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Shimaa A. Abdellatef, Francesca Bard, [Jun Nakanishi](https://orcid.org/0000-0003-4457-6581)

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[Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteins](https://mdr.nims.go.jp/datasets/1755a70f-6197-4573-bddb-36ed89e9d244)

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Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteinsBiomaterialsSciencePAPERCite this: Biomater. Sci., 2024, 12,3446Received 13th February 2024,Accepted 20th May 2024DOI: 10.1039/d4bm00225crsc.li/biomaterials-sciencePhotoactivatable substrates show diversephenotypes of leader cells in collective migrationwhen moving along different extracellular matrixproteins†Shimaa A. Abdellatef,*a Francesca Barda,b and Jun Nakanishi *a,c,dIn cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells thatmove through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECMprotein types on leader cells and migrating clusters is unknown. To address this, we investigated theresponse of leader cells and migrating clusters upon moving from one ECM protein to another using aphotoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since theyare abundant in two distinct regions in living tissues, namely basement membrane and connective tissue.Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areaswas achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled thedeposition of the second ECM protein in the new irradiated regions, resulting in region-selective hetero-geneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation fromlaminin into laminin compared to those migrating from laminin into collagen were observed. The formationof focal adhesion and actin structures for cells within the same cluster in the ECM proteins respondedaccording to the underlying ECM protein type. Finally, integrin β1 was crucial for the appearance of leadercells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin,integrin β1 was not responsible. This highlights the correlation between leader cells in collective migrationand the biochemical signals that arise from underlying extracellular matrix proteins.1. IntroductionCollective cell migration is an intriguing process that plays acrucial role in several biological processes, such as morpho-genesis, tissue repair, and cancer metastasis. It demonstratesthe remarkable coordination of cells as they move together.The coordination of movement ranges from multicellularstreaming until it reaches tightly connected cells that migrateas a cohesive unit, known as supracellular migration.1 Basedon the topology of migrating populations and tissue-scalekinetics, the “leader-follower” model has been proposed2 withother models3 to understand the persistent directional collec-tive movement. The “leader-follower” model is based on theidea that cell populations are discriminated into two distinctcell populations. These two groups are distinguished by theirposition within the moving cell group. Leader cells are special-ized cells located at the front of the collective. On the otherhand, follower cells constitute the majority of the collectiveand are located in the cell reservoir. The leader-follower for-mation is associated with various cellular changes, includinggenetic,4 epigenetic,5 biochemical,6,7 and mechanical factors.8Leader cells are distinguishable by their position at the frontof the migrating group, actin-rich lamellipodia, higher meta-bolic demands,7 and ability to guide migration direction andspeed.9,10 They are also more sensitive to chemoattractantsthan follower cells.11,12 Follower cells that migrate after theleaders can also contribute passively or actively to collectivemigration.13 They can push, pull, or even compete with leadersfor their positions. Leader cells play a significant role incancer metastasis, where cells migrate from one organ toanother distant organ. During this journey, leader cells, fol-†Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4bm00225caMechanobiology group, Research Centre for Macromolecules and Biomaterials,National Institute for Materials Science (NIMS), Tsukuba, Japan.E-mail: nims.email.Shimaa@gmail.com, NAKANISHI.Jun@nims.go.jpbDepartment of Material Science and Engineering, Cornell University, Ithaca, NY,USAcWaseda University Graduate School of Advanced Science and EngineeringDepartment of Nanoscience and Engineering, Tokyo, JapandTokyo University of Science, advanced Graduate School of Engineering MaterialsInnovation Engineering, Japan3446 | Biomater. Sci., 2024, 12, 3446–3457 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View Issuehttp://rsc.li/biomaterials-sciencehttp://orcid.org/0000-0003-4457-6581https://doi.org/10.1039/d4bm00225chttps://doi.org/10.1039/d4bm00225chttps://doi.org/10.1039/d4bm00225chttp://crossmark.crossref.org/dialog/?doi=10.1039/d4bm00225c&domain=pdf&date_stamp=2024-06-20http://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225chttps://pubs.rsc.org/en/journals/journal/BMhttps://pubs.rsc.org/en/journals/journal/BM?issueid=BM012013lowed by other cells, move through the extracellular matriceswith tremendously different characteristics. Leader cells pavethe way in front of migrating clusters by biochemically andphysically altering the surrounding environment. This isachieved through matrix deposition, physical remodeling, andproteolysis.14 However, before this can happen, it is not knownhow leader cells first sense and respond to the immediatechanges in their environment. In recent years, advancedmaterial fabrication has been used to mimic the differentchemical and mechanical cues of the