Determining the degree of chromosomal instability in breast cancer cells by atomic force microscopy.

Chromosomal instability (CIN) is a source of genetic variation and is highly linked to the malignance of cancer. Determining the degree of CIN is necessary for understanding the role that it plays in tumor development. There is currently a lack of research on high-resolution characterization of CIN and the relationship between CIN and cell mechanics. Here, a method to determine CIN of breast cancer cells by high resolution imaging with atomic force microscopy (AFM) is explored. The numerical and structural changes of chromosomes in human breast cells (MCF-10A), moderately malignant breast cells (MCF-7) and highly malignant breast cells (MDA-MB-231) were observed and analyzed by AFM. Meanwhile, the nuclei, cytoskeleton and cell mechanics of the three kinds of cells were also investigated. The results showed the differences in CIN between the benign and cancer cells. Also, the degree of structural CIN increased with enhanced malignancy of cancer cells. This was also demonstrated by calculating the probability of micronucleus formation in these three kinds of cells. Meanwhile, we found that the area of the nucleus was related to the number of chromosomes in the nucleus. In addition, reduced or even aggregated actin fibers led to decreased elasticities in MCF-7 and MDA-MB-231 cells. It was found that the rearrangement of actin fibers would affect the nucleus, and then lead to wrong mitosis and CIN. Using AFM to detect chromosomal changes in cells with different malignancy degrees provides a new detection method for the study of cell carcinogenesis with a perspective for targeted therapy of cancer.

Cancer Development in Hepatocytes by Long-Term Induction of Hypoxic Hepatocellular Carcinoma Cell (HCC)-Derived Exosomes In Vivo and In Vitro.

Hypoxic tumor cell-derived exosomes play a key role in the occurrence, development, and metastasis of tumors. However, the mechanism of hypoxia-mediated metastasis remains unclear. In this study, hypoxic hepatocellular carcinoma cell (HCC-LM3)-derived exosomes (H-LM3-exos) were used to induce hepatocytes (HL-7702) over a long term (40 passages in 120 days). A nude mouse experiment further verified the effect of H-LM3-exos on tumor growth and metastasis. The process of cancer development in hepatocytes induced by H-LM3-exos was analyzed using both biological and physical techniques, and the results showed that the proliferation and soft agar growth abilities of the transformed cells were enhanced. The concentration of tumor markers secreted by transformed cells was increased, the cytoskeleton was disordered, and the migration ability was enhanced and was accompanied by epithelial-mesenchymal transition (EMT). Transcriptome results showed that differentially expressed genes between transformed cells and hepatocytes were enriched in cancer-related signaling pathways. The degree of cancer development in transformed cells was enhanced by an increase in H-LM3-exos-induced passages. Nude mice treated with different concentrations of H-LM3-exos showed different degrees of tumor growth and liver lesions. The physical properties of the cells were characterized by atomic force microscopy. Compared with the hepatocytes, the height and roughness of the transformed cells were increased, while the adhesion and elastic modulus were decreased. The changes in physical properties of primary tumor cells and hepatocytes in nude mice were consistent with this trend. Our study linking omics with the physical properties of cells provides a new direction for studying the mechanisms of cancer development and metastasis.

Study on Calceolarioside A Anti-Aß25-35 -Induced Damage in SH-SY5Y†cells by Atomic Force Microscopy.

Calceolarioside A is a phenylethyl glycoside compound, originally isolated from the bark of Fraxinus mandshurica Rupr. In this work, the protective effect of Calceolarioside A on beta Amyloid protein induced toxicity in SH-SY5Y cells was studied. DPPH experiment, MTT assay, SEM, Atomic force microscopy and Colony formation were used to study the activity of Calceolarioside A. The results in this article show that the survival rate of the cells with Calceolarioside A (20-40 mg/mL) was significantly higher than that of the model cells without Calceolarioside A. Calceolarioside A could protect SH-SY5Y cells by improving some parameters in cells, such as the cell height, Young's modulus, adhesion and branch. In summary, Calceolarioside A can reduce Aß25-35 -induced damage in SH-SY5Y cells. It can be a potential medicine to treatment with AD.

Electrochemical sensors based on platinum-coated MOF-derived nickel-/N-doped carbon nanotubes (Pt/Ni/NCNTs) for sensitive nitrite detection.

As excess nitrite has a serious threat to the human health and environment, constructing novel electrochemical sensors for sensitive nitrite detection is of great importance. In this report, platinum nanoparticles were deposited on nickel-/N-doped carbon nanotubes, which were obtained through a self-catalytically grown process with Ni-MOF as precursors. The as-prepared Pt/Ni/NCNTs were applied as amperometric sensors and presented superior sensing properties for nitrite detection. Benefiting from the synergy of Pt and Ni/NCNTs, Pt/Ni/NCNTs displayed much wider detection ranges (0.5-40 mM and 40-110 mM) for nitrite sensing. The sensitivity is 276.92 µA mM-1 cm-2 and 224.39 µA mM-1 cm-2, respectively. The detection limit is 0.17 µM. The Pt/Ni/NCNTs sensors also showed good feasibility for nitrite sensing in real samples (milk and peach juice) analysis. The active Pt/Ni/NCNTs composites and facile fabrication technique may provide useful strategies to develop other sensitive nitrite sensors.

