Automated Fluorescence Microscopy Research Articles

Design, Synthesis, and Bioactivation of O-Glycosylated Prodrugs of the Natural Nitric Oxide Precursor N(ω)-Hydroxy-l-arginine.

Naturally occurring N(ω)-hydroxy-l-arginine (NOHA, 1) is the best substrate of NO synthases (NOS). The development of stable and bioavailable prodrugs would provide a pharmacologically valuable strategy for the treatment of cardiovascular diseases that are associated with endothelial dysfunction. To improve NOHAs druglike properties, we demonstrate that O-substitution by (glycosylic) acetal formation greatly increased the chemical stability of the hydroxyguanidine moiety and provided a nontoxic group that could be easily bioactivated by glycosidases. A straightforward synthetic concept was devised and afforded a series of diversely substituted prodrugs by O-conjugation of the hydroxyguanidine moiety with different monosaccharides. Systematic exploration of their bioactivation profile revealed that glucose-based prodrugs were more efficiently bioactivated than their galactose counterparts. NOS-dependent cytosolic NO release was quantified by automated fluorescence microscopy in a cell-based assay with murine macrophages. Glucose-based prodrugs performed particularly well and delivered cellular NO levels comparable to 1, demonstrating proof-of-concept.

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB)

Purpose: Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise.Materials and methods: Whole blood and separated lymphocyte samples were homogenously irradiated with 60Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples.Results: Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty.Conclusions: This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response.

The Synthesis of L-Alanyl and β-Alanyl Derivatives of 2-Aminoacridone and Their Application in the Detection of Clinically-Important Microorganisms.

In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445–450 nm for the substrate, λem 550 nm for the product). Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino)-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our β-alanyl aminopeptidase substrate, 2-(N- β-alanylamino)-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.

High-Content Screening for Quantitative Cell Biology.

High-content screening (HCS), which combines automated fluorescence microscopy with quantitative image analysis, allows the acquisition of unbiased multiparametric data at the single cell level. This approach has been used to address diverse biological questions and identify a plethora of quantitative phenotypes of varying complexity in numerous different model systems. Here, we describe some recent applications of HCS, ranging from the identification of genes required for specific biological processes to the characterization of genetic interactions. We review the steps involved in the design of useful biological assays and automated image analysis, and describe major challenges associated with each. Additionally, we highlight emerging technologies and future challenges, and discuss how the field of HCS might be enhanced in the future.

Shear Stress Modulates Endothelial VCAM‐1 Expression via Endoplasmic Reticulum Stress Response Pathways

IntroductionComplications from atherosclerosis remain the leading cause of death and disability in the U.S. This inflammatory disease is characterized by the accumulation of lipid‐rich plaques within the artery wall at predictable locations, which correlate strongly with the local arterial geometry and associated hemodynamic environment, notably low magnitude and reversing fluid shear stress (SS). Mechano‐responsive endothelial cells (EC) moderate vascular inflammation, including cell adhesion molecule (CAM) presentation and subsequent leukocyte recruitment. Endoplasmic reticulum (ER) stress and associated responses have emerged as signaling pathways linking cellular stress to inflammation and cardiovascular risk. Chronic activation of the ER stress response in endothelium has been observed in regions of disturbed flow in the arteries of lesion free swine, and in response to disturbed flow in cultured EC, demonstrating its importance as a possible signature of endothelial dysfunction and atherosusceptibility. The objective of this study was to determine the extent to which ER stress serves as a primary signaling pathway by which SS mediates inflammatory responses in endothelium of functional importance to atherosclerosis.MethodsTo recapitulate flow characteristics of the atherogenic niche in vivo, human aortic EC (HAEC) were exposed to a linear gradient of SS (0 to 16 dynes/cm2) in a microfluidic device and stimulated with the pro‐inflammatory cytokine TNFα in the presence and absence of the ER stress inhibitor 4‐phenylbutyric acid (PBA) for 4 hr. ER morphology was examined by immunofluorescence staining for the ER luminal protein calreticulin. Expression of VCAM‐1, ICAM‐1, and the ER stress sensors eIF2α, XBP1, and ATF6 were examined using automated fluorescence microscopy. HAEC were transfected with shRNA against eIF2α, XBP1, or ATF6 to assess each pathway's contribution to VCAM‐1 expression. To determine if the changes in VCAM‐1 were of functional importance, isolated human monocytes were sheared over HAEC treated as above and adhesion events quantified.ResultsTNFα‐induced VCAM‐1 expression varied significantly with SS magnitude, peaking at ~3‐fold of static expression at 2 dynes/cm2. Peak VCAM‐1 expression under low magnitude SS (LSS) correlated with an increase in the CV for calreticulin pixel intensity, a marker for ER expansion and activity. PBA abrogated the peak in VCAM‐1 expression under LSS, effectively normalizing its expression over the entire gradient, without affecting ICAM‐1 expression. Peak VCAM‐1 expression under LSS was abrogated by knock‐down of eIF2α or XBP1, but not ATF6. At 2 dynes/cm2, there was a 78% decrease in monocytes adhered to the monolayer treated with PBA compared to TNFα alone.ConclusionsThese data support a primary role for ER stress response pathways in the regulation of cytokine‐induced VCAM‐1 expression by SS. This study enhances our understanding of the mechanisms by which SS locally modulates EC inflammatory phenotype. Further elucidation of the pathways involved may have important ramifications for understanding and managing atherosclerosis.Support or Funding InformationSupported by National Institute of Health grant HL082689 (SIS and AGP)

