Direct Micropatterning of Extracellular Matrix Proteins on

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Characterization, Synthesis, and Modifications

Direct micropatterning of ECM proteins on functionalized polyacrylamide hydrogels shows geometric regulation of cell-cell junctions Bapi Sarker, Christopher Walter, and Amit Pathak ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00331 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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ACS Biomaterials Science & Engineering

Direct micropatterning of ECM proteins on functionalized polyacrylamide hydrogels shows geometric regulation of cell-cell junctions

Bapi Sarker1, Christopher Walter2, and Amit Pathak1,2,*

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Department of Mechanical Engineering & Materials Science, Washington University, St. Louis, MO 63130, USA

2

Department of Biomedical Engineering, Washington University, St. Louis, MO 63130, USA

* To whom correspondence should be addressed: Amit Pathak, Ph.D. One Brookings Dr, CB 1185 St. Louis, MO 63130 Ph: (314) 935-7585 Email: [email protected]

KEYWORDS: micro-patterning; matrix topography; E-cadherin; matrix stiffness; confinement; cell shape; cell clusters; mechanobiology.

ACS Paragon Plus Environment

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Abstract Micro-contact printing of extracellular matrix (ECM) proteins in defined regions of a substrate allows spatial control over cell attachment and enables the study of cellular response to irregular ECM geometries. Over the last decade, numerous micropatterning techniques have emerged that conjugate ECM proteins on hydrogel substrates of tunable stiffness, which have revealed a range of cellular responses to varying matrix stiffness and geometry. However, micropatterning of ECM proteins on polyacrylamide (PA) hydrogel remains inconsistent due to its unreliable conjugation with the commonly used protein crosslinkers, particularly at low stiffness. To address these problems, we have developed a micropatterning technique in which the PA gel is functionalized by incorporating oxidized N-hydroxyethylacrylamide (HEA), which allows direct protein binding through reactive aldehyde groups without any exogenous crosslinkers. As a result, a uniform and consistent protein transfer onto the hydrogel substrates of defined geometries is achieved, even for soft PA gels. We formed square, rectangular, and triangular patterns of two constant areas on soft and stiff PA gels that provide large and small adhesive areas for the MCF10A human mammary epithelial cell pairs. We measured intercellular Ecadherin expression and found that cell-cell junctions could be deteriorated independently by either the stiff ECM of any shape or the elongated cell morphology, accompanied by increased cell-generated tractions, on rectangular soft ECM patterns. Inhibition of non-muscle myosin II reduced the E-cad junctional localization in cell pairs. When the cell spreading was restricted by reducing the adhesive area of the patterns, we observed an overall rise in E-cad expression at cell-cell junctions. Our findings present an improved micropatterning technique, which reveals a geometric regulation of cell-cell junctions in epithelial cell-pairs.


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Introduction Living cells sense and respond to their surrounding extracellular matrix (ECM) through adhesions, protrusions, and intracellular forces, all of which are fundamental regulators of key cellular functions such as migration, proliferation, and apoptosis.1 When attached to stiffer matrix, cells are known to undergo a mechano-activation through increased adhesions and actinmyosin contractility.2-4 It is also well-recognized that matrix geometry can alter subcellular adhesion localization and actin-myosin stress fiber orientation,5-7 which lead to variations in cell spreading, proliferation, and migration.8-9 These studies have been possible by forming defined geometric patterns of ECM proteins on cell culture substrates through a variety of micro-contact printing techniques.5,

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Over the years, polyacrylamide (PA) hydrogel has been a popular

candidate for micropatterning because of its tunable stiffness over two orders of magnitude.11-13 The micro-contact printing of ECM proteins in defined regions of the PA surface can be implemented because the PA gel possesses antifouling properties that prevent non-specific protein binding and cell adhesion.14 However, the unfavorable wettability and absence of protein-binding functional groups in the PA composition have continued to pose challenges in micropatterning ECM proteins at desired consistency and efficiency. To surmount the nonbinding properties of PA, different external crosslinkers haven introduced, such as the Nhydroxysuccinimide (NHS)-ester15 and NHS-ester-containing heterobifunctional cross-linker sulfosuccinimidyl

6-[40-azido-20-nitro-phenylamino]hexanoate

(sulfo-SANPAH).16

These

strategies have also been adopted for the micropatterning of ECM proteins on PA gels. In addition to these crosslinkers, hydrazine and protein oxidation by periodate17 and deep UV


