Characterization of Cancer Cell Mechanics by Measuring Active Deformation Behavior.

Active deformation behavior reflects cell structural dynamics adapting to varying environmental constraints during malignancy progression. In most cases, cell mechanics is characterized by modeling using static equilibrium systems, which fails to comprehend cell deformation behavior leading to inaccuracies in distinguishing cancer cells from normal cells. Here, a method is introduced to measure the active deformation behavior of cancer cells using atomic force microscopy (AFM) and the newly developed deformation behavior cytometry (DBC). During the measurement, cells are deformed and allows a long timescale relaxation (≈5 s). Two parameters are derived to represent deformation behavior: apparent Poisson's ratio for adherent cells, which is measured with AFM and refers to the ratio of the lateral strain to the longitudinal strain of the cell, and shape recovery for suspended cells, which is measured with DBC. Active deformation behavior defines cancer cell mechanics better than traditional mechanical parameters (e.g., stiffness, diffusion, and viscosity). Additionally, aquaporins are essential for promoting the deformation behavior, while the actin cytoskeleton acts as a downstream effector. Therefore, the potential application of the cancer cell active deformation behavior as a biomechanical marker or therapeutic target in cancer treatment should be evaluated.

AFM-Based Poroelastic@Membrane Analysis of Cells and its Opportunities for Translational Medicine.

Cell mechanics is an emerging field of research for translational medicine. Here, the cell is modeled as poroelastic cytoplasm wrapped by tensile membrane (poroelastic@membrane model) and is characterized by the atomic force microscopy (AFM). The parameters of cytoskeleton network modulus EC , cytoplasmic apparent viscosity ηC , and cytoplasmic diffusion coefficient DC are used to describe the mechanical behavior of cytoplasm, and membrane tension γ is used to evaluate the cell membrane. Poroelastic@membrane analysis of breast cells and urothelial cells show that non-cancer cells and cancer cells have different distribution regions and distribution trends in the four-dimensional space composed of EC , ηC . From non-cancer to cancer cells, there is often a trend of γ, EC , ηC decreases and DC increases. Patients with urothelial carcinoma at different malignant stages can be distinguished at high sensitivity and specificity by analyzing the urothelial cells from tissue or urine. However, sampling directly from tumor tissues is an invasive method, may lead to undesirable consequences. Thus, AFM-based poroelastic@membrane analysis of urothelial cells from urine may provide a non-invasive and no-bio-label method to detecting urothelial carcinoma.

Substrate stiffness affects the morphology, proliferation, and radiosensitivity of cervical squamous carcinoma cells.

Cervical cancer is associated with the highest morbidity rate among gynecological cancers. Radiotherapy plays an important role in the treatment of cervical cancer. However, a considerable number of patients are radiation resistant, leading to a poor prognosis. Matrix stiffness is related to the occurrence, development, and chemoresistance of solid tumors. The association between matrix stiffness and radiosensitivity in cervical cancer cells remains unknown. Here, we sought to determine the effect of matrix stiffness on the phenotype and radiosensitivity of cervical cancer cells. Cervical squamous carcinoma SiHa cells were grown on substrates of different stiffnesses (0.5, 5, and 25 kPa). Cell morphology, proliferation, and radiosensitivity were examined. Cells grown on hard substrates displayed stronger proliferative activity, larger size, and higher differentiation degree, which was reflected in a more mature skeleton assembly, more abundant pseudopodia formation, and smaller nuclear/cytoplasmic ratio. In addition, SiHa cells exhibited stiffness-dependent resistance to radiation, possibly via altered apoptosis-related protein expression. Our findings demonstrate that matrix stiffness affects the morphology, proliferation, and radiosensitivity of SiHa cells. Tissue stiffness may be an indicator of the sensitivity of a patient to radiotherapy. Thus, the data provide insights into the diagnosis of cervical cancer and the design of future radiotherapies.

Substrate Stiffness Modulates the Growth, Phenotype, and Chemoresistance of Ovarian Cancer Cells.

