Microrheological comparison of melanoma cells by atomic force microscopy.

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Comparison of Rheological Properties of Healthy versus Dupuytren Fibroblasts When Treated with a Cell Contraction Inhibitor by Atomic Force Microscope.

Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.

Mechanical Properties of Colorectal Cancer Cells Determined by Dynamic Atomic Force Microscopy: A Novel Biomarker.

Colorectal cancer (CRC) has been addressed in the framework of molecular, cellular biology, and biochemical traits. A new approach to studying CRC is focused on the relationship between biochemical pathways and biophysical cues, which may contribute to disease understanding and therapy development. Herein, we investigated the mechanical properties of CRC cells, namely, HCT116, HCT15, and SW620, using static and dynamic methodologies by atomic force microscopy (AFM). The static method quantifies Young's modulus; the dynamic method allows the determination of elasticity, viscosity, and fluidity. AFM results were correlated with confocal laser scanning microscopy and cell migration assay data. The SW620 metastatic cells presented the highest Young's and storage moduli, with a defined cortical actin ring with distributed F-actin filaments, scarce vinculin expression, abundant total focal adhesions (FAK), and no filopodia formation, which could explain the lessened migratory behavior. In contrast, HCT15 cells presented lower Young's and storage moduli, high cortical tubulin, less cortical F-actin and less FAK, and more filopodia formation, probably explaining the higher migratory behavior. HCT116 cells presented Young's and storage moduli values in between the other cell lines, high cortical F-actin expression, intermediate levels of total FAK, and abundant filopodia formation, possibly explaining the highest migratory behavior.

Mechanobiology of Colorectal Cancer.

In this review, the mechanobiology of colorectal cancer (CRC) are discussed. Mechanotransduction of CRC is addressed considering the relationship of several biophysical cues and biochemical pathways. Mechanobiology is focused on considering how it may influence epithelial cells in terms of motility, morphometric changes, intravasation, circulation, extravasation, and metastization in CRC development. The roles of the tumor microenvironment, ECM, and stroma are also discussed, taking into account the influence of alterations and surface modifications on mechanical properties and their impact on epithelial cells and CRC progression. The role of cancer-associated fibroblasts and the impact of flow shear stress is addressed in terms of how it affects CRC metastization. Finally, some insights concerning how the knowledge of biophysical mechanisms may contribute to the development of new therapeutic strategies and targeting molecules and how mechanical changes of the microenvironment play a role in CRC disease are presented.

Melanoma in the Eyes of Mechanobiology.

Skin is the largest organ of the human body with several important functions that can be impaired by injury, genetic or chronic diseases. Among all skin diseases, melanoma is one of the most severe, which can lead to death, due to metastization. Mechanotransduction has a crucial role for motility, invasion, adhesion and metastization processes, since it deals with the response of cells to physical forces. Signaling pathways are important to understand how physical cues produced or mediated by the Extracellular Matrix (ECM), affect healthy and tumor cells. During these processes, several molecules in the nucleus and cytoplasm are activated. Melanocytes, keratinocytes, fibroblasts and the ECM, play a crucial role in melanoma formation. This manuscript will address the synergy among melanocytes, keratinocytes, fibroblasts cells and the ECM considering their mechanical contribution and relevance in this disease. Mechanical properties of melanoma cells can also be influenced by pigmentation, which can be associated with changes in stiffness. Mechanical changes can be related with the adhesion, migration, or invasiveness potential of melanoma cells promoting a high metastization capacity of this cancer. Mechanosensing, mechanotransduction, and mechanoresponse will be highlighted with respect to the motility, invasion, adhesion and metastization in melanoma cancer.

Direct evidence that tumor cells soften when navigating confined spaces.

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.

Stiffness-Induced Endothelial DLC-1 Expression Forces Leukocyte Spreading through Stabilization of the ICAM-1 Adhesome.

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.

Mechanical and migratory properties of normal, scar, and Dupuytren's fibroblasts.

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-ß1 (TGF-ß1) 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-ß1. 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-ß1 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.

The role of the microenvironment in the biophysics of cancer.

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.

Comparison of viscoelastic properties of cancer and normal thyroid cells on different stiffness substrates.

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.

Comparison of mechanical properties of normal and malignant thyroid cells.

Cancer is a disease of uncontrolled cell proliferation causing approximately 13% of deaths worldwide. Cancer cell mechanics is currently an important topic of investigation in cancer diagnostics as a possible tool to distinguish malignant cells from normal cells in addition to increasing our understanding of pathophysiology of the disease. Our study, based on Atomic Force Microscopy (AFM) measurements on cells, shows that malignant thyroid cells are 3- to 5-fold softer in comparison to primary normal thyroid cells depending on duration between cell seeding and AFM experiments. These results reveal cultivation period as an important factor that influences cell mechanics and which must be considered when comparing cells. Investigation of actin cytoskeleton by fluorescent labelling revealed differences in organization of actin between malignant and normal thyroid cells, which may be directly contributing to alteration of cell mechanics in cancer cells.