整合素分布变化与肝癌细胞迁移取向调节的相关性

目的采用机械拉伸方法并通过裱衬纤连蛋白(胞外基质成份)调节整合素受配体结合,探索整合素再分布变化对肝癌细胞黏附运动行为的影响,以及骨架重排对整合素再分布变化的影响。方法运用免疫荧光染色、激光共聚焦显微技术和定量形态学分析法,观察整合素分布变化和细胞骨架装配调整,及对细胞运动变形能力进行检测和定量分析。结果 (1)不同形态细胞的整合素表达和分布特征不同,铺展细胞β1整合素表达高于未铺展的圆细胞,分布峰值为基底面,而圆细胞游离面的整合素分布反较基底面多。(2)机械拉伸5h后不同形态细胞β1整合素表达均升高,分布带增宽;卸载1h整合素表达下调,出现弥散分布倾向,其中以圆细胞最为明显。(3)裱衬FN后β1整合素表达增加,不同形态细胞整合素分布均向基底转移,并使肝癌细胞净迁移距离减小。(4)机械拉伸5h后60%的细胞长轴取向集中在70°～110°之间,细胞骨架倾向垂直于拉伸方向排列,卸载1h细胞骨架明显解聚。结论肝癌细胞整合素分布变化受胞内骨架调整和胞外配体数量的影响,单个圆细胞的整合素分布特征与其较强的转移浸润能力相关。     

肝癌细胞"重叠生长"局部成因初探

探索肝癌细胞"重叠生长"形成过程及其成因.运用显微形态学观察、计算机图像处理技术和统计学分析,以及细胞力学和生化检测等研究手段对肝癌细胞原位生长信息进行量化表征.结果发现:(1)肝癌细胞变形调节能力较正常肝细胞强;(2)对"重叠生长"斑进行负压吸吮时,斑片从基底脱落,其下方未见铺展细胞;(3)肝癌细胞株HepG2的整合素表达明显高于正常肝细胞株L02;(4)纤维连接蛋白(Fibronectin, Fn)对肝癌细胞表达整合素β1有下调作用,Fn裱衬基底后,细胞黏附力和形态稳定性增高,圆细胞产生与聚集减少.由此得出(1)所谓"重叠生长层"实际上是由大量圆细胞聚集排列构成;(2)圆细胞产生与整合素β1表达异常及其对细胞黏附特性的影响相关;(3)肝癌细胞"重叠层"的形成与细胞频发形变诱导的圆细胞聚集相关.     

基底硬度对肝细胞和肝癌细胞融合生长的影响

目的通过比较融合生长肝细胞(L02)与肝癌细胞(HCCLM3)对不同硬度基底(0.5、4 kPa和玻璃)的响应,探查两类细胞融合生长差异的成因。方法运用实时显微摄影技术、免疫荧光染色、流式细胞技术及Western Blot-ting等试验方法分别检测融合生长L02和HCCLM3细胞在不同硬度基底上的形态特征、骨架构象、E-cad和Integrinβ1表达分布,以及Src激酶活性水平的变化。结果 (1)融合生长L02细胞呈圆形或立方形,HCCLM3细胞呈多角形,铺展和极化更为明显;随基底硬度增加,L02细胞圆度随时间变化幅度较小,HCCLM3细胞变化幅度较大。(2)融合生长的L02细胞皮质下呈现环形骨架,E-cad定位于细胞-细胞接触处,HCCLM3中皮质骨架环不完整,胞内骨架沿基底呈放射状分布,E-cad呈弥散分布于细胞质中。(3)随基底硬度增加,L02和HCCLM3细胞中E-cad表达显著降低(P<0.01),而p-Src和Integrinβ1的表达量则显著增加(P<0.01),HCCLM3较L02细胞变化明显。结论皮质下环形骨架装配与E-cad在细胞-细胞定位呈正相关,基底硬度对肝癌细胞钙黏素与整合素黏附系统平衡调节的影响较对肝细胞的影响明显。     

