JAK-2 dysregulation plays an important role as an oncogenic driver, and is thus a promising therapeutic target in hematological malignancies. Ruxolitinib is a pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3. Vorinostat is an HDAC inhibitor that reduces JAK-2 expression, thus affecting JAK-2 mRNA expression and increasing JAK-2 proteasomal deterioration. Here we hypothesized that the combination of ruxolitinib and vorinostat could have synergistic effects against hematological disease. We tested combinations of low doses of ruxolitinib and vorinostat in 12 cell lines, and observed highly synergistic cytotoxic action in six cell lines, which was maintained for up to 120 h in the presence of stromal cells. The sensitivity of the six cell lines may be explained by the broad effects of the drug combination, which can affect various targets. Treatment with the combination of ruxolitinib and vorinostat appeared to induce a possible reversal of the Warburg effect, with associated ROS production, apoptotic events, and growth inhibition. Decreased glucose metabolism may have markedly sensitized the six more susceptible cell lines to combined treatment. Therapeutic inhibition of the JAK/STAT pathway seems to offer substantial anti-tumor benefit, and combined therapy with ruxolitinib and vorinostat may represent a promising novel therapeutic modality for hematological neoplasms. During hematopoietic ontogenesis, cytokines play key roles by initiating the intracellular signals that govern cell fate decisions, such as proliferation and differentiation. Most cytokine receptors lose intrinsic kinase activity and, therefore, often employ Janus kinase (JAK) as a signaling intermediate to facilitate downstream signaling. Receptor-related non-receptor tyrosine kinases, such as JAK1, JAK2, JAK3, and TYK2, are triggered after cytotoxic receptor activation . HematolRuxolitinib INCB018424) is an orally administered, biologically available pyrrolo[2.3-d]pyrimidine derivative that shows inhibitory activity against JAK1 and JAK2, moderate activity against TYK2, and minor activity against JAK3 , 8. Clin424 is anHistone deacetylase (HDAC) inhibitors are a promising group of therapeutic agents for various malignancies , 18. His50) in the 12 hematological cell lines after 24 and 48 h of treatment with ruxolitinib and vorinostat as single agents. We used the MTT assay to quantify the inhibition of cell viability. In all cell lines, the IC50 of ruxolitinib ranged from 12–20 µM after 24 h of incubation, and from 1–5 µM after 48 h. The IC50 of vorinostat in all cell lines ranged from 15–25 µM after 24 h, and from 5–10 µM after 48 h.First, we evaluated the cytotoxic effects  in the WSU-NHL, Granta 519, Jeko1, U266, HUT78, and L540 cell lines after 24 h of simultaneous exposure to ruxolitinib and vorinostat. In the remaining cell lines  the drug combination induced a synergistic effect (CI < 1) after 24 h. More specifically, the CI values ranged from 0.1–0.3, indicating strong synergy according to the Chou-Talalay theory . After 24 h of single-drug treatment with ruxolitinib (5 µM) and vorinostat (10 µM), the percentage of apoptotic cells was not more than 5–10%. On the other hand, combined treatment with these drugs for 24 h led to an increase of the apoptotic fraction to 40–50% Figure . AdditioTo better clarify the apoptosis mechanism induced by ruxolitinib and vorinostat, we evaluated the expressions of some apoptosis-regulating proteins that induce programmed cell death , and of apoptosis inhibitors (BCL-2 and MCL-1). Expression levels were measured as mean fluorescence intensity (MFI) by flow cytometry after 24 h of incubation. In all cell lines, treatments with both single drugs and with the drug combination were associated with increased expressions of the BAX, BID, and BAD pro-apoptotic proteins. With regards to the anti-apoptotic proteins BCL-2 and MCL-1, the combination of ruxolitinib with vorinostat was associated with downregulation of both proteins in the six most sensitive cell lines, while single-drug treatment did not have this effect  secretion of IL-10 into the media  increase of ROS production in the six more sensitive cell lines. Co-treatment with NAC effectively blocked ROS generation in all 12 cell lines.We previously found that the combination of ruxolitinib and vorinostat triggers the mitochondria-mediated signaling pathway. Alterations in the redox state of apoptotic cells could be related to activation of the final stage of the caspase cascade , 35. ROSin vitro model with cultured cells, thus limiting fluctuations in lactate levels. Based on our results, we further examined whether cells treated with the combined drugs would show decreased expression of GLUT1, the key enzyme of the glycolytic pathway. After 24 h of incubation with ruxolitinib plus vorinostat, we detected decreased GLUT1 expression in all 12 cell lines, with no significant difference between the most sensitive cell lines and the remaining cell lines . The success of ruxolitinib has inspired the rapid development of additional JAK inhibitors, extending the armamentarium for targeted therapy of malignancies driven by JAK signaling [JAK-2 dysregulation plays an important role as an oncogenic driver, prompting increasing interest in JAK-2 inhibitors for therapy against hematological malignancies , 38. Theignaling . Howeverignaling . Since Jignaling –43. Combin vitro model of hematological disease. We found that a combination of the two drugs showed greater efficacy compared to low-dose single-agent treatment with ruxolitinib and vorinostat in 12 cell lines, including two B-cell lymphomas, two T lymphoma, one chronic B-cell leukemia, two myeloma multiple, two Hodgkin lymphoma, two mantle lymphoma, one non-Hodgkin lymphoma. The results of combination treatment were additive in both mantle cell lymphoma cell lines, in one LH cell line, in one myeloma cell line, in one T-cell lymphoma cell line, and in one B-cell lymphoma cell line. In the remaining cell lines, the combination of the two drugs had highly synergistic effects. These results led us to postulate that this combination may have the potential to overcome protective effects of a tumor microenvironment. Indeed, tumor cells treated with the combination of ruxolitinib and vorinostat for 48 h were not protected by co-culture with stromal cells. The synergistic cytotoxic action of this drug combination was maintained for up to 120 h. We also found that the ruxolitinib and vorinostat combination influenced the cell cycle distribution, increasing G2-M phase arrest after 24 h in all 12 cell lines. Correspondingly, the combined treatment affected the related cell cycle proteins. In all 12 cell lines, treatment with ruxolitinib and vorinostat for 24 h resulted in upregulation of the cell cycle regulators p21 and p27, and downregulation of CCND1 and AURORA A, which are important in regulating the cell cycle control points. These results confirmed that this combination of drugs induced tumor growth inhibition.Our present results show that combination treatment with ruxolitinib and vorinostat had improved effects on proliferation and apoptosis in an After 24 h of single-agent treatment with ruxolitinib or vorinostat, less than 10% of cells were apoptotic annexin V-positive cells. However, this proportion of apoptotic cells increased to 50% in all 12 cell lines after treatment with ruxolitinib combined with vorinostat. Caspases play a very important role in programmed cell death and are thus attractive targets for the development of new cancer therapies. We chose to study the caspase initiator caspase-8, and the caspase effectors caspase-9 and caspase-3. After 24 of combined drug treatment, we detected cleavage of caspase-8 and caspase-3 in all 12 cell lines, suggesting activation of the extrinsic apoptotic pathway. Moreover, only the six most sensitive cell lines showed increases in the cleaved form of caspase-9, suggesting caspase activation of mitochondrial events regulating intrinsic apoptosis. The most sensitive cell lines also differed from the other cell lines in terms of the regulation of anti-apoptotic proteins. After treatment with the drug combination, all 12 cell lines showed increased expression of the pro-apoptotic proteins BAX, BID and BAD, but only the six more sensitive cell lines showed downregulation of the apoptotic inhibitors BCL-2 and MCL-1. These results confirmed the possibility that ruxolitinib combined with vorinostat may differentially target signaling pathways in the six more sensitive cell lines.JAK/STAT activation has been demonstrated in hematological malignancies, providing the rationale for various therapeutic approaches involving JAK kinase inhibitors –46. HereJAK is reportedly involved in autophagy induction for immune regulation in various cancer cells –52. Thusin vitro and ex vivo conditions to clinical practice. Our present results offer evidence of synergistic interaction between ruxolitinib and vorinostat in hematological tumor cells, and provide the rationale to support clinical studies using the combination of both agents in patients.In conclusion, here we show that treatment with a combination of ruxolitinib and vorinostat led to decreased glucose metabolism, and that this partial reversion of the Warburg effect was associated with ROS production, apoptosis, and cell growth inhibition. Testing these drugs under different cell culture conditions provided information regarding drug efficacy, and could help predict pharmacological effects that may be difficult to translate from http://www.dsmz.de/human_and_animal_cell_lines/main). The B-cell lymphoma cell line WSU-NHL was kindly provided by Dr. M. Introna . GRANTA519 and MEC1 cells were cultured in Iscove’s MEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL). All other cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL) . The human mesenchymal stem cells (hMSC) were purchased from Tebu Bio and cultured in complete MSC expansion media (Tebu Bio). Cells in the logarithmic growth phase were used for experiments.We purchased the mantle cell lymphoma cell lines GRANTA519 and Jeko1, multiple myeloma cell lines U266 and RPMI8266, B-cell lymphoma cell lines Karpas422 and RL, cutaneous T-cell lymphoma cell line HUT78, anaplastic cell lymphoma cell line Karpas 299, chronic B-cell leukemia cell line MEC1, and Hodgkin lymphoma cell lines LH540 and LH1236 from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. Cell line characteristics are available online  to create 1050) using Calcusyn software  applying the median-effect method. Viability was assessed by exclusion assay with 0.2% Trypan Blue (Euroclone). DMSO was directly added to control samples and used as a drug solvent for drug-treated samples, and the DMSO concentration was kept constant (0.1%) among treatments for all experiments. The maximum final concentration of DMSO (< 0.1%) did not affect cell proliferation and did not induce cytotoxicity in the tested cell lines.In dose-response experiments, cell lines were treated with increasing concentrations of ruxolitinib and vorinostat for 24 and 48 h. Relative viable cells were determined by MTT assay . The absorbance at 550 nm was measured