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Atomic Force Microscopy (AFM) is a powerful technique widely employed in biophysics, for instance to study topography of living cells and cell mechanics. Cell mechanics is a very interesting, biophysical parameter of cells, because it is strongly changed by various cellular processes, for example during cell division, cell movement, differentiation, aging, and also various diseases. Since cancer is a prominent example of changes in mechanical properties of diseases, the concept of the mechanical fingerprint has developed, which makes it possible to distinguish between healthy and diseased cells. In this article we report on various studies of cell mechanics with the AFM. We will first give a brief introduction on AFM principles and operational modes and then we will report on some applications of AFM in the field of cellular biophysics, like discriminating between healthy and cancer cells, as well as distinguishing cancer cells at different stages of malignancy. Overall, we will show that AFM has made a significant contribution in studying the biophysics of cancer and the concept of mechanical fingerprints could find a wide variety of applications in biomedicine and medical diagnostics.

Extracellular matrix (ECM), as a dynamic component of the tissue, influences cell behavior and plays an important role in cell mechanics and tissue homeostasis. Reciprocally, this three-dimensional scaffold is dynamically, structurally and mechanically modified by cells. In the field of biophysics, the independent role of cell and ECM mechanics has been largely investigated; however, there is a lack of experimental data reporting the interdependent interplay between cell and ECM mechanics, measured simultaneously. Here, using Atomic Force Microscopy (AFM) we have characterized five different decellularized matrices diverse in their topography, ECM composition and stiffness and cultured them with normal and pathological fibroblasts (scar and Dupuytren’s). We investigated the change in topography and elasticity of these matrices due to cell seeding, by using AFM peak force imaging and mechanical mapping, respectively. We found normal fibroblasts soften these matrices more than pathological fibroblasts, suggesting that pathological fibroblasts are profoundly influencing tissue stiffening in fibrosis. We detected different ECM composition of decellularized matrices used here influences fibroblast stiffness, thus highlighting that cell mechanics not only depends on ECM stiffness but also on their composition. We used confocal microscopy to assess fibroblasts invasion and found pathological fibroblasts were invading the matrices deeper than normal fibroblasts.

Current knowledge about cell-biomaterial interactions is often based on two-dimensional (2D) cell culture systems like protein-coated glass slides. However, such smooth surfaces cannot mimic the nanofibrous environment of the native extracellular matrix (ECM). It is therefore a major challenge to transfer the results from 2D surfaces to 3D protein scaffolds with biomimetic nanofiber architecture. To understand the influence of different protein topographies on the cell response we introduce a new process to fabricate binary collagen scaffolds of variable thickness with spatially controlled regions of nanofibrous and smooth topography. We used pH-induced self-assembly to prepare collagen nanofibers with diameters between 130 and 150 nm on glass surfaces, which were partly covered with a polymer mask. After cross-linking with glutaraldehyde, smooth collagen films were prepared on the remaining glass regions. Atomic force microscopy revealed a much lower surface roughness of smooth collagen compared to nanofibers. Subsequently, we studied the viability, morphology and migration of 3T3 fibroblasts on both collagen topographies. We found small, elongated fibroblasts with few, long filopodia on collagen nanofibers whereas large, flat fibroblasts with many short filopodia were observed on smooth collagen. Actin stress fibers on collagen nanofibers were substantially reduced in comparison to smooth collagen. Live cell tracking revealed that fibroblasts on thin nanofibrous collagen migrated faster than on smooth collagen. In summary, binary collagen scaffolds enabled us for the first time to study cell responses to topographical cues on a single protein scaffold. In future, it will be intriguing to transfer our patterning process to other proteins to study fundamental principles of topography-dependent cell recognition processes.

The mechanical properties of cells strongly regulate many physiological and pathological processes. For example, in cancer, invasive and metastatic tumor cells have often been reported to be softer than nontumor cells, raising speculation that cancer cells might adaptively soften to facilitate migration through narrow tissue spaces. Despite growing interest in targeting cell softening to impede invasion and metastasis, it remains to be directly demonstrated that tumor cells soften as they migrate through confined spaces. Here, we address this open question by combining topographically patterned substrates with atomic force microscopy (AFM). Using a polydimethylsiloxane open-roof microdevice featuring tapered, fibronectin-coated channels, we followed the migration of U2OS cells through various stages of confinement while simultaneously performing AFM indentation. As cells progress from unconfined migration to fully confined migration, cells soften and exclude Yes-associated protein from the nucleus. Superresolution imaging reveals that confinement induces remodeling of actomyosin stress fiber architecture. Companion studies with flat one-dimensional microlines indicate that the changes in cytoarchitecture and mechanics are intrinsically driven by topographical confinement rather than changes in cellular aspect ratio. Our studies represent among the most direct evidence to date that tumor cells soften during confined migration and support cell softening as a mechanoadaptive mechanism during invasion.

