), scale bar 500 μm. Quantification of wound width (right panel) at 0 and 24 h incubation (*: p < 0.05 and **: p < 0.01).

expression of TβRI for cells cultured only in the stiff matrix. The most evident expression of TβRI existed in the conditions of i-TGF and it exhibited a homogeneous and punctuate distribution in the perinuclear regions (Figure 5C), revealing the effective internalization after TβRI activation by i-TGF. Additionally, there was barely significant difference in TβRI expression between soft i-TGF and stiff i-TGF, which was also confirmed by the Western blotting analysis (Figure 5D). Thus, i-TGF could specifically lead to high level of TβRI in a stiffness-independent manner. Otherwise, β1 integrin expression showed commonly higher for cells cultured on the stiff matrix in comparison to that on the soft one (p < 0.01), in agreement with the previous finding.34 No significant differences were found between No TGF and s-TGF on the soft matrix, whereas s-TGF induced a slightly higher expression of β1 integrin on the stiff matrix (Figure 5D). Strikingly, i-TGF was found to nobly boost β1 integrin expression both on soft

migration, and they could function in a certain collaboration manner. 2.5. Interactions between Receptors of Sensing Biochemical and Stiffness Cues. We next investigated whether matrix stiffness and TGF-β1 presentation modes would impact the level of mechanotransduction receptors and the TGF-β1 receptor. TβRI is known to be the major TGF-β1 receptor to initiate intracellular signal propagation in HCC cells.32 Integrins are mechano-sensors that transduce stiffnesssensing from ECM into biochemical events and stimulate cytoskeletal remodeling.33 The ECM receptor β1 integrin induces focal adhesion formation and supports a forcedependent stiffening response.34 We observed the expression level and spatial distribution of TβRI (Figure 5A) and β1 integrin (Figure 5B) through immunostaining. HCC cells on both soft and stiff matrix displayed a low expression of TβRI without TGF-β1 stimulation. s-TGF could induce a slight E


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Figure 5. Expression of TGF-β1 receptor TβRI and the mechano-sensor β1 integrin on HCC cells cultured for 48 h in different matrix. (A) Immunostaining of TβRI (red) and nuclei (blue), scale bar 20 μm. (B) Immunostaining of β1 integrin (green) and nuclei (blue), scale bar 20 μm. (C) High-magnification images of immunostaining for TβRI (red), β1 integrin (green) and nuclei (blue) in the condition of Stiff i-TGF, scale bar 20 μm. (D) Western blot analysis of TβRI and β1 integrin expression. The ratios of receptor/β-actin bands were calculated (*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ns, not significant).

Figure 6. Activation of signaling pathways on HCC cells cultured in different matrix. (A) Immunostaining of vinculin (green) and nuclei (blue), white arrow: small vinculin dots, yellow arrows: large vinculin plaques, white circles: thin vinculin fibrils, scale bar: 20 μm. Western blot analysis of p-FAK and p-PI3K activation (B) without any inhibition as control, (C) after incubation with TβRI inhibitor, or (D) after incubation with β1 integrin neutralizing antibody. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ns, not significant).

and stiff matrix. β1 integrin showed a homogeneous distribution all over the cells with certain focuses at the cell periphery (Figure 5C), indicating formation of some focal adhesions (FAs) to stimulate cell elongation and migration. The results might explain why cells in the i-TGF condition

exhibited more advanced EMT phenotype than No TGF and s-TGF. 2.6. Crosstalk of Biochemical and Stiffness Cues in Activating Signaling Pathways. FAs and its downstream signaling molecule focal adhesion kinase (FAK) have been F


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Figure 7. Scheme summarizing the major differences in the EMT response of hepatocellular carcinoma cells to biochemical and stiffness cues in different matrix.

