GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B‐lineage ALL GC‐sensitive cell line, PreB 697, and its GC‐resistant sub‐line, R3F9, pre‐ and post‐dexamethasone exposure. PAX5, a transcription factor critical to B‐cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC‐sensitive parent and confirmed to be lower in other GC‐resistant sub‐lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC‐resistance was associated with differentiation from preB‐II to an immature B‐lymphocyte stage. GC‐resistant sub‐lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re‐sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach.Glucocorticoid ( Acute lymphoblastic leukaemia (ALL) is the most common cancer in children and is characterized by the clonal expansion of poorly differentiated lymphoid precursors within the bone marrow. The treatment of ALL is a highly complex protocol comprising several chemotherapeutic agents, of which glucocorticoids (GCs) are integral due to their ability to specifically induce apoptosis in immature lymphoid cells , causing impaired receptor function , coupled with two‐dimensional liquid chromatography/tandem mass spectrometry (LC‐LCMS/MS). The iTRAQ method relies on differentially labelling peptides from individual proteolytic digests by covalent binding of isobaric tags and allows identification and quantification of peptides in multiple samples in a single experiment  at 96 h, as measured by MTS ‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium) assay, are 37 nmol/l (PreB 697), 86 nmol/l (R3C3), 1·32 μmol/l (R3G7), 1·43 μmol/l (R4C10) and >10 μmol/l . Other cell lines were obtained from European Collection of Cell Cultures  or the American Tissue Culture Collection  and all were cultured in RPMI 1640 medium  supplemented with 10% (v/v) foetal bovine serum (Invitrogen) at 37°C and 5% (v/v) carbon dioxide. All cell lines were routinely tested for mycoplasma contamination using MycoAlert® .The PreB lineage childhood ALL cell line PreB 697  supplemented with protease inhibitor cocktail . Antibodies to detect PAX5 (E‐9) , α‐Tubulin (Sigma‐Aldrich), IRF4, phospho‐SAPK/JNK (Thr183/Tyr185), SAPK/JNK  were used. Secondary antibodies used were horseradish peroxidase conjugates of either anti‐rabbit or anti‐mouse IgG . Quantification was carried out by imaging the enhanced chemiluminescence (ECL)‐plus exposed polyvinylidene difluoride membranes using the Fuji LAS‐3000 Luminescent Image Analyser System  and analysing the intensity of the protein bands using an Automatic Image Data Analysis (AIDA) software image analysis program (Fujifilm). PAX5 basal protein levels in the cell lines were calculated by dividing the intensity of the PAX5 protein band by the intensity of the loading control, α‐tubulin, band, thereby normalizing for any loading inaccuracies.PAX5 (Hs00277134_m1) and TATA‐binding protein (TBP) were purchased from Assays‐On‐Demand . Samples were assayed in duplicate on three independent occasions with the ABI 7500 Fast Real‐Time PCR System (Applied Biosystems) using the TaqMan®universal PCR MasterMix (Applied Biosystems). To quantify mRNA levels the 2−ΔCT or 2−ΔΔCT method was used with TBP as the endogenous control, as indicated.Briefly, total RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit  and cDNA synthesis was carried out using the Applied Biosystem High‐Capacity cDNA Reverse Transcriptase kit according to the manufacturer's instructions. Primers and probes for et al  and control ATP10 gene. Assays were performed in duplicate with 50 ng of genomic DNA from each cell line per 20 μl reaction, using an ABI 7500 Fast Real‐Time PCR System (Applied Biosystems). PAX5 gene dosage for each exon was calculated by dividing the value obtained for PAX5 by the corresponding value for ATP10A.This assay was performed as described in An et al  with ATPPAX5 exons 2‐10 were mutationally screened by direct DNA sequencing. Briefly, genomic DNA was extracted from cell lines using the Qiagen Mini kit (Qiagen) and amplified by PCR. Primer sequences and annealing temperatures are shown in Table SI. DNA sequencing was performed by purifying 100 μl of PCR product using a QIAquick PCR Purification kit (Qiagen) with a final elution volume of 30 μl and then sequenced using both forward and reverse primers with the ABI Version 3 BigDye Terminator Cycle Sequencing kit and analysed on an ABI Prism DNA sequencer (Applied Biosystems).et al, Cell line DNA was analysed using the SALSA MLPA kit P335‐A2  as described previously  or 2‐5 nmol/l Bortezomib (Selleckchem). Following a 96‐h drug exposure, cytotoxicity was assessed using the CellTiter 96 Aqueous One kit , also known as MTS assay, which assesses the capacity of cells to reduce formazan and thus is a measure of metabolically active cells. The resulting absorbances were averaged and expressed as a percentage of the control vehicle. Survival curves were plotted using GraphPad Prism software . Drug interactions were assessed using the Chou‐Talalay method, which is based on the median effect equation  and CD10‐phycoerythrin (PE) or the corresponding isotype control. Ten thousand events for each sample were acquired using a FACSCalibur flow cytometer  and analysed using CellQuest software (BD Biosciences) on three independent occasions.Cells were stained with Annexin V  and evaluated for apoptosis by flow cytometry following the manufacturer's protocol. Ten thousand events for each sample were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analysed using CellQuest software (BD Biosciences).et al, http://www.broadinstitute.org/gsea/msigdb/index.jsp). Genes were pre‐ranked according to their Pearson correlation with 50% inhibitory concentration (IC50) values for individual PreB 697 cell lines.Total RNA was harvested and extracted using the Qiagen RNeasy Mini kit according to the manufacturer's intructions and subjected to expression profiling using Affymetrix Human Exon 1.0 ST GeneChips . Exon microarray hybridization and pre‐processing was carried out as previously described . Each experiment was repeated at least three times, unless indicated. The To identify differentially expressed, low abundance, nuclear proteins related to GC‐resistance, nuclear lysates from untreated and dexamethasone‐treated (DEX) or ethanol control vehicle (CV) were prepared from PreB 697 and R3F9 cells. Lysates were labelled with a distinct isobaric tag for relative quantification and analysed by two dimensional liquid chromatography/tandem mass spectrometry . In total, 811 unique proteins were identified at greater than 95% confidence, most of which did not show any differential expression between samples (Tables SII and SIII).versus’ control vehicle for each cell line. Data mining of the Excel spreadsheets  identified a number of proteins differentially expressed between cell lines and treated ‘versus’ control. Figure P = 0·001) and PAX5 (P = 0·02). For PAX5, a single peptide (amino acid sequence: ANLASPTADIGSSVPGPQSYPIVTGR) was identified in all four samples . A second B cell transcription factor, IRF4, based on two peptide sequences (LFDTQQFLSELQAFAHHGR and LITAHVEPLLAR) was also identified to be differentially unregulated in the GC‐sensitive cell line, but did not achieve statistical significance , while the IRF4 levels in the treated R3F9 cell line remained unchanged . This differential upregulation of IRF4 and PAX5 in response to GC in the sensitive compared to the resistant line was verified by Western blot  and protein level at 24 h, while R3F9 cell line failed to upregulate PAX5 significantly at either the mRNA (P = 0·299) or protein level  also showed reduced basal and induced PAX5 levels, relative to the GC‐sensitive parent  but this cell line, as we have previously shown, is GR deficient  and Western blotting analyses showed that PreB 697 cells significantly upregulated PAX5 in response to dexamethasone, both at the mRNA   in exons 10, 14, and 16 but showed a normal copy number for exon 1 for all 6 cell lines .Recent studies have shown that PAX5 showed that mRNA levels were similar in all cell lines (p = >0·2251)  but not for R3G7 (P = 0·219). Again, the GR‐deficient RD11 cell line with normal levels of PAX5 had similar CD19 levels to the GC‐sensitive parent.RQ‐RT‐PCR of 251) Fig D, sugges251) Fig E. As exp50 values for the individual sub‐lines. A set of genes whose expression describes the transition from a preB‐II differentiation state to immature B‐lymphocytes, was significantly enriched in the GC‐resistant sub‐lines; Normalized Enrichment Score = 1·96, P = 0·001, q = 0·16 . All cell lines were found to be negative for CD20 expression, a mature B cell differentiation marker, and also the stem cell marker, CD34 (data not shown).As PAX5 is integral to B‐cell commitment and differentiation, we investigated whether the reduced levels of PAX5 are a reflection of an altered maturation state by using gene expression arrays. GSEA was used to analyse the state of differentiation of GC‐resistant sub‐lines and genes were ranked according to the correlation of gene expression with dexamethasone IC0·16 Fig . and a modest sensitization was also seen in the sensitive parent line, 80–24 nmol/l (P < 0·0001)   by JNK, which triggered proteasomal degradation; JNK inhibitors were similarly able to re‐sensitize to this drug  agonist, was shown to resensitize GC response via activation of both p38 and JNK . 932.8, triply charged ion. The inset shows the peak area at the low mass/charge (m/z) region with the iTRAQ reporter ions.Figure S2. Differential induction of IRF4 protein in PreB 697 compared to R3F9 cell lines in response to dexamethasone exposure.Figure S3. Reduced induction of PAX5 in GC‐resistant sublines.Figure S4. MLPA result for the R3C3 cell line using the MRC Holland P335‐IKZF1 ALL kit.Figure S5. PAX5 protein stability in GC sensitive and resistant sublines.Figure S6. CD10 expression is lower in GC resistant sub lines.Figure S7. Phospho‐JNK levels are higher basally in GC‐resistance sub lines.Figure S8. Determining a non‐toxic dose of JNKi.Figure S9. JNK inhibition significantly sensitises to dexamethasone in PreB 697 GC resistant sublines lines and is synergistic.Figure S10. Bortezomib does not mimic JNKi in GC resensitisation.Figure S11. JNK inhibition significantly sensitises to dexamethasone in T‐ALL cell lines.Table SI. PAX5 Primer sequences.Table SII. (A) List of differentially up‐regulated proteins in PreB 697 in response to 24 h dexamethasone exposure as quantified in iTRAQ experiment. (B) List of differentially down‐regulated proteins in PreB 697 in response to 24 h dexamethasone exposure as quantified in iTRAQ experiment.Table SIII. (A) List of differentially up‐regulated proteins in R3F9 in response to 24 h dexamethasone exposure as quantified in iTRAQ experiment. (B) List of differentially down‐regulated proteins in R3F9 in response to 24 h dexamethasone exposure as quantified in iTRAQ experiment.Table SIV. PAX5 protein identification based on a single peptide.Click here for additional data file.