using an ELISA reader. We determined the drug concentration required for 50% inhibition of cell proliferation , following the manufacturer’s instructions. At least 20,000 events were acquired using a FACSCalibur cytometer , which were then analyzed using FlowJo Software .In preparation for intracellular marker staining, the cells were washed with 1% v/v FBS-PBS (staining buffer), fixed in 4% w/v paraformaldehyde (20 min at 4°C), and permeabilized with 0.1% saponin in PBS. Then the cells were incubated for 1 h at 4°C with primary antibodies against p-BAD (Ser112), BID, BAX, MCL-1 and BCL-2 . Finally, the cells were incubated with fluorophore-tagged secondary antibodies for 45 min at 4°C, and the cells were analyzed using a FACSCalibur flow cytometer. The results were analyzed using the CellQuest program. Whenever possible, the immunophenotype results were expressed as the percentage of positive cells.We measured caspase-3, caspase-8, and caspase 9 activity using colorimetric assay kits purchased from Enzo Life Sciences, in accordance with the manufacturer’s instructions. Samples were analyzed using an ELISA reader.For cell cycle distribution analysis, cells were harvested after 24 h of drug treatment. These cells were fixed in ethanol (95%) containing RNase (10 g/mL), stained for 15 min with propidium iodide , and then analyzed with a FACSCalibur cytometer. We calculated the percentages of cells in the subG1/G0 (dead cells), G1/G0, S, and G2/M phases of the cell cycle  using Modifit LT software .We quantified autophagy in cell lysates using the quantitative immunometric detection method provided by the p62 (sequestosome 1) ELISA kit  following the manufacturer’s instructions.Reactive oxygen species (ROS) production was analyzed by incubating cells with 2′,7′-dicholorodihydrofluorescein diacetate  in complete medium for 30 minutes at 37°C. Fluorescence was quantified with a FACSCalibur cytometer (Becton Dickinson), and these data were analyzed using FlowJo Software (Tree Star).We measured lactate and ATP levels using an L-lactate assay colorimetric kit and an ATP assay colorimetric kit (Abcam), following the manufacturer’s instructions. In the L-lactate assay kit, lactate is oxidized by lactate dehydrogenase to generate a product that interacts with a probe, producing a color (OD = 450 nm). The ATP assay kit is based on the phosphorylation of glycerol, generating a product that can be colorimetrically quantified (OD = 570 nm).6 cells were pelleted and then lysed using the Mammalian Cell Extraction Kit  following the manufacturer’s instructions. Proteins (100 µg/lane) were electrophoresed on 4–20% (w/v) Miniprotean TGX Precast Gels , and then transferred to nitrocellulose membranes . Membranes were immunoblotted using the following primary antibodies: total JAK2, p-JAK2, total STAT3, p-STAT3, total STAT5, p-STAT5, CCND1, AURORA A, p21, p27 and GLUT-1. Next, the membranes were incubated with species-specific horseradish peroxidase (HRP)-conjugated secondary antibody. All above-mentioned antibodies were purchased from Euroclone. Blots were developed using SuperSignal West Pico Chemiluminescent Substrate , and images were acquired using Chemidoc XRS+ and analyzed with Image Lab Software v.3.0 (Bio-Rad Laboratories).After drug treatments, 1 × 105 cells/mL for 24 h with ruxolitinib and vorinostat, alone and in combination. Then the supernatants were collected and analyzed for levels of the cytokines IL-6, IL-10, and IL-17a using a Human Magnetic Luminex Assay (R&D Systems), following the manufacturer’s instructions.Cells were cultured at a density of 5 × 10P values less than .05 were assigned significance. Data were analyzed using the Stata 8.2/SE package .All experiments were independently repeated three times, with multiple replicates within each run. Data are expressed as mean ± standard error. We analyzed statistical differences between control and drug-treated cells using one-way ANOVA, and


This panel of 150 genomically validated cancer cell lines represents a valuable resource for the cancer research community and will advance our understanding of the disease by providing a standard reference for cell lines that can be used for biological as well as preclinical studies.In recent years, biological research involving human cell lines has been rapidly developing in China. However, some of the cell lines are not authenticated before use. Therefore, misidentified and/or cross-contaminated cell lines are unfortunately commonplace. In this study, we present a comprehensive investigation of cross-contamination and misidentification for a panel of 278 cell lines from 28 institutes in China by using short tandem repeat profiling method. By comparing the DNA profiles with the cell bank databases of ATCC and DSMZ, a total of 46.0% (128/278) cases with cross-contamination/misidentification were uncovered coming from 22 institutes. Notably, 73.2% (52 out of 71) of the cell lines established by the Chinese researchers were misidentified and accounted for 40.6% of total misidentification (52/128). Further, 67.3% (35/52) of the misidentified cell lines established in laboratories of China were HeLa cells or a possible hybrid of HeLa with another kind of cell line. Furthermore, the bile duct cancer cell line HCCC-9810 and degenerative lung cancer Calu-6 exhibited 88.9% match in the ATCC database (9-loci), indicating that they were from the same origin. However, when we used 21-loci to compare these two cell lines with the same algorithm, the percent match was only 48.2%, indicating that these two cell lines were different. The SNP profiles of HCCC-9810 and Calu-6 also revealed that they were different cell lines. 150 cell lines with unique profiles demonstrated a wide range of  Although cell line authentication has been widely recommended for many years, misidentification, including cross-contamination, remains an unresolved issue , 2. WithDNA from 278 cell lines was analyzed by STR profiling . By perfThe authenticity test of non-Chinese cell model has been performed by cell repositories and their corresponding STR profiles that are available for comparison. 129 instances out of 193 characterized samples had distinct STR profiles and consisted of 78 individual cell lines. A total of 64 instances were unequivocally misidentified or cross-contaminated , including four examples that were notably false cell lines  for which the actual identity had been previously reported [Compared to the non-Chinese group, extremely high levels of contamination were found among cell lines established in China. In the Chinese model, only 19 instances out of 71 had distinct STR profiles (26.8%) and consisted of 11 individual cell lines . The remThe amelogenin locus on the X and Y chromosomes were used for gender identification. Since Y chromosome was unstable, some cell lines and even normal cells from older male donors lost the Y chromosome , such asIn most instances (89/128), the cross-contaminating intruder could be identified via comparison in the databases provided by cell banks. The remaining 39 cell lines could not be identified by source. Cross-contamination of cell lines causes mischaracterization, which has been demonstrated in several studies. HeLa  and its The prolific contaminants varied in each group. 25 instances of 64 contaminants in the non-Chinese model were HeLa, with an incidence rate of 39.1%. Contamination caused by non-human cell lines contributed a minor proportion . The remaining contaminants identified in the non-Chinese model are described as follows: HCT-15 (n = 3), AGS (n = 1), U-87MG (n = 1), H226 (n = 1), SNB19 (n = 1), ECV304 (n = 1), T24 (n = 3), CCRF-CEM (n = 2), H460 (n = 1), 769-P (n = 1), Saos-2 (n = 1) and unknown (n = 14) . In contIn general, 69 HeLa cell lines showed one or more alterations in STR profile compared with the prototype cell. The differences among most of the samples from different laboratories were likely attributable to different culture conditions or passage number. This suggested HeLa cell line had remarkable degree of genetic instability as defined by STR profile. In sharp contrast, the subclones generated from K562 showed a high concordance of the clonotype homogeneity, i.e., one different clonotype was observed among the six K562 cell lines and the profile of the subclones differed from the parental culture by a single change of STR.Comparison of original cell lines with the sublines derived from them revealed that nine other cell lines including 293, BxPC-3, HT-29, DLD-1, HCT-116, Jurkat, Raji, HeLa and 5637, exhibited variable STR profiles after multiple passages in culture. The subclones of these nine cell lines shared 80–94.12% identity to the original cell lines and appeared to bear null allele and mutations . SeveralIn contrast to the loss of heterozygosity, microsatellite instability , charactHigh degree of similarity (88.9%) was observed in one pair of cell lines, HCCC-9810 and Calu-6, by using ATCC STR database (9-loci STR database). This percent match indicated that these two cell lines were from the same origin. The HCCC-9810 cell line shared 4 same loci with the Calu-6 cell line at AMEL, D5S818, D13S317 and TH01, and differed from the Calu-6 cell line at other 5 loci by using the ATCC 9-loci STR database for comparison. However, when comparing these two cell lines by using 21-loci STR, we found that they still shared the same 4 loci as found by using 9-loci STR comparison, while the remaining 17 loci were different. The alleles of D21S11, D3S1358, D2S441, Penta E, D12S391 and FGA were totally different. When the same algorithm as used in 9-loci was adopted in 21-loci, the percent match of these two cell lines was 48.2%, indicating that they were from different origin . To confWe presented a panoptic survey of cell line cross-contamination among human tumor cell lines using molecular genetic methods. The STR profiling technique uses fluorescence and multiplex PCR based technology to detect STR loci with overlapping size range. The specificity and polymorphism advantages make it method of choice for the authentication of human cell lines.The misidentification and cross-contamination of cell lines are deemed to be a widespread problem in research, dating back as far as 45 years ago. Our lab started using STR for cell line authentication in 2009 with 16-STR loci. An alarming rate of cross-contamination among human cell lines used in China was reported by Fang Ye and colleagues . The simUndue success in cell line cultures from primary material provides valid ground for suspicion of cell line cross-contamination. Normal diploid cell lines, namely CCC-HIE-2, FHC and FSH-74int, established by the Chinese Academy of Medical Sciences , 33, resClose to 67% of the misidentified cell lines established in China were contaminated by the HeLa cell line. In addition to their use as research tools, false or misidentified cell lines such as HL-7702, TSCCA and BEL-7402 have been used in anti-cancer drug screening programs in respected centers in China. WISH, which has been reported as a HeLa contaminant for decades , was alsThe grave problem of cell line misidentification in China has been neglected. Lack of information