Differential Depth Sensing Reduces Cancer Cell Proliferation via Rho

Differential Depth Sensing Reduces Cancer Cell Proliferation via Rho-Rac-Regulated Invadopodia Parthiv Kant Chaudhuri,† Catherine Qiurong Pan,† Boon Chuan Low,*,†,‡,∥ and Chwee Teck Lim*,†,§ †

Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411, Singapore Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore § Department of Biomedical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore ∥ University Scholars Programme, National University of Singapore, Singapore 138593, Singapore ‡

S Supporting Information *

ABSTRACT: Bone, which is composed of a porous matrix, is one of the principal secondary locations for cancer. However, little is known about the effect of this porous microenvironment in regulating cancer cell proliferation. Here, we examine how the depth of the pores can transduce a mechanical signal and reduce the proliferation of noncancer breast epithelial cells (MCF-10A) and malignant breast cancer cells (MDA-MB-231 and MCF-7) using micrometer-scale topographic features. Interestingly, cells extend actin-rich protrusions, such as invadopodia, to sense the depth of the matrix pore and activate actomyosin contractility to decrease MCF-10A proliferation. However, in MDA-MB-231, depth sensing inactivates Rho-Rac-regulated actomyosin contractility and phospho-ERK signaling. Inhibiting contractility on this porous matrix using blebbistatin further reduces MDA-MB-231 proliferation. Our findings support the notion of mechanically induced dormancy through depth sensing, where invadopodia-mediated depth sensing can inhibit the proliferation of noncancer and malignant breast cancer cells through differential regulation of actomyosin contractility. KEYWORDS: cancer microenvironment, cancer cell proliferation, topographic cues, actomyosin contractility, mechanotransduction

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Bone is one of the principal secondary locations for metastatic relapse and is composed of a porous matrix with embedded cells. The fluid-filled porous matrix comprises a hydroxyapatite and collagen meshwork that provides ductile strength.5,6 Bone is a complex mechanical microenvironment that undergoes constant remodeling due to the activity of boneproducing osteoblasts, bone-decaying osteoclasts, and matrixembedded osteocytes. The tumor cells colonize the marrow spaces of cancellous or spongy bone and depending on the microenvironmental cues either start proliferating immediately or enter into an indefinite dormancy. Subsequently, when the environment becomes favorable for growth, a fraction of the dormant cells become reactivated and proliferate to form macrometastases.7 Therefore, the architecture or the topography of the bone undergoes dynamic remodeling in the

lood-borne metastasis of cancer cells from a primary tumor to a secondary location is one of the leading causes of cancer-related deaths worldwide. The disseminated tumor cells that are principally found in the secondary locations such as bone marrows either stop their proliferation ability due to the hostile microenvironment and enter a temporary dormancy for an indefinite period or grow slowly into micrometastases as the amount of cell death counterbalances the proliferation rate.1 Therefore, the microenvironment that surrounds the tumor cells in their secondary niche plays a decisive role in determining the final fate of the metastatic disease. Over the past decade, it has increasingly been observed that mechanical cues from the microenvironment such as topography (mechanically induced dormancy, MID),2 stiffness,3 and shear stress from the circulatory system4 can influence the phenotypic behavior of healthy and cancer cells. However, it is not yet understood what the roles of the mechanical cues are in the specific secondary sites and how these cues are transduced into biochemical signals and thereby control the proliferation of the cancer cells. © 2017 American Chemical Society

Received: May 17, 2017 Accepted: June 27, 2017 Published: June 27, 2017 7336

DOI: 10.1021/acsnano.7b03452 ACS Nano 2017, 11, 7336−7348

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Figure 1. Micropit depth reduces noncancer breast epithelial cell proliferation. (a) Scanning electron microscopic (SEM) representative images of micropits of different widths (2, 3, 4 μm) and depths (1.5, 3.8, 5.7, 7.0, 9.0 μm), respectively. Width scale is 10 μm and depth scale is 5 μm. (b) Schematic representation of the micropits’ topographic pattern and morphology of the cells seeded on top of it. (c) Micropit depth greater than 1.5 μm decreases MCF-10A proliferation after 24 h of cell seeding. Data are means ± SEM (n = 3). For each experiment, an average of 300 cells were considered. P values were obtained using Student’s unpaired t test. **p < 0.01 with respect to planar. (d) Proliferation reduction induced by micropits is not affected by altering the width of the micropits (2, 3, 4 μm) at a constant depth (9 μm). (e) Cell protrusion length increases as the depth of the micropits is increased. (f) Protrusion length is not affected by the width of the micropits at a constant depth. N.S. denotes nonsignificant differences between the protrusion lengths for the different micropit widths. Confocal microscopy representative images of the protrusion lengths of MCF-10A cells cultured on micropits of different depths [1.5 μm (g) and 9.0 μm (h)]. (Phalloidin: green, region of interest indicated by Imaris software: red; scale 10 μm.)

porous matrix dimensions along with the progression of the metastatic disease. Invadopodia are actin-rich mechanosensing structures that are produced by cancer cells to degrade and remodel the surrounding extracellular matrix (ECM) by secretion of metalloproteinases. The formation of invadopodia by cancer cells is correlated with greater invasive potential in both in vivo and in vitro studies.8,9 ECM stiffness and cellular traction forces can regulate invadopodia numbers and activity, suggesting the role in sensing mechanical cues from the microenvironment.10 For breast cancer cells, invadopodia-induced ECM degradation peaks around 30 kPa, which is in the same stiffness range as that of the desmoplastic stroma.11 Multiple adhesive and contractile molecules form an adhesive ring at the base of the invadopodia, which are involved in probing and remodeling the environment using myosin-II-based contractile forces. In fact, the formation of adhesion rings has been strongly correlated with invadopodial activity.12,13 Nonmuscle myosin IIA colocalizes with the adhesion ring in 40% of the cells and is absent in these actin-rich structures, thereby suggesting the importance of global actomyosin-based contractile forces in invadopodiamediated mechanosening and matrix proteolysis.12

To understand how the topographic cues induced by the dynamic porous matrix of the bone affect cancer cell proliferation, we fabricated well-defined micrometer-scale pit structures (known as micropits) of varying width and depth using microfabrication tools. We observed that the micropit depth (but not width) reduces the proliferation of both noncancer breast epithelial cells (MCF-10A) and malignant breast cancer cells (MDA-MB-231 and MCF-7). Our results further support the concept of mechanically induced dormancydepth sensing (MIDDS), where invadopodia could sense different depths of the porous matrix and cause reduction in the proliferation of both noncancer breast epithelial cells and malignant breast cancer cells via differential regulation of RhoRac-mediated actomyosin contractility.