Trypanosoma brucei is dependent on the tetracycline inducible system for the precise control over the expression of both genes and dsRNA. Another independent inducible system for trypanosomes would enable the control of the activities of two different genes in the same cell, providing greater experimental sophistication. Here, I describe the development of the vanillic acid based inducible expression system for T. brucei, which operates independently of, and can be used in parallel with the tetracycline inducible system.Reverse genetics in  Trypanosoma brucei has a well annotated genome and well developed reverse genetic tools, making it a highly tractable experimental organism used for the study of a large range of biological processes such as antigenic variation and the eukaryotic flagellum T. brucei inducible expression is controlled by the tetracycline inducible system in the tetON configuration  pJ1271 was integrated into the 927 pJ1173 cells to give a cell line with the expression of eGFP under vanillic acid control. To determine whether this cell line (927 pJ1173 pJ1271) was able to express eGFP a range of vanillic acid concentrations were added to the cells before analysing the eGFP expression level by flow cytometry D. WithouTo compare the relative expression levels between the vanillic acid and tetracycline inducible systems the pDEX877 plasmid was integrated into the 927 pJ1173 cell line to create 927 pJ1173 pDEX877. After 24 h of vanillic acid induction the 927 pJ1173 pJ1271 cell line had ∼18 fold increase in the fluorescent signal over the uninduced cell line; however, after 24 h of doxycycline induction the 927 pJ1173 pDEX877 cell line had ∼250 fold increase in the fluorescent signal over the uninduced cell line E, F. TheTo show that the two induction systems could operate simultaneously, a modified version of pDEX777 called pJ1225 with eGFP replaced with dTomFP was integrated into the 927 pJ1173 pJ1271 cell line. This cell line was then treated with no chemical inducer, vanillic acid only, doxycycline only or both vanillic acid and doxycycline for 24 h; the cells were then fixed and imaged A. When eTetracycline, IPTG and vanillic acid inducible systems have now all been described for use in PCFs


BMPR2), E1A binding protein p300 (EP300), transforming growth factor-β2 (TGFβ2), and tumor necrosis factor, and alpha-induced protein 3 (TNFAIP3) gene expression patterns in B-cell malignancies were studied.The results of a previous study showed that a clear dysregulation was evident in the global gene expression of the BCL11A-suppressed B-lymphoma cells. In this study, the bone morphogenetic protein receptor, type II  with SYBR Green Dye. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference.The relative expression levels of TGFβ2 mRNA in B-lymphoma cell lines was significantly higher than those in the cells from the healthy control (P<0.05). However, the expression level of TNFAIP3 mRNA in B-malignant cells was significantly lower than that of the healthy control (P<0.05). The expression levels of BMPR2 and EP300 mRNA showed no significant difference between B-malignant cell lines and the healthy group (P>0.05). In B-lymphoma cell lines, correlation analyses revealed that the expression of BMPR2 and TNFAIP3  had significant positive relation. The expression levels of BMPR2, EP300, and TNFAIP3 mRNA in cell lines from myeloid leukemia were significantly lower than those in the cells from the healthy control (P<0.05). The expression levels of TGFβ2 mRNA showed no significant difference between myeloid leukemia cell lines and the healthy control or B-malignant cell lines (P>0.05). The expression levels of BMPR2, EP300, and TNFAIP3 mRNA in B-lymphoma cells were significantly higher than those of the myeloid leukemia cells (P<0.05).The expression level of BMPR2, EP300, TGFβ2, and TNFAIP3 genes in B-lymphoma cells exist.Different expression patterns of  Deregulation of BMPs signaling pathways has been reported in certain human cancers. BMPR2, EP300, TGFβ2, and TNFAIPI3 have been studied in lymphoma or leukemia, no report has compared the expression of the four genes among B-lymphoma cells, the healthy control, and myeloid leukemia. In this study, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to analyze the expression levels of BMPR2, EP300, TGFβ2, and TNFAIPI3 in B-lymphoma and myeloid leukemia cell lines.Although 2 in RPMI-1640 medium supplemented with penicillin (100 U/mL), streptomycin (0.1 mg/mL), and 10% fetal calf serum.Human B-lymphoma cell lines  were kindly provided by Prof. Ailin Guo . Human B-lymphoma cell lines  and myeloid leukemia cell lines  were purchased from a cell bank in Shanghai, China. The cells were cultured at 37 °C in an atmosphere containing 5% COTen healthy volunteers served as control . All procedures were conducted in accordance with the guidelines of Medical Ethics committees of the health bureau of Guangdong Province, PR China. Peripheral blood was collected by heparin anticoagulation. Peripheral blood mononuclear cells (PBMNCs) were separated with the use of the Ficoll-Hypaque gradient centrifugation method.GAPDH) gene by qRT-PCR was used to determine the quality of the synthesized cDNA.RNA was extracted using the Trizol kit  and reversely transcribed into the first-strand cDNA with the use of random hexamer primers and the reverse transcriptase Superscript II Kit , according to the manufacturer’s instructions. RNA purity and concentration was measured with a spectrophotometer. The integrity of RNA was checked on a 1% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase  were determined by linear regression analysis. Briefly, the primers were designed and synthesized . The following primer sequences used in PCR reactions were shown in The 2P<0.05.Nonparametric test analysis of two independent samples was used for the relative expression levels of gene mRNA in different samples, whereas the Mann-Whitney U test was used for non-normally distributed data using the SPSS 13.0 statistical software. Differences were considered statistically significant at BMPR2, EP300, TGFβ2, and TNFAIP3 reactions are above 0.99. The amplification efficiencies of the four genes and the GAPDH control gene were above 95%. The high amplification efficiency of the four genes was consistent with that of the GAPDH reference gene. The PCR products from the GAPDH control gene and the four genes were confirmed using 2% gel electrophoresis (data not shown).R2 values of standard curves for GAPDH). The ∆Ct was calculated for all four genes. According to the relative qRT-PCR formula: 2–∆Ct ×100%, the relative expression level of TGFβ2 mRNA in cell lines from B-cell malignancies was significantly higher than that in the cells from the healthy control (P<0.05). On the other hand, the expression level of TNFAIP3 mRNA in B-malignant cells was significantly lower than that of the healthy control (P<0.05). As shown in TGFβ2 and TNFAIP3 expression in these B-cell lines are 6.25% and 4.78%, respectively, whereas the relative median expression levels of the two genes in the healthy group are 0.12% and 164.74%. The relative expression levels of BMPR2 and EP300 mRNA indicated no significant difference between B-cell malignant cell lines and PBMNCs of the healthy group (P>0.05).Ten malignancy hematology cell lines  were assayed for RNA expression levels. B-malignancy cells include Burkitt’s lymphoma cell lines  and GCB DLBCL-derived cell lines (OCI-LY-3 and SUDHL6). The mRNA expression level for each of the four genes was normalized to the reference gene  had significant positive relation.In B-lymphoma cell lines, correlation analyses revealed that the expression of BMPR2, EP300, and TNFAIP3 mRNA in cell lines from myeloid leukemia were significantly lower than those in the cells from the healthy people (P<0.05). As shown in BMPR2, EP300, and TNFAIP3 expression in the myeloid leukemia cell lines are 0.10%, 0.56%, and 0.34%, respectively. Furthermore, the relative median expression levels of these genes in the healthy control are 2.04%, 19.05%, and 164.74%, respectively. The relative expression levels of TGFβ2 mRNA showed no significant difference between myeloid leukemia cell lines and the healthy control (P>0.05).The relative expression levels of BMPR2, EP300, and TNFAIP3 mRNA in the cell lines from B-cell malignancy were significantly higher than those cell lines from myeloid leukemia (P<0.05), whereas the relative expression levels of TGFβ2 mRNA showed no significant difference between B-cell malignant cell lines and myeloid leukemia cell lines (P>0.05).As shown in BMPR2, EP300, TGFβ2, and TNFAIP3 mRNA expression were determined by qRT-PCR using SYBR Green I dye. Results showed that the high amplification efficiency of these genes was consistent with that of GAPDH, suggesting that the 2–∆Ct ×100% method can be used in quantifying the relative expression levels of the gene mRNA. This study revealed the expression of TGFβ2 and TNFAIP3 mRNA in B-malignant cells were significantly different from those of the healthy control. Meanwhile, the expression levels of BMPR2, EP300, and TNFAIP3 mRNA in myeloid leukemia cells were significantly lower than those of healthy people.In this study, et al.TGFβ2 was expressed in normal B-cell differentiation of the germinal center, especially from the follicular center cell stage to the postfollicular plasma cell stage. However, there was no expression of TGF-β in specimens from patients with neoplastic NHL. In this study, the expression level of TGFβ2 mRNA in PBMNCs from the healthy control is almost lacking. The expression level of TGFβ2 mRNA in the cell lines from B-malignancies was significantly higher than those in the cells from the healthy control. Furthermore, TGF-β functions as either suppressing or promoting the effect on tumors need to be investigated. TNFAIP3 inactivation has been reported in various B-cell lymphomas. In GCB DLBCL, the expression of TNFAIP3 was lower than that in ABC DLBCLet al.TNFAIP3 deletion was in similar frequencies in GCB and non-GCB/ABC DLBCL using fluorescence in situ hybridization. These contradictory observations may reflect the diversified roles of TNFAIP3 in DLBCL. These results showed that the expression level of TNFAIP3 mRNA in B-malignant cells was significantly lower than that of the healthy control, suggesting possible deletion of TNFAIP3 gene in the B-lymphoma cells, which is in accordance with previous observationsTNFAIP3 gene was expressed at lower levels in GCB DLBCL cell lines than in Burkitt’s lymphoma cell lines.Deregulated or aberrant TGF-β signaling has been strongly implicated in the pathogenesis of human solid tumors, while less is known about the role of this pathway in lymphoma pathogenesisEP300 mutations were recently identified as a major pathogenetic mechanism shared by common forms of B-cell NHLsBMPR2 and EP300 mRNA had no significant difference between B-cell malignant cell lines and PBMNCs of the healthy people.BMPR2, EP300, and TNFAIP3 mRNA in myeloid leukemia cell lines were significantly lower than those of healthy people or B-malignant cell lines. BMPR2, EP300, and TNFAIP3 mRNA have been observed to be almost absent in myeloid leukemia cell lines. This observation indicates that the expression patterns of BMPR2, EP300, and TNFAIP3 in B-cell lymphoma may be significantly different from myeloid leukemia cell lines. Although the median quantity of TGFβ2 expression in B-lymphoma cell lines is obviously higher than that of the myeloid leukemia cell lines, no statistically significant difference is observed. This finding might denote a large variance of TGFβ2 expression level (three orders of magnitude) in the B-lymphoma cell lines. Additionally, in the different B-lymphocyte malignant cell lines, the expression levels of BMPR2, EP300, TGFβ2, and TNFAIP3 mRNA were quite different, which may be related to the heterogeneity of B-cell lymphoma.Results indicate that the expression levels of BMPR2 and TNFAIP3 genes in B-cell lymphoma. This study showed that the expression of BMPR2 and TNFAIP3 had a significant positive relation in B-lymphoma cell lines. These results indicate that a positively correlated expression pattern may be a feature in B-cell lymphoma. However, increasing the number of samples for validating the expression relation of the two genes is needed.Little is known about the expression correlation between BMPR2, EP300, TGFβ2, and TNFAIP3 genes were characterized in B-lymphoma cells. Meanwhile, the differential expression pattern needs to increase the sample numbers to be further studied in B-cell malignancies. In addition, further biological studies are needed to determine if TGFβ2 and TNFAIP3 are therapeutic targets for B-cell lymphoma.In conclusion, the expression patterns of