基底硬度与细胞因子TGF-β1协同作用对肝细胞表型的影响

目的通过建立与肝组织生理和病理硬度相当的体外培养模型,探讨基底硬度和TGF-β1协同作用对肝细胞表型变化的影响。方法采用免疫荧光显微观测技术及Western Blotting等试验方法,研究肝细胞在不同硬度梯度的聚丙烯酰胺基底膜上的细胞形态调整、运动变化特征、骨架构象以及整合素、E-钙粘素、白蛋白和alpha-平滑肌动蛋白表达的差别,并通过图像分析软件对上述结果进行定量分析。结果 3.6 kPa基底膜上单个分散细胞运动变形活跃,但群体细胞极化变小,肌动蛋白沿皮质下呈环状排列,E-钙粘素和白蛋白表达高,整合素和alpha-平滑肌动蛋白的表达水平较低,加药组与对照组变化趋势一致;30 kPa基底膜上细胞运动变形欠活跃,加药组与对照组相比,E-钙粘素和白蛋白表达均下调,alpha-平滑肌动蛋白表达上调;30 kPa与3.6 kPa对照组相比、30 kPa与3.6 kPa加药组相比,E-钙粘素及白蛋白表达均下调(P<0.05),alpha-平滑肌肌动蛋白的表达上调(P<0.05)。10 kPa基底膜上对照组和加药组与30 kPa和3.6 kPa对照组和加药组相比,均无显著性差异。结论基底硬度增加可诱导肝细胞表型转化,并促进TGF-β1对肝细胞代谢行为的影响。     

整合素介导低剪切力诱导的肝癌细胞迁移

低剪切力是肿瘤体内微环境的组成部分,是调控肿瘤细胞迁移、侵袭的重要因素。作为细胞膜表面受体,整合素(integrin)不仅介导细胞与细胞外基质之间的黏附,而且作为细胞的重要力学感受器起到整合细胞内的力学、化学信号的作用。本研究的目的是探讨低剪切力引起肝癌细胞整合素的变化及其对肝癌细胞迁移的影响。采用低剪切力(1.4dyn/cm2)作用于肝癌HepG2细胞,划痕法测定在0、1、2、4h细胞的迁移情况,F-actin染色观察低剪切力作用2h和4h后细胞骨架的变化,Western blot测定integrinα亚基在瞬时(15s和30s)、短时(5、15和30min)和长时(1、2和4h)的表达规律。结果表明:与对照组比较,低剪切力刺激HepG2细胞后,细胞迁移距离显著增加,应力纤维丝排列规则,integrinα亚基在不同的时间点的表达呈现出时序性的差异,提示integrin的不同亚基在剪切力诱导肝癌细胞迁移中具有不同的调控作用。     

不同转移潜能肝癌细胞株GRP78的表达及其对肝癌细胞生物学行为的影响

目的: 研究葡萄糖调节蛋白78(GRP78)在不同转移潜能肝癌细胞株内的表达及其对肝癌细胞生物学行为的影响。方法: 应用GRP78反义寡核苷酸(GRP78 ASODN)转染肝癌细胞株HepG2和MHCC97-H。逆转录聚合酶链式反应(RT-PCR)技术检测肝癌细胞内GRP78 mRNA的表达,Transwell小室实验和细胞黏附实验分别检测2种肝癌细胞的侵袭、迁移及黏附能力。结果: 2种细胞内均有GRP78的表达,MHCC97-H细胞表达较HepG2高(P<0.05)。GRP78 ASODN可以有效地抑制2种肝癌细胞株内GRP78的表达。GRP78 ASODN转染的高侵袭潜能的MHCC97-H肝癌细胞的侵袭、迁移和黏附能力较对照组有明显的下降(P<0.05),而低侵袭潜能的HepG2肝癌细胞侵袭、迁移及黏附能力下降不明显(P>0.05)。结论: 抑制肝癌细胞内GRP78的表达可以削弱其侵袭、迁移及黏附能力,GRP78可能成为肝癌治疗上有效的分子靶点。     