RESULTS AND DISCUSSION Micropit Depth Reduces Noncancer Breast Epithelial Cell Proliferation. Bone is one of the principal metastatic locations for breast cancer. The mechanical microenvironment of the bone undergoes dynamic changes along with the progression of metastasis. When the cancer cells arrive in their new secondary niche, they remain dormant for some time, and 7337


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Figure 2. Micropits induce the greatest reduction of MCF-10A proliferation after 24 h of cell seeding independent of biochemical cues. (a) Greatest reduction of MCF-10A proliferation occurred after 24 h of cell seeding (highlighted by a red box). Growth curves of cells seeded on a planar surface and micropits (W = 2 μm, D = 9 μm) are indicated by blue- and red-colored lines, respectively. (b) Fold-change calculation of proliferating MCF-10A cells as a function of time. (c) Protrusion length remains constant as time increases. N.S. denotes nonsignificant differences between the protrusion lengths for the different time points. (d) Micropits reduce MCF-10A proliferation across different extracellular matrix coatings such as collagen and poly-L-lysine (PLL). (e) MCF-10A proliferation is reduced by membranes of 2 μm pore size. Blue bar indicates planar, and red indicates micropits (W = 2 μm, D = 9 μm). Bright field images of the membrane pore in random orientation. Confocal microscopy images of the protrusion lengths (indicated with white arrows) stained with phalloidin (green) for actin filaments (scale 10 μm). Data are means ± SEM (n = 3). For each experiment, an average of 300 cells were considered. P values were obtained using Student’s unpaired t test. **p < 0.01 and *p < 0.05 with respect to planar surface.

subsequently, they remodel the microenvironment by activating osteoclasts, thereby leading to the degradation of the porous matrix of the bone. Therefore, the pores widen (from a few micrometers to hundreds of micrometers) and the patient becomes more prone toward severe bone pain and fracture. In order to better mimic the porous microenvironment of the bone in vitro using well-defined geometric structures, we used microfabrication techniques to fabricate micropits of different widths and depths. To replicate the dynamic microenvironment, we fabricated the micropits across a range of steadily increasing pore width (W) of 2, 3, and 4 μm and depth (D) of 1.5, 3.8, 5.7, 7.0, and 9.0 μm (Supplementary Table 1). The successful replication of the micropit feature was verified using

scanning electron microscopy (SEM), as illustrated in Figure 1a. Micropits were coated with a collagen matrix to facilitate cell adhesion and spreading and to better mimic the in vivo condition, since collagen is one of the principal constituents of the bone environment.5 Cells were seeded on top of these collagen-coated structures, and actin-rich protrusions of varying lengths were found along the depth of the micropits, as indicated by the schematic diagram (Figure 1b). Therefore, the cells may use these actin-rich structures to sense the depth of the micropits, and these structures serve as our region of interest (ROI). In order to have a clear view of the microscopic images of these structures, the subsequent images will be 7338


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distribution and heterogeneous length of the actin-rich protrusions. F-actin is stained with phalloidin (green), and the protrusion lengths were outlined in red using Imaris image analysis software. Dynamics of Micropit-Induced Dormancy of MCF-10A Cells Is Independent of Biochemical Cues. To further characterize the dynamics of the micropit-induced depthsensing mechanism, MCF-10A cells were cultured on micropits of 2 μm width and 9 μm depth for varying periods of time (Figure 2a). The proliferation efficiency decreased for all the time points from 12 to 72 h, which indicates that at least 12 h is required for the transduction of depth-sensing cues, thereby leading to reduced proliferation. Interestingly, the maximum reduction in proliferation occurred after 24 h of cell seeding (Figure 2b). The doubling time for MCF-10A cells is around 20 h.15 However, the proliferation fold change gradually increased from 36 h to 72 h, which shows that the cells were steadily acquiring resistance against these depth-sensing cues and approaching their normal proliferation rate, as observed on a planar surface. In other words, the depth-sensing mechanism could induce a temporary dormancy or stoppage in the proliferation of MCF-10A cells, and with the passage of time, the cells would acquire resistance against these mechanical cues. By “dormancy” we mean the cells are not proliferating for the particular time frame of study, while they are still metabolically active and secreting biochemical factors. We then characterized the dynamics of the protrusion length with the passage of time (Figure 2c). We observed that the protrusion length does not change as the time increased from 12 h to 72 h and maintained an average length of around 1.5 μm. Thus, the cells were sustaining an equilibrium protrusion length that remained constant with the passage of time by maintaining a steady state between actin polymerization and depolymerization rates. Subsequently, we investigated whether the depth-sensinginduced reduction in proliferation of MCF-10A is dependent on the biochemical cues provided by the ECM such as collagen. To test this possibility, we seeded cells on poly-L-lysine (PLL)coated micropits, where cells adhere via electrostatic interactions instead of integrin-mediated adhesion.16 The proliferation of MCF-10A was observed to reduce similarly on both collagen- and PLL-coated micropits, which indicates that the depth-sensing cues dominate over the biochemical cues provided by the collagen matrix in decreasing MCF-10A proliferation (Figure 2d). Another possible reason for the reduced cell proliferation on the micropits could be the secretion of various soluble factors from the cells and concentration as a gradient within the individual micropockets of the micropit structure, which might be responsible for the proliferation reduction. To rule out this possibility, we seeded cells on collagen-coated membranes of 2 μm pores arranged in a random orientation, where the bottom of the membrane is in contact with the culture media (Figure 2e). The proliferation still decreased on the membrane compared to the planar surface, and actin-rich protrusions were formed at the base of the membrane. This shows that depth-sensing cues reduce MCF-10A proliferation in a manner independent of biochemical factors and orientation of the pores. Interestingly, when MCF-10A cells were cultured on membranes across different time points ranging from 12 to 48 h, we observed a sustained reduction in proliferation (Figure S1c). Therefore, it is plausible that the secreted factors from the cells are responsible for acquiring resistance against these depth-sensing