However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future.Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past,  Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. SurvivaKRAS, BRAF, CTNNB1, PTEN, ARID1A, FBXW74, PIK3CA, PPP2R1A, and TGFBR2[TP53, BRCA1, and BRCA2[TP53 mutations and significantly recurring somatic mutations in NF1, BRCA1, BRCA2, RB1, and CDK12[Among the different tumours arising from the ovary 90% are of epithelial origin. The majnd TGFBR2. The morand BRCA2. A more and CDK12.TP53 and IL-6 mutations[TP53 mutations, DNA damage, and secretory cells, suggesting the tubal fimbria as the precursor for high grade serous ovarian cancers[TP53 mutations, show elevated γH2AX expression, a marker of DNA damage, and express Ki-67 and PAX2, two proliferation markers also expressed in serous tubal intraepithelial carcinomas and high grade serous ovarian carcinomas[Brca, Tp53, Pten genetic mouse model that de novo high grade serous ovarian carcinoma are originated from the fallopian tube secretory epithelium and that these tumours are correlated with high grade serous carcinoma tumour markers and genomic alterations of the human TCGA data set[The monolayer of epithelial cells covering the outer surface of the ovary (OSE) has traditionally been thought to be the site of origin of epithelial ovarian cancer. This isutations,18. Thisutations-21. Thos cancers,22,23. Trcinomas-27. In crcinomas. Recentldata set.in vitro models to investigate the molecular nature and the pathological processes underlying the development of ovarian, tubal, and peritoneal tumours, and their progression to advanced diseases, and even to search for diagnostic or prognostic tumour markers as well as for therapeutic targets.Serous ovarian- (SOC), tubal- (STC), and peritoneal- (SPC) cancers are remarkably similar in term of morphology,31, genein vivo animal models, cancer cell lines as in vitro models have proven invaluable experimental tools for many decades in basic research. Cancer cell lines can be grown continuously in culture, allowing countless experiments to be performed without the necessary restrictions required for in vivo models. However, due to few regulations for the development and testing of these cell lines, the question arises as to the quality of long-time established ovarian cancer cell lines. Often laboratories obtain cell lines from collaborating groups and trust in their identification of cells. Conducting research on the basis of such cell lines means not only a waste of a great deal of money and time but also a risk to steer research in an undesired direction.Falling short of the use of It is therefore of great importance to define and establish a world-wide standard applicable to all cell lines that are commercially available for research, in order to ensure that only high-quality cancer cell lines with an unequivocal molecular identity and source are distributed to the research community.http://www.ncbi.nlm.nih.gov/) was also searched to retrieve references provided by these cell banks reporting additional details of the stocked cell lines. We also included a recent publication in which the copy-number changes, mutations, and mRNA expression profiles in ovarian cancer cell lines were compared to those of high grade SOC [We performed a web search for currently available banks for cells and cell lines using the terms ‘cell bank’, ‘cell lines’, and ‘cell line bank’. Only web pages in English and containing normal or cancer ovarian, tubal, and peritoneal cell lines were included in the study. PubMed (ih.gov/).Five cell banks worldwide that stock and distribute normal and/or ovarian cancer cell lines were identified. These are the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC), the German Collection of Microorganisms and Cell Cultures (DSMZ), the Japanese Collection of Research Bioresources (JCRB), and the National Cell Bank of Iran (NCBI) Table . RemarkaCricetulus griseus) origin. About 3% originate from mice (Mus musculus) and 4.5% from various species such as Spodoptera frugiperda , Esox lucius (Northern pike fish), Ictalurus punctatus (Channel catfish), and Sus domesticus (Domestic pig). Strikingly, one third of the 104 described human ovarian cancer-derived cell lines were in reality not from ovarian tissue but from peritoneal ascites (21.2%), pleural fluid (3.8%), or metastatic masses (6.7%).Our search algorithm retrieved 153 cell lines. ECAAC distributes almost 40% of all publicly available cell lines, followed by JCRB (19%). A number of cell lines 7.2%) are distributed by two or more cell banks. A listing of the ID number, cell line designation (name), origin, and source of the retrieved normal and malignant ovarian, tubal, and peritoneal cell lines is presented in Tables % are disIt is noteworthy that cell line banks do not stock human cell lines described originating from primary tubal or peritoneal origin. However, only recently the isolation and culturing of normal ovarian and fallopian tube epithelial cells from the same healthy female has been described. This fiin vitro to display chemo resistance (e.g. cisplatin-resistant A2780CP) or different cellular factors. In addition, the verification of information given by the cell bank is difficult, because not all cell lines are linked to their original publications and their depositors are rarely mentioned.The re-naming of cell lines causes constant confusion as respective annotations are often not found in cell banks. For example, 41 M cells are the same as OAW28 cells. Some cell lines have similar names and require caution in the selection of the cell line of choice: a majority of the animal cell lines and several human cell lines are derived from a parental line  and have been modified One apparent shortcoming is that the ethnicity of the ovarian cancer patient from whom the tumour is derived is indicated in only 30.5%. Apart from the JCRB cell bank where all the deposited cell lines were derived from Japanese females (48.3%), the majority of samples where ethnical details are provided were from Caucasian females.BRCA and APC genes of Ashkenazi Jews[Since we know that different ethnic groups can have a propensity for specific genetic mutations, for example in the azi Jews,37, it iThe (molecular) characterization of EOC in the clinics significantly depends on the presence and type of genetic alterations in the cancer and may define the treatment options and the patients’ outcome. The tumor origin where the cell lines derived from was not precisely provided in 51.2% Table . ConsideTP53 mutations (4.2%), Her2/neu (3.2%), EpCAM (3.2%), and cytokines 7, 8, 17, 18, and 19 (ranging from 5.3% to 8.4%).We also collected and evaluated data provided by cell banks in regards to molecular markers. This information was very limited and only few cell lines were evaluated for expression of progesterone (7.4%) and oestrogen (6.3%) receptors, vimentin (5.3%), et al. 2013 reported that the popular and frequently used KU7 urothelial carcinoma cell line was cross-contaminated years ago with HeLa cells[The misidentification and cross-contamination of cell lines is problematic in research and may increase the risk for false results and misinterpretations. The extent of misidentification is documented in a recent study wherein a panel of ovarian and endometrial cell lines was analysed by DNA profiling. The autLa cells. Cross-cBacterial/fungal/yeast/mycoplasma contamination presents another problem adversely affecting research results. Of these, mycoplasma species are most likely to be detrimental to cell functioning. Unlike most bacterial, fungal or yeast infections, mycoplasma are macroscopically and microscopically undetectable; it may remain in culture for extended periods of time affecting cell growth, gene expression and overall cell functioning. This main vitro cell line models to the research community is also an issue. The problem is two-fold: firstly, there is no quality control of cells generated in individual laboratories when they are not deposited in a professional cell bank. Even when these cells are meticulously generated and cultured, independent quality checks and verifications are not possible. This flaw is overcome by directly contacting the laboratory where the cell lines were generated. This, however, can lead to the second problem; the passing on of cell lines from laboratory to the other, thereby bypassing the critical quality control cell banks. In the past it has been common practice to obtain cells from collaborating groups, and with the required permission, to again distribute these to other laboratories. Whilst this practice is in the spirit of research collaborations, it increases the risk of receiving contaminated or misidentified cell lines that, in turn, can be detrimental to research.The unavailability of a considerable number of http://www.atcc.org), China Center for Type Culture Collection , Australian Cell Bank , European Culture Collection of Cell Cultures , or German Cell Culture Collection . From a recent study that histotyped standard ovarian cancer cell lines by short tandem repeats, immunohistochemistry, and mutation analysis it was concluded that the knowledge of the mutation status of cancer genes such as ARID1A and TP53 and of the general immunoprofile would be beneficial for the determination of the histotype of ovarian cancer cells[To ensure a unique quality of cancer research around the world we recommend that all cell lines used for research should be deposited in a cell bank and be readily accessible for all researchers. Ovarian cancer cell bank operators should provide development protocols and comprehensive clinical data for all commercially available cell lines. Depositors of cell lines should ensure that they have carefully collected all relevant clinical information from the donor individuals. This information includes: the exact origin of the cells, the stage during disease progression the cells were taken, the type of therapy the patient underwent prior to sample collection, the data on the patient’s survival, the ethnicity and family history , and the preoperative plasma CA125 levels currently provided by only 5.3% of all human ovarian cancer cell lines. Additionally, we recommend that all cell bank operators conform to the same style of reporting the cell line information and only bank cells where all necessary information is available. This will ensure that the highest standard of research is maintained worldwide. Short tandem repeat (STR) profiling, a highly-sensitive method to detect cellular cross-contamination, should be performed by researchers for all newly generated cell lines and should be confirmed by the cell bank once deposited and prior to the sale of the cells. The service for STR profiling is provided by various laboratories, e.g. American Type Cell culture Collection (ATCC-USA,er cells. FollowiThe authors declare that they have no competing interests.FJ carried out literature research, data analysis and drafted the manuscript. SN carried out literature research drafted the manuscript. NFH drafted the manuscript. VAS conceived the review and drafted the manuscript. All authors read and approved the final manuscript.


