Analyzing the Roles of Rho GTPases in Cancer Cell Adhesion to Endothelial Cells Under Flow Conditions.

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is an important event in tumor progression and metastasis formation. The expression of Rho GTPases is frequently altered in cancers, and they are known to regulate cell migration through their effects on adhesion and cytoskeletal dynamics. Several different types of assays are used to investigate how cancer cells attach to and cross the endothelium. Here, we describe an in vitro technique to study the effects of Rho GTPases on human cancer cell adhesion to endothelial cells under shear stress coupled to live cell imaging.

3DeeCellTracker, a deep learning-based pipeline for segmenting and tracking cells in 3D time lapse images.

Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and 'straightened' freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze.

Microscopes have been used to decrypt the tiny details of life since the 17th century. Now, the advent of 3D microscopy allows scientists to build up detailed pictures of living cells and tissues. In that effort, automation is becoming increasingly important so that scientists can analyze the resulting images and understand how bodies grow, heal and respond to changes such as drug therapies. In particular, algorithms can help to spot cells in the picture (called cell segmentation), and then to follow these cells over time across multiple images (known as cell tracking). However, performing these analyses on 3D images over a given period has been quite challenging. In addition, the algorithms that have already been created are often not user-friendly, and they can only be applied to a specific dataset gathered through a particular scientific method. As a response, Wen et al. developed a new program called 3DeeCellTracker, which runs on a desktop computer and uses a type of artificial intelligence known as deep learning to produce consistent results. Crucially, 3DeeCellTracker can be used to analyze various types of images taken using different types of cutting-edge microscope systems. And indeed, the algorithm was then harnessed to track the activity of nerve cells in moving microscopic worms, of beating heart cells in a young small fish, and of cancer cells grown in the lab. This versatile tool can now be used across biology, medical research and drug development to help monitor cell activities.

Spatiotemporal Characterizations of Spontaneously Beating Cardiomyocytes with Adaptive Reference Digital Image Correlation.

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine.

Distinguishing perforin-mediated lysis and granzyme-dependent apoptosis.

Perforin is an indispensable effector protein of primary cytotoxic lymphocytes (CTL or NK cells) that typically defend the host against virus infection, or gene-modified (chimeric antigen receptor-CAR) anticancer T cells. Perforin's pore-forming activity is necessary for the delivery of proapoptotic serine proteases, granzymes, into the cytosol of infected or cancerous target cells. The complete loss of perforin function is detrimental for the function of cytotoxic lymphocytes, and leads to fatal immune dysregulation in infants and predisposes the carriers of hypomorphic perforin mutations to various chronic inflammatory sequelae and blood cancers. Here, we describe several optimized and validated functional assays using purified effector proteins and cytotoxic lymphocytes that enable detailed analysis of perforin-mediated target cell death pathways.

Coincidence Analysis of Molecular Dynamics by Raster Image Correlation Spectroscopy.

The dynamics of cellular processes is a crucial aspect to consider when trying to understand cell function, particularly with regard to the coordination of complex mechanisms involving extensive molecular networks in different cell compartments. Thus, there is an urgent demand of methodologies able to obtain accurate spatiotemporal information on molecular dynamics in live cells. Different variants based on fluorescence correlation spectroscopy have been used successfully in the analysis of protein diffusion and complex or aggregation status. However, the available approaches are limited when simultaneous spatial and temporal resolutions are required to analyze fast processes. Here we describe the use of raster image correlation spectroscopy to analyze the spatiotemporal coincidence of collaborating proteins in highly dynamic molecular mechanisms.

Correlation of Capacitance and Microscopy Measurements Using Image Processing for a Lab-on-CMOS Microsystem.

We present a capacitance sensor chip developed in a 0.35-µm complementary metal-oxide-semiconductor process for monitoring biological cell viability and proliferation. The chip measures the cell-to-substrate binding through capacitance-to-frequency conversion with a sensitivity of 590 kHz/fF. In vitro experiments with two human ovarian cancer cell lines (CP70 and A2780) were performed and showed the ability to track cell viability in realtime over three days. An imaging platform was developed to provide time-lapse images of the sensor surface, which allowed for concurrent visual and capacitance observation of the cells. The results showed the ability to detect single-cell binding events and changes in cell morphology. Image processing was performed to estimate the cell coverage of sensor electrodes, showing good linear correlation and providing a sensor gain of 1.28 ± 0.29 aF/µm2, which agrees with values reported in the literature. The device is designed for unsupervised operation with minimal packaging requirements. Only a microcontroller is required for readout, making it suitable for applications outside the traditional laboratory setting.

Methods for the Study of Entotic Cell Death.

Entosis is a mechanism of cell competition occurring in cancers that involves the engulfment and killing of neighboring cells. The death of ingested cells, called entotic cell death, usually occurs in a non-apoptotic, autophagy protein-dependent manner, where microtubule-associated protein light chain 3 (LC3) is lipidated onto entotic vacuoles. Here we present methods to quantify entotic cell death and its associated LC3 lipidation.