To justify the crosstalk between FAK and PI3K signaling pathways induced by biochemical and stiffness cues, we performed the inhibition study of TβRI and β1 integrin receptors and observed changes of the phosphorylation of FAK and PI3K. After the inhibition of TβRI receptor (Figure SI5), i-TGF-triggered FAK and PI3K activation were blocked in both soft and stiff matrix (Figure 6C). The stiffness-dependent activation was kept in a certain extent, because some β1 integrin could be stimulated to express in the stiff matrix, but its mechanotransduction effect was far less than that in stiff iTGF without TβRI inhibition (Figure 6B). Otherwise, FAK phosphorylation almost disappeared in all the conditions after β1 integrin inhibition, which might indicate the direct activation relationship of β1 integrin/vinculin/p-FAK pathway in FA formation (Figure 6D). It was not surprising to find that stiffness-dependent PI3K activation impeded after β1 integrin inhibition (no significant difference between soft “No TGF” and stiff “No TGF” groups). However, i-TGF-triggered PI3K activation was not obviously affected by β1 integrin inhibition, and p-PI3K expression in the condition of i-TGF exhibited significantly higher than that of s-TGF (p < 0.01) or “No TGF” (p < 0.05) in both soft and stiff matrix. 2.7. Possible Mechanism of Immobilized TGF-β1 Enhancing Mechanotransduction in EMT. β1 integrin and TβRI will be in a low expression level, when there is deficient stimulation of stiffness or immobilized TGF-β1 in the matrix. A negligible PI3K phosphorylation is mainly induced by a slight effect of mechanotransduction through the β1 integrin/vinculin/p-FAK pathway, which will confine HCC cells in an inactive phenotype with a round morphology lacking of the capacity to spread and migrate (Figure 7A). When HCC cells are incubated in the stiff matrix, mechanotransduction will be moderately enhanced due to the increasing expression of β1 integrin. The modest activation of PI3K will induce the appearance of epithelial phenotype of a cobblestone morphology with the elevated cell area (Figure 7B). i-TGF can not only stimulate TβRI expression but also activate the i-TGF-TβRI-dependent PI3K pathway. Meanwhile, i-TGF-TβRI interactions promote HCC cell adhesion to enlarge the contact area for stiffness sensing, resulting in the raising expression of β1 integrin. Then PI3K activation was

demonstrated to exert a direct effect on cell spreading, migration, and differentiation.35 Vinculin interconnects signals in FAs, which further induces and reflects FAs formation.36 Thus, to gain further insight into the roles of stiffness and iTGF in FA formation, we then aimed at characterizing localization of the pan-focal adhesion marker protein vinculin (Figure 6A). Cells cultured in the soft matrix with No TGF or s-TGF showed no clear vinculin plaques, whereas i-TGF triggered some small focal adhesions localizing regularly along the cell periphery (indicated by a white arrow). In contrary, cells cultured in the stiff matrix with i-TGF showed numerous and large vinculin plaques, especially in proximity to the polarized cell protrusions (indicated by yellow arrows), which would consequently enhance cellular migration as a hallmark of mesenchymal phenotype. This phenomenon could not be found in the stiff matrix with No TGF or s-TGF (only several thin fibrils indicated by white circles), consistently with estimation of mainly epithelial phenotype from cell morphology. The expression of phosphorylated FAK (p-FAK) located at FA complexes was subsequently examined by Western blotting analysis (Figure 6B). The results exhibited that FAK phosphorylation was in a stiffness-dependent manner based on the quantitive analysis of bands. p-FAK was greatly upregulated for HCC cells cultured on both the soft and stiff i-TGF, while its expression kept in low levels in the No TGF and s-TGF conditions. As expected, the most significant FAK phosphorylation occurred in the stiff i-TGF, consistently with the most obvious FAs formation. FAK, regulated by matrix rigidity, is known to directly mediate the activation of phosphatidylinositol 3-kinase (PI3K) in HCC cells,37 and also potentiates growth factor dependent PI3K signaling.34 Therefore, we next determined the effects of stiffness and i-TGF on the activation of PI3K (Figure 6B). The soft matrix resulted in a negligible effect on inducing PI3K activation in the No TGF and s-TGF conditions, when i-TGF slightly increased the expression of p-PI3K. The stiff matrix was able to induce more obvious activation of PI3K due to the enhanced expression of p-FAK. Not surprisingly, the most significant activation of PI3K existed in the stiff i-TGF condition, where both β1 integrin and p-FAK were in the highest expression levels. G


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ACS Applied Materials & Interfaces greatly improved through the β1 integrin/vinculin/p-FAK pathway (Figure 7C, D). The most significant cooperation effect of mechanotransduction and i-TGF signaling occurs when HCC cells are cultured in the stiff matrix, and the strongest PI3K activation results in the mesenchymal phenotype of a fibroblastic morphology with large cell area and accelerated migration (Figure 7D).