Responses of INS-1 cells to glucose stimulation patterns.

Diabetes has become a major public health problem in the world for many years, and it is driving us to probe into its complex mechanism of insulin secretion in pancreatic ß cells. The nanoscale resolution characterization of pancreatic ß cells in response to glucose led to insights into diverse mechanical and functional processes at the single cell level. Recent advances allowed the direct observations of cytoskeleton dynamics which were quantitatively determined. Here, we firstly performed the glucose stimulation with multiple physiologically relevant glucose patterns. Atomic force microscopy (AFM) produced high spatial resolution mechanical images together with the insulin secretions linking the physical interactions to the biochemical process of INS-1 cells. Altered material properties of the INS-1 cells revealed the regulation of multiple glucose stimulation patterns. Rapidly responded to high glucose (HG), INS-1 cells presented the unique meshing networks of elasticities. The decreases of Young's modulus (YM) and insulin secretion suggested that mechanical changes affected the insulin release. Furthermore, the frequency and gradient of glucose patterns induced nanomechanical and secreting changes of the INS-1 cells and gained the knowledge on the potential controllability of glucose. The relationships between the cellular mechanics and insulin secretion of INS-1 cells could contribute to establish a mechanical cell model for the study of ß cells in diabetes. The results also indicated the cell mechanics as promising mechanical biomarkers for ß cells, and promoted the understanding of specific mechanical mechanism of glucose regulation, which lighted on the further application of functional glucose regulation in therapy.

Atomic force microscopy-based assessment of multimechanical cellular properties for classification of graded bladder cancer cells and cancer early diagnosis using machine learning analysis.

Cellular mechanical properties (CMPs) have been frequently reported as biomarkers for cell cancerization to assist objective cytology, compared to the current subjective method dependent on cytomorphology. However, single or dual CMPs cannot always successfully distinguish every kind of malignant cell from its benign counterpart. In this work, we extract 4 CMPs of four different graded bladder cancer (BC) cell lines by AFM (atomic force microscopy)-based nanoindentation to generate a CMP database, which is used to train a cancerization-grade classifier by machine learning. The classifier is tested on 4 categories of BC cells at different cancer grades. The classification shows split-independent robustness and an accuracy of 91.25% with an AUC-ROC (ROC stands for receiver operating characteristic, and ROC curve is a graphical plot which illustrates the performance of a binary classifier system as its discrimination threshold is varied) value of 97.98%. Finally, we also compare our proposed method with traditional invasive diagnosis and noninvasive cancer diagnosis relying on cytomorphology, in terms of accuracy, sensitivity and specificity. Unlike former studies focusing on the discrimination between normal and cancerous cells, our study fulfills the classification of 4 graded cell lines at different cancerization stages, and thus provides a potential method for early detection of cancerization. STATEMENT OF SIGNIFICANCE: We measured four cellular mechanical properties (CMPs) of 4 graded bladder cancer (BC) cell lines using AFM (atomic force microscopy). We found that single or dual CMPs cannot fulfill the task of BC cell classification. Instead, we employ MLA (Machine Learning Algorithm)-based analysis whose inputs are BC CMPs. Compared with traditional cytomorphology-based prognoses, the non-invasive method proposed in this study has higher accuracy but with many fewer cellular properties as inputs. The proposed non-invasive prognosis is characterized with high sensitivity and specificity, and thus provides a potential tumor-grading means to identify cancer cells with different metastatic potential. Moreover, our study proposes an objective grading method based on quantitative characteristics desirable for avoiding misdiagnosis induced by ambiguous subjectivity.

Facile fabrication of micropattern surfaces with controlled wettability on PDMS-modified fiber membranes for cell patterning.

Various cell culture substrates have been developed for cell patterning to control cell distributions and orientations in tissue engineering, drug screening and regenerative medicine. In this study, a preparation method of modified fiber membranes was applied in the field of cell patterning, and the obtained fiber membranes guided the cell distributions and orientations flexibly. The aligned electrospinning fiber membranes were dip-coated with polydimethylsiloxane (PDMS) to improve the stability of wettability, and then it was treated with oxygen plasma with a photomask to obtain a hydrophilic-hydrophobic surface micropattern. The morphologies, wettabilities and chemical structures of the membranes were analyzed by using a scanning electron microscope (SEM), drop shape analysis instrument, energy dispersive spectrometer (EDS) and Fourier transform infrared (FTIR) spectrometer. The L929 cells were cultured on the obtained membranes to observe the controlled cell distributions and orientations by using a SEM and fluorescence microscope. The results indicate that the treated membranes have the ability to control both cell distributions and orientations simultaneously. This method offers a novel approach to develop cell culture substrates for cell patterning in tissue engineering.

