Quantitative phase imaging for cell culture quality control.

The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7.

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell-cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles.

Effects of decitabine on the expression of selected endogenous control genes in human breast cancer cells.

To quantify gene expression levels, appropriate controls have to be used to adjust for experimental variation. Endogenous control genes are widely used as they are stably expressed independent of cell cycle and experimental conditions, however, they can be altered upon drug treatment. DNA methylation is widely studied in chemotherapy drug resistance and the DNA methylation inhibitor decitabine showed promising results reversing drug resistance in cancer. We aimed to investigate the effect of different decitabine concentrations on the expression of selected endogenous control genes (GAPDH, 18S rRNA, PPIA, RPL13A, OAZ1) in two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) compared to untreated cells. In MCF-7 cells, 18S rRNA remained stable, however, GAPDH, PPIA and OAZ1 gene expression was increased after treatment. RPL13A was stably expressed at 8 muM decitabine but was increased at lower drug concentrations. In MDA-MB-231 cells, GAPDH levels remained relatively stable following decitabine treatment and so was PPIA expression at low decitabine concentrations. Decitabine increased 18S rRNA, RPL13A and OAZ1 gene expression. In this study, we observed cell line specific effects of decitabine and suggest that 18S rRNA is most suitable to use in MCF-7 cells, while GAPDH is recommended to use in MDA-MB-231 cells during decitabine treatment.

Quantitative phase imaging of cells in a flow cytometry arrangement utilizing Michelson interferometer-based off-axis digital holographic microscopy.

We combined Michelson-interferometer-based off-axis digital holographic microscopy (DHM) with a common flow cytometry (FCM) arrangement. Utilizing object recognition procedures and holographic autofocusing during the numerical reconstruction of the acquired off-axis holograms, sharply focused quantitative phase images of suspended cells in flow were retrieved without labeling, from which biophysical cellular features of distinct cells, such as cell radius, refractive index and dry mass, can be subsequently retrieved in an automated manner. The performance of the proposed concept was first characterized by investigations on microspheres that were utilized as test standards. Then, we analyzed two types of pancreatic tumor cells with different morphology to further verify the applicability of the proposed method for quantitative live cell imaging. The retrieved biophysical datasets from cells in flow are found in good agreement with results from comparative investigations with previously developed DHM methods under static conditions, which demonstrates the effectiveness and reliability of our approach. Our results contribute to the establishment of DHM in imaging FCM and prospect to broaden the application spectrum of FCM by providing complementary quantitative imaging as well as additional biophysical cell parameters which are not accessible in current high-throughput FCM measurements.

Discrimination of skin cancer cells using Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy is a highly versatile tool for cell and tissue analysis. Modern commercial FTIR microspectroscopes allow the acquisition of good-quality hyperspectral images from cytopathological samples within relatively short times. This study aims at assessing the abilities of FTIR spectra to discriminate different types of cultured skin cell lines by different computer analysis technologies. In particular, 22700 single skin cells, belonging to two non-tumoral and two tumoral cell lines, were analysed. These cells were prepared in three different batches that included each cell type. Different spectral preprocessing and classification strategies were considered, including the current standard approaches to reduce Mie scattering artefacts. Special care was taken for the optimisation, training and evaluation of the learning models in order to avoid possible overfitting. Excellent classification performance (balanced accuracy between 0.85 and 0.95) was achieved when the algorithms were trained and tested with the cells from the same batch. When cells from different batches were used for training and testing the balanced accuracy reached values between 0.35 and 0.6, demonstrating the strong influence of sample preparation on the results and comparability of cell FTIR spectra. A deep study of the most optimistic results was performed in order to identify perturbations that influenced the final classification.

Enhanced expression of thioredoxin-interacting-protein regulates oxidative DNA damage and aging.