has prevented researchers from keeping up to date with literature reports of cell line misidentification. Although the international cell repositories have compiled a list of cross-contaminated cell lines to alert their users, the grave problem continues to fester due to the negligence of cell line users and originators as well as journal editors and funding agencies. Making the Chinese researchers aware of the situation is the most essential step. For those scientists working with cell lines, basic authentication test such as STR profiling, isoenzyme analysis, cytochrome C oxidase subunit I (COI) barcoding and mycoplasma contamination tests should be carried out and transferring cell lines to colleagues should be avoided.STR-based profiling methods have been used for the authentication of cell lines for more than 24 years. However, there are variations in the numbers of loci and in the specific loci comprising the published profiling. We observed that HCCC-9810 and Calu-6 did not reliably discriminate between same or different origin within 9 markers. Obviously, authentication was greatly improved when we employed the 21-loci STR profiling and SNP genotyping method. Over the last years, several STR profiling assays have been published for developmental validation of new STR system. Compared to the current STR profiling, which included larger number of strategically selected markers for the human population, the discrimination power of 9 STR markers is satisfactory. Obviously, authentication was much more improved when we employed the 21-loci STR profiling and SNP genotyping method. Unfortunately, currant STR databases such as ATCC, DSMZ and JCRB only provide 9-loci STR profiles.www.CCTCC.org) appealed to the scientific community to bring attention to the importance of cell line authentication. In 2016, a group of cell biologists led by CCTCC developed a program for cell line authentication. CCTCC repository generated a new 21-loci STR database (www.CCTCC.org/STR.html)). It was designed to represent a unique reference for validated molecular authentication of human cell lines, using the 21-marker commercial kit. End users could compare their STR profiles of cell lines from the website. The schema of the database included data tables, which consisted of a general table, where information on STR loci value could be entered, and match criteria have been set to 50–100% at 5% interval in accordance with ATCC, DSMZ and CCTCC standards. We hope that the verified cell lines available in CCTCC STR verification-database can strengthen the reproducibility and comparability of cell lines in different laboratories.CCTCC  emphasized the need for careful and frequent authentication of cell lines, possibly by more than one method. STR-based profiling method has been a great benefit to the scientific community, which has allowed cell line-based studies to be carried out without false premise resulted from misidentified or contaminated cell lines. To prevent exacerbation of the problem and reduce the frequency of cell-line misidentification and cross-contamination, the whole scientific community needs to get involved.2.The authenticity of 278 tumor cell lines deposited in CCTCC by the 28 institutes was examined. The individual cell lines were supplied with duplicate, triplicate and quadruplicate collections from an independent source. The cell lines were grown in respective media at 37°C in a humidified incubator filled with 5% CODNA was isolated from cells using a genomic DNA purification kit . Mutiplex PCR reactions were performed on a T-Gradient thermal cycler . Briefly, DNA from each cell line was amplified using the Microreader TM21D System  according to the manufacturer’s instructions. The TM21D system uses primers to co-amplify 20 STR loci  and amelogenin. The reaction mixture contained 2 ng genomic DNA, 10× PCR buffer, 2.5 mM dNTPs, 0.2 μM of each primer, and 2.5 U of Taq DNA polymerase. The PCR protocol was as follows: denaturing at 94°C for 5 min, denaturing at 94°C for 30 s, annealing at 60°C for 60 s, extension at 70°C for 60 s in 30 cycles and final extension at 60°C for 30 min. One μl aliquot of amplicon was mixed with 7.5 μl HiDiformamide,  and 0.5 μl ROX-500 internal-lane size standards. The mixture was denatured at 95°C for 5 min and placed on ice immediately for 5 min, finally added 1 μl allelic ladder  and loaded on a POP-7 polymer gel  for electrophoresis. The samples were electrophoresed with the GeneScan program on the ABI 3130 Genetic Analyzer . The amplicon sizes were determined using GeneMapper 3.2 . Alleles were designated by comparison to the allelic ladder. Each sample was repeated at least twice to confirm the results. If the sample could not be amplified in three time repeats, it will be considered as non-human origin. For data comparison, well-characterized and validated reference data obtained from ATCC and DSMZ were used. The algorithm of ATCC STR database was used for STR profile comparison. Percent match = Number of shared alleles between query sample and database profile/Total number of alleles in the database profile. Cell lines with ≥ 80% match are considered to be related, derived from a common ancestry. Cell lines with < 55% match are considered to be not related. Cell lines with 55% ~ 80% match require further authentication of relatedness.To test the sensitivity of detecting cross-contamination, AsPC-1:HeLa cell mixtures at fixed ratios of 99:1, 95:5, 90:10 and 80:20 were generated. DNA was extracted from the cell mixtures using the genomic DNA purification kit. DNA concentrations were determined by a NanoDrop 8000 spectrophotometer. Contaminated samples were created by mixing each 50 ng/μl sample into the other to create 20%, 15%, 10%, 5% and 1% contamination by volume.DNA was isolated from cells using a genomic DNA purification kit . DNA was amplified by PCR using 10 species-specific primer sets designed to amplify a specific product, respectively. The primers for 10 species were listed in the HCCC-9810 and Calu-6 cell lines were obtained from the cell bank at the Chinese Academy of Science. The genetic identification of HCCC-9810 and Calu-6 cell lines was further characterized by SNP genotyping using direct SNP sequencing and standard PCR method. The 24 selected tagging SNPs were rs5742909, rs231775, rs1800682, rs2234767, rs1800896, rs1800872, rs1805010, rs1805015, rs1801275, rs1554606, rs1800797, rs1041981 and rs7208422 in immune response genes, rs9344, rs1801270, rs1042522 in cell cycle genes, and rs8177374, rs5743305, rs3775291, rs3788935, rs3761623, rs5743836, rs352139 and rs352140 in innate response genes. Genotyping was performed on ABI Prism 3100 Genetic Analyzer in conjunction with Clustal software. Details of PCR primer sequences for SNP genotyping were listed in S1 Fig(A) Species screen for HIBEC cell line. (B) Confirmation of rat origin for HIBEC cell line. (C) Species screen for C201441 cell line. (D) Confirmation of mouse origin for C201441 cell line.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Richtersius coronifer under repeated cycles of desiccation, and the potential effect of repeated desiccation on size, shape and number of storage cells. We also analyzed potential change in body size, gut content and frequency of mitotic storage cells. Specimens were kept under non-cultured conditions and desiccated under controlled relative humidity. After each desiccation cycle 10 specimens were selected for analysis of morphometric characteristics and mitosis. The study demonstrates that tardigrades may survive up to 6 repeated desiccations, with declining survival rates with increased number of desiccations. We found a significantly higher proportion of animals that were unable to contract properly into a tun stage during the desiccation process at the 5th and 6th desiccations. Also total number of storage cells declined at the 5th and 6th desiccations, while no effect on storage cell size was observed. The frequency of mitotic storage cells tended to decline with higher number of desiccation cycles. Our study shows that the number of consecutive cycles of anhydrobiosis that R. coronifer may undergo is limited, with increased inability for tun formation and energetic constraints as possible causal factors.Tardigrades represent one of the main animal groups with anhydrobiotic capacity at any stage of their life cycle. The ability of tardigrades to survive repeated cycles of anhydrobiosis has rarely been studied but is of interest to understand the factors constraining anhydrobiotic survival. The main objective of this study was to investigate the patterns of survival of the eutardigrade  Water availability is one of the most important ecological factors and evolutionary pressures on terrestrial life. Despite the fact that water is crucial for all life forms, numerous organisms  survive temporary drying to equilibrium with the air humidity by entering a highly stable and reversible state called anhydrobiosis, a special form of the ametabolic life state known as cryptobiosis ,2,3,4,5.Among animals, tardigrades represent one of the main groups in which a capacity for anhydrobiosis is widespread. Tardigrades are microscopic aquatic animals found in a variety of habitats worldwide , and theThe state of anhydrobiosis, characterized as an ametabolic condition, is not connected with any energy consumption, and this explains why tardigrades may stay in this state for many years, even decades, and still be able to revive ,14. HoweMacrobiotus (species not given). The results showed that single specimens were able to survive up to 9 repeated desiccations, but already after 5 desiccations about 50% of the animals had died. The only other study where tardigrades have been exposed to several sequential cycles of anhydrobiosis is that by Hengherr et al. [Milnesium tardigradum were repeatedly desiccated with intermediate 7-day periods of hydration under cultured conditions allowing feeding. The study found no decline in anhydrobiotic performance over 9 consecutive desiccations.Very few previous studies have evaluated how many consecutive periods of anhydrobiosis that tardigrades are able to survive. According to Baumann , Lance  was the r et al. , in whicThe purpose of the current study was to investigate the patterns of survival in a tardigrade under repeated cycles of desiccation/rehydration and the potential effect of repeated desiccation on size, shape and number of storage cells. We also analyzed if body size, gut content and frequency of mitosis in storage cells change over the course of repeated desiccation.Richtersius coronifer . The population consists almost exclusively of females [Orthotrichum cupulatum) containing tardigrades were collected dry and stored at room temperature for 3 days until use. Moss cushions were hydrated for about 2 hours in tap water and within the following 3 hours 400 active specimens were extracted with sieves (mesh size 250 and 40 μm) under running tap water. Extracted animals were washed thoroughly with distilled water to remove adherent particles.In our desiccation experiment we used medium sized  specimens of the eutardigrade  females . No cult females  for descThe