Differential Expression Pathway Research Articles

The effects of exosomes originating from different cell sources on the differentiation of bone marrow mesenchymal stem cells into Schwann cells

BackgroundBone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms.Materials and methodsWe extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways.ResultsBMSCs were cultured by three media: conventional (no induction), pre-induction or pre-induction + original induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The induction ability was ranked as follows: pre-induction + ODM + exosome group > pre-induction + exosome group > non-induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups.ConclusionsOur analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.

Psychosocial stressors and breast cancer gene expression in the Black Women's Health Study.

Prior studies indicate that the physiologic response to stress can affect gene expression. We evaluated differential gene expression in breast cancers collected from Black women with high versus low exposure to psychosocial stressors. We analyzed tumor RNA sequencing data from 417 Black Women's Health Study breast cancer cases with data on early life trauma and neighborhood disadvantage. We conducted age-adjusted differential gene expression analyses and pathway analyses. We also evaluated Conserved Transcriptional Response to Adversity (CTRA) contrast scores, relative fractions of immune cell types, T cell exhaustion, and adrenergic signaling. Analyses were run separately for estrogen receptor positive (ER+; n = 299) and ER- (n = 118) cases. Among ER+ cases, the top differentially expressed pathways by stress exposure were related to RNA and protein metabolism. Among ER- cases, they were related to developmental biology, signal transduction, metabolism, and the immune system. Targeted analyses indicated greater immune pathway enrichment with stress exposure for ER- cases, and possible relevance of adrenergic signaling for ER+ cases. CTRA contrast scores did not differ by stress exposure, but in analyses of the CTRA components, ER- breast cancer cases with high neighborhood disadvantage had higher pro-inflammatory gene expression (p = 0.039) and higher antibody gene expression (p = 0.006) compared to those with low neighborhood disadvantage. There are multiple pathways through which psychosocial stress exposure may influence breast tumor biology. Given the present findings on inflammation and immune response in ER- tumors, further research to identify stress-induced changes in the etiology and progression of ER- breast cancer is warranted.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

Differential gene expression associated with flower development of mango (Mangifera indica L.) varieties with different shelf-life

Mango (Mangifera indica L.) is one of the most important commercial fruit crop grown in many parts of the world. Major challenges affecting mango trade are short shelf-life, high susceptibility to chilling injury, post-harvest diseases and consumer demand for improved fruit quality. The objective of the present study was to reveal the key regulators present in bud and flower tissues during flower development stage, associated with fruit development and affect the shelf-life of the mango fruit. RNA-sequencing of contrasting genotypes having short and long shelf-life, was carried out. Comparative differential expression pathway studies of long shelf-life (Totapuri) and short shelf-life (Bombay Green) mango genotypes revealed a total of 177 highly differentially expressed genes. Out of 177 total genes, 101 genes from endoplasmic reticulum pathway and very few from gibberellins (3) and jasmonic acid (1) pathway were identified. Genes from endoplasmic reticulum pathway like hsp 90, SRC2, DFRA, CHS, BG3 and ASPG1 mainly up regulated in Bombay Green. Uniprotein B9R8D3 also shows up regulation in Bombay Green. Ethylene insensitive pathway gene EIL1 up regulated in Bombay Green. Gene CAD1 from phenylpropanoid pathway mainly up regulated in Bombay Green. A total of 4 SSRs and 227 SNPs were mined from these pathways specific to the shelf-life. Molecular studies of endoplasmic reticulum, phenylpropanoid, ethylene, polygalacturonase and hormone pathways at the time of bud and flower formation revealed key regulators that determine the shelf-life of mango fruit.

IBD Subtype-Regulators IFNG and GBP5 Identified by Causal Inference Drive More Intense Innate Immunity and Inflammatory Responses in CD Than Those in UC.