extracellular matrix(ECM) to understand how these cues affect the formation ofleader cells and collective migration. However, most previousstudies have compared collective cell migration and leader cellformation using different substrates with different ECMcharacteristics, assuming that cells migrate in the same ECMcharacteristics and record their behaviours in these differentsubstrates. In reality, migrating clusters and leader cells facechanges in the ECM characteristics and respond to themaccordingly. Therefore, it is crucial to develop substrates thatenable us to have different ECM characteristics while the cellsare migrating. By doing so, we can gain a more comprehensiveunderstanding of how leader cells respond to changes in theirenvironment and develop more effective strategies to harnesstheir migratory potential.To tackle this challenge, in this study, we used a photoacti-vatable surface that changes from protein-repellent to protein-adhesive in response to light.15 Several types of photoactivata-ble substrates based on photocleavable poly(ethylene glycol)(PCP) have been previously developed not only on microscopi-cally friendly glass coverslip16 but also on stiffness-definedhydrogels17 to demonstrate cell culturing in specific patterns18or the development and coculture systems.19 The use of photo-activatable surfaces in this study can greatly aid our researchin creating surfaces coated with various ECM proteins. Thisenables us to study the collective migration of cells from oneECM to another and compare it to their migration on thesame ECM (Fig. 1A). Additionally, as we demonstrated pre-viously, by exposing the surface to UV light through a photo-mask, we can establish cellular constraints with preferred geo-metries20 to standardize cell morphology6 before the onset ofmigration, even in an inverted configuration.20 This remotecontrollability of cell migration induction would provide morereliable and cell-friendly assessments by eliminating the limit-ations associated with traditional tests that often impede thecollective migration process due to damaged cells at theboundaries.This study aims to examine the response of leader cellswhen moving across different types of extracellular matrix(ECM). We chose laminin and collagen I as simple examples ofdifferent ECM types since it is widely recognized that a combi-nation of different ECM proteins forms the distinct structureof ECM in various organs and tissues.21 Therefore, it can reallymimic what the leader cells could face during cancer meta-stasis. In detail, laminins (LM) are crucial components of base-ment membranes and play a vital role in the healing processof wounds. They are heterodimeric glycoproteins. Several iso-forms of laminins have been identified.22 Laminin isoformLM332 promotes the persistent migration of keratinocytes,23 avital step in wound re-epithelialization. Knocking downlaminin LM-511, another isoform of laminin, inhibits thedirectional migration of MDCK cells.24 While collagen I is themost abundant ECM protein in connective tissue and bone,18this protein comprises a long continuous triple helix thatassembles into highly organized fibrils that mainly determinethe ECM mechanics. Upregulation of collagen I is an earlyevent in renal fibrosis.25 We have chosen these two proteinsfor our study since they are abundant in two distinct regions inliving tissues: basement membrane and connective tissue.This will allow us to gain valuable insights into the role andfunction of alterations of ECM proteins in leader cell appear-ances in collective migration.2. Results and discussion2.1. Photoactivatable substrates as a testing platform tostudy collective cell migration on different ECMsWe have utilized glass surfaces functionalized with a photo-cleavable monolayer of PEG, as previously reported.26 ThesePEG groups were covalently linked to the glass, which made itresistant to cell adhesion and protein adsorption (Fig. 1B). Weused a photocleavable 2-nitrobenzyl (2-NB) group to link PEGand glass surfaces, which can be easily cut by irradiation thesurface with UV light (λ = 365 nm). Under this process, glasssurfaces that were once passivated can now allow protein depo-sition and cell adhesion (Fig. 1B). Next, the stepwise depo-sition of ECM proteins and the subsequent cellular culture is afacile method to examine the effect of various ECM proteinson collective cell migration compared to conventionalmethods such as wound scratch assays. Fig. 1C presents avisual representation of the experimental procedure. Circularclusters with a diameter of 266 µm were created on PEG-coatedglass surfaces using photomasks and UV light. The first ECMprotein, such as collagen or laminin, was deposited for5 minutes. Following that, cells were seeded to the circular pat-terns coated with the specific ECM protein in serum-freemedium to reduce the risk of non-specific adhesion of serumproteins during cellular attachment. We seeded the cells for abrief period of 3–4 hours before the second irradiation. Wechose this short incubation time because our group has pre-viously observed that shorter culture time is positively associ-ated with the higher appearance of leader cells and cellularexpansion.6,20 A second irradiation was performed using theopposite photomask to shield the cells from UV hazards. Next,the second ECM protein (collagen or laminin) is deposited inthe irradiated area for another 5 minutes. Finally, the expan-sion and migration of cell sheets were observed using liveimaging. We have confirmed the adsorption of ECM proteinsto glass surfaces after photoirradiation using Alexa Fluor-labelled antibodies (Fig. 