High-content Fluorescence Microscopy Research Articles

Optimizing miRNA transfection for screening in precision cut lung slices.

Precision cut lung slices(PCLS) are complex 3D lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules(e.g. miRNA). The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. 4mm diameter PCLS were generated using agarose-filled rodent lungs and a vibratome. TYE665 labelled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualised using live high content fluorescence microscopy, followed by higher resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using WST-1 and lactate dehydrogenase(LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2TM, we established that cellular membranes in PCLS are permeable in the initial 24 hours of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 hours after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared to other LNPs, with detection possible as low as 25nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs.

Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

A versatile method for the identification of senolytic compounds.

The increased burden of senescent cells is as a well-established hallmark of aging and age-related diseases. This finding sparked significant interest in the identification of molecules capable of selectively eliminating senescent cells, so-called senolytics. Here, we fine-tuned a method for the identification of senolytics that is compatible with high-content fluorescence microscopy. We used spectral detector imaging to measure the emission spectrum of unlabeled control or senescent cells. We observed that senescent cells exhibited higher levels of autofluorescence than their non-senescent counterparts, particularly in the cytoplasmic region. Building on this result, we devised a senolytic assay based on co-culturing quiescent and senescent cells, fluorescently tagged in the nuclear region through the overexpression of H2B-GFP and H2B-RFP, respectively. We validated this approach by showing that first generation senolytics were effective in reducing the number of RFP+ nuclei leaving the count of GFP+ nuclei unaffected. The result was confirmed by flow cytometry analysis of nuclei isolated from these quiescent-senescent cell co-cultures. We found that this system enables to capture cell type-specific effects of senolytics as in the case of fisetin, which kills senescent Mouse Embryonic Fibroblasts but not senescent human melanoma SK-MEL-103 cells. This approach is amenable to genetic and chemical screening for the discovery of senolytic compounds in that it overcomes the limitations of current methods, which rely upon costly chemical reagents or fluorescence microscopy using cells labeled with fluorescent cytoplasmic probes that overlap with the autofluorescence signal emitted by senescent cells.

Cytokine-directed cellular cross-talk imprints synovial pathotypes in rheumatoid arthritis

IntroductionStructural reorganisation of the synovium with expansion of fibroblast-like synoviocytes (FLS) and influx of immune cells is a hallmark of rheumatoid arthritis (RA). Activated FLS are increasingly recognised as a...

Viability Analysis and High-Content Live-Cell Imaging for Drug Testing in Prostate Cancer Xenograft-Derived Organoids

Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. Moreover, most drug tests are restricted to whole-well viability as the sole read-out, thereby losing important information about key biological aspects that might be impacted due to the use of administered drugs. These bulk read-outs also discard potential inter-organoid heterogeneity in drug responses. To tackle these issues, we developed a systematic approach for processing organoids from prostate cancer (PCa) patient-derived xenografts (PDXs) for viability-based drug testing and identified essential conditions and quality checks for consistent results. In addition, we generated an imaging-based drug testing procedure using high-content fluorescence microscopy in living PCa organoids to detect various modalities of cell death. Individual organoids and cell nuclei in organoids were segmented and quantified using a dye combination of Hoechst 33342, propidium iodide and Caspase 3/7 Green, allowing the identification of cytostatic and cytotoxic treatment effects. Our procedures provide important insights into the mechanistic actions of tested drugs. Moreover, these methods can be adapted for tumor organoids originating from other cancer types to increase organoid-based drug test validity, and ultimately, accelerate clinical implementation.

Preparing Planarian Cells for High-Content Fluorescence Microscopy Using RNA in Situ Hybridization and Immunocytochemistry.

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. Here we describe a modular collection of assays adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. These include protocols for RNA fluorescent in situ hybridization (RNA FISH) as well as immunocytochemical protocols for quantifying proliferating cells targeting phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine(BrdU) incorporated into the nuclear DNA. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single-cell suspension before fixation and staining. By sharing many reagents with established planarian whole-mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment.

MODL-15. High-throughput combination drug screening identifies synergism between retinoic acid treatment and BCL-XL-inhibition in MYC(N)-driven medulloblastoma and neuroblastoma models

Abstract PURPOSE: Despite remarkable improvements in childhood cancer survival, certain high-risk entities including malignancies of the nervous system still carry dismal prognoses. Retinoic acid (RA) treatment has long been administered in high-risk neuroblastoma patients and preclinical data suggest a benefit in applying retinoids in selected brain tumor entities. However, limited single-agent efficacy and developing treatment resistance provide a rationale for investigating improved combination treatments. METHODS: We assessed the RA sensitivity of 16 cell lines and patient-derived cultures of pediatric nervous system tumors from the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) registry. Bulk RNA sequencing was performed for five sensitive models undergoing RA treatment. All models were screened for drug sensitivity to 76 clinically relevant drugs alone and combined with RA. Beneficial hit combinations were further investigated for synergy, as well as their effect on differentiation and apoptosis by high-content fluorescence microscopy and caspase activity assay. Results from in vivo experiments are pending. RESULTS: Group 3 medulloblastoma and high-risk neuroblastoma models were most sensitive to RA treatment leading to significant changes in gene expression of apoptotic regulation. Drug sensitivity screening revealed similar patterns of response to combination treatments in MYC-driven Group 3 medulloblastomas and MYCN-amplified neuroblastomas. Apoptosis regulating BCL-XL-inhibitors (A-1155463, navitoclax) were identified as top hits across RA sensitive models, whereas epigenetic BET family inhibitor I-BET151 and histone deacetylase inhibitor entinostat suggested entity-specific combination benefits in neuroblastoma and medulloblastoma models, respectively. In both entities, combining RA with navitoclax was synergistic and significantly increased caspase-3 activity. The combination led to a morphological shift from differentiation under RA treatment to cell death. CONCLUSION: Exploring combinations for RA treatment in pediatric nervous system tumors, we found that MYC(N)-driven medulloblastoma and neuroblastoma not only share responsiveness to RA treatment, but also demonstrate enhanced vulnerability in combination with BCL-XL-inhibition resulting in increased apoptosis.