This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding β-chemokines.Results of our studies showed that HTLV-1Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L. Anotin vitro. This intein Tax . Tax, intein Tax . Selecti+ T cell lines. We report herein for the first time that HTLV-1+ T cell lines express a biologically active form of OX40L while the OX40 molecule appears biologically inactive or masked. The OX40L expressed by HTLV-1+ T cell lines was capable of inhibiting R5 HIV-1 infection of activated PBMCs via production of CCR5-binding β-chemokines. These findings suggest that autologous HTLV-1-immortalized T cell lines can be utilized as a readily available convenient source of natural OX40L in large quantities for various immunological studies.Studies were therefore carried out in efforts to examine whether OX40L and OX40 were expressed in a biologically active form by HTLV-1+ T cell lines were biologically active, we examined their capacities to bind biotinylated rec-OX40 and rec-OX40L, respectively. The finding that rec-OX40 and rec-OX40L bound specifically to the OX40L-transfected CEM cells (CEM/OX40L) and the OX40-transfected CEM/OX40 cells, respectively, demonstrated the specificity of the assay being utilized  was stained double positive with anti-OX40L (clone 5A8) and anti-OX40 (clone B-7B5) mAbs, they bound only rec-OX40 but not rec-OX40L. This finding indicated that while OX40L was expressed in an active form on MT-2 cells, the OX40 was likely to be expressed in an inactive form. Similar results were obtained by the testing of a number of additional HTLV-1+ T cell lines, including T cell lines spontaneously established from a HTLV-1-infected patient with adult T cell leukemia (ILT-H2) and a HTLV-1-associated myelopathy (HAM/TSP) patient (ILT-M1), and various in vitro-HTLV-1-immortalized CD4+ or CD8+ T cell lines from different healthy donors   were analyzed in parallel with the HTLV-1+ T cell line MT-2 using standard Western Blot techniques. Results of these studies displayed in Figure + T cell lines.A series of studies were subsequently conducted in efforts to identify the potential reason(s) for the failure of HTLV-1+ T cell lines to bind rec-OX40L, we utilized an additional anti-OX40 specific mAb (W4-54 mAb) along with B-7B5 mAb. While the clone W4-54 anti-OX40 mAb inhibited the binding of OX40 and OX40L, the clone B-7B5 failed to show any detectable inhibition  were first captured with the use of immobilized anti-OX40L (clone HD-1) or anti-OX40 (clone B-7B5) mAb, respectively. The levels of captured antigens were assayed with the use of HRP-labeled anti-OX40 mAb or anti-OX40L mAb. Although it is reasonable to assume that the natural interaction between OX40 and OX40L on the living cell surface may be dissociated by the detergent treatment, as shown in Figure To further probe for the molecular basis for the inability of the OX40 expressed by the HTLV-1+ T cell lines is biologically functional, we performed co-culture experiments using the experimental in vitro infection of autologous activated PBMCs with HIV-1 as a read out. PBMCs activated with anti-CD3/anti-CD28 mAbs for 24 hours were washed and infected with either R5 HIV-1JR-FL or X4 HIV-1NL4-3 at a low m.o.i., and then co-cultured with paraformaldehyde (PFA)-fixed autologous HTLV-1+ T cell line in the presence or absence of anti-OX40L mAb or a mixture of the three CCR5-binding chemokine-blocking mAbs . The reasons why we utilized autologous PFA-fixed HTLV-1+ T cell lines were to avoid any allogeneic stimuli and minimize the secretion of any anti-HIV-1 factors by the HTLV-1+ T cell lines. As shown in Figure + T cells in the cultures were reduced by co-culture with not only autologous HTLV-1+ T cell line but also with the addition of soluble rec-OX40L. This inhibition was mediated by OX40L-OX40 interaction since the addition of the anti-OX40L blocking mAb (clone 5A8) and/or the addition of a mixture of the anti-β-chemokine mAbs reversed the level of reduction. It is worthy to note that, similar to data we have previously reported with the use of recombinant OX40L [+ T cell line, suggesting the CCR5-specificity of this antiviral effect.To confirm that the OX40L expressed on the HTLV-1nt OX40L , X4 HIV-+ T cell lines with that of soluble rec-OX40L to inhibit R5 HIV-1 infection by the quantitation of p24 production in the culture supernatants. As shown in Figure + T cell line could inhibit more effectively at even an HTLV-1+ T cell to PBMCs ratio as low as 0.3. The maximum inhibition reached with rec-OX40 was around 65% of the maximum inhibition reached with HTLV-1+ T cell line, with similar IC50. Altogether, these data demonstrate that indeed, the OX40L expressed by HTLV-1+ T cell lines is biologically active.Finally, we compared the potential of membrane bound OX40L of the fixed HTLV-1+ T cell lines. The expression of active forms of OX40L is not unique to HTLV-1+ T cell lines, since similar conditions have also been observed in normal T cells when they are activated under mild DNA damaging conditions or cultured for long-term in IL-2 containing media with periodic stimulation [+ T cell lines are immortal and thus can provide unlimited amounts of OX40L.In the present study, we revealed that the cell surface expressed OX40L on T cell lines immortalized by HTLV-1 is biologically active in concert with the co-expression of an inactive form of OX40. As far as we know, this is the first study to report the polarized “OX40L-active/OX40 inactive” expression by HTLV-1mulation ,24. Howe+ T cell lines to bind OX40L remains to be clearly defined. Based on our previous paper showing that functional OX40L can be transferred to OX40 intercellularly [cis and/or trans mode. Indeed, the WB analysis showed that the OX40-OX40L blocking mAb W4-54 that did not stain living HTLV-1+ T cells reacted to the p50 OX40 molecule , supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin . Anti-human CD3 mAb (clone OKT-3) and agonistic anti-CD28 mAb were purchased from the American Type Culture Collection  and Biolegend , respectively. Neutralizing mAbs against human RANTES, MIP-1α, and MIP-1β were purchased from R&D systems . The mouse mAbs produced in our laboratory included anti-OX40L  (1 ml/well) pre-coated with 5 μg/ml OKT3 for 1 hour and cultured in the presence of soluble 0.1 μg/ml anti-CD28 mAb. After 24 hours at 37°C in a 5% CO2 humidified atmosphere, the activated PBMCs were harvested and washed once. These activated PBMCs (1 × 106 cells/ml) were mixed with an equal number of ILT-M1 cells that were pretreated with 50 μg/ml MMC for 30 min at 37°C and cultured in RPMI media supplemented with 20 U/ml IL-2 (culture media). The cultures were performed in 24-well plates (BD) (2 ml/well) and the culture media was replenished every 3–4 days. After 1 ~ 2 months when HTLV-1 Tax+ T cells appeared and started to grow continuously, they were split every 3 to 5 days using the culture medium.The HTLV-1-producing T cell lines used included the MT-2, HUT102 and the IL-2 dependent T cell lines ILT-M1 and ILT-H2 that had been generated from a HTLV-1-associated myelopathy (HAM) and an adult T cell leukemia (ATL) patient, respectively. Additional cell lines utilized included the CEM cell lines transfected with either human OX40L or OX40 (CEM/OX40L and CEM/OX40) . T cells+ cells were performed by FCM using a cell counting kit (BD) according to the manufacturer’s protocol. For staining of Tax antigen, cells were fixed with PBS containing 4% PFA followed by washing in FACS buffer containing 0.5% saponin. Aliquots of these cells were stained with Cy5-conjugated mouse anti-Tax mAb (Lt-4) [FCM analysis of live cells was carried out as described previously. Briefly, cells to be analyzed were Fc-blocked with 2 mg/ml normal human pooled IgG on ice for 15 min. Aliquots of these cells were then subjected to staining using pre-determined optimum concentrations of fluorescent dye-conjugated mAbs for 30 min on ice. The cells were then washed using FACS buffer (PBS containing 2% FCS and 0.1% sodium azide), fixed in 1% paraformaldehyde (PFA) containing FACS buffer and analyzed using a FACS Calibur, and the data obtained were analyzed using the Cell Quest software (BD). In order to determine whether cell surface OX40 or OX40L is functional, aliquots of Fc-blocked cells were incubated with either biotinylated recombinant-OX40L (rec-OX40L) or rec-OX40 at a concentration of 2.5 μg/ml for 30 minutes on ice, followed by staining with PE-labeled streptavidin (Beckman Coulter) for 30 minutes on ice and then analyzed by FCM. For detection of HIV-1 infected cells, PBMCs were fixed with PBS containing 4% PFA followed by washing twice in FACS buffer containing 0.5% saponin. These cells were Fc-blocked with 2 mg/ml normal human pooled IgG on ice for 15 min, and aliquots of these cells were stained with FITC- or Cy5-conjugated anti-HIV-1 p24 mAb (clone 2C2) for 30 min on ice. The cells were then washed using FACS buffer and absolute cell counts of p24b (Lt-4)  for 30 mFor the quantitation of OX40L and OX40 by ELISA, anti-OX40L capture mAb (clone HD1)/ HRP-labeled detector mAb (clone 8F4) and anti-OX40 (clone B-7B5)/HRP-labeled detector mAb (clone 17D8), respectively, were used together with recombinant standard proteins purchased from R&D systems. Immunoprecipitation followed by Western blot analysis of OX40 was performed as reported previously [JR-FL and HIV-1NL4-3 viral stocks were produced as described previously [In vitro activated PBMCs were prepared as described above, washed once and infected with either R5 HIV-1JR-FL or X4 HIV-1NL4-3 at a multiplicity of infection (m.o.i.) of 0.005 for 2 hours. After washing 3 times, PBMCs were re-suspended at 1 × 106 cells/ml in 20 U/ml IL-2-containing RPMI medium, dispensed into individual wells of 48-well plates (BD) (0.5 ml/well) and cultured in the presence or absence of 1 μg/ml of rec-OX40L or graded numbers of autologous HTLV-1+ T cells (HTLV-1+ T cells : PBMCs ratio of 10 to 0.15) that had been previously inactivated with 4% paraformaldehyde (PFA). Production of HIV-1 was determined by either the measurement of HIV-1 core p24 levels produced in the culture supernatants using our in-house formulated and standardized kits or FCM using Cy5 labeled anti-HIV-1 p24 mAb [HIV-1eviously . In vitr p24 mAb .t test by using Prism software (GraphPad Software).Data were tested for significance using the Student’s The authors declare no competing financial interests.