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exposure18 strategies have also been applied for the micropatterning of ECM proteins onto the PA gels. In popular micropatterning methods, an external chemical crosslinker, such as sulfoSANPAH19 or NHS–ethyl(dimethylaminopropyl)carbodiimide (EDC),18 is required to bind ECM proteins to the PA gel network. Although these crosslinkers have been used for tethering proteins to the PA gel, they present several fundamental limitations in micropatterning. Namely, these crosslinkers are unstable in aqueous environments, which may lead to rapid decrease of protein binding activity when solubilized and cause inconsistent crosslinking efficiency.14,

17

The

successful transfer of proteins to the substrate requires a dry surface, which is particularly difficult to achieve for soft and viscous PA gels. Since the half-life of the NHS-ester in sulfoSANPAH is very short (less than 20 min) at neutral pH,13 the time window of achieving a dry gel after sulfo-SANPAH treatment is quite narrow. As an alternative to the sulfo-SANPAH-based crosslinking of proteins, Grevesse et al.20 incorporated N-hydroxyethylacrylamide (HEA) monomers into the PA gel, resulting in the presence of hydroxyl groups that could form hydrogen bonds with proteins. Using HEA in PA gel opens new possibilities of functionalization of PA gel network to achieve successful micropatterning with strong protein adhesion. Since both the geometry of ECM proteins and the substrate stiffness have fundamental implications for intercellular junctions of epithelial cells and related physiology,11,

18, 21

it is

important to combine both these matrix properties to study cellular responses. From tumor metastasis to morphogenesis, grouped cells often find themselves in tissue microenvironments of varying confinements and geometries.8, 22-23 To deconstruct these complex scenarios of cell-cell versus cell-matrix interactions, focusing on a pair of cells in contact with a tunable matrix substrate can serve as a useful reductionist system. In previous studies of the role of cell-cell


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junctions in cell pairs, the micro-patterns of ECM proteins have been fabricated by using deepUV activation and two-steps protein transferring methods.21,24

On non-continuous micro-

patterns of fibronectin on PA, the intercellular tension in the cell-pair was found to depend on the position and orientation of the intercellular junction on ECM protein.21 However, it remains unclear how the intercellular junctions in cell pairs would behave on soft and stiff adhesive substrates of varying shapes and sizes that alter cell spreading, symmetry of cell placements, and orientation of cell-cell junctions. In our current work, we have developed a method for functionalizing the entire PA gel through oxidized HEA, which contains reactive aldehyde groups that facilitate improved protein binding to the PA gel substrate. Our method of protein-PA covalent bonds through aldehyde groups should provide stronger protein conjugation, compared to the prior approaches of proteinPA hydrogen bonds through hydroxyl groups from pristine HEA-based functionalization. We use this PA gel system of two different elasticities to pattern collagen type I in three different shapes of identical area and investigate the geometric regulation of intercellular junctions in a cell-pair. We monitor the localization of E-cadherin (E-cad), an epithelial transmembrane protein, at the intercellular junction of a cell-pair grown on the collagen patterns of various shapes and sizes. We found that cell-cell junctions can be weakened independently by either increasing the PA stiffness or using an elongated adhesive pattern. The E-cad localization is further reduced in cases where the cell-pair did not cover the entire micro-pattern area. Collectively, our findings present a new method of micropatterning ECM proteins on PA substrates of tunable stiffness and demonstrate the regulation of cell-cell junctions by both matrix geometry and stiffness.


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Experimental Fabrication of PDMS stamps Silicon masters with defined embedded geometries were fabricated using a standard photolithographic technique, in which silicon wafers were spin-coated with a photoresist (SU-8 2050, Microchem) to a thickness of 40 µm and exposed to 365 nm UV light through a transparency photo-mask of defined topographical features. Three geometries of specific areas (2500 and 1600 µm2) were used: a square, an equilateral triangle, and a rectangle of 4:1 aspect ratio. Polydimethylsiloxane (PDMS) stamps of these defined geometries were fabricated by polymerizing PDMS on the silicon master mold. In this step the PDMS prepolymer solution was prepared by mixing base and curing agent of Sylgard 184 Silicone Elastomer (Dow Corning, Midland, MI) in 10:1 weight ratio. The prepolymer solution was then degassed and poured onto the master mold and subsequently cured at 70 °C for an hour. The stamps were cut into cubes of dimension 5 mm × 5 mm × 2 mm.