Mechanical factors in the tumor microenvironment play an important role in response to a variety of cellular activities in cancer cells. Here, we utilized polyacrylamide hydrogels with varying physical parameters simulating tumor and metastatic target tissues to investigate the effect of substrate stiffness on the growth, phenotype, and chemotherapeutic response of ovarian cancer cells (OCCs). We found that increasing the substrate stiffness promoted the proliferation of SKOV-3 cells, an OCC cell line. This proliferation coincided with the nuclear translocation of the oncogene Yes-associated protein. Additionally, we found that substrate softening promoted elements of epithelial-mesenchymal transition (EMT), including mesenchymal cell shape changes, increase in vimentin expression, and decrease in E-cadherin and ß-catenin expression. Growing evidence demonstrates that apart from contributing to cancer initiation and progression, EMT can promote chemotherapy resistance in ovarian cancer cells. Furthermore, we evaluated tumor response to standard chemotherapeutic drugs (cisplatin and paclitaxel) and found antiproliferation effects to be directly proportional to the stiffness of the substrate. Nanomechanical studies based on atomic force microscopy (AFM) have revealed that chemosensitivity and chemoresistance are related to cellular mechanical properties. The results of cellular elastic modulus measurements determined by AFM demonstrated that Young's modulus of SKOV-3 cells grown on soft substrates was less than that of cells grown on stiff substrates. Gene expression analysis of SKOV-3 cells showed that mRNA expression can be greatly affected by substrate stiffness. Finally, immunocytochemistry analyses revealed an increase in multidrug resistance proteins, namely, ATP binding cassette subfamily B member 1 and member 4 (ABCB1 and ABCB4), in the cells grown on the soft gel resulting in resistance to chemotherapeutic drugs. In conclusion, our study may help in identification of effective targets for cancer therapy and improve our understanding of the mechanisms of cancer progression and chemoresistance.

Cytotoxicity and Cellular Responses of Gold Nanorods to Smooth Muscle Cells Dependent on Surface Chemistry Coupled Action.

Gold nanorods (AuNRs), with their unique physicochemical properties, are recognized as promising materials for biomedical applications. Chemical modification of their surfaces is attracting increasing attention with regard to cytotoxicity and cellular uptake. Herein, the toxicological effects of three types of polymer-coated AuNRs, which are cetyltrimethylammonium bromide-coated AuNRs, polystyrene sulphonate-coated AuNRs, and poly(diallyldimethyl ammonium chloride-coated AuNRs (PDDAC-AuNRs), on vascular smooth muscle cells (VSMCs) are investigated. The results show significantly different effects on VSMCs with different surface coatings. PDDAC-AuNRs, which were nontoxic in cancer cells in previous reports, display extreme toxicity to VSMCs. Initial contact between AuNRs and cell membranes is the important step in AuNRs cellular uptake. Force spectroscopy based on atomic force microscopy is exploited to study interactions between AuNRs and VSMCs membrane in the absence or presence of a corona on the AuNRs surface. The results show that the binding force and binding probability between AuNRs and membranes are closely related to cytotoxicity and cellular responses. These findings highlight the importance of assessing nanoparticle cytotoxicity in somatic cells for medical applications.

Decorating gold nanostars with multiwalled carbon nanotubes for photothermal therapy.

Gold nanoparticles and carbon nanotubes have attracted substantial attention in recent years for their potential applications in photothermal therapy (PTT) as an emerging breakthrough in cancer treatment. Herein, a hybrid nanomaterial of gold nanostars/multiwalled carbon nanotubes (MWCNTs) was synthesized by two-step reduction via the control of several synthetic conditions such as the reducing agent, pH value, concentration and ratio of reagents. The material shows good biocompatibility and high photothermal conversion efficiency, demonstrating its applicability in PTT. The lack of surfactant in the synthesis process made the hybrid nanomaterial cell-friendly, with no effects on viability in vitro. The MWCNT/gold nanostars hybrid nanomaterial presented 12.4% higher photothermal efficiency than gold nanostars alone and showed a 2.4-fold increase over gold nanospheres based on a heating test under 808 nm laser irradiation. Moreover, the MWCNTs/gold nanostars at low concentration (0.32 nM) exhibited remarkably improved photothermal cancer cell-killing efficacy, which may be attributed to the surface plasmon resonance absorption of the gold nanostars and the combined effects of enhanced coupling between the MWCNTs and gold nanostars. Collectively, these results demonstrate that the MWCNTs/gold nanostars developed herein show prominent photothermal value, and thus may serve as a novel photothermal agent for cancer therapy.