1D). The patterned substrates wereimmersed in ECM protein for 5 minutes, forming a circularpattern of fluorescently labelled protein surrounded by non-Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 3446–3457 | 3447Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225cirradiated areas (Fig. 1D). The photoactivatable surfaces notonly allow for the creation of well-defined cellular confine-ments but also allow for the precise deposition of ECM inthese patterns before the cellular seeding. Conventionalmethods like scratch wound healing assays for performingthese sequential steps of extracellular matrix (ECM) depositionand cellular patterning are limited. However, photoactivatablesurfaces provide a more practical and straightforward platformto explore collective cell migration on several ECM proteins.This emphasizes the potential of our system for further investi-gating the relationship between collective cell migration andother extracellular matrix cues.2.2. The leader cell appearance and expansion area arecorrelated to the underlying ECM proteinsNext, we examined the migratory characteristics of MDCK cellsmigrating collectively on different ECM proteins. MDCK cellsare normal epithelial cells and are a widely studied model ofcollective migration;25,26 they naturally differentiate betweenleaders and followers.27,28 Our study defines leader cells bytheir position and unique characteristics (Fig. S1†). Theseleader cells are located at the front of the migrating clusters;they are larger in size relative to their follower cells, possess afan-shaped morphology, and show active ruffled lamellipodia.Leader cells could lead the migration for several hours or bereplaced with another leader. After a longer duration of cellmigration, we observe a similar cellular arrangement as thefinger-like structures reported in several studies.28,29Our study used two indicators to measure cellularmigration velocity and leader cell appearances. Firstly, we uti-lized the cluster area expansion ratio to indicate cellularmigration velocity (Fig. 2A). Secondly, we used cluster circular-ity to indicate leader cell appearances. Since we started withcircular clusters due to the utilization of photoactivatable sur-Fig. 1 (A) cartoon depicts the migration of cells on heterogeneous ECM protein-coated glass compared to a single ECM protein-coated glass. (B) Thescheme depicts the PEG-coated surfaces to block the protein adsorption and cellular adhesion, while upon the UV irradiation, the surface allows stepwiseECM protein adsorption followed by cellular seeding. (C) The scheme describes the experimental procedure for our study. (D) A fluorescence image of acircular cluster after UV irradiation and fibronectin is adsorbed for 5 minutes, then visualization occurs using Anti-fibronectin Alex-fluor-488.Paper Biomaterials Science3448 | Biomater. Sci., 2024, 12, 3446–3457 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225cfaces, we were able to easily determine the appearance ofleader cells by observing the decrease in cluster circularity(Fig. 2B). Movie 1† shows the expansion and collectivemigration of MDCK on various ECM proteins. Fig. 2C–F showsthe cellular cluster expansion from 0 h compared to 3.5 h. Wechose four conditions for cell migration. Our first conditioninvolves the movement of cells from laminin-coated areasunderneath cell clusters to the irradiated regions outside theclusters, which are also coated with laminin (Fig. 2C). In thesecond condition, we focus on cells migrating from collagen-coated areas underneath the clusters to irradiated areas coatedwith collagen (Fig. 2D). The third condition involves observingthe migration of cells from laminin-coated areas into collagen-coated regions (Fig. 2E). Finally, in the fourth condition, weexamined the migration of cells from collagen-coated areasinto laminin-coated regions (Fig. 2F). For convenience, thesefour conditions will be mentioned in the rest of the study asLam–Lam, Col–Col, Lam–Col, and Col–Lam, respectively. Thecircular clusters that moved on Lam–Lam show a higher expan-sion ratio than those that moved on Col–Col (Fig. 2G, lamininblue vs. collagen orange). This highlights the importance ofthe types of ECM proteins as chemical cues in determining theexpansion of clusters and migration velocity. When we com-pared the Lam–Lam to the Lam–Col, we found no significantdifference in the migration expansion area (Fig. 2G, lamininblue vs. laminin orange). In the alternative case, when wecompare Col–Col and Col–Lam, we could not find also any sig-nificant difference in the migration expansion area (Fig. 2G,collagen blue vs. collagen orange). This suggests that the sec-ondary ECM, whether collagen or laminin, did not play anyrole in the cluster’s expansion ratio or migration velocity;rather, the primary ECM underneath the clusters representsthe starting point for cluster expansion and migration. Inother word, our results suggest the role of the follower cells tosteer the migration from behind based on their underlyingECM proteins. Several studies have shown that the contri-Fig. 2 Cartoons illustrate the change in (A) area expansion and (B) cellular circularity. Phase contrast images for cells at 0 h and after 3.5 h of induc-tion of migration for surfaces (C) laminin–laminin, (D) collagen–collagen, (E) laminin–collagen, and (F) collagen–laminin. (G) Average calculation ofarea expansion ratio at 3.5 h for migrating clusters (N = 8–13 clusters from three different experiments, results showed as Mean ± SD). (H) Averagecluster circularity at 3.5 h for migrating clusters (N = 8–13 clusters from three different experiments). (I and J) Leader cell trajectories for clustersmigrating on (I) laminin–laminin and (J) laminin–collagen. (K) The calculated velocity of leader cells migrating on laminin–laminin and laminin–col-lagen (N = 8 cells).Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 3446–3457 | 3449Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225cbution of the follower cells to the migrating clusters goesbeyond simply being dragged along passively.2,13,30 During themigration of zebrafish polster cells, follower cells guide theirmigration by pulling the leader cells from behind throughE-cadherin/a-catenin mechanotransduction.31 Another workshows that followers push the leader cells during the migrationof posterior lateral line primordium (PLLp) migrating withinthe zebrafish embryo.32 Therefore, the migration of cells isdriven by biochemical signals originating from their inter-actions with primary extracellular matrix (ECM-1), rather thanby the new chemical environment (ECM-2) to which they aremigrating. After that, when we compared the change in circu-larity upon the cluster migration, we observed exciting results;first of all, when we compared the Lam–Lam and Col–Col cir-cularity, we observed that cells migrating on Lam–Lam showeda decrease of circularity compared to cells migrating collagenCol–Col (Fig. 2H, laminin blue vs. collagen orange). Thissuggests that there are more leader cells for Lam–Lam com-pared to Col–Col. We concluded that the type of ECM proteinscan be utilized as an ECM cue to modify the appearance ofleader cells. Previously, several ECM cues have been shown toimpact the appearance of leader cells, including chemical cuessuch as ECM concentration,6 geometrical cues,20,33,34 andmechanical cues such as compressive stress,8 substratestiffness35 and, gravity vector.36 Interestingly, we found thatthe circularity of cells migrating on Lam–Lam was significantlylower than those migrating from Lam–Col (Fig. 2H, lamininblue vs. laminin orange). Despite having the same expansionratio (Fig. 2G, laminin blue vs. laminin orange). On the otherhand, the circularity of cells migrating from collagen to col-lagen or laminin remained equal and larger (Fig. 2H, collagenblue vs. collagen orange), with a lower expansion ratio (Fig. 2G,collagen blue vs. collagen orange). Based on these results, wecan conclude that the secondary ECM is important in deter-mining the appearance of leader cell formation in highlymigrating cells such as Lam–Lam and Lam–Col. However, forslow-migrating cells, this difference was not significantlyobserved. In order to distinguish between the phenotypes ofleader cells, we monitored the trajectories and velocity ofhighly migrating cells, specifically Lam–Lam and Lam–Col(Fig. 2I–K). Our observations revealed that the leader cellsmigrating on Lam–Lam exhibited a greater number of direc-tional changes when compared to those migrating on Lam–Col(Fig. 2I and J). Moreover, the velocity of the leader cells wasfaster on laminin and secondary ECM compared to collagen(Fig. 2K). Despite these differences, when we think about themovement of epithelial cells, it is important to understandthat this motion results from a global tug-of-war within clus-ters. This tug-of-war integrates each local force generated byleaders and submarginal followers, working together toproduce the required tensile stress.37 It is worth noting thatleaders guide migrating follower cells during migration and donot drag them along. Therefore, when comparing Lam–Lamand Lam–Col during cluster expansion, it is expected that theoverall force generated would remain unchanged since theaverage cellular expansion was identical for both migratingclusters. This could be due to the fact that most of the followercells remained in laminin during our experiments. However, itwas observed that the number of leader cells, which act astrack-generating cells, decreased for cells migrating to collagencompared to those migrating to laminin. This suggests thatmigration into collagen hinders the transformation of externalcells into leader cells. This suggests that the expansion of cel-lular clusters and the generation of leader cells are completelydifferent processes controlled by different biochemical signalsand factors.2.3. The variations in the formation of cytoskeletal and focaladhesion structures when cells migrate in different types ofextracellular matrix proteinsSince we have identified that the tendency of leader cellappearance depends on the type of underlying ECM proteins,we planned to describe the cytoskeletal structures and for-mation of focal adhesion variations in these different ECMproteins. Our initial observations of single cells cultured onirradiated glass substrates coated with laminin and collagenrevealed the presence of altered focal adhesions and actinstructures formed for each ECM protein (Fig. S2†). Vinculinstaining was used to visualize the shape and size of formedfocal adhesions, and phalloidin staining was used to visualizethe actin structures. In laminin, single cells exhibited smallersizes and fewer vinculin and actin filaments compared tosingle cells on collagen, which exhibited larger sizes andhigher numbers of vinculin and actin filaments (Fig. S2†). Thesame trend in the formation of focal adhesions was observedacross cells of different origins, such as cervical human cancer(HeLa) and human breast cancer (MDA MB-468), when cul-tured on laminin and collagen (Fig. S3†). By utilizing the pat-terned photoactivatable surface, we could observe the for-mation of focal adhesions (FA) and actin structures for cellswithin the same cluster that exists in the heterogenous orhomogenous