Gene Expression Profiling Methods Research Articles

Identifying potential maternal genes of Bombyx mori using digital gene expression profiling.

Maternal genes present in mature oocytes play a crucial role in the early development of silkworm. Although maternal genes have been widely studied in many other species, there has been limited research in Bombyx mori. High-throughput next generation sequencing provides a practical method for gene discovery on a genome-wide level. Herein, a transcriptome study was used to identify maternal-related genes from silkworm eggs. Unfertilized eggs from five different stages of early development were used to detect the changing situation of gene expression. The expressed genes showed different patterns over time. Seventy-six maternal genes were annotated according to homology analysis with Drosophila melanogaster. More than half of the differentially expressed maternal genes fell into four expression patterns, while the expression patterns showed a downward trend over time. The functional annotation of these material genes was mainly related to transcription factor activity, growth factor activity, nucleic acid binding, RNA binding, ATP binding, and ion binding. Additionally, twenty-two gene clusters including maternal genes were identified from 18 scaffolds. Altogether, we plotted a profile for the maternal genes of Bombyx mori using a digital gene expression profiling method. This will provide the basis for maternal-specific signature research and improve the understanding of the early development of silkworm.

Comparison of Immunohistochemistry Assay Results with Gene Expression Profiling Methods for Diffuse Large B-Cell Lymphoma Subtype Identification in Matched Patient Samples

Background: The cell of origin (COO) in diffuse large B-cell lymphoma (DLBCL) has prognostic importance. While the COO was originally classified into germinal center B-cell (GCB) or activated B-cell (ABC) subtypes by microarray analysis, routine use was not practical. Immunohistochemistry (IHC)–based methods are widely used with varying results due to lack of standardization. Several classification methods have been developed recently; understanding the concordance between these and existing methods is essential to their practical application. Therefore, we evaluated concordance between 3 commercial assays: a standardized Hans-based IHC method and 2 gene expression profiling (GEP) methods and compared these to the accepted microarray classification.Methods: 137 DLBCL-confirmed tumor samples were evaluated using a standardized Hans-based IHC method for GCB or non-GCB subtype, by a published microarray-based assay, a digital gene expression-based Lymphoma Subtyping Test (LST) and a next-generation sequencing-based assay (EdgeSeq COO). Subtype calls from the 3 GEP methods were harmonized to “GCB” or “non-GCB” and assessed for concordance.Results: Concordance between the Hans-based IHC assay and the microarray-based assay, LST assay and EdgeSeq assay was 79.6% (N=137), 80.0% (N=125) and 78.2% (N=64), respectively; the positive percent agreement (PPA) in non-GCB was 88.9%, 87.0% and 78.1%, respectively. Concordance for the Hans-based IHC assay versus GEP methods was especially high for direct GCB calls (91.0%, 88.3% and 78.1% for microarray, LST and EdgeSeq COO methods, respectively). The newly developed GEP assays performed well against the microarray GEP method, against which they were calibrated (concordance 93.7% and 87.5% and PPA 94.3% and 92.9%, respectively, for LST and EdgeSeq COO).Conclusion: These results demonstrated good consistency between various platforms for stratification of DLBCL into COO subtype classifications. Application of a standardized Hans-based IHC assay offers a robust, rapid and easily accessible platform to classify DLBCL into prognostically important subtypes.

Preparation of Small RNA NGS Libraries from Biofluids.

Next generation sequencing (NGS) is a powerful method for transcriptome analysis. Unlike other gene expression profiling methods, such as microarrays, NGS provides additional information such as splicing variants, sequence polymorphisms, and novel transcripts. For this reason, NGS is well suited for comprehensive profiling of the wide range of extracellular RNAs (exRNAs) in biofluids. ExRNAs are of great interest because of their possible biological role in cell-to-cell communication and for their potential use as biomarkers or for therapeutic purposes. Here, we describe a modified protocol for preparation of small RNA libraries for NGS analysis. This protocol has been optimized for use with low-input exRNA-containing samples, such as plasma or serum, and has modifications designed to reduce the sequence-specific bias typically encountered with commercial small RNA library construction kits.

