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.

Dual targeting of melanoma translation by MNK/eIF4E and PI3K/mTOR inhibitors.

Melanoma is relatively resistant to chemotherapy, and no targeted therapies are fully effective. The most common mutations in melanoma result in hyperactivation of the mitogen-activated protein kinase (MAPK) and PI3K/AKT/ mTOR pathways responsible for initiating and controlling oncogenic protein translation. This makes both the signaling pathways potentially important therapeutic targets in melanoma. Our studies were carried out on human melanoma cell lines WM793 and 1205 LU with similar genomic alteration (BRAFV600E and PTEN loss). We used a highly specific PI3K/mTOR inhibitor, dactolisib (NVP-BEZ235), and Mnk inhibitor - CGP57380 alone and in combination. Here, we explore the mechanism of action of these drugs alone and in combination, as well as their effect on the viability and invasiveness of melanoma cells. Although when used independently, both drugs suppressed cell proliferation and migration, their combination has additional antitumor effects. We demonstrate that simultaneous inhibition of both pathways may prevent possible drug resistance.

Discriminating bladder cancer cells through rheological mechanomarkers at cell and spheroid levels.

The stiffening or softening of cancers observed in nanoindentation experiments has been recognized as a marker of cancer-related changes. In bladder cancers, continuous stretching/destretching is observed due to its functionality, indicating that shear forces dominate the mechanical response of these cells. Thus, nanoindentation and microrheological measurements conducted in parallel allow for a fully reliable mechanomarker of cancer progression. Here, bladder cancer cell lines, i.e., non-malignant cell cancer of the ureter (HCV29), bladder carcinoma (HT1376), and transitional cell carcinoma (T24), were studied. Nanoindentation and microrheological experiments were conducted on individual cells, cell monolayers, and spheroids that were formed using non-adherent surface plates. The results show that nanoindentation experiments can only differentiate between non-malignant HCV29 (stiffer) and cancerous HT1376 and T24 (softer) cells. Applying microrheology recognizes the type of grade 3 bladder cancers (carcinoma HT1376 or transitional cell carcinoma T24 cells). We showed that actin filaments are a vital element defining the rheological properties of spheroids. Differences in mechanical properties of cell monolayers could be associated with thick actin bundles and intercellular connections, with some extracellular matrix (ECM) contributing to the stiffening of such monolayers. Our findings demonstrate that a complete image of how cancer cells respond to mechanical stress (compressive and shear forces) can only be obtained after microrheological measurements using the transition frequency separating elastic and viscous regimes as a non-labeled biomarker of bladder cancer progression.

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD).

Although complex, the biological processes underlying ischemic stroke are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24 h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1 h of OGD and with longer OGD duration, and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.

Discrimination between NSIP- and IPF-Derived Fibroblasts Based on Multi-Parameter Characterization of Their Growth, Morphology and Physic-Chemical Properties.

BACKGROUND: The aim of the research presented here was to find a set of parameters enabling discrimination between three types of fibroblasts, i.e., healthy ones and those derived from two disorders mimicking each other: idiopathic pulmonary fibrosis (IPF), and nonspecific interstitial pneumonia (NSIP). METHODS: The morphology and growth of cells were traced using fluorescence microscopy and analyzed quantitatively using cell proliferation and substrate cytotoxicity indices. The viability of cells was recorded using MTS assays, and their stiffness was examined using atomic force microscopy (AFM) working in force spectroscopy (FS) mode. To enhance any possible difference in the examined parameters, experiments were performed with cells cultured on substrates of different elasticities. Moreover, the chemical composition of cells was determined using time-of-flight secondary ion mass spectrometry (ToF-SIMS), combined with sophisticated analytical tools, i.e., Multivariate Curve Resolution (MCR) and Principal Component Analysis (PCA). RESULTS: The obtained results demonstrate that discrimination between cell lines derived from healthy and diseased patients is possible based on the analysis of the growth of cells, as well as their physical and chemical properties. In turn, the comparative analysis of the cellular response to altered stiffness of the substrates enables the identification of each cell line, including distinguishing between IPF- and NSIP-derived fibroblasts.