The "free radical theory of aging" suggests that reactive oxygen species (ROS) are responsible for age-related loss of cellular functions and, therefore, represent the main cause of aging. Redox regulation by thioredoxin-1 (TRX) plays a crucial role in responses to oxidative stress. We show that thioredoxin-interacting protein (TXNIP), a negative regulator of TRX, plays a major role in maintaining the redox status and, thereby, influences aging processes. This role of TXNIP is conserved from flies to humans. Age-dependent upregulation of TXNIP results in decreased stress resistance to oxidative challenge in primary human cells and in Drosophila. Experimental overexpression of TXNIP in flies shortens lifespan due to elevated oxidative DNA damage, whereas downregulation of TXNIP enhances oxidative stress resistance and extends lifespan.

Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers.

We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystem's influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers.

TNF-a stimulation enhances ROS-dependent cell migration via NF-?B activation in liver cells.

Development of hepatocellular carcinoma (HCC) is accompanied by a continuous increase in generation of reactive oxygen species (ROS). TNF-a was used in murine hepatocytes as stimulus to identify the primary source of ROS generation. Using specific inhibitors targeting the different complexes of the respiratory chain we detected the mitochondria as main producer of ROS. TNF-a altered mitochondrial integrity by mimicking a mild uncoupling effect in liver cells. siRNA mediated downregulation of essential assembly factors for complex I and complex III led to an inhibition of ROS production. Therefore, ROS is generated by the mitochondrial respiratory chain upon TNF-a stimulation. ROS activated NF-?B and subsequently enhanced migration of liver cells. Thus, we identified complex I and complex III of the respiratory chain as point of ROS release after TNF-a treatment in hepatocytes which enhances cell migration by activating NF-?B signaling.

Rhenium complexes with visible-light-induced anticancer activity.

Shedding light on the matter: Rhenium(I) indolato complexes with highly potent visible-light-triggered antiproliferative activity (complex 1: EC50 light=0.1 µM vs EC50 dark=100 µM) in 2D- and 3D-organized cancer cells are reported and can be traced back to an efficient generation of singlet oxygen, causing rapid morphological changes and an induction of apoptosis.

Multiple internalization pathways of polyelectrolyte multilayer capsules into mammalian cells.

Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.

Altered DNA methylation is associated with docetaxel resistance in human breast cancer cells.

Docetaxel is an effective chemotherapy drug to treat breast cancer but the underlying molecular mechanisms of drug resistance are not fully understood. DNA methylation is an epigenetic event, involved in the control of gene expression, which is known to play an important role in cancer and chemotherapy drug resistance. To investigate the role of DNA methylation in docetaxel resistance in breast cancer we used two human breast cancer cell lines (MCF-7 and MDA-MB-231) that were made resistant to docetaxel. Docetaxel-resistant sub-lines were treated with different concentrations of decitabine. Global methylation and DNA methyltransferase (DNMT) activity was measured using an ELISA-based assay. Quantitative real-time PCR was used to study DNMT gene expression. Cell viability was studied by MTT assay. Global methylation was increased in MCF-7 but not significantly changed in MDA-MB-231 docetaxel-resistant cells. Decreased DNMT activity and decreased DNMT1 and DNMT3b mRNA expression was associated with docetaxel resistance in both cell lines. To investigate how the components of the DNA methylation machinery may contribute towards docetaxel resistance, decitabine (5-aza-2'-deoxycytidine), an inhibitor of DNA methylation, was used. Decitabine treatment decreased global methylation, DNMT activity and DNMT1, DNMT3a and DNMT3b mRNA expression in MDA-MB-231 docetaxel-resistant cells. In contrast, decitabine-treated MCF-7 docetaxel-resistant cells showed increased DNMT1, DNMT3a and DNMT3b mRNA expression indicating a cell line specific effect of decitabine. Decitabine treatment increased resistance in MCF-7 docetaxel-resistant cells and in the parental MCF-7 and MDA-MB231 docetaxel-sensitive cell lines, however, it did not alter response to docetaxel in MDA-MB-231 docetaxel-resistant cells. This study demonstrates that changes in the DNA methylation machinery are associated with resistance to docetaxel in breast cancer cells. The use of epigenetic therapies, as a strategy to overcome drug resistance, needs to be investigated more fully to determine their effectiveness in different cancers and for different chemotherapy drugs.