Insights into cell classification based on combination of multiple cellular mechanical phenotypes by using machine learning algorithm.

Although cellular elastic property (CEP, also known as cellular elastic modulus) has been frequently reported as a biomarker to distinguish some cancerous cells from their benign counterparts, it cannot be adopted as a universal hallmark to be applied to every kind cell. In the present study, we report that insignificant difference is observed between normal gastric cell and its cancer counterpart which is one of the common human malignancies, in terms of CEP statistical distribution. In this regard, we propose multiple cellular mechanical phenotypes (CMPs) to differentiate the above two cell types, which is realized by machine learning algorithm (MLA). The results show that the cellular classification effect proves better with more CMPs adopted, regardless of the exact MLA employed. Moreover, the MLA-based method remains effective if we add two more cell lines to the above two cell categories. Our study indicates that MLA-based cellular classification can potentially serve as an efficient and objective means to assist or even validate cancer prognostics.

Bi-layered disulfiram-loaded fiber membranes with antibacterial properties for wound dressing.

In this study, the bi-layered disulfiram-loaded fiber membranes with the antibacterial activity and different surface wettabilities are prepared using electrospinning technology. In the application of wound dressing, the hydrophilic surface of fiber membranes is beneficial for cell adhesion and drug release to heal the wound. Meanwhile, the outside hydrophobic surface is able to block water penetration to reduce the probability of wound infection. The obtained bi-layered drug-loaded fiber membranes are composed of polyvinylidene fluoride (PVDF) bottom surface and disulfiram (DSF)/polylactic acid (PLA) top surface. To modify the top surface wettability, the oxygen plasma modification of bi-layered membranes was carried out. The morphology, wettability, and chemical compositions of bi-layered drug-loaded fiber membranes were analyzed using the scanning electronic microscope (SEM), drop shape analysis instrument, X-ray diffractometer (XRD), and X-ray photoelectron spectrometer (XPS). The bi-layered disulfiram-loaded membranes showed the potent antibacterial activity in vitro against both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). It was found that the bi-layered membranes had good biocompatibility with L929 cells. Thus, the obtained bi-layered disulfiram-loaded fiber membranes are suitable for wound dressing application.

Effects of Tricholoma matsutake (Agaricomycetes) Extracts on Promoting Proliferation of HaCaT Cells and Accelerating Mice Wound Healing.

Tricholoma matsutake is popular in Asian countries because of its edibility and medicinal use. T. matsutake is a precious natural medicinal fungus, and it is widely used in food and biological products. This study aimed to explore the mechanism of T. matsutake on promoting proliferation of human immortalized keratinocyte (HaCaT) cells and accelerating wound healing in mice. The MTT assay was used to test the effects of three different T. matsutake extracts (0, 62.5, 125, 250, 500, and 1000 µg/mL) on HaCaT cell viability. HaCaT cells were treated with the three T. matsutake extracts (100, 500 µg/mL) and morphological and biophysical properties were detected by atomic force microscopy with JPK data processing. Western blot analysis detected Notch signaling pathways of HaCaT cells treated with 50% ethanol extract of T. matsutake (50%T) for 24 h (100, 500 and 1000 µg/mL). Mouse wounds were treated with 50%T for 15 days. Wound healing effects were observed on the back skin of mice at different times. The quality of wound healing was estimated by histological staining (hematoxylin and eosin and Masson's trichrome). All data were counted by GraphPad Prism 5 software. The increased concentration of T. matsutake remarkably promoted HaCaT cell proliferation. The Young's modulus of HaCaT cells showed the biggest increase from 1.73 ± 0.13 kPa (0 µg/mL) to 4.57 ± 0.16 kPa (500 µg/mL) in the 50%T group. The Notch1/Jagged1 pathways were upregulated with an increase in concentration (0, 100, 500, and 1000 µg/mL). Moreover, compared with the negative and positive control groups, T. matsutake promoted wound healing in mice by epidermal regeneration, subepidermal tissue formation, and collagen deposition. The results showed that T. matsutake promotes not only proliferation of HaCaT cells but also wound healing in mice.

Investigation of the mechanical effects of targeted drugs on cancerous cells based on atomic force microscopy.

Cancer is currently drawing more and more attention as the leading factor in death worldwide. However, little research has been directed towards investigating the micro/nanoscale mechanical properties of cancer cells treated by targeted drugs to evaluate the model systems of targeted drugs using atomic force microscopy (AFM) nano-indentation, especially in light of the multiple drugs targeting various cancerous cells. This paper aims to compare the mechanical effects of sorafenib tosylate and osimertinib mesylate on hepatoma carcinoma cells and lung cancerous cells using atomic force microscopy from the perspective of a model system based on nano-indentation at the micro/nanoscale, which has rarely been investigated. The Sneddon model is applied to fit the force-distance curves, and the mechanical properties, i.e., Young's moduli, can then be calculated. For the SMMC-7721 cells, osimertinib mesylate is a more effective inhibitor than sorafenib tosylate. For the A549 cells, osimertinib mesylate and sorafenib tosylate both have an obvious inhibitory effect. The experimental results may make possible contributions to the diagnosis and treatment of early-stage cancers.

Study of NSCLC cell migration promoted by NSCLC-derived extracellular vesicle using atomic force microscopy.

Extracellular vesicles (EVs) secreted by cancer cells play a key role in the cancer microenvironment and progression. Previous studies have mainly focused on molecular functions, cellular components and biological processes using chemical and biological methods. However, whether the mechanical properties of cancer cells change due to EVs remains poorly understood. This work studies the effects of mechanical changes in non-small cell lung cancer (NSCLC) cells after treatment with EVs on migration by atomic force microscopy (AFM). Different concentrations of EVs were added into the experimental groups based on co-culture experiments, while the control group was cultured without EVs for 48 h. Cellular migration was evaluated by wound healing experiments. The cellular morphology, cell stiffness and surface adhesion were investigated by AFM. Cytoskeleton changes were detected by fluorescence staining assay. By comparison to the control group, the cell migration was enhanced. After treatment with EVs, the cell length and height show an upward trend, and the adhesion force and Young's modulus show a downward trend, and filopodia were also detected in the cells. Overall, the EVs promoted the migration of NSCLC cells by regulating cells' physical properties and skeletal rearrangement.

Migration of BEAS-2B cells enhanced by H1299 cell derived-exosomes.

Previous studies reported that exosomes (Exos) secreted by tumor cells could affect the tumor cells themselves and normal cells. However, the effects of exosomes derived from tumor cells on normal cells' migration and mechanical characteristics are rarely reported. This work explores the effects of H1299 cell-derived exosomes (H1299-Exos) on the migration of BEAS-2B cells, and analyzes possible mechanical mechanisms. In the experiments, exosomes were isolated from the culture supernatants of H1299 cells by ultracentrifugation. The H1299-Exos were confirmed by scanning electron microscope (SEM) and western blotting (WB). The BEAS-2B cell migration was assessed using scratch assays. Cytoskeletal structure changes were detected by immunofluorescence. Surface morphology and mechanical properties were measured by atomic force microscopy (AFM). After incubation with H1299-Exos for 48 h, BEAS-2B cells enhanced migration ability, with increased filopodia and cytoskeletal rearrangements. The changes in the morphology and mechanical properties of the cells caused by H1299-Exos were detected using AFM, including the increase in cell length and the decrease in cell height, Young's modulus and adhesion. In short, H1299-Exos promoted the BEAS-2B cell migrations. It indicates that the morphological and mechanical properties can be used as a means to assess normal cell alterations induced by tumor cell derived-exosomes. This provides a method for studying the effects of exosomes secreted by tumor cells on normal cells and the changes in their physical properties.

Dynamic mechanics of HK-2 cell reaction to HG stimulation studied by atomic force microscopy.

Renal tubular cell injury by exposure to high glucose (HG) stimulation mainly accounts for diabetic nephropathy (DN). To understand the mechanism of injury by HG, quantitative characterization has commonly focused on the cells that are already impaired, which ignores the signals for the process of being injured. In this study, the architecture and morphology of HK-2 cells were observed dynamically by multiple imaging methods. AFM (atomic force microscopy)-based single-cell force spectroscopy was employed to investigate the dynamic mechanics quantitatively. The results showed that the Young's modulus increased continuously from 2.44 kPa up to 4.15 kPa for the whole period of injury by HG, while the surface adhesion decreased from 2.43 nN to 1.63 nN between 12 h and 72 h. In addition, the actin filaments of HK-2 cells exposed to HG depolymerized and then nucleated with increasing Young's modulus. The absence of cell pseudopodia coincided with the reduced cell adhesion, strongly suggesting close relationships between the cell architecture, morphology and mechanical properties. Furthermore, the stages of cell reactions were identified and assessed. Overall, the dynamic mechanics of the cells facilitate the identification of injured cells and the assessment of the degree of injury for accurate diagnoses and treatments.

[Immunity to parasitic infection: the role of dendritic cells].

Dendritic cells are a major class of professional antigen-presenting cells which play an important role in the immune response of parasite-infected host. This article summarizes the role of dendritic cells in the immunity to parasitic infection by analyzing the studies of protozoan, nematode, and trematode infections.