β2整合素介导的人中性粒细胞在ICAM-1裱衬表面的铺展动力学

目的研究由表达于人中性粒细胞表面的β2整合素与胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)裱衬表面相互作用介导人中性粒细胞铺展的动力学过程。方法以裱衬2%人血清白蛋白(human serumalbumin,HSA)和空白的底板为对照,分别考察裱衬了10、20和100μg/mL ICAM-1的底板上人中性粒细胞铺展比例随时间的变化规律。通过流式细胞仪检测人中性粒细胞β2整合素的表达量,以及采用抗体阻断β2整合素的CD11a或CD11b亚基,观察其在100μg/mL ICAM-1裱衬表面细胞铺展比例随时间的改变情况。结果中性粒细胞在裱衬2%HSA的表面不铺展,在裱衬ICAM-1表面的铺展动力学依赖于ICAM-1浓度,并与β2整合素表达量相关;抗CD11b抗体阻断后,中性粒细胞在裱衬ICAM-1表面的铺展明显减少。结论人中性粒细胞在ICAM-1表面的铺展是由β2整合素与其配体ICAM-1特异性相互作用介导的,并且CD11b亚基对铺展过程起主要调控作用。     

流动剪切应力下体外培养人体滋养层细胞的迁移和β1整合素表达

目的在人胎盘发育过程中,滋养层细胞群体侵入子宫内膜的毛细血管,并沿母体脉管系统逆流迁移,最终停留在母体螺旋动脉对其结构进行重构。本文目的是验证我们的推测:血液流动剪切力可以调控人体滋养层细胞黏附分子β1整合素的表达,从而影响滋养层细胞的迁移。方法体外培养人体胚胎滋养层细胞,测量流动剪切力下滋养层细胞的迁移和β1整合素表达,所加载的剪切力大小为0,75,150和300Pa4个水平,时间24h。结果在剪切力作用下,滋养层细胞迁移活性随剪切力的增高而显著增加,在流动剪切力方向存在优势性的迁移。同时,剪切力还增加了β1整合素的表达。结论流动剪切力的参与调节了滋养层细胞β1整合素的表达,从而可影响滋养层细胞的迁移。     

基底硬度对肝细胞与肝癌细胞迁移行为的影响

目的通过比较不同硬度基底对肝细胞和肝癌细胞迁移特征的影响,研究肿瘤细胞迁移行为变化的成因。方法运用免疫荧光染色、形态学分析法和Transwell侵袭实验,观察不同硬度基底上HCCLM3和L02细胞的形态特征及对细胞运动能力进行检测和定量分析。结果 (1)相对于极软(0.5 kPa)和极硬(玻璃)基底,HCCLM3和L02细胞在较软基底(4 kPa)上具有较高的迁移速率和迁移净距离,且L02细胞表现出高的迁移效率。(2)HC-CLM3和L02细胞在不同硬度基底上均方位移趋势一致,较软基底上L02细胞具有较高的方向持续能力。(3)0.5和1 mg/mL三维胶原基质中,HCCLM3细胞穿透基底膜的个数分别显著多于L02细胞穿透基底膜的个数;加入40μg/mL水解酶抑制剂GM6001后,HCCLM3细胞穿透基底膜的个数显著增加,而L02细胞穿透基底膜的个数显著减少。结论 (1)二维较软基底上,L02细胞因有较高的方向持续能力而表现出高的迁移效率。(2)三维胶原基质中,HCCLM3细胞以不同迁移模式适应周围环境,从而表现出更大的侵袭能力。     

冬凌草甲素对人肝癌MHCC-97H细胞系侵袭和迁移的作用

目的:探讨冬凌草甲素(oridonin)对肝癌细胞侵袭和迁移的影响及其机制。方法:采用细胞培养技术培养人肝细胞癌MHCC-97H细胞,用不同浓度的冬凌草甲素处理肝癌细胞,采用划痕实验检测细胞的迁移能力;Transwell实验测定细胞的侵袭能力;黏附实验评价细胞的黏附能力;Western blot法检测LIM激酶1(LIMK-1)、丝切蛋白(cofilin)和磷酸化cofilin(p-cofilin)蛋白水平的改变。结果:冬凌草甲素可明显降低肝癌细胞的体外侵袭、迁移及黏附能力(P0.05),且在一定浓度范围内具有明显的量效关系。冬凌草甲素干预处理细胞后,cofilin的蛋白水平无明显变化,LIMK-1和p-cofilin的蛋白水平明显下调。结论:冬凌草甲素体外具有抑制肝癌MHCC-97H细胞侵袭和迁移的作用,其机制可能与调控LIMK-1/cofilin信号通路有关。     