Nanomechanics in Monitoring the Effectiveness of Drugs Targeting the Cancer Cell Cytoskeleton.

Increasing attention is devoted to the use of nanomechanics as a marker of various pathologies. Atomic force microscopy (AFM) is one of the techniques that could be applied to quantify the nanomechanical properties of living cells with a high spatial resolution. Thus, AFM offers the possibility to trace changes in the reorganization of the cytoskeleton in living cells. Impairments in the structure, organization, and functioning of two main cytoskeletal components, namely, actin filaments and microtubules, cause severe effects, leading to cell death. That is why these cytoskeletal components are targets for antitumor therapy. This review intends to describe the gathered knowledge on the capability of AFM to trace the alterations in the nanomechanical properties of living cells induced by the action of antitumor drugs that could translate into their effectiveness.

Indenting soft samples (hydrogels and cells) with cantilevers possessing various shapes of probing tip.

The identification of cancer-related changes in cells and tissues based on the measurements of elastic properties using atomic force microscopy (AFM) seems to be approaching clinical application. Several limiting aspects have already been discussed; however, still, no data have shown how specific AFM probe geometries are related to the biomechanical evaluation of cancer cells. Here, we analyze and compare the nanomechanical results of mechanically homogenous polyacrylamide gels and heterogeneous bladder cancer cells measured using AFM probes of various tip geometry, including symmetric and non-symmetric pyramids and a sphere. Our observations show large modulus variability aligned with both types of AFM probes used and with the internal structure of the cells. Altogether, these results demonstrate that it is possible to differentiate between compliant and rigid samples of kPa elasticity; however, simultaneously, they highlight the strong need for standardized protocols for AFM-based elasticity measurements if applied in clinical practice including the use of a single type of AFM cantilever.

Biophysical and Biochemical Characteristics as Complementary Indicators of Melanoma Progression.

The multistep character of cancer progression makes it difficult to define a unique biomarker of the disease. Interdisciplinary approaches, combining various complementary techniques, especially those operating at a nanoscale level, potentially accelerate characterization of cancer cells or tissue properties. Here, we study a relation between the surface and biomechanical properties of melanoma cells, measured by mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). In total, seven cell lines have been studied. Six of them were melanoma cells derived from various stages of tumor progression: (1) WM115 cells derived from a 55 year old female skin melanoma at a vertical growth phase (VGP) in the primary melanoma site, (2) WM793 cells established from the vertical growth phase (VGP) of a primary skin melanoma lesion, (3) WM266-4 cells established from a cutaneous skin metastasis detected in the same patient as WM115 cells, (4) WM239 cells derived from a cutaneous skin metastasis, (5) 1205Lu cells originated from a lung metastasis diagnosed in the same patient as WM793 cells, and (6) A375P-cells were derived from a solid malignant tumor located in the lung. As a reference cell line, human epidermal melanocytes from adult skin (primary cell line HEMa-LP) were used. Results reveal low, medium, and large deformability of melanoma cells originating from vertical growth phase (VGP), and skin and lung metastasis, respectively. These changes were accompanied by distinct outcome from principal component analysis (PCA). In relation to VGP melanoma cells, cells from skin and lung metastasis reveal similar or significantly different surface properties. The largest deformability difference observed for cells from VGP and lung metastasis was accompanied by the largest separation of unspecific changes in their surface properties. In this way, we show the evidence that biomechanical and surface biochemical properties of cells change in parallel, indicating a potential of being used as nanobiophysical fingerprints of melanoma progression.

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity.

BACKGROUND: Morphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization. METHODS: In this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells. RESULTS: In our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation. CONCLUSIONS: The presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers. GENERAL SIGNIFICANCE: Our findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.

Measuring Elastic Properties of Single Cancer Cells by AFM.