Development of Methods for Selective Gene Expression Profiling in Tertiary Lymphoid Structure Using Laser Capture Microdissection.

Tertiary lymphoid structures (TLS) are de novo lymphoid formations that are induced within tissues during inflammatory episodes. TLS have been reported at various anatomic sites and in many different contexts like cancer, infections, autoimmunity, graft rejection, and idiopathic diseases. These inducible, ectopic, and transient lymphoid structures exhibit the prototypical architecture found within secondary lymphoid organs (SLO) and have been recently appreciated as a major driver of the local adaptive immune reaction. As TLS emerge within tissues, the isolation in situ and the molecular characterization of these structures are challenging to operate. Laser capture microdissection (LCM) is a powerful tool to isolate selective structural components and cells from frozen or paraffin-embedded tissues. We and other groups previously applied LCM to decipher the molecular network within TLS and uncover their intrinsic connection with the local microenvironment. In this chapter, we describe a detailed LCM method for selecting and isolating TLS in situ to perform comprehensive downstream molecular analyses.

Abstract 4990: High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities

Abstract Monolayer, two-dimensional (2D) cell cultures have been a predominant in vitro model in anticancer drug discovery and high-throughput screening (HTS). However, 2D cultures of cancer cells lack numerous properties of in vivo tumors, such as tissue-like structure, cell-cell interactions and nutrient/oxygen gradients. Thus, in recent years there has been an increased interest in 3D cell cultures, such as multicellular tumors spheroids (MCTS), to address some of these limitations. Recently, we and others have applied MCTS for HTS and identified oxidative phosphorylation (OXPHOS) as a selective vulnerability of quiescent cancer cells persisting in hypoxic and nutrient-deprived milieu. However, prolonged continuous exposure to OXPHOS inhibitors is necessary for the cytotoxic effect. Thus, there is a need to identify processes that could be co-targeted for enhanced anticancer activity. Here, we present two distinct HTS approaches to identify combination partner molecules for OXPHOS inhibitors. Since we were interested in targeting non-dividing nutrient-deprived cancer cells, we used quiescent MCTS (Q-MCTS), as an in vitro model. Cells in Q-MCTS experience glucose concentrations and pH similar to those observed in deep tumor parenchyma in vivo. In our first screening approach, we have applied high-throughput gene-expression profiling to study drug effects in MCTS at a large scale. Using L1000 Gene Expression Profiling method, we generated a dataset of over 1000 drug-induced gene-expression profiles and found that co-targeting of OXPHOS and the mevalonate pathway results in selective synergistic toxicity in quiescent cancer cells. In the other approach, we screened a library of 1650 biologically active compounds, with or without addition of the FDA-approved anthelmintic agent nitazoxanide (an OXPHOS inhibitor with high drug repurposing potential). After the screen, we selected molecules that demonstrated pronounced synergy when combined with nitazoxanide, but not when used alone. Then, we validated the hits in an extensive dose-response combination experiments in Q-MCTS and chose 14 compounds that demonstrated strong synergistic interaction with nitazoxanide at broad range of concentrations. These included antifungal agents, kinase inhibitors and others. In summary, we here report on novel approaches, utilizing 3D cell cultures, to identify drug combinations targeting quiescent cancer cells. By high-throughput gene-expression profiling and large-scale combinatorial drug screens, we were able to identify drug combinations preferentially toxic to quiescent cells. This work also demonstrates how 3D cell cultures yield functional insights that are not accessible through standard 2D cultures. Citation Format: Wojciech Senkowski, Madiha Nazir, Malin Jarvius, Jenny Rubin, Johan Lengqvist, Mats G. Gustafsson, Peter Nygren, Kim Kultima, Rolf Larsson, Mårten Fryknäs. High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2017-4990

Antibody targeting of claudin-1 as a potential colorectal cancer therapy

BackgroundMetastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb).MethodsGene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells.ResultsCompared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation.ConclusionOur data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target.