presented with the cell in an inverted position, that is, the basal side pointing upward. To understand the effect of micropit depth on the proliferation of noncancer breast epithelial cells, MCF-10A cells were cultured on these collagen-coated structures of gradually increasing depth from 1.5 to 9.0 μm with a constant width of 2 μm (Figure 1c). Cells were cultured for 24 h on these structures and subsequently fixed and stained with EdU, which labels the nucleus of the proliferating cells. The proliferation of MCF-10A cells did not change for micropits with a 1.5 μm depth compared to the planar case. This signifies that cells could not sense the difference between the planar and 1.5 μm depth of micropits, thereby leading to change in proliferation. Interestingly, when the depth was further increased from 3.8 μm to 9.0 μm, MCF-10A proliferation decreased significantly. Therefore, cells could sense the depth of 3.8 μm or higher and reduce proliferation. Additionally, the depth-sensing mechanism reaches a saturation level for a depth of 3.8 μm, and a further increase in micropit depth does not lead to a further decrease in proliferation. Next, we investigated the effect of the width of the micropits on MCF-10A proliferation, by seeding the cells on micropits of gradually increasing width from 2 to 4 μm, keeping a constant depth of 9.0 μm (Figure 1d). The proliferation of MCF-10A decreased for all the widths of the micropits when the depth is kept constant at 9.0 μm. Therefore, the depth of the micropits plays a dominant role compared to their width in the reduction of MCF-10A proliferation, and this depth-sensing mechanism approaches a saturation value for a depth of 3.8 μm. To further characterize whether the micropit-induced depthsensing mechanism is dependent on the different ECMs that are present in the tumor microenvironment, we cultured cells on collagen- and fibronectin-coated substrates (Figure S1a). Interestingly, MCF-10A proliferation decreases across both matrices, and the greatest reduction occurred on collagencoated micropits. Collagen is one of the most abundant ECMs within the bone microenvironment.14 Therefore, cells cultured on collagen-coated micropits might sense the physical and chemical cues from the collagen matrix and reduce proliferation. In addition, micropit-induced reduction of MCF-10A cell proliferation was further confirmed using the MTS assay (Figure S1b). Subsequently, we characterized how the length of the protrusions changes with respect to the alteration of depth and width of the micropits. We observed that as the micropit depth increases from 1.5 μm to 9.0 μm, the protrusion length also increases proportionally with a mean and highest protrusion length of 1.57 and 8.10 μm, respectively, for 9.0 μm deep micropits (Figure 1e). It is interesting to note here that the mean protrusion length for 3.8 μm deep micropits is 0.90 μm, which signifies that at least a 0.90 μm length of the actin-rich protrusion is required for the cells to sense the topographic depth and thereby reduce proliferation. However, when the width of the micropits was increased from 2 μm to 4 μm at a constant depth of 9.0 μm, the protrusion length did not change significantly, and it maintained a constant mean value around 1.55 μm (Figure 1f). Thus, a minimum mean protrusion length of 0.90 μm is required for depth sensing and reduction of MCF-10A proliferation, and the width of the micropits does not have any effect on the protrusion length. Confocal microscopy representative images of the protrusion length of MCF-10A cells cultured on micropits of 1.5 μm (Figure 1g) and 9.0 μm (Figure 1h) depth, which depicts the 7339


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Figure 3. Invadopodia decrease MCF-10A proliferation on micropits. (a) Protrusion length of MCF-10A cells cultured on 9 μm deep micropits was immunostained for cortactin (green) and actin (phalloidin, red) (scale 10 μm). (b) Paxillin (immunostained in red) forms a ring at the base of the protrusions for a micropit depth of 9 μm. Actin filaments are costained with phalloidin (green) (scale bar 5 μm). (c) Arp2/3 inhibitor (CK-666; 50 μM) prevents the formation of invadopodia. White arrow indicates accumulation of actin filaments (phalloidin, green) at the base of the cell without forming the protrusions (top view; scale bar 10 μm) (side view; region of interest (red) indicated by Imaris software, scale bar 5 μm). Arp2/3 inhibitor (CK-666; 10, 50, 100 μM) prevents depth-induced proliferation reduction of MCF-10A cells. Data are means ± SEM (n = 3). For each experiment, an average of 300 cells were considered. P values were obtained using Student’s unpaired t test. **p < 0.01 with respect to planar surface. Blue bar indicates planar surface, and red indicates micropits (W = 2 μm, D = 9 μm). N.S. denotes nonsignificant difference compared to planar surface.

adhesive ring at the base of the protrusions comprising the adhesion-related molecules such as paxillin, vinculin, focal adhesion kinases (FAK), Src, and β1 integrins.18 The adhesive ring formation has been previously corelated with invadopodial activity, and nonmuscle myosin IIA localizes along these structures to provide contractile forces for sensing mechanical cues from the environment.12,13 However, for 1.5 μm deep micropits, paxillin accumulated at the edges of the pores but did not form the adhesive ring (Figure S2c). The depth-sensing mechanical cues provided by the 1.5 μm depth might not be sufficient to lead to the formation of functional invadopodia, and this result is consistent with the proliferation of MCF-10A, where at least a 3.8 μm depth is required for the reduction in proliferation. To examine whether the depth-sensing-induced invadopodia formation is responsible for the reduction of MCF-10A proliferation on the micropits, we inhibited invadopodia formation using the Arp 2/3 complex (actin-related protein 2 and 3) inhibitor CK-666 and observed the proliferation of MCF-10A in the presence of different concentrations of the drug (10, 50, 100 μM). The Arp 2/3 complex has previously been shown to play a critical role in invadopodia formation, and reduced expression of the Arp2/3 complex decreases invadopodia formation.9,19 In the presence of 50 μM CK-666,

cues and inducing a temporary dormancy, the mechanism of which remains to be further investigated. Invadopodia Decrease MCF-10A Proliferation on Micropits. Subsequently, we wanted to characterize these structures and identify the molecules that colocalize in these mechanosensitive actin-rich protrusions. To answer these questions, we performed cortactin and paxillin immunostaining of these protrusions at different depths. Interestingly, we found that cortactin strongly colocalizes with these actin-rich protrusions in micropits of both 9.0 μm (Figure 3a) and 1.5 μm depth (Figure S2a), which indicates that these protrusions could be invadopodia. Colocalization of cortactin and actin in these protrusive structures is considered one of the key features of invadopodia, which the cell uses mainly to degrade the ECM.9,17 Therefore, the depth-sensing cues provided by the micropits that mimic the porous microenvironment of bones are inducing the invasive potential of MCF-10A cells, which results in the formation of invadopodia. To further confirm our findings, paxillin was observed to form a ring-like structure at the base of the protrusions in 9 μm deep micropits, and the actin cytoskeleton passed through the center of this ring (Figure 3b). But paxillin staining did not colocalize along the depth of the protrusive structures (Figure S2b). Another important characteristic of invadopodia is the formation of an 7340


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Figure 4. Micropits decrease MCF-10A proliferation by activating actomyosin contractility and inactivating p-ERK signaling. Micropits reduce MCF-10A proliferation via inactivation of Rac and activation of the Rho pathway. (a) Acto-myosin contractility modulating drug treatment: Rac1 inhibitor (NSC23766; 50, 100 μM), blebbistatin (5, 20 μM), and (b) Rho activator II (0.1, 0.25, 0.5 μg/mL). Data are means ± SEM (n = 3). For each experiment, 300 cells were considered on average. The number on top of the respective bar indicates mean fold change with respect to planar substrate. (c) Immunofluorescence images of p-ERK (green) and nucleus (DAPI, blue) of MCF-10A cells grown on a planar substrate and micropits in the absence and presence of different drugs, respectively: Rho activator II (0.25 μg/mL); Arp2/3 inhibitor (CK666; 50 μM) (scale bar 25 μm). (d) p-ERK fluorescence intensity quantification in the nucleus/cytoplasm of MCF-10A cells. Over 25 cells were analyzed for each condition. P values were obtained using Student’s unpaired t test. **p < 0.01 with respect to the planar substrate. Blue bar indicates planar substrate, and red indicates micropits (W = 2 μm, D = 9 μm). The number on top of the respective bar indicates mean fold change with respect to the planar substrate. N.S. denotes nonsignificant difference compared to a planar surface.