Polyacrylamide gel preparation and micropatterning of ECM proteins Polyacrylamide (PA) gels were chemically modified to facilitate protein conjugation onto its surface. Chemically modified PA gels with desired stiffness were fabricated on a glass coverslip (diameter 15 mm, Fisher Scientific). First, N-hydroxyethylacrylamide (HEA, Sigma Aldrich, St. Louis, MO, USA) was oxidized by adding 0.02 M sodium metaperiodate (Sigma Aldrich, St. Louis, MO, USA) into 67 and 200 µL of HEA for soft and stiff PA gels, respectively. The mixtures were incubated at room temperature under dark conditions and continuous shaking for


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4 hours to facilitate free aldehydes generation in HEA. PA precursor solutions were prepared by mixing varying amounts of acrylamide (A, Bio-Rad) and bis-acrylamide (B, Bio-Rad) according to the previous stiffness characterization of PA gels as the monomer (A) : crosslinker (B) percentages of 4% A : 0.2% B and 12% A : 0.145% B for soft and stiff PA gels, respectively.12-13 The freshly prepared 67 and 200 µL of oxidized HEA was added to 5 mL of PA precursor solutions for soft and stiff PA gels, respectively. To initiate polymerization, 0.5% ammonium persulfate (APS, Sigma) and 0.05% tetramethylethylenediamine (TEMED, Sigma) were added to the precursor mixtures. On an activated glass coverslip, 35 µL of gel precursor solution was pipetted and flattened by a hydrophobic surface. The coverslip containing PA gels were gently detached after polymerization. Gel surface was air-dried for 30-40 mins prior to micropatterning. Collagen solution (rat tail type-I, Santa Cruz Biotechnology, USA) at a concentration of 50 µg/mL in phosphate buffered saline (PBS), was pooled onto the patterned side of PDMS stamps for 20 mins. Collagen solution was then aspirated and any excess solution was removed by gently blowing nitrogen gas. As shown in Fig. 1, the stamp was brought in contact with the PA gel surface and a pressure was applied by placing a weight of 10 g onto the PDMS stamp. After 30 mins, the stamp was removed and the protein was transferred from the PDMS stamp to the PA gel surface through covalent conjugation of primary amine groups of proteins to the reactive free aldehyde groups on the modified PA gel. Micropatterning of fibronectin was performed in the same manner as stated above using 50 µg/mL solution of human plasma fibronectin (EMD Millipore). To visualize the fibronectin patterns, the fixed fibronectinpatterned PA gels were incubated overnight at 4 °C with mouse monoclonal anti-fibronectin (Santa Cruz Biotechnology; 1:200 dilution). Secondary antibody labelling was performed using the same procedure with Alexa Fluor 488-conjugated anti-mouse (Cell signaling; 1:500 dilution).


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The micropatterns were imaged using a Zeiss LSM 880 laser-scanning confocal microscope (Carl Zeiss Microscopy).

Mechanical characterization of PA gels Mechanical characterization of PA gels were performed using an MFP-3D-BIO atomic force microscope (AFM, Asylum Research, Santa Barbara, CA), as we have done earlier.25-26 Olympus TR400PB AFM probes with an Au/Cr coated silicon nitride cantilever and pyramidal tip were used. Measurements were acquired by indenting the gels using the AFM probe with spring constants of 20–30 pN⁄nm, as measured by thermal calibration. Elastic moduli were analyzed from force curves using a modified Hertz model.27

Cell culture and immunofluorescence The micropatterned PA gels were transferred to a non-tissue culture 12-multiwell plate and washed three times with PBS and sterilized with UV light in the tissue culture (TC) hood for 2 hours and washed subsequently with sterile PBS. The unpatterned regions were passivated with bovine serum albumin (BSA, Fraction V, Merck, USA) solution (1 vol%) overnight at 4 °C. BSA was aspirated and the gels were washed three times with sterile PBS. Human mammary epithelial cells (MCF-10A) were seeded onto the gel at a density of 3,000 cells per cm2 of total bottom-surface area of a well of 12-multiwell plate. MCF10A cells were grown in DMEM/F12 (Invitrogen), supplemented with 5% (v/v) horse serum (Invitrogen), 20 ng/mL epidermal growth factor (EGF, Miltenyi Biotec Inc), 0.5 mg/mL hydrocortisone (Sigma-Aldrich), 100 ng/mL cholera toxin (Sigma-Aldrich), 10 ug/mL insulin (Sigma-Aldrich), and 1% (v/v) penicillin-