Pex14p Phosphorylation Modulates Import of Citrate Synthase 2 Into Peroxisomes in Saccharomyces cerevisiae.

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.

Computational structured illumination for high-content fluorescence and phase microscopy.

High-content biological microscopy targets high-resolution imaging across large fields-of-view (FOVs). Recent works have demonstrated that computational imaging can provide efficient solutions for high-content microscopy. Here, we use speckle structured illumination microscopy (SIM) as a robust and cost-effective solution for high-content fluorescence microscopy with simultaneous high-content quantitative phase (QP). This multi-modal compatibility is essential for studies requiring cross-correlative biological analysis. Our method uses laterally-translated Scotch tape to generate high-resolution speckle illumination patterns across a large FOV. Custom optimization algorithms then jointly reconstruct the sample's super-resolution fluorescent (incoherent) and QP (coherent) distributions, while digitally correcting for system imperfections such as unknown speckle illumination patterns, system aberrations and pattern translations. Beyond previous linear SIM works, we achieve resolution gains of 4× the objective's diffraction-limited native resolution, resulting in 700 nm fluorescence and 1.2 μm QP resolution, across a FOV of , giving a space-bandwidth product (SBP) of 60 megapixels.

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae).

Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High-content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.

A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression.Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes.Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism (“energetic stress”).The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control.To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses.

Evaluation of the use of imaging parameters for the detection of compound-induced hepatotoxicity in 384-well cultures of HepG2 cells and cryopreserved primary human hepatocytes

Drug-induced liver injury (DILI) is a major cause of failed drug development, withdrawal and restricted usage. Therefore screening assays which aid selection of candidate drugs with reduced propensity to cause DILI are required. We have investigated the toxicity of 144 drugs, 108 of which caused DILI, using assays identified in the literature as having some predictivity for hepatotoxicity. The validated assays utilised either HepG2 cells, HepG2 cells in the presence of rat S9 fraction or isolated human hepatocytes. All parameters were quantified by multiplexed and automated high content fluorescence microscopy, at appropriate time points after compound administration (4, 24 or 48h). The individual endpoint which identified drugs that caused DILI with greatest precision was maximal fold induction in CM-H2DFFDA staining in hepatocytes after 24h (41% sensitivity, 86% specificity). However, hierarchical clustering analysis of all endpoints provided the most sensitive identification of drugs which caused DILI (58% sensitivity, 75% specificity). We conclude that multi-parametric high content cell toxicity assays can enable in vitro detection of drugs that have high propensity to cause DILI in vivo but that many DILI compounds exhibit few in vitro signals when evaluated using these assays.

MiRNAs to Regenerate the Heart

### Study Hypothesis MicroRNAs (miRNAs) are small nucleic acid products that simultaneously modulate expression levels of several different genes by partial complementation, leading to posttranscriptional regulation. Among their functions, miRNAs have been shown to regulate cardiac development and differentiation. During heart development, immature cardiomyocytes proliferate actively to accommodate increasing heart size and function; however, this proliferation is abruptly abrogated shortly after birth, leaving the heart with a limited regenerative capacity insufficient to replace substantial amounts of tissue lost after injury. In this article, Eulalio et al hypothesize that neonatal proliferation capacity can be reactivated in adult cardiomyocytes by the exogenous administration of selected miRNAs, leading to cardiac repair. ### How Was the Hypothesis Tested? The authors used a synthetic whole-genome human miRNA library to induce individual expression of miRNAs in neonatal cardiomyocytes. To assess the effect of the miRNA mimics, they immunostained transfected cells for cardiac-specific proteins together with markers of DNA synthesis and proliferation. High-content fluorescence microscopy enabled quantification of proliferating cardiomyocytes and selection of the miRNAs with the highest potential to induce cell cycle reentry. To ensure that DNA replication led to nuclear division (karyokinesis) and cytoplasm division (cytokinesis), characteristic markers for late mitosis and …

Deciphering cellular morphology and biocompatibility using polymer microarrays

A quantitative and qualitative analysis of cellular adhesion, morphology and viability is essential in understanding and designing biomaterials such as those involved in implant surfaces or as tissue-engineering scaffolds. As a means to simultaneously perform these studies in a high-throughput (HT) manner, we report a normalized protocol which allows the rapid analysis of a large number of potential cell binding substrates using polymer microarrays and high-content fluorescence microscopy. The method was successfully applied to the discovery of optimal polymer substrates from a 214-member polyurethane library with mouse fibroblast cells (L929), as well as simultaneous evaluation of cell viability and cellular morphology. Analysis demonstrated high biocompatibility of the binding polymers and permitted the identification of several different cellular morphologies, showing that specific polymer interactions may provoke changes in cell shape. In addition, SAR studies showed a clear correspondence between cellular adhesion and polymer structure. The approach can be utilized to perform multiple experiments (up to 1024 single experiments per slide) in a highly reproducible manner, leading to the generation of vast amounts of data in a short time period (48–72 h) while reducing dramatically the quantities of polymers, reagents and cells used.