A single-cell micro-trench platform for automatic monitoring of cell division and apoptosis after chemotherapeutic drug administration.

Cells vary in their dynamic response to external stimuli, due to stochastic fluctuations and non-uniform progression through the cell cycle. Hence, single-cell studies are required to reveal the range of heterogeneity in their responses to defined perturbations, which provides detailed insight into signaling processes. Here, we present a time-lapse study using arrays of micro-trenches to monitor the timing of cell division and apoptosis in non-adherent cells at the single-cell level. By employing automated cell tracking and division detection, we precisely determine cell cycle duration and sister-cell correlations for hundreds of individual cells in parallel. As a model application we study the response of leukemia cells to the chemostatic drug vincristine as a function of cell cycle phase. The time-to-death after drug addition is found to depend both on drug concentration and cell cycle phase. The resulting timing and dose-response distributions were reproduced in control experiments using synchronized cell populations. Interestingly, in non-synchronized cells, the time-to-death intervals for sister cells appear to be correlated. Our study demonstrates the practical benefits of micro-trench arrays as a platform for high-throughput, single-cell time-lapse studies on cell cycle dependence, correlations and cell fate decisions in general.

Live imaging of stem cells in the germarium of the Drosophila ovary using a reusable gas-permeable imaging chamber.

Live imaging of stem cells and their support cells can be used to visualize cellular dynamics and fluctuations of intracellular signals, proteins, and organelles in order to better understand stem cell behavior in the niche. We describe a simple protocol for imaging stem cells in the Drosophila ovary that improves on alternative protocols in that flies of any age can be used, dissection is simplified because the epithelial sheath that surrounds each ovariole need not be removed, and ovarioles are imaged in a closed chamber with a large volume of medium that buffers oxygen, pH, and temperature. We also describe how to construct the imaging chamber, which can be easily modified and used to image other tissues and non-adherent cells. Imaging is limited by follicle cells moving out of the germarium in culture around the time of egg chamber budding; however, the epithelial sheath delays this abnormal cell migration. This protocol requires an hour to prepare the ovarioles, followed by half an hour on the confocal microscope to locate germaria and set z limits. Successful imaging time depends on germarial morphology at the time of dissection, but we suggest 10-11 h to encompass all specimens.

Dissecting single-cell molecular spatiotemporal mobility and clustering at focal adhesions in polarised cells by fluorescence fluctuation spectroscopy methods.

Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexible and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous communication, these structures can be highly dynamic depending on the extracellular characteristics. By using a previously well-characterised model like paxillin, we examine the powerful sensitivity and some limitations of RICS and N&B analyses. We show that cells upon contact to different surfaces show differential self-assembly dynamics in terms of molecular diffusion and oligomerisation. In addition, single-cell studies show that these dynamics change gradually following an antero-posterior gradient.

Correlative Live Imaging and Immunofluorescence for Analysis of Chromosome Segregation in Mouse Preimplantation Embryos.

Chromothripsis is a phenomenon observed in cancer cells, wherein a single or few chromosome(s) exhibit vast genomic rearrangements. Recent studies elucidated a striking series of events in which defective segregation of chromosomes causes their incorporation into micronuclei, where they are subject to extensive DNA damage prior to re-joining the main mass of chromosomes in a subsequent cell cycle, which provide an appealing mechanism for the etiology of chromothripsis. Micronuclei are well known to be common in human preimplantation embryos. We recently showed that, unlike in cancer cells, in mouse preimplantation embryos the micronuclei are maintained during multiple cell generations and apparently fail to re-join the main set of chromosomes. This unexpected finding could safeguard the early embryonic genome from chromothripsis. Here, we describe an approach that combines live and immunofluorescence imaging methods that was pivotal in that study to reveal the lack of a functional kinetochore in chromosomes from mouse embryo micronuclei.

Assessing the Influence of a Protease in Cell Migration Using the Barrier-Migration Assay.

Proteases play crucial roles in all steps of tumor progression including cancer cell migration. In fact, uncontrolled proteolytic activity could lead to the degradation of different components of the extracellular matrix which facilitates dissemination of tumor cells. However, numerous studies have revealed that proteases may also exert tumor-protective actions which could impede progression of malignant cells. Consequently, it is crucial to distinguish those situations in which proteases promote tumor growth from those in which exhibit tumor-suppressive effects. In this regard, analysis of the influence of a particular protease on the capacity of a cell line to migrate can be employed as an approach to better understand its involvement in tumorigenesis. Different experimental designs have been developed to investigate cell migration. Herein, we describe a barrier assay to monitor cell migration, which overcomes some disadvantages of traditional methods such as the Boyden chamber or the wound healing assays. The version of the barrier assay explained in this chapter allows to examine cell migration through the analysis of the closure of a premade 500 µm wound. This method also facilitates comparison between two different situations in a given cell line (i.e., gene up- or downregulation) in the same assay and under the same conditions. Additionally, migration can be monitored and measured using a time lapse microscope which facilitates further analysis through different softwares.

