Effects of electrode surface modification with chlorotoxin on patterning single glioma cells

A microchip patterned with arrays of single cancer cells can be an effective platform for the study of tumor biology, medical diagnostics, and drug screening. However, patterning and retaining viable single cancer cells on defined sites of the microarray can be challenging. In this study we used a tumor cell-specific peptide, chlorotoxin (CTX), to mediate glioma cell adhesion on arrays of gold microelectrodes and investigated the effects of three surface modification schemes for conjugation of CTX to the microelectrodes on single cell patterning, which include physical adsorption, covalent bonding mediated by N-hydroxysuccinimide (NHS), and covalent bonding via crosslinking succinimidyl iodoacetate and Traut's (SIA-Traut) reagents. The CTX immobilization to microelectrodes was confirmed by high-resolution X-ray photoelectron spectroscopy. Physically adsorbed CTX showed better support for cell adhesion and is more effective in confining adhered cells on the electrodes than covalently-bound CTX. Furthermore, cell adhesion and spreading on microelectrodes were quantified in real-time by impedance measurements, which revealed an impedance signal from physically adsorbed CTX electrodes four times greater than the signal from covalently-bound CTX electrodes.     

Cancer,pre-cancer and normal oral cells distinguished by dielectrophoresis

Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C _Eff) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C _Eff of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C _Eff and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.     

Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference

A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between normal epithelial and cancer cells, flat or nanostructured polymer surfaces (400 nm pillars, 400 nm perpendicular, or 400 nm parallel lines) were constructed on the bottom of polydimethylsiloxane (PDMS) microfluidic channels in a parallel fashion using a UV-assisted capillary moulding technique. The adhesion of human breast epithelial cells (MCF10A) and cancer cells (MCF7) on each channel was independently measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., one, two, and four hours). It was found that MCF10A cells showed higher adhesion than MCF7 cells regardless of culture time and surface nanotopography at all flow rates, resulting in label-free separation and enrichment of cancer cells. For the cell types used in our study, an optimum separation was found for 2 hours pre-culture on the 400 nm perpendicular line pattern followed by flow-induced detachment at a flow rate of 200 microl min(-1). The fraction of MCF7 cells was increased from 0.36 +/- 0.04 to 0.83 +/- 0.04 under these optimized conditions.     

Characterization of newly established oral cancer cell lines derived from six squamous cell carcinoma and two mucoepidermoid carcinoma cells

Since genetic abnormalities of human cancer are greatly geographically dependent, cultural and environmental backgrounds are thought to be closely related to the carcinogenic process. In the present study, eight human cell lines were established by culture from untreated carcinomas of the oral cancer, of which five were from primary oral squamous cell carcinomas (OSC), one from a mucoepidermoid carcinoma (MEC) and one each originating from metastatic OSC and MEC. All the studied tumor lines grew as monolayers, and showed: i) an epithelial origin by the presence of cytokeratin, and ii) tumorigenic potential in nude mice. Western blot analysis revealed i) over expression of EGFR in six of the cell lines ii) decreased expression of E-cadherin in six cell lines compared to normal human oral mucosa. A mutational analysis showed: point mutations of p53 at exon 7, with transversion, and at exon 8, with transition. These well-characterized human YD cell lines should serve as useful tools in the study of the molecular pathogenesis and biological characteristics of head and neck cancer cells, and in the future testing of new therapeutic reagents for oral cancer.     

Real-time characterization of cytotoxicity using single-cell impedance monitoring

Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.     

Cisplatin-induced alteration on membrane composition of A549 cells revealed by ToF-SIMS

Cisplatin has been clinically used for treatment of solid tumors such as non–small-cell lung cancer for decades. However, tumor resistance may be acquired with losing the antitumor activity of cisplatin. As cellular membrane is the first barrier that cisplatin has to overcome before its further action inside the cells, the membrane composition must play a vital role in the cisplatin uptake and excretion, which further influences cisplatin sensitivity. In this work, we applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) surface analysis combined with principle component analysis to distinguish the differences of cell membrane composition between non–small-cell lung cancer cells (A549) and its cisplatin resistant counterpart A549/DDP cells. The decreased phosphatidylcholine content and more abundant cholesterol were observed in the drug resistant cell surfaces, indicating the decreased membrane fluidity of A549/DDP cells. Moreover, we further compared membrane composition of A549 and A549/DDP cells after being treated with different concentrations of cisplatin. A higher composition level of proteins was discovered on all groups of A549/DDP cell membranes. The altered surface chemistry of cellular membranes induced by cisplatin indicates the significance of membrane structures in the drug resistance, which deserves further investigations to this regard.     