Bioinspired One-Dimensional Nano-Wrinkles Guide Liquid Behaviors at the Liquid-Solid Interfaces.

Learning from nature concerning how nanostructured surfaces interact with liquids may provide insight into better understanding of inside living biological interfaces bearing these nanostructures and further development of innovative materials contacting water. Here we investigate the dynamic behaviour of water droplet interacting with one-dimensional nano-wrinkles of different size on polydimethylsiloxane (PDMS) surface. The structure design of the variationally one-dimensional nano-wrinkles is inspired by in vivo responding topographic changes in aortic intima, which was characterized with liquid-phase atomic force microscopy. We show here that increasing the amplitude of the wrinkles promotes the spreading and energy dissipation of liquid droplets on the wrinkled interfaces. This result suggests a possible bio-protection mechanism of blood vessels via its structural changes on the aortic intima against elevated flowing blood, and provides a basis for tuning interfacial nanostructure of optimal durability against wearing by the liquid behaviors.

Nanomechanical clues from morphologically normal cervical squamous cells could improve cervical cancer screening.

Applying an atomic force microscope, we performed a nanomechanical analysis of morphologically normal cervical squamous cells (MNSCs) which are commonly used in cervical screening. Results showed that nanomechanical parameters of MNSCs correlate well with cervical malignancy, and may have potential in cancer screening to provide early diagnosis.

Mechanical characterization of cervical squamous carcinoma cells by atomic force microscopy at nanoscale.

To investigate the nanoscale mechanical properties of exfoliated cervical epithelial cells from patients to further reveal the pathogenesis of cervical cancer and help early diagnose. Exfoliated cells were collected from nine patients with chronic cervicitis or CIN1(control group), 30 patients with CIN2-3 (CIN 2-3 group), and 13 patients with cervical cancer (cervical cancer group). Stiffness of the cells was determined by atomic force microscope (AFM). Expression of P16INK4A was studied by immunocytochemistry. Environmental scanning electron microscopy was performed to observe the surface microtopography of the exfoliated cells. Young's modulus was measured for cells exfoliated from control and patients with CIN 2-3 and cervical cancer by AFM. The results showed that with increasing cervical lesions, the Young's modulus of the exfoliated cervical cells increased (P < 0.05). The modulus of the exfoliated cells was significantly decreased in the three patients 1 year after the surgery compared with the value before the surgery. Expression of P16INK4A in the exfoliated cells had not been statistically significant. Squamous cells from cervical cancer group had dense and disordered microvilli without clear microridges compare to other groups. The Young's modulus is increased from the control group, to CIN2-3 and cervical cancer groups, suggesting that the stiffness of cervical epithelial cells increases gradually with increasing cervical lesions. The changes in the mechanical properties of the exfoliated cells occur earlier than the changes in cell morphology. Therefore, analysis of mechanical properties of the exfoliated cells may be used to aid early diagnosis of the cancer.

Topographical binding to mucosa-exposed cancer cells: pollen-mimetic porous microspheres with tunable pore sizes.

Mucoadhesives have been perceived as an effective approach for targeting the mucosa-associated diseases, which relied on the adhesive molecules to enhance the specificity. Here, topographical binding is proposed based on the fabrication of surface pore size tunable pollen-mimetic microspheres with phase separation and electrospray technology. We proved that microspheres with large-pores (pore size of 1005 ± 448 nm) were the excellent potential candidate for the mucoadhesives, as they not only possessed better adhesion ability, but also could topographically bind cervical cancer cells. Our methods of topographical binding offered a new way of designing the mucoadhesives for treating the mucosa-associated diseases.

Local thermal injury induces general endothelial cell contraction through p38 MAP kinase activation.