Screening for biofilm formation of E. coli pathotypes and determination of the effect of antimicrobial peptides on biofilm formation by automated fluorescence microscopy

Screening for biofilm formation of E. coli pathotypes and determination of the effect of antimicrobial peptides on biofilm formation by automated fluorescence microscopy

Abstract P2-02-11: Detection of activating estrogen receptor 1 (ESR1) mutation on single circulating tumor cells from metastatic breast cancer patients

Abstract Background: 65% of primary breast cancers express the estrogen receptor α (ERα) and the mainstay of treatment are therapies that result in selective estrogen receptor modulation (SERM) of estrogen deprivation (aromatase inhibitors, AIs). Even thought endocrine therapy resulted in reduced recurrence and mortality, a significant portion of patients relapse with a metastatic disease and subsequently progress while of therapy for advanced disease (endocrine resistance). Recent evidence showed that activating hot spot mutation in the ligand binding domain of the ERα are acquired on treatment (frequency of 20%) and can drive resistance to endocrine therapy. Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material and the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies. These features suggest that the detection of ESR1 mutation on single CTC may be a useful biomarker for therapy guidance. Purpose: Investigate the incidence and heterogeneity of ESR1 mutational status within single CTCs isolated from individual metastatic breast cancer patients (mBCs), combining the FDA approved CellSearch® system for enumeration of CTCs with the DEPArrayTM technologies. Methods: CTCs were enriched and enumerate by CellSearch® in 7.5 ml blood samples collected from 21 mBCs according to standard protocol. Each CTC-enriched sample with at least 20 CTCs was recovered from Veridex cartridge and loaded into the DEPArrayTM A300K chip, since the DEPArrayTM analyzed only the 66% of the sample volume loaded, according to the manufacturer's instructions. The chip scanning was performed by automated fluorescence microscope. The loaded cells were recovered as single cell and subdivided in tree different group: Cytokeratin (CK) positive ( Dapi+, CK+, ER-, CD45-); ER positive (Dapi+, ER+, CK+, CD45-); White Blood cells (WBCs) (Dapi+, CD45+, CK-, ER-). Single CTCs and WBCs were then submitted to whole genome amplification (WGA) using the Single Cell WGA kit (Yikon Genomics) according the manufacturer's instructions. Detection of target 14 ESR1 hot spot mutations was performed on ABI PRISM® 3700 genetic analyzer by target Sanger sequencing. Results: 3 out of 21 mBCs with ≥20 CTCs were sorted and a total of 65 cells were recovered. WGA and ESR1 mutational status were performed on a total of 25 cells (respectively 11 ER+, 6 CK+ and 8 WBCs). In 1 of the 3 patients, that failed 2 lines of chemotherapy and previous single agent endocrine therapy, molecular heterogeneity was detected among its ER+ cells. 4 of 5 ER+ cells were heterozygote for the Y537S while one cell was homozygous, maybe due to a loss of heterozygosity. Y537S is one of the most common mutations that leads to a ligand independent ER transcriptional activity that does not respond to endocrine manipulation. No mutations were reported in all the CK+ and WBC cells analyzed. Conclusions: This study demonstrates the feasibility of a non-invasive approach based on liquid biopsy in mBCs. Evaluation of ER status and early identification of ESR1 mutation in ER+ CTCs might allow to predict effect of the endocrine therapies and switching to other treatments before the emergence of metastatic disease. Citation Format: Paolillo C, Mu Z, Austin L, Nguyen T, Capoluongo E, Fortina P, Cristofanilli M. Detection of activating estrogen receptor 1 (ESR1) mutation on single circulating tumor cells from metastatic breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-11.

