Role of galectin-3 in the elastic response of radial growth phase melanoma cancer cells.

Melanoma is originated from the malignant transformation of the melanocytes and is characterized by a high rate of invasion, the more serious stage compromising deeper layers of the skin and eventually leading to the metastasis. A high mortality due to melanoma lesion persists because most of melanoma lesions are detected in advanced stages, which decreases the chances of survival. The identification of the principal mechanics implicated in the development and progression of melanoma is essential to devise new early diagnosis strategies. Cell mechanics is related with a lot of cellular functions and processes, for instance motility, differentiation, migration and invasion. In particular, the elastic modulus (Young's modulus) is a very explored parameter to describe the cell mechanical properties; most cancer cells reported in the literature smaller elasticity modulus. In this work, we show that the elastic modulus of melanoma cells lacking galectin-3 is significantly lower than those of melanoma cells expressing galectin-3. More interestingly, the gradient of elastic modulus in cells from the nuclear region towards the cell periphery is more pronounced in shGal3 cells. RESEARCH HIGHLIGHTS: AFM imaging and force spectroscopy were used to investigate the morphology and elasticity properties of healthy HaCaT cells and melanoma cells WM1366, with (shSCR) and without (shGal3) expression of galectin-3. It is shown the effect of galectin-3 protein on the elastic properties of cells: the cells without expression of galectin-3 presents lower elastic modulus. By the results, we suggest here that galectin-3 could be used as an effective biomarker of malignancy in both melanoma diagnostic and prognosis.

Mechanical profile of human keratinocytes expressing HPV-18 oncogenes.

During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.

Actin Stress Fibers Response and Adaptation under Stretch.

One of the many effects of soft tissues under mechanical solicitation in the cellular damage produced by highly localized strain. Here, we study the response of peripheral stress fibers (SFs) to external stretch in mammalian cells, plated onto deformable micropatterned substrates. A local fluorescence analysis reveals that an adaptation response is observed at the vicinity of the focal adhesion sites (FAs) due to its mechanosensor function. The response depends on the type of mechanical stress, from a Maxwell-type material in compression to a complex scenario in extension, where a mechanotransduction and a self-healing process takes place in order to prevent the induced severing of the SF. A model is proposed to take into account the effect of the applied stretch on the mechanics of the SF, from which relevant parameters of the healing process are obtained. In contrast, the repair of the actin bundle occurs at the weak point of the SF and depends on the amount of applied strain. As a result, the SFs display strain-softening features due to the incorporation of new actin material into the bundle. In contrast, the response under compression shows a reorganization with a constant actin material suggesting a gliding process of the SFs by the myosin II motors.

Nuclear elongation correlates with neurite induced cellular elongation during differentiation of SH-SY5Y neural cells / El alargamiento del núcleo se correlaciona con el alargamiento celular inducido por neuritas durante la diferenciación de las células neuronales Sh-Sy5Y

SUMMARY: Cellular differentiation is a highly regulated process that has vast implications for the mechanics of the cell. The interplay between differentiation induced cytoskeletal mechanical changes and strain on the nucleus is a potential cause of gene level changes. This study explores mechanical changes in SH-SY5Y neural cells during differentiation mediated by Retinoic Acid (RA) across Days 0 through 9. Findings suggest that cellular elongation increases 1.92-fold over a 10-day differentiation period, from 48.97 ±16.85µm to 93.96 ± 31.20 µm over 3 repeated trials and across multiple cells analyzed on ImageJ. Nuclear elongation increases less substantially from 17.51 ± 2.71 µm to 23.26 ± 3.10 µm over 3 repeated trials and across multiple cells. Results are statistically significant at a significance level of α = 0.05. This study is one of the first studies to show that during the process of RA mediated neural differentiation in SH-SY5Y neural cells, nuclear elongation is initially not significantly correlated with cellular elongation, but it becomes correlated during the differentiation process with an overall correlation coefficient of 0.4498 at a significance level of α = 0.05. Given the time course of the mechanical changes and the known coupling between the cytoskeleton and nuclear lamina, this study suggests a causative and correlative relationship between neurite-driven cellular elongation and nuclear elongation during neural differentiation.