general procedure of our study consisted of repeated 24 hour periods of desiccation at 95% relative humidity (RH) followed by 5 hours of rehydration. These times were assumed to provide enough time for the animal to enter anhydrobiosis and to rehydrate and adjust physiologically for the hydrated state, respectively. After each desiccation cycle, 10 revived specimens were selected for analysis of mitosis in storage cells and morphometric characteristics, and the remaining animals that were alive were mixed and prepared for a new period of desiccation. This procedure was repeated until there were too few alive animals to continue.st cycle = 40 specimens/replicate, 2nd = 38.4, 3rd = 31.5, 4th = 22.9, 5th = 14.9, 6th = 3.5). In all desiccation cycles the number of specimens in the 10 replicates differed by maximum one animal.At the start of the experiment, 400 randomly chosen specimens were put individually on ten replicate filter papers (5 x 2 cm) in petri dishes and transferred into a desiccator with a saturated potassium nitrate (KNO3) salt solution providing a relative humidity of 95%. The 24h desiccation allowed the animals to enter anhydrobiosis and equilibrate with the surrounding humidity condition. Since the total number of animals available for each new desiccation cycle steadily declined , the mean number of animals in each replicate was a decreasing function of the number of desiccation cycles . Animals were assumed to be dead if there was no visible body movement. After each rehydration period, alive specimens were put together in a single dish and transferred individually with an Irwin loop to 10 new filter papers, followed by a new period of desiccation. For each replicate sample within a desiccation-rehydration cycle the proportion of surviving animals was calculated and used in the analysis.The 10 animals randomly selected after each desiccation/rehydration cycle were transferred in a drop of water to an object slide, covered with a cover slip, and observed under light microscope (Olympus BX60) fitted with a digital camera . Images of the whole animal, buccal tube and storage cells were taken for later measurements using the image analysis software . The time for collection of all images for a single specimen was about 10 minutes. Images for measurement of buccal tube and storage cells were taken at 40X magnification.in toto in a drop of acetic-lactic orcein according to the method used by Rebecchi [After initial observation specimens were transferred into Carnoys´ solution for 3 hours and stained Rebecchi . Slides Measured morphometric variables were body length, buccal tube length, gut content, size and shape of storage cells and presence of mitotic storage cells. Measurement of total body length was taken from anterior to posterior part of the body excluding the fourth pair of legs, and buccal tube length was measured according to the description by Pilato . The meaStorage cell shape and size were estimated from 20 randomly selected cells per individual (and from different parts of the bodies). Cell size was calculated as the mean of two diagonal right angle diameter measurements for each cell. From the diameter measurement cell volume was calculated  and used in the analysis. Shape of the cells was characterized for each examined individual as regular  or irregular (crescent or borders not straight). There were no specimens with mixed cell shapes; all cells in an animal were either regular or irregular.In order to calculate the mitotic index  at the individual level, the total number of clearly recognizable storage cells was estimated manually at 100X magnification with phase contrast and oil, using image analysis software (see above). Smashed cells, without clear borders, were not counted. Mitotic storage cells  were ideIn total 420 specimens were used in the experiment, 400 specimens in the repeated desiccation sequence and 20 as controls. Mitosis and morphometric analyses were performed on 80 specimens.Statistical effects on survival of repeated desiccations were tested using ANOVA and linear regression analysis (IBM SPSS Statistics v.23). Proportion data were arcsin-transformed before statistical analyses. In tests where parametric assumptions were not met, non-parametric statistics were used. Associations between mitosis occurrence and desiccation cycles/phenotypic traits were analyzed with logistic regression. Statistical tests were considered significant when P < 0.05, and marginally significant when P = 0.05.5,54 = 20.96, P < 0.001; 1,58 = 87.62, r = –0.78, P < 0.001). The highest survival rate was recorded after the first desiccation (98.5%) and the lowest non-zero survival rate was recorded after the sixth desiccation (28.6%). After the 6th desiccation only 10 specimens were alive and all of these were used for morphometric and mitosis analyses.Survival rates differed significantly among the repeated desiccations , with desiccation groups 5 and 6 having significantly more semi-tuns than most of the other groups , and there was no trend in gut content in relation to number of desiccation cycles .There was an overall significant difference in the mean number of storage cells among desiccation cycle groups, including also the control group  of all examined specimens. The average percentage of mitotic storage cells among all cells (“mitotic index”) was 0.28%  when all analyzed animals  were included, and 1.60%  when only animals where mitosis was found were included. The proportion of animals with mitotic storage cells in each desiccation cycle varied between 0 and 0.4 . OccurreR. coronifer are able to survive a maximum of 6 repeated desiccations under non-cultured conditions, with declining survival rates as the number of desiccations increased. This result is slightly lower than in the study by Baumann [initial population of animals surviving after different numbers of desiccation cycles. Since we removed 10 specimens after each desiccation cycle, the proportion of survivals in relation to the initial population were calculated based on the specific desiccation cycle survival rate . He also used a different desiccation-hydration schedule, with 24 hour desiccations (at 35–45% relative humidity), and 15 minute rehydrations. His results must therefore be taken with some caution. In contrast to Baumann [Our study shows that tardigrades of the species  Baumann  who repo Baumann . However Baumann  only use Baumann  and the  Baumann  did not Dactylobiotus dispar Murray, 1907 (formerly: Macrobiotus dispar). Howeverowever, data from another experiment in R. coronifer in which animals were starved for several days under continuously hydrated conditions at room temperature did not show a significant decline in number of storage cells . It is therefore not likely that the reduced storage cell number in this study was related to starvation during the short (5 hour) repeated periods of hydration.The decline in the probability of a tardigrade to survive a desiccation cycle with number of previous desiccations suggests a progressive deterioration in the functions underlying anhydrobiosis. One plausible explanation for this limitation on multiple desiccations is that the animals eventually reach an energetic constraint, where the physiological processes necessary for the transition to and from the anhydrobiotic state is not supported by available energy. In support of this explanation our analyses showed a significant reduction in the number of storage cells after the fifth desiccation, indicating reabsorption of cells. Reabsorption of storage cells was also reported by Węglarska  as an efR. coronifer, corresponding to a decrease in cell volume by 22%. Decreased storage cell size has also been documented in the late phase of oogenesis, when energy investment into the eggs are high [M. tardigradum, Paramacrobiotus tonollii and Macrobiotus sapiens) in response to 7 days of starvation, and in M. tardigradum in response to one cycle of anhydrobiosis. However, the number of storage cells in that study [R. coronifer show that this species is able to survive continuous starvation at room temperature for many days and even weeks [R. coronifer therefore remains unclear.Energy depletion connected to anhydrobiosis is also expected to lead to reduced size of the storage cells, as previously shown by Jönsson and Rebecchi . In thatare high ,26. At tare high  also repat study  was not at study  in his sen weeks ,28. The th and 6th cycle, corresponded with a higher proportion of animals that were unable to contract properly and create tuns when they were desiccated. This was also observed by Baumann [The decline in survival with more desiccation cycles, particularly clear in the 5 Baumann , who sug Baumann , but we R. coronifer, and a mitotic index of 1.47% . In that study it was also found that a higher frequency of mitosis was connected with the period of moulting which usually corresponds to the late phase of egg development. As mentioned above, that period is also characterized by smaller storage cells [The overall frequency of mitosis found in this study was similar to that reported in Czernekova and Jönsson , who fouge cells ,26, but ge cells  nor in tRichtersius coronifer to enter and successfully leave the anhydrobiotic state declines with the number of previous anhydrobiotic cycles experienced, thus verifying a limit of how many times this tardigrade can enter anhydrobiosis in a row. The causal explanation behind this decline in anhydrobiotic performance is unclear, but increased inability to morphologically rearrange the body into a proper tun seems to be involved. The ultimate physiological or energetic reason for this remains to be documented.In conclusion, our study clearly shows that the ability of Ethical approval: This chapter does not contain any studies with human participants performed by any of the authors.S1 FigRichtersius coronifer after proper tun formation. b) A desiccated specimens of Richtersius coronifer after semi-tun formation. c) A desiccated specimens of Richtersius coronifer in an extended (non-tun) state.a) Two desiccated specimens of (JPG)Click here for additional data file.S2 FigScale bar: 10μm.(TIF)Click here for additional data file.S1 FileTable A. Survival estimates for different number of repeated desiccation cycles, and the number and percentage of specimens that were in semi-tun or extended state.a The total number of specimens were divided into 10 replicate samples. b Survival was evaluated after 3 and 5 hours of rehydration, except for specimens that were chosen for morphometry and mitosis analyses, which were evaluated 3 hours after rehydration. c Specimens with irregular tun were characterized as “semi-tun stage”. d Specimens that did not contract during desiccation. See Table B. Mean number and size (diameter) of storage cells after repeated cycles of desiccations. Estimates for group 0 (controls) were based on 19–20 specimens, while estimates for the other groups were based on 10 specimens. Four outliers with cell diameters > 20 um were removed from the data; two from group 0 (5440 and 8662 um3), one from group 1 (4571 um3), and one from group 6 (10409 um3).(DOC)Click here for additional data file.