Observational study of association between salivary alpha amylase seriated, fibromyalgia degree and rage

The exquisite orchestration of gene expression at the transcriptional level and beyond is responsible for the differentiation of cells and tissues and, ultimately, for conferring the phenotype of the organism. The phenotype of cells is ultimately governed by the modulation of gene expression; it is logical to focus on the mRNA population of a cell and how the complexity and composition of that population changes in response to experimental challenge, compared to control or baseline conditions. Two types of methods are used to understand the biochemical changes that occur within a cell, namely, subtractive methods (suppression subtractive hybridization) and nonsubtractive methods (mRNA differential display). The overall efficiency of subtractive methods depends on maintaining high stringency conditions while favoring efficient forward hybridization kinetics, both of which are dependent upon salt concentration, hybridization temperature, hybridization time, and the presence of excess driver throughout, whereas the strategy of nonsubtractive methods for gene expression profiling is more about selective amplification rather than subtraction. Regardless of the method utilized, there are terms and concepts common to all approaches that must be clearly understood. The tester is the cDNA population containing induced sequences that are to be sequestered. The driver is the control or reference population of cDNA sequences against which the tester is being compared. During the subtraction hybridization, the driver is always present in a large molar excess over the tester. This will ensure first-order hybridization kinetics, resulting in complete hybridization and concomitant removal of all sequences common to both populations. These methods are contemporary examples of whole-transcriptome approaches.

Gene Expression Profiling of Large Cell Lung Cancer Links Transcriptional Phenotypes to the New Histological WHO 2015 Classification

IntroductionLarge cell lung cancer (LCLC) and large cell neuroendocrine carcinoma (LCNEC) constitute a small proportion of NSCLC. The WHO 2015 classification guidelines changed the definition of the debated histological subtype LCLC to be based on immunomarkers for adenocarcinoma and squamous cancer. We sought to determine whether these new guidelines also translate into the transcriptional landscape of lung cancer, and LCLC specifically. MethodsGene expression profiling was performed by using Illumina V4 HT12 microarrays (Illumina, San Diego, CA) on samples from 159 cases (comprising all histological subtypes, including 10 classified as LCLC WHO 2015 and 14 classified as LCNEC according to the WHO 2015 guidelines), with complimentary mutational and immunohistochemical data. Derived transcriptional phenotypes were validated in 199 independent tumors, including six WHO 2015 LCLCs and five LCNECs. ResultsUnsupervised analysis of gene expression data identified a phenotype comprising 90% of WHO 2015 LCLC tumors, with characteristics of poorly differentiated proliferative cancer, a 90% tumor protein p53 gene (TP53) mutation rate, and lack of well-known NSCLC oncogene driver alterations. Validation in independent data confirmed aggregation of WHO 2015 LCLCs in the specific phenotype. For LCNEC tumors, the unsupervised gene expression analysis suggested two different transcriptional patterns corresponding to a proposed genetic division of LCNEC tumors into SCLC-like and NSCLC-like cancer on the basis of TP53 and retinoblastoma 1 gene (RB1) alteration patterns. ConclusionsRefined classification of LCLC has implications for diagnosis, prognostics, and therapy decisions. Our molecular analyses support the WHO 2015 classification of LCLC and LCNEC tumors, which herein follow different tumorigenic paths and can accordingly be stratified into different transcriptional subgroups, thus linking diagnostic immunohistochemical staining–driven classification with the transcriptional landscape of lung cancer.

Discovery of novel cold-induced CISP genes encoding small RNA-binding proteins related to cold adaptation in barley

To adapt to cold conditions, barley plants rely on specific mechanisms, which have not been fully understood. In this study, we characterized a novel barley cold-induced gene identified using a PCR-based high coverage gene expression profiling method. The identified gene encodes a small protein that we named CISP1 (Cold-induced Small Protein 1). Homology searches of sequence databases revealed that CISP1 homologs (CISP2 and CISP3) exist in barley genome. Further database analyses showed that the CISP1 homologs were widely distributed in cold-tolerant plants such as wheat and rye. Quantitative reverse transcription PCR analyses indicated that the expression of barley CISP genes was markedly increased in roots exposed to cold conditions. In situ hybridization analyses showed that the CISP1 transcripts were localized in the root tip and lateral root primordium. We also demonstrated that the CISP1 protein bound to RNA. Taken together, these findings indicate that CISP1 and its homologs encoding small RNA-binding proteins may serve as RNA chaperones playing a vital role in the cold adaptation of barley root. This is the first report describing the likely close relationship between root-specific genes and the cold adaptation process, as well as the potential function of the identified genes.