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streptomycin (Sigma-Aldrich) as previously described.26 After 2 hours of culturing, the nonadhered cells were washed off with sterile PBS and fresh cell culture media was added, as described earlier.11, 28-29 To investigate the effect of nonmuscle myosin II inhibition on cell-cell junctional E-cad expression, blebbistatin (Sigma-Aldrich) was added to the cell culture medium at the final concentration of 10 µM after 16 h of cell seeding. Cells were fixed with 4% paraformaldehyde in PBS after 36 hours of culturing, followed by permeabilization of cell membrane with 0.1% Triton- X 100 (Sigma, USA) and blocking with 2% BSA in PBS. Primary antibody labelling was performed in 1% BSA in PBS overnight at 4 °C with mouse monoclonal E-cadherin (E-cad, BD Biosciences; 1:400 dilution). Secondary antibody labelling was performed using the same procedure with Alexa Fluor 488-labeled goat anti-mouse (Invitrogen; 1:500 dilution). Cellular F-actin and nuclei were stained with rhodamine phalloidin (Invitrogen; 1:200 dilution) and 4́,6-diamidino-2-phenylindole (DAPI; 1:500 dilution), respectively.

Confocal microscopy and image analysis Confocal microscopy was performed using a Nikon A1Rsi confocal microscope (Nikon Instruments Inc.), where z-stacks were acquired at 1 µm interval and combined with the Zprojection tool in ImageJ (NIH). All images were taken using same acquisition parameters including laser power, scan speed, and pixel resolution to ensure accurate quantitative image analysis. Only the patterns that had two cells were considered for analysis. E-cad expression at the junction of the two cells on each pattern was computed by analyzing peak fluorescence intensity, width, and area of the junction of adjoining cells as described ahead (Fig. 3a). The junctional and cytoplasmic E-cad expression were calculated along several lines drawn through


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the adjoining cells. As a separate method, integrated intercellular junctional E-cad expression was also analyzed after drawing a region of interest (ROI) around the cell-cell junction, as illustrated in Fig. 3b. Mean integrated E-cad expression was presented as the integrated fluorescent intensity per micron length of the cell-cell junction.

Traction microscopy Fluorescent beads of size 0.2 µm (dark red, F-8807; Invitrogen) were mixed in our soft (~1 kPa) PA gel precursor solution at 0.3% (v/v) from a stock solution. Collagen micropatterning was performed and cells were seeded as previously described. Images of fluorescent beads and DIC images of cells were taken with a Zeiss LSM 880 laser-scanning confocal microscope (Carl Zeiss Microscopy, Germany), where z-stacks were acquired at 0.3 µm interval. Another set of images was taken after trypsinization of cells followed by washing with PBS. Drift correction was made using an ImageJ plug-in, Template Matching and Slice Alignment.21 Bead displacements were detected using the Particle image velocimetry ImageJ plug-in based on the accompanying crosscorrelation algorithm.21 The traction field was obtained using regularized Fourier transform traction cytometry (FTTC) ImageJ plug-in. Mean traction for each cell pair on the micropatterns was calculated from the traction heat map using ImageJ by drawing ROI around the adhesive pattern.


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Fluorescent labelling of collagen and imaging of micropatterns To fluorescently label collagen I, we modified a previously described method.30 Briefly, 1.5 mL volume of a 3 mg mL-1 collagen I solution (pH~7.5) was added to a well of a 12-multiwell plate and gelled at 37 °C. The collagen gel was incubated with 0.2 M sodium bi-carbonate buffer (pH 9.0, Sigma Aldrich, USA) for about 10 min to maintain the pH of the reaction mixture. The buffer was aspirated and replaced by 500 µL of Sulfo-Cyanine5 NHS ester dye (Lumiprobe Co. USA) solution (dissolved in DMSO) and incubated at room temperature in the dark for 1 h. The concentration of the dye solution (in DMSO) was used according to the calculation based on the molar excess of the NHS-ester provided in supplier’s protocol. The dye solution was aspirated and 3 mL of 50 mM TRIS buffer (pH 7.5, Sigma Aldrich, USA) was added subsequently to the gel to quench remaining NHS ester. The stained collagen gel was then washed 6 times with PBS over 2 hours. 200 mM HCl (Sigma Aldrich, USA) was added to the collagen gel and mixed until the gel was completely solubilized. The resulted collagen solution was then dialyzed against 20 mM acetic acid (Sigma Aldrich, USA) at a ratio of 1:1000 (protein solution: dialysis buffer) in melting ice with continuous stirring for 4 h using a 10000 MWCO dialysis cassette (Thermo Scientific, USA). The labelled collagen solution was kept at 4 °C in the dark. About 4% of the unlabeled collagen I solution (50 µg/mL) was removed and replaced with same amount of fluorescently labelled collagen I solution for micropatterning on PA gel. The micropatterns were imaged (Fig. 2) using a Zeiss LSM 880 laser-scanning confocal microscope (Carl Zeiss Microscopy, Germany). Uniformity of the micropatterns was investigated by analyzing gray level intensities along a line across the patterns after background subtraction using ImageJ software. The analysis was performed along the multiple lines and representative plot of the gray values are shown in Figure 2. Integrated fluorescent intensity of the patterns of three different