Widely established targeted therapies directed at triple negative breast cancer (TNBC) are missing. Classical chemotherapy remains the systemic treatment option. Cisplatin has been tested in TNBC but bears the disadvantage of resistance development. The purpose of this study was to identify resistance mechanisms in cisplatin-resistant TNBC cell lines and select targeted therapies based on these findings.The TNBC cell lines HCC38 and MDA-MB231 were subjected to intermittent cisplatin treatment resulting in the 3.5-fold cisplatin-resistant subclone HCC38CisR and the 2.1-fold more resistant MDA-MB231CisR. Activation of pro-survival pathways was explored by immunostaining of phospho-receptor tyrosine kinases. Targeted therapies  against activated pathways were investigated regarding cancer cell growth and cisplatin sensitivity.In HCC38CisR and MDA-MB231CisR, phosphorylation of epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) was observed. In HCC38CisR, treatment with NVP-AEW541 increased potency of lapatinib almost seven-fold, but both compounds could not restore cisplatin sensitivity. However, the dual phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 acted synergistically with cisplatin in HCC38CisR and fully restored cisplatin sensitivity. Similarly, NVP-BEZ235 increased cisplatin potency in MDA-MB231CisR. Furthermore, NVP-AEW541 in combination with lapatinib restored cisplatin sensitivity in MDA-MB231CisR.Simultaneous inhibition of EGFR and IGF1R in cisplatin-resistant TNBC cell lines was synergistic regarding inhibition of proliferation and induction of apoptosis. Co-treatment with NVP-BEZ235 or with a combination of NVP-AEW541 and lapatinib restored cisplatin sensitivity and may constitute a targeted treatment option for cisplatin-resistant TNBC.The online version of this article (10.1186/s12885-017-3695-5) contains supplementary material, which is available to authorized users. Breast cancer is the second most common cancer in the world and the incidence of female breast cancer has continuously increased . In 2013We have previously shown that resveratrol or ellagic acid prevented the development of cisplatin resistance in the ovarian cancer cell line A2780. This effect is at least in part based on the prevention of activation of ErbB2 and ErbB3 in the course of long-term cisplatin treatment . In 1998The purpose of our study was to identify resistance mechanisms in a cisplatin-resistant TNBC cell line leading to targeted therapies as treatment options in this cancer type. Evaluation of the phosphorylation status of receptor tyrosine kinases revealed activation of IGF1R and EGFR as a result of cisplatin resistance. Therefore, inhibitors of these two receptors (NVP-AEW541 and lapatinib) and an inhibitor of downstream acting PI3K/Akt/mTOR (NVP-BEZ235) were evaluated regarding their effects on cancer cell growth and cisplatin sensitivity. Indeed, co-treatment of NVP-AEW541 with lapatinib increased potency of lapatinib in the cisplatin-resistant TNBC cell line HCC38CisR but did not increase cisplatin sensitivity. On the other hand, NVP-BEZ235 acted synergistically with cisplatin and fully restored cisplatin sensitivity in HCC38CisR. Furthermore, in the highly cisplatin-resistant TNBC cell line MDA-MB231CisR, treatment with NVP-BEZ235 or co-treatment of NVP-AEW541 with lapatinib increased potency of cisplatin up to 4.8-fold.NVP-AEW541 and NVP-BEZ235 were gifts from Novartis . Lapatinib, KU0063794 and LY294002 were from Cayman Chemical . Cisplatin was purchased from Sigma-Aldrich . 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva . Propidium iodide was from PromoCell . Roswell Park Memorial Institute (RPMI) media 1640, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin  and trypsin-EDTA  were purchased from PAN Biotech . Primary antibodies were purchased from R&D Systems   or Santa Cruz Biotechnology  . HRP-conjugated secondary antibodies were from R&D Systems. All other reagents and chemicals were from VWR BDH PROLABO .2. HCC38CisR and MDA-MB231CisR, the cisplatin resistant subclones of HCC38 and MDA-MB231, respectively, were generated by intermittent treatment of HCC38 or MDA-MB231 with cisplatin for 40 cycles according to methods previously published [The triple negative breast cancer cell line HCC38 was obtained from ATCC  and cultivated in RPMI-1640 medium supplemented with 10% FBS, 120 μg/ml streptomycin and 120 U/ml penicillin. The TNBC cell line MDA-MB231  was cultivated in DMEM supplemented with 15% FBS, 120 μg/ml streptomycin and 120 U/ml penicillin. Cells were grown at 37 °C in a humidified atmosphere containing 5% CO50 of the resistant cell line and IC50 of the sensitive cell line. To investigate the effect of the small molecule inhibitors on cisplatin cytotoxicity, compounds were added 48 h prior to 72 h cisplatin treatment. For combination index analysis, cell viability was determined from each well relative to the average absorbance of control wells. The combination indexes (CIs) were calculated using CalcuSyn 2.1 software  based on the Chou − Talalay method [Cell viability was determined using the MTT assay as previously described . Resistay method . CI > 1 2 × 2 H2O in 1% formaldehyde solution. After a second washing step, cells were lysed with a 1:1 mixture of ethanol and 1% acetic acid. Absorbance was measured at 544 and 690 nm in a FLUOstar microplate reader .To exclude compound effects potentially influencing mitochondrial activity, neutral red cell viability assay instead of MTT assay was performed as previously described . BrieflyThe assay was performed as previously described . Cells wFor western blotting, standard procedures were used as previously described .The tyrosine-kinase phospho-proteom was investigated by a human phospho-receptor tyrosine kinase antibody array (Cat# ARY001) from R&D Systems according to the manufacturer’s protocol. Cell lysate containing 300 μg protein was used.Distribution of cell cycle phases of the different cell lines was analyzed by flow cytometry using standard procedures as previously described .Apoptotic cells were determined by propidium iodide staining as previously described .Scratch assay was performed according to standard procedures as previously described . Cell-frp < 0.05. pIC50 ± SEM leading to the reported IC50 values are shown in Additional file Assays were performed at least in three independent experiments. Concentration effect curves were then generated by nonlinear regression curve fitting using the 4-parameter logistic equation with variable hill slope . Data presented are mean ± SEM if not otherwise stated. Statistical significance was assessed by two-tailed Student’s t-test or ANOVA and considered significant if 50 of cisplatin for 6 h. After 40 cycles, the IC50 (determined by MTT) has shifted from 2.7 μM to 9.4 μM corresponding to a resistance factor of 3.5 . IGF1R and EGFR phosphorylation was increased after 6 h cisplatin stress and 24 h recovery, and in HCC38CisR. If HCC38 treated 6 h with cisplatin could recover from cisplatin stress for one week, receptor phosphorylation decreased nearly to the initial state. Evaluating the expression of growth factor receptors, there was hardly any difference between untreated HCC38 and HCC38CisR. Akt expression and phosphorylation was enhanced in HCC38CisR compared to HCC38 either untreated or short-term treated with cisplatin. Long-term cisplatin treatment resulting in HCC38CisR further increased proliferation rate and decreased doubling time significantly from 24 h to 17 h as displayed in Fig. The cisplatin resistant cell line HCC38CisR was generated by weekly exposure to the IC3.5 Fig. . This re50 of both inhibitors was lower in HCC38CisR than in HCC38  than in HCC38CisR (69.3 nM)  sensitize HCC38CisR for cisplatin treatment by a factor of approximately 2. If both inhibitors LY294002 and KU0063794 were combined in 48 h preincubation prior to cisplatin treatment in HCC38CisR, the cisplatin IC50 of the parental cell line HCC38 was nearly restored  stronger than the effect of the combination of KU0063794 and LY294002.It has been shown that cancer cells can easily switch membrane-bound RTK pathways upon inhibition of a particular RTK and still use the same downstream signaling pathways . FurtherSynergism between NVP-BEZ235 and cisplatin was observed in MTT Table  and apop2/M phase slightly increased compared to control (28.3% versus 23.6%) accompanied by a slight decrease of cells in G1 phase , we found a 4.8-fold resensitization for cisplatin upon pretreatment with NVP-AEW541 and lapatinib  [1 phase. This might be one possible mechanism leading to reduced cell proliferation. These results are in accordance with studies performed on adrenocortical carcinomas applying EGFR and IGF1R inhibitors [Among breast cancer, TNBC has a poor prognosis due to the lack of targeted hormone or HER2-directed therapy and resistance development against classical cytostatics including cisplatin currently under clinical investigation for TNBC . We haveold Fig. . This ef.7–4 μM) , 24. In hibitors . Anotherhibitors , drugs rhibitors , the in hibitors . Taking Lapatinib and NVP-AEW541 were ineffective to restore cisplatin sensitivity in HCC38CisR Fig. . HoweverIn our study, NVP-BEZ235 could fully restore cisplatin sensitivity in the cisplatin-resistant TNBC cell line HCC38CisR and acted synergistically with cisplatin Fig. . CombiniTaken together, activation of EGFR and IGF1R and their downstream signaling pathway kinase Akt is associated with resistance induced by long-term treatment with cisplatin in the TNBC cell line HCC38 and in MDA-MB231. Based on these results, two approaches for treating cisplatin resistant cell lines are presented: 1) Simultaneous inhibition of EGFR and IGF1R