Development of a novel protein identification approach to define mitochondrial proteomic signatures in glioblastoma oncogenesis: T98G vs U87MG cell lines model

AbstractGlioblastoma Multiforme is a cancer type with an important mitochondrial component. Here was used mitochondrial proteome Random Sampling in 2D gels from T98G (oxidative metabolism) and U87MG (glycolytic metabolism) cell lines to obtain and analyze representative spots (regardless of their intensity, size, or difference in abundance between cell lines) by Principal Component Analysis for protein identification. Identified proteins were ordered into specific Protein-Protein Interaction networks, to each cell line, showing mitochondrial processes related to metabolic change, invasion, and metastasis; and other nonmitochondrial processes such as DNA translation, chaperone response, and autophagy in gliomas. T98G and U87MG cell lines were used as glioblastoma transition model; representative proteomic signatures, with the most important biological processes in each cell line, were defined. This pipeline analysis describes the metabolic status of each line and defines clear mitochondria performance differences for distinct glioblastoma stages, introducing a new useful strategy for the understanding of glioblastoma carcinogenesis formation.Biological significanceThis study defines the mitochondria as an organelle that follows and senses the carcinogenesis process by an original proteomic approach, a random sampling in 2DE gels to obtain a representative spots sample and analyzing their relative abundance by Principal Components Analysis; to faithfully describe glioblastoma cells biology.

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Quantitative cell fusion: the fusion sensitivity (FS) potential

Journal of Cell Science ◽

10.1242/jcs.49.1.87 ◽

1981 ◽

Vol 49 (1) ◽

pp. 87-97

Author(s):

D. Rohme

Keyword(s):

Cell Line ◽

Cell Fusion ◽

Cell Lines ◽

Dose Response ◽

Sendai Virus ◽

Biological Significance ◽

Diploid Cell ◽

Mammalian Cell Lines ◽

Virus Dose ◽

A Cell

The dose response of Sendai virus-induced cell fusion was studied in 10 mammalian cell lines, comprising 5 continuous and 5 diploid cell lines originating from 5 species. The extent of fusion was calculated using a parameter directly proportional to the number of fusion events (t-parameter). At lower levels of fusion the dose response was found to be based on the same simple kinetic rules in all cell lines and was defined by the formula: t = FS. FAU/(I + FS. FAU), where FS (fusion sensitivity) is a cell-specific constant of the fusion rate and FAU (fusion activity units) is the virus dose. The FS potential of a cell line was determined as the linear regression coefficient of the fusion index (t/(I - t)) on the virus dose. At higher levels of fusion, when the fusion extent reached cell-line-specific maximal levels, the dose response was not as uniform. In general, and particularly in the cases of the diploid cell lines, these maximal levels were directly proportional to the FS potentials. Thus, it was concluded that the FS potential is the basic quantitative feature, which expresses the cellular fusion efficiency. The fact that FS varied extensively between cell lines, but at the same time apparently followed certain patterns (being higher in continuous compared to diploid cell lines and being related to the species of origin of the cells), emphasizes it biological significance as well as its possible usefulness in studies of the efficiency of various molecular interactions in the cell membrane/cytoskeleton system.

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Regulatory landscapes of specific miRNAs are conserved between cell lines and primary tumors

F1000Research ◽

10.12688/f1000research.52478.1 ◽

2021 ◽

Vol 10 ◽

pp. 633

Author(s):

Hanwen Zhu ◽

Boting Ning

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Mirna Expression ◽

Regulatory Network ◽

Human Cancer ◽

Cancer Cell Line ◽

Cancer Type ◽

Cancer Tissues

Background: MicroRNAs are essential gene expression regulators and play important roles in various biological processes, such as cancer. They have shown great translational promise as either diagnostic biomarkers or therapeutic targets. While the similarities between transcriptomic profiles from The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia have been thoroughly studied before, less is known on the microRNA side. This project aims to provide critical biological knowledge on the extent of consensus microRNA expression and regulation between cell line models and primary human tumors. Method: First, we examined the similarity of miRNA expression profiles between CCLE cell lines and TCGA tumor samples for each cancer type. Next, we compared the expression of miRNAs associating the hallmarks of cancer pathways. Finally, we constructed miRNA-mRNA regulatory network for each cancer type and evaluated whether the regulatory role of each miRNA is conserved between cell lines and tumor samples. Results: Our results indicate that, similar to gene expression, how well cancer cell line microRNA expression would capture the transcriptomic profile of human cancer tissues is greatly affected by the tumor type and purity. The cell-type composition for a cancer type also affects how accurately cancer cell lines could reflect the miRNA expression in tumor tissues. Furthermore, through network analysis, we show that certain microRNAs, not all, regulate the same set of target genes in both the cell line and human cancer tissues. Conclusions: Through systematically comparing the miRNA expression profile and the regulatory network, our study highlights the biological differences between cell line and tumor samples and provides resources for future miRNA and cancer studies.