Background: The pathological differences between Crohn’s disease (CD) and ulcerative colitis (UC) are substantial and unexplained yet. Here, we aimed to identify potential regulators that drive different pathogenesis of CD and UC by causal inference analysis of transcriptome data. Methods: Kruskal–Wallis and Dunnett’s tests were performed to identify differentially expressed genes (DEGs) among CD patients, UC patients, and controls. Subsequently, differentially expressed pathways (DEPs) between CD and UC were identified and used to construct the interaction network of DEPs. Causal inference was performed to identify IBD subtype-regulators. The expression of the subtype-regulators and their downstream genes was validated by qRT-PCR with an independent cohort. Results: Compared with the control group, we identified 1,352 and 2,081 DEGs in CD and UC groups, respectively. Multiple DEPs between CD and UC were closely related to inflammation-related pathways, such as NOD-like receptor signaling, IL-17 signaling, and chemokine signaling pathways. Based on the priori interaction network of DEPs, causal inference analysis identified IFNG and GBP5 as IBD subtype-regulators. The results with the discovery cohort showed that the expression level of IFNG, GBP5, and NLRP3 was significantly higher in the CD group than that in the UC group. The regulation relationships among IFNG, GBP5, and NLRP3 were confirmed with transcriptome data from an independent cohort and validated by qRT-PCR. Conclusion: Our study suggests that IFNG and GBP5 were IBD subtype-regulators that trigger more intense innate immunity and inflammatory responses in CD than those in UC. Our findings reveal pathomechanical differences between CD and UC that may contribute to personalized treatment for CD and UC.

Abstract 2333: EMT programs ovarian cancer cells to survive the adipocyte-rich microenvironment

Abstract Background: Metabolic pathways are non-canonical targets of the EMT program. The relevance of re-programming metabolism during the metastasis cascade is underscored in ovarian cancer cells, which primarily metastasizes to the adipocyte-rich omentum. Ovarian cancer-adipocyte interaction has been shown to promote fatty acid (FA) uptake into the cancer cells. These FA are then catabolized via β-oxidation thus providing the energy required for rapid tumor growth. β-oxidation of FA requires efficient mitochondria and oxygen processing. The objective of this study is to determine if E/M status determine adaptation and survival in adipocyte-rich microenvironment. Materials and Methods: RNA seq was performed in Ck18+/Twist- epithelial and Ck18-/Twist+ mesenchymal human ovarian cancer cells. Pathway and Gene Ontology analyses were performed on differentially expressed (DEX) genes (FC&amp;gt;2 and p&amp;lt;0.05) using iPathway Guide. Protein levels were determined by western blot. Oxygen consumption rate was measured using Seahorse XF Analyzer. Cell viability was quantified using CelltoxTM Green viability dye. Results: Pathway analysis of DEX genes with focus on metabolic pathways showed the following DEX pathways: glycolysis/gluconeogenesis (p=0.008); pyruvate metabolism (0.019); fatty acid degradation (p=0.01); fatty acid elongation (0.008). Gene ontology analysis showed significant difference in GO:0006979: response to oxidative stress (p=0.007). Further analysis of DEX genes within these pathways showed upregulation in mesenchymal cancer cells of both pyruvate dehydrogenase subunits, PDHA1 (p = 0.011) and PDHB (p=0.014) and upregulation of carnitine palmitoyltransferase (CPT1; p=0.011), the rate limiting enzyme in FA β-oxidation. The upregulation of these genes was validated at the protein level by western blot analysis, which also showed increased levels of COX-IV in mesenchymal cancer cells compared to epithelial cancer cells. Moreover, mesenchymal cells exhibited enhanced maximal respiration and enhanced spare respiratory capacity. Finally, viability studies conclusively demonstrate the metabolic flexibility in mesenchymal cancer cells, which maintained viability upon nutrient deprivation suggesting metabolic plasticity. In contrast, cell death was observed epithelial cancer cells upon nutrient deprivation, which was rescued by the addition of glucose but not the FA, oleic acid. Conclusion: Ovarian cancer cells acquire enhanced pyruvate shuttling to the TCA cycle, a more efficient FA β-oxidation machinery, as well as more efficient oxygen processing during EMT. These phenotypes are relevant in supporting survival in the adipocyte microenvironment. Further elucidation of EMT related pathways that re-program lipid metabolism may lead to better control of carcinomatosis in ovarian cancer patients. Citation Format: Hussein Chehade, Alexandra Fox, Roslyn Tedja, Radhika Gogoi, Gil Mor, Ayesha B. Alvero. EMT programs ovarian cancer cells to survive the adipocyte-rich microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2333.