(NSC23766; prevents Rac1 activation) (50, 100 μM) (Figure 4a). Interestingly, in the presence of Rac1 inhibitor the proliferation of MCF-10A further decreased on micropits, as shown by the mean fold change of 0.56 and 0.45 for a Rac1 inhibitor concentration of 50 and 100 μM, respectively, compared to 0.66 in the case of the planar surface. Thus, Rac1 is already inhibited by depth-sensing cues from micropits, and when we further inhibited it using pharmacological drugs such as NSC23766, the proliferation decreases even more. We further confirmed this finding using Rac1-FRET (fluorescence resonance energy transfer) biosensor in MCF-10A cells and observed that the FRET ratio throughout the entire cell decreases for cells grown on micropits compared to a planar surface. All these data support the notion that micropits reduce Rac1 activity in MCF-10A cells (Figure S3a). In contrast, blebbistatin prevents micropit-induced reduction in proliferation, which signifies the activation of Rho-ROCK-myosinbased contractility in sensing depth. In other words, in the presence of contractility inhibitory drugs, the cell cannot sense the difference between a planar surface and micropits in terms of their proliferation response. Interestingly, on micropits cells expressed a lesser amount of myosin light chain (MLC) compared to the planar surface (Figure S3b). However, they

we noticed complete retraction of invadopodia with actin accumulations at the base of the cells, but these structures did not extend along the depth (Figure 3c). Interestingly, inhibition of the Arp 2/3 complex prevented depth-sensing-induced reduction of MCF-10A proliferation across the different concentrations of the drug used. In the presence of Arp 2/3 complex inhibitor that inhibits the invadopodia formation, cells cannot sense the difference between planar and micropitted surface, which shows the involvement of invadopodia in sensing the depth of the micropits and reducing MCF-10A proliferation. Micropit Depth Reduces MCF-10A Proliferation by Activating Actomyosin Contractility and Inactivating ERK Signaling. Previous studies have shown the involvement of nonmuscle myosin-II-based contractility in regulating invadopodia activity and mechanosensing.12,13 We hypothesized that depth-sensing-induced temporary dormancy of MCF10A via invadopodia formation might be due to the generation of Rho-Rac-regulated actomyosin contractility within the cell. To test this hypothesis, we treated cells cultured on micropits with various concentrations of drugs that affect actomyosin contractility, such as blebbistatin (inhibitor of nonmuscle myosin-II-based contractility) (5, 20 μM) and Rac1 inhibitor 7341


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mean fold change of 0.51 for Rho activator II compared to 0.66 for the no inhibitor condition. This indicates that in the presence of Rho activator II p-ERK resides more in the cytoplasm for cells grown on micropits compared to no inhibitor. This predicts lower proliferation on micropits in the presence of Rho activator II, which is consistent with our previous proliferation data. In contrast, p-ERK localization did not show any significant difference between a planar substrate and micropits in the presence of Arp 2/3 inhibitor. Thus, when invadopodia formation is inhibited using Arp 2/3 inhibitor, cells cannot realize the difference between a planar substrate and micropits, which is observed by no difference in p-ERK shuttling and proliferation efficiency. Cells cultured on micropits expressed a lesser amount of p-ERK compared to the ones cultured on planar substrates (Figure S3e). However, in the presence of blebbistatin, there is no difference in the expression of p-ERK between the cells grown on a planar substrate and micropits, which indicates that micropits reduce MCF-10A proliferation by activating cellular contractility and by decreasing p-ERK expression as well as preventing it from translocating into the nucleus. Depth Sensing Reduces Malignant Breast Cancer Cell Proliferation by Inactivating Actomyosin Contractility and ERK Signaling. We subsequently explored how malignant breast cancer cells (MCF-7, MDA-MB-231) interact with depth-sensing cues arising from micropits to control their proliferation. We seeded nonmetastatic breast cancer cells (MCF-7) on the micropits with different depths and widths and found that 9 μm deep micropits of different width (2, 3, and 4 μm) reduce MCF-7 proliferation (Figure S4a). However, 1.5 μm deep micropits with varying width did not cause any reduction in proliferation, which signifies the dominant role played by the depth of the micropits instead of the width in reducing MCF-7 proliferation, which is consistent with MCF10A observations. We also found that MCF-7 cells extended actin-rich protrusions to sense the micropits’ depth, and the lengths of the protrusions were higher for a 9.0 μm depth (mean protrusion 1.72 μm) compared to a 1.5 μm depth (mean protrusion 0.79 μm) (Figure S4b). We then investigated the role of actomyosin contractility in controlling invadopodiamediated depth sensing in MCF-7 cells (Figure S4c). In the presence of Rac1 inhibitor, the difference in proliferation between a planar substrate and micropits diminishes. However, with blebbistatin treatment, the proliferation further decreased on the micropits compared to that of the planar substrate. When cells were treated with both blebbistatin and Rac1 inhibitor, there was no difference in proliferation between the planar substrate and micropits compared to blebbistatin alone. Therefore, in contrast to MCF-10A, depth-sensing cues inhibit Rho-ROCK-myosin-based contractility and promote Rac1 activity in MCF-7, and there is a cross-talk between them. Subsequently, we characterized the depth-sensing response of metastatic breast cancer cells (MDA-MB-231), and consistent with our previous findings for MCF-10A and MCF-7, MDA-MB-231 proliferation was reduced by the 9 μm depth of the micropits instead of the varying width (Figure S5a). We seeded MDA-MB-231 cells on collagen- and fibronectin-coated substrates and found that proliferation decreased on both the ECM proteins and the maximum decrease occurred on collagen-coated micropits (Figure S5b). Additionally, micropit-induced reduction in MDA-MB-231 proliferation was further validated using the MTS assay (Figure S5c). We noticed actin-rich protrusions of varying length and