The nitric oxide synthase 2 pathway is targeted by both pro- and anti-inflammatory treatments in the immature human intestine

Background and AimNO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine. MethodsGene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 μM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes. ResultsIn the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO. ConclusionThese results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.

Transcriptome Analysis Reveals Silver Nanoparticle-Decorated Quercetin Antibacterial Molecular Mechanism.

Facile and simple method is developed to synthesize silver-nanoparticle-decorated quercetin nanoparticles (QA NPs). Modification suggests that synergistic quercetin (Qe) improves the antibacterial effect of silver nanoparticles (Ag NPs). Characterization experiment indicates that QA NPs have a diameter of approximately 10 nm. QA NPs show highly effective antibacterial activities against drug-resistant Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). We explore antibacterial mechanisms using S. aureus and E. coli treated with QA NPs. Through morphological changes in E. coli and S. aureus, mechanisms are examined for bacterial damage caused by particulate matter from local dissociation of silver ion and Qe from QA NPs trapped inside membranes. Moreover, we note that gene expression profiling methods, such as RNA sequencing, can be used to predict discover mechanisms of toxicity of QA NPs. Gene ontology (GO) assay analyses demonstrate the molecular mechanism of the antibacterial effect of QA NPs. Regarding cellular component ontology, "cell wall organization or biogenesis" (GO: 0071554) and "cell wall macromolecule metabolic process" (GO: 0044036) are the most represented categories. The present study reports that transcriptome analysis of the mechanism offers novel insights into the molecular mechanism of antibacterial assays.

Transcriptomics Analysis of Apple Leaves in Response to Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease of apple (Malus × domestica Borkh.), caused by the apple pathotype of Alternaria alternata, is one of the most serious fungal diseases to affect apples. To develop an understanding of how apples respond to A. alternata apple pathotype (AAAP) infection, we examined the host transcript accumulation over the period between 0 and 72 h post AAAP inoculation. Large-scale gene expression analysis was conducted of the compatible interaction between “Starking Delicious” apple cultivar and AAAP using RNA-Seq and digital gene expression (DGE) profiling methods. Our results show that a total of 9080 differentially expressed genes (DEGs) were detected (>two-fold and FDR < 0.001) by RNA-Seq. During the early phase of infection, 12 h post inoculation (HPI), AAAP exhibited limited fungal development and little change in the transcript accumulation status (950 DEGs). During the intermediate phase of infection, the period between 18 and 36 HPI, increased fungal development, active infection, and increased transcript accumulation were detected (4111 and 3838 DEGs detected at each time point, respectively). The majority of DEGs were detected by 72 HPI, suggesting that this is an important time point in the response of apples' AAAP infection. Subsequent gene ontology (GO) and pathway enrichment analyses showed that DEGs are predominately involved in biological processes and metabolic pathways; results showed that almost gene associated with photosynthesis, oxidation-reduction were down-regulated, while transcription factors (i.e., WRKY, MYB, NAC, and Hsf) and DEGs involved in cell wall modification, defense signaling, the synthesis of defense-related metabolites, including pathogenesis-related (PRs) genes and phenylpropanoid/cyanoamino acid /flavonoid biosynthesis, were activated during this process. Our study also suggested that the cell wall defensive vulnerability and the down-regulation of most PRs and HSP70s in “Starking Delicious” following AAAP infection might interpret its susceptible to AAAP.