by lapatinib and NVP-AEW541 is highly synergistic and results in the induction of apoptosis. Furthermore, co-treatment with lapatinib and NVP-AEW541 may increase cisplatin sensitivity as seen in MDA-MB231CisR. 2) Co-treatment of cisplatin-resistant TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 and cisplatin is synergistic, fully reversed acquired cisplatin resistance, and may thus constitute a targeted treatment option for cisplatin-resistant TNBC.Additional file 1:pIC50 values and standard error of the mean. pIC50 values, errors, and IC50 values of all MTT assays performed in this study are listed. (DOC 70 kb)Additional file 2:cell cycle distribution. The cell cycle distribution in HCC38 after treatment with NVP-AEW541, lapatinib or both compounds is displayed as bar graph. (DOCX 30 kb)Additional file 3:MTT assay of combination of RTK inhibitors with cisplatin. Influence of 48 h preincubation with 1.5 μM NVP-AEW541, 1 μM lapatinib or a combination of both compounds on cisplatin sensitivity in HCC38. (DOCX 173 kb)Additional file 4:MTT assay of NVP-BEZ235 . Effect of NVP-BEZ235 on cell viability determined by MTT assay. (DOCX 29 kb)Additional file 5:Western blot of cleaved PARP upon NVP-BEZ235 and cisplatin treatment. Western Blot on cleaved PARP after treatment of HCC38 with 20 nM NVP-BEZ235 or 2 μM cisplatin or a combination of both compounds. (DOCX 80 kb)Additional file 6:induction of apoptosis in HCC38 upon NVP-BEZ235 and cisplatin treatment. Induction of apoptotic nuclei in HCC38 after treatment with 2 μM cisplatin, 20 nM NVP-BEZ235 or a combination of both compounds. (DOCX 25 kb)Additional file 7:Characterization of MDA-MB231 and cisplatin-resistant MDA-MB231CisR. MDA-MB231 and cisplatin-resistant MDA-MB231CisR cells were characterized by MTT assay, phospho-RTK status, and induction of apoptosis upon kinase inhibitor and cisplatin treatment. (DOCX 141 kb)


A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. What's new?A variety of analytical approaches have indicated that melanoma cell line M14 and breast carcinoma cell line MDA‐MB‐435 originate from a common donor, but there is ongoing debate regarding which is the misidentified cell line. Here, authentication testing of M14 from 1975 (prior to the establishment of MDA‐MB‐435), with comparison to donor serum and lymphoblastoid cell line ML14, shows that M14 is the authentic cell line and MDA‐MB‐435 is a misidentified derivative. With clear provenance information and authentication testing of early samples, debates regarding the origins of problematic cell lines that are widely used in cancer research can be resolved. Questions were first raised regarding the tissue origin of MDA‐MB‐435 in 2000, when cDNA microarray analysis of the NCI‐60 panel showed that the expression pattern of the claimed breast carcinoma cells closely resembled patterns seen in melanoma cell lines.The authenticity of two cell lines, MDA‐MB‐435 and UCLA‐SO‐M14 (commonly referred to as M14), has been debated in the scientific literature over many years  analysis and bioinformatics analysis of gene expression) have demonstrated that the MDA‐MB‐435 cell line shares a common origin with the M14 melanoma cell line.This conclusion, however, has been debated on the basis of phenotypic evidence. MDA‐MB‐435 can express breast‐specific and epithelial‐specific markers, leading to the conclusion that it is derived from breast carcinoma and that expression of melanocyte‐specific markers may be caused by lineage infidelity,The provenance of M14 and MDA‐MB‐435 was reviewed in a 2009 letter, resulting in further concerns regarding the authenticity of M14.MDA‐MB‐435 and derivatives such as MDA‐MB‐435S  continue to be used extensively and described as “breast carcinoma” in the scientific literature.b). Many laboratories have used MDA‐MB‐435 as a breast cancer model, because of its rapid growth and ability to metastasize in nude mice, to develop treatments for breast cancer, or to help understand how breast cancers metastasize.A recent search of PubMed using the search term “MDA‐MB‐435” shows that the cell line was referred to in the title or abstract of 1,205 publications between 1982 and 2016. MDA‐MB‐435 was described as a breast cell line in 56% (123/221) of recent publications between 2013 and 2016  obtained frozen samples corresponding to the M14 donor (serum and cell lines), MDA‐MB‐435 and derivative MDA‐MB‐435S. Samples were used for DNA extraction or culture.One tube of donor serum, dated December 21, 1973.Two vials of M14 melanoma, labeledOne vial of ML14 (synonymous lymphoblastoid cell lineSamples from the donor of M14 were requested from the John Wayne Cancer Institute (JWCI) Sample Repository. The Sample Repository informed us that serum and cell lines, but not tissue samples, were maintained from this donor. Samples from the M14 donor were first deposited in June 1973. The Specimen Repository provided the following samples for testing:Requests for M14 or ML14 cells may be made to the JWCI Specimen Repository.2 in RPMI 1640  with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Mycoplasma testing was performed using the e‐Myco plus Mycoplasma PCR Detection kit , a PCR‐based assay containing internal and sample controls. Mycoplasma were not detected in M14 or ML14 cultures.M14 (adherent culture) and ML14 cells (suspension culture) were grown at 37°C and 5% COA sample of MDA‐MB‐435 was requested from the authors of an earlier study that demonstrated breast‐differentiation specific markers in MDA‐MB‐435 cells.M14 cells at passage 16 (one passage after thawing) were plated on a slide and grown to 80% confluence. Slides were fixed with formalin and immunohistochemistry performed using the Melanoma Triple Cocktail stain , a mixture of three mouse monoclonal antibodies directed against melanosome (HMB45), MART‐1/Melan A (A103) and tyrosinase (T311).DNA extraction for STR analysis from cultured cells was performed using the Quick‐gDNA MiniPrep kit . DNA extraction for ABO sequencing was performed using the DNeasy Blood & Tissue kit, using the protocol for Total DNA from Animal Blood or Cells . Pre‐treatment was performed using 25 µL RNase A  with incubation at 56°C for 10 min prior to purification. DNA was eluted in 10 mM Tris‐HCl, pH 8 and its concentration and quality assessed by gel electrophoresis and spectrophotometric measurement.DNA extraction from serum was performed using a different method that was previously shown to be effective for serum samples. A detailed procedure and references are supplied in the Supporting Information Methods section.To examine the ABO gene locus, primers were designed to amplify exon 6 of the human ABO locus. This region includes nucleotide 261; deletion of this nucleotide occurs in individuals that carry the O allele, resulting in a frameshift mutation.DNA from the M14 cell line was amplified using primers at a final concentration of 0.4 µM and 300 ng of genomic DNA. Cycle conditions were as follows: initial hold at 95°C, 2 min; followed by 25 cycles at 94°C, 30 sec; 64°C, 1 min; 72°C, 30 sec; followed by 15 cycles at 94°C, 30 sec; 53°C, 1 min; 72°C, 1 min; followed by 10 min at 72°C, with final hold at 10°C. The PCR products were treated with ExoSAP‐IT  and submitted for DNA sequencing. Amplicons were sequenced using a 3130 Genetic Analyzer with a BigDye® Terminator v3.1 Cycle sequencing Kit  and using POP7 .® kit, with a reduced volume modification of the manufacturer's instructions . Identifiler® data were obtained using an ABI 3730 genetic analyzer and analyzed using GeneMapper 4.0 software . Percent match comparisons were made using the Tanabe algorithmSTR profiling to examine the core set of loci used for cell line authenticationX‐Chromosome‐specific STR loci were analyzed using a DXS multiplex PCR, developed at the DSMZ. A detailed procedure for the DXS multiplex PCR is set out in the Supporting Information Methods. Control samples were included for comparison from the male cell line CAKI‐2, and the female cell line A‐204 (Supporting Information Table S3).® Y23 and PowerPlex® Fusion 6C Systems , in accordance with the manufacturer's instructions.Y‐Chromosome‐specific STR loci and autosomal STR loci were analyzed using PowerPlexSNP analysis was performed using the Fluidigm SNP Trace™ Panel  as previously reported.Locations of all Y‐STR and Y‐SNP loci evaluated in the current study are shown in Supporting Information Figure S1.Cells were harvested after incubation with Colcemid (0.05 µg/mL) for 2 hrs. Cells were detached using trypsin‐EDTA, treated with KCl hypotonic solution, fixed, and dropped onto a pre‐cleaned slide. FISH was performed with the Vysis CEP X SpectrumOrange/Yq12 SpectrumGreen probe set  using standard methodology, as previously described.G‐banding was performed also with standard methodologyhttp://iclac.org/databases/cross-contaminations/). The debate, and ensuing confusion surrounding M14 and MDA‐MB‐435, prompted the committee to review the scientific literature in an attempt to verify the provenance of these two cell lines.The International Cell Line Authentication Committee (ICLAC) maintains a list of known misidentified cell lines based on testing and known provenance  from a 33‐year‐old patient with metastatic melanoma; tissue was removed from an amelanotic lesion on the right buttock. A specific date was not recorded in the literature for commencement of M14 culture. The first journal article to describe the M14 cell line was submitted for publication on July 7, 1975.Further review of M14 publications showed that at least two other synonymous cell lines were established from the same donor. ML14 was cultured from the donor's lymphoblasts and immortalized by transfection with EBV; MF14/SV40 was