+ T cell lines and carried out the FCM and ELISA, performed the statistical analysis and drafted the manuscript. AT performed WB and FCM analyses. AK produced R5 and X4 HIV-1 and titrated. RT produced and labeled antibodies, confirmed their specificities and made in-house ELISA. AAA participated in the design of the study and helped to draft the manuscript. YT conceived of the study, participated in its design and coordination, carried out the HIV-1 infection experiments and drafted the manuscript. All authors read and approved the final manuscript.DK and YTak generated HTLV-1Characterization of two anti-human OX40 mAbs. In the presence of B-7B5, W4-54 or isotype control mAbs, the OX40 and OX40L co-expressing control CEM cells were singly stained either with biotinylated recombinant OX40L (rec-OX40L) or rec-OX40, respectively, followed by PE-streptavidin. Data shown are representative profiles of 3 independent experiments.Click here for file+ T cell line (YT/cM1) by Western Blot with B-7B5 and W4-54 mAbs. Cell lysates of HTLV-1+ T cell line, YT/cM1, were subjected to 10% PAGE and blotted onto nitrocellulose sheets. The sheets were then probed with anti-OX40 mAbs (B-7B5 or W4-54) or isotype controls followed by goat anti-mouse IgG or anti-rat IgG. Mol. Wt. markers are shown on the right. Data shown are representative of 2 independent experiments.Detection of OX40 expressed by HTLV-1Click here for filePhenotype of HUT-102 cell line. Phenotype of HUT-102 cells were examined by FCM using anti-OX40 mAbs (FITC-labeled B-7B5 and Cy5-labeled W4-54), anti-OX40L (Cy5-labeled 5A8), biotinylated OX40 (rec-OX40) and OX40L (rec-OX40L) followed by PE-streptavidin. Intracellular Tax antigen was stained by mouse anti-Tax Lt-4 mAb.Click here for file


Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors.Twenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 μM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women.All 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors.The distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease. ESR1 gene [ESR1 gene [Approximately 80 % of primary breast cancers express estrogen receptor α (ER), a nuclear transcription factor that is the product of the SR1 gene . AromataSR1 gene . Many poSR1 gene , chromatSR1 gene , and disSR1 gene , 8 have SR1 gene –11. We hSR1 gene .Despite their insensitivity to tamoxifen, most resistant breast cancers still express ER . ResistaDue to the frequency of acquired endocrine resistance and the lack of treatment for this type of breast cancer, many researchers have studied biological mechanisms that predict acquired endocrine resistance. One recently recognized marker of acquired endocrine resistance is E74-liked factor 5 (ELF5) . ELF5 isAdditionally, expression and methylation of docking protein 7 (DOK7) were measured due to the protein’s potential role as a tumor suppressor. A recent study of identical twins with differential breast cancer statuses identified DOK7 gene methylation as an indicator of breast cancer risk . IncreasIn this study, we sought to characterize tamoxifen-selected MCF-7 derivative cell lines based on changes in the expression and methylation of DOK7, ELF5, and ERα, and the protein levels of ERα, PR, and HER2. To extend these findings clinically, we examined the relationship between hormone receptor status, anti-estrogen treatment and promoter methylation of DOK7 and ELF5 in a set of matched primary and recurrent breast tumors from 24 women.During the initial expansion of the tamoxifen-selected cell lines, a few of the cell lines appeared to grow either in unusual clumps or in suspension. However, after several passages in T-75 flasks all of the tamoxifen-selected lines displayed a phenotype similar to that of MCF-7 Fig. .Fig. 1PhBlinded observers (pathologists RMJ and CNO) assessed IHC-stained slides for ER, PR and HER2. ER protein expression in the 21 tamoxifen-selected cell lines was similar to that observed in the parental MCF-7 Figs. a. NineteHaving established with IHC that the novel tamoxifen-selected cell lines were ER-positive, PR-negative (18 of 21), and HER2-negative, we wanted to examine the expression of additional genes that might play a role in endocrine resistance, and compare the expression in the novel cell lines with the expression in several previously described tamoxifen-resistant cell lines, TMX2-28, TMX2-11, and TMX2-4 , 23. We We next compared the expression of DOK7 mRNA in MCF-7 with the expression in all of the tamoxifen-selected lines and the mass culture TMX3A. DOK7 was significantly overexpressed in eleven of the 21 novel tamoxifen-selected cell lines p < 0.05) and was not significantly altered in any of the other cell lines or in the mass culture Fig. . Interes and was decrease in methylation associated with the up-regulation observed in the novel tamoxifen-selected cell lines, and an increase in DNA methylation associated with the down-regulation observed in the TMX2-28 cell line.Given that the expression of DOK7 and ELF5 was significantly increased in roughly half of the novel tamoxifen-selected, ER–positive cell lines, and decreased  in the only ER-negative cell line, TMX2-28, we wanted to determine the extent to which DNA methylation in the promoter region of DOK7 and ELF5 controlled gene expression. If DNA promoter methylation was controlling expression we would expect to see a We first examined DNA methylation data available from the Illumina human methylation 450K bead chip (HM450BC) for MCF-7, TMX2-11 and TMX2-28 . The HM4To confirm the methylation results observed with the HM450BC we designed pyrosequencing assays to interrogate six CpG sites within the TSS of DOK7 and three CpG sites within the TSS of ELF5. Figure Likewise, increased DNA methylation of ELF5 was observed only for TMX2-28 Fig. b. AveragWe next examined DNA methylation in primary and second/recurrent breast tumors from three groups of women Table  to deterComparison of DOK7 promoter methylation across the primary and second tumors of women in the three groups showed no differences Fig. a; medianWidespread resistance to endocrine therapies limits their usefulness and reduces patient remission. Previous studies have identified tamoxifen-induced cellular changes. Here we characterize novel clonal cell lines whose diversity could represent drug resistant cancers in women. All of the tamoxifen-selected lines remained ER positive, which is consistent with results from past experiments , 13, yetHowever, we found the promoter region of ELF5 to be highly methylated in primary PR-positive tumors and that methylation was vastly reduced in second tumors occurring after anti-estrogen treatment. This finding is consistent with Kalyuga et al.  who founThe novel tamoxifen-resistant cell lines described here display molecular differences from the parental MCF-7 that include decreased PR protein levels, increased ELF5 and DOK7 gene expression and will likely be useful in further mechanistic studies of endocrine-resistance. Examination of paired primary and second tumors from 24 women, 19 of whom received anti-estrogen treatment, revealed a subset of primary PR-positive tumors with distinct ELF5 promoter methylation indicating the possibility of stratification of women for tailored treatment in the early stages of disease.2. Cells were grown in Falcon T-75 flasks, given fresh media twice per week, and split once or twice per week when 70–90 % confluency was obtained. The tamoxifen-selection was conducted in triplicate for six months resulting in three populations (referred to as mass cultures), one of which, TMX3A, was used for generating clonal lines. Cloning by limiting dilution was performed to assess the diversity of cells within the tamoxifen-selected mass culture. Tamoxifen-selected TMX3A cells were seeded at one cell/well and allowed to grow in 96-well plates for 6–7 weeks. Twenty-one cell lines were selected , expanded and cryopreserved.MCF-7 cells were purchased from the American Type Culture Collection (ATCC) and grown as previously described , with ta® Ribonuclease Inhibitor . Quantitative RT-PCR (qRT-PCR) was performed on this cDNA using FastStart Universal SYBR Green Master mix . Primers used for qRT-PCR are listed in Table RNA isolation was performed using Tri-reagent . DNA was isolated from frozen cells using the QIAmp DNA Mini Kit (Qiagen Cat. No. 51304). RNA and DNA were quantified using a NanoDrop 8000 (Thermo Scientific). cDNA was created from isolated RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with added RNasin500 ng of DNA was bisulfite treated using the EZ DNA methylation-Lightning Kit (Zymo Research Cat # D5030). Bisulfite-treated DNA was pyrosequenced using the Pyromark Q24 system (Qiagen) and manufacturers protocol (Qiagen), and gene methylation levels were detected as cysteine to thymidine ratios at specific loci. Primers used for pyrosequencing are listed in Table l-lysine coated-slides (Polysciences #22247) at a density of 200,000 cells/mL/slide and fixed the following day using 100 % ice-cold methanol. Slides were stained on a BenchMark Ultra platform using the UltraView Universal DAB Detection Kit with previously optimized antibodies for ER , PR  and HER2 . Stained slides were dehydrated in ethanol and xylene and coverslips were added manually. Slides were examined by a pathologist and ER and PR were recorded as Allred scores [Cells were seeded onto poly-d scores , HER2 waCollection and processing of human breast tumors was conducted with IRB approval from Baystate Medical Center and the University of Massachusetts as previously described . Breast Q RT-PCR data represent biological duplicates for each cell line run in technical duplicate. The data were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reference gene. One-way ANOVAs with Bonferroni post hoc tests were run to determine significance at p < 0.05. For presentation purposes, each value was normalized to the average MCF-7 value for the gene of interest. Data were then log transformed to provide the expression levels relative to MCF-7, with values greater than 0 indicating higher expression, and values below 0 indicating reduced expression. The average of 4 biological replicates was used to determine the standard deviation. Error bars on Figs.