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Matching cell lines with cancer type and subtype of origin via mutational, epigenomic and transcriptomic patterns

10.1101/809400 ◽

2019 ◽

Author(s):

Marina Salvadores ◽

Francisco Fuster-Tormo ◽

Fran Supek

Keyword(s):

Cell Line ◽

Cell Lines ◽

Experimental Work ◽

Drug Sensitivity ◽

Tissue Cell ◽

Cancer Type ◽

Tumor Type ◽

Cell Type ◽

Cancer Subtypes ◽

The One

AbstractCell lines are commonly used as cancer models. Because the tissue and/or cell type of origin provide important context for understanding mechanisms of cancer, we systematically examined whether cell lines exhibit features matching the cancer type that supposedly originated them. To this end, we aligned the mRNA expression and DNA methylation data between ∼9,000 solid tumors and ∼600 cell lines to remove the global differences stemming from growth in cell culture. Next, we created classification models for cancer type and subtype using tumor data, and applied them to cell line data. Overall, the transcriptomic and epigenomic classifiers consistently identified 35 cell lines which better matched a different tissue or cell type than the one the cell line was originally annotated with; we recommend caution in using these cell lines in experimental work. Six cell lines were identified as originating from the skin, of which five were further corroborated by the presence of a UV-like mutational signature in their genome, strongly suggesting mislabelling. Overall, genomic evidence additionally supports that 22 (3.6% of all considered) cell lines may be mislabelled because we predict they originate from a different tissue/cell type. Finally, we cataloged 366 cell lines in which both transcriptomic and epigenomic profiles strongly resemble the tumor type of origin, designating them as ‘golden set’ cell lines. We suggest these cell lines are better suited for experimental work that depends on tissue identity and propose tentative assignments to cancer subtypes. Finally, we show that accounting for the uncertain tissue-of-origin labels can change the interpretation of drug sensitivity and CRISPR genetic screening data. In particular, in brain, lung and pancreatic cancer cell lines, many novel determinants of drug sensitivity or resistance emerged by focussing on the cell lines that are best matched to the cancer type of interest.

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A Study of the Lipidomic Profiles of the CAL-27 and HOK Cell Lines Using EMS Spectra

Frontiers in Oncology ◽

10.3389/fonc.2021.771337 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xue-ying Wang ◽

Ting Zhang ◽

Wei-qun Guan ◽

Hua-zhu Li ◽

Ling Lin

Keyword(s):

Cell Line ◽

Cell Lines ◽

Metabolic Pathway ◽

High Performance ◽

Bioinformatics Analysis ◽

Tongue Cancer ◽

Cancer Cell Line ◽

Principal Component ◽

Metabolic Pathway Analysis ◽

Human Tongue

ObjectiveThe aim of this study was to explore the lipidomic profiles of the CAL-27 human tongue cancer cell line and the human oral keratinocyte (HOK) cell line.MethodsThe lipidomic differences between the CAL-27 and the HOK cell lines were investigated using non-targeted high-performance liquid chromatography–mass spectrometry lipidomic analysis. The resulting data were then further mined via bioinformatics analysis technology and metabolic pathway analysis was conducted in order to map the most affected metabolites and pathways in the two cell lines.ResultsA total of 711 lipids were identified, including 403 glycerophospholipids (GPs), 147 glycerolipids, and 161 sphingolipids. Comparison of the enhanced MS (EMS) spectra of the two cell lines in positive and negative ionization modes showed the lipid compositions of HOK and CAL-27 cells to be similar. The expressions of most GP species in CAL-27 cells showed an increasing trend as compared with HOK, whereas a significant increase in phosphatidylcholine was observed (p < 0.05). Significant differences in the lipid composition between CAL-27 and HOK cells were shown as a heatmap. Through principal component analysis and orthogonal partial least squares discriminant analysis, noticeably clear separation trends and satisfactory clustering trends between groups of HOK and CAL-27 cells were identified. The numbers of specific lipid metabolites that could distinguish CAL-27 from HOK in positive and negative modes were 100 and 248, respectively. GP metabolism was the most significantly altered lipid metabolic pathway, with 4 metabolites differentially expressed in 39 hit products.ConclusionThis study demonstrated the potential of using untargeted mass spectra and bioinformatics analysis to describe the lipid profiles of HOK and CAL-27 cells.

