Vascular smooth muscle cell durotaxis depends on extracellular matrix composition

Christopher D. Hartmana, Brett C. Isenberga, Samantha G. Chuaa, and Joyce Y. Wonga,1

aDepartment of Biomedical Engineering, Boston University, Boston, MA 02215

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved August 9, 2016 (received for review July 14, 2016) Mechanical compliance has been demonstrated to be a key de- by an increase in collagen I concentration (21), and in breast cancer terminant of cell behavior, directing processes such as spreading, an increase in stiffness from the tumor core to the periphery is migration, and differentiation. Durotaxis, directional migration from associated with increased levels of collagen I and laminin (12). In softer to more stiff regions of a substrate, has been observed for a atherosclerosis, a disease characterized by the thickening of the variety of cell types. Recent stiffness mapping experiments have intimal region of the arterial wall, changes in the mechanics and shown that local changes in tissue stiffness in disease are often composition of the intimal matrix occur in conjunction with the accompanied by an altered ECM composition in vivo. However, accumulation of smooth muscle and inflammatory cells (22–24). the importance of ECM composition in durotaxis has not yet been Stiffness mapping experiments have shown that plaque stiffness is explored. To address this question, we have developed and charac- spatially heterogeneous, and that these changes can be histo- terized a polyacrylamide hydrogel culture platform featuring highly logically related to the ECM composition of the plaque (20, 25). tunable gradients in mechanical stiffness. This feature, together with Given the increasing number of examples for which changes in the ability to control ECM composition, allows us to isolate the effects ECM composition in disease are coupled to changes in me- of mechanical and biological signals on cell migratory behavior. Using chanical properties of the diseased tissue, there is a need for this system, we have tracked vascular smooth muscle cell migration in vitro experimental systems that allow for systematic explora- in vitro and quantitatively analyzed differences in cell migration as a tion of how the cellular response to stiffness gradients is mod- function of ECM composition. Our results show that vascular smooth ulated by ECM composition. muscle cells undergo durotaxis on mechanical gradients coated with Recent experimental work has demonstrated that the effect of SCIENCES fibronectin but not on those coated with laminin. These findings

matrix stiffness on cell behaviors such as differentiation, spreading, APPLIED BIOLOGICAL indicate that the composition of the adhesion ligand is a critical and motility is modulated by the composition of the ECM on which determinant of a cell’s migratory response to mechanical gradients. the cells are grown (26–30). Though the role of matrix composition has been investigated on uniformly stiff substrates, previous work durotaxis | cell migration | extracellular matrix | polyacrylamide investigating the cellular response to mechanical gradients has been limited to substrates coated with a single type of matrix protein, typically collagen or fibronectin (31), and the behavioral response of ell migration is essential to numerous biological processes, SCIENCES including development, angiogenesis, wound healing, and a given cell type to different matrix compositions has yet to be ex-

