Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

A dataset of images and morphological profiles of 30 000 small-molecule treatments using the Cell Painting assay.

Background: Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. Findings: This microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at "The Cell Image Library" (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied. Conclusions: Because computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics.

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes.

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, which is a morphological profiling assay that multiplexes six fluorescent dyes, imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multiwell plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Next, an automated image analysis software identifies individual cells and measures ∼1,500 morphological features (various measures of size, shape, texture, intensity, and so on) to produce a rich profile that is suitable for the detection of subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes 2 weeks; feature extraction and data analysis take an additional 1-2 weeks.

Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay.

BACKGROUND: The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder. OBJECTIVES: To investigate SOX10 as a potential biomarker for melanoma and vitiligo. METHODS: In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes. RESULTS: The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically. CONCLUSIONS: We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

Toward performance-diverse small-molecule libraries for cell-based phenotypic screening using multiplexed high-dimensional profiling.

High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.

Multiplex cytological profiling assay to measure diverse cellular states.

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

Expression and functional role of sprouty-2 in breast morphogenesis.

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.

Sensitive detection of Aß protofibrils by proximity ligation--relevance for Alzheimer's disease.

BACKGROUND: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aß peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aß protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. RESULTS: For specific detection of Aß protofibrils we have used a monoclonal antibody, mAb158, selective for Aß protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aß aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aß protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aß protofibril detection by up to 25-fold. The assay was used to measure soluble Aß aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aß aggregation. CONCLUSIONS: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aß aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells.

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo. Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice. The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.

Sensitive plasma protein analysis by microparticle-based proximity ligation assays.

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

Use of proximity ligation to screen for inhibitors of interactions between vascular endothelial growth factor A and its receptors.

BACKGROUND: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. METHODS: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. RESULTS: The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. CONCLUSIONS: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus.

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.

In vitro analysis of DNA-protein interactions by proximity ligation.

Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.

Proximity ligation: a specific and versatile tool for the proteomic era.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

Autocrine/paracrine platelet-derived growth factor regulates proliferation of neural progenitor cells.

Growth factors play an important role in regulating neural stem cell proliferation and differentiation. This study shows that platelet-derived growth factor (PDGF) induces a partial differentiation of neural stem/progenitor cells (NSPCs) in the absence of other mitogens in vitro. NSPCs thus acquire an immature morphology and display markers for both neurons and glia. In addition, these cells do not readily mature in the absence of further stimuli. When NSPC cultures treated with PDGF were exposed to additional differentiation factors, however, the differentiation proceeded into neurons, astrocytes, and oligodendrocytes. We find that NSPC cultures are endowed with an endogenous PDGF-BB production. The PDGF-BB expression peaks during early differentiation and is present both in cell lysates and in conditioned medium, allowing for autocrine as well as paracrine signaling. When the NSPC-derived PDGF was inhibited, progenitor cell numbers decreased, showing that PDGF is involved in NSPC expansion. Addition of a PDGF receptor (PDGFR) inhibitor resulted in a more rapid differentiation. Neurons and oligodendrocytes appeared earlier and had more elaborate processes than in control cultures where endogenous PDGFR signaling was not blocked. Our observations point to PDGF as an inducer of partial differentiation of NSPC that also sustains progenitor cell division. Such an intermediate stage in stem cell differentiation is of relevance for the understanding of brain tumor development because autocrine PDGF stimulation is believed to drive malignant conversion of central nervous system progenitor cells.

Detection of individual microbial pathogens by proximity ligation.

BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity. METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents. RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium. CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes.

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.

Cytokine detection by antibody-based proximity ligation.

Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.

Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities.

Platelet-derived growth factor-B (PDGFB) is necessary for normal cardiovascular development, but the relative importance of different cellular sources of PDGFB has not been established. Using Cre-lox techniques, we show here that genetic ablation of Pdgfb in endothelial cells leads to impaired recruitment of pericytes to blood vessels. The endothelium-restricted Pdgfb knockout mutants also developed organ defects including cardiac, placental and renal abnormalities. These defects were similar to those observed in Pdgfb null mice. However, in marked contrast to the embryonic lethality of Pdgfb null mutants, the endothelium-specific mutants survived into adulthood with persistent pathological changes, including brain microhemorrhages, focal astrogliosis, and kidney glomerulus abnormalities. This spectrum of pathological changes is reminiscent of diabetic microangiopathy, suggesting that the endothelium-restricted Pdgfb knockouts may serve as models for some of the pathogenic events of vascular complications to diabetes.