expressed an increased amount of phosphorylated-MLC when normalized to the total pool within the cell. This finding is consistent with the notion that despite having a lower amount of myosin (due to an unknown feedback mechanism), micropits could activate Rho/ROCK signaling. Subsequently, we wanted to investigate whether there is any interplay or cross-talk between the activation of Rho-ROCK-myosin-based contractility and Rac1 inhibition as shown in previous studies.20,21 To test this question, we used Rac1 inhibitor in the presence of blebbistatin and found that in the presence of this double inhibition the proliferation of MCF-10A again was reduced on micropits to 0.73 and 0.62 mean fold change as the concentrations of the drug increased, respectively. Therefore, when both the drugs were added simultaneously, there is a rescue of phenotype compared to that observed in the case of only blebbistatin. These results imply that there is cross-talk between Rho-ROCK-myosin and Rac1 in depth-sensinginduced MCF-10A proliferation reduction. To further confirm our findings, we used other drugs that perturb actomyosin contractility, such as Rho activator II (activator of Rho GTPases) (0.10, 0.25, 0.50 μg/mL) and Y-27632 (ROCK inhibitor) (1, 10 μM). Consistent with our earlier results, in the presence of Rho activator II the proliferation was further reduced to mean fold changes of 0.60, 0.58, and 0.57 along with the increasing concentration of the drug, respectively (Figure 4b). However, in the presence of Y-27632, there is no reduction of proliferation on micropits, similar to what was observed in the presence of blebbistatin (Figure S3c). Therefore, invadopodia-mediated depth-sensing cues could activate RhoROCK-myosin-based contractility, which in turn inactivates Rac1, and there is cross-talk between them. Next, we looked into the effect of actomyosin contractility on the protrusion length of MCF-10A because myosin-II-based contractile forces regulate the activity and mechanosensing ability of invadopodia, as shown in previous studies.12,22 We found that Rac1 inhibitor reduced the protrusion length; however, in the presence of blebbistatin the protrusion length increased (Figure S3d). Thus, increasing contractile “pulling” forces using Rac1 inhibitor causes a retraction of the protrusions; however, when the contractile forces were released using blebbistatin, the invadopodia length increased further. Additionally, when the two inhibitors were added simultaneously, the protrusion decreased compared to that observed in the case of blebbistatin alone, which highlights the crucial interplay between Rho and Rac1 in controlling the length of invadopodia. Previous studies have shown that mechanical cues can control translocation of p-ERK between the nucleus and cytoplasm to regulate gene expression, while higher nuclear localization of p-ERK has been related to increased proliferation and vice versa.23−25 We further explored whether depth-sensing-induced reduction of MCF-10A proliferation is also mediated via differential translocation of p-ERK to the nucleus. To examine this possibility, we immunostained the cells seeded on micropits with p-ERK antibody. We observed that p-ERK has a diffused cytoplasmic distribution for cells cultured on micropits; however, it shuttles predominantly into the nucleus for cells on a planar substrate (Figure 4c). Additionally, in the presence of Rho activator II, we observed a similar trend of higher nuclear localization of p-ERK for cells grown on a planar substrate compared to micropits, which is quantified in Figure 4d. Interestingly, the p-ERK fluorescence intensity ratio between the nucleus and cytoplasm has a lower 7342


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Figure 5. Depth sensing decreases malignant breast cancer cell proliferation by inactivating actomyosin contractility and p-ERK signaling. Micropits decrease MDA-MB-231 proliferation by inactivating actomyosin contractility and p-ERK signaling. (a) Acto-myosin contractility modulating drug treatment: Rac1 inhibitor (NSC23766; 50, 100 μM) and blebbistatin (5, 20 μM). Data are means ± SEM (n = 3). For each experiment, an average of 300 cells were considered. P values were obtained using Student’s unpaired t test. **p < 0.01, *p < 0.05 with respect to planar substrate. Blue bar indicates planar substrate, and red indicates micropits (W = 2 μm, D = 9 μm). The number on top of the respective bar indicates mean fold change with respect to the planar substrate. N.S. denotes nonsignificant difference compared to a planar substrate. (b) Greatest reduction of MDA-MB-231 proliferation occurred after 24 h of cell seeding (highlighted by the red box) in the absence and presence of blebbistatin (20 μM). Growth curves of cells seeded on a planar substrate and micropits (W = 2 μm, D = 9 μm) are indicated by blue- and red-colored lines, respectively. (c) Fold change calculation of proliferating MDA-MB-231 cells as a function of time in the absence and presence of blebbistatin (20 μM). (d) Immunofluorescence images of p-ERK (green) and nucleus (DAPI, blue) of MDA-MB-231 cells grown on a planar substrate and micropits in the absence and presence of different drugs, respectively: Rho activator II (0.25 μg/mL); Arp2/3 inhibitor (CK-666; 50 μM) (scale bar 10 μm).

μm) compared to a 1.5 μm depth (mean protrusion 0.87 μm) (Figure S7a). Additionally, MDA-MB-231 proliferation was reduced by a 2 μm pore membrane compared to the planar case, and formation of actin-based protrusions was observed at the base of the membrane. This shows that MDA-MB-231 proliferation was reduced by invadopodia-mediated depthsensing cues independent of biochemical cues and orientation of the pores (Figure S7b). Next, we examined the localization of cortactin and paxillin in the invadopodia structures of different depths using immunostaining. For 1.5 μm depth micropits, cortactin localizes with actin in the shorter invadopodia (Figure S7c) and paxillin staining was observed at the edges of the pores (Figure S7d), similar to that of MCF10A. However, for a 9 μm depth, paxillin formed a ring-like structure around the pores with the actin cytoskeleton passing through the middle, which indicates the formation of functional invadopodia for micropits of greater depth (Figure S7e). Invadopodia formation has been previously correlated with the invasive potential of the cells.8,9 So, we quantified the length of the invadopodia for the three different breast cell lines according to their increasing invasive characteristics, such as MCF-10A, MCF-7, and MDA-MB-231. Interestingly, for 9 μm deep micropits, the protrusion length increases proportionally

identified these protrusions as invadopodia by the colocalization of cortactin and actin for 9 μm deep micropits (Figure S6a). Interestingly, in contrast to MCF-10A, the actin-rich protrusions in MDA-MB-231 were not completely inhibited (mean protrusion 1.06 μm) in the presence of the Arp 2/3 complex inhibitor (CK-666; 50 μM) compared to no inhibitor (mean protrusion 2.03 μm) (Figure S6b). One previous study has shown that three members of the diaphanous-related formin (DRF) family (DRF1−DRF3) are involved in the formation and activation of invadopodia in MDA-MB-231 cells.26 Therefore, in the presence of the Arp 2/3 complex inhibitor, only the Arp 2/3-mediated branched actin network is disrupted, but the linear filaments formed by formins are not affected, thereby causing a partial retraction of the protrusions. However, in the presence of different concentrations of Arp 2/3 complex inhibitor (CK-666; 10, 50, 100 μM), the proliferation of MDA-MB-231 did not decrease on micropits compared to the planar substrate, which indicates that invadopodia formation is essential for depth sensing and reducing MDAMB-231 proliferation (Figure S6c). We further characterized the invadopodia of MDA-MB-231 cells by quantifying their protrusion lengths. Cells extended longer invadopodia for a 9.0 μm depth (mean protrusion 2.03 7343


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Figure 6. Schematic representation of differential depth sensing regulated by mechanically induced dormancydepth sensing (MIDDS) of cancer cells. Cells could sense the differential depth of the matrix using actin-enriched protrusions such as invadopodia. For MCF-10A cells, depthsensing cues could activate actomyosin contractility and prevent translocation of p-ERK into the nucleus, thereby causing proliferation reduction. In contrast, MDA-MB-231 cell proliferation is reduced via inactivation of myosin-II-based contractility and p-ERK signaling. (+) sign, green color, and point-ended arrows denote activation of the signaling process and (−) sign, red color, and blunt-ended arrows indicate inactivation of the phenomenon.