C APPLIED PHYSICAL cancer metastasis (1–4). The movement of cells in these processes plored in the same study. Previous work in our laboratory has is determined by a complex assessment of environmental cues that demonstrated that vascular smooth muscle cell (VSMC) adhesion include soluble factors, ECM composition, orientation, and stiff- rate, spread area, cytoskeletal assembly, and focal adhesion signaling ness. Numerous experiments have demonstrated that directional undergo opposing responses to substrate stiffness depending on cell migration can result from gradients in these environmental whether they are seeded on fibronectin- or laminin- coated sub- cues: for example, chemotaxis (cell migration in response to gra- strates (27), and furthermore that VSMCs will preferentially migrate dients of soluble signals) and haptotaxis (cell migration in response toward stiffer regions (durotaxis) when exposed to mechanical gra- to gradients of bound ligands) have been established in both dients on fibronectin substrates (14, 16). Additionally, there is in vitro and in vivo experimental systems (5–7). More recently, it has been demonstrated that cells are also capable of directed mi- Significance gration in response to gradients in substrate stiffness, a process termed durotaxis (8). Though there have been limited reports of Many cell types have been observed to migrate toward stiffer specifically measured in vivo gradients (9), a number of recent regions of mechanical gradients in a process termed durotaxis. stiffness mapping measurements imply the presence of stiffness Tissue stiffness gradients are being discovered in an increasing gradients in both healthy and diseased tissues spanning a wide number of diseases, including cancer and fibrotic diseases. – range of stiffnesses (10 13). In vitro experiments have demon- However, the role of ECM composition, which often changes in strated that directed migration in response to stiffness gradients diseased tissues with stiffness, in directing this behavior has can be observed in numerous cell types, using various materials as not previously been investigated. To understand how stiffness – substrates, and across various stiffness levels (8, 14 19). However, gradients and changing matrix composition may affect cell the role of ECM composition in mediating this behavior has not migration in disease, we have designed a mechanical gradient been thoroughly investigated. platform that allows for independent control of absolute The interplay between mechanical stiffness and matrix compo- stiffness, gradient steepness, and ECM coating. We demon- sition in normal and pathological physiology is only now becoming strate that smooth muscle cells will undergo durotaxis on appreciated. Recent studies in which tissue stiffness was mapped mechanical gradients coated with fibronectin but not laminin. by atomic force microscopy (AFM) indentation have identified heterogeneities that indicate the presence of mechanical stiffness Author contributions: C.D.H., B.C.I., and J.Y.W. designed research; C.D.H., B.C.I., and S.G.C. gradients in both healthy and diseased tissues. These measurements performed research; C.D.H. analyzed data; and C.D.H. and J.Y.W. wrote the paper. indicate the presence of a wide range of absolute stiffnesses and The authors declare no conflict of interest. gradient strengths in vivo (9–13, 20). Importantly, such stiffness This article is a PNAS Direct Submission. gradients have been demonstrated to accompany changes in ECM 1To whom correspondence should be addressed. Email: [email protected]. composition in a number of diseases. For instance, in lung fibrosis, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. local increases in lung parenchymal tissue stiffness are accompanied 1073/pnas.1611324113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1611324113 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 evidence that both stiffness and ECM composition can modulate VSMCs between a quiescent, contractile phenotype typical of healthy tissue and a synthetic, proliferative, and migratory phenotype observed in vascular disease (32–34). Fibronectin and laminin have opposing effects on VSMC phenotype, with fibronectin driving cells away from the contractile phenotype in vitro, whereas laminin has been shown to conserve it (35–38). A loss of laminin and an increase in fibronectin surrounding smooth muscle cells has been observed during the progression of neointima formation in vascular disease, suggesting these proteins may play an important role in regulation of cell phenotype in disease (39). Additionally, increasing stiffness has been shown to drive smooth muscle cells toward the synthetic, mi- gratory phenotype observed in atherosclerosis (40, 41), This evidence that VSMC phenotype is modulated by both matrix stiffness and composition, coupled with our previous observations of matrix type- dependent responses to stiffness by VSMCs, led us to hypothesize that the durotactic migratory behavior of VSMC s on substrates with mechanical gradients may also be differentially modulated by fi- bronectin and laminin. To test this hypothesis, we have developed a method of gener- ating polyacrylamide hydrogels with tunable gradients in substrate stiffness and independent control of matrix composition. Previously described methods to fabricate polyacrylamide mechanical gradient gels have been limited due to the difficulty of independently con- Fig. 1. Schematic of gradient generator device preparation and use. (A)Glass trolling the absolute stiffness range and gradient steepness (8, 14–16, slide coated in S1818 photoresist (orange). (B) Maskless lithography with visible 19, 42–44). However, by carefully controlling hydrogel geometry, light exposes device pattern onto photoresist-coated slide. (C) Photoresist re- cross-linker diffusion, and UV photopolymerization (Fig. 1), we are moved by development in TMAOH. (D) Exposed glass is coated with OTS to form a able to easily and independently adjust both absolute stiffness and hydrophobic barrier. (E) Remaining photoresist is removed with acetone. (F)Ex- gradient steepness in our system. Maskless lithography, which unlike posed glass is backfilled with hydrophilic