Microwell-Based Live Cell Imaging of NK Cell Dynamics to Assess Heterogeneity in Motility and Cytotoxic Response.

NK cell heterogeneity has primarily been studied either on the population level, measuring average responses, or on the single cell level by flow cytometry, providing static snapshots. These approaches have certain drawbacks, not enabling dynamic observations of single cells over extended periods of time. One of the primary limitations of single cell imaging has been throughput; it has been challenging to collect data for many cells due to their dynamic nature and migrating out of the field of view. Spatially confining cells combined with automated fluorescence microscopy enables the simultaneous monitoring of many NK cells in parallel for extended periods of time (>12 h). Such an approach allows us to dissect how the sum of individual NK cell responses translates to the global average response typically observed.

Scrape Loading/Dye Transfer Assay.

The scrape loading/dye transfer (SL/DT) technique is a simple functional assay for the simultaneous assessment of gap junctional intercellular communication (GJIC) in a large population of cells. The equipment needs are minimal and are typically met in standard cell biology labs, and SL/DT is the simplest and quickest of all the assays that measure GJIC. This assay has also been adapted for in vivo studies. The SL/DT assay is also conducive to a high-throughput setup with automated fluorescence microscopy imaging and analysis to elucidate more samples in shorter time, and hence can serve a broad range of in vitro pharmacological and toxicological needs.

Valproate Disturbs Morphology and Mitochondrial Membrane Potential in Human Neural Cells

Abstract Valproic acid (VPA) is one of the main anticonvulsive drugs used worldwide for the treatment of epilepsy. Its use and clinical benefits are acknowledged; however, apparent effects of VPA on mitochondria vary among cell types and methods of analysis. Automated fluorescence microscopy offers stable, reproducible, and expandable assays useful for identifying mitochondrial responses to drugs. Here we have used human neural progenitor cells (NPCs) derived from embryonic stem cells to evaluate neural mitochondrial responses to VPA. We have analyzed the effects of different concentrations of VPA (from 0.01 to 1 mM) on mitochondria morphology and inferred membrane potential, as well as shape and content distribution. Our results show that following exposure to VPA the morphological and functional alterations observed in NPC mitochondria were consistent with prior reports for such effects. Changes detected include mitochondria shape from filamentous to globular and increases in mitochondrial membrane pote...

Abstract 5486: Identification of novel candidate genes associated with sorafenib cytotoxicity

Abstract Background: Sorafenib is an oral multikinase inhibitor that decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. Novel pathways and targets of sorafenib activity remain to be identified, and no predictive biomarkers of sorafenib activity exist to help guide clinicians. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains were genomically characterized previously (PMID: 21623374). MEFs were treated with ten concentrations (0-300 μM) of sorafenib, incubated for 72 h, and then fixed and stained. Cytochrome C was immunolabeled in the fixed cells and imaged using an automated fluorescence microscope. An image analysis software assessed sorafenib-induced cytochrome C release from mitochondria. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the EMMA algorithm, was performed on cytochrome C EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values ≤ 1.0×10−7 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 72 h (17.2-44.5 μM) sorafenib incubations. We identified one peak (chromosome 9 from 51-52 Mb; p = 1.0×10−8), which reached genome-wide significance and significantly associated with cytochrome C release. From this peak, we have identified candidate genes that may underlie variability in sorafenib-induced cytochrome C release from mitochondria. A total of nine genes expressed in MEF cells at mRNA level are present in this QTL. Of particular interest, the identified locus contains Rdx, a gene that encodes radixin. Radixin is a component of the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) complex, and is conserved between humans and mice. In humans, radixin, as part of the EBP50 complex, has been shown to act as a tumor suppressor and to promote apoptosis in hepatocellular carcinoma by modulating β-catenin/E-cadherin (PMID: 23483729). It has also been shown to potentially enhance the metastatic potential of renal cell carcinoma (PMID: 20395446). Conclusions: Our high-throughput cellular genetics approach has detected robust interstrain cellular differences in sorafenib activity, and identified one region, which reached genome-wide significance, that potentially associates with sorafenib-induced cytochrome C release from mitochondria. Functional validation of Rdx and other promising candidates is currently ongoing. Citation Format: Daniel James Crona, Oscar Suzuki, O. Joseph Trask, Bethany Parks, Amber Frick, Timothy Wiltshire, Federico Innocenti. Identification of novel candidate genes associated with sorafenib cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2015-5486

Assessment of modulated cytostatic drug resistance by automated γH2AX analysis.