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Synthetic lethality of LP-184, a next generation acylfulvene, in ex vivo PDX models with homologous recombination defects.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15064 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15064-e15064

Author(s):

Aditya Kulkarni ◽

Diana Restifo ◽

Igor A. Astsaturov ◽

Umesh Kathad ◽

Joseph McDermott ◽

...

Keyword(s):

Homologous Recombination ◽

Cell Line ◽

Cell Lines ◽

Synthetic Lethality ◽

Ex Vivo ◽

Clinical Success ◽

Tumor Growth Inhibition ◽

Cytotoxic Action ◽

Cancer Type ◽

Pdx Models

e15064 Background: The clinical success of PARP inhibitors (PARPi) in homologous recombination (HR) deficient (HRD+) solid tumors has broadened the scope of identifying additional agents and vulnerabilities in cancers with DNA repair deficiencies. However, with more than 40% of BRCA1/2-deficient patients failing to respond to PARPi or acquiring resistance with prolonged PARPi administration, newer agents are also needed. LP-184, an acylfulvene, is a prodrug activated by PTGR1. Threshold expression levels of PTGR1 are higher in several tumors, providing a window of specificity for its cytotoxic action. DNA damage inflicted by acylfulvene (AF) agents is reliant upon HR pathway genes including BRCA1 for correction and removal. We hypothesized that tumors with high PTGR1 expression and HR deficiency will therefore be uniquely targeted and demonstrate synthetic lethality when exposed to LP-184. Methods: We evaluated ex vivo antitumor activity of LP-184 in selected PDX models representing lung, pancreatic and prostate cancers with high PTGR1 and known HR defects. Dissociated tumor fragments were treated with LP-184 across a concentration range of 5 nM to 36 uM for 5 days. Cell viability was quantified by CellTiter Glo. LP-184 IC50s were compared with PARPi efficacy. We further confirmed the dependency of PTGR1 in HR deficient tumor cells by comparing LP-184 sensitivity in the BRCA2 deficient cell line CAPAN-1 and the ATM mutant cell line PANC03.27, with and without PTGR1 suppression following an engineered CRISPR knockout of PTGR1. We also analyzed TCGA data to estimate the percentage of tumors with elevated PTGR1 and co-occurring damaging mutations in a panel of 60 HR genes. Results: The mean LP-184 IC50 across 15 HRD+ PDX models tested was 288 nM (range 31 - 2900 nM). LP-184 turned out to be 6 - 340X more potent ex vivo than the PARPi Olaparib in these models. 9 of 15 models were associated with no clinical response to or initial response followed by progression on approved standard of care (SOC) agents. 6 of 15 models showed < 10% tumor growth inhibition in vivo with SOC treatment. Regardless of cancer type, models with high-impact, loss-of-function mutations in ATM, ATR and BRCA1 showed exquisite sensitivity to LP-184 (mean IC50 ̃ 60 nM). CRISPRi-mediated stable suppression of PTGR1 in the pancreatic cancer cell lines CAPAN-1 and PANC03.27 entirely abrogated LP-184 sensitivity relative to isogenic parental cell lines. 17.6% of lung adenocarcinomas (n = 517), 4.5% of pancreatic adenocarcinomas (n = 179) and 9.6% of prostate adenocarcinomas (n = 498) displayed elevated PTGR1 along with damaging HR related mutations, and are likely to be responsive to LP-184 based on analysis of TCGA data. Conclusions: LP-184 is broadly effective in HRD+ tumors that may be less responsive to SOC including PARPi and could be useful clinically in a subset of tumors with high PTGR1 and HR defects.

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An In Vitro and In Vivo Evidence That Downregulation of Leukemia Inhibitory Factor (LIF) Receptor (LIF-R) Decreases the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Blood ◽

10.1182/blood.v108.11.2561.2561 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2561-2561

Author(s):

Marcin Wysoczynski ◽

Katarzyna Miekus ◽

Anna Marcinkowska ◽

Anna Janowska-Wieczorek ◽

Mariusz Z. Ratajczak

Keyword(s):

Skeletal Muscle ◽

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Leukemia Inhibitory Factor ◽