An EAV-HP insertion in the promoter region of SLCO1B3 has pleiotropic effects on chicken liver metabolism based on the transcriptome and proteome analysis

Solute carrier organic anion transporter 1B3 (SLCO1B3) is an important liver primarily highly expressed gene, its encoded protein (OATP1B3) involved in the transport of multi-specific endogenous and exogenous substances. We previously reported that an EAV-HP inserted mutation (IM+) in the 5ʹ flanking region of SLCO1B3 was the causative mutation of chicken blue eggs, and a further research showed that IM+ significantly reduced the expression of SLCO1B3 in liver. Herein, we confirmed a cholate response element (IR-1) played an important role in activating SLCO1B3 and in vitro experiments showed that the activation of IR-1 can be significantly reduced by the EAV-HP IM+ . We performed transcriptome and proteomic analysis using the same set of IM+ and IM− liver tissues from Yimeng hens (a Chinese indigenous breed) to study the effect of SLCO1B3 and OATP1B3 expression reduction on chicken liver function. The results showed that common differential expression pathways were screened out from both transcriptome and proteome, in which fatty acid metabolism and drug metabolism—cytochrome P450 were significantly enriched in the KEGG analysis. The lipid-related metabolism was weakened in IM+ group, which was validated by serum biochemical assay. We unexpectedly found that EAV-HP fragment was highly expressed in the liver of the IM+ chickens. We cloned the EAV-HP full-length transcript and obtained the complete open reading frame. It is worth noting that there was some immune related differential expressed genes, such as NFKBIZ, NFKBIA, and IL1RL1, which were higher expressed in the IM+ group, which may due to the high expression of EAV-HP. Our study showed that EAV-HP IM+ reduced the expression of SLCO1B3 in liver, resulting in the decrease of fatty metabolism and exogenous substance transport capacity. The mutation itself also expressed in the liver and may be involved in the immune process. The mechanism needs further study.

Abstract 5362: Analysis of circulating tumor cells from prostate cancer patients

Abstract Background: Intermediate and high-risk prostate cancer patients are candidates for radiotherapy (RT). Evidence has shown that the presence of circulating tumor cells (CTCs) correlates with poor overall survival for patients with metastatic prostate cancer. CTCs have been reported as a better earlier predictor of treatment response than evaluation of prostate-specific antigen (PAS). The aim of our study was to identify and quantify circulating tumor cells from the peripheral blood of patients enrolled in two randomized Phase II radiotherapy clinical trials (BLaStM, NCT02307058 and CoMBINe, NCT02997709). Moreover, we investigated the genomic profiling of single CTCs using 10X Genomics to explore prognostic factors. Design/Methods: CTCs were successfully harvested and enumerated from more than 215 patients and 500 blood samples using a microfilter system, the Circulogix FaCTChecker, and the cells of interest enriched onto CyteCatch slides. Circulating tumor cells were visualized by staining, identified and quantified for epithelial and prostate specific markers. The number of single CTCs, CTC clusters and their sum were recorded. Unfixed CTCs were enriched from whole blood from 13 prostate cancer patients at high risk of metastasis (median age of 63.5 years and median CTC value of 12 (range 0-42)) using The Parsortix™ Cell Separation System (ANGLE Inc.), a microfluidic-based platform able to capture and harvest CTCs, and single-cell RNA sequencing was then performed with Chromium (10X Genomics) and Illumina NGS sequencing technologies. Initial bioinformatic and data analysis was performed by the Biostatistics and Bioinformatics Shared Resource (BBSR) at SCCC. Subsequent principal component analysis (PCA) and gene set enrichment analysis (GSEA) were performed. Meanwhile, patient demographic and disease characteristic variables (PSA, Gleason Score (GS), T stage, % tumor in biopsies, age, race/ethnicity) were also collected and compared. Results: Single CTCs or CTC clusters were evident in over 70% of patients analyzed using Immunofluorescence staining, with a median number of 30 (range 0-346). CTC transcriptomes at the single cell level was performed using the 10X platform, and principal component analysis discovered 16 distinct cell clusters of differential gene signatures within these cells in a t-SNE plot. Gene ontology analysis found some of these cell clusters contain differential expression pathways such as mTOR signaling (p=5.82E-05), EIF2 signaling (p=7.90E-05) and regulation of eIF4 and p70S6K signaling (p= 1.04E-04) which have been associated with prostate cancer. Gene Set Enrichment Analysis also identified distinct differential expression pathways in prostate CTCs comparing with non-CTCs. Conclusion: We have developed a novel pipeline to harvest CTCs and analyze transcriptomes at the single-cell level. Our results suggest that gene expression profiling of CTCs can be a promising way to explore prognostic markers for predicting radiation sensitivity and metastatic risk. Citation Format: Teresa Giret, Wensi Tao, Robert Suter, Saba Ansari, Sion Williams, Nagi Ayad, Radka Stoyanova, Brian Marples, Alan Pollack. Analysis of circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5362.