In this era of precision medicine, the deep and comprehensive characterization of tumor phenotypes will lead to therapeutic strategies beyond classical factors such as primary sites or anatomical staging. Recently, “-omics” approached have enlightened our knowledge of tumor biology. Such approaches have been extensively implemented in order to provide biomarkers for monitoring of the disease as well as to improve readouts of therapeutic impact. The application of metabolomics to the study of cancer is especially beneficial, since it reflects the biochemical consequences of many cancer type-specific pathophysiological processes. Here, we characterize metabolic profiles of colon and ovarian cancer cell lines to provide broader insight into differentiating metabolic processes for prospective drug development and clinical screening.We applied non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography and gas chromatography for the metabolic phenotyping of four cancer cell lines: two from colon cancer  and two from ovarian cancer . We used the MetaP server for statistical data analysis.A total of 225 metabolites were detected in all four cell lines; 67 of these molecules significantly discriminated colon cancer from ovarian cancer cells. Metabolic signatures revealed in our study suggest elevated tricarboxylic acid cycle and lipid metabolism in ovarian cancer cell lines, as well as increased β-oxidation and urea cycle metabolism in colon cancer cell lines.Our study provides a panel of distinct metabolic fingerprints between colon and ovarian cancer cell lines. These may serve as potential drug targets, and now can be evaluated further in primary cells, biofluids, and tissue samples for biomarker purposes.The online version of this article (doi:10.1186/s12967-015-0576-z) contains supplementary material, which is available to authorized users. The treatment of complex diseases like cancer still remains a major challenge, both for patients and for the healthcare system. Better characterization of tumor identity through a comprehensive “-omics” approach has modified paradigms in translational cancer research. By combining several analyses, major consortiums have been driven to describe tumor-specific landscapes. Transcriptomic studies have led to the definition of several tumor-specific subtypes, leading to optimal staging as well as tailored treatment. Finally, the characterization of epigenetic changes has also recently informed clinicians about tumor plasticity as a mechanism that supports therapeutic escape. Aside from the large body of clinical work, most of these novel techniques have been optimized using model cancer cell lines. The use of model cell lines has clearly culminated in the cancer cell line encyclopedia (CCLE) project, in which multiple cancer cell lines have been characterized in detail using several “-omics” platforms.Metabolomics is the study of the small molecule composition  in bio-fluids, tissue samples, and cell lines. By measuring the consequences of all changes in gene expression, protein abundance, and environmental influence, metabolomics has been recognized as the “-omics” technology that provides readouts that are closest to the clinical endpoint . MetabolMetabolomics has already been used to identify biomarkers of ovarian and colon carcinomas in plasma , 7 and tThe main goal of this study was to determine the metabolic signatures of colon and ovarian cancer cell lines, which might serve several purposes. First, we endeavored to determine the metabolic signatures of ovarian and colon cancer cell lines, which could be evaluated in greater detail to determine metabolic fingerprints for cell identity purposes. The identified metabolic signatures and pathways will provide insight into the pathophysiology of ovarian and colon cancer cell lines. Second, we attempted to identify metabolic processes and pathways that distinguish ovarian and colon carcinomas that might be targetable to control neoplastic disease, with potential clinical applications. We applied non-targeted metabolomics to profile four different cell lines from colorectal  and ovarian . We identified metabolic signatures and pathways that clearly demonstrated differences between the two tumor types.l-glutamine (Sigma), 1× non-essential amino acids . The human colorectal adenocarcinoma cell lines HCT15 (ATCC CCL 225) and HCT116 (CCL247) were maintained in McCoy’s 5A medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, and 1% penicillin–streptomycin solution. For the experiments, cells were trypsinized and washed with PBS, and all cell lines were cultivated in M199 medium to avoid the effect of culture milieu on metabolic profile. After incubation overnight, cells were thoroughly washed, medium was changed, and cells were cultivated in fresh medium for the next 24 h. Once cells reached 80% confluence (approximately 48 h after cell were seeded), samples were collected by trypsinization. One million cells were counted and washed with PBS; cell pellets were frozen at −80°C. Ten replicates were prepared for each cell line.Established cancer cell lines were obtained from the American Type Culture Collection . Upon receipt, cells were expanded; at <10 passages, aliquots of cells were stored frozen in liquid nitrogen. Cells from an aliquot were kept in culture for <2 months. Human ovarian adenocarcinoma cell lines SKOV3 (HTB-77) and OVCAR3 (HTB-161) were grown in Dulbecco’s modified Eagle medium high glucose  supplemented with 10% fetal bovine serum , 1% penicillin–streptomycin solution (Sigma), 2 mM ® system from Hamilton Company. In a first step recovery standards were added for quality control (QC) purposes. Samples were extracted with series of organic and aqueous solvents to remove the protein fraction, allowing the maximum recovery of small molecules (metabolites). Each sample extract was split into equal parts for GC and LC analysis. After the removal of organic solvent, samples were frozen and vacuum-dried.Frozen pellets were sent to Metabolon Inc.  for metabolic profiling, where all procedures were performed according to Metabolon’s standard protocols. All samples were maintained at −80°C after processing with the MicroLab STAR2 scans using dynamic exclusion.Each sample extract designated for LC–MS analysis was split into two aliquots, dried, and reconstituted in acidic or basic LC-compatible solvents, each of which contained eight or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. The analysis of LC/MS samples performed on the platform using a Waters ACQUITY UPLC system and a Thermo Fisher Scientific Orbitrap Elite high resolution/accurate mass spectrometer, which was composed of a heated electrospray ionization (HESI) source and an orbitrap mass analyzer operating at 30,000 mass resolution. Samples were analyzed using acidic positive ion optimized conditions  and basic negative ion optimized conditions  in two independent injections on separate dedicated columns. Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts used water/methanol containing 6.5 mM ammonium bicarbonate. The MS analysis alternated between MS and data-dependent MSN,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) under dried nitrogen conditions. The GC column was 5% phenyl, and the temperature ramp was from 40 to 300°C over a 16-min span. The measurements were performed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer instrument using electron impact ionization, which was tuned and calibrated daily for mass resolution and mass accuracy.Samples assigned for GC–MS analysis were re-dried under vacuum for at least 24 h prior to derivatization. The derivatization process was performed using Peaks obtained after measurements were identified using Metabolon’s propriety peak integration software, and component parts were stored in a separated and specifically designed complex data structure. Metabolites were identified by comparing the obtained data to the library entries of purified standards or unknown recurrent entities. On the date of data evaluation, more than 3,000 commercially available purified standard compounds were acquired and registered in the Laboratory Information Management System (LIMS) for distribution to both LC and GC platforms for the determination of their analytical characteristics. The combination of chromatographic properties and mass spectra provided an indication of a match to the specific compound or isobaric entity. To achieve the highest data quality for statistical analysis, series of QC and curation processes were conducted. This system enables accurate and consistent identification of true chemical entities with simultaneous removal of system artifacts, mis-assignments, and background noise. Library matches for each compound were checked for each sample, and manually corrected if necessary.Because sample measurements were obtained during a 5-day period, data were normalized to correct variations resulting from inter-day tuning differences in the instrument. Essentially, each compound was corrected in a run-day by registering the medians to equal one, proportionally normalizing each data point.In total, we identified 533 metabolites, including 435 metabolites of known identity and 98 molecules of unknown identity. The median number of metabolites detected in any single sample in the whole data set was 405, the median number of metabolites detected in a single sample per cell line were as follows: HCT15, 372; HCT116, 391; OVCAR, 438; SKOV3, 467. Metabolites with >20% missing (N = 308) values were removed from the data set, and metabolites with <20% missing values (N = 225) were imputed to the average in the group.metaP-server at the Helmholtz Center Munich (http://metabolomics.helmholtz-muenchen.de/metap3), which provides automated and standardized data analysis for quantitative metabolomics data [metaP-server creates PCA plots, bar plots, and box plots [Statistical data analysis was performed using the web-based tool ics data . The serics data . For visox plots .6 cells). PCA was applied to identify outliers. Two outliers in the SKOV3 cell line were identified and removed, possibly caused by some non-traceable experimental or technical errors , but no differences within groups of cell lines from the same origin, (p > 2.22 × 10−4 for HCT116 vs. HCT15 and OVCAR3 vs. SCOV3).The non-parametric Kruskal–Wallis test for multi-class categorical phenotypes was applied to test the association of metabolite concentration with cell line. We used the Mann–Whitney, non-parametric hypothesis test for two-class phenotype. Metabolites that enable differentiation between cancer types were identified based on the following criteria: significant difference between ovarian and colon cell lines (p < 2.22 × 10−4). The full data set