(yhe calculated isoelectric point of TGF-β1 is 8.9, whereas that of serum proteins is ∼4.7) before it attached to the surface. According to the study of Gullotti et al.,45 the surface charge is negative after pDA-coating (zeta potential: −11.7 ± 2.1 mV). Thus, our pDA-coating surface should prevent serum aggregates containing TGF-β1 to deposit onto it. ECM stiffness has been noted as a contributing factor to cancer progression,46 and also regulates many cellular behaviors including EMT.47−50 TGF-β1 contributes to tumor progression and metastasis through induction of EMT with control of cytoskeletal rearrangements and cell migration,51 suggesting a role in mechanotransduction.52 However, little is known about how mechanotransduction and TGF-β1 signaling pathways interact in EMT progression, which receptor initiates signaling, and whether such cross-talk involves membrane− proximal interactions or cooperation in the downstream signaling pathways. Using our stiffness-tunable matrix, we found either stiff matrix or i-TGF in the soft matrix could stimulate HCC cells to form typical well-polarized epithelial phenotype with intercellular adhesion, different from the initial inactive state showing by the round morphology. This highlights that local mechanical or biochemical stimulation at the ventral side of the cell would lead to similar cell responses, which indicates the possible potential of a synergic effect of coordination and cooperation by two types of signals. As expected, the stiff i-TGF drastically increased cell area (1.85fold to the stiff “No TGF” and 1.21-fold to the soft i-TGF) and migration speed (2.08-fold to the stiff “No TGF” and 1.54-fold to the soft i-TGF). Additionally, the stiff i-TGF was the only condition in which HCC cells acquired a highly motile fibroblastic or mesenchymal phenotype, with obvious expression of N-cadherin and vimentin but negative in Ecadherin (Figure 3). Our data demonstrated that either stiff matrix or i-TGF was able to initiate EMT of HCC cells, but they were both required to efficiently accelerate EMT progression in cancer metastasis. The subsequent question addressed identity of interactions between mechano-receptors and TGF-β1 receptor TβRI. The integrin family plays a critical role in many cellular processes that include cell adhesion, spreading, migration and signaling pathways toward stimulants/growth factors.33 β1 integrin, reported to exhibit a positive correlation with ECM mechanical stiffness in the development of hepatic cirrhosis and HCC,53 was chosen as a candidate to study its functions in the stiff iTGF induced HCC cell behaviors. The topology of our matrix was optimal for the proximity between TβRI and β1 integrin to favor their cross-talks. We found that β1 integrin expression obviously increased in the presence of i-TGF, especially on the stiff matrix (Figure 5), but β1 integrin did not significantly stimulate TβRI expression inversely. In addition, i-TGF triggered vinculin plaques disappeared after TβR1 inhibition, while a few small vinculin dots or fibrils could still be found along the cell periphery in the stiff conditions. However, vinculin expression was largely diminished in all the conditions and FAs could barely be found after β1 integrin inhibition (Figure SI6). Consequently, we propose i-TGF was able to provide two anchorage points for HCC cells. When cells started to contact with matrix surface, i-TGF-TβRI interaction functioned as the initial step to increase contact area for better sensing stiffness, which would enhance β1 integrin expression and develop further anchorage (Figure 7). Two types of mechanisms have been proposed to explain the relationships between mechanotransduction and growth factor