Influence of 5-amino-3-methyl-4-isoxazolecarbohydrazide on selective gene expression in Caco-2 cultured cells

The 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide (HIX) is a synthetic isoxazole derivative with a potential for development as an anti-inflammatory drug candidate. The goal of this study was to explore in vitro autoimmune and inflammatory gene modulation by HIX in human Caco-2 cultured cells. The effect of low dose of HIX was tested on the expression level of RNA in 24 h Caco-2 cultures using the QIAGEN Th17 for Autoimmunity & Inflammation RT2 Profiler PCR Array. We choose the PCR technology as the most reliable and sensitive gene expression profiling method for analyzing specific gene regulatory networks. In all experiments, Leflunomide (5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide), an immuno-suppressive disease-modifying antirheumatic drug was used, as a reference to clinical utility of the isoxazole derivatives. Changes in RNA levels were analyzed and differentially expressed genes with at least 2-fold change were identified. For the majority of genes tested, the effects of HIX and Leflunomide were similar, including up-regulation of CX3CL1 and IL-17F, and down-regulation of IL-10 and TLR4. However twelve genes were were differently regulated by the two compounds: interleukins (IL) IL-1B, IL-6 and a chemokine CCL22 were upregulated by HIX and significantly supressed by Leflunomide. In contrary, IL-2 and IL-27 were upregulated by Leflunomide and suppressed by HIX. The network search by Ingenuity Pathway Analysis showed, that majority of differentially expressed genes were involved in cellular inflammatory responses. These results suggest that 5-amino-3-methyl-4-isoxazolecarbohydrazide has a potential for future clinical developments with structure modification as a disease modifying agent in different than Leflunomide applications.

Large-Scale Gene Expression Profiling Platform for Identification of Context-Dependent Drug Responses in Multicellular Tumor Spheroids

Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of invivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.

A new ten-gene risk fraction model serving as prognostic indicator for clinical outcome of multiple myeloma.

Multiple myeloma (MM) is a kind of aggressive tumor prevalent with high heterogeneity. Abnormal expression of certain genes may lead to the occurrence and development of MM. Nowadays, it is not commonly seen in clinical research to predict the prognostic circumstances of patients with MM by multiple gene expression profiling method. Identification of potential genes in prognostic process could be beneficial for clinical management of MM. Therefore, we aimed to build a risk fraction model to screen out the prognostic indicator for clinical outcome of MM. Microarray data were downloaded from the Genome Expression Omnibus (GEO) datasets with accession numbers GSE24080 and GSE57317. A total of 279 samples were selected out randomly. Besides, a risk formula was constructed and verified in the dataset. Time-dependent receiver operating characteristic (ROC) curve was applied in evaluating the accurate prognostic conditions of patients. Finally, a ten genes model in the training dataset was found to be closely related to the survival condition of MM patients. Patients with MM were divided into two groups, high-risk and low-risk, by the expression of these ten genes, and significant statistical difference was found between the two groups. Furthermore, the result of multivariate cox regression and stratified analysis indicated that this model was independent of other clinical phenotypes. ROC curves also showed its feasibility to predict the survival status of MM patients. Our results demonstrated that the fraction risk model constructed by the selected ten genes could be used to predict survival status of multiple myeloma patients, which could also help in improvement of prognostic and therapeutic tool of MM.

TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways.

BackgroundTransducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/β-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce.MethodsGene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR.ResultsTLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1.ConclusionThis study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0426-8) contains supplementary material, which is available to authorized users.

Abstract 3187: Concordance of immunohistochemistry (IHC) assay results with gene expression profiling (GEP) methods for diffuse large-B-cell lymphoma (DLBCL) subtype identification