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double-strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug-induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter-individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P-gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB-inducing agent etoposide (ETP) are known P-gp substrates. In this respect, modulation of MDR by P-gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB-positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB-inducing cytostatics..

Comprehensive assessment of germline chemical toxicity using the nematode Caenorhabditis elegans.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals. We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Quantitative analysis of transferrin cycling by automated fluorescence microscopy.

Surface receptors are transported between the plasma membrane and intracellular compartments by various endocytic mechanisms and by recycling via different pathways from sorting or recycling endosomes. The analysis of cellular components involved in mediating or regulating these transport steps is of high current interest and requires quantitative methods to determine rates of endocytosis and/or recycling. Various biochemical procedures to measure uptake of labeled ligand molecules or internalization and reappearance of surface-labeled receptors have been developed. Here, we describe a quantitative method based on fluorescence microscopy of adherent cells taking advantage of the transferrin (Tf) receptor as the prototype of cycling transport receptors. Tf is endocytosed with bound Fe(3+) and, upon release of the iron ion in endosomes, recycled as apo-Tf together with the receptor. To follow the ligand-receptor complex, fluorescently labeled Tf is used and detected microscopically with or without releasing Tf from cell surface receptors by acid stripping. To go beyond the observation of a few individual cells, automated fluorescence microscopy is employed to image thousands of cells at different time points and in parallel with different treatments (such as chemical inhibitors, siRNA silencing, or transfection of candidate genes) in a 96-well format. Computer-assisted image analysis allows unbiased quantitation of Tf content of each cell and to distinguish between different cell populations.

Clinical performance evaluation of a novel, automated chemiluminescent immunoassay, QUANTA Flash CTD Screen Plus.

The QUANTA Flash(®) CTD Screen Plus is a chemiluminescent immunoassay (CIA) for the detection of the major antinuclear antibodies (ANA) on the BIO-FLASH(®) platform. NOVA View(®) is an automated fluorescence microscope that acquires digital images of indirect immunofluorescent assay (IFA) slides. Our goal was to evaluate the clinical performance of the two automated systems and compare their performance to that of traditional IFA. Sera from patients with systemic autoimmune rheumatic diseases (SARD, n = 178), along with disease and healthy controls (n = 204), were tested with the CTD CIA and with NOVA Lite(®) HEp-2 ANA, using both the manual method of reading the IFA slides and the NOVA View instrument. The CTD CIA showed 78.1% sensitivity for SARD, coupled with 94.1% specificity. Manual IFA and NOVA View showed somewhat higher sensitivity (81.5 and 84.8% in SARD, respectively), but significantly lower specificity (79.4 and 64.7%, respectively). Both automated systems displayed somewhat different performance, due to the different principals of ANA detection: IFA with NOVA View digital image interpretation had higher sensitivity, while the CTD CIA showed higher specificity. With the added benefits of full automation, the new CTD CIA is an attractive alternative to traditional ANA screening.

Single-cell in situ imaging of palmitoylation in fatty-acylated proteins.

Dissecting the subcellular distribution of a fatty-acylated protein is key to the understanding of the molecular mechanisms regulating protein movement and function in a cell. This protocol describes how to perform single-cell imaging of palmitoylation in a fatty-acylated protein of interest with high sensitivity using click chemistry, proximity ligation and fluorescence microscopy. The initial steps in this protocol involve optimization of conditions for (i) metabolic incorporation of an alkynyl analog of palmitic acid into cellular proteins coupled with click chemistry and (ii) detecting a specific protein of interest with primary antibodies using automated fluorescence microscopy, followed by (iii) imaging palmitoylation of the target fatty-acylated protein of interest, such as Wnt, Sonic Hedgehog or H-Ras. Furthermore, we outline strategies for imaging specific fatty-acylated proteins with subcellular organelles and/or total proteome palmitoylation, and we discuss special considerations that need to be given depending on the experimental design. The use of clickable fatty acids with proximity ligation may have promising applications to the investigation of fatty acylation cell biology. The entire protocol takes ∼3 weeks to complete.