Summary: Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration. : Leukocyte extravasation depends on local cellular and substrate stiffness. Schimmel et al. identified endothelial DLC-1 as a mediator to translate stiffness to leukocyte behavior. DLC-1 is crucial for the ICAM-1 adhesome, which allows leukocytes to switch from the rolling to the spreading and crawling phase, followed by diapedesis. Keywords: ICAM-1, DLC-1, spreading, leukocyte, transmigration, diapedesis, rolling, stiffness, mechanosignaling, endothelial

The tumor microenvironment highly influences cancer cell modes and dynamics, above all during invasive and metastatic processes. When aiming at studying cancer cell behavior in vitro, the use of conventional cell culture systems, such as Petri dishes, fails in recapitulating the mechanical and topographical properties of the natural extracellular matrix (ECM). Here the versatility of stiffness-tunable hydrogels and the efficacy of the replica molding technique with silicone polymers are exploited, aiming at studying cancer and normal cell behavior with platforms able to capture the heterogeneities of the natural in vivo context. We compared the mechanical properties of normal and cancer renal cells on different stiffness value gels (with Young's moduli of 3, 17 and 31 kPa) by using atomic force microscopy (AFM) and investigated cell indentation phenomena on compliant gels with confocal microscopy. Moreover, we studied cell mechanics, morphology and migration on isotropic linear structures, spaced at 1.5 μm, aiming at mimicking the aligned fiber bundles typically observed at tumor borders. By using this approach, we could highlight differences in the way healthy and cancer renal cells react to changes in their microenvironment. Our results may potentially pave the way to unravel the complex processes involved in cancer progression, especially in tissue invasion and migration during metastasis formation.

Cell response to exogenous cues is the result of a complex integration of multiple biochemical/biophysical signals, which might occur simultaneously and might be characterized by specific spatial and temporal patterns. Among these signals, surface topography plays an important role in affecting cell functions and fate. However, the current understanding of the interplay between cells and topography relies on static environment. Here the intrinsic light-responsive properties of azopolymers and the versatility of laser-based confocal microscope technique is exploited, aiming to induce spatio-temporal dynamic topographic changes in situ during cell culture. Diverse patterns can be designed on cell-populated azopolymer films with high control on time, space, and on-off signal modification. The technique proposed in this study enables the development of synthetic platforms that finely control cell orientation and migration both in time and space. The results may pave the way to unravel complex processes involved in cell-topography interactions, thus allowing to define the spatio-temporal features that most effectively influence cell functions.

Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor-β1 (TGF-β1) is known to induce expression of α-smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF-β1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF-β1 enhances the level of α-smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.

During the last decades, cell mechanics has been recognized as a quantitative measure to discriminate between many physiological and pathological states of single cells. In the field of biophysics of cancer, a large body of research has been focused on the comparison between normal and cancer mechanics and slowly the hypothesis that cancer cells are softer than their normal counterparts has been accepted, even though in situ tumor tissue is usually stiffer than the surrounding normal tissue. This corroborates the idea that the extra-cellular matrix (ECM) has a critical role in regulating tumor cell properties and behavior. Rearrangements in ECM can lead to changes in cancer cell mechanics and in specific conditions the general assumption about cancer cell softening could be confuted. Here, we highlight the contribution of ECM in cancer cell mechanics and argue that the statement that cancer cells are softer than normal cells should be firmly related to the properties of cell environment and the specific stage of cancer cell progression. In particular, we will discuss that when employing cell mechanics in cancer diagnosis and discrimination, the chemical, the topographical and - last but not least - the mechanical properties of the microenvironment are very important.

We measured the mechanical properties of living neonatal mouse skin tissue using atomic force microscopy. Moreover, to discriminate between the contribution of cells and extracellular matrix (ECM), we treated the tissue with the bacterial collagenase to degrade the ECM and measure the mechanical properties of the exposed cells. After collagenase treatment, we could observe a softening of the tissue samples: typical elastic moduli for the intact tissue were around 10 kPa, whereas, after ECM degradation, we measured elastic moduli of about 400 Pa. Thus, while studying mechanical properties of tissues we found that the ECM plays a major role in determining the stiffness of tissues: cells are much softer than the surrounding material, emphasizing the importance of cell’s environment in tissue mechanics.