Abstract Introduction: The survival of DLBCL patients who receive immunochemotherapy is correlated with the molecular signature of their tumors. The two most common subtypes which are defined by cell of origin (COO) are the germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes which have 3-year OS rates of 80% and 45%, respectively when treated with R-CHOP. Several methods have been developed to categorize DLBCL into these sub-types as the treatment they receive directly affects their outcome. Microarray GEP was first used to classify DLBCL into ABC or GCB, with a minority unclassified (UNC), however this is difficult to implement widely and has been replaced by more recent technologies. The most commonly used IHC-based Hans method profiles patients into GCB and non-GCB. The primary focus of this study was to evaluate concordance between current GEP and IHC methods to designate DLBCL subtypes. Methods: For this study, the IHC assay used was a standardized IHC assay based on the Hans algorithm and for the GEP-based assay; the NanoString Technologies Lymphoma Subtyping Test (LST) was used. Commercially available DLBCL samples (N = 138) were confirmed for tumor content and sent to a central laboratory for evaluation by the IHC assay. RNA was extracted from each sample and sent to NanoString for GEP evaluations. To compare methods homogeneously, calls for each method were converted to either “GCB” or “non-GCB”. All “ABC” and “UNC” subtype calls from the NanoString platform was converted to “non-GCB” and any “did not pass” (DNP) calls were converted to not available (NA). All NA values were excluded from the tables and concordance calculations. Results: The Positive Percent Agreement [95% CI] in calling non-GCB between IHC and LST was 87% [78% - 96%] (47/54). Overall concordance between IHC and LST was 80% [73% - 87%] (N = 125, 12 LST samples DNP, 1 IHC sample DNP). The Negative Percent Agreement between IHC and LST (i.e., in calling the GCB sub-type) was 75% [65% - 85%] using the LST result as a non-reference standard (53/71). Conclusions: Given that the success of targeted therapy for the treatment of DLBCL is contingent on the correct identification of sub-type of DLBCL, it is important to adopt a method that accurately and consistently identifies the sub-type in the majority of patients. Given the good concordance between the GEP and IHC methods, DLBCL can be stratified into COO sub-types using either platform. The results show that an optimized Hans IHC assay standardized in a central laboratory offers a robust, relatively inexpensive and accessible platform for cell of origin sub-typing in DLBCL and, with clinical validation, may offer an excellent tool for the selection of optimal therapy. Citation Format: Michael Schaffer, Shalini Chaturvedi, Sandy Frans, Regina Aquino, Jaehong Park, Brett Hall, Sriram Balasubramanian. Concordance of immunohistochemistry (IHC) assay results with gene expression profiling (GEP) methods for diffuse large-B-cell lymphoma (DLBCL) subtype identification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3187.

Abstract 213: Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells

Abstract Inhibition of mitochondrial oxidative phosphorylation (OXPHOS) has recently emerged as a promising strategy for treatment of therapy-resistant cancer cells. These cells often reside within hypoxic tumor regions, where nutrient concentrations are low. Recently, OXPHOS inhibitors have been demonstrated to be toxic to quiescent, nutrient-deprived cells in multicellular tumor spheroids. Such spheroids, formed without medium exchange over the culture period, can serve as an appropriate model to mimic quiescent in vivo tumor regions. These spheroids exhibit low cell proliferation and comprise necrotic cores, contrary to commonly used spheroids cultured with frequent medium change. We here aimed to characterize how quiescent cells respond to OXPHOS inhibition and thereby identify processes that could be co-targeted for enhanced toxicity. We treated HCT116 colon cancer cell line, grown as monolayer cultures and spheroids, with a range of OXPHOS inhibitors (n = 10, including FDA-approved drugs, e.g. nitazoxanide) and other compounds (n = 14) at escalating doses and in 4 biological replicates. Then, we obtained global gene expression profiles (n = 1149, including 144 vehicle controls) of all treatment conditions using L1000 Gene Expression Profiling method. We found that upon exposure to OXPHOS inhibitors cells grown as nutrient-deprived spheroids significantly and in dose-dependent manner upregulate expression of genes involved in biosynthesis of cholesterol. This response was not observed for spheroids cultured with medium change or monolayer cell cultures. Thus, we were interested if simultaneous exposure to OXPHOS inhibitors and statins, inhibitors of mevalonate (cholesterol precursor) synthesis, would result in enhanced cytotoxic effects in quiescent, metabolically stressed cells. We here demonstrate that combination of OXPHOS inhibitors and statins results in pronounced synergistic cytotoxicity in metabolically stressed spheroids. This effect was observed for various classes of OXPHOS inhibitors and different types of statins, indicating that the observed synergy was not a result of off-target effects. This notion was further strengthened by the finding that mevalonate largely abrogated the synergistic effects. In conclusion, we here report that statins enhance the toxic effects of OXPHOS inhibitors in quiescent, metabolically stressed cells. Our results can serve as a foundation for further studies on targeting therapy-resistant and nutrient-deprived cancer cells by inhibition of OXPHOS. We also demonstrate, for the first time, that the L1000 Gene Expression Profiling can be used to study 3D cell cultures. Importantly, our findings underscore the importance of using a relevant cellular model for target discovery endeavors. Citation Format: Wojciech Senkowski, Malin Jarvius, Kim Kultima, Jenny Rubin, Mats Gustafsson, Peter Nygren, Rolf Larsson, Mårten Fryknäs. Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 213.

