High-content Analysis Approach Research Articles

Novel immortalization approach defers senescence of cultured canine adipose-derived mesenchymal stromal cells.

In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery, and increased activity of the senescence-associated β-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac™ Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.

RNAi-Screening in Knochenbildenden Zellen

Osteoporosis, the condition of decreased bone density resulting in bone porosity, brittleness, and a increased fracture risk is rising with aging. Current treatments slow-down bone loss but cannot increase bone formation and quality. Using osteoblasts–the bone forming cells–and RNAi libraries, we developed a high-content analysis approach which identified osteoblast modulators. Several among them are potential drug targets, to induce increase in bone formation thus efficiently combating osteoporosis.

MAARS: a novel high-content acquisition software for the analysis of mitotic defects in fission yeast.

Faithful segregation of chromosomes during cell division relies on multiple processes such as chromosome attachment and correct spindle positioning. Yet mitotic progression is defined by multiple parameters, which need to be quantitatively evaluated. To study the spatiotemporal control of mitotic progression, we developed a high-content analysis (HCA) approach that combines automated fluorescence microscopy with real-time quantitative image analysis and allows the unbiased acquisition of multiparametric data at the single-cell level for hundreds of cells simultaneously. The Mitotic Analysis and Recording System (MAARS) provides automatic and quantitative single-cell analysis of mitotic progression on an open-source platform. It can be used to analyze specific characteristics such as cell shape, cell size, metaphase/anaphase delays, and mitotic abnormalities including spindle mispositioning, spindle elongation defects, and chromosome segregation defects. Using this HCA approach, we were able to visualize rare and unexpected events of error correction during anaphase in wild-type or mutant cells. Our study illustrates that such an expert system of mitotic progression is able to highlight the complexity of the mechanisms required to prevent chromosome loss during cell division.

Modulation of CYP3A4 activity alters the cytotoxicity of lipophilic phycotoxins in human hepatic HepaRG cells

The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in human HepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72h treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and γ-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity.

Automation Highlights from the Literature

Automation Highlights from the Literature

Structural profiling and biological performance of phospholipid–hyaluronan functionalized single-walled carbon nanotubes

In spite of significant insolubility and toxicity, carbon nanotubes (CNTs) erupt into the biomedical research, and create an increasing interest in the field of nanomedicine. Single-walled CNTs (SWCNTs) are highly hydrophobic and have been shown to be toxic while systemically administrated. Thus, SWCNTs have to be functionalized to render water solubility and biocompatibility. Herein, we introduce a method for functionalizing SWCNT using phospholipids (PL) conjugated to hyaluronan (HA), a hydrophilic glycosaminoglycan, with known receptors on many types of cancer and immune cells. This functionalization allowed for CNT solubilization, endowed the particles with stealth properties evading the immune system, and reduced immune and mitochondrial toxicity both in vitro and in vivo. The CNT–PL–HA internalized into macrophages and showed low cytotoxicity. In addition, CNT–PL–HA did not induce an inflammatory response in macrophages as evidenced by the cytokine profiling and the use of image-based high-content analysis approach in contrast to non-modified CNTs. In addition, systemic administration of CNT–PL–HA into healthy C57BL/6 mice did not alter the total number of leukocytes nor increased liver enzyme release as opposed to CNTs. Taken together, these results suggest an immune protective mechanism by the PL–HA coating that could provide future therapeutic benefit.

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

BackgroundEpidermal Growth Factor (EGF) plays an important function in the regulation of cell growth, proliferation, and differentiation by binding to its receptor (EGFR) and providing cancer cells with increased survival responsiveness. Signal transduction carried out by EGF has been extensively studied at both transcriptional and post-transcriptional levels. Little is known about the involvement of microRNAs (miRNAs) in the EGF signaling pathway. miRNAs have emerged as major players in the complex networks of gene regulation, and cancer miRNA expression studies have evidenced a direct involvement of miRNAs in cancer progression.ResultsIn this study, we have used an integrative high content analysis approach to identify the specific miRNAs implicated in EGF signaling in HeLa cells as potential mediators of cancer mediated functions. We have used microarray and deep-sequencing technologies in order to obtain a global view of the EGF miRNA transcriptome with a robust experimental cross-validation. By applying a procedure based on Rankprod tests, we have delimited a solid set of EGF-regulated miRNAs. After validating regulated miRNAs by reverse transcription quantitative PCR, we have derived protein networks and biological functions from the predicted targets of the regulated miRNAs to gain insight into the potential role of miRNAs in EGF-treated cells. In addition, we have analyzed sequence heterogeneity due to editing relative to the reference sequence (isomiRs) among regulated miRNAs.ConclusionsWe propose that the use of global genomic miRNA cross-validation derived from high throughput technologies can be used to generate more reliable datasets inferring more robust networks of co-regulated predicted miRNA target genes.

