Single-cell tracking as a tool for studying EMT-phenotypes.

This article demonstrates that label-free single-cell video tracking is a useful approach for in vitro studies of Epithelial-Mesenchymal Transition (EMT). EMT is a highly heterogeneous process, involved in wound healing, embryogenesis and cancer. The process promotes metastasis, and increased understanding can aid development of novel therapeutic strategies. The role of EMT-associated biomarkers depends on biological context, making it challenging to compare and interpret data from different studies. We demonstrate single-cell video tracking for comprehensive phenotype analysis. In this study we performed single-cell video tracking on 72-h long recordings. We quantified several behaviours at a single-cell level during induced EMT in MDA-MB-468 cells. This revealed notable variations in migration speed, with different dose-response patterns and varying distributions of speed. By registering cell morphologies during the recording, we determined preferred paths of morphological transitions. We also found a clear association between migration speed and cell morphology. We found elevated rates of cell death, diminished proliferation, and an increase in mitotic failures followed by re-fusion of sister-cells. The method allows tracking of phenotypes in cell lineages, which can be particularly useful in epigenetic studies. Sister-cells were found to have significant similarities in their speeds and morphologies, illustrating the heritability of these traits.

Initial refinement of data from video-based single-cell tracking.

Background: Video recording of cells offers a straightforward way to gain valuable information from their response to treatments. An indispensable step in obtaining such information involves tracking individual cells from the recorded data. A subsequent step is reducing such data to represent essential biological information. This can help to compare various single-cell tracking data yielding a novel source of information. The vast array of potential data sources highlights the significance of methodologies prioritizing simplicity, robustness, transparency, affordability, sensor independence, and freedom from reliance on specific software or online services. Methods: The provided data presents single-cell tracking of clonal (A549) cells as they grow in two-dimensional (2D) monolayers over 94 hours, spanning several cell cycles. The cells are exposed to three different concentrations of yessotoxin (YTX). The data treatments showcase the parametrization of population growth curves, as well as other statistical descriptions. These include the temporal development of cell speed in family trees with and without cell death, correlations between sister cells, single-cell average displacements, and the study of clustering tendencies. Results: Various statistics obtained from single-cell tracking reveal patterns suitable for data compression and parametrization. These statistics encompass essential aspects such as cell division, movements, and mutual information between sister cells. Conclusion: This work presents practical examples that highlight the abundant potential information within large sets of single-cell tracking data. Data reduction is crucial in the process of acquiring such information which can be relevant for phenotypic drug discovery and therapeutics, extending beyond standardized procedures. Conducting meaningful big data analysis typically necessitates a substantial amount of data, which can stem from standalone case studies as an initial foundation.

Single-Cell Tracking of A549 Lung Cancer Cells Exposed to a Marine Toxin Reveals Correlations in Pedigree Tree Profiles.

Long-term video-based tracking of single A549 lung cancer cells exposed to three different concentrations of the marine toxin yessotoxin (YTX) reveals significant variation in cytotoxicity, and it confirms the potential genotoxic effects of this toxin. Tracking of single cells subject to various toxic exposure, constitutes a conceptually simple approach to elucidate lineage correlations and sub-populations which are masked in cell bulk analyses. The toxic exposure can here be considered as probing a cell population for properties and change which may include long-term adaptation to treatments. Ranking of pedigree trees according to a measure of "size," provides definition of sub-populations. Following single cells through generations indicates that signaling cascades and experience of mother cells can pass to their descendants. Epigenetic factors and signaling downstream lineages may enhance differences between cells and partly explain observed heterogeneity in a population. Signaling downstream lineages can potentially link a variety of observations of cells making resulting data more suitable for computerized treatment. YTX exposure of A549 cells tends to cause two main visually distinguishable classes of cell death modalities ("apoptotic-like" and "necrotic-like") with approximately equal frequency. This special property of YTX enables estimation of correlation between cell death modalities for sister cells indicating impact downstream lineages. Hence, cellular responses and adaptation to treatments might be better described in terms of effects on pedigree trees rather than considering cells as independent entities.

Mitotic Catastrophe in BC3H1 Cells following Yessotoxin Exposure.