and adhesive silane mixture (teal). μ traditional lithographic techniques does not require a custom-made, (G) The gradient generator device is assembled with 250- m Teflon spacers be- physical photomask and thus allows one to rapidly modify pattern tween the silane-coated generator slide and a top sacrificial slide coated in DCDM. Solutions of acrylamide solutions with high (blue) and low (white) concentrations dimensions with ease, is used to micropattern glass slides with hy- of bis-acrylamide cross-linker are injected into the reservoir regions through drophobic and hydrophilic silanes (45); this difference in hydro- 32-gauge needles. (H) The two acrylamide solutions meet in the middle of the phobicity gives the geometric constraint necessary for fine control device with their boundary constrained by silane patterning. The needles are re- over the location and degree of pregel solution mixing before po- moved and diffusion of cross-linker proceeds until (I) the acrylamide solutions are lymerization. Accordingly, a dumbbell- shape pattern was developed polymerized by exposure to UV light. (J) The sacrificial slide and spacers are re- in which the “weights” of the dumbbell consisted of large reservoir moved leaving a gradient hydrogel with a well-defined linear gradient in elasticity. regions filled with a high-concentration cross-linker solution on one side and low-concentration cross-linker solutions on the other (Fig. 1), and the “bar” of the dumbbell acted as a mixing region for cross- concentration of acrylamide monomer in solution and the extent linker diffusing between the two weights. After polymerization, the of cross-linking, which can be controlled by the concentration of resultant polyacrylamide gels were then functionalized with either bis-acrylamide cross-linker. In preliminary studies, we produced – fibronectin or laminin through an NHS–ester linker. The advantage uniform stiffness gels with 8 15% (wt/vol) acrylamide and bis- – of this design is that the cell movements observed in the constant acrylamide cross-linker concentrations ranging from 0.1 1%. We stiffness regions at the ends can serve as controls for cell migration found that after polymerization by photoinitiation, the elastic observed in the stiffness gradient region between them. Atomic modulus of the resulting gels was linearly proportional to the bis- force microscope nanoindentation of the end-product cell substrates acrylamide concentration over these ranges. We took advantage produced in this manner demonstrated the presence of linear and of this linear proportionality to generate hydrogels with linear highly reproducible mechanical gradients, and fluorescence imaging gradients in elastic modulus (Fig. 2). Briefly, the time allowed for demonstrated that homogeneous surface coatings of Alexa Fluor diffusion of cross-linker between reservoirs of high and low bis- 488-labeled fibronectin or laminin could be achieved. acrylamide concentration solutions was adjusted to yield linear We then used this system to directly compare the durotactic mi- gradients in bis-acrylamide across the region connecting the two gration of VSMCs on mechanical gradients coated with either fi- reservoirs. Nanoindentation was performed using an AFM in bronectin or laminin. The extent to which cells exhibited biased force contact mode to measure the stiffness of the gels as a migration in the direction of increasing substrate stiffness was com- function of position. It was determined that for gradient gels pared across all conditions by calculating a durotactic index, defined with 2- and 1-mm-long gradient regions, 40 and 10 min of diffu- as the length of migration in the direction of increasing substrate sion time, respectively, was sufficient to generate a linear gradient stiffness divided by the total migration path distance, for each cell in cross-linker concentration between the two reservoir regions. track. We found that cells seeded on fibronectin-coated gradient Results from the gels tested demonstrate that the gradient substrates exhibited durotactic behavior, whereas cells seeded on steepness can be changed both by altering the composition of the laminin-coated gradient substrates were capable of random move- pregel solutions while maintaining the same gradient geometry ment but did not exhibit durotaxis. This finding indicates that ECM and adjusting the gradient length. The stiffness gradients between composition is capable of modulating a cell’s motile response to the high- and low-stiffness regions (0.5–10 and 7–170 kPa, re- mechanical gradients. spectively, for the range of gel solutions tested) ranged from 2.9 to 142.6 kPa/mm (Fig. 2). Notably, an effective doubling of the Results gradient steepness was achieved by halving the distance between Generation and Characterization of Hydrogels with Defined Stiffness the reservoir regions, demonstrating that the stiffness gradient of Gradients. Polyacrylamide hydrogel stiffness is determined by the the gels can be tuned by adjusting the gel dimensions.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1611324113 Hartman et al. Downloaded by guest on September 27, 2021 200 trajectory, where x is defined as parallel to the direction of in- creasing gradient stiffness (or a random direction in the case of 1mm Gradient 150 uniform controls), was calculated from migration tracks as a 142.6 kPa/mm function of time (Fig. 4). Cells on uniform stiffness control gels experienced an average x-directional displacement of zero as time 100 increased, with a wider range of displacements reflected in an increase in the SD of displacement as stiffness is increased. Cells 50 on gradient gels coated with fibronectin displayed a steady in- 18.6 kPa/mm crease in x-directional displacement as a function of time, reaching an average value of 37 μm after 18-h migration, whereas cells on 0 5.8 kPa/mm laminin-coated gradients migrated to an average displacement of 200 −2.63 μm after 18 h. Statistical testing of the displacements of cells at all time points by ANOVA and Tukey’s honest significant dif- 2mm Gradient 150 71.7 kPa/mm ference test showed that the mean displacement of cells on fibronectin-coated gradient gels was significantly different from