ECM proteins (Fig. 3). For the Lam–Lam sub-strate, we observed the formation of small and scattered FA inboth leader cells and cells within initial circular clusterscoated with laminin. Fewer actin stress fibers were observedfor both leading cells (migrating on laminin), and those fol-lower cells remained in laminin (Fig. 3A). On the other hand,in cells migrating in Col–Col, we observed the formation oflarger FAs and higher numbers, as visualized by vinculin stain-ing by both leading cells (migrating on collagen) and the fol-lower cells that remained on collagen (Fig. 3B). Additionally,both types of cells formed a higher number of stress fibers(Fig. 3B). Afterward, we examined the focal adhesion (FA) andactin structures of cells migrating in heterogeneous ECM pro-teins such as Lam–Col (Fig. 3C) and Col–Lam (Fig. 3D). Weobserved that each cell type maintained its original FA andactin stress fiber type based on the underlying ECM proteintype. For instance, in the case of Lam–Col, leader cells thatmigrate on collagen showed the formation of larger FA and alot of stress fibers, while the follower cells inside the clustersmigrating on laminin displayed the formation of small FA andless actin stress fibers (Fig. 3C). Conversely, for cells on Col–Paper Biomaterials Science3450 | Biomater. Sci., 2024, 12, 3446–3457 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225cLam, where leader cells that migrate on laminin showed theformation of small FA and less actin stress fibers. On the otherhand, the follower cells that remained in collagen showed theformation of larger FA and a lot of stress fibers. This resultdemonstrates the feasibility of our system to analyze changesin ECM protein types and how leader and follower cellsrespond to underneath biochemical signal alterations.Moreover, despite the observed differences in the size of focaladhesions, it’s not possible to establish a direct correlationbetween the size of focal adhesions and the observedmigratory characteristics. This is because there is a biphasicrelationship between the size of focal adhesions and migrationspeed. Initially, the migration speed increases as the size offocal adhesions increases until a certain threshold, after whichthe migration speed decreases.38 However, our findings indi-cate that leader cells and followers that emerge during collec-tive cell migration display distinct traits based on the extra-cellular matrix (ECM) protein type present. This implies thatbiochemical signals in the ECM could be linked to the devel-opment of different subtypes of leader cells, each with uniquestructural and morphological features. Finally, the utilizationof photoactivatable substrates to monitor collective cellmigration on various extracellular matrix (ECM) proteins couldbe incredibly valuable in understanding the impact of alteredECM proteins or other cues in scenarios like cancer metastasisor morphogenesis.2.4. Leader cell subtypes are different for cells migratingfrom laminin into laminin or collagenIn the previous section, we demonstrated that the leader cellsexhibit distinct appearance, structural, and morphologicalcharacteristics when they migrate from laminin to collagencompared to when they migrate from laminin to laminin. Thisobservation suggests that the biochemical signals transmittedto the cells are different. Previous reports discuss theexpression of integrin β1 specifically to the leading edges ofmigrating clusters,39,40 another report indicated that integrinβ1 is upregulated in leader cells that migrate in collagen gel.28This upregulation plays a crucial role in activating Rac, whichis essential for driving collective migration and leader cellappearance.28 Therefore, if the signals that cause leader cellsto appear are different between laminin and collagen, and weknow for certain that integrin β1 is involved in the appearanceof leader cells for collagen,28 then blocking integrin β1 onmigrating clusters would result in different tendencies ofleader cell appearance. To test this hypothesis, we used integ-rin β1 blocking antibodies (AIIB2) during the migrationprocess (Movie 2†). In Fig. 4A and B, the phase contrastimages of two types of cell clusters, Lam–Lam and Lam–Col,both with and without blocking antibodies, at 0 and 3.5 hoursof migration were presented. We observed the change incluster circularity and found that for the migrating clusters,Lam–Lam did not show any significant change with or withoutblocking antibodies (Fig. 4B). This indicates that integrin β1does not play a role in the appearance of leader cells when thecells migrate within laminin. However, for Lam–Col substrates,we observed a significant increase in the cluster circularity,which indicates a decrease in the formation of leader cells inthe presence of blocking antibodies (Fig. 4C). Additionally, thearea expansion ratio decreased for both migrating clustersafter treatment with antibodies (Fig. 4D), which likely suggeststhat different biochemical mechanisms control the expansionFig. 3 Fluorescence images of MDCK cell stained for actin (red), nucleus (blue), and vinculin (green) for migrating clusters on surfaces (A) laminin–laminin, (B) collagen–collagen, (C) laminin–collagen, and (D) collagen–laminin.Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 3446–3457 | 3451Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225cratio and leader cell formation. This indicates that while integ-rin β1 contributed to the cluster expansion, it is not involvedin leader cell formation for cells migrating in Lam–Lam sub-strates. However, it is engaged in both migration and leadercell formation in the other case (Lam–Col). In general, cellscan express many integrin heterodimers that interact with par-tially overlapping sets of ECM molecules. MDCK cells, forinstance, express a range of integrins, including β1, β3, β5, b6,β8, α5, and αV-integrins which alters their adhesion withdifferent ECM proteins41 and associated functionalities.42Although integrin β1 has an affinity to laminin as well as col-lagen I, other integrins are involved in the case of laminin. Itwas previously reported that the knockdown integrin β1 con-tributes to the spreading of MDCK on collagen and laminin115, yet to a different extent.41 This result supports our findingsince the expansion rate is delayed in both migrating clusters(Lam–Col) and (Lam–Lam), which could represent the contri-bution of integrin β1 in spreading, while leader cell formationFig. 4 Phase contrast images for cells at 0 h and after 3.5 h of migration with and without integrin β1 blocking antibody (AIIB2) for surfaces (A)laminin–laminin, (B) laminin–collagen. (C) Average cluster circularity at 3.5 h for migrating clusters (N = 3–11 clusters from 3 different experiments).(D) Average calculation of area expansion ratio at 3.5 h for migrating clusters (N = 3–11 clusters from 3 different experiments). Fluorescence imagesof MDCK cell stained for vinculin (green), actin (red), nucleus (blue) for migrating clusters with and without integrin β1 blocking antibody (AIIB2) onsurfaces (E) laminin–laminin, (F) laminin–collage. (G) Fluorescence images of leader cells for p-FAK 861 (green), and nucleus (blue) after theirmigration in laminin–laminin and laminin–collagen. (H) Calculated fluorescence intensity of p-FAK-861 of leader cells after their migration inlaminin–laminin and laminin–collagen (N = 13–16 cells).Paper Biomaterials Science3452 | Biomater. Sci., 2024, 12, 3446–3457 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225crequires the contribution of another type of integrins for thosemigrating on laminin. Our study has not identified the specificintegrins involved in leader cell formation for laminin;however, based on previous literature, integrin α4β6 may be apromising candidate, as it is known to bind to laminin.43Additionally, another study finds that the knockdown of α4β6impedes collective migration, while the forced expression ofintegrin α4β6 in leading edge cells has been observed torestore this activity.44 Another interesting observation fromthis experiment is that even though the follower cells werefound in the laminin-coated areas in both cases, this did notmaintain the appearances of leaders for Lam–Col substrates, itseems that the primary factor determining external cell dis-crimination into leaders is the external cells themselves andtheir underlying environment. This is particularly true sincethe expansion rate is impeded for both cases, and backwardfollowers could not push migrating clusters forward in eithercase. After that, we stained and characterized vinculin andactin for leader cells, with and without the blocking anti-bodies. In the formed leaders, we did not observe any signifi-cant difference in the vinculin formation for Lam–Lam sur-faces with and without AIIB2 treatment (Fig. 4E). On the otherhand, we observed a decrease in the localization of vinculin inthe focal adhesions for Lam–Col surfaces after AIIB2 treat-ment, and it seemed to be distributed in the cytoplasm(Fig. 4F). This is in line with previous reports that suggest adefective localization of vinculin to FA upon the knockdown ofintegrin β1 in MDCK cultured in collagen I-coated surfaces.41After demonstrating that the appearance of leader cells inextracellular matrix (ECM) proteins originates from differentintegrin subtypes, we investigated how this affects FAK phos-phorylation. By staining the migrating clusters, we analyzedthe phosphorylation levels of FAK at two phosphorylationsites, autophosphorylation at Tyr-397 (Fig. S4A†) and thesecond one at Tyr-861 (Fig. 4G). The results showed that thelevel of p-FAK-861 was higher in the leader cells migrating inLam–Lam compared to those migrating in Lam–Col(Fig. 4H),while the FAK-397 levels remained the same (Fig. S4B†). Thesefindings support our hypothesis that the leader cell formationis controlled by the underlying ECM protein composition,which is associated with different integrin subtypes and levelsof FAK-861 phosphorylation. FAK-861 plays a vital role in theacquisition of metastatic potentials for a lot of cancercells.45,46 Thus, we showed that integrin β1 is crucial for thefocal adhesions in leader cells that are migrating into collagenI. However, when it comes to laminin, integrin β1 is notresponsible for this function which was associated withdifferent FAK activation. Finally, our study has achieved a sig-nificant milestone in understanding the link between leadercells and the different of ECM proteins.2.5. The leader cells’ appearance and ECM compositionsOur research aimed to provide a better understanding of therole of ECM composition as a biochemical cue in shaping thebehavior of leader cells. Using photoactivatable surfaces haveshed light on how leader cells respond during migration onheterogeneous ECM proteins compared to homogenous types(Fig. 5). Our findings also have highlighted the pivotal role ofintegrin β1 in the formation of leader cells in MDCK cells thatmigrate into collagen I. Furthermore, we have opened up excit-ing avenues for further research into the formation of leadercells in MDCK cells that migrate into laminin and other ECMproteins, what specific integrins are involved in this process,how FAK-861 involved, what are the underlying biochemicalsignals that regulate it. Understanding