to the invasive potential of the cells (Figure S8). This result implies that as the cell becomes more metastatic, it can extend longer actin protrusions to degrade the matrix using metalloproteinase, and the length of the invadopodia can be a good indicator of the invasive potential of cancer cells. Next, we investigated how the actomyosin contractility is regulated by invadopodia-mediated depth sensing for MDAMB-231 cells. We treated the cells cultured on micropits with varying concentrations of drugs that regulate cellular contractility, such as blebbistatin (5, 20 μM) and Rac1 inhibitor (NSC23766) (50, 100 μM) (Figure 5a). In the presence of Rac1 inhibitor, there is no difference in proliferation between the planar substrate and micropits, which indicates that Rac1 is activated within the cell, and when we prevented this activation using drugs, the cell could not respond to the depth-sensing cues. We further validated this finding using the Rac1-FRET biosensor in MDA-MB-231 cells and found that the FRET ratio increased for cells cultured on micropits compared to on planar substrate (Figure S9a). This signifies that micropits induce Rac1 activity in MDA-MB-231 cells. Surprisingly, blebbistatin treatment further reduces the proliferation of MDA-MB-231 on micropits with mean fold changes of 0.68 and 0.59 for 5 and 20 μM concentrations of blebbistatin, respectively, as compared to 0.76 for no inhibitor control. Thus, in contradiction to MCF10A, depth-sensing cues are inhibiting acto-myosin contractility of MDA-MB-231 cells, and in the presence of pharmaceutical drugs that inhibit contractility the proliferation rate decreases further on micropits. Cells seeded on micropits express a higher amount of MLC compared to on planar substrate (Figure S9b). However, they express a reduced level of phosphorylated-MLC when normalized to the total pool within the cell, which further confirms that micropits inhibit Rho/ROCK signaling. We then

examined whether there is any interplay between Rac1 activation and inhibition of Rho-ROCK-myosin-based contractility as was previously observed with MCF-10A. To test this possibility, we treated the cells with both blebbistatin and Rac1 inhibitor and found that there was a rescue in phenotype in the presence of the double inhibitors compared to that of blebbistatin alone. In other words, with both inhibitors, the difference in proliferation between the planar substrate and micropits gradually diminished as the concentration of the drugs increased. This indicates that there is cross-talk between actomyosin contractility and Rac1 in reducing MDA-MB-231 proliferation in response to depth-sensing cues. To further confirm our observations, we used other drugs that regulate actomyosin contractility, such as Y-27632 (1, 10 μM) and Rho activator II (0.10, 0.25, 0.50 μg/mL). Consistent with our previous results, inhibiting contractility using Y-27632 further reduced MDA-MB-231 proliferation on micropits to mean fold changes of 0.71 and 0.65 for 1 and 10 μM concentrations of the drug compared to 0.77 for the no inhibitor condition (Figure S10a). However, activating contractility using Rho activator II prevented micropit-induced reduction in proliferation (Figure S10b). Therefore, for MDA-MB-231 cells, invadopodiamediated depth-sensing cues could inhibit actomyosin contractility, which in turn activates Rac1, and there is crosstalk between them. Subsequently, we investigated the dynamics of blebbistatin-mediated greater reduction of MDA-MB-231 proliferation on micropits for different periods of time. Similar to MCF-10A, the greatest reduction of proliferation occurred after 24 h of cell seeding for both no inhibitor and the blebbistatin treatment condition (Figure 5b). The doubling time for MDA-MB-231 cells is around 28 h.27 Interestingly, 24 h of blebbistatin treatment reduced MDA-MB-231 proliferation 7344


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features that are less than the dimension of a single cell play a crucial role in single-cell mechanosensing. Previous studies have highlighted the effect of these types of surface topographies in regulating the behavior of various bone cells such as osteoblasts,34−36 osteoclasts,37 and osteocytes.38 Therefore, topographic features that are smaller than the size of a cell play an essential role in mechanotransduction in the bone microenvironment, and the dimensions of the micropits in this study were less than 10 μm so that the effect of topographic cues on the single cell can be monitored. When the cancer cells reach the bone niche through the circulatory system, either they undergo long-term dormancy (for breast cancer39,40) by adapting to the new microenvironment or they start proliferating immediately (for lung cancer),41 depending on the particular cancer type. While adapting to their “new home”, the cancer cells can reprogram the preosteoblasts using soluble factors to make the environment favorable for their own growth.7 As a result, the activity of the osteoclasts increases, which results in bone degradation and increased porosity of the matrix, subsequently leading to severe bone pain and fracture in the advance stages of the metastatic disease. However, it was not known previously the effect of the constantly evolving pore dimensions on the proliferation efficiency of cancer cells. In our previous work, we introduced the concept of mechanically induced dormancy, whereby anisotropic cues provided by the aligned collagen fibers of the primary breast tumor environment could reduce the proliferation of noncancer breast epithelial cells via activation of actomyosin contractility, but the malignant breast cancer cells somehow bypass these growth inhibitory cues and continue their uncontrolled proliferation.2 In this study, we examined the effect of the dynamic mechanical niche of the bone on cancer cell proliferation using micropits of specific dimensions to mimic the physiological porous environment. We found that the depth of the micropits (but not the width and the orientation) could reduce the proliferation of both normal and malignant breast cancer cells using mechanosensing structures such as invadopodia. Therefore, these findings show the role of invadopodia as a depth sensor and their ability to regulate the proliferation ability of cells, apart from their well-known function of matrix degradation and remodeling. Noncancer breast epithelial cells respond to these depth-sensing cues via activation of Rho-Rac-regulated actomyosin contractility, which subsequently inactivates the translocation of p-ERK into the nucleus. In contrast, metastatic breast cancer cells inactivate Rho-Rac-controlled actomyosin contractility and p-ERK shuttling into the nucleus to cause reduced proliferation on micropits. Previous studies have shown a positive correlation between the expression of RhoA and ROCK1 and metastatic potential, which signifies the importance of the contractile forces during metastasis of breast cancer.42,43 Interestingly, the expressions of contractile proteins such as RhoA, ROCK1, and phospho-myosin light chain are lowered in MCF-10A compared to MDA-MB-231. Therefore, depending on their metastatic potential, cells use different signaling mechanisms to sense identical microenvironmental cues to cause proliferation changes. This presents an opportunity for potential therapeutic intervention, where contractility-inhibiting drugs could selectively reduce the proliferation ability of the cancer cells and induce dormancy while the healthy cells’ proliferation remains unaffected. Chemotherapeutic drugs targeting myosin-II-based contractility and matrix adhesion machinery such as the integrin