Design,Synthesis and Antitubulin Activity of Novel Podophyllotoxin Derivatives as Potent Anticancer Agent

Twenty novel podophyllotoxin derivatives(1―20) were designed and synthesized. The anti-proliferation activities of these compounds were evaluated against three human cancer cell lines(HepG2, Calu-1 and MCF-7) using podophyllotoxin and Combretastatin A4(CA-4) as positive controls. Among all the compounds, compound 2 displayed more significant anti-proliferation activities against MCF-7 and Calu-1 cell lines and showed lower toxicity towards non-cancer cells. Furthermore, the cell cycle and apoptosis analysis results revealed that compound 2 can cause cell arrest at G2/M phase, leading to cancer cell apoptosis. Meanwhile, it can also reduce the adhesive ability of Calu-1 cells to fibronectin and laminin. The docking simulation results demonstrated that compound 10 can nicely bind to the colchicine site of tubulin. The podophyllotoxin derivatives are worthy to be further investigated to obtain more potent anti-cancer drugs.     

A label-free indicator for tumor cells based on the CH2-stretch ratio

In this paper, we assess the potential of a label-free infrared absorbance based measurement method for determination of the CH(2)-symmetric to CH(2)-antisymmetric stretch ratio, to aid in the detection of the presence of cancer cells and to differentiate between various cancer cells. For this study a normal epithelial kidney cell line, two carcinoma epithelial kidney cell lines, an adult primary human melanocyte cell line, and three human melanoma cell lines were investigated. For the measurements we used a self-designed IR sensor which has the potential to be further developed in a point-of-care instrument. To investigate the mechanism influencing the CH(2)-stretch ratio of mammalian cell membranes, a normal epithelial kidney cell line was exposed to the plasma membrane bound cholesterol reducing agent methyl-β-cyclodextrin. This methodology yielded statistically significant CH(2)-stretch ratio differences between the individual cell lines, normal and tumorous, of both epithelial kidney and melanocyte origin. Measurement results of normal epithelial kidney cells exposed to methyl-β-cyclodextrin indicate that an increase in the CH(2)-stretch ratio arises when there is a decrease in, or redistribution of, the membrane stabilizing agent cholesterol. This study proves that the proposed cell type discrimination method, based on the CH(2)-symmetric to CH(2)-antisymmetric stretch ratio, allows the discrimination between normal and tumor cells. In addition, the method shows high potential for improvement of staging of suspicious tissues.     

Mitotic slippage in non-cancer cells induced by a microtubule disruptor,disorazole C<Subscript>1</Subscript>

Background

Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C₁ was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C₁ in both non-cancer and cancer cells and compared our results to vinblastine and taxol.

Results

In non-cancer cells, disorazole C₁ induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C₁. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C₁.

Conclusion

Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C₁. In contrast, cancer cells induce the apoptotic pathway after disorazole C₁ treatment, indicating a possibly significant therapeutic window for this compound.

     

Cell-based sensor for analysis of EGFR biomarker expression in oral cancer

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. To address this clinical need, we have developed a cell-based sensor to detect oral cancer biomarkers, such as the epidermal growth factor receptor (EGFR) whose over-expression is associated with early oral tumorigenesis and aggressive cancer phenotypes. The lab-on-a-chip (LOC) sensor utilizes an embedded track-etched membrane, which functions as a micro-sieve, to capture and enrich cells from complex biological fluids or biopsy suspensions. Once captured, "on-membrane" immunofluorescent assays reveal the presence and isotype of interrogated cells via automated microscopy and fluorescent image analysis. Using the LOC sensor system, with integrated capture and staining technique, EGFR assays were completed in less than 10 minutes with staining intensity, homogeneity, and cellular localization patterns comparable to conventional labeling methods. Further examination of EGFR expression in three oral cancer cell lines revealed a significant increase (p < 0.05) above control cells with EGFR expression similar to normal squamous epithelium. Results obtained in the microfluidic sensor system correlated well with flow cytometry (r(2) = 0.98), the "gold standard" in quantitative protein expression analysis. In addition, the LOC sensor detected significant differences between two of the oral cancer cell lines (p < 0.01), accounting for disparity of approximately 34 000 EGFR per cell according to quantitative flow cytometry. Taken together, these results support the LOC sensor system as a suitable platform for rapid detection of oral cancer biomarkers and characterization of EGFR over-expression in oral malignancies. Application of this technique may be clinically useful in cancer diagnostics for early detection, prognostic evaluation, and therapeutic selection. Having demonstrated the functionality of this integrated microfluidic sensor system, further studies using clinical samples from oral cancer patients are now warranted.     

Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts. The first two authors contributed equally to this paper     

An ultrasensitive electrochemical biosensor for detection of DNA species related to oral cancer based on nuclease-assisted target recycling and amplification of DNAzyme

A simple, label-free, ultra-highly sensitive and selective electrochemical sensor based on nuclease-assisted target recycling and DNAzyme for the detection of DNA species related to oral cancer in saliva is developed.     