Relative Rigidity of Cell–Substrate Effects on Hepatic and Hepatocellular Carcinoma Cell Migration

Abstract

Polyacrylamide gels with different stiffness and glass were employed as substrates to investigate how substrate stiffness affects the cellular stiffness of adherent hepatocellular carcinoma (HCCLM3) and hepatic (L02) cells. The interaction of how cell-substrate stiffness influences cell migration was also explored. An atom force microscope measured the stiffness of HCCLM3 and L02 cells on different substrates. Further, F-actin assembly was analyzed using immunofluorescence and Western blot. Finally, cell-surface expression of integrin β1 was quantified by flow cytometry. The results show that, while both HCCLM3 and L02 cells adjusted their cell stiffness to comply with the stiffness of the substrate they were adhered to, their tuning capabilities were different. HCCLM3 cell stiffness complied when substrate stiffness was between 1.1 and 33.7 kPa, whereas the analogous stiffness for L02 cells occurred at a higher substrate stiffness, 3.6 kPa up to glass. These ranges correlated with F-actin filament assembly and integrin β1 expression. In a migration assay, HCCLM3 cells migrated faster on a relatively soft substrate, while L02 cells migrated faster on substrates that were relatively rigid. These findings indicate that different tuning capabilities of HCCLM3 and L02 cells may influence cell migration velocity on substrates with different stiffness by regulating cy- toskeleton remodeling and integrin β1 expression.     

The motility of normal and cancer cells in response to the combined influence of the substrate rigidity and anisotropic microstructure

Cell adhesion and migration are strongly influenced by extracellular matrix (ECM) architecture and rigidity, but little is known about the concomitant influence of such environmental signals to cell responses, especially when considering cells of similar origin and morphology, but exhibiting a normal or cancerous phenotype. Using micropatterned polydimethylsiloxane substrates (PDMS) with tunable stiffness (500 kPa, 750 kPa, 2000 kPa) and topography (lines, pillars or unpatterned), we systematically analyse the differential response of normal (3T3) and cancer (SaI/N) fibroblastic cells. Our results demonstrate that both cells exhibit differential morphology and motility responses to changes in substrate rigidity and microtopography. 3T3 polarisation and spreading are influenced by substrate microtopography and rigidity. The cells exhibit a persistent type of migration, which depends on the substrate anisotropy. In contrast, the dynamic of SaI/N spreading is strongly modified by the substrate topography but not by substrate rigidity. SaI/N morphology and migration seem to escape from extracellular cues: the cells exhibit uncorrelated migration trajectories and a large dispersion of their migration speed, which increases with substrate rigidity.     

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.     

Effect of substrate stiffness on pulmonary fibroblast activation by TGF-β

Peptide crosslinkers containing the sequence C-X-CG (X represents various adhesive peptides) were incorporated into poly(ethylene glycol) (PEG) hydrogel networks with different mechanical properties. Pulmonary fibroblasts (PFs) exhibit increased adhesion to rigid hydrogels modified with X=RGDS, DGEA and IKVAV (0.5 and/or 5 mM) compared with a scrambled control (X=HRPNS). PFs exhibit increased adhesion to softer hydrogels when X=DGEA at low (0.5 mM) peptide concentration. PFs seeded onto hydrogels modified with X=RGDS produce alpha-smooth muscle actin (α-SMA), a myofibroblast marker, and form an extensive cytoskeleton with focal adhesions. Decreasing substrate stiffness (achieved through hydrolytic degradation) results in down-regulation of α-SMA expression by PFs. Substrate stiffness increases the sensitivity of PFs to exogenously applied transforming growth factor beta (TGF-β1); PFs on the most rigid gels (E=900 kPa) express α-SMA when treated with low concentrations of TGF-β1 (1 ng ml(-1)), while those on less rigid gels (E=20-60 kPa) do not. These results demonstrate the importance of both mechanical and chemical cues in studying pulmonary fibroblast activation, and establish PEG hydrogels as a viable material for further study of IPF etiology.     