and metaP analysis are freely and fully available at the following websites: ovarian vs. colon: http://metap.helmholtz-muenchen.de/metap3/run.cgi?ID=1415435214597630; HTC15 vs. HTC116: http://metap.helmholtz-muenchen.de/metap3/run.cgi?ID=1415362867314825; SKOV3 vs. OVCAR: http://metap.helmholtz-muenchen.de/metap3/run.cgi?ID=1415431362747244; all cell lines HTC15, HTC116, OVCAR3, and SKOV3: http://metap.helmholtz-muenchen.de/metap3/run.cgi?ID=141536220398464.We adopted the most conservative approach to inferring significant differences between cancer types; statistical significance between cancer types and between cell lines was inferred using stringent Bonferroni correction to account for testing 225 metabolites  was performed using the MultiExperiment Viewer (MeV) v. 4.9 software , based oWe applied non-targeted metabolomics in order to determine the metabolic signatures of four widely used cancer cell lines, two from colorectal carcinoma (HCT15 and HTC116) and two from ovarian carcinoma (OVCAR3 and SKOV3). The obtained dataset resulted in 225 individual metabolites, which in turn were used in the analysis , but not within groups of cell lines from a common origin (p > 2.22 × 10−4 for HCT116 vs. HCT15 and OVCAR3 vs. SCOV3) , we identified molecules that differed significantly between ovarian and colon cell lines  isoleucine and valine and the aromatic amino acids (AAA) tyrosine and phenylalanine were higher in ovarian cancer cell lines. BCAAs can be catabolized to propionyl-CoA, and then to methylmalonyl-CoA and succinyl-CoA, which in turn can be incorporated into the TCA cycle. In addition, phenylalanine and tyrosine can enter the TCA cycle after conversion to fumarate Figure a. Colon Fatty acid (FA) and glycerophospholipid (GPL) signatures strongly differentiated colon from ovarian cancer cell lines, but are similar between cell lines of a shared origin , which suggests the de novo synthesis and elongation of FAs and GPLs (which requires the acetyl-CoA molecule), as well as their accumulation. Because citrate serves as an acetyl-CoA donor for FA synthesis and elongation  and GPLsN-acetylputrescine, beta-alanine (both products of putrescine metabolism), and trans-4-hydroxyproline (a product of proline metabolism).Among the urea cycle intermediates, the level of citrulline was higher in ovarian cancer cell lines. Other metabolites connected to the urea cycle were higher in colon cancer- cell lines, such as Metabolic differences between cancer cells and healthy cells were recognized by Warburg as early as the 1920s . FurtherAmong the 225 metabolites analyzed in this study, we found strong metabolic individuality among the different cell lines, with concomitant identity in lines with a shared tissue of origin (colon vs. ovary). To ensure robustness, we normalized metabolic data on cell number as described previously , 13; we ). For example, previous reports indicate that TCA cycle metabolism, which we observed as lower in colon cancer cell lines, down-regulated in both tissue and plasma samples from patients with colorectal cancer [We have found 67 metabolites that significantly differentiate colon from ovarian cancer cell lines, supporting the idea that cancers originating from different tissues can be metabolically distinct. These findings are preliminary and cannot be directly translated to clinical applications, and validation in human tissue and biofluids samples is required; however, we compared our data with previous reports comparing healthy controls with colorectal or ovarian cancer patients. Interestingly, our findings were concordant with those of previous studies reporting differences in metabolites and metabolic pathways in biofluids and tissue samples Figure b. For exl cancer , 27–32  for FA synthesis and elongation, it may justify the demand of increased TCA cycle metabolism observed in ovarian cancer cell lines. Furthermore, higher phosphatidylethanolamine and phosphatidylcholine levels suggest increases in the de novo synthesis of these GPLs, known as the Kennedy pathway . Our obsN-acetylputrescine. Significant elevation of the metabolite N-acetylputrescine has been reported previously in colorectal cancer tissue [N-acetylputrescine may be associated with urea cycle metabolism. Ornithine decarboxylase (ODC) is a first and rate-limiting factor in polyamine biosynthesis; it catalyzes the conversion of ornithine to putrescine [N-acetylputrescine. Increased ODC activity in colorectal cancer has been reported [Colon cancer cell lines displayed higher levels of beta-alanine and r tissue , 30. A btrescine , which ireported . Our obsreported , 45  that should be extensively examined. The ovarian cancer cell lines were mainly characterized by elevated TCA cycle intermediates and elevated lipid synthesis. This information might assist novel drug design or drug repurposing, focusing on enzymes/transporters involved in lipid and TCA cycle metabolism. In turn, the metabolic signatures of colon cancer cell lines suggest increased β-oxidation and increased urea cycle activity, which may indicate a demand for the removal of excess ammonia after rapid amino acid catabolism. These observations suggest that targeting β-oxidation and/or urea cycle metabolism in colon cancer might be successful strategies to control this neoplastic disease. Although there are incontestable benefits to the use of cell culture as a model system for metabolomics studies , additio


Comparison of cell line and patient sample data identified new candidate drugs for CML BC, such as vascular endothelial growth factor receptor and nicotinamide phosphoribosyltransferase inhibitors. Our results indicate that these drugs in particular warrant further evaluation by analyzing a larger set of primary patient samples. The results also pave way for designing rational combination therapies.Chronic myeloid leukemia in blast crisis (CML BC) remains a challenging disease to treat despite the introduction and advances in tyrosine kinase inhibitor (TKI) therapy. In this study we set out to identify novel candidate drugs for CML BC by using an unbiased high-throughput drug testing platform. We used three CML cell lines representing different types of CML blast phases  and primary leukemic cells from three CML BC patients. Profiling of drug responses was performed with a drug sensitivity and resistance testing platform comprising 295 anticancer agents. Overall, drug sensitivity scores and the drug response profiles of cell line and primary cell samples correlated well and were distinct from other types of leukemia samples. The cell lines were highly sensitive to TKIs and the clinically TKI-resistant patient samples were also resistant  The discovery of the Philadelphia chromosome and the role of BCR-ABL1 kinase in CML pathogenesis led to the development of the first approved TKI imatinib, which has remarkably improved the prognosis and survival of CML patients.4 Owing to the more aggressive nature of advanced phase CML, patients tend to respond less favorably to TKI treatment. When first-line TKI treatment fails the options available are few, from higher dosages of TKI to allogeneic hematopoietic stem cell transplantation. None of the current treatment options are significantly effective in CML BC. Hence, there is a clear need for identification of novel drug therapies for CML BC patients.Despite the treatment breakthroughs in chronic phase CML, advanced phase and blast crisis (BC) remain a therapeutic challenge.5 presents a new concept of individualized systems medicine, which emphasizes high-throughput drug sensitivity and resistance testing (DSRT) of primary patient samples to profile patient cancer cells and identify personalized treatment strategies. With this novel approach we characterized drug responses in CML BC cell lines and primary patient samples, and aimed to identify new potential candidates for the treatment of CML BC. Our results indicate that both primary CML BC and cell line samples display a unique drug sensitivity pattern, which differs from other leukemia types. Furthermore, novel candidate drugs for CML BC such as vascular endothelial growth factor receptor (VEGFR) and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors were discovered from the DSRT analysis.Sequencing of cancer genomes has become a popular choice in characterizing individual cancer patients in the search for new druggable targets. However, genetic findings do not always translate to efficacious drug therapy. Our recent study in acute myeloid leukemia (AML)The bone marrow aspirates or peripheral blood samples (leukemic cells) and skin biopsies (noncancerous cells for germline genomic information) from three CML patients and nine different healthy donors (controls) were obtained after informed consent with approval from local Institutional Review Boards  and in accordance with the Declaration of Helsinki. Patient characteristics are summarized in http://www.dsmz.de/). K562 cell line has erythroleukemic (resembles both undifferentiated granulocytes and erythrocytes), MOLM-1 cell line megakaryocytic and EM-2 myeloid features.8 Cells were maintained according to the manufacturer's instructions at 37 °C and 5% CO2 in RPMI 1640 supplemented with 10% (K562 and EM-2) or 20% (MOLM-1) fetal bovine serum, L-glutamine and penicillin–streptomycin. Collection of drug sensitivity score (DSS) values from >150 cell lines served as a control group for CML BC cell line results .Human CML BC cell lines  were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh . Mononuclear cells were counted using a hemocytometer and suspended in mononuclear cell medium  supplemented with 0.5 μg/ml gentamicin and 2.5 μg/ml amphotericin B before DSRT. Blast counts in tested patient samples 1, 2 and 3 were 40%, 86% and 75%, respectively.5 Mononuclear cell medium was used with patient primary cells and healthy controls. Cell line assays were done with the corresponding cell culture medium.DSRT protocol has been described previously.2 for 72 h. Cell viability was measured using CellTiter-Glo luminescent assay  according to the manufacturer's instructions with a PHERAstar FS  or Paradigm  plate reader. Dose–response curves were generated on the basis of the viability readouts.The drug collection used in these studies contained 295 different substances and covered the majority of US Food and Drug Administration/European Medicines Agency-approved anticancer drugs, as well as emerging investigational and preclinical compounds covering a wide range of molecular targets . The com9 DSS values were further normalized against the median values from healthy controls or from cell line reference data for patient and cell line samples, respectively, to obtain selective DSS, which was then used to measure leukemia-specific