呫吨酮并吡啶季铵盐的合成及其生物活性

以邻碘苯甲酸和8-羟基喹啉为原料,通过Ullmann反应合成了呫吨酮并吡啶(2),再将其进行季铵化反应合成了它的甲基及乙基季铵盐化合物3a和3b,用IR、NMR、MS及元素分析等测试技术对其结构进行了表征。运用四甲基偶氮唑盐微量酸反应比色法(MTT法)测得化合物3a和3b对体外培养人卵巢癌(A2780)、宫颈癌(Hela)、肺癌(SPC-A)和口腔上皮癌(KB)细胞的抑制作用均优于阳性对照药5-氟尿嘧啶(5-Fu)。溴乙锭置换荧光探针法测得化合物3a和3b与小牛胸腺DNA(CT-DNA)的表观结合常数分别为3.91×105和2.72×105L/mol,揭示目标化合物的抗癌活性可能与其跟DNA的相互作用有关。化合物3a和3b对乙酰胆碱酯酶(AChE)和丁酰胆碱酯酶(BuChE)均具有良好的选择性抑制活性,IC50值达μmol/L以下,与阳性对照药氢溴酸加兰他敏(Galantamine.HBr)近似,它们对AChE的抑制作用为非竞争性抑制。     

Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug–cell interactions

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800–3050 cm⁻¹) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug–cell interactions at the subcellular scale.

Stimulated Raman scattering microscopy with spectral phasor analysis provides a label-free approach for phenotypic evaluation of drug-induced effects.     

Synthesis and biological evaluation of 3-substituted-indolin-2-one derivatives containing chloropyrrole moieties

Eighteen novel 3-substituted-indolin-2-ones containing chloropyrroles were synthesized and their biological activities were evaluated. The presence of a chlorine atom on the pyrrole ring was crucial to reduce cardiotoxicity. The presence of a 2-(ethyl-amino)ethylcarbamoyl group as a substituent at the C-4' position of the pyrrole enhanced the antitumor activities notably. IC50 values as low as 0.32, 0.67, 1.19 and 1.22 μM were achieved against non-small cell lung cancer (A549), oral epithelial (KB), melanoma (K111) and large cell lung cancer cell lines (NCI-H460), respectively.     

Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging

Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors, pathologically classified as fibroepithelial tumors. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Here, a combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. The epithelial to stromal area ratio and the collagen SHG signal strength were investigated for their ability to distinguish fibroepithelial lesions. An image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network showed the accurate separation of epithelial and stromal regions. A further investigation, to determine if scoring the epithelial to stromal area ratio and the SHG signal strength within the stromal area could be a marker for differentiating fibroepithelial tumors, showed accurate classification. Therefore, molecular and morphological changes, detected through the assistance of computational and label-free multi-photon imaging techniques, enable us to propose quantitative signatures for epithelial and stromal alterations in breast tissues.     

Rapid detection of apoptosis in mammalian cells by using intact cell MALDI mass spectrometry

Detection of cell death has extensive applications and is of great commercial value. However, most current high-throughput cell viability assays cannot distinguish the two major forms of cell death: apoptosis and necrosis. Many apoptosis-specific detection methods exist but they are time consuming and labour intensive. In this work, we proposed a novel approach based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) for the specific detection of apoptosis in cultured mammalian cells. Buffer washed cells were directly mixed with a matrix solution and subsequently deposited onto the stainless steel target for MALDI analysis. The resulting mass spectrometric profiles were highly reproducible and can be used to reflect cell viability. Remarkably, the mass spectrometric profiles generated from apoptotic cells were distinct from those from either normal or necrotic cells. The apoptosis-specific features of the mass spectra were proportional to the percentage of apoptotic cells in the culture, but are independent of the drugs used to stimulate apoptosis. This is the first report on the utilization of intact cell MALDI mass spectrometry in detecting mammalian cell apoptosis, and can be used as a basis for the development of a reliable, fast, label-free and high-throughput method for detecting apoptotic cell death.     

Suppressed Migration and Enhanced Cisplatin Chemosensitivity in Human Cancer Cell Lines by Tuning the Molecular Mobility of Supramolecular Biomaterials

Cancer cells recognize physical cues transmitted from the surrounding microenvironment, and accordingly alter the migration and chemosensitivity. Cell adhesive biomaterials with tunable physical properties can contribute to the understanding of cancer cell responses, and development of new cancer therapies. Previously, it was reported that polyrotaxane-based surfaces with molecular mobility effectively modulate cellular functions via the yes-associated protein (YAP)-related signaling pathway. In the present study, the impact of molecular mobility of polyrotaxane surfaces on the migration and chemosensitivity of lung (A549), pancreatic (BxPC-3), and breast cancer (MDA-MB-231) cell lines is investigated, and it is found that the cellular spreading of adherent A549 and BxPC-3 cells and nuclear YAP translocation are promoted on low-mobility surfaces, suggesting that cancer cells alter their subcellular YAP localization in response to molecular mobility. Furthermore, low-mobility surfaces suppress cellular migration more than high-mobility surfaces. Additionally, low-mobility surfaces promote the cisplatin chemosensitivity of each cancer cell line to a greater extent than high-mobility surfaces. These results suggest that the molecular mobility of polyrotaxane surfaces suppresses cellular migration and enhances chemosensitivity via the subcellular translocation of YAP in cancer cells. Biointerfaces based on polyrotaxanes can thus be a new platform for elucidating cancer cell migration and chemoresistance mechanisms.     

Controlled viable release of selectively captured label-free cells in microchannels

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.     

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging

Adoptive T lymphocyte (T cell) transfer and tumour-specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label-free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour-specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour-specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR-antigen specificity and screen both of them for immunotherapies.