RESUMEN: La diferenciación celular es un proceso altamente regulado que tiene vastas implicaciones para la mecánica de la célula. La interacción entre los cambios mecánicos citoesqueléticos inducidos por la diferenciación y la tensión en el núcleo es una causa potencial de cambios a nivel genético. Este estudio explora los cambios mecánicos en las células neurales SH-SY5Y durante la diferenciación mediada por el ácido retinoico (RA) durante los días 0 a 9. Los resultados sugieren que el alargamiento celular aumenta 1,92 veces durante un período de diferenciación de 10 días, de 48,97 ± 16,85 µm a 93,96 ± 31,20 µm en 3 ensayos repetidos y en múltiples células analizadas en Image J. El alargamiento nuclear aumenta menos sustancialmente de 17,51 ± 2,71 µm a 23,26 ± 3,10 µm durante 3 ensayos repetidos y en múltiples células. Los resultados son estadísticamente significativos a un nivel de significancia de α = 0,05. Esta investigación es uno de los primeros estudios en demostrar que durante el proceso de diferenciación neural mediada por RA en las células neurales SH-SY5Y, el alargamiento nuclear inicialmente no se correlaciona significativamente con el alargamiento celular, pero se correlaciona durante el proceso de diferenciación con un coeficiente de correlación global de 0,4498 a un nivel de significancia de α = 0,05. Dado el curso temporal de los cambios mecánicos y el acoplamiento conocido entre el citoesqueleto y la lámina nuclear, este estudio sugiere una relación causal y correlativa entre el alargamiento celular impulsado por neuritas y el alargamiento nuclear durante la diferenciación neural.

Effects of myofiber isolation technique on sarcolemma biomechanics.

Isolated myofibers are commonly used to understand the function of skeletal muscle in vivo. This can involve single isolated myofibers obtained from dissection or from enzymatic dissociation. Isolation via dissection allows control of sarcomere length and preserves tendon attachment but is labor-intensive, time-consuming and yields few viable myofibers. In contrast, enzymatic dissociation is fast and facile, produces hundreds of myofibers, and more importantly reduces the number of muscles/animals needed for studies. Biomechanical properties of the sarcolemma have been studied using myofibers from the extensor digitorum longus, but this has been limited to dissected myofibers, making data collection slow and difficult. We have modified this tool to perform biomechanical measurements of the sarcolemma in dissociated myofibers from the flexor digitorum brevis.

Evaluation of the elastic Young's modulus and cytotoxicity variations in fibroblasts exposed to carbon-based nanomaterials.

BACKGROUND: The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. These assays are strongly dependent on the properties of the nanomaterials, for example; specific surface area (SSA), size, surface defects, and surface charge, and the host response. The NMs properties can also interfere with the reagents of the biochemical and optical assays leading to skewed interpretations and ambiguous results related to the NMs toxicity. Here, we proposed a structured approach for cytotoxicity assessment complemented with cells' mechanical responses represented as the variations of elastic Young's modulus in conjunction with conventional biochemical tests. Monitoring the mechanical properties responses at various times allowed understanding the effects of NMs to the filamentous actin cytoskeleton. The elastic Young's modulus was estimated from the force volume maps using an atomic force microscope (AFM). RESULTS: Our results show a significant decrease on Young's modulus, ~ 20%, in cells exposed to low concentrations of graphene flakes (GF), ~ 10% decrease for cells exposed to low concentrations of multiwalled carbon nanotubes (MWCNTs) than the control cells. These considerable changes were directly correlated to the disruption of the cytoskeleton actin fibers. The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Applying both conventional biochemical approach and cells mechanics, we were able to detect differences in the actin networks induced by MWCNT inside the cells and GF outside the cell's membrane. These results contrast with the conventional live/dead assay where we obtained viabilities greater than 80% after 24 h; while the elasticity dramatically decreased suggesting a fast-metabolic stress generation. CONCLUSIONS: We confirmed the production of radical oxygen species (ROS) on cells exposed to CBNs, which is related to the disruption of the cytoskeleton. Altogether, the changes in mechanical properties and the length of F-actin fibers confirmed that disruption of the F-actin cytoskeleton is a major consequence of cellular toxicity. We evidenced the importance of not just nanomaterials properties but also the effect of the location to assess the cytotoxic effects of nanomaterials.