3. DISCUSSION Understanding how cancer cells integrate multiple types of stimulation to achieve specific progression is a challenging question in cancer studies. The difficulty comes from used experimental conditions that do not possess the potential to copresent several ECM properties. For example, in the published studies of TGF-β1 on EMT in HCC cells, cells were usually cultured on the tissue culture plastic or glass coverslips, and then exposed to soluble TGF-β1 in the medium. The induced mesenchymal changes were found,38 even associated with a switch in the expression of stem genes as well as the enhanced stemness potential and migratory/ invasive capacity.39 However, there would be two problems existing in this manner of cellular studies. First, stiffness of common cultural materials is too much higher (Young’s modulus of tissue culture polystyrene is ∼3 GPa).40 Besides, these synthetic materials are not representative of the actual ECM components encountered in tumors. Therefore, an appropriate biomaterial model is required to harbor tunable mechanical properties to promote localized growth factor signaling, to better mimic the actual in vivo ECM conditions. Here we used a thin biomaterial made by self-assembly of HA and PLL, and obtained low cross-linked (soft matrix) and high cross-linked films (stiff matrix). Previously, growth factors (such as BMP-2, SDF-1α) was postloaded onto the films to obtain a matrix-bound manner presentation, with tunable localized concentrations as the highest as 1 μg/cm2 for BMP218 and 5 μg/cm2 for SDF-1α.19,41 It was found that BMP-2 diffused throughout the entire (PLL/HA)12 film, thus leading to its homogeneous distribution in the films.18 The major proportion of growth factors were inside of films but not on the surface, which could not directly contact with cells to perform their bioactivities. Thus, the previous process leaves significant room for improvement to enhance functional efficiency of immobilized growth factors. A facile surface modification method can be applied to versatile solid materials from metal to synthetic polymers by simple coating with dopamine.42 Dopamine can undergo oxidative polymerization in slightly basic conditions, to create a stable layer that is adherent to the surfaces, without affecting the mechanical properties of materials (up to 960 min coating).24 Consistently, we found no evident changes in Young’s modulus either on soft or stiff matrix after pDA coating (Figure 1B). The pDA coating time was chosen 1 h to avoid the formation of aggregates, and the deposited pDA were homogeneously distributed throughout the area without significant change in surface topography (Figure 1D). Postmodification of pDA layer allows for the functionalization of bioactive molecules containing thiols or primary amines.43 The high efficiency of growth factor immobilization (90%) (Figure 1C) was observed by using pDA coating as the anchoring layer, in comparison to that of physical entrapment (less than 20%).19,41,44 Additionally, in our culture conditions (FBS-containing growth medium, pH 7.4), soluble TGF-β1 was easily masked by massive FBS in the medium due to electrostatic interactions H


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Mili-Q water. TGF-β1 was diluted to desired initial concentrations, deposited, and left overnight at 4 °C. The washing process by HepesNaCl buffer was repeated to keep only immobilized TGF-β1. To quantify the amount of TGF-β1 immobilized on films, we measured the concentration of TGF-β1 in the remaining solution after loading and in combined washing solution by using an enzymelinked immunosorbent assay (ELISA) kit (LiankeBio, Hangzhou, China). The density of immobilized TGF-β1 was calculated from the difference between the initial and remaining amounts of TGF-β1. 5.3. Surface Characterization. Surface atomic composition of pDA coated films was analyzed with X-ray photoelectron spectroscopy. An ESCA LAB 220I (Thermo VG Scientific) spectrometer was used with a magnesium anode source producing Mg−K (1253.6 eV photons) X-ray with pass energy of 20 eV for high-resolution narrow scans. To measure stiffness, silicon wafers coated with the films were characterized by an atomic force microscope (multimode 8, Bruker). The probe (SNL-10, Bruker) had a nominal spring constant of 0.06 N/m. The samples were immersed in a drop of 0.15 M NaCl solution, and the tests were performed in a liquid environment. For each sample, measurements were obtained at eight random sites, and the data was analyzed by NanoScope Analysis software using the Sneddon model (a better model for soft or biological samples indicated by the manufacturer) as modulus fit model. The fitting range was from 30 to 90%. To visualize surface topography, the oscillation mode was used on a Tap190Al-G (Budget sensors) probe. The resonance frequency was 180 kHz. The image was obtained using SPIP image processing software (Image Metrology, Denmark). 5.4. Cell Culture. Human HCC cells HepG2 were obtained from Chinese Academy of Sciences and cultured in DMEM medium supplemented with 10% FBS and 1% P/S antibiotics, at 37 °C in a humidified atmosphere containing 5% CO2. Cells with 90% confluence were harvested by using trypsin-EDTA and passaged or seeded onto the substrates for further experiments. 5.5. Quantification of Cell Adhesion and Morphology. HCC cells were seeded in matrix without TGF-β1 (No TGF), with 10 ng/ mL soluble TGF-β1 added in cell culture media (s-TGF)56 and with immobilized TGF-β1 (i-TGF) at a density of 15,000 cells/cm2. After culture, cells were fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized in TBS containing 0.1% Triton X-100 for 10 min. After rinsing 3 times with TBS, the slides were blocked with 0.1% BSA in TBS for 1 h. Then cells were incubated with Actin Tracker Green (Beyotime, China) in TBS with 0.1% BSA for 1 h. After the cells were rinsed 3 times, the nuclei were stained with DAPI. All samples were mounted onto glass slides with antifade reagent and viewed under a fluorescence microscope. The images were analyzed with ImageJ software (NIH, USA) to determine four different parameters: cell number, cell area, circularity, and aspect ratio. 5.6. Quantification of Cell Migration. For individual cell migration on films, HCC cells were seeded at 15 000 cells/cm2. After 4 h incubation at 37 °C, cells were imaged every 10 min for 16 h using a confocal microscope (LSM, Carl Zeiss) in a 5% CO2 atmosphere at 37 °C. Time-lapse images and movies were assembled using ImageJ software. Individual cell tracking was performed using the “Manual tracking” plugin. All data was analyzed using the “Chemotaxis tool” plugin to acquire cell migration speeds. For crowded cell migration on films, wound healing assay was performed by using a culture insert. HCC cells were seeded at a high density of 60 000 cells/cm2. After overnight incubation, the culture insert was removed and washed twice with PBS, and a cell-free gap of 600 μm remained. A fresh growth medium was added to initiate cell migration, then the cells were washed with PBS twice after 24 h incubation. The cells were imaged using an inverted microscope before and after wound healing. 5.7. Immunofluorescence. Cells were labeled with antibodies against TGFR (Abcam), β1 integrin (Abcam), vinculin (Sigma), E cadherin (CST) and Vimentin (Sigma). Primary antibodies were detected using Alexa Fluor 488-conjugated AffiniPure goat antirabbit or Alexa Fluor 568-conjugated AffiniPure goat antimouse antibodies

