| 1. |
- Godoy, Patricio, et al.
(författare)
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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME
- 2013
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 87:8, s. 1315-1530
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Forskningsöversikt (refereegranskat)abstract
- This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4 alpha, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4 alpha), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
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| 2. |
- Krebs, Alice, et al.
(författare)
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The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods
- 2020
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 94:7, s. 2435-2461
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Tidskriftsartikel (refereegranskat)abstract
- Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.
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| 3. |
- Botling, Johan, et al.
(författare)
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Biomarker Discovery in Non-Small Cell Lung Cancer : Integrating Gene Expression Profiling, Meta-analysis, and Tissue Microarray Validation
- 2013
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Ingår i: Clinical Cancer Research. - 1078-0432 .- 1557-3265. ; 19:1, s. 194-204
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multigene signatures in clinical practice is unclear, and the biologic importance of individual genes is difficult to assess, as the published signatures virtually do not overlap.Experimental Design: Here, we describe a novel single institute cohort, including 196 non-small lung cancers (NSCLC) with clinical information and long-term follow-up. Gene expression array data were used as a training set to screen for single genes with prognostic impact. The top 450 probe sets identified using a univariate Cox regression model (significance level P < 0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n = 860).Results: The meta-analysis revealed 14 genes that were significantly associated with survival (P < 0.001) with a false discovery rate < 1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on tissue microarrays from 2 independent NSCLC cohorts, altogether including 617 NSCLC samples. Low CADM1 protein expression was significantly associated with shorter survival, with particular influence in the adenocarcinoma patient subgroup.Conclusions: Using a novel NSCLC cohort together with a meta-analysis validation approach, we have identified a set of single genes with independent prognostic impact. One of these genes, CADM1, was further established as an immunohistochemical marker with a potential application in clinical diagnostics. Clin Cancer Res; 19(1); 194-204. (c) 2012 AACR.
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| 4. |
- Figueroa, Jonine D., et al.
(författare)
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Identification of a novel susceptibility locus at 13q34 and refinement of the 20p12.2 region as a multi-signal locus associated with bladder cancer risk in individuals of European ancestry
- 2016
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Ingår i: Human Molecular Genetics. - : Oxford University Press. - 0964-6906 .- 1460-2083. ; 25:6, s. 1203-1214
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Tidskriftsartikel (refereegranskat)abstract
- Candidate gene and genome-wide association studies (GWAS) have identified 15 independent genomic regions associated with bladder cancer risk. In search for additional susceptibility variants, we followed up on four promising single-nucleotide polymorphisms (SNPs) that had not achieved genome-wide significance in 6911 cases and 11 814 controls (rs6104690, rs4510656, rs5003154 and rs4907479, P < 1 × 10−6), using additional data from existing GWAS datasets and targeted genotyping for studies that did not have GWAS data. In a combined analysis, which included data on up to 15 058 cases and 286 270 controls, two SNPs achieved genome-wide statistical significance: rs6104690 in a gene desert at 20p12.2 (P = 2.19 × 10−11) and rs4907479 within the MCF2L gene at 13q34 (P = 3.3 × 10−10). Imputation and fine-mapping analyses were performed in these two regions for a subset of 5551 bladder cancer cases and 10 242 controls. Analyses at the 13q34 region suggest a single signal marked by rs4907479. In contrast, we detected two signals in the 20p12.2 region—the first signal is marked by rs6104690, and the second signal is marked by two moderately correlated SNPs (r2 = 0.53), rs6108803 and the previously reported rs62185668. The second 20p12.2 signal is more strongly associated with the risk of muscle-invasive (T2-T4 stage) compared with non-muscle-invasive (Ta, T1 stage) bladder cancer (case–case P ≤ 0.02 for both rs62185668 and rs6108803). Functional analyses are needed to explore the biological mechanisms underlying these novel genetic associations with risk for bladder cancer.
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| 5. |
- Keller, Magdalena, et al.