drug sensitivity. Drugs with selective DSS values >5 were considered selective and >10 highly selective to tested cells.DSS is an integrative and robust drug response model based on normalized area under the curve by taking all four curve fitting parameters into account.n=10, Ph+ ALL n=3, T-ALL n=3 and B-ALL n=3).In addition to CML BC samples, we used the DSS of different types of leukemia samples in clustering analysis (AML BCR-ABL1 mutations after diagnosis. Identified mutations were confirmed by exome sequencing for patient samples 1 and 2 at the time of DSRT sampling.All patient primary cell samples were Sanger sequenced for 10 and Cancer Cell Line Encyclopedia (CCLE)11 studies were further analyzed. DSS was calculated for each drug response profile to compare data from our study with CGP and CCLE data based on the standardized drug response metrics used in our study. The nonparametric Spearman's rank correlation coefficient was used to evaluate the similarity of drug response profiles across the three data sets.The drug response data from Cancer Genome Project (CGP)P-values for all cell line and patient primary cell sample correlation pairs were <0.0001. Clustering of the drug sensitivity profiles across the cell line and patient samples were performed using unsupervised hierarchical complete-linkage clustering using Spearman and Euclidean distance measures of the drug and sample profiles, respectively. All correlation and clustering analyses were performed using DSS profiles of the cell line and primary patient cell samples.The nonparametric Spearman's rank correlation coefficient was calculated with SPSS Statistics software . To identify novel therapeutic drugs for CML BC, we screened three CML cell lines representing blast phase disease. All cell lines proliferated at similar rates during the 72 h incubation period . As the analyzed cell lines lacked a specific control group , we used DSS values of >150 cell lines as a control. To determine the CML BC-specific sensitivity of tested drugs, we chose the 30 most selective drugs for closer comparison . Drug reThe cell lines shared several drugs that fell into the top 30 selective drugs, although many individual differences occurred . All celThere was no common drug selectivity pattern when comparing classical cytotoxic drugs between three tested CML cell lines . EM-2 ceBCR-ABL1 gene in patients 1 and 2 , whereas no BCR-ABL1 mutations were found in patient 3. Compared with the cell line data, drug responses in the primary patient cells exhibited more inter-individual variability, which could be due to the specific mutations  were among the top 30 most selective drugs in all three primary CML BC cases. VEGFR inhibitors were also effective at inhibiting the growth of cells derived from patient samples, whereas almost no effect was observed in the cell lines. In addition to MEK and VEGFR inhibitors, AZD8055 (mTOR kinase inhibitor) and navitoclax were effective in all patient samples, AZD8055 being within six most selective drugs in all cases. Daporinad was highly selective in two patient samples and also selective in the sample of patient 3.Leukemia cells from patient 2 displayed sensitivity to glucocorticoids. In concordance, this patient had a bi-phenotypic leukemia (both B-lymphoid and myeloid markers), whereas other patients had myeloid BC.Between individual patients a large number of shared selective drugs  were found among the top 20 when drug responses of classical cytotoxic drugs were compared . Howeverr=0.887; EM-2 vs MOLM-1, r=0.816; K-562 vs MOLM-1, r=0.775), but DSS of primary patient cells exhibited more variability . Similarly, the correlation between individual patient samples and cell lines showed high degree of variability: DSRT results from patient 3 had the best  and from patient 2 the worst correlation with the cell lines . Thus, patient samples with higher overall drug sensitivity seemed to correlate better with cell lines.The DSS values of individual cell lines correlated closely with each other  samples were compared with primary samples from different types of leukemia.For the first cluster analysis we focused only on a small set of BCR-ABL1 and VEGFR inhibitors . In secoIn the first analysis CML BC cell line and patient samples clustered relatively close to each other and separately from the majority of other type of leukemia samples . EM-2, Kr=0.68, P<0.0001) but the drug responses of K-562 correlated poorly with our data   (The CGP study screened two cell lines (EM-2 and K-562) and 47 drugs that matched with our study. The CCLE study had only 1 matching screened cell line (EM-2) and 14 matching drugs. The drug responses of CGP EM-2 had moderate correlation (=0.0626) . EM-2 dr<0.0001) .In this study we assessed drug response profiles of CML BC samples by high-throughput drug testing using 295 anticancer compounds. The average drug responses of the CML BC cell lines and primary patient samples correlated well, although each sample had unique responses to specific drugs. Detailed comparison of cell line and patient sample results revealed a number of compounds  with high activity across all samples.11 In both studies the EM-2 and K-562 cell lines were included in the analysis and also common TKIs were tested. A meta-analysis of these two studies has shown some inconsistencies between the two data sets.12 On the basis of the comparison between our results of the EM-2 and K-562 cell lines with these two data sets, our results appeared to correlate better with the CCLE data, but the comparison was not balanced as the CGP data contained 2 matching CML BC cell lines and 47 matching drugs, whereas with CCLE data only 14 matching drugs and 1 cell line was common.Recently, the Sanger and Broad institutes performed two separate large studies  where they linked genomic profiles of various cancer cell lines to pharmacological responses.8 The high response to TKIs of K-562 cells was similar to EM-2 cells but otherwise K-562 cells were more resistant. The drug responses of MOLM-1 differed most from the other two cell lines, as TKI responses were clearly lower than in EM-2 or K-562. MOLM-1 is characterized as a mega-karyocytic cell line and in addition to the Ph chromosome with an e13-a2 BCR-ABL1 fusion gene, it carries the inv(3)(q21q26) chromosomal aberration, which leads to overexpression of ecotropic viral integration site 1 (EVI1) gene.13 High EVI1 expression has been linked with poorer prognosis in myeloid malignancies and TKI resistance, which could be one explanation for poorer TKI sensitivity in MOLM-1 cells.14 Instead, MOLM-1 cells were highly sensitive to glucocorticoids, which has not been described previously.Our cell line results indicate that BCR-ABL1-positive CML cell lines are mostly sensitive to TKIs targeting ABL1. EM-2, a myeloid CML cell line with multiple copies of Philadelphia (Ph) chromosome and low tendency for differentiation, was not only highly sensitive to TKIs, but also in general was more sensitive to other drugs than other cell lines. K-562 cell line undergoes erythroid and mega-karyocytic differentiation, although it does not carry a classical Ph chromosome.15 Furthermore, lower levels of VEGF at the diagnosis have been shown to be associated with better prognosis in CML.16 Recently axitinib was shown to possess high affinity to T315I-mutated BCR-ABL1,17 which may explain the high activity in patient sample 2.Comparison of drug responses across all samples revealed drugs and drug classes, which deserve further investigation in a clinical setting. VEGFR inhibitors as a drug class exhibited activity in all samples, although the effect of individual VEGFR inhibitors varied from sample to sample. In our study, the most potent VEGFR inhibitors were tivozanib and axitinib, both of which are used in the treatment of renal cell carcinoma. Food and Drug Administration has approved axitinib but tivozanib is still in phase III studies. Elevated plasma levels of VEGF have been found in patients with AML and CML, and increased bone marrow vasculature has especially been noted in CML.19A small number of other drugs with various mechanisms of action proved to be effective in most samples. Daporinad (AKA APO866), a NAMPT inhibitor, was a top 10 hit in most samples. It is currently in phase II for various types of cancer, but not yet clinically tested in CML. Daporinad was the only NAMPT inhibitor tested in our assay but new promising compounds with the same mechanism are emerging from other screenings.20 Most of the samples were highly sensitive to danusertib, including the T315I-mutated sample of patient 2. The lack of other aurora inhibitors among most selective drugs suggests that the activity of danusertib is mostly due to BCR-ABL1 inhibition. Danusertib has been tested in clinical trials on imatinib refractory CML patients but no results have been published yet.Danusertib (AKA PHA-739358) was the most effective aurora kinase inhibitor in our study. It has a dual role as it also inhibits BCR-ABL1 with affinity also to T315I-mutated isoform.21 and that the combination of TKIs and BCL2 inhibitors may have synergistic effects in TKI-resistant patients.22Interestingly, navitoclax exhibited the best activity with the patient samples compared with other BCL2 inhibitors in our assay. Similarly, venetoclax (ABT-199) was also highly effective in two samples (MOLM-1 and patient 3), but it was not tested in patient samples 1 and 2. The efficacy of BCL2 inhibitors in BC CML has recently been suggested also in other studies, which have shown that the inhibition of Bcl-2/Bcl-xL induce apoptosis of quiescent CML progenitor cells23 MEK1/2 was more phosphorylated in cell lines but the phosphorylation of downstream targets ERK1/2 and CREB was more pronounced in patient sample. It is difficult to draw conclusions from only one patient sample, but this may implicate a MAPK/ERK pathway having a more central role in primary patient cells.MEK inhibitors were highly selective in all patient samples but they lacked activity in the cell lines. We compared the phosphorylation of MAPK/ERK pathway of patient sample 2 (data not shown) with our recently published CML cell line phosphorylation data.ex vivo drug responses of CML BC cell line and patient primary samples using a high-throughput drug testing method. The drug responses in TKI-resistant patient samples matched the clinical characteristics and disease course. New promising candidate compound classes such as VEGFR, NAMPT and MEK inhibitors were identified for BC CML. These drugs were highly sensitive in most samples (including TKI resistant patient samples) warranting their further evaluation as combination regimens and paving the way for proof-of-concept clinical studies.In conclusion, we characterized