signaling: (1) Matrix stiffness may regulate mechanotransduction in parallel to TGF-β1 signaling because it does not affect the Smad2/3 signaling pathway.54 (2) Integrin mechanotransduction potentiates TGF-β1-dependent PI3K signaling.34 Hintz et al. also suggested that the TGF-β1/LTBP-1 binding domains acted as a sensor in a mechanical model of integrinmediated TGF-β1 activation.55 In our study, we found that mechanotransduction and TGF-β1 signaling barely functioned in a parallel manner, but in a sequential order. First, i-TGF can increase the TβRI expression to activate the PI3K pathway, then i-TGF-TβRI interactions promote HCC cell adhesion to enlarge contact area for stiffness sensing, resulting in the raising expression of β1 integrin. More β1 integrin will strengthen the β1 integrin/vinculin/p-FAK pathway, and finally nobly enhance PI3K signaling in turn. Of note, a novel point-ofview has been provided to demonstrate immobilized growth factors enhancing mechanotransduction in the EMT.

4. CONCLUSIONS In this study, we engineered a tumoral ECM-mimicking matrix by biopolymeric thin films with tunable mechanical properties and used dopamine’s ability to immobilize the growth factor TGF-β1 in a high efficiency. Immobilized TGF-β1 nobly increased cell spreading, migration and percentage of epithelial phenotype when HCC cells were incubated in the soft matrix, while HCC cells were found to exhibit the similar responses to the stiff matrix itself without any TGF-β1 stimulation. Furthermore, the stiff matrix with immobilized TGF-β1 was the only condition to induce mesenchymal phenotype and promote the EMT progression of HCC cells. We evidenced only in the immobilized TGF-β1 condition, the TGF-β1 receptor TβRI could enhance the expression of an important mechano-sensor β1 integrin, following by a profound activation effect on FAK and PI3K phosphorylation. Thus, our model offers great potential for investigating how multiple properties of ECM interact on the same dimension in a membrane proximal manner and reveals hidden molecular mechanisms of cancer progression that cannot be achieved by the commonly used cell culture condition. 5. EXPERIMENTAL SECTION 5.1. (PLL/HA) Film Preparation and Cross-Linking. HA (MW 360 000 g/mol) was purchased from Lifecore (Chaska, USA). PLL (P2636) and PEI (polyethylenimine, 7 × 104 g/mol) were purchased from Sigma (St. Louis, USA). (PLL/HA) film building and crosslinking were done as previously described.41 Briefly, PLL (0.5 mg/ mL) and HA (1 mg/mL) were dissolved in Hepes-NaCl buffer. Glass slides were immersed in the PLL solution for 8 min. After being rinsed with 150 mM NaCl solution for 3 times, the slides were immersed in the HA solution for 8 min. The slides were then rinsed again, and the sequence was repeated 12 times. For 96-well plates, films fabrication was started with a first layer of PEI at 5 mg/mL followed by the deposition of a HA-(PLL/HA)12 film. Films were cross-linked for 18 h at 4 °C using 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide at 30 or 70 mg/mL and sulfo N-hydrosulfosuccinimide at 11 mg/mL. Final washing was performed with the Hepes-NaCl buffer for 1 h. 5.2. pDA Coating and TGF-β1 Immobilization. The crosslinked films were immersed in dopamine solution (2 mg/mL in 10 mM Tris−HCl buffer, pH 8.5), and then gently shaken on a rocker for 1 h at room temperature. Following the reaction, the substrates were vigorously washed with Mili-Q deionized water three times, and the pDA coated films were stabilized at 40 °C for 2 h. Recombinant human TGF-β1was purchased from PeproTech (Rocky Hill, USA) and the storage concentration was 1 μg/mL in I


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ACS Applied Materials & Interfaces Notes

(Proteintech). All the slides were mounted onto coverslips with antifade reagent. Fluorescence images were obtained using a confocal microscope. 5.8. Inhibition of Receptors. For inhibition of β1 integrin-FAK pathway using the anti-β1 integrin antibody (ab24693, Abcam), HCC cells (1 × 105/mL) were preincubated for 20 min at room temperature with blocking antibodies (0.5 μg/mL). Then, the cells were seeded at 15 000 cells/cm2 for all groups and incubated for 48h in the presence of 0.5 μg/mL antibodies. For inhibition of TβRI receptor, the specific inhibitor (LY2157299) was dissolved in DMSO at 10 mM. Then HCC cells for all groups were seeded at 15 000 cells/ cm2 and incubated for 48h with LY2157299 at a final concentration of 1 μM. 5.9. Immunoblotting. HCC cells were seeded at 15 000 cells/ cm2 for 48 h, and then scraped, rinsed in PBS, lysed with RIPA buffer containing a protease and phosphatase inhibitor cocktail (Roche) for 30 min on ice followed by centrifugation at 14,000 rpm for 10 min. The total protein content was determined using a BCA protein assay kit (Thermo Fisher Scientific). Twenty μg of total protein samples were loaded and run on polyacrylamide gels before transfer onto PVDF membranes (Merck Millipore). Membranes were then saturated in 5% BSA in TBS with 0.1% Tween 20 for 1 h and subsequently incubated with antibodies against β1 integrin (Abcam), TβR1(Abcam), E-cadherin (CST), N-cadherin (Abcam), vimentin (Sigma), FAK (CST), p-FAK (CST), PI3K (Abcam), p-PI3K (Abcam), and β-actin (Sungene Biotech). Membranes were washed and incubated with peroxidase-conjugated antimouse, antirat, or antirabbit secondary antibodies (Thermo Fisher Scientific), respectively. Peroxidase activity was visualized by ECL (Thermo Fisher Scientific) using a GeneGnome XRQ-Chemiluminescence imaging system (Syngene). 5.10. Statistical Analysis. Data for at least 50 cells are presented as box plots (1st quartile, median, third quartile, the limits being 10 and 90% and the extreme values 5 and 95%). Experiments were conducted at least three times, and data are presented as mean ± SD. Statistical analysis was performed between more than two groups using analysis of variance (ANOVA) and pair wise comparisons to obtain p values. p ≥ 0.05 was considered not significant with an abbreviation as “ns”, whereas p < 0.05 was considered significant (0.01≤ p < 0.05 was indicated by *, 0.001≤ p < 0.01 was indicated by **, and p < 0.001 was indicated by ***).

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Dr. Hong Li of Xi’an Shiyou University for fruitful discussions. We also thank the Analysis and Testing Center of Huazhong University of Science and Technology for technical assistance. This work was supported by National Natural Science Foundation of China (81803108), Fundamental Research Fund for Central Universities (2018KFYYXJJ023), Natural Science Foundation of Hubei Province (2018CFB113) and the starting grant from Huazhong University of Science and Technology (3004514110).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b03572.

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REFERENCES

Release of TGF-β1 in the soft and stiff i-TGF conditions after 24 and 48 h (Figure SI1); adhesion and morphology of HCC cells cultured in the matrix with and without pDA coating (Figure SI2); morphology and spreading area of HCC cells cultured in the soft and stiff i-TGF matrix with different amount of immobilized TGF-β1 (Figure SI3); proliferation rate analysis of HCC cells by MTT assay after 12 and 48 h incubation (Figure SI4); inhibition results of TβRI and β1 integrin receptors (Figure SI5); effect of inhibition of TβR1 and β1 integrin on vinculin (Figure SI6) (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (X.Q.L). ORCID

Xi-Qiu Liu: 0000-0002-4842-8175 Author Contributions †

R.Z.T. and S.S.G. contributed equally to this work. J


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Immobilized Transforming Growth Factor-Beta 1 in a Stiffness

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