cultured from skin cells and immortalized with SV40 virus.MDA‐MB‐435 was reported to be established at the MD Anderson Hospital and Tumor Institute from a 31‐year‐old female patient with metastatic breast carcinoma and were placed into culture on January 23, 1976.a). Although authentication testing was performed in the 1970s using HLA analysis, showing that M14 corresponded to two synonymous cell lines,e.g., blood type) was not available from the MD Anderson Cancer Center.Based on our literature review of provenance, we concluded that M14 was in culture for at least 6 months before MDA‐MB‐435 was established .Two vials of M14 (passages 14 and 15) and other samples from this donor were provided by the JWCI Specimen Repository for analysis. The M14 vials were both dated December 2, 1975, 7 weeks before MDA‐MB‐435 cells were first placed into culture , which is consistent with the original sample being from an amelanotic lesion. A melanoma tissue sample, used as the positive control, resulted in strong staining (data not shown).Immunostaining was performed on the M14 cell line (one passage after thawing) to evaluate expression of melanoma‐specific markers. A pan‐melanoma antibody cocktail was used, which is commonly employed for melanoma diagnosis.e), typically seen in individuals who carry the O allele.DNA was extracted from M14 cells (December 1975 samples and one passage after thawing) to evaluate donor characteristics. The donor was reported to be male, a finding that did not concur with karyotypic analysisM14 DNA was also tested for a BRAF mutation, V600E, which is present in 50–70% of melanoma cases.We concluded that M14 immunostaining, BRAF status and blood type are consistent with the reported tissue type and donor characteristics. However, these characteristics alone were insufficient to conclude that M14 cells were derived from the reported donor. To exclude misidentification, it is important to use authentication testing to compare M14 to other samples from the same donor.® kit, which includes 15 autosomal STR loci plus amelogenin for sex determination. Despite containing low amounts of DNA, DNA was successfully extracted from donor serum and an STR profile generated (see Supporting Information Methods for detail).We asked JWCI if synonymous cell linesSerum DNA from the melanoma donor was found to correspond to M14 and ML14 . STR profiles included an early sample from the MD Anderson Characterized Cell Line Core Facility, tested in 2009. All STR profiles corresponded to serum from the M14 donor; none of the STR profiles from MDA‐MB‐435 exhibited a different donor origin.Variations were observed at some STR loci across our cell line dataset. Loss of heterozygosity was present in all cultured samples compared to donor serum, which is a common observation for cell line STR profiles.Sex‐specific markers are of particular interest, considering that M14 is reported to come from a male donor and MDA‐MB‐435 from a female donor. STR profiles typically include amelogenin, using AMELX and AMELY for sex determination.To assess concordance between test methods, SNP genotyping was performed on M14, ML14 and MDA‐MB‐435S DNA. The SNP Trace™ System was used because it contains sex‐specific SNP loci and published data are available for MDA‐MB‐435.Sex‐specific SNP loci were examined in closer detail. One X‐specific locus, hu103X (rs525869), was called as heterozygous in ML14, M14 and MDA‐MB‐435S . We explored this finding further by performing PCR amplification and sequencing of the hu103X locus. Sequences from ML14, M14 and MDA‐MB‐435S were found to contain a single SNP variant , indicating that the allele call from the SNP Trace™ System was incorrect in this instance. Two other X‐specific SNP loci, hu107X and hu109X, were called as homo‐ or hemizygous and these results were confirmed by sequencing. The SNP Trace™ System also contains three Y‐specific SNP loci, which were previously detected in a sample of MDA‐MB‐435.We concluded that the STR and SNP profiles previously reported for M14 and MDA‐MB‐435Cytogenetic analysis of the M14 cell line was undertaken to better understand the finding that MDA‐MB‐435 has an XX karyotype.a–d). Interphase analysis of 100 nuclei showed an average count of 2.4 copies per nucleus for centromere X signals and 1.9 copies for Yq12 signals. Metaphase analyses identified two distinct derivative chromosomes carrying the Yq12 sequences , the shorter derivative being more common than the longer derivative. Sequential G‐banding and FISH analysis revealed the shorter derivative as der(22)t, with the Yq12 material translocated to the short arm of chromosome 22 . The rare, longer derivative, also carrying Yq12 sequences, was not present in the metaphases analyzed by GTL‐banding and FISH.Both centromeric X‐ and Yq12‐chromosomal material were detected in M14 Fig. a–3d. Id–f. FISH analysis with the CEP X/Yq12 probe set indicated that each metaphase of the ML14 lymphoblastoid cell line contained a normal X chromosome, a derivative X chromosome with a large unknown fragment added on to its short arm, and a normal Y chromosome. Metaphases were typically tetraploid with numerous rearranged chromosomes.To determine whether the M14 Y/22 translocation is specific to that cell line, ML14 cells were examined by G‐banding and FISH analysis. Eleven metaphases were karyotyped; representative images are shown in Figures a). Counting of interphase nuclei suggested that the Y/22 translocation was present in a minority of cells .MDA‐MB‐435S cells were also examined to confirm findings from M14. MDA‐MB‐435S cells displayed two X chromosomes and the Y/22 translocation first seen in M14 . We then performed sex‐specific STR analysis to see if other loci were detectable.For the X chromosome, four additional STR loci were examined: DXS‐7132, DXS‐HPRTB, DXS‐7423 and DXS6807. All four X‐specific STR loci were hemi‐ or homozygous in M14 . We concluded that these Y‐specific STR loci are absent from the portion of the Y chromosome that remains in the M14 cell line. Alternatively, STR primer binding site mutations or deletions might be preventing their amplification. It is likely that apart from Yq12, all other Y‐chromosomal material has been lost from M14, either as part of the disease process or during cell line establishment.For the Y chromosome, PowerPlexAuthentication testing of M14 from 1975 (prior to the establishment of MDA‐MB‐435), with comparison to donor serum and lymphoblastoid cell line ML14, shows that M14 is the authentic cell line and MDA‐MB‐435 is a misidentified derivative.Misidentification of MDA‐MB‐435 is likely to have occurred prior to 1981–1982.Our results are consistent with studies performed previously, using STR profiling to examine authenticity, as well as expression profiling and mutation status to examine tissue origin.Expression of markers that are inconsistent with tissue identity has been explored previously in M14 and MDA‐MB‐435.Work on M14 melanoma cells rests on a strong foundation laid down by Dr. Donald Morton and his colleagues in the 1970s. Their preservation of serum and other cell lines from this donor, which were authenticated using methods available at that time, made our subsequent detective work possible. Documentation of cell line provenance, and retention of donor material and early passage samples are essential to prove authenticity. New cell lines should always have donor tissue stored for testing and comparison to later passage samples. Deposit of samples in a core facility or cell line repository will ensure that valuable samples are retained for future use. Deposit of STR profiles into a shared database of cell line STR profiles, such as BioSample,Early authentication testing of M14 was performed using HLA typing.SNP profiling is a complementary approach to STR analysis, although at least one incorrect allele call was detected here, showing the need to optimize the SNP Trace™ System for cell line analysis. Cytogenetic analysis can also be highly informative. For this study, it was the only method to detect the Y‐chromosomal material in M14 cells.Consideration of sex in the design and interpretation of preclinical studies is important and has been highlighted by NIH and others.Donor sex is therefore relevant, but can be difficult to determine in cell lines. Loss of Y‐chromosomal material is frequently seen in cell culture. A recent study of 1,843 human cell lines demonstrated that 331 were annotated as male but called as female through absence of AMELY on STR analysis.The absence of Y‐specific loci suggests that the majority of the Y chromosome has been lost from M14 and MDA‐MB‐435 cells. Y chromosomal rearrangements may have occurred during cell line establishment or ongoing culture. Variable Y‐chromosome numbers have been observed while examining A549 cells from different sources using cytogenetic analysis.X‐chromosomal uniparental disomy is the most likely mechanism for the XX karyotype previously reported for M14 and MDA‐MB‐435.We conclude that the M14 cell line is authentic, established from a male donor with melanoma. Currently available stocks of MDA‐MB‐435 are misidentified and were derived from M14. Unless authentic stocks of MDA‐MB‐435 can be identified, investigators should be aware that MDA‐MB‐435 is not a female cell line and is not a suitable model for breast carcinoma. Cell lines are living materials and can be complex, changeable, and difficult to understand. Clear provenance and careful characterization of widely used cell lines, such as M14 and MDA‐MB‐435, are essential if we are to understand the tumors from which they were established.Supplementary MaterialClick here for additional data file.Supplementary TablesClick here for additional data file.