Atomic force microscopy (AFM) enables the measurements of the elastic properties of individual cells in liquids that mimic natural conditions. The single-cell deformability, quantitatively described through the Young's (elastic) modulus, can be used as a marker of pathological alterations, particularly those observed in cancer progression. Here, the AFM-based measurements and data analysis of mechanical properties of single cancerous cells are presented.

Fibroblasts change spreading capability and mechanical properties in a direct interaction with keratinocytes in conditions mimicking wound healing.

Keratinocytes are predominant in the uppermost layer of the skin, while fibroblasts dominate in the dermal layer. These cells interact with each other directly when fibroblasts migrate to a region of the wound where they induce keratinocytes proliferation through double paracrine signalling. Since a response from both keratinocytes and fibroblasts dominates during the inflammatory and proliferative phases, the exact knowledge how these two types of cells interact with each other is crucial for deeper understanding of mechanisms involved in the wound healing process. The aim of this study was to quantify alterations in mechanical properties of cells, i.e. fibroblasts and keratinocytes, in conditions mimicking direct cellular interactions observed in wound healing. Single cell elasticity was measured using atomic force microscope. To verify the influence of keratinocyte neighbors on fibroblasts elasticity (and vice versa), the effect of cellular confluency was studied in parallel. Our results enabled us to distinguish cellular density-related effects from intercellular interactions occurring between fibroblasts and keratinocytes. While the presence of keratinocytes affects fibroblasts spreading capability and mechanical properties, the keratinocytes remain unaffected by the fibroblasts. These results highlight the importance of the cellular deformability in understanding of the role of biomechanics in double paracrine signalling as fibroblast-keratinocyte interaction can change the potential of the wound healing.

Atomic force microscopy as a tool for assessing the cellular elasticity and adhesiveness to identify cancer cells and tissues.

From the first experiments of the atomic force microscopy (AFM) with biological samples, the range of its potential applications grows extensively. One of them is the use of AFM to characterize biophysical fingerprints of cancer progression in search of non-labelled biomarkers of the disease. The technique offers various functionalities, starting from surface imaging to detection of interaction forces, delivering quantitative parameters that can describe changes characteristic for various diseases, including cancer. In this review, the special emphasis was laid on these studies that compare the AFM-derived properties of reference and cancerous cells using all functionalities from cellular deformability measurements to quantification of the interaction forces at the single-molecule and single-cell levels. Despite the large effort and evidence of the microscope applicability to detect pathologically altered cells, there are still practical challenges remained to be solved before AFM can be implemented for routine cancer tracking and diagnosis. To-date, the AFM can be used to achieve a better understanding of cancer-related processes and mechanisms that could be further employed to design high-resolution clinical assays in a quantitative way.

Protocol of single cells preparation for time of flight secondary ion mass spectrometry.

There are several techniques like time of flight secondary ion mass spectrometry (ToF SIMS) that require a special protocol for preparation of biological samples, in particular, those containing single cells due to high vacuum conditions that must be kept during the experiment. Frequently, preparation methodology involves liquid nitrogen freezing what is not always convenient. In our studies, we propose and validate a protocol for preparation of single cells. It consists of four steps: (i) paraformaldehyde fixation, (ii) salt removal, (iii) dehydrating, and (iv) sample drying under ambient conditions. The protocol was applied to samples with single melanoma cells i.e. WM115 and WM266-4 characterized by similar morphology. The surface and internal structures of cells were monitored using atomic force, scanning electron and fluorescent microscopes, used to follow any potential protocol-induced alterations. To validate the proposed methodology for sample preparation, ToF SIMS experiments were carried out using C60(+) cluster ion beam. The applied principal component analysis (PCA) revealed that chemical changes on cell surface of melanoma cells were large enough to differentiate between primary and secondary tumor sites. Subject category: Mass spectrometry.

Differentiation between single bladder cancer cells using principal component analysis of time-of-flight secondary ion mass spectrometry.

Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) mass spectra measurements combined with an appropriate sample preparation protocol are the powerful tools to obtain unique information about the chemical composition of biological materials. In our studies, two questions were addressed, i.e., whether it is possible to develop a fixative-based sample preparation protocol and whether it allows one to distinguish between cells originating from various stages of cancer progression. Therefore, four human bladder cancer cell lines (with distinct malignancy degree) have been investigated. A chemical fixation protocol has been used for TOF-SIMS measurements, and mass spectra were obtained using a Bi3(+) primary ion beam. The principal component analysis (PCA) has been applied to analyze the whole range of mass spectra (without preselection of any particular masses) using two approaches of data preprocessing, namely, mean centering and autoscaling. The PC3 versus PC2 plot has showed significant differences between nonmalignant cancer cells and the cancerous ones for both of preprocessing approaches. The analysis of mass spectra of human bladder cells allows one to find a list of mass peaks with intensities significantly larger in cancerous bladder cells compared to nonmalignant cell cancer of the ureter (HCV29 cells). These findings show that TOF-SIMS in combination with PCA can be used to identify reference, human bladder cells from cancerous ones.

PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells.

A deep understanding of the interaction between cancerous cells and surfaces is particularly important for the design of lab-on-chip devices involving the use of polydimethylsiloxane (PDMS). In our studies, the effect of PDMS substrate stiffness on mechanical properties of cancerous cells was investigated in conditions where the PDMS substrate is not covered with any of extracellular matrix proteins. Two human prostate cancer (Du145 and PC-3) and two melanoma (WM115 and WM266-4) cell lines were cultured on two groups of PDMS substrates that were characterized by distinct stiffness, i.e. 0.75 ± 0.06 MPa and 2.92 ± 0.12 MPa. The results showed the strong effect on cellular behavior and morphology. The detailed analysis of chemical and physical properties of substrates revealed that cellular behavior occurs only due to substrate elasticity.

Nano-characterization of two closely related melanoma cell lines with different metastatic potential.

Cutaneous malignant melanoma is one of the most lethal types of skin cancer. Its progression passes through several steps, leading to the appearance of a new population of cells with aggressive biological potential. Here, we focused on the nano-characterization of two different melanoma cell lines with similar morphological appearance but different metastatic potential, namely, WM115 from vertical growth phase (VGP) and WM266-4 derived from metastasis to skin. The first cell line represents cells that progressed to the VGP, while the WM266-4 cell line denotes cells from the metastasis to skin. Exploring with a combination of atomic force and fluorescence microscopes, our goal was to identify cell surface characteristics in both cell lines that may determine differences in the cellular nano-mechanical properties. Cell elasticity was found to be affected by the presence of F-actin-based flexible ridges, rich in F-actin co-localized with ß1 integrins in the studied cell lines. These results point out how progressive changes in the surface structure of melanoma cells can affect their bionanomechanical properties.

The softening of human bladder cancer cells happens at an early stage of the malignancy process.

Various studies have demonstrated that alterations in the deformability of cancerous cells are strongly linked to the actin cytoskeleton. By using atomic force microscopy (AFM), it is possible to determine such changes in a quantitative way in order to distinguish cancerous from non-malignant cells. In the work presented here, the elastic properties of human bladder cells were determined by means of AFM. The measurements show that non-malignant bladder HCV29 cells are stiffer (higher Young's modulus) than cancerous cells (HTB-9, HT1376, and T24 cell lines). However, independently of the histological grade of the studied bladder cancer cells, all cancerous cells possess a similar level of the deformability of about a few kilopascals, significantly lower than non-malignant cells. This underlines the diagnostic character of stiffness that can be used as a biomarker of bladder cancer. Similar stiffness levels, observed for cancerous cells, cannot be fully explained by the organization of the actin cytoskeleton since it is different in all malignant cells. Our results underline that it is neither the spatial organization of the actin filaments nor the presence of stress fibers, but the overall density and their 3D-organization in a probing volume play the dominant role in controlling the elastic response of the cancerous cell to an external force.