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shapes on soft and stiff PA gels was analyzed by drawing region of interest (ROI) around the individual pattern after background subtraction. 60 patterns of each condition were analyzed and mean fluorescent intensity is plotted.

Statistical analysis Data are presented as the mean ± standard error, unless noted otherwise. Statistical significance was determined by one-way analysis of variance (ANOVA). The pairwise comparison of the means was performed with a Bonferroni’s test (post hoc comparison). Differences were considered as statistically significant for p-values < 0.05. Means were calculated from at least 10 micropatterns for all conditions.

Results & Discussion Whole-gel functionalization allows uniform protein transfer during micropatterning. Since the PA network does not have binding sites for ECM proteins, its functionalization is necessary to facilitate protein-substrate adhesion during micropatterning. In this study, we synthesized PA hydrogels of varying stiffness (soft and stiff) by co-polymerizing acrylamide (A) and bis-acrylamide (B) in different ratios, as indicated earlier. The compositions, 4% A : 0.2% B and 12% A : 0.145% B resulted in the Young’s Modulus of 0.97±0.03 and 6.9±0.5 kPa, respectively, both based on AFM measurements (Fig. 2a). We refer to the ~1 kPa gel as ‘soft’ and ~7 kPa gel as ‘stiff’ henceforth. In these gels, oxidized N-hydroxyethylacrylamide (HEA)


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containing reactive aldehyde groups (Fig. 1) resulted in the whole-gel functionalization. The aldehyde groups can bind to the ɛ-amino groups of the ECM protein by forming Schiff’s base bond.31 This covalent bonding facilitated micropatterning of collagen onto the PA substrate. We printed collagen micropatterns of various shapes (square, triangle and rectangle) and a constant surface area (2500 µm2) on the functionalized soft and stiff PA gels, as shown in Fig. 2b. To confirm a uniform transfer of protein to the soft and stiff PA gels, we acquired confocal images of fluorescent-labelled collagen micropatterns (Fig. 2). Here, the deep contrast between the patterned and unpatterned (BSA-passivated) regions can restrict cell spreading in defined geometries. The uniformity of protein micropatterning on the functionalized PA gels was verified by plotting fluorescence intensity of the Sulfo-Cyanine5 NHS ester-labelled collagen along a line (Fig. 2c). To further verify that protein transfer onto soft and stiff PA gels was similar, we quantified mean integrated fluorescent intensity on micro-patterns of various shapes on soft and stiff PA gels (Fig. 2d). To assess the efficacy of our method for another ECM protein, we performed micropatterning of fibronectin and visualized these patterns by immunostaining for fibronectin, as described earlier. The representative images provided as Supporting Information (Fig. S1) show that the presented method can work for proteins other than collagen, which is used for the epithelial cell-pair analyses in this study.

ECM geometry alters the stiffness-dependent E-cad localization in cell-pairs. In an epithelial monolayer, multiple cells form interconnected junctions in varying shapes, which makes it difficult to analyze how matrix geometry could influence cell-cell junctions. To this end, we focused our attention to a pair of cells (MCF-10A) forming a single line of cell-cell