The roles of Qishen granules recipes, Qingre Jiedu, Wenyang Yiqi and Huo Xue, in the treatment of heart failure

Recipes (Qingre Jiedu (QJ), Wenyang Yiqi (WYYQ) and Huo Xue (HX)) in Qishen granules (QSG) are believed to synergistically exert cardio-protective effects. However, the underlying pattern of each decomposed recipe in QSG and their synergistic effects in the treatment of heart failure (HF) are not clear. The purpose of this study is to explore the biological contributions of decomposed recipes to therapeutic effects of QSG and reveal the pharmacological mechanism of QSG in treating HF. The therapeutic effects of QSG or its recipes on heart failure were examined in wet-lab at both transcription and phenotypic level using HF Sprague-Dawley rats. Sequencing and transcriptome analyses were performed using in silico approaches including identification of differentially expressed genes, pathway enrichment and protein-protein interaction network studies. Specially, an optimized in silico quantitative pathway analysis that maximally extracted gene expression information was developed to reveal differentially expressed pathways (DEPs) among various groups, and is publicly available as R package QPA on GitHub (https://github.com/github-gs/QPA). Finally, the HF-related genes predicted using DEP approach were validated by quantitative real-time polymerase chain reaction and western blot. Multiple key genes and the associated signaling pathways were shown to be highly relevant for the therapeutic effect of QSG. Decreased expression of Spp1 gene required for inflammatory signaling and profibrotic signaling were observed in failing hearts treated with QJ, WYYQ and HX. Decreased expression of Cx3cr1 gene required for inflammatory signaling was observed in failing hearts treated with WYYQ and HX. Decreased expression of Myc gene required for oxidative stress and Fgfr2 gene required for profibrotic signaling were observed in failing hearts treated with HX and WYYQ, respectively. Increased expression of Adcy1 gene required for cAMP-PKA signaling cascade was observed in failing hearts treated with WYYQ and HX. Our study suggests that QJ, WYYQ and HX recipes in QSG achieve synergistic and complementary therapeutic effects through alleviating inflammatory responses, attenuating ventricular remodeling and enhancing myocardial energy supply.

Screening pathways and hub genes involved in osteoclastogenesis by gene expression analysis of circulating monocytes based on Gibbs sampling.

Differential expression pathways and hub genes in circulating monocytes from healthy Chinese women with high peak bone mass (PBM) vs. low PBM were explored using a Markov chain Monte Carlo (MCMC) algorithm. Human circulating monocytes transcription profiling (containing 14 samples with high PBM and 12 samples with low PBM) and KEGG pathways were all downloaded from the public database. Initial state of all the pathways were constructed and Gibbs sampling was performed to obtain a Markov chain and the posterior values of all the pathways were calculated. The probability (α) of occurrence of each pathway was calculated based on the posterior value and it was adjusted by taking gene expression variation into account. Pathways with αadj >0.8 were considered as differentially expressed pathways. Then, these steps were performed again on all the genes in the differentially expressed pathways to find the hub genes in the differential pathways. After Gibbs sampling, neuroactive ligand-receptor interaction (hsa04080) with αadj = 0.986 was screened out as the differentially expressed pathway. Analyzing the genes in this pathway, three genes (neurotensin, tachykinin receptor 3 and follicle-stimulating hormone receptor) with αadj >0.8 were identified as hub genes in circulating monocytes which may associate with osteoporosis development. One pathway and three genes which may possess close relationship with osteoporosis development were found in this study. These results provide insights into our understanding of the role of circulating monocytes in osteoporosis development.