基质刚度通过调节YAP活化控制乳腺癌细胞的耐药性

目的探究肿瘤细胞外基质刚度调控细胞耐药性的详细分子机制。方法制备不同基质刚度的软基底(10 kPa)、硬基底(38 kPa)和刚性基底(57 kPa)聚丙烯酰胺水凝胶,模拟体内乳腺癌不同阶段的物理基质刚度。结果硬基底刚度上的乳腺癌细胞增殖率明显高于软基底和刚性基底。乳腺癌细胞对阿霉素的内吞在硬基底上显著低于软基底和刚性基底。在硬基底上YAP的入核水平相比软基底和刚性基底显著增加,证实YAP是参与肿瘤细胞耐药性的关键分子。结论基质刚度可通过YAP活化调节乳腺癌细胞的耐药性。研究结果不仅为阐明乳腺癌细胞耐药机制提供新方向,也为开发乳腺癌治疗的药物传递系统奠定新基础。     

Chemokine receptor 7 promotes cell migration and adhesion in metastatic squamous cell carcinoma of the head and neck by activating integrin αvβ3

The mechanisms leading to squamous cell carcinoma of the head and neck (SCCHN) metastasis are incompletely understood. Although evidence shows that the chemokine receptor 7 (CCR7) and its ligand CCL19 may regulate tumor dissemination, their role in SCCHN is not clearly defined. CCR7 has been shown to regulate integrins, which facilitate adhesion of cancer cells to and/or migration through the extracellular matrix (ECM). To investigate the relationship between CCR7 and integrin αvβ3 in metastatic SCCHN, we used adhesion and migration assays, immunofluorescence staining and western blotting to determine whether integrin αvβ3 can be activated by CCL19 in the metastatic SCCHN cell line PCI-37B, which was pre-incubated with CCL19 or the integrin αvβ3 inhibitor, IS201. Our results demonstrate that CCR7 favors PCI-37B cell adhesion and migration, induces reorganization of the actin cytoskeleton and induces integrin αvβ3 phosphorylation. The integrin αvβ3 inhibitor, IS201, blocked all of these effects. CCR7 and integrin αvβ3 expression significantly and positively correlated with tumor size, clinical stage and nodal metastasis. Taken together, our data indicate that CCR7 regulates cell adhesion and migration via integrin αvβ3 in metastatic SCCHN. These results should provide the groundwork for new strategies aimed at preventing SCCHN metastasis.     

The influence of substrate stiffness on the behavior and functions of Schwann cells in culture

Solid tissues in the body possess a range of stiffness and provide cells with an instructive microenvironment. Scaffolds in tissue engineering should be rationally designed to become an adhesion substrate friendly to cells. Schwann cells are the principal glial cell in the peripheral nervous system and used as support cells for generating tissue-engineered nerve grafts. Although an important mechanical cue, substrate stiffness, has been documented to make significant effects on many types of cells cultured on the substrate, such a study for Schwann cells is still lacking. In this study, we investigated cell adhesion, survival, proliferation, migration, cytoskeleton, and neurotrophic actions of Schwann cells cultured on polyacrylamide gel substrates with different stiffness, and determined an optimal elastic modulus value for these substrates. Our data not only highlight the importance of substrate stiffness in the crosstalk between Schwann cells and surrounding microenvironment, but also introduce a new parameter, in addition to biocompatibility, biodegradability, and neuroaffinity, for designing scaffolds in nerve tissue engineering.     

The effects of substrate stiffness on the in vitro activation of macrophages and in vivo host response to poly(ethylene glycol)-based hydrogels