these differences willgive us insights into the relationship between collectivemigration, extracellular matrix, and cancer metastasis.Especially when cancer metastasis is triggered by the modifi-cation of the ECM and the creation of very complex structuresaround the tumor.47 This involves altering the density, organiz-ation, composition, and structure of different ECM proteinsand macromolecules, which in turn modify the chemical andmechanical properties of the ECM. This can be achieved bydepositing ECM proteins, chemically modifying existing pro-teins through post-translational modifications, or using remo-deling enzymes like proteases to release bioactive proteins. Forexample, basement membranes usually comprise laminin andcollagen IV, and their remodeling is associated with tumorprogression.48 Melanoma cells are involved in matrix remodel-ing by expression of specific MMP type, disposition, and clea-vage of laminin.49 Collagen I is a substrate that hinders thecollective migration of cancer cells that originate from epi-thelial tissue. However, the presence of fibroblasts can initiatethe movement of these cancer cells by remodeling the matrixand creating a pathway for their movement.50 If we comparethese results with our findings, we can observe a similar trend;MDCK, as epithelial cells, experience inhibited movement dueto the presence of collagen I, while its presence in a heteroge-nous ECM has different outcomes. These unique results wereobtained by utilizing photoactivatable systems. If thesesystems continue to be used, there are tremendous choicesand combinations of proteins/peptides that could be explored.From another perspective, changes in the composition ofextracellular matrix (ECM) in nature are often linked tochanges in its mechanical properties. For example, an increasein the ratio of collagen I to collagen III leads to stiffening ofheart muscles.51,52 However, in our research, we used physicallyadsorbed collagen I/laminin on stiff glass surfaces to eliminatethe effect of alteration of mechanical properties of ECM pro-teins. Our findings revealed the distinct formation of FAs,Fig. 5 Cartoon depicting how switching from laminin to collagen inexternal ECM protein alters leader cell formation and integrin involved.Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 3446–3457 | 3453Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225crearrangement of the cytoskeleton, migratory phenotypes, andleader cell formation, highlighting the importance of biochemi-cal signals in ECM composition. Nonetheless, it raises an inter-esting question about cellular behavior on compliant surfaceswith different ECM compositions. Would we observe similar cel-lular behavior associated with compliant surfaces? Could theextensively studied mechanisms for mechanosensing be altered,especially those involving the YAP/TAZ53 and integrin cluster-ing54? To explore this area, further development of photoactiva-table functionalization technology is needed to better under-stand the mechanisms for mechanosensing concerning theunderlying ECM chemical as well as mechanical cues.3. ConclusionsWe have used photoactivatable substrates to investigate theimportance of different type of extracellular matrix (ECM) pro-teins in collective cell migration and leader cell formation.Traditional methods, such as wound scratch assays, areineffective in inducing ECM protein switching. In our research,we have focused on using Laminin and Collagen I as ourprimary EM proteins. We found that the secondary ECM playsa crucial role in determining the formation of leader cells inhighly migrating cells, such as laminin to laminin substrateand laminin to collagen substrate. However, this differencewas not significantly observed in slow-migrating cells such ascollagen to collagen substrate and collagen to laminin sub-strate. Additionally, cells maintained their original type offocal adhesion (FA) and actin stress fiber based on the under-lying ECM protein type. We also found that integrin β1 contrib-utes to cluster expansion but is not involved in leader cell for-mation for cells migrating from laminin to laminin. However,it is engaged in both migration and leader cell formation inthe other case such as laminin to collagen. Our findings high-light the promising potential of utilizing photoactivatable sur-faces as a more efficient approach to explore collective cellmigration across different and heterogeneous ECM proteins.4. Experimental4.1. Photopatterning and ECM adsorptionPhotoactivatable substrate functionalized with PCP was pre-pared according to the procedure reported previously.55Photoirradiation occurred as previously reported with a slightmodification;6,16 shortly, glass surfaces in PBS were exposed toa dose of 30 J cm−2 with a wavelength of 365 nm with a photo-mask in the field diaphragm of Olympus microscope (IF81-PAFM, Olympus, Tokyo, Japan) using HBO mercury arc lamp(Olympus) and focusing UV light through an objective(UPlanSApo 10×/0.4, Olympus). After cleaving the PEG12-NH,the primary extracellular matrix protein such as Laminin(from Engelbreth-Holm-Swarm murine sarcoma basementmembrane, Sigma-Aldrich, MO, USA) or Collagen Type I (Rattail, Discovery Labware, MA, USA) was diluted in PBS and de-posited on irradiated surfaces (2 μg cm−2) for 5 minutes at37 °C. Then, the surfaces were washed 3–4 times with PBS toremove non-adsorbed proteins. After that, cells were seeded onthese patterned substrates at a density of 75 × 103 cells per cm2in serum-free medium. After cellular attachment to irradiatedclusters (∼1 h), the medium was changed into fresh medium(+) FBS. The second irradiation