Elastic Modulus (kPa) the mean displacement of cells in all other experimental condi- 100 tions after 6 h (P < 0.05) of migration. The extent to which each cell exhibited biased migration in 50 the direction of the gradient can be further quantified using a 10.0 kPa/mm durotactic index (16), defined as the displacement of a cell in the 0 2.9 kPa/mm gradient direction divided by its cumulative displacement (i.e., total path length). The distribution of durotactic indices for each 01234 population of cells observed is plotted in Fig. 5. For both cells on Position along gradient gel (µm) uniform stiffness control gels and laminin-coated gradients, the mean durotactic index is zero, whereas for the population of cells migrating on fibronectin-coated gradients, the distribution of

P < SCIENCES 15% Acrylamide, 0.1−1.0% bis−acrylamide durotactic indices is significantly shifted to a mean of 0.17 ( 0.005). There is thus a clear shift toward biased migration in the Gel Composition 10% Acrylamide, 0.1−0.5% bis−acrylamide cell population when these cells are seeded on fibronectin- APPLIED BIOLOGICAL coated substrates having a stiffness gradient. 8% Acrylamide,0.1−0.50% bis−acrylamide Discussion Fig. 2. Mechanical characterization of polyacrylamide gradient gels with dif- Generation of Hydrogels with Controlled Gradients in Substrate fering gel compositions and dimensions by AFM nanoindentation as a function Rigidity. Here we present a method to generate gradients in me- of position along the gradient. Gel compositions tested were as follows: chanical compliance in hydrogels based on rapid prototyping and SCIENCES 15% (wt/vol) acrylamide:0.1–0.1% bis-acrylamide, 10% (wt/vol) acrylamide:0.1– 0.5% bis-acrylamide, 8% (wt/vol) acrylamide:0.1–0.5% bis-acrylamide with gra- UV-initiated photopolymerization. An important feature of this APPLIED PHYSICAL dient lengths of 1 and 2 mm for each gel composition. Gel elastic modulus was method is that it allows for independent selection of absolute high calculated as a function of position from a linearized Hertzian contact model, and low stiffness values and gradient steepness, accomplished by and the elastic modulus profile was fitted to a linear model in across the gra- varying gel solution composition and gel dimensions, respectively. dient region of the gel to determine the steepness of the gradient. In previous studies where hydrogels with gradients in mechanical compliance were used to investigate durotaxis (31), the methods of generating mechanical gradients relied on juxtaposition of pregel Functionalization of Gradient Hydrogels with ECM Substrates. To solutions with varying concentrations of cross-linker (8, 15, 16), ensure cell migration on gradient gel substrates was not biased by varying UV exposure in photopolymerization with a photomask stiffness-induced changes in coating density, substrates with stiffness gradients between 10- and 170-kPa reservoir regions were functionalized with Alexa Fluor 488-labeled fibronectin or laminin-1 and were imaged under epifluorescence for quantifi- 10 kPa 170 kPa 72 kPa/mm Gradient cation. The results indicate the average fluorescence intensity of 100 µm attached fibronectin did not change either as a function of po- Increasing sition along gradient gels or as a function of stiffness (Fig. S1). Stiffness Fibronectin

Effect of Substrate Stiffness on Cell Migration. Cell migration on gradient gels and uniform control gels coated with either fibro- nectin or laminin was qualitatively assessed by plotting cell tra- jectories relative to a common origin (Fig. 3). On gels of uniform stiffness, there is a stiffness-dependent increase in the average migration velocity on both fibronectin- and laminin-coated gels, Increasing Stiffness but there was no apparent directionality to the movement. Ad- Laminin ditionally, there were no significant differences in the migration velocities of cells on fibronectin compared with on laminin for both the control and gradient conditions (Fig. S2). In contrast to the uniform stiffness controls, cells migrating on fibronectin- coated gradient gels appear to migrate preferentially toward the stiffer end of the gradient, whereas cells on laminin-coated Fig. 3. Representative migration tracks for VSMCs migrating on mechanical gradients do not display a bias in migration to any direction. gradient and uniform stiffness gels coated with fibronectin or laminin. Cell To more quantitatively assess whether there is biased migration centroid position was tracked at 20-min intervals for 18 h. n = 15 randomly of cells in the direction of the gradient, the average x-direction selected cells per condition.