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contact, which is made possible by our micropatterns of defined area. Since E-cad is a key constituent of intercellular junctions, we measured its localization at the cell-cell contact for cellpairs on three different ECM geometries (square, triangle and rectangle) with two different elasticities of the underlying PA gels. From the plot profile of E-cad expression across a line perpendicular to the cell-cell contact (Fig. 3a), we calculated the peak pixel intensity, the width of the cell-cell contact, and the area under the curve to integrate the width and peak expressions. To calculate E-cad expression over the entire cell-cell junction, we analyzed integrated fluorescent intensity of E-cad at the junction by drawing ROI around the cell-cell junction (as shown in Fig. 3b) using ImageJ and plotted the E-cad expression per micron-length of the cellcell junction. According to the data presented ahead, these different methods of measuring E-cad junctional expression agree with each other. To understand the roles of matrix stiffness and geometry on cell-cell junctions, we first analyzed micropatterns of varying shapes on which cellpairs covered the entire pattern. For soft PA substrates, we observed significantly higher intercellular junctional E-cad intensity on square-shaped patterns as compared to the triangular or rectangular ones (Fig. 4a,b). Similar outcomes were observed for the junctional E-cad width and area (Figs. 4c,d). It is possible that a symmetric arrangement of the two cells on square shapes preserves cell-cell junctions better than the elongated configuration of the rectangles.24 On the triangles, the two cells in the pair would generally occupy different shapes and areas, which might break the symmetry and deteriorate cell-cell junctions. Notably, no significant difference was found for the intercellular E-cad peak intensity, junction width, or the E-cad area between triangular and rectangular micropatterns on soft PA gel. We also observed that cell pairs arranged themselves in different shapes and spreading areas on the square, triangular and rectangular patterns (Fig. 4g).


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We argued that an asymmetric arrangement of the two cells on a pattern could affect the cell-cell junction. Therefore, we quantified this ‘asymmetry’ in terms of the difference in the spread areas of the two cells on a given micropattern. Here, the lesser difference in cell areas would correspond to higher degree of symmetry. As shown in Fig. 4e, the square patterns had the least difference in cell area, i.e., more symmetric spreading of the cell-pairs. In comparison, cell pairs on triangular patterns showed significantly higher cell area difference and asymmetric spreading. This asymmetric behavior of cell pairs could cause instability in cell-cell junction. We also quantified cell elongation in terms of aspect ratio. Although the cell spreading was not very asymmetric on rectangular patterns (Fig. 4e), the cells were highly elongated, as shown through high aspect ratio, compared to squares or triangles (Fig. 4f). These results indicate that both asymmetric cell spreading and cell elongation can be associated with reduced E-cad expression in cell-cell junctions. Since the cell pairs spread and maintain cell-cell junctions differentially on the three shapes, we next asked whether their ability to generate forces also varies with shape geometry. To assess tractions generated by the cell pairs, we plated cells on collagen-micropatterned soft PA gels suitable for high resolution traction microscopy.32 As shown in Fig. 4h, the tractions generated by the cell pairs were localized at the corners of the micropatterns. By averaging across multiple patterns, we show that the cell pairs generated the highest and the lowest tractions on rectangular and square patterns, respectively (Fig. 4i), which is consistent with earlier findings of higher forces for more elongated shapes.24 These findings suggest a correlation between higher tractions and lower junctional E-cad levels on more elongated patterns (Figs. 4a,i).


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On stiff PA substrates, the intercellular junctional E-cad expression was significantly reduced on square patterns, which is expected given the known mechanosensitive regulation of cell-cell junctions by matrix stiffness through an integrin-cadherin crosstalk.11, 33-37 These results reveal that the known stiffness-sensitive regulation of cell-cell junctions can be altered by matrix geometry. Namely, while a symmetric arrangement of epithelial cells (square) preserve stiffnesssensitivity, the elongated (rectangle) and asymmetric (triangle) shapes are detrimental to cell-cell junctions even on soft ECMs that otherwise protect epithelial integrity.

Reduced cell-cell junctions in partially spread cell-pairs. Given the known importance of cellular protrusions and spreading in the scattering of epithelial cells,33, 38 we used our micropatterns to assess cell-cell junctions in cell-pairs that occupied only 60-70% of the available adhesive area on the pattern. We found that the length of cell-cell contact reduced in partially spread cells (Fig. 5b), as compared to the coverage of at least 90% of the patterns discussed above in Fig. 4. The cells on rectangular patterns exhibited comparatively smaller contact length, which however, was not significant. Since the aspect ratio of rectangular pattern is very high (1:4) compared to square (1:1) and triangular (1:1) patterns, it is expected to allow a smaller intercellular contact length. In these partially spread cells, the intercellular E-cad localization was significantly lower compared to that of the >90% covered patterns (Figs. 5a,b). These cells may still be exploring the surrounding adhesive area, which could cause instability around the intercellular contact and create unfavorable conditions for the assembly of cell-cell junctions. Indeed, a previous study has shown that active protrusions of partially spread cells cause the dissociation of E-cad