Poly(ethylene glycol) (PEG) hydrogels, modified with RGD, are promising platforms for cell encapsulation and tissue engineering. While these hydrogels offer tunable mechanical properties, the extent of the host response may limit their in vivo applicability. The overall objective was to characterize the effects of hydrogel stiffness on the in vitro macrophage response and in vivo host response. We hypothesized that stiffer substrates induce better attachment, adhesion, and increased cell spreading, which elevates the macrophage classically activated phenotype and leads to a more severe foreign body reaction (FBR). PEG-RGD hydrogels were fabricated with compressive moduli of 130, 240, and 840 kPa, and the same RGD concentration. Hydrogel stiffness did not impact macrophage attachment, but elicited differences in cell morphology. Cells retained a round morphology on 130 kPa substrates, with localized and dense F-actin and localized α(V) integrin stainings. Contrarily, cells on stiffer substrates were more spread, with filopodia protruding from the cell, a more defined F-actin, and greater α(V) integrin staining. When stimulated with lipopolysaccharide, macrophages had a classical activation phenotype, with increased expression of TNF-α, IL-1β, and IL-6, however the degree of activation was significantly reduced with the softest hydrogels. A FBR ensued in response to all hydrogels when implanted subcutaneously in mice, but 28 days postimplantation the layer of macrophages at the implant surface was significantly lower in the softest hydrogels. In conclusion, hydrogels with lower stiffness led to reduced macrophage activation and a less severe and more typical FBR, and therefore are more suited for in vivo tissue engineering applications.     

The influence of GFP-actin expression on the adhesion dynamics of HepG2 cells on a model extracellular matrix

Integrins belong to a family of important cell surface receptors which mediate the adhesion of most anchorage-dependent cells to nature extracellular matrix (ECM) and biomaterials. It is known that the binding of integrin with ECM proteins triggers mechanochemical responses of cytoskeleton. To date, the intricate interplay between integrin-ECM interaction and cytoskeleton dynamics leading to the regulation of cell morphogenesis on biomaterials remains largely unknown. In this study, green fluorescence protein (GFP)-actins were expressed in HepG2 cells for the temporal visualization of cytoskeletal structure of adherent cells on naturally derived materials. By combining confocal reflectance contrast microscopy and fluorescence microscopy, the adhesion contact dynamics, cytoskeleton remodeling and two-dimensional spreading of intact and GFP-actin expressing HepG2 cells on collagen and fibronectin-coated substrates are simultaneously probed during the initial cell seeding. First of all, our results show that the evolution of adhesion contact of HepG2 cells upon integrin-collagen or integrin-fibronectin interaction is impaired by GFP-actin expression. Also, the initial rate of cell deformation is reduced by 70% and 43% on fibronectin and collagen, respectively, upon GFP-actin expression. Interestingly, the steady-state adhesion energy of HepG2 cells remains unchanged and increases on fibronectin- and collagen-coated substrate, respectively, upon GFP-actin expression. Our highly integrated biophysical approach demonstrates that GFP-actins diffusively concentrate in the cytoplasmic cortex during initial cell seeding while adhesion contact evolves and cell spreads. Kinetics analysis on the adhesion contact formation demonstrates the intricate interplay between cytoskeleton property and ECM proteins in cell adhesion.     

Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers

Polyelectrolyte multilayer coatings have emerged as substrates to control a variety of cell behaviour, including adhesion, proliferation and differentiation. In particular, it is possible to modulate film stiffness by physical or chemical cross-linking. In this study, we evaluate the adhesive behaviour of skeletal muscle cells (C2C12 myoblasts) during the initial steps of spreading on layer-by-layer films of controlled stiffness made of poly(l-lysine) and hyaluronan as model biomaterial surfaces for muscle tissue engineering. We show that integrin clustering, integrin actin cytoskeleton connection and focal adhesion formation for cell spreading can be decoupled by controlling film stiffness. This made it possible to switch the cells morphologically between round and spreading shapes depending on the stiffness of the microenvironment. Although hyaluronan is one of the main components of cross-linked multilayer films, the HA receptor CD44 did not appear to mediate early adhesion as suggested by the use of blocking antibodies. In contrast, integrins were found to play a pivotal role in early adhesion: their activation significantly enhanced C2C12 myoblast spreading on soft films, where they were otherwise round. Integrin clustering was also induced by the softer films and enhanced on the stiffest films. Conversely, the use of soluble inhibitors or blocking antibodies directed against integrins induced a round phenotype on stiff films, where cells were well spread out in control conditions. We show that specific integrins were involved in the adhesion process as blocking β₃, but not β₁, integrins inhibited cell adhesion. These soft, stiff films can thus be used to tune the adhesion of C2C12 myoblasts, an early key event in myogenesis, via integrin clustering and subsequent signalling. They may be further used to decorticate the signalling pathways associated with β₃ integrins.