Eliciting Anti-Tumor T Cell Immunity in Chronic Lymphocytic Leukemia (CLL) with PD-L1/PD-1 Blockade Is Enhanced By Avadomide Immunotherapy through the Triggering of Immunogenic Interferon Signaling

Immune checkpoint blockade has demonstrated potential to reactivate anti-tumor immunity and regress tumors. However, response rates in B-cell non-Hodgkin lymphoma patients have been lower compared to Hodgkin lymphoma, with no activity reported in a recent trial of anti-PD-1 immunotherapy in relapsed CLL. This suggests that certain lymphoma subtypes may harbor non-immunogenic tumor microenvironments (TME).Our preliminary studies revealed that avadomide (CC-122), a cereblon modulator, can enhance T cell immune synapse signaling with autologous CLL cells, resulting in a concomitant increase in PD-1/PD-L1 expression at repaired synapses, indicating that avadomide may represent a complementary treatment partner for checkpoint inhibition. Here, we have extended our pre-clinical studies to investigate how these immunotherapy drugs alter the function and gene signatures of previously exhausted T cells from treatment naïve CLL patients (representing disease heterogeneity).Cytotoxicity assays revealed that treating primary T cells and autologous CLL cells with avadomide (1 μM, 48h) activated anti-tumor T cell killing function (P<.01, n=16), while combining avadomide with anti-PD-1 or anti-PD-L1 treatment significantly enhanced (P<.01) T cell killing function compared to these immunotherapies alone. Notably, anti-PD-1 monotherapy induced comparatively modest improvements of effector function.We next performed RNA sequencing on highly purified T cells from treatment naïve CLL patients, representing extremes of prognosis (n=6 good and n=6 poor including disease harboring TP53 abnormalities), following 18 h treatment with avadomide (100 nM) or anti-PD-1 (nivolumab) or PD-L1 (durvalumab) alone (10 μg/ml) or in combinations. Differential expression pathway analysis revealed that the top functional gene categories common for all the avadomide and combination treated samples (independent of anti-PD-1 or anti-PD-L1 monotherapy) were related to the response to type I and II IFN signaling, as well as inflammatory/stimulatory cytokine TNF-α, proliferative IL-6/JAK/STAT3, and IL-2/STAT5 responses. Type I IFN drives expression of chemokines CXCL10 and CXCL9, which are linked to enhanced tumor-infiltrated lymphocyte recruitment, and these chemoattractant genes were significantly upregulated in the avadomide and combination treated patient samples. In addition to their immunostimulatory roles, type II IFN (IFN-γ) and chronic type I IFN signaling have been linked to T cell resistance/exhaustion. We detected upregulated PD-L1 transcript in avadomide and combination treated T cells - supporting blockade of this inhibitory ligand.To investigate the ability of avadomide to modulate T cell migration, we performed comparative quantitative time-lapse microscopy analysis of pretreated patient T cells (n=12). These assays revealed that avadomide, as well as anti-PD-1 or anti-PD-L1 monotherapies, significantly enhanced (P<.01) T cell migration, and this response was significantly augmented by combination immunotherapy. Luminex protein analysis and cell migration assays confirmed the transcriptome data, highlighting the ability of avadomide and combination immunotherapy to significantly (P<.01) induce the release of CXCL10 (as well as IFN-γ, IL-2, GM-CSF and IL-8) from patient T cells, thereby attracting autologous T cells (CXCR3+). Avadomide as well as its pairing with anti-PD-L1 were also found to increase the proliferation of patient T cells (P<.01, n=6), particularly CD8+ T cells.Patient-derived xenograft models demonstrate that the therapeutic treatment of established tumors (3 weeks post-engraftment) with avadomide treatment (0.5 mg/kg) can activate anti-tumor T cells and significantly reduce disease volume (P<.01) that was associated with increased infiltration of PD-L1+ CD8+ T cells. Pairing avadomide with anti-PD-L1 durvalumab led to maximal activation of anti-tumor T cell immunity.In conclusion, our findings support the concept that PD-L1/PD-1 blockade in CLL could be enhanced when combined with avadomide through the promotion of immunogenic IFN signaling that enhances T cell infiltration and function. We believe these data support the rationale for combination immunotherapy that could convert a non-immunogenic TME into a ‘hot’ T cell inflamed TME which would be sensitive to checkpoint blockade. DisclosuresHagner:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Patten:L Hoffman La Roche: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, Research Funding; AbbVie Inc: Honoraria, Other: travel; Janssen: Honoraria, Other: travel. Vardi:Gilead: Research Funding; Janssen: Honoraria. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding.