An open RNA-Seq data analysis pipeline tutorial with an example of reprocessing data from a recent Zika virus study

RNA-seq analysis is becoming a standard method for global gene expression profiling. However, open and standard pipelines to perform RNA-seq analysis by non-experts remain challenging due to the large size of the raw data files and the hardware requirements for running the alignment step. Here we introduce a reproducible open source RNA-seq pipeline delivered as an IPython notebook and a Docker image. The pipeline uses state-of-the-art tools and can run on various platforms with minimal configuration overhead. The pipeline enables the extraction of knowledge from typical RNA-seq studies by generating interactive principal component analysis (PCA) and hierarchical clustering (HC) plots, performing enrichment analyses against over 90 gene set libraries, and obtaining lists of small molecules that are predicted to either mimic or reverse the observed changes in mRNA expression. We apply the pipeline to a recently published RNA-seq dataset collected from human neuronal progenitors infected with the Zika virus (ZIKV). In addition to confirming the presence of cell cycle genes among the genes that are downregulated by ZIKV, our analysis uncovers significant overlap with upregulated genes that when knocked out in mice induce defects in brain morphology. This result potentially points to the molecular processes associated with the microcephaly phenotype observed in newborns from pregnant mothers infected with the virus. In addition, our analysis predicts small molecules that can either mimic or reverse the expression changes induced by ZIKV. The IPython notebook and Docker image are freely available at: http://nbviewer.jupyter.org/github/maayanlab/Zika-RNAseq-Pipeline/blob/master/Zika.ipynband https://hub.docker.com/r/maayanlab/zika/.

Development of a multitissue microfluidic array for assessing changes in gene expression associated with channel catfish appetite, growth, metabolism, and intestinal health

Large-scale, gene expression profiling methods allow for high throughput analysis of physiological pathways at a fraction of the cost of individual gene expression analysis. Systems, such as the Fluidigm quantitative PCR array described here, can provide powerful assessments of the effects of diet, environment, and management on physiological pathways affecting production parameters. A targeted microfluidic PCR array was designed and validated, for channel catfish (Ictalurus punctatus) representing key pathways involved in appetite, growth, metabolism, and intestinal inflammation for their potential to provide insight into the effects of diet and dietary supplements on these important physiological processes regulating feed efficiency and growth. With few exceptions, PCR primers were designed from Ictaluridae gene sequences published in GenBank. PCR amplicons from primers designed outside of Ictaluridae were sequenced to verify gene identity. All target gene primers were initially validated via conventional real-time qPCR (RT-qPCR). Combined hypothalamus/pituitary, hepatic, stomach, and intestinal tissue were used validate a 48.48 microfluidic PCR array to analyze multitissue gene expression. Use of the Fluidigm array resulted in reliable cycle threshold levels (Ct), efficiencies (E), and quality threshold scores (QS) for all but eight genes examined. Of the potential reference genes included in the panel, alpha-tubulin (TUBA) had a high QS, E, and acceptable Ct. The high throughput application of this technology, relative to conventional RT-qPCR, for assessing dietary effects on these pathways is demonstrated. Development of this targeted multi-tissue microfluidic array paves the way for the rapid evaluation of regulatory pathways in response to alternative feeding strategies, dietary formulations, and supplementation, as well as environmental and management effects for improving channel catfish culture and validates a cost-effective, dynamic, gene expression platform for use with other cultured fishes.