Beyond the Proteolytic Activity: Examining the Functional Relevance of the Ancillary Domains Using Tri-Dimensional (3D) Spheroid Invasion Assay.

In this chapter, we describe a straightforward protocol to generate multicellular tumor spheroids (MTSs) and evaluate the role of specific genes in regulating cell invasiveness in real-time and tridimensional (3D) matrices. This approach provides advantages over other conventional invasion assays by offering intimate cell-cell and cell-ECM contacts and by mimicking the pathophysiological characteristics observed in tumor microenvironments (e.g., microregional gradients in glucose and O2 concentrations and metabolic and proliferative tumor heterogeneity). We also provide an original and semiautomated approach to quantify MTS invasion using the freely available ImageJ software and plugins.

Randomized controlled trial comparing embryo culture in two incubator systems: G185 K-System versus EmbryoScope.

OBJECTIVE: To study whether the closed culture system, as compared with a benchtop incubator with similar culture conditions, has a positive impact on intracytoplasmic sperm injection (ICSI) outcomes. DESIGN: Randomized controlled trial. SETTING: University hospital. PATIENT(S): A total of 386 patients undergoing ICSI cycles with at least six mature oocytes were randomized. INTERVENTION(S): Of these patients, 195 were assigned to the group with culture in a time-lapse imaging (TLI) system (EmbryoScope) and 191 to the group with culture in the G185 K-System (G185). MAIN OUTCOME MEASURE(S): Rate of implantation (primary endpoint) and embryo morphology grade. RESULT(S): No significant differences were found in the implantation rates. The proportion of high-grade embryos on day 2 was significantly higher in the TLI group compared with the G185 group (40.4% vs. 35.2%). The impact of the incubator on embryo morphology remained significant in multivariate analysis, which took into account the woman's age, the rank of attempt, and the smoking status (TLI vs. G185: odds ratio = 1.27; 95% confidence interval, [1.04-1.55]). No difference was found in the mean number of frozen embryos, even though the total proportion of frozen embryos was significantly higher in the TLI group than in the G185 group (29.5% vs. 24.8%). CONCLUSION(S): No difference in implantation rate was found between the two incubators for fresh cycles. It remains to be determined whether the observed differences in embryo morphology and the total number of embryos cryopreserved would translate into higher cumulative outcomes with subsequent frozen embryo transfers. CLINICAL TRIAL REGISTRATION NO: NCT02722252.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

Time-Lapse Imaging to Examine the Growth Kinetics of Arabidopsis Seedlings in Response to Ethylene.

Ethylene is well known to inhibit the growth of dark-grown eudicot seedlings. Most studies examine this inhibition after several days of exposure to ethylene. However, such end-point analysis misses transient responses and the dynamic nature of growth regulation. Here, high-resolution, time-lapse imaging is described as a method to gather data about ethylene growth kinetics and movement responses of the hypocotyls of dark-grown seedlings of Arabidopsis thaliana. These methods allow for the characterization of short-term kinetic responses and can be modified for the analysis of roots and seedlings from other species.

Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser.

Time-lapse morphokinetic assessment has low to moderate ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account with the use of clustered data analysis.

OBJECTIVE: To study whether time-lapse morphokinetic (TLM) assessment predicts ploidy status when patient- and ovarian stimulation-related factors are taken into account. DESIGN: Retrospective cohort study. SETTING: Private IVF clinic. PATIENT(S): In total, 103 consecutive patients (415 blastocysts) were included. All embryos were individually cultured in a time-lapse incubator from intracytoplasmic sperm injection up to trophectoderm biopsy. Following trophectoderm biopsy on day 5 or 6, blastocysts were vitrified and 23 TLM parameters were analyzed. INTERVENTION(S): Correlations between patient- and ovarian stimulation-related factors and TLM parameters were tested in a multilevel mixed-effects linear regression model and assessed by means of intraclass correlation coefficient (ICC). MAIN OUTCOME MEASURE(S): Predictive ability of TLM parameters for euploidy. RESULT(S): The majority of TLM parameters had ICCs of 16%-47%. None of the patient- or ovarian stimulation-related factor had any systematic effect on any TLM parameter; however, body mass, total FSH dose, duration of infertility, number of previous cycles, antral follicle count, ovarian stimulation protocol, and E2 on the trigger day had a significant impact on some TLM parameters. With the use of multilevel mixed-effects logistic regression analysis, of the ten TLM parameters that were initially noted to be significantly different among euploid and aneuploid blastocysts in the univariate analysis, only five remained significant. However, the areas under the receiver operating characteristic curves at regression analysis were low, ranging from 0.55 to 0.63. CONCLUSION(S): Five TLM parameters, all related to timing of blastocyst development, have limited ability to predict euploidy when patient- and ovarian stimulation-related factors are taken into account.

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics.

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.