The degree of postpartum metabolic challenge in dairy cows is associated with peripheral blood mononuclear cell transcriptome changes of the innate immune system

Dairy cows undergo a nutrient deficit immediately postpartum when lactational demands exceed nutrient intake. This occurs concurrently to an increased challenge due to bacterial and viral infections, yet ability for pathogen clearance is reduced despite a heightened and often host-damaging inflammatory response. We hypothesized that nutrient stress is associated with differences in the immune cell transcriptome. Our objective was therefore to investigate differentially expressed pathways (DEP) by RNA-seq in peripheral blood mononuclear cells harvested 3 weeks before and 1 week after calving from Holstein cows in low (L, n = 3) or high (H, n = 3) postpartum metabolic stress situations. Metabolic stress was defined by differences in circulating concentrations of glucose, fatty acids, and ketones postpartum. Cows in group H showed several upregulated DEP in relation to myeloid cell function and inflammatory response, as well as downregulation of the Th2 pathway. Principal components analysis showed that the transcriptome of group H postpartum samples was most different from all other samples. Differences in DE genes were noted even prepartum albeit fewer DE genes were identified and myeloid cell pathways in group H were generally downregulated at this time compared with group L. Samples within group L showed little difference between the two time points. We conclude that the metabolic phenotype of cows allowed us to identify differences in immune-regulatory pathways and that myeloid immune cells could play a dominant role in identifying these metabolically-associated differences that were demonstrated among a mixed mononuclear cell population.

DynSig: Modelling Dynamic Signaling Alterations along Gene Pathways for Identifying Differential Pathways.

Although a number of methods have been proposed for identifying differentially expressed pathways (DEPs), few efforts consider the dynamic components of pathway networks, i.e., gene links. We here propose a signaling dynamics detection method for identification of DEPs, DynSig, which detects the molecular signaling changes in cancerous cells along pathway topology. Specifically, DynSig relies on gene links, instead of gene nodes, in pathways, and models the dynamic behavior of pathways based on Markov chain model (MCM). By incorporating the dynamics of molecular signaling, DynSig allows for an in-depth characterization of pathway activity. To identify DEPs, a novel statistic of activity alteration of pathways was formulated as an overall signaling perturbation score between sample classes. Experimental results on both simulation and real-world datasets demonstrate the effectiveness and efficiency of the proposed method in identifying differential pathways.

CtPath: Demixing pathway crosstalk effect from transcriptomics data for differential pathway identification

Identifying differentially expressed pathways (DEPs) plays important roles in understanding tumor etiology and promoting clinical treatment of cancer or other diseases. By assuming gene expression to be a sparse non-negative linear combination of hidden pathway signals, we propose a pathway crosstalk-based transcriptomics data analysis method (ctPath) for identifying differentially expressed pathways. Biologically, pathways of different functions work in concert at the systematic level. The proposed method interrogates the crosstalks between pathways and discovers hidden pathway signals by mapping high-dimensional transcriptomics data into a low-dimensional pathway space. The resulted pathway signals reflect the activity level of pathways after removing pathway crosstalk effect and allow a robust identification of DEPs from inherently complex and noisy transcriptomics data. CtPath can also correct incomplete and inaccurate pathway annotations which frequently occur in public repositories. Experimental results on both simulation data and real-world cancer data demonstrate the superior performance of ctPath over other popular approaches. R code for ctPath is available for non-commercial use at the URL http://micblab.iim.ac.cn/Download/.