The marine toxin yessotoxin (YTX) can cause various cytotoxic effects depending on cell type and cell line. It is well known to trigger distinct mechanisms for programmed cell death which may overlap or cross-talk. The present contribution provides the first evidence that YTX can cause genotoxicity and induce mitotic catastrophe which can lead to different types of cell death. This work also demonstrates potential information gain from non-intrusive computer-based tracking of many individual cells during long time. Treatment of BC3H1 cells at their exponential growth phase causes atypical nuclear alterations and formation of giant cells with multiple nuclei. These are the most prominent morphological features of mitotic catastrophe. Giant cells undergo slow cell death in a necrosis-like manner. However, apoptotic-like cell death is also observed in these cells. Electron microscopy of treated BC3H1 cells reveal uncondensed chromatin and cells with double nuclei. Activation of p-p53, p-H2AX, p-Chk1, p-ATM, and p-ATR and down-regulation of p-Chk2 indicate DNA damage response and cell cycle deregulation. Micronuclei formation further support this evidence. Data from tracking single cells reveal that YTX treatment suppresses a second round of cell division in BC3H1 cells. These findings suggest that YTX can induce genomic alterations or imperfections in chromosomal segregation leading to permanent mitotic failure. This understanding extends the list of effects from YTX and which are of interest to control cancer and tumor progression.

Computer-assisted image processing to detect spores from the fungus Pandora neoaphidis.

This contribution demonstrates an example of experimental automatic image analysis to detect spores prepared on microscope slides derived from trapping. The application is to monitor aerial spore counts of the entomopathogenic fungus Pandora neoaphidis which may serve as a biological control agent for aphids. Automatic detection of such spores can therefore play a role in plant protection. The present approach for such detection is a modification of traditional manual microscopy of prepared slides, where autonomous image recording precedes computerised image analysis. The purpose of the present image analysis is to support human visual inspection of imagery data - not to replace it. The workflow has three components:•Preparation of slides for microscopy.•Image recording.•Computerised image processing where the initial part is, as usual, segmentation depending on the actual data product. Then comes identification of blobs, calculation of principal axes of blobs, symmetry operations and projection on a three parameter egg shape space.

Autophagic activity in BC3H1 cells exposed to yessotoxin.

The marine toxin yessotoxin (YTX) can induce programmed cell death through both caspase-dependent and -independent pathways in various cellular systems. It appears to stimulate different forms of cellular stress causing instability among cell death mechanisms and making them overlap and cross-talk. Autophagy is one of the key pathways that can be stimulated by multiple forms of cellular stress which may determine cell survival or death. The present work evaluates a plausible link between ribotoxic stress and autophagic activity in BC3H1 cells treated with YTX. Such treatment produces massive cytoplasmic compartments as well as double-membrane vesicles termed autophagosomes which are typically observed in cells undergoing autophagy. The observed autophagosomes contain a large amount of ribosomes associated with the endoplasmic reticulum (ER). Western blotting analysis of Atg proteins and detection of the autophagic markers LC3-II and SQSTM1/p62 by flow cytometry and immunofluorescence verified autophagic activity during YTX-treatment. The present work supports the idea that autophagic activity upon YTX exposure may represent a response to ribotoxic stress.

Lifetime Distributions from Tracking Individual BC3H1 Cells Subjected to Yessotoxin.

This work shows examples of lifetime distributions for individual BC3H1 cells after start of exposure to the marine toxin yessotoxin (YTX) in an experimental dish. The present tracking of many single cells from time-lapse microscopy data demonstrates the complexity in individual cell fate and which can be masked in aggregate properties. This contribution also demonstrates the general practicality of cell tracking. It can serve as a conceptually simple and non-intrusive method for high throughput early analysis of cytotoxic effects to assess early and late time points relevant for further analyzes or to assess for variability and sub-populations of interest. The present examples of lifetime distributions seem partly to reflect different cell death modalities. Differences between cell lifetime distributions derived from populations in different experimental dishes can potentially provide measures of inter-cellular influence. Such outcomes may help to understand tumor-cell resistance to drug therapy and to predict the probability of metastasis.

Assessment of potential transport of pollutants into the Barents Sea via sea ice--an observational approach.

The present estimates of ice drift in the Arctic include utilization of satellite imagery data (special sensor microwave/imager) and a reconstruction of air pressure for the period 1899-1998. A significant part of the sea ice in the Arctic Ocean has its origin in the Kara Sea and melts in the Greenland and the Barents Sea (BS). Consequently there may be a particular risk of pollutants in the Kara Sea entering the food webs of the Greenland and BS. The ice export from the Kara Sea between 1988 and 1994 was about 208,000 km2 (154 km3) per year. The import of ice into the BS was during the same period 161,000 km2 (183 km3) per year while the ice drift through the Fram Strait into the Greenland Sea was 583,000 km2 (1859 km3) per year. Ice which formed adjacent to the Ob and Yenisey rivers in early January, drifted into the BS within two years (with a probability of about 50%.