Endothelial cells (ECs) of thin-walled blood vessels form a barrier between blood and tissue. As a response to inflammation, the EC junctions widen and gaps form, resulting in compromised barrier functions. Although the mechanisms behind the establishment of these changes are still incompletely understood, one known reason is actomyosin-dependent actin rearrangement. Here, by using atomic force microscopy and a combination of confocal microscopy methods, we are the first to report that thermal injury induces general venular hyperpermeability and that serum from burned rats induces EC actin rearrangement, contraction, as well as tight-junction damage. Inhibition of the p38 mitogen-activated protein kinase (p38MAPK) largely ameliorates resulting vascular dysfunction by significantly reducing EC stress-fiber formation, contraction, volume changes and tight-junction damage, thereby greatly reducing the appearance of EC gaps. The findings may be of importance for the design of future pharmacotherapies aiming to ease the severe general vascular dysfunction that follows extensive burns.

Validation of reliable reference genes for real-time PCR in human umbilical vein endothelial cells on substrates with different stiffness.

BACKGROUND: The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness. METHODOLOGY/PRINCIPAL FINDINGS: Four algorithms, GeNorm(PLUS), NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes. CONCLUSION/SIGNIFICANCE: Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study.

High-speed AFM for scanning the architecture of living cells.

We address the modelling of tip-cell membrane interactions under high speed atomic force microscopy. Using a home-made device with a scanning area of 100 × 100 µm(2), in situ imaging of living cells is successfully performed under loading rates from 1 to 50 Hz, intending to enable detailed descriptions of physiological processes in living samples.

Substrate stiffness influences the outcome of antitumor drug screening in vitro.

Substrate stiffness has been proven to play a critical role in vitro tumor proliferation; however, pharmacological studies on antitumor drug screening are still routinely carried out in regular plastic culture plates. In the article, polydimethylsiloxane (PDMS) substrates with different stiffness (mimicking articular cartilage, collagenous bone and mammary tumor respectively) and plastic substrate were employed to establish the mechanical microenvironment for the in vitro drug screening platform. We studied the influences of stiffness on the responses of MCF-7 cells to typical antitumor drugs, cisplatin and taxol. Results showed that for both the treatment IC50 value to MCF-7 cells decreased significantly (p < 0.01) on the rigid substrate, indicating that MCF-7 cells on soft substrate have a resistance to cytotoxicity of antitumor drugs. The sensitivity of MCF-7 cells on rigid substrate to drug cytotoxicity was attributed to the increased cell cycle progression, implying that agents proven to be effective in vitro by conventional screening approach might be inefficient in a soft microenvironment in vivo. We conclude that stiffness of the substrates, as a critical mechanical factor, should be concerned for screening antitumor agents in vitro. As an extrapolation, the extensively used drug screening system needs to be revalued and redesigned.

In situ mechanical analysis of cardiomyocytes at nano scales.

Nanomechanical behaviors of single living cardiomyocytes are quantitatively observed using calculated torsions and deflections of an AFM cantilever. The lateral contractions are related to the calcium intensity within rather than the vertical beating power of the cardiomyocytes. Drug-induced nanomechanical changes of cardiomyocytes were further investigated by measuring lateral contractions in real time.

Math1 gene transfer based on the delivery system of quaternized chitosan/Na-carboxymethyl-beta-cyclodextrin nanoparticles.

Mammalian cochlear hair cells don't regenerate naturally after injury, which usually leave permanent hearing loss. Math1 gene is a positive regulator of hair cell differentiation during cochlear development and was proved to be very critical in hair cell regeneration in deaf animals. Generating new cochlear hair cells by forced Math1 expression may be a cure for hearing loss. However, satisfying gene delivering vectors in gene therapy are not available. We combined quaternized chitosan (QCS) with Na-carboxymethyl-beta-cyclodextrin (CM-beta-CD) as novel non-viral vector, which adsorbs pRK5-Math1-EGFP perfectly at the mass ratio of 4:1. In vitro cell transfection can reach a 40% transfect efficiency and relatively low cytotoxity than liposomes. These results suggest that QCS/CM-beta-CD nanoparticle complexes could be a novel non-viral gene carrier in further clinical application.

Sodium alginate as viscosity modifier may induce aggregation of red blood cells.