(författare)
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Inhibiting the glycerophosphodiesterase EDI3 in ER-HER2+breast cancer cells resistant to HER2-targeted therapy reduces viability and tumour growth
- 2023
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Ingår i: Journal of Experimental & Clinical Cancer Research. - : BioMed Central (BMC). - 1756-9966. ; 42
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Tidskriftsartikel (refereegranskat)abstract
- Background: Intrinsic or acquired resistance to HER2-targeted therapy is often a problem when small molecule tyrosine kinase inhibitors or antibodies are used to treat patients with HER2 positive breast cancer. Therefore, the identification of new targets and therapies for this patient group is warranted. Activated choline metabolism, characterized by elevated levels of choline-containing compounds, has been previously reported in breast cancer. The glycerophosphodiesterase EDI3 (GPCPD1), which hydrolyses glycerophosphocholine to choline and glycerol-3-phosphate, directly influences choline and phospholipid metabolism, and has been linked to cancer-relevant phenotypes in vitro. While the importance of choline metabolism has been addressed in breast cancer, the role of EDI3 in this cancer type has not been explored.Methods: EDI3 mRNA and protein expression in human breast cancer tissue were investigated using publicly-available Affymetrix gene expression microarray datasets (n = 540) and with immunohistochemistry on a tissue microarray (n = 265), respectively. A panel of breast cancer cell lines of different molecular subtypes were used to investigate expression and activity of EDI3 in vitro. To determine whether EDI3 expression is regulated by HER2 signalling, the effect of pharmacological inhibition and siRNA silencing of HER2, as well as the influence of inhibiting key components of signalling cascades downstream of HER2 were studied. Finally, the influence of silencing and pharmacologically inhibiting EDI3 on viability was investigated in vitro and on tumour growth in vivo.Results: In the present study, we show that EDI3 expression is highest in ER-HER2 + human breast tumours, and both expression and activity were also highest in ER-HER2 + breast cancer cell lines. Silencing HER2 using siRNA, as well as inhibiting HER2 signalling with lapatinib decreased EDI3 expression. Pathways downstream of PI3K/Akt/mTOR and GSK3 beta, and transcription factors, including HIF1 alpha, CREB and STAT3 were identified as relevant in regulating EDI3 expression. Silencing EDI3 preferentially decreased cell viability in the ER-HER2 + cells. Furthermore, silencing or pharmacologically inhibiting EDI3 using dipyridamole in ER-HER2 + cells resistant to HER2-targeted therapy decreased cell viability in vitro and tumour growth in vivo.Conclusions: Our results indicate that EDI3 may be a potential novel therapeutic target in patients with HER2-targeted therapy-resistant ER-HER2 + breast cancer that should be further explored.
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| 6. |
- Mattsson, Johanna Sofia Margareta, 1985-, et al.
(författare)
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Prognostic impact of COX-2 in non-small cell lung cancer : a comprehensive compartment-specific evaluation of tumor and stromal cell expression
- 2015
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Ingår i: Cancer Letters. - : Elsevier BV. - 0304-3835 .- 1872-7980. ; 356:2, s. 837-845
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Tidskriftsartikel (refereegranskat)abstract
- Cyclooxygenase-2 (COX-2) is an enzyme that has been extensively investigated as a prognostic marker in cancer. In non-small cell lung cancer (NSCLC) previous results regarding the prognostic impact of COX-2 expression are inconsistent. Therefore we evaluated the association between transcript levels and overall survival in nine publicly available gene expression data sets (total n = 1337) and determined in situ compartment-specific tumor and stromal cell protein expression in two independent cohorts (n = 616). Gene expression did not show any correlation with clinical parameters or with overall survival. Protein expression in tumor and stromal cells did not correlate with any clinical parameter or with overall survival in one of the analyzed cohorts, while a significant association of high stromal expression with longer survival was observed in both univariate and multivariate analysis in the other cohort. Stromal expression of COX-2 has not been separately evaluated in NSCLC previously and may be a subject of further investigation, whereas the presented findings from this comprehensive compartment specific evaluation clearly reject the hypothesis of COX-2 tumor cell expression having a prognostic value in NSCLC. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
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| 7. |
- Rafnar, Thorunn, et al.
(författare)
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European genome-wide association study identifies SLC14A1 as a new urinary bladder cancer susceptibility gene.
- 2011
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Ingår i: Human molecular genetics. - : Oxford University Press (OUP). - 1460-2083 .- 0964-6906. ; 20:21, s. 4268-81
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Tidskriftsartikel (refereegranskat)abstract
- Three genome-wide association studies in Europe and the USA have reported eight urinary bladder cancer (UBC) susceptibility loci. Using extended case and control series and 1000 Genomes imputations of 5 340 737 single-nucleotide polymorphisms (SNPs), we searched for additional loci in the European GWAS. The discovery sample set consisted of 1631 cases and 3822 controls from the Netherlands and 603 cases and 37 781 controls from Iceland. For follow-up, we used 3790 cases and 7507 controls from 13 sample sets of European and Iranian ancestry. Based on the discovery analysis, we followed up signals in the urea transporter (UT) gene SLC14A. The strongest signal at this locus was represented by a SNP in intron 3, rs17674580, that reached genome-wide significance in the overall analysis of the discovery and follow-up groups: odds ratio = 1.17, P = 7.6 × 10(-11). SLC14A1 codes for UTs that define the Kidd blood group and are crucial for the maintenance of a constant urea concentration gradient in the renal medulla and, through this, the kidney's ability to concentrate urine. It is speculated that rs17674580, or other sequence variants in LD with it, indirectly modifies UBC risk by affecting urine production. If confirmed, this would support the 'urogenous contact hypothesis' that urine production and voiding frequency modify the risk of UBC.