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junctions and enable epithelial disintegration.38 In this study, when the cells fully covered the circular adhesive islands, even a mechano-stimulation through hepatocyte growth factor (HGF) treatment was not able to dissociate the epithelial cell-cell contacts.38 Consistent with this study, we have found that the cells that covered at least 90% of the patterns are more likely to preserve cell-cell junctions compared to the partially spread ones. Notably, the E-cad junction area on square patterns was higher compared to rectangles or triangles on soft PA gels. Thus, the cellcell junctions remain sensitive to both matrix stiffness and geometry even in case of partially spread cells.

Non-muscle myosin II inhibition reduces intercellular junctional E-cad expression. Non-muscle myosin II (NMII) does not only control integrin-mediated cell-matrix adhesion and cell migration, it also regulates cell polarization, morphogenesis and epithelial cell-cell junctions.39-40 To understand whether myosin activity affects cell-cell adhesion between a pair of cells in a confined region of various geometries, we inhibited NMII by blebbistatin treatment, which is known to abrogate the generation of actomysosin forces in cells. After myosin inhibition, junctional E-cad intensity decreased in the cell pairs on square and triangular patterns on both soft and stiff PA gels (Fig. 6). Interestingly, no change in E-cad expression was observed in the cell pairs on rectangular patterns on soft and stiff gels. Our observations are consistent with previous studies, in which NMII activity is found to be important for stable cell-cell contact formation and cadherin clustering.39-40

Notably, myosin-inhibition did not cause further

reduction in E-cad expression on rectangular patterns (Fig. 6), which could be because the cellcell junctions are already weakened by cell elongation on this geometry.


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Reducing the pattern size reduces cell spreading and enhances junctional E-cad expression. We have shown that the junctional E-cad expression decreased in the partially spread cell pairs that covered 60-70% of the total available adhesive patterned area on a pattern (Fig. 5). Next, to better delineate the role of cell spreading area relative to the available adhesive area, we fabricated smaller patterns with the adhesive area of 1600 µm2, which is 64% of the larger patterns (2500 µm2) used in prior experiments. In this study, we only considered the cells that covered the entire 1600 µm2 patterns for E-cad analysis. As reported in Fig. 7, the intercellular junctional E-cad expression increased in the cell pairs in all conditions compared to the cell pairs the covered at least 90% of the area of large patterns (2500 µm2). We found significantly lower E-cad localization at the cell-cell junction on rectangular patterns compared to square and triangular patterns on soft gels. On the rectangles, we found more asymmetric spreading, as indicated by higher cell area difference (Fig. 7b), and more elongated cell shapes (Fig. 7c). Thus, even with more restricted cell spreading on these smaller patterns, the shape of the pattern plays a role in regulating the state of cell-cell junctions. These findings provide new insights into the regulation of cell-cell contact by cell-ECM forces and anisotropic spreading caused by ECM shapes. 21, 24, 32


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Conclusions In summary, we have developed a functionalized PA gel system of tunable stiffness that facilitates precise micropatterning of desired ECM proteins. The outcomes of this study show that the transfer of proteins from the PDMS stamps of defined geometry to the surface of our functionalized PA gel can be achieved with uniform fidelity in a gel stiffness-independent manner. We combined ECM geometry with substrate stiffness, a well-recognized mechanotransduction cue, to understand how the integrity of cell-cell junctions is regulated by matrix properties. We found that asymmetric and elongated arrangements of cell-pairs on triangles and rectangles can hamper cell-cell junctions, as compared to the square patterns, even on soft PA gels that are known to protect epithelial integrity. The cell-generated tractions increased progressively as the pattern shapes change from squares to triangles and rectangles of a constant area. These findings indicate that as the cell-pairs adopt a more elongated morphology the rise in cellular forces occurs concurrently with a reduction in intercellular junctions. When the cells did not cover the entire available area on the pattern, the E-cad expression on cell-cell junctions was significantly reduced, compared to the fully spread cells on large patterns. Furthermore, reducing the adhesive area available for cell spreading enhances E-cad expression at the cell-cell junctions. Taken together, our results indicate that cell-cell junctions can be impaired by any of these factors: stiffer matrices, more elongated cell shapes, more asymmetric arrangements of cell-pairs, and less restricted spreading within a given area. These results demonstrate that our micropatterning methodology can be used to reveal new cellular responses to varying mechanical properties of the extracellular matrix, such as geometry and elasticity.