was done using an Axiovert200 microscope (Zeiss, Oberkochen, Germany) equipped witha xenon lamp and a mercury arc lamp through an objective(Plan-APOCHROMAT 10×/0.45, Zeiss). Next, the medium waschanged into a PBS solution with ECM proteins at the sameconcentration for 5 minutes at 37 °C. The surfaces werewashed 3–4 times with PBS to remove non-adsorbed proteins.Finally, fresh medium (+) FBS was added to start the time-lapse observation using the same microscope. The image wascaptured using a Cool SNAP MYO camera (Photometrics,Tucson, AZ, USA). All systems were controlled usingMetamorph software (Molecular Devices, Sunnyvale, CA, USA);captured images were processed using Image J.4.2. Cell culture and immunostainingMDCK cells (RCB0995, RIKEN cell bank) were cultured inMEM (Sigma, St Louis, MO, USA) containing 10% FBS (heat-inactivated FBS; BioWest, EU origin), 100 units per mL penicil-lin and 100 ug mL−1 streptomycin (Nacalai, Japan), 1% MEM-nonessential amino acids (Nacalai, Japan), 1% sodium pyru-vate (Nacalai, Japan), and 1% L-glutamine (Nacalai, Japan) at37 °C in a humidified atmosphere containing 5% CO2 at 75%confluency of cell subculture. Cells were collected by trypsin/EDTA (Wako, Japan) and seeded on photo-irradiated glass. Thecells were initially seeded in a medium without FBS for onehour, after which the medium was changed to a completemedium for the rest of the experiments. HeLa andMDA-MB468 cells were obtained from American Type culturecollection (ATCC, Manassa, VA, USA). HeLa was maintained ina state of continuous growth in MEM containing 10% FBS, 100units per mL penicillin, and 100 μg mL−1 streptomycin at37 °C in a humidified atmosphere containing 5% CO2 andsubcultured every 2 or 3 days. MDA MB-468 was maintained ina state of continuous growth in DMEM-high glucose contain-ing 10% FBS, 1% glutamine, 100 units per mL penicillin, and100 μg mL−1 streptomycin at 37 °C in a humidified atmo-sphere containing 5% CO2 and subcultured every 2 or 3 days.Confocal images were obtained by Olympus microscope (IF81-PAFM, Olympus, Tokyo, Japan) using a disk-scan unit (CSU-10,Yokogawa, Tokyo, Japan) and Andro CCD camera (SONA4BV6U, UK), captured images were processed using Fiji (ImageJ, USA). For leader cell trajectory tracking and velocity calcu-lations, each leader cell was manually outlined every30 minutes, and then the centroid was determined using Fiji.In the immunofluorescence staining for ECM-coated surfaces,Fibronectin diluted in PBS with deposited (2 μg cm−2) in theirradiated glass for 5 min, followed by fixation with 4% paraf-ormaldehyde (Nacalai, Japan), blocked with bovine serumalbumin (Wako, Japan) for 30 min, then incubation with fibro-nectin-Alex Fluor-488 Antibody (A1918, Santa Cruz biotechnol-Paper Biomaterials Science3454 | Biomater. Sci., 2024, 12, 3446–3457 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 30 May 2024. Downloaded on 7/4/2024 3:46:48 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by/3.0/http://creativecommons.org/licenses/by/3.0/https://doi.org/10.1039/d4bm00225cogy). For cell staining, Fixation was done with 4% paraformal-dehyde (Nacalai, Japan) for 15 min, quenched with 5% glycine(Wako, Japan) in PBS for 5 min, permeabilized with 0.5%Triton X-100 for 5 min, and blocked with bovine serumalbumin (Wako, Japan) for 30 min; cells were then incubatedwith Alexa Fluor™ 565 Phalloidin (1 : 1000, ThermoFisherScientific, USA), mouse anti-vinculin (1 : 400, Sigma), anti-mouse IgG Alexa Fluor 488 (1 : 1000, ThermoFisher Scientific,USA), and Hoechst 33342 (1 : 1000, Life Technologies, Eugene,OR, USA), Rabbit p-FAK 397 (1 : 400, Invitrogen, USA) andRabbit p-FAK 861 (1 : 400, Abcam) anti-rabbit IgG Alexa Fluor488 (1 : 1000, ThermoFisher Scientific, USA). To calculate thefluorescence intensities for p-FAK 861 and 397, each cell wasmanually outlined, and then the integrated density was calcu-lated by selecting the integrated density option under themeasurement function in Fiji. After that, the background wassubtracted, where the background represents the cell area mul-tiplied by the mean grey value of the non-fluorescent back-ground.56 Integrin β1 (AIIB2) antibody (University of Iowa,Hybridoma bank, USA) was used in concentration (1.5 µgml−1) for the integrin β1 blocking experiment. The statisticalanalyses were performed for the presented data using astudent t-test.Author contributionsShimaa A. Abdellatef: conceptualization, data acquisition,formal analysis, investigation, methodology, funding acqui-sition, writing – original draft, writing – review & editing.Francesca Bard: data acquisition, formal analysis. JunNakanishi: project administration, supervision, methodology,validation, funding acquisition, writing – original draft,writing – review & editing.Conflicts of interestThere are no conflicts to declare.AcknowledgementsThis study was partly supported by the Japan Society for thePromotion of Science, KAKENHI (22H00596, 23K17481,21J40229), and the KAO-Crescent Award for women research-ers. The authors are grateful to Mrs Elham Elmasry for herhelp in experiments and data analysis.References1 P. Rørth, Fellow travellers: emergent properties of collectivecell migration, EMBO Rep., 2012, 13(11), 984–991.2 L. Qin, D. Yang, W. Yi, H. Cao and G. Xiao, Roles of leaderand follower cells in collective cell migration, Mol. Biol.Cell, 2021, 32, 1267–1272.3 P. Friedl and D. Gilmour, Collective cell migration in mor-phogenesis, regeneration and cancer, Nat. Rev. Mol. CellBiol., 2009, 10(7), 445–457, DOI: 10.1038/nrm2720.4 E. L. Zoeller, B. Pedro, J. Konen, B. Dwivedi, M. Rupji,N. 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