Biological Significance ◽

Human Cells ◽

Inhibitory Factor

Abstract Rhabdomyosarcoma (RMS) and skeletal muscle-derived tumors frequently infiltrate bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1-CXCR4 receptor (Blood2002;100:2597) and hepatocyte growth factor (HGF)-c-Met receptor (Cancer Res. 2003;63:7926) play an important role in RMS metastasis to BM. Leukemia inhibitory factor (LIF) is a well known factor that plays an important role in skeletal muscle development/regeneration and similarly as SDF-1 and HGF is secreted by BM stroma. This prompted us to examine whether the LIF-LIF receptor (LIF-R) axis affects the biology/metastasis of RMS cells. We employed in our studies, human established RMS cell lines, as well as RMS samples isolated from patients and noticed that LIF-R was expressed not only on established human RMS cell lines (7/7) but more importantly, it was also detectable in patient samples (23/23). We also found that in RMS cells LIF stimulatesphosphorylation of MAPKp42/44, AKT and STAT3,chemotaxis and adhesion andincreases resistance to cytostatics (e.g., etoposide). These LIF-mediated effects were inhibited after downregulating the LIF-R by siRNA. To learn more on the biological significance of the LIF-LIF-R axis in vivo we employed two models. First, human RMS cells (RH-30) were exposed or not exposed to LIF-R siRNA and subsequently injected into SCID™-Beige immunodeficient mice. To estimate the number of RMS cells that seed to BM and liver in these animals, we isolated DNA and using real- time RT-PCR, amplified human a-satellite sequences and murine b-actin. The number of human cells present in murine organs was subsequently calculated from a standard curve derived from mixing varying numbers of human cells with a constant number of murine cells. We noticed that downregulation of LIF-R by siRNA significantly decreased the number of human RMS cells in murine BM and liver (x4 and x2 respectively). In a second model, the RH30 cell line was selected by repetitive chemotaxis for cells that are highly responsive to LIF (RH-30 L) and subsequently the cells from parental RH-30 cell line and RH-30 L cells were injected intramuscularly. Six weeks after tumour inoculation, we detected more metastasis in bone marrow and lungs in mice injected with RH-30L cells as compared to parental RH-30 clone (x6 and x3 respectively). In conclusion, we present evidence for the first time that the inhibition of LIF-LIF-R axis may decrease the invasive potential of human RMS both in vitro and in vivo. Hence, molecular targeting of LIF-LIF-R axis could possibly become a more effective new strategy to control the progression and metastasis of RMS.

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Induction of CD45 Expression and Proliferation in U-266 Myeloma Cell Line by Interleukin-6

Blood ◽

10.1182/blood.v92.10.3887.422a48_3887_3897 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3887-3897 ◽

Cited By ~ 1

Author(s):

Maged S. Mahmoud ◽

Hideaki Ishikawa ◽

Ryuichi Fujii ◽

Michio M. Kawano

Keyword(s):

Cell Line ◽

Expression Pattern ◽

Cell Lines ◽

Interleukin 6 ◽

Cellular Proliferation ◽

Tyrosine Phosphatase ◽

Biological Significance ◽

Myeloma Cell ◽

Myeloma Cells ◽

Gradual Loss

Recently, there has been an increasing interest in the expression pattern and biological significance of the CD45 molecule in myeloma cells. In this study, we have further defined the phenotypic pattern of CD45 expression on myeloma cells. Using a panel of myeloma cell lines, we showed that CD45 showed a remarkably heterogeneous pattern of expression. Whereas some cell lines were CD45+ and others were CD45−, the U-266 cell line, although predominantly CD45−, still had a considerable subpopulation of CD45+ cells. Among the myeloma cell lines examined, there was a direct correlation between interleukin-6 (IL-6) dependency and CD45 positivity. Moreover, we showed that IL-6 stimulation led to the induction of expression of CD45 and cellular proliferation. Using independent experimental approaches, we could show that the IL-6–induced effects were closely linked to CD45 expression. First, sorting out CD45+ and CD45− subsets of U-266 cell line followed by IL-6 stimulation, only the CD45+ cells showed a proliferative advantage after IL-6 stimulation. Second, IL-6 stimulation of sorted CD45−cells was gradually followed by phenotypic conversion to CD45+ cells that started after 2 days as judged by the detection of CD45 mRNA by reverse transcription polymerase chain reaction (RT-PCR) and immunophenotypic analysis by flow cytometry. Withdrawal of IL-6 from the medium led to gradual loss of CD45 expression in CD45+ flow-sorted U-266 cells. Third, the use of vanadate, a potent inhibitor of protein tyrosine phosphatase (PTP), abrogated the IL-6–induced proliferation in the CD45+ myeloma cells. On the other hand, cellular proliferation induced by IL-6 was not affected by the serine-threonine phosphatase inhibitor okadaic acid. Our data show that the expression pattern of CD45 in myeloma cell lines is heterogeneous and show for the first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in myeloma by determining the proliferative population among myeloma cells.