Abstract 3558: Single cell-based image analysis of nuclear receptor levels and activity in breast cancer cells reveals heterogeneous subpopulations: Development of biological response fingerprints for ligands and endocrine disruptors.

Abstract Modern microscopy- and flow cytometry-based analyses indicate tumor cell populations can be markedly heterogeneous. Yet, the vast majority of approaches used to investigate tumorigenic mechanisms and therapeutic responses are based upon average single-point analysis of bulk populations of cells, where subpopulation responses are undetectable and therefore unexplored. For example, breast cancers are generally classified by IHC as either estrogen receptor-alpha positive (ER+) or negative (ER-); however, in ER+ tumors, there is a broad range of ER levels in individual cells. Despite a growing awareness of the importance of tumor cell heterogeneity and outlier biology in cancer therapeutics, there is a paucity of data on the potential interplay between multiple nuclear receptors and coregulators, their heterogeneity of expression and functions, and their combinatorial role in determining therapeutic response, or in sensing environmental stressors (e.g., endocrine disruptors). To specifically quantify biological responses on an individual cell basis rather than averaged responses from bulk populations, we performed single cell-based high content analysis (HCA) to examine MCF-7 breast cancer cells in terms of simultaneous expression of several type-1 nuclear receptors (e.g., Estrogen, Androgen and Glucocorticoid receptors - ER, AR and GR) and coregulators (e.g., SRC-2 and SRC-3). Quantitative immunofluorescence reveals a wide range of heterogeneity in the expression and ratios of ER, AR, and GR. Multi-parametric analyses describing NR expression and spatial distribution were used to evaluate ligand dose responses and time pattern of the three nuclear receptors, simultaneously. Next, we are expanding our analysis to endogenous target gene expression via mRNA fluorescence in situ hybridization (RNA FISH) and to cell cycle marker analysis to link heterogeneity of NRs/coregulators expression with transcriptional output and cell cycle phases. We are also implementing robust analysis pipelines to describe and quantify NRs and coregulators patterns of phenotypic heterogeneity to create “biological response fingerprints,” which we are using to classify the effects of individual or mixed ligand treatments with particular focus on chemotherapeutic drugs and endocrine disruptors. We are also starting to expand our HCA approach to other breast cancer cell lines moving towards primary tumors microarrays analysis to evaluate the prognostic/diagnostic significance of NR heterogeneity. Collectively, these high throughput-amenable approaches are poised to greatly aid in the understanding the role of heterogeneity in breast cancer, and serve as a robust model for next-generation drug screening and endocrine disruptor assays moving toward personalized medicine. Citation Format: Fabio Stossi, Alexander Renwick, Radhika D. Dandekar, Justin Y. Newberg, Arvind Rao, Michael A. Mancini. Single cell-based image analysis of nuclear receptor levels and activity in breast cancer cells reveals heterogeneous subpopulations: Development of biological response fingerprints for ligands and endocrine disruptors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2013-3558

Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling.

Aims/hypothesisKnowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size.MethodsElectron micrographs of rat beta cells fixed either chemically or by high-pressure freezing were compared using a high-content analysis approach. These data were used to develop three-dimensional in silico beta cell models, the slicing of which would reproduce the experimental datasets.ResultsAs previously reported, chemically fixed insulin secretory granules appeared as hollow spheres with a mean diameter of ∼350 nm. Remarkably, most granules fixed by high-pressure freezing lacked the characteristic halo between the dense core and the limiting membrane and were smaller than their chemically fixed counterparts. Based on our analyses, we conclude that the mean diameter of rat insulin secretory granules is 243 nm, corresponding to a surface area of 0.19 μm2. Rat beta cells have a mean volume of 763 μm3 and contain 5,000–6,000 granules.Conclusions/interpretationA major reason for the lower mean granule number/rat beta cell relative to previous accounts is a reduced estimation of the mean beta cell volume. These findings imply that each granule contains about twofold more insulin, while its exocytosis increases membrane capacitance about twofold less than assumed previously. Our integrated approach defines new standards for quantitative image analysis of beta cells and could be applied to other cellular systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2438-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a highcontent analysis approach

Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleicacids. Although most of them are considered safe, there is confusing evidencein the literature regarding their potential cellular toxicities. Moreover, little isknown about the recovery process cells undergo after a cytotoxic insult. We havepreviously studied the systemic effects of common LNPs with different surfacecharge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by actingas an agonist of Toll-like receptor 4 on immune cells. In this study, we focusedon the response of human fibroblasts exposed to LNPs and their cellularrecovery process. To this end, we used image-based high content analysis(HCA). Using this strategy, we were able to show simultaneously, in severalintracellular parameters, that fibroblasts can recover from the cytotoxic effects of(+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicityand may become a valuable tool for screening safe materials for drug delivery and tissueengineering.

Laser scanning cytometry for automation of the micronucleus assay

Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure.

Androgen Receptor Mutations Associated with Androgen Insensitivity Syndrome: A High Content Analysis Approach Leading to Personalized Medicine

Androgen insensitivity syndrome (AIS) is a rare disease associated with inactivating mutations of AR that disrupt male sexual differentiation, and cause a spectrum of phenotypic abnormalities having as a common denominator loss of reproductive viability. No established treatment exists for these conditions, however there are sporadic reports of patients (or recapitulated mutations in cell lines) that respond to administration of supraphysiologic doses (or pulses) of testosterone or synthetic ligands. Here, we utilize a novel high content analysis (HCA) approach to study AR function at the single cell level in genital skin fibroblasts (GSF). We discuss in detail findings in GSF from three historical patients with AIS, which include identification of novel mechanisms of AR malfunction, and the potential ability to utilize HCA for personalized treatment of patients affected by this condition.

A new mechanism of gastric epithelial injury induced by acid exposure: The role of Egr‐1 and ERK signaling pathways

The molecular mechanisms by which gastric acid causes epithelial injury in the stomach and initiates an inflammatory reaction are poorly understood. We aimed in the present study to investigate the role of the early growth response gene Egr-1 and ERK in gastric epithelial cells following acid exposure, and the signaling pathways involved. Western blotting was used to assess Egr-1 protein levels in AGS cells. A quantitative measurement of acid-induced Egr-1 and ERK translocation was performed using a high content analysis approach. Egr-1 functionality was assessed by transient transfection with Egr-1 antisense oligonucleotide. Exposure of AGS cells to acidic conditions induced Egr-1 protein expression in a pH- and time-dependent manner. Egr-1 expression was markedly increased as the pH was reduced from pH 7.4 to 6.4. High content analysis of Egr-1 activation showed acid-induced Egr-1 nuclear translocation; a maximum observed at 1-2 h followed by a decline to basal levels beyond 4 h. Acidic pH also activated ERK1/2 phosphorylation, whereas ERK1/2 inhibitors PD98059 and U0216 blocked both acid-induced Egr-1 and ERK translocation and expression. Moreover, acid exposure up-regulated VEGF expression, which was inhibited by the Egr-1 antisense oligonucleotide. Our results also demonstrate that exposure to acid induces Egr-1 via MEK-ERK1/2 pathway. These data suggest that Egr-1 activation might play a crucial role in gastric mucosal inflammation and associated epithelial injury.

The use of Cellomics to study enterocyte cytoskeletal proteins in coeliac disease patients

Abstract Coeliac disease is characterised by inflammation of small intestinal mucosa accompanied by abnormal villous architecture. It is now accepted that some patients with positive coeliac serology tests may have minor mucosal lesions that may not be apparent on routine histopathological analysis. The aim of the study was to perform detailed examination of enterocyte morphology and cytoskeletal structures using a high content analysis technology. Duodenal biopsies from 14 untreated and 10 treated coeliac patients and from 20 non-coeliac controls were examined. Tissue sections from six patients (study group subjects) before and after the development of gluten-sensitive enteropathy were also investigated. Immunohistochemical studies were performed on paraffin-embedded sections using an anti-α-tubulin antibody. Significant differences in enterocyte morphology and intracellular cytoskeletal structures were demonstrated in patients with proven coeliac disease and in the study group subjects. These changes were present in study group biopsies before evidence of enteropathy, as assessed by routine microscopy. This is the first study to demonstrate detailed characteristics of enterocyte morphology in coeliac patients using a high content analysis approach. The use of this technology allows a quantitative analysis of enterocyte intracellular structures from routine biopsy material and permits detection of subtle changes that precede the characteristic histological lesion.

Multiparametric Cell Cycle Analysis by Automated Microscopy

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.