Hartman et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 any desired gradient profile to be produced in easily customized Fibronectin Laminin 50 geometries (43). Though this method of gradient gel production is highly tun- able, there are practical limits on the mechanical properties and 25 dimensions of gels that may be produced. Polyacrylamide gels have 10 kPa been produced with stiffnesses ranging from ∼200 Pa to 350 kPa, 0 consistent with the mechanical data presented in this study (43, 47). The length of the gradient region can be made longer or −25 shorter to alter the gradient steepness, but for extremely low gradient rates, limited directional migration is expected (16, 46), −50 whereas at extremely high gradient steepness the system mimics 50 step gradient methods (8, 15) and the observation region of in- terest becomes extremely small. The absolute stiffness range and ms) 25 gradient rate range is sufficient to model the stiffnesses of a wide 170 kPa variety of soft tissues that are likely to contain mechanical gradi- – 0 ents (9 13). Gels can be made thinner or thicker as desired by changing the size of the spacers used, but there are practical limits on the achievable upper and lower thicknesses. A gel that is too −25

x position ( µ thin will begin to appear stiffer to a cell due to mechanical cou- pling to the underlying glass substrate (42). Additionally, because −50 the gradient generator system relies on surface tension between 50 * two slides to control the gel geometry, using spacers that are too 72 kPa/mm Gradient thick can result in surface tension failing to maintain the pregel 25 solution between both slides, and thus the gel dimensions and contact point between the two solutions cannot be controlled. The 0 limits of the width of the gradient region are related to the size of the reservoir region and imaging area. As the gradient region is −25 made thinner, the available area for cells to migrate on the gra- dient region is reduced, and it becomes less likely that cells will −50 migrate without interacting with the sides of the gel, which greatly 0 5 10 15 0 5 10 15 reduces the areas in the gel from which data can be reliably ac- Time (hrs) quired. The width of the gradient area can be increased provided there is also an increase in the size of the reservoir regions to Fig. 4. Average x displacement of cells migrating on polyacrylamide gels compensate and prevent depletion of cross-linker from the res- coated with fibronectin (red) or laminin (blue) as a function of time. Values ervoir during diffusion. The volume of a reservoir region needs to presented as mean ± 95% confidence interval. be sufficiently large such that it is largely unaffected by diffusion