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Genomic data integration by WON-PARAFAC identifies interpretable factors for predicting drug-sensitivity in vivo

Nature Communications ◽

10.1038/s41467-019-13027-2 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Yongsoo Kim ◽

Tycho Bismeijer ◽

Wilbert Zwart ◽

Lodewyk F. A. Wessels ◽

Daniel J. Vis

Keyword(s):

Data Integration ◽

Cell Line ◽

Cell Lines ◽

Cancer Biology ◽

Drug Response ◽

Genomic Data ◽

Response Prediction ◽

Molecular Data ◽

Cancer Type

Abstract Integrative analyses that summarize and link molecular data to treatment sensitivity are crucial to capture the biological complexity which is essential to further precision medicine. We introduce Weighted Orthogonal Nonnegative parallel factor analysis (WON-PARAFAC), a data integration method that identifies sparse and interpretable factors. WON-PARAFAC summarizes the GDSC1000 cell line compendium in 130 factors. We interpret the factors based on their association with recurrent molecular alterations, pathway enrichment, cancer type, and drug-response. Crucially, the cell line derived factors capture the majority of the relevant biological variation in Patient-Derived Xenograft (PDX) models, strongly suggesting our factors capture invariant and generalizable aspects of cancer biology. Furthermore, drug response in cell lines is better and more consistently translated to PDXs using factor-based predictors as compared to raw feature-based predictors. WON-PARAFAC efficiently summarizes and integrates multiway high-dimensional genomic data and enhances translatability of drug response prediction from cell lines to patient-derived xenografts.

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Multiple Localization of Endogenous MARK4L Protein in Human Glioma

Analytical Cellular Pathology ◽

10.1155/2009/345781 ◽

2009 ◽

Vol 31 (5) ◽

pp. 357-370

Author(s):

Ivana Magnani ◽

Chiara Novielli ◽

Melissa Bellini ◽

Gaia Roversi ◽

Lorenzo Bello ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Glioma Cell ◽

Cellular Localization ◽

Protein Identification ◽

Glioblastoma Cell Line ◽

Glioblastoma Cell ◽

Human Glioma ◽

Glioma Cell Lines ◽

Biochemical Fractionation

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma.Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines.Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines.Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.

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Proteomic Comparison of Malignant Human Germ Cell Tumor Cell Lines

Disease Markers ◽

10.1155/2019/8298524 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14

Author(s):

Felix Bremmer ◽

Hanibal Bohnenberger ◽

Stefan Küffer ◽

Thomas Oellerich ◽

Hubert Serve ◽

...

Keyword(s):

Germ Cell ◽

Cell Line ◽

Cell Lines ◽

Germ Cell Tumor ◽

Embryonal Carcinoma ◽

Germ Cell Tumors ◽

Cell Tumor ◽

Antiproliferative Effect ◽

Correct Identification ◽

Proteomic Approach

Malignant germ cell tumors (GCT) are the most common malignant tumors in young men between 18 and 40 years. The correct identification of histological subtypes, in difficult cases supported by immunohistochemistry, is essential for therapeutic management. Furthermore, biomarkers may help to understand pathophysiological processes in these tumor types. Two GCT cell lines, TCam-2 with seminoma-like characteristics, and NTERA-2, an embryonal carcinoma-like cell line, were compared by a quantitative proteomic approach using high-resolution mass spectrometry (MS) in combination with stable isotope labelling by amino acid in cell culture (SILAC). We were able to identify 4856 proteins and quantify the expression of 3936. 347 were significantly differentially expressed between the two cell lines. For further validation, CD81, CBX-3, PHF6, and ENSA were analyzed by western blot analysis. The results confirmed the MS results. Immunohistochemical analysis on 59 formalin-fixed and paraffin-embedded (FFPE) normal and GCT tissue samples (normal testis, GCNIS, seminomas, and embryonal carcinomas) of these proteins demonstrated the ability to distinguish different GCT subtypes, especially seminomas and embryonal carcinomas. In addition, siRNA-mediated knockdown of these proteins resulted in an antiproliferative effect in TCam-2, NTERA-2, and an additional embryonal carcinoma-like cell line, NCCIT. In summary, this study represents a proteomic resource for the discrimination of malignant germ cell tumor subtypes and the observed antiproliferative effect after knockdown of selected proteins paves the way for the identification of new potential drug targets.

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