to a 0.55 mean fold change compared to a 0.70-fold change for the no inhibitor condition (Figure 5c). This provides a potential opportunity for therapeutic intervention, where contractility-inhibiting drugs and a depth-sensing mechanism could selectively induce 0.55-fold reduction of malignant cancer cell proliferation without affecting the proliferation of noncancer epithelial cells. However, with the passage of time, MDA-MB-231 cells acquire resistance against these depthsensing cues and gradually approach their normal proliferation efficiency as that on a planar surface. We then examined the effect of contractility on the invadopodia length of MDA-MB231 and found that, similar to MCF-10A, in the presence of Rac1 inhibitor the protrusions retracted due to increased contractile forces and blebbistatin treatment leads to further extension of the protrusions due to the release of contractile forces (Figure S10c). However, in the presence of double inhibition, there is a reduction in invadopodia length compared to blebbistatin alone, thereby highlighting the interplay between Rho and Rac1 in regulating the protrusion length. We then investigated whether the differential depth-induced reduction of MDA-MB-231 proliferation is coupled with the translocation of p-ERK into the nucleus. To explore this further, we immunostained MDA-MB-231 cells cultured on micropits with p-ERK antibody. We observed that p-ERK has a predominant cytoplasmic localization for cells cultured on micropits, which is in contrast to the nuclear localization of pERK for cells cultured on a planar substrate (Figure 5d). However, in the presence of Rho activator II, there is no difference in the p-ERK fluorescence intensity ratio between the nucleus and cytoplasm for a planar substrate and micropits (Figure S10d). This indicates that when the contractility of the cell is increased using Rho activator II, the cell cannot sense the difference between the planar substrate and micropits, and this results in no difference in the p-ERK localization and proliferation efficiency. Additionally, when the invadopodiamediated depth-sensing mechanism is inhibited using the Arp2/3 complex inhibitor, there is no significant difference in p-ERK intensity between the planar substrate and micropits. However, there is no difference in p-ERK expression between the cells cultured on a planar substrate and micropits for no inhibitor and blebbistatin treatment (Figure S10e). Therefore, MDA-MB-231 senses matrix depth using invadopodia, which subsequently inhibits actomyosin contractility and reduces proliferation predominantly by preventing p-ERK translocation into the nucleus, rather than by decreasing the overall p-ERK protein expression.

CONCLUSIONS In summary, we investigated the existence of mechanically induced dormancy through depth sensing, where cells can sense the different depths of the ECM using actin-enriched protrusions such as invadopodia and transduce a mechanical cue to regulate the contractile machinery, thereby reducing proliferation (Figure 6). The mechanical microenvironment of the bone, which is one of the principal metastatic sites, undergoes continuous remodeling due to the regulated activity of bone-forming osteoblasts and bone-degrading osteoclasts. Bone is composed of a complex hierarchical structure that varies within a wide range: from a few nanometers (∼100−300 nm)28,29 to the dimension of a single cell (∼15−20 μm)30,31 to macroscale structures (∼100−600 μm).32,33 The macroscale structures help in the diffusion of essential nutrients and oxygen for cell survival and bone tissue growth. The topographic 7345


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cells were stained with DAPI (Invitrogen, D1306) and imaged using a Nikon Confocal A1R microscope. The ratio between the number of proliferating cells (labeled with EdU) and the total number of cells (labeled with DAPI) was obtained to determine the percentage of proliferating cells. Fold change of proliferation was calculated as the ratio of the proliferation percentage on micropits to that on the planar substrate. Viable cells in proliferation were quantified using a colorimetric method (CellTiter 96 AQueous One Solution cell proliferation assay; MTS assay) according to the manufacturer’s protocol. Percentages of viable cells on micropits were normalized to that on the planar substrate. To visualize the actin-rich protrusions, cells were fixed with 4% PFA and permeabilized with 0.01% Triton X100. F-actin was then stained using Green 488 phalloidin (Invitrogen) and imaged using a Nikon Confocal A1R microscope. Protrusion length was quantified by selecting the region of interest along the zaxis using Imaris (Bitplane, Zurich, Switzerland). A total of 300 cells were taken on average for each experiment. Inhibitor Treatment. Inhibitor drugs were added to the cell culture media after 6 h of cell seeding, to allow sufficient time for the cells to spread properly. The cells were cultured in the presence of the drug for the required amount of time as indicated in the text and subsequently fixed and stained. The drugs used were blebbistatin (nonmuscle myosin II inhibitor; Sigma-Aldrich) (5, 20 μM), Y-27632 (ROCK inhibitor; Sigma-Aldrich) (1, 10 μM), Rho activator II (activator of Rho GTPases; Cytoskeleton) (0.10, 0.25, 0.50 μg/mL), Rac1 inhibitor (prevents Rac1 activation; NSC23766; Sigma-Aldrich) (50, 100 μM), and Arp 2/3 complex inhibitor (inhibitor of Arp2/3 mediated actin assemble; CK-666; Sigma-Aldrich) (10, 50, 100 μM). Immunostaining and Image Analysis. After culturing the cells on the micropits for the required amount of time, cells were fixed with 4% PFA and permeabilized with 0.01% Triton X-100 as mentioned previously. Subsequently, nonspecific binding was blocked using 1% bovine serum albumin (BSA; Sigma-Aldrich) in 1× PBS for 1 h. Cell were then incubated with primary antibody in 1% BSA for 1 h at room temperature. Primary antibody was then washed three times with PBS, and cells were incubated with fluorescent-tagged secondary antibody in 1% BSA for 1 h at room temperature. Cells were then washed three times with PBS, and nuclei of the cells were stained with DAPI. The primary antibodies used were anti-cortactin (Cell Signaling Technology) at 1:500, anti-paxillin (Invitrogen) at 1:400, and anti-phosphoERK (1/2) (Cell Signaling Technology) at 1:500. Secondary antibodies from Alexa Fluor (Invitrogen) were used. Cells were immunostained and imaged using a Nikon Confocal A1R microscope. Protrusion length was visualized by selecting the ROI along the z-axis using Imaris (Bitplane, Zurich, Switzerland). The pERK protein expression intensity of the nucleus and cytoplasm was quantified using ImageJ software. More than 25 cells were analyzed under each condition. Western Blot Analysis. Cell lysates were separated by gradient SDS-PAGE gels followed by Western blotting. The blot was blocked with blocking buffer (5% BSA in 0.1% Tween in TBS) for 1 h at room temperature. Primary antibody was incubated overnight at 4 °C prior to three washes of 10 min each at room temperature. Blots were subsequently incubated with secondary antibody for 1 h at room temperature and washed three times for 10 min each at room temperature. Immunoblots were developed using Pierce Pico ECL (Thermo Scientific). Antibodies used were polyclonal phospho-MLC of Thr18/Ser19 (CST), polyclonal MLC (CST), polyclonal phosphoERK1/2 (CST), monoclonal ERK1/2 (Santa Cruz), and monoclonal α-GAPDH (Santa Cruz). Statistics. Statistical significance was obtained using Student’s unpaired t test. Data represent ±SEM (standard error of mean) for independent experiments (n = 3). Statistical significance was considered at p < 0.05 and p < 0.01. All statistical comparisons were based on experiments performed on the same day.