Viscosity of blood substitutes is among the important determinants to restore microcirculation. Sodium alginate (SA) is always mentioned as "viscosity modifier" in creating blood substitutes. In the present study, the whole blood was diluted using SA solutions to final hematocrits of 10%, 20%, and 35%, respectively. The whole blood viscosity (WBV) at different shear rates, plasma viscosity (PV), and rheological behavior of red blood cells (RBCs) was studied in vitro. The results show that SA may induce RBCs aggregation in a dose-dependent manner. Furthermore, the effect of SA on RBCs aggregation maybe involve the regulation of microcirculation.

AFM and fluorescence imaging of nanomechanical response in periodontal ligament cells.

Most biologists think that AFM has only a limited use in biological research due to its inability to study other than surface structures. Therefore, a BIO-AFM system has been developed to combine both AFM imaging and fluorescence detection, which acts as a powerful tool for a better understanding of dynamic cell processes. In this study, based on a custom-made BIO-AFM system, the elasticity and ultrastructure of living periodontal ligament cells (PDLCs) were investigated. The cantilever probe with a micron-sized bead was used to exert nano-loading force onto the PDLCs. The related signal of NO was then recorded simultaneously. The results show that PDLCs hold strong networks of stress fibers as well as high elastic modulus value, exhibiting the ability for better counteracting the external forces. In the mechano-transduction studies, an initial increase and subsequent drop in intracellular NO response was found. Furthermore, NO may diffuse from a stimulated cell to adjacent cells. In conclusion, our single-cell nano-mechanical study provides a significant advancement in elucidating the magnitude, location, time scale, and biomolecular mechanisms underlying cell mechano-transduction.

Time-series investigation of fused vesicles in microvessel endothelial cells with atomic force microscopy.

Vesicles or caveolae within endothelial cells, fusing together to form vacuolar organelles, are implicated in macromolecular transport and cellular element transmigration across the blood-brain barrier (BBB) during inflammation and ischemia. Vacuolar organelles have been described by transmission electron microscopy and immunofluorescence, but the details of their dynamics have not been well addressed yet. Herein, by using tapping mode atomic force microscopy (AFM), we observed the time-series changes of fused vesicles within the serum-free cultured rat cerebral microvessel endothelial cells. The fused vesicles were certainly proved by fluorescent staining of Fm4-64 combining simultaneous AFM imaging, as well as the field emission scanning electron microscopy technique. And energy dispersive spectrum results additionally implied that there may be specific structure and compositions around the vesicle region. These results indicate that increased vesicles in BBB may contribute to the formation of fused vesicles and a higher probability to construct the trans-endothelial channel across endothelium layer. Furthermore, the AFM application may open up a new approach to investigate the details of transcellular process by fused vesicles.

Fullerene nanoparticles selectively enter oxidation-damaged cerebral microvessel endothelial cells and inhibit JNK-related apoptosis.

There is a dearth in fundamental cellular-level understanding of how nanoparticles interact with the cells of the blood brain barrier (BBB), particularly under the oxidative environment. The apoptosis of cerebral microvessel endothelial cells (CMECs) induced by oxidative stress injury plays a key role in the dysfunction of BBB. By use of CMECs as an in vitro BBB model, we show for the first time that C(60)(C(COOH)(2))(2) nanoparticles can selectively enter oxidized CMECs rather than normal cells, and maintain CMECs integrity by attenuating H(2)O(2)-induced F-actin depolymerization via the observation of several state-of-the art microscopic techniques. Additionally, we have found that C(60)(C(COOH)(2))(2) nanoparticles greatly inhibit the apoptosis of CMECs induced by H(2)O(2), which is related to their modulation of the JNK pathway. C(60)(C(COOH)(2))(2) nanoparticles can regulate several downstream signaling events related to the JNK pathway, including reduction of JNK phosphorylation, activation of activator protein 1 (AP-1) and caspase-3, and inhibition of polyADP-ribose polymerase (PARP) cleavage and mitochondrial cytochrome c release. Our results indicate that C(60)(C(COOH)(2))(2) nanoparticles possess a novel ability of selectively entering oxidation-damaged cerebral endothelial cells rather than normal endothelial cells and then protecting them from apoptosis.