We used atomic force microscopy (AFM) technique to measure the viscoelastic response of cancer and normal thyroid cells on different stiffness polyacrylamide gels. After applying a step in contact we recorded the stress relaxation of cells in order to measure their viscous and elastic properties. With the help of an extended version of the Hertz model, we could quantify for the first time by AFM the elastic modulus and the dynamic viscosity of cells on substrates with different stiffnesses. We have cultured anaplastic carcinoma and normal thyroid cells on three different substrates: polyacrylamide gels with elastic modulus in a range of 3–5 and 30–40 kPa and “infinitely” stiff Petri dishes. Whereas normal thyroid cells adapted their mechanical properties to different stiffness substrates, cancer cells were less affected by the surrounding stiffness. Normal cells changed the elastic modulus from 1.2 to 1.6 and to 2.6 kPa with increasing substrate stiffness; the dynamic viscosity values varied from 230 to 515 and to 470 Pa·s, accordingly. By contrast, the values for cancer cells were rather constant regardless of substrate stiffness (in average the elastic modulus was 1.3 kPa and the dynamic viscosity was 300 Pa·s). This difference in sensing and reacting to the mechanical properties of the substrate shows that normal and cancer cells interact differently with the neighboring tissue, which may be related to the ability of cancer cells to form metastases.

Many diseases are related to changes in cell mechanics. Atomic Force Microscopy (AFM) is one of the most suitable techniques allowing the investigation of both topography and mechanical properties of adherent cells with high spatial resolution under physiological conditions. Over the years the use of this technique in medical and clinical applications has largely increased, resulting in the notion of cell mechanics as a biomarker to discriminate between different physiological and pathological states of cells. Cell mechanics has proven to be a biophysical fingerprint able discerning between cell phenotypes, unraveling processes in aging or diseases, or even detecting and diagnosing cellular pathologies. We will review in this report some of the works on cell mechanics investigated by AFM with clinical and medical relevance in order to clarify the state of research in this field and to highlight the role of cell mechanics in the study of pathologies, focusing on cancer, blood and cardiovascular diseases.At...

Understanding the response to illumination at a molecular level as well as characterising polymer brush dynamics are key features that guide the engineering of new light-stimuli responsive materials. Here, we report on the use of a confocal microscopy technique that was exploited to discern how a single molecular event such as the photoinduced isomerisation of azobenzene can affect an entire polymeric material at a macroscopic level leading to photodriven mass-migration. For this reason, a set of polymer brushes, containing azobenzene (Disperse Red 1, DR) on the side chains of poly(methacrylic acid), was synthesised and the influence of DR on the polymer brush dynamics was investigated for the first time by Fluorescence Correlation Spectroscopy (FCS). Briefly, two dynamics were observed, a short one coming from the isomerisation of DR and a long one related to the brush main chain. Interestingly, photoinduced polymer aggregation in the confocal volume was observed.

Mechanical properties of living cancer and normal thyroidal cells were investigated by atomic force microscopy (AFM). Cell mechanics was compared before and after treatment with ML7, which is known to reduce myosin activity and induce softening of cell structures. We recorded force curves with extended dwell time of 6 seconds in contact at maximum forces from 500 pN to 1 nN. Data were analyzed within different frameworks: Hertz fit was applied in order to evaluate differences in Young’s moduli among cell types and conditions, while the fluctuations of the cantilever in contact with cells were analyzed with both conventional algorithms (probability density function and power spectral density) and multifractal detrended fluctuation analysis (MF-DFA). We found that cancer cells were softer than normal cells and ML7 had a substantial softening effect on normal cells, but only a marginal one on cancer cells. Moreover, we observed that all recorded signals for normal and cancer cells were monofractal with small...

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nano-patterning techniques have been employed in order to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated NIH-3T3 cell response to reversible topographic signals encoded on light responsive azopolymer films. Switchable patterns were fabricated by means of a well established holographic set-up. Surface relief gratings (SRGs) were realized with Lloyd's mirror system and erased with circular polarized or incoherent light. Cell cytoskeleton organization and focal adhesions assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as triggers. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way towards an in-situ and real-time investigation of the material-cytoskeleton crosstalk owing to the intrinsic proprieties of azopolymers.

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry.