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.

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.

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.

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.