(14, 19, 43), or overlaying a uniform gel solution on a rigid material with varying height to achieve a gradient in apparent rigidity at the surface (42, 44). Though these methods effectively generate 1.0 stiffness gradients, they are limited because they do not easily allow * independent control of both the absolute stiffness range of the gels and the steepness of the generated gradient. We overcame this limitation by using an approach to gradient generation based on carefully controlled hydrogel geometry, cross-linker diffusion time, 0.5 and UV photopolymerization time. As demonstrated in Fig. 2, the absolute stiffness range of the gradient gels produced using the method presented here can be tuned by changing the composition of the acrylamide gel, and the steepness of the mechanical gradients 0.0 produced using this method can be predictably controlled by adjusting the length of the gradient region and cross-linker diffusion time. This flexibility will allow gradient profiles and material elasticity Durotactic Index to be tailored to mimic stiffness gradients between tissues or cellular −0.5 layers in vivo and also allows for simple modulation of gradient properties to further study the roles of absolute stiffness and gradient steepness in the cellular response to mechanical gradients (16, 46). In addition to the ease of gradient customization, gels produced using this method contain built-in uniform stiffness regions on the −1.0 same sample to act as controls, allowing for easier experimental setup and greater throughput from a smaller number of independent samples. For this set of experiments we have optimized the gradient 10 kPa 170 kPa 72 kPa/mm Gradient profile generated to be linear between two reservoir solutions, but it Gel Condition is also possible to produce gels with step-like gradients or sigmoid Fig. 5. Distribution of durotactic index values for VSMCs migrating on sub- shaped gradients by reducing the time allowed for cross-linker dif- strates coated with fibronectin (red) or laminin (blue) with overlaid boxplots fusion. Additionally, this method is compatible with photomask- representing median and quartile values for each distribution. n = 120 cells based techniques of stiffness modulation, theoretically allowing for from n > 5 gels for each condition. *P < 0.05 relative to all other conditions.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1611324113 Hartman et al. Downloaded by guest on September 27, 2021 from the much smaller gradient region and thus maintains a nearly how cells respond to variations in stiffness in their local environ- constant cross-linker concentration; this ensures that a pseudos- ment in vivo. Assessing the manner in which cells respond to these teady gradient profile can form in the gradient region. variations in stiffness when presented with different ECM mole- cules will be critical in understanding diseases in which the presence VSMC Durotaxis Is ECM-Type Dependent. Though durotaxis behav- of a gradient in local tissue mechanical compliance is accompanied ior has been observed in numerous experimental systems, to our by alterations in the ECM composition, such as breast cancer, lung knowledge, the role of matrix composition in durotactic behavior fibrosis, and atherosclerosis (12, 20, 21). has not yet been investigated. To that end, we evaluated the migration of VSMCs on gradient substrates coated with either Future Directions. The observation that VSMCs are capable of fibronectin or laminin, matrix proteins that have previously been undergoing durotaxis on mechanical gradients coated with fibro- demonstrated to evoke opposing responses to stiffness changes nectin but do not exhibit this behavior on laminin indicates that the in smooth muscle cells and drive smooth muscle cells toward ability of cells to sense and respond to variations in substrate different phenotypes (27, 35–38). This model system was chosen stiffness may be ECM-type dependent, but the mechanisms un- both because of its potential relevance to atherosclerosis and its derlying this matrix-dependent response are currently unknown. It use in previous studies in our laboratory. Interestingly, directed is well documented that different integrin subtypes engage different migration up mechanical gradients was observed when cells were matrix proteins, and thus the mechanical interaction between cells seeded on fibronectin, but no bias in migration was observed on and the underlying substrates are mediated by different integrin– gradient substrates coated with laminin (Figs. 3–5). To verify that ECM pairings, which can vary in their bond strengths and dynamics the lack of observable biased migration on gradient substrates of adhesion formation and turnover (48–52). Though beyond the coated with laminin was not due to a decrease in the overall scope of this present study, it is possible that such differences in the migration rates of VSMCs on laminin relative to those observed dynamics of integrin–ECM bond pairs could lead to variations in on fibronectin, the velocities of cells migrating on gradients, as the response of VSMCs to stiffness gradients coated with fibro- well as uniform high- and low-stiffness control gels, were mea- nectin or laminin. Recent work by Elosegui-Artola et al. (53) has sured. The matrix selection was not found to have a significant shown that, even for a single type of matrix, cell attachment via effect on the cell migration velocities (Fig. S2) and, as expected, different integrins can result in changes in the traction forces randomly directed migration was observed on all uniform stiff- exerted by cells as a function of stiffness, leading to the hypothesis “ ”

ness control regions (Fig. 4). Collectively, these results indicate that different integrins may be tuned to different environmental SCIENCES that VSMCs were capable of adhering on substrates coated with stiffnesses. Future experiments to evaluate whether VSMCs exhibit

either fibronectin or laminin and that the ability of cells to move this integrin-type–dependent behavior and whether differences in a APPLIED BIOLOGICAL on these substrates in regions of uniform stiffness did not vary cell’s ability to generate tension on different types of matrix could significantly with matrix choice. However, though we observed lead to matrix-dependent changes in a cell’s sensitivity to stiffness biased migration toward the stiffer region of gradient gels coated gradients would help clarify the mechanisms underlying the results with fibronectin, migration on laminin-coated gradients appeared reported here. to be random (Fig. 4), with cells exhibiting a distribution of tactic Additionally, the experimental platform detailed here can be