Abstract PR02: Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is composed of skeletal myoblast-like cells that have lost the capacity to terminally differentiate. This suggests that RMS cells may contain a factor that blocks normal muscle differentiation. Because cell cycle arrest is coupled to muscle differentiation, identifying putative negative regulators of differentiation could lead to novel therapeutic approaches aimed at fostering terminal differentiation. To gain insight into the events that normally trigger the initial phase of muscle differentiation, we carried out a high content cell-based screen using detection of Myogenin, an early marker of muscle differentiation, as a readout that we measured by automated fluorescence microscopy. We focused on identifying kinases that hinder this developmental transition because of the potential to develop pharmacological inhibitors of “hit” kinases. Using an siRNA library targeting over 600 kinases, we identified 56 as putative negative regulators of myogenic differentiation: 43 with and 19 without PD332991, a Cdk4/6 inhibitor included as a sensitizer. Network analysis showed that 47% of the hits identified without the sensitizer, including SRC family kinases Src and Fyn, were just one interaction node away from MyoD- or Mef2-family myogenic regulatory factors. Although some of the hit kinases were previously implicated in myogenesis, many others were not. Further, 9 (16%) of the hits are causally linked with cancer, based on KEGG and COSMIC databases. Pathway analyses highlighted certain cancer-associated pathways, like EPHA/Ephrin B signaling, the MAPK kinase pathway, de novo pyrimidine synthesis, and mTOR signaling. Among the hits relevant to RMS, Mtor was particularly interesting because this gene encodes a kinase regarded to positively regulate skeletal muscle differentiation. We confirmed our screen findings that Mtor blocks muscle differentiation by showing that its knockdown increases the transcription of a panel of muscle-specific genes in an established myoblast cell line and primary mouse myoblasts. Induction of muscle genes by Mtor knockdown correlated with G0/G1 cell cycle arrest that normally accompanies differentiation. Rapamycin mimicked the effects of Mtor knockdown on muscle gene expression and cell proliferation, implying a role for mTorc1. Finally, preliminary analysis of RMS gene expression data demonstrated that higher expression of muscle differentiation genes correlates with improved survival. These data highlight the potential for mTOR inhibitors to foster the expression of muscle specific genes, pushing the myoblast-like tumor cells to a more differentiated state. Altering their biology in this way may improve outcome. On-going efforts are exploring the mechanisms acting downstream of Mtor to impede muscle differentiation and directly evaluating pro-differentiation effects of mTOR inhibition in RMS models. This abstract is also presented as Poster A1. Citation Format: Raphael A. Wilson, Jing Liu, Lin Xu, Yanbin Zheng, Stephen X. Skapek. Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR02.

Is there a gain in chromosome 3q in the pathway to anal cancer?

Chromosome 3q gain has been identified in human papillomavirus-infected cervical cancer cells. We sought to identify the presence of chromosomal 3q gain in anal neoplasia. This was a retrospective cohort. The study was conducted in a group colorectal surgery practice. Fifty-two patients with no dysplasia, low-grade dysplasia, high-grade dysplasia, or anal cancer were studied. Pairs of biopsy specimens were paraffin embedded and reviewed. One of each slide pair was stained with hematoxylin and eosin and the second processed for fluorescence in situ hybridization. The hybridized set was deparaffinized first and then hybridized with a probe for the chromosome 3q26 region. Then, slides were scanned using an automated fluorescence microscopy system that analyzed defined areas of the tissue to enumerate all of the nuclei for hybridized probe signals to detect chromosome 3q gain. We measured for gain in chromosome 3q26. We identified chromosome 3q gain in 7 (78%) of 9 patients with squamous-cell cancer, 8 (53%) of 15 high-grade dysplasia samples, 0 of 12 low-grade dysplasia samples, and 0 of 16 samples with no dysplasia. The sensitivity for high-grade or invasive neoplasia was 58%, with a specificity of 100%. The positive predictive value of the test was 100% for detecting high-grade dysplasia and/or squamous-cell cancer from no dysplasia, and the negative predictive value of the test was 62%. This study was limited by its small sample size and retrospective design. Chromosome 3q gain represents an important shared pathway to tumorigenesis in cervical and anal neoplasia. Multiple potential diagnostic roles exist for this easily performed test in the evaluation of anal neoplasia.

Automated Fluorescence Microscope for Tuberculosis Detection1

Automated Fluorescence Microscope for Tuberculosis Detection1