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| 8. |
- Djureinovic, Dijana, et al.
(författare)
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Profiling cancer testis antigens in non-small-cell lung cancer
- 2016
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Ingår i: JCI INSIGHT. - : American Society for Clinical Investigation. - 2379-3708. ; 1:10
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Tidskriftsartikel (refereegranskat)abstract
- Cancer testis antigens (CTAs) are of clinical interest as biomarkers and present valuable targets for immunotherapy. To comprehensively characterize the CTA landscape of non-small-cell lung cancer (NSCLC), we compared RNAseq data from 199 NSCLC tissues to the normal transcriptome of 142 samples from 32 different normal organs. Of 232 CTAs currently annotated in the Caner Testis Database (CTdatabase), 96 were confirmed in NSCLC. To obtain an unbiased CTA profile of NSCLC, we applied stringent criteria on our RNAseq data set and defined 90 genes as CTAs, of which 55 genes were not annotated in the CTdatabase, thus representing potential new CTAs. Cluster analysis revealed that CTA expression is histology dependent and concurrent expression is common. IHC confirmed tissue-specific protein expression of selected new CTAs (TKTL1, TGIF2LX, VCX, and CXORF67). Furthermore, methylation was identified as a regulatory mechanism of CTA expression based on independent data from The Cancer Genome Atlas. The proposed prognostic impact of CTAs in lung cancer was not confirmed, neither in our RNAseq cohort nor in an independent meta-analysis of 1,117 NSCLC cases. In summary, we defined a set of 90 reliable CTAs, including information on protein expression, methylation, and survival association. The detailed RNAseq catalog can guide biomarker studies and efforts to identify targets for immunotherapeutic strategies.
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| 9. |
- Edlund, Karolina, et al.
(författare)
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Prognostic Impact of Tumor Cell Programmed Death Ligand 1 Expression and Immune Cell Infiltration in NSCLC
- 2019
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Ingår i: Journal of Thoracic Oncology. - : Elsevier BV. - 1556-0864 .- 1556-1380. ; 14:4, s. 628-640
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Infiltration of T and B/plasma cells has been linked to NSCLC prognosis, but this has not been thoroughly investigated in relation to the expression of programmed death ligand 1 (PD-L1). Here, we determine the association of lymphocytes and PD-L1 with overall survival (OS) in two retrospective cohorts of operated NSCLC patients who were not treated with checkpoint inhibitors targeting the programmed death 1/PD-L1 axis. Moreover, we evaluate how PD-L1 positivity and clinicopathologic factors affect the prognostic association of lymphocytes.Methods: Cluster of differentiation (CD) 3 (CD3)-, CD8-, CD4-, forkhead box P3 (FOXP3)-, CD20-, CD79A-, and immunoglobulin kappa constant (IGKC)-positive immune cells, and tumor PD-L1 positivity, were determined by immunohistochemistry on tissue microarrays (n = 705). Affymetrix data was analyzed for a patient subset, and supplemented with publicly available transcriptomics data (N = 1724). Associations with OS were assessed by Kaplan-Meier plots and uni- and multivariate Cox regression.Results: Higher levels of T and B plasma cells were associated with longer OS (p = 0.004 and p < 0.001, for CD8 and IGKC, respectively). Highly proliferative tumors with few lymphocytes had the worst outcome. No association of PD-L1 positivity with OS was observed in a nonstratified patient population; however, a significant association with shorter OS was observed in never-smokers (p = 0.009 and p = 0.002, 5% and 50% cutoff). Lymphocyte infiltration was not associated with OS in PD-L1–positive tumors (50% cutoff). The prognostic association of lymphocyte infiltration also depended on the patients’ smoking history and histologic subtype.Conclusions: Proliferation, PD-L1 status, smoking history, and histology should be considered if lymphocyte infiltration is to be used as a prognostic biomarker.
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| 10. |
- Godoy, Patricio, et al.
(författare)
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Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells
- 2015
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Ingår i: Journal of Hepatology. - : Elsevier. - 0168-8278 .- 1600-0641. ; 63:4, s. 934-942
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND & AIMS: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories.METHODS: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified.RESULTS: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC.CONCLUSIONS: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation.
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