Numeric reconstruction of 2D cellular actomyosin network from substrate displacement

Introduction: One of the fundamental structural elements of the cell is the cytoskeleton. Along with myosin, actin microfilaments are responsible for cellular contractions, and their organization may be related to pathological changes in myocardial tissue. Due to the complexity of factors involved, numerical modeling of the cytoskeleton has the potential to contribute to a better understanding of mechanical cues in cellular activities. In this work, a systematic method was developed for the reconstruction of an actomyosin topology based on the displacement exerted by the cell on a flexible substrate. It is an inverse problem which could be considered a phenomenological approach to traction force microscopy (TFM). Methods An actomyosin distribution was found with a topology optimization method (TOM), varying the material density and angle of contraction of each element of the actomyosin domain. The routine was implemented with a linear material model for the bidimensional actomyosin elements and tridimensional substrate. The topology generated minimizes the nodal displacement squared differences between the generated topology and experimental displacement fields obtained by TFM. The structure resulting from TOM was compared to the actin structures observed experimentally with a GFP-attached actin marker. Results The optimized topology reproduced the main features of the experimental actin and its squared displacement differences were 11.24 µm2, 27.5% of the sum of experimental squared nodal displacements (40.87 µm2). Conclusion This approach extends the literature with a model for the actomyosin structure capable of distributing anisotropic material freely, allowing heterogeneous contraction over the cell extension.

Modes of deformation of walled cells.

The bewildering morphological diversity found in cells is one of the starkest illustrations of life's ability to self-organize. Yet the morphogenetic mechanisms that produce the multifarious shapes of cells are still poorly understood. The shared similarities between the walled cells of prokaryotes, many protists, fungi, and plants make these groups particularly appealing to begin investigating how morphological diversity is generated at the cell level. In this review, I attempt a first classification of the different modes of surface deformation used by walled cells. Five modes of deformation were identified: inextensional bending, equi-area shear, elastic stretching, processive intussusception, and chemorheological growth. The two most restrictive modes-inextensional and equi-area deformations-are embodied in the exine of pollen grains and the wall-like pellicle of euglenoids, respectively. For these modes, it is possible to express the deformed geometry of the cell explicitly in terms of the undeformed geometry and other easily observable geometrical parameters. The greatest morphogenetic power is reached with the processive intussusception and chemorheological growth mechanisms that underlie the expansive growth of walled cells. A comparison of these two growth mechanisms suggests a possible way to tackle the complexity behind wall growth.

SU-E-J-134: Motion Modeling of Non-Small Cell Lung Nodules Based on Respiratory Mechanics.

PURPOSE: To quantify the movements of non-small cell lung nodules using 4D cone-beam computed tomography (4D-CBCT) that is automatically registered with planning CT, and to develop a mathematical model to predict the motion trajectory. Modeling the tumor motion may reduce the PTV and ultimately increase the therapeutic ratio. METHODS: Absolute coordinates of the lung nodules in 15 patients were quantified for each phase of 4D-CBCT scans using auto-registration methods. Assuming respiration follows an elliptical pattern spatially in the lung, these coordinates were fitted to trigonometric functions in each x-y-z direction. Adjusting for phase dependence, the motion could be compared quantitatively for inter-fractional and intra-patient variations to determine if this model is universally applicable and has predictive value. RESULTS: Examination of over 36 sets of 4D-CBCT data shows acceptable agreement (< 2mm) with the elliptical model for both individual scans and over the course of treatment. Some inter-fractional variations in amplitude and cycling periods indicate the need to remodel as patients' conditions change. The intra-patient variations are significant and strongly dependent on the patient lung volume and tumor location, thus individual modeling of tumor motion is expected. CONCLUSIONS: The model indicates good agreement and clinical relevance with non-small cell lung nodule motion, and it appears to be potentially relevant over the course of treatment. Most re-acquired 4D-CBCT images inter-fractionally were within the baseline spatial resolution of the auto- registration technique. However, if remodeling is necessary inter-fractionally, this model still has the potential for significant motion margin reduction over the course of treatment.