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Supporting Information Fluorescence images of micropatterned fibronectin, DIC images corresponding to the traction maps.

Acknowledgements This work was in part supported by grants to A.P. from the National Science Foundation (CAREER Award 1454016) and the Edward Mallinckrodt, Jr. Foundation (New Investigator Award). Lithography facilities were provided by the Institute of Materials Science & Engineering (IMSE) at Washington University in St. Louis.


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FIGURES

Figure 1. Functionalized PA hydrogels and micropatterning. Copolymerization of acrylamide, bis-acrylamide and oxidized N-hydroxyethylacrylamide (HEA) leads to PA gel with reactive aldehyde groups. Schematic describing the steps of micropatterning by stamping collagen-inked PDMS stamp onto the prepared functionalized PA gel.


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Figure 2. Uniform transfer of protein from PDMS stamp to PA hydrogel. (a) Young’s moduli of functionalized PA gels with varying acrylamide and bis-acrylamide ratio, where oxidized HEA was added to the PA gel precursor mixtures at a volume ratio of 1:7.5 (oxidized HEA : acrylamide). Gels with the stiffness of ~1 kPa and ~7 kPa are termed as ‘soft PA’ and ‘stiff PA’, respectively. N>30. Data from at least 3 PA gels of each composition is reported. (b) Fluorescence images of micropatterned Sulfo-Cyanine5 NHS ester-labelled collagen of various shapes on the functionalized PA gels of varying stiffness. Scale bar=100 µm. (c) Fluorescence intensity profile corresponding to the line of the fluorescence image, showing the uniformity of the collagen micropatterning. (d) Mean integrated fluorescent intensity of the micropatterns of various shapes on soft and stiff PA gels, exhibiting similar protein transfer onto soft and stiff PA gels. N>60 patterns. Values presented as mean ± SD.


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Figure 3. Measurement of cell-cell junctions from E-cad expression. (a) Schematic illustrating a method of quantifying E-cad junctional localization in which the E-cad expression along a line drawn between two cells through the cell-cell junction varies such that it reaches a peak value at the intercellular boundary, denoted as peak intensity. Here, the height of the peak is calculated as the intensity difference between peak value (junctional E-cad intensity) and average base values (start and end points of the junctional E-cad intensity curve). Width of the E-Cad junction is computed as the distance between the start and end points of the junctional E-cad intensity curve. The area of the intercellular junctional E-cad intensity curve is approximated as a triangle. (b) Schematic illustrating another method of quantifying overall intercellular junctional E-cad expression in which integrated fluorescent intensity of E-cad at the junction was analyzed by drawing ROI around the cell-cell junction and the integrated E-cad expression was expressed as the total fluorescent intensity per unit length of the cell-cell junction.


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Figure 4. Geometric regulation of stiffness-sensitive cell-cell contact. E-cad localization at cell-cell junctions in cell-pairs that cover at least 90% region of the square, triangular and rectangular collagen micropatterns. E-cad localization is presented as (a) peak intensity computed using line-scan method and (b) integrated E-cad intensity per unit length of cell-cell junction length analyzed using the second method. N>10 patterns. Line scan method was also use to compute (c) E-cad junction width, and (d) E-cad junction area. N>10 patterns. Degree of asymmetry in cellular morphology on the patterns is presented as (e) difference between the cell areas in each cell pair. N>20 patterns. Cell elongation is presented as (f) aspect ratio of the cells. N>20 patterns.(g) Representative immunofluorescence images of E-cad (green) expression with


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DAPI (blue) and F-actin (red) in MCF10A cells on the collagen micropatterns of three different shapes on functionalized soft and stiff PA gels. Scale bars=25 µm. (h) Distribution of traction magnitude for cell pairs on square, triangular and rectangular micropatterns on soft PA gels, measured from the displacement of beads embedded within the PA gels. Here, white dash-lines depict the outer-edge of micropatterns. Corresponding DIC images of cells are provided in Figure S2. (i) Mean traction magnitude for cell pairs on the micropatterns. N>10 patterns. *p

Direct Micropatterning of Extracellular Matrix Proteins on

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