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.

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.

Porous Matrix Stiffness Modulates Response to Targeted Therapy in Breast Carcinoma.

Porous matrix stiffness modulates response to targeted therapy. Poroelastic behavior within porous matrix may modulate the molecule events in cell-matrix and cell-cell interaction like the complex formation of human epidermal growth factor receptor-2 (HER2)-Src-α6ß4 integrin, influencing the targeted therapy with lapatinib.

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.

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.

Effects of Panax notoginseng saponins on alleviating low shear induced endothelial inflammation and thrombosis via Piezo1 signalling.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng saponins (PNS) are the major effective components of Panax notoginseng (burk) F.H.Chen which is one of the classic promoting blood circulation herbs in traditional Chinese medicine. PNS is widely used in China for the treatment of cerebral ischemic stroke. Pathological low shear stress is a causal factor in endothelial inflammation and thrombosis. However, the mechanism of PNS against low shear related endothelial inflammation is still unclear. AIM TO THE STUDY: This study aims to investigate the effects of PNS against endothelial inflammation induced by low shear stress and to explore the underlying mechanical and biological mechanisms. MATERIALS AND METHODS: Mouse model of carotid partial ligation for inducing low endothelial shear stress was established, the pharmacodynamic effect and mechanism of PNS against endothelial inflammation induced by low shear stress through Piezo1 were explored. Yoda1-evoked Piezo1 activation and expression in human umbilical vein endothelial cells (HUVECs) were determined at static condition. Microfluidic channel systems were used to apply shear stress on HUVECs and Piezo1 siRNA HUVECs to determine PECAM-1, p-YAP and VCAM-1 expression. And platelet rich plasma (PRP) was introduced to low shear treated endothelial cells surface to observe the adhesion and activation by fluorescence imaging and flowcytometry. RESULTS: PNS attenuated endothelial inflammation and improved blood flow in a reasonable dose response pattern in carotid partial ligation mouse model by influencing Piezo1 and PECAM-1 expression, while suppressing yes-associated protein (YAP) nuclear translocation. We found Piezo1 sensed abnormal shear stress and transduced these mechanical signals by different pathways in HUVECs, and PNS relieved endothelial inflammation induced by low shear stress through Piezo1. We also found Piezo1 signalling has interaction with PECAM-1 under low shear stress, which were involved in platelets adhesion to endothelial cells. Low shear stress increased YAP nuclear translocation and increased VCAM-1 expression in HUVECs which might activate platelets. PNS inhibited low shear induced Piezo1 and PECAM-1 expression and YAP nuclear translocation in HUVECs, furthermore inhibited platelet adhesion and activation on dysfunctional endothelial cells induced by low shear stress. CONCLUSION: PNS ameliorated endothelial inflammation and thrombosis induced by low shear stress through modulation of the Piezo1 channel, PECAM-1 expression, and YAP nuclear translocation. PNS might serve as a potential therapeutic candidate for ameliorating endothelial inflammation induced by abnormal blood shear stress.

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.

Exploration of the Effects of Substrate Stiffness on Biological Responses of Neural Cells and Their Mechanisms.

Substrate stiffness, as a critical mechanical factor, has been proven to be an important regulator of biological responses, cellular functions, and disease occurrence. However, the effects of substrate stiffness on the phenotypes and drug responses of neural cells remain largely unknown. In this study, polydimethylsiloxane (PDMS) substrates with different stiffnesses were employed to establish the mechanical microenvironment of tissues of different organs. We studied the influences of stiffness on neural cell phenotypes, including cell viability, cell cycle, cytoskeleton structures, cell stiffness, and drug responses of neural cells for hormesis and therapeutic efficacy in neurodegenerative disorders (NDD). The results showed that the greater the range of maximum stimulatory responses, the bigger the width of the stimulatory dosage and the higher the range of maximum neuroprotective activities of hormetic chemicals in neural cells grown on the soft substrate commensurable to the stiffness of the brain, indicating that neural cells on a rigid substrate are resistant to hormetic and neuroprotective effects of hormetic chemicals against 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model. The sensitivity of neural cells on the soft substrate to drug response was attributed to the increased cell viability rate, cell cycle progression, actin stress fibers, focal adhesion formation, and decreased cell stiffness. The promoting effect of the soft substrate and the enhanced hormetic and neuroprotective effect of hormetic chemicals on soft substrates in PC12 cells were confirmed to be mediated by the upregulated EGFR/PI3K/AKT signaling pathway by RNA-Seq and bioinformatics analysis. This study demonstrates that the biomechanical properties of the neural microenvironment play important roles in cell phenotypes and drug responses of neural cells in vitro and suggests that substrate stiffness should be considered in the anti-NDD drug design and treatment.

Characterization of a NDM-1- Encoding Plasmid pHFK418-NDM From a Clinical Proteus mirabilis Isolate Harboring Two Novel Transposons, Tn6624 and Tn6625.