EXPERIMENTAL METHODS Microfabrication of Micropit Topographic Features. The micropit features were fabricated using SU-8 on silicon wafers. Each feature was of size 1 × 1 cm dimensions, and the adjacent flat region served as a planar control. The curing agent was mixed homogeneously with polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning) in a ratio of 1:10 and poured on top of the silanized silicon wafer. The air bubbles formed during mixing were removed by degassing, and PDMS was allowed to solidify for 2 h at 80 °C. Solidified PDMS was taken off from the silicon wafer and then used for cell seeding. To examine the proper replication of the topographic features, 11-nm-thick platinum (JEOL JFC 1600 Auto Fine Coater) was sputter-coated on the PDMS substrate and visualized using a field emission scanning electron microscope (JEOL). Cell Culture. Noncancer human breast epithelial cells (MCF-10A; American Type Culture Collection (ATCC)) were maintained in mammary epithelial growth medium (MEGM Bullet Kit; Lonza Corporation) in the presence of cholera toxin (100 ng mL−1). Before seeding on the micropit pattern, cells were synchronized by culturing them to 100% confluence in the same medium for 1−3 days.50 Synchronized cells were then detached from the culture dish using 1× trypsin and cultured under subconfluence for 18−24 h prior to seeding on the micropit features. The human metastatic breast cancer cell line (MDA-MB-231; ATCC) and the human nonmetastatic breast cancer cell line (MCF-7; ATCC) were maintained in DMEM, 10% fetal bovine serum, and 1% penicillin and streptomycin in a 37 °C incubator in the presence of 5% CO2. Cells were detached using trypsin after approaching 80% confluence. Before seeding on the micropit pattern, cells were synchronized by serum starvation for 18−24 h. MCF-10A and MDA-MB-231 cells were transfected using Lipofectamine 2000 (Invitrogen). Rac1 biosensor (pTriEx4-Rac1-2G) was a gift from Olivier Pertz (University of Basel). The FRET ratio of the Rac1 biosensor was analyzed as described.51 The PDMS features were made hydrophilic by air-plasma treatment for 3 min (model PDC-002, Harrick Scientific Corp.) and UV sterilized for 5 min in the presence of 70% 2-propanol. They were then washed three times with sterile phosphate-buffered saline (PBS, SigmaAldrich) and coated with 10 μg mL−1 fibronectin (Sigma-Aldrich), 20 μg mL−1 collagen I (bovine; Nutragen), or poly-L-lysine (SigmaAldrich) for 1 h in a 37 °C incubator. Subsequently, they were washed with PBS three times, and 70 000 cells were seeded on each feature. Cells were cultured until subconfluence for 12−72 h. Membranes of 2 μm pore size (Millipore) were coated with 20 μg mL−1 collagen I, and cells were seeded on top of them and cultured for 12−48 h, as indicated in the text. Cell Proliferation Assay and Fluorescent Microscopy Staining. Cells were cultured on micropits for definite periods of time and then washed with PBS and fixed with 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich) for 10 min. Cells were then permeabilized using 0.01% Triton X-100 for 5 min. The proliferating cell nucleus was then labeled with EdU stain from a Click-iT EdU Alexa Fluor 555 imaging kit (Invitrogen, C10338). Nuclei of all the 7346


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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/acsnano.7b03452. Micropit reduces MCF-10A proliferation across different ECM coatings and time points, Nonmetastatic breast cancer cell (MCF-7) proliferation decreases on 9.0 μm depth micropits but not on 1.5 μm depth, Protrusion length increases proportionally to metastatic potential, Dimension of micropit patterns (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (B. C. Low): [email protected]. *E-mail (C. T. Lim): [email protected]. ORCID

Chwee Teck Lim: 0000-0003-4019-9782 Author Contributions

P.K.C., B.C.L., and C.T.L. designed the research; P.K.C. performed the experiments and data analysis; C.Q.P. performed the Western blot experiment and analysis; B.C.L. and C.T.L. supervised the project. All authors wrote the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research is supported by National Research Foundation Singapore under its Research Center of Excellence programme and administrated by Mechanobiology Institute Singapore (MBI), National University of Singapore. P.K.C. acknowledges support from the MBI for the Graduate Scholarship. We also thank M. Ashraf and S. Vaishnavi from the MBI Microfabrication Core for their assistance in fabrication and Mr. Wong Chun Xi (MBInfo) for preparing the illustrations. REFERENCES (1) Nguyen, D. X.; Bos, P. D.; Massagué, J. Metastasis: From Dissemination to Organ-Specific Colonization. Nat. Rev. Cancer 2009, 9, 274−284. (2) Chaudhuri, P. K.; Pan, C. Q.; Low, B. C.; Lim, C. T. Topography Induces Differential Sensitivity on Cancer Cell Proliferation via RhoROCK-Myosin Contractility. Sci. Rep. 2016, 6.10.1038/srep19672 (3) Ulrich, T. A.; de Juan Pardo, E. M.; Kumar, S. The Mechanical Rigidity of the Extracellular Matrix Regulates the Structure, Motility, and Proliferation of Glioma Cells. Cancer Res. 2009, 69, 4167−4174. (4) Chang, S.-F.; Chang, C. A.; Lee, D.-Y.; Lee, P.-L.; Yeh, Y.-M.; Yeh, C.-R.; Cheng, C.-K.; Chien, S.; Chiu, J.-J. Tumor Cell Cycle Arrest Induced by Shear Stress: Roles of Integrins and Smad. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3927−3932. (5) Ferreira, A. M.; Gentile, P.; Chiono, V.; Ciardelli, G. Collagen for Bone Tissue Regeneration. Acta Biomater. 2012, 8, 3191−3200. (6) Patino, M. G.; Neiders, M. E.; Andreana, S.; Noble, B.; Cohen, R. E. Collagen: An Overview. Implant Dent. 2002, 11, 280−285. (7) Croucher, P. I.; McDonald, M. M.; Martin, T. J. Bone Metastasis: The Importance of the Neighbourhood. Nat. Rev. Cancer 2016, 16, 373−386. (8) Coopman, P. J.; Do, M.; Thompson, E. W.; Mueller, S. C. Phagocytosis of Cross-Linked Gelatin Matrix by Human Breast Carcinoma Cells Correlates With Their Invasive Capacity. Clin. Cancer Res. 1998, 4, 507−515. (9) Yamaguchi, H.; Lorenz, M.; Kempiak, S.; Sarmiento, C.; Coniglio, S.; Symons, M.; Segall, J.; Eddy, R.; Miki, H.; Takenawa, T.; Condeelis, 7347


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Differential Depth Sensing Reduces Cancer Cell Proliferation via Rho

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