Combination of lentivirus-mediated silencing of PPM1D and temozolomide chemotherapy eradicates malignant glioma through cell apoptosis and cell cycle arrest.

Temozolomide (TMZ) is approved for use as first-line treatment for glioblastoma multiforme (GBM). However, GBM shows chemoresistance shortly after the initiation of treatment. In order to detect whether silencing of human protein phosphatase 1D magnesium dependent (PPM1D) gene could increase the effects of TMZ in glioma cells, glioma cells U87-MG were infected with lentiviral shRNA vector targeting PPM1D silencing. After PPM1D silencing was established, cells were treated with TMZ. The multiple functions of human glioma cells after PPM1D silencing and TMZ chemotherapy were detected by flow cytometry and MTT assay. Significantly differentially expressed genes were distinguished by microarray-based gene expression profiling and analyzed by gene pathway enrichment analysis and ontology assessment. Western blotting was used to establish the protein expression of the core genes. PPM1D gene silencing improves TMZ induced cell proliferation and induces cell apoptosis and cell cycle arrest. When PPM1D gene silencing combined with TMZ was performed in glioma cells, 367 genes were upregulated and 444 genes were downregulated compared with negative control. The most significant differential expression pathway was pathway in cancer and IGFR1R, PIK3R1, MAPK8 and EP300 are core genes in the network. Western blotting showed that MAPK8 and PIK3R1 protein expression levels were upregulated and RB1 protein expression was decreased. It was consistent with that detected in gene expression profiling. In conclusion, PPM1D gene silencing combined with TMZ eradicates glioma cells through cell apoptosis and cell cycle arrest. PIK3R1/AKT pathway plays a role in the multiple functions of glioma cells after PPM1D silencing and TMZ chemotherapy.

Bioinformatics analysis of differentially expressed pathways related to the metastatic characteristics of osteosarcoma.

In this study, gene expression data of osteosarcoma (OSA) were analyzed to identify metastasis-related biological pathways. Four gene expression data sets (GSE21257, GSE9508, GSE49003 and GSE66673) were downloaded from Gene Expression Omnibus (GEO). An analysis of differentially expressed genes (DEGs) was performed using the Significance Analysis of Microarray (SAM) method. Gene expression levels were converted into scores of pathways by the Functional Analysis of Individual Microarray Expression (FAIME) algorithm and the differentially expressed pathways (DEPs) were then disclosed by a t-test. The distinguishing and prediction ability of the DEPs for metastatic and non-metastatic OSA was further confirmed using the principal component analysis (PCA) method and 3 gene expression data sets (GSE9508, GSE49003 and GSE66673) based on the support vector machines (SVM) model. A total of 616 downregulated and 681 upregulated genes were identified in the data set, GSE21257. The DEGs could not be used to distinguish metastatic OSA from non-metastatic OSA, as shown by PCA. Thus, an analysis of DEPs was further performed, resulting in 14 DEPs, such as NRAS signaling, Toll-like receptor (TLR) signaling, matrix metalloproteinase (MMP) regulation of cytokines and tumor necrosis factor receptor-associated factor (TRAF)-mediated interferon regulatory factor 7 (IRF7) activation. Cluster analysis indicated that these pathways could be used to distinguish between metastatic OSA from non-metastatic OSA. The prediction accuracy was 91, 66.7 and 87.5% for the data sets, GSE9508, GSE49003 and GSE66673, respectively. The results of PCA further validated that the DEPs could be used to distinguish metastatic OSA from non-metastatic OSA. On the whole, several DEPs were identified in metastatic OSA compared with non-metastatic OSA. Further studies on these pathways and relevant genes may help to enhance our understanding of the molecular mechanisms underlying metastasis and may thus aid in the development of novel therapies.