Acquisition of the bla NDM- 1 gene by Proteus mirabilis is a concern because it already has intrinsic resistance to polymyxin E and tigecycline antibiotics. Here, we describe a P. mirabilis isolate that carries a pPrY2001-like plasmid (pHFK418-NDM) containing a bla NDM- 1 gene. The pPrY2001-like plasmid, pHFK418-NDM, was first reported in China. The pHFK418-NDM plasmid was sequenced using a hybrid approach based on Illumina and MinION platforms. The sequence of pHFK418-NDM was compared with those of the six other pPrY2001-like plasmids deposited in GenBank. We found that the multidrug-resistance encoding region of pHFK418-NDM contains ΔTn10 and a novel transposon Tn6625. Tn6625 consists of ΔTn1696, Tn6260, In251, ΔTn125 (carrying bla NDM- 1), ΔTn2670, and a novel mph(E)-harboring transposon Tn6624. In251 was first identified in a clinical isolate, suggesting that it has been transferred efficiently from environmental organisms to clinical isolates. Genomic comparisons of all these pPrY2001-like plasmids showed that their relatively conserved backbones could integrate the numerous and various accessory modules carrying multifarious antibiotic resistance genes. Our results provide a greater depth of insight into the horizontal transfer of resistance genes and add interpretive value to the genomic diversity and evolution of pPrY2001-like plasmids.

Synthesis of Hierarchical ZnFe2O4@SiO2@RGO Core-Shell Microspheres for Enhanced Electromagnetic Wave Absorption.

Hierarchical structured ZnFe2O4@SiO2@RGO core-shell nanocomposites were prepared via a "coating-coating" route, and its structure, composition and electromagnetic properties were characterized. Compared with the binary composites of ZnFe2O4@SiO2, the hierarchical ZnFe2O4@SiO2@RGO ternary composites exhibited enhanced electromagnetic wave (EMW) absorption properties in terms of the effective bandwidth and minimum reflection loss (RL). Furthermore, EMW absorption properties of the prepared samples can be tuned by changing RGO content and thickness of SiO2 layer to reach the best impedance match. The minimum RL of the sample with a thickness of 2.8 mm can reach -43.9 dB at 13.9 GHz, and its effective bandwidth (RL ≤ -10 dB) was up to 6 GHz. Hence, the obtained products can be a new candidate for lightweight EMW absorbing materials.

Substrate stiffness modulates mRNA expression profiling in breast cancer cells.

Identifying effective targets induced by ECM stiffness is of critical importance for treating metastatic cancer diseases, which are followed by changes in the mechanical microenvironment in cancer cells. In this study, polyacrylamide hydrogel substrates with different stiffnesses were prepared and mRNA microarrays were performed to analyze the mRNA expression profiles in breast cancer cell line SK-BR-3 grown on different stiffness substrates. The results indicated that the expressions of 1831 genes were changed significantly in the SK-BR-3 cells on the different stiffness substrates. GO and KEGG pathway analyses of the differently expressed genes in five significant profiles annotated that the most significant pathways were cell cycle, ubiquitin mediated proteolysis RNA transport and pathways in cancer. Finally, the network of genes and gene interaction based on these differently expressed genes was established, and the phosphorylation of AKT and ERK, respectively the downstreams of the PI3K and Ras signal pathways, was further validated. The genes identified in this study may represent good therapeutic targets, and further study of these targets may help to increase our understanding of the mechanisms underlying the pathological processes and therapy for metastatic breast cancer disease.

Substrate stiffness of endothelial cells directs LFA-1/ICAM-1 interaction: A physical trigger of immune-related diseases?

In blood vessels, substrate stiffness of the endothelium varies between different body locations and increases during the progression of multiple sclerosis. As a crucial step of the immune response, lymphocyte function associated antigen-1 (LFA-1)/ intercellular adhesion molecule-1 (ICAM-1) interaction occurs in various tissues and plays a pivotal role in atherosclerosis. However, the contribution of the physical property of endothelium substrate, such as the stiffness, to LFA-1/ICAM-1 interaction and immune-related diseases progression remains largely unknown. In this study, we investigated the influence of substrate stiffness on the adhesion force of LFA-1/ICAM-1 bond and ICAM-1 expression on the endothelial cell apical surface with an improved in vitro model. A silica microsphere-functionalized atomic force microscopy (AFM) tip was linked to LFA-1 via a polyethylene glycol (PEG) chain, and then approached toward human aortic endothelial cells (HAECs) on polyacrylamide gels of different stiffnesses. The results showed that the adhesion force was elevated on stiff substrates, while the expression of ICAM-1 on the HAECs surface was not influenced by substrate stiffness. A low-dose blebbistatin treatment (5µmol/L) reduced the adhesion force on both substrates while a high dose blebbistatin treatment (50µmol/L)) eliminated the adhesion between LFA-1 and ICAM-1, indicating that endothelium substrate stiffness directs the LFA-1/ICAM-1 interaction in a myosin II-dependent manner. These results help to describe the relationship between substrate stiffness and myosin II-dependent LFA-1/ICAM-1 interaction, and may increase the understanding of the pathogenesis and treatment of immune-related diseases.

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.

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.

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.

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.

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.