indices similar to that observed for migration on the uniform used to determine whether this phenomenon can be observed in SCIENCES stiffness control gels (Fig. 5). other cell types and what the effect of additional matrix proteins APPLIED PHYSICAL The distribution of tactic indices for cells on fibronectin-coated beyond fibronectin and laminin are on directed migration; it gels was found to shift to a mean of 0.17, but it is interesting to note could also potentially play a significant role in generating a li- that after 18 h of migration, ∼20% of cells still had a negative tactic brary of cell responses to aid in the design of complex engineered index. To assess whether this might indicate a subpopulation of cells tissues, for which control over the positioning and migration of was not able to sense or respond to the mechanical gradient by one or more cell types would be desirable. Moreover, one can exhibiting directed migration, we looked at the change in the dis- use this system to better understand complex dynamic environ- tribution of x displacements over time (Fig. S3). For cells migrating ments in vivo. For example, the changing matrix environment in randomly, we expect the distribution to widen over time but remain diseased states in terms of both composition and mechanics centered about x = 0, as observed for cells on laminin-coated gra- could work in concert to modulate cell migratory behavior. This dients. If a subpopulation of cells on fibronectin exhibited this experimental platform is ideally suited to assess whether such random migration behavior, a peak in the distribution centered at changes induce a migratory response and allows for the testing of x = 0 should emerge at later time points. Rather, as time increases, therapeutics targeted to prevent or diminish such behavior. there is a shift in the distribution toward positive x displacements corresponding to biased migration. Additionally, the left tail of the Materials and Methods distribution does not continue to spread to more negative values, Substrate Preparation. Polyacrylamide gels featuring gradients in mechanical but rather moves toward more positive values than observed at compliance between uniform stiffness control regions were prepared using earlier time points. A similar trend is observed for the fraction of surface tension-based glass microfluidic device (Fig. 1). Briefly, clean glass slides cells with positive net displacements as a function of time. For were coated with Microposit S1818 photoresist (Shipley) and were micro- x patterned to feature a dumbbell shape (Fig. 1) using maskless lithography (45). randomly migrating cells, the fraction of cells with positive dis- The pattern was developed in 2.5% (wt/wt) tetramethylammonium hydroxide in placements fluctuates around 0.5, whereas for cells exhibiting di- water (TMAOH; Sigma), and the exposed region was then treated with octa- rected migration, this value increases from over time. The rate of decyltrichlorosilane (OTS; Sigma) to form a hydrophobic region. The remaining this increase slows at later time points, but has not leveled off to an photoresist was removed using acetone, and newly exposed portion of the

asymptote at a fraction <1. Taken together, these observations surface was backfilled with a 2:1 ratio of 2[methoxy(poly(ethyleneoxy)n = 6–9) suggest that the negative tactic indices calculated for some cells propyl]trimethoxysilane (Gelest) and 3-(trimethoxysilyl)propyl methacrylate are not necessarily indicative of a subpopulation of cells that does (Sigma) to provide a hydrophilic, gel-adhesive surface. A micropatterned slide not respond to the gradient, but rather that migration would need and a sacrificial glass slide coated with dichlorodimethylsilane (DCDM; Sigma) μ to be observed for a longer duration to see the remainder of the were stacked and separated by 250- m spacers. Acrylamide solutions with x varying monomer and cross-linker concentrations, adjusted to pH 6.0, consisting population approach a positive displacement. of acrylamide (Bio-Rad), bisacrylamide (Bio-Rad), amine-reactive cross-linker Collectively, the data presented here indicate that the ability of NHS–ester acrylic acid (Sigma), I2959 photoinitiator (Irgacure), and hydrochloric VSMCs to detect and respond to a gradient in substrate stiffness acid were injected into each end of the gradient device until the solutions may be subject to the type of matrix they are seeded on, and that contacted in the center of the device. The solutions were allowed to diffuse for matrix composition could therefore be an important determinant of 40 min for 2-mm gradient gels and 10 min for 1-mm gradient gels to establish

Hartman et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 a linear gradient in cross-linker concentration before polymerization under UV at 5 μg/cm2 inPBSadjustedtopH8.0for2hat room temperature. The uni- light for 240 s. Hydrogels were stored PBS adjusted to pH 6.0 until protein formity of ECM attachment across gradient gels was assessed from fluorescent functionalization was performed. micrographsofgradientgelsfunctionalizedwithAlexaFluor488NHSester(Life Technologies)-labeled fibronectin or laminin. The average fluorescence intensity Mechanical Characterization. Characterization of mechanical gradient gels was measured in 33-μm-wide bins across the gel oriented parallel to the direction was performed as previously described (16); complete details of the method of the gradient. are given in SI Materials and Methods. Cell Culture and Motility. Cell culture and tracking was performed as described Substrate Functionalization. Polyacrylamide gradient gels were functionalized in SI Materials and Methods. with ECM proteins via NHS–ester acrylic acid, which incorporates into the hydrogel backbone during polymerization and can subsequently be reacted with ACKNOWLEDGMENTS. This work was supported by NIH Grants R01 primary amines to covalently attach proteins to the gel surface. Polyacrylamide HL072900 and R01 HL124280 (to J.Y.W.); NIH Predoctoral Training Grant gels were incubated in a solution of fibronectin (Millipore) or laminin-1 (Sigma) NIGMS 5T32 GM008764 (to C.D.H.); and a Lutchen fellowship (to S.G.C.).

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