Novel therapeutic approaches are required to treat ovarian cancer and dependency on glycolysis may provide new targets for treatment. This study sought to investigate the variation of expression of molecular components  of the glycolytic pathway in ovarian cancers and the effectiveness of targeting this pathway in ovarian cancer cell lines with inhibitors.Expression of GLUT1, HKII, PKM2, LDHA were analysed by quantitative immunofluorescence in a tissue microarray (TMA) analysis of 380 ovarian cancers and associations with clinicopathological features were sought. The effect of glycolysis pathway inhibitors on the growth of a panel of ovarian cancer cell lines was assessed by use of the SRB proliferation assay. Combination studies were undertaken combining these inhibitors with cytotoxic agents.Mean expression levels of GLUT1 and HKII were higher in high grade serous ovarian cancer (HGSOC), the most frequently occurring subtype, than in non-HGSOC. GLUT1 expression was also significantly higher in advanced stage (III/IV) ovarian cancer than early stage (I/II) disease. Growth dependency of ovarian cancer cells on glucose was demonstrated in a panel of ovarian cancer cell lines. Inhibitors of the glycolytic pathway  attenuated cell proliferation in platinum-sensitive and platinum-resistant HGSOC cell line models in a concentration dependent manner. In combination with either cisplatin or paclitaxel, 3PO (a novel PFKFB3 inhibitor) enhanced the cytotoxic effect in both platinum sensitive and platinum resistant ovarian cancer cells. Furthermore, synergy was identified between STF31 (a novel GLUT1 inhibitor) or oxamic acid (an LDH inhibitor) when combined with metformin, an inhibitor of oxidative phosphorylation, resulting in marked inhibition of ovarian cancer cell growth.The findings of this study provide further support for targeting the glycolytic pathway in ovarian cancer and several useful combinations were identified.The online version of this article (10.1186/s12885-018-4521-4) contains supplementary material, which is available to authorized users. Ovarian cancer is the 7th most common female cancer worldwide with an estimated 239,000 new diagnoses worldwide each year . StandarMany cancer cells rely on glycolysis as their primary source of energy regardless of oxygen availability; the persistence of glycolysis in cancer cells even under aerobic conditions is termed aerobic glycolysis or the Warburg effect. This metabolic alteration in tumours has been extensively demonstrated in a wide variety of cancers and considered a ‘hallmark’ of advanced malignancy –5. It haGlucose transporter 1 (GLUT1) is the first component of the glycolysis pathway, transporting glucose into the cell, and is up-regulated in many tumour types. High expression has been associated with poor clinical outcome and adverse prognosis –20. STF3Hexokinase catalyses the first rate-controlling irreversible reaction of the glycolytic pathway; phosphorylating glucose to glucose-6-phosphate coupled with ATP de-phosphorylation. The mitochondrial-bound isoform HKII is considered to play a pivotal role in carcinogenesis and is overexpressed in many tumours , 24.6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase (3PFKFB3), which converts fructose-6-phosphate to fructose-2,6-bisP , is downstream of HKII. PFKFB3 overexpression has been documented in several tumour types including ovarian cancers . In 2008Further downstream is the M2 isozyme of pyruvate kinase (PKM2) which catalyses the irreversible conversion of phosphoenolpyruvate (PEP) to pyruvate coupled with ADP phosphorylation and is found overexpressed in various tumour types and plays a pivotal role in carcinogenesis , 28.Lactate dehydrogenase A (LDHA) is the enzyme catalysing the reduction of pyruvate in the final step of the glycolytic pathway. LDHA upregulation has been reported in ovarian cancers when compared to normal tissues . LDHA ovSeveral successful combinations of glycolytic inhibitors with cytotoxic drugs have recently been identified and glycolytic inhibitors have been demonstrated to resensitise drug-resistant cells to conventional regimens , 37–39.We have previously demonstrated antitumour activity of glycolytic inhibitors against panels of ovarian and breast cancer cell lines . In the Primary Ovarian cancer patients treated at the Edinburgh Cancer Centre between 1991 and 2006 were retrospectively identified from the Edinburgh Ovarian Cancer Database. Tissues were formalin-fixed and paraffin-embedded. Haematoxylin-eosin stained slides were reviewed by a subspecialist gynaecological pathologist, and histological classification of tumour type confirmed. Three separate Tissue Microarray (TMA) replicates containing cores of 380 ovarian tumours were constructed. The number of samples available for histology and stage analysis is shown in Additional file No informed consent was obtained for use of retrospective tissue samples from the patients within this study, most of whom were deceased, since this was not deemed necessary by the Ethics Committee. The TMA material was kindly provided by the Edinburgh Experimental Cancer Medicine Centre (ECMC ID: SR319). Ethical approval for the use of tumour material and correlation with associated clinical data was obtained from South East Scotland Human Annotated Bioresource (East of Scotland Research Ethics Service Reference 15/ES/0094).Microscope slides of TMA sections were deparaffinised and rehydrated followed by heat-induced antigen retrieval being performed in sodium citrate buffer at pH 6. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min and non-specific binding was blocked by a 10 min incubation in serum-free protein block (DAKO). Primary antibodies were diluted in antibody diluent (DAKO) and were applied overnight at 4 °C. The following primary rabbit antibodies, validated for the protocol, were used: GLUT1 (Merck Millipore), HKII , LDHA  and PKM2 . The following day, tissue sections were washed with 0.05% PBS Tween 20 (PBS-T), and were then incubated with primary mouse anti-cytokeratin antibody (M3515/DAKO) diluted 1:25 in the same antibody diluent in order to mask the tumour areas. This incubation was performed at room temperature, lasted 1 h and was followed by PBS-T washes. To enable epithelial mask visualisation, slides were then incubated with the secondary goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo Fisher Scientific) diluted 1:25 in the goat anti-rabbit peroxidase-conjugated Envision reagent (DAKO). This incubation was conducted at room temperature protected from light for 90 min and was followed by PBS-T washes. Target visualisation was implemented by a 10 min incubation with Cyanine 5 (Cy5) Tyramide, diluted at 1:50 in amplification diluent (PerkinElmer), at room temperature protected from light. Subsequently, tissue sections were washed with PBS-T and dehydrated. Finally, slides were counterstained with 45 μl Prolong Gold Antifade Mountant with DAPI  (Thermo Fisher Scientific) to visualise the nuclei and a coverslip was mounted.Protein expression in the ovarian tumour cores was quantitatively evaluated by Automated Quantitative Analysis (AQUA) . High rehttp://www.tmanavigator.org/). For this analysis, expression data of different markers had been log2 transformed, mean-centred and quantile-normalised to compensate for differences in the staining. The expression of examined glycolytic targets was compared across the different pathological stages and histological types of ovarian tumours using one-way ANOVA and statistical significance was determined by the Tukey’s multiple comparisons test. The Spearman correlation coefficient was calculated for each pair of markers and statistical significance was determined using the Algorithm AS89 [P-values were adjusted for multiple hypothesis testing according to Benjamini-Yekutieli FDR correction. The P-value significance threshold was set at 0.01.Target expression in the cytoplasmic compartment of each core was quantified and assigned an AQUA score. Data were filtered and only samples that had at least two replicate values were considered. Expression values were averaged from either two or three replicates. Spearman non-parametric correlation and network analysis were conducted using TMA Navigator . Correlathm AS89 . SpearmaA panel of four ovarian cancer cell lines were used initially. OVCAR5, OVCAR3 and CAOV3 are HGSOC cell lines  while TOAll cell line work was conducted in sterile conditions in a class II Laminar Air Flow hood at room temperature. Cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C. The panel of four ovarian cancer cell lines  were all maintained in Dulbecco’s Modified Eagle Medium without HEPES modification , containing glucose (5.56 mM), Sodium Pyruvate (1 mM) and L-glutamine (3.97 mM). The two ovarian cancer cell line pairs  were maintained in RPMI 1640 (Thermo Fisher Scientific) containing 11.11 mM glucose and 2 mM L-glutamine. In both cases the media contained phenol red and were supplemented with 10% heat inactivated fetal bovine serum FBS  and 1% Penicillin-Streptomycin .In the deprivation experiments where the effect of glucose availability on cell growth of different cell lines was examined, medium without glucose was used . Phenol red free media were supplemented with 10% heat inactivated dialysed fetal bovine serum (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin. In the glucose depleted medium the desired concentration of D-Glucose (Sigma Aldrich) was added along with a standard 4 mM L-Glutamine (Sigma Aldrich) concentration.3 tissue culture flasks and were sub-cultured at least once a week, when reaching 70–80% confluence as described below. Medium was discarded and cells were washed with preheated phosphate buffered saline. Cells were then incubated for a few minutes with a trypsin/EDTA solution  to cause cell detachment and cell suspension was centrifuged at 1200 rpm for 5 min. Pelleted cells were resuspended in fresh media and transferred into new flasks. When setting up an experiment cells were counted using a Neubauer hemocytometer and were seeded in cell culture plates or dishes at the desired dilution.Cells were routinely maintained in T175cmhttps://patents.google.com/patent/WO2014187922A1/en).The SRB assay is a colorimetric cell density assay based on the quantification of cellular protein content . Cells wCisplatin (Teva UK Limited) and paclitaxel (Actavis) were obtained as formulated drugs. Stock solutions of compounds were prepared in DMSO except for oxamic acid and metformin which were dissolved in PBS. A series of 10 dilutions with 1:2 steps of each inhibitor in six replicates was applied. Once the treatment period was completed, cell monolayers were fixed on the day of treatment (Day 0 control) and on selected time points thereafter with cold 25% trichloroacetic acid . Then cell monolayers were stained with 0.4% SRB dye solution (Sigma Aldrich) and unbound excess dye was removed by 1% glacial acetic acid  washes. The protein bound stain was solubilised in 10 mM Tris buffer solution pH 10.5 (Sigma Aldrich). Finally absorbance was measured at 540 nm using a plate reader.50), indicating the concentration needed to reduce cell viability by half, was used as a quantitative indication of the effectiveness of each compound as a cancer cell growth inhibitor. IC50 values were generated through sigmoidal concentration response curves fitted using the XL fit tool within Microsoft Excel.Measurements were corrected for background absorbance and values are presented as percentage of absorbance of untreated control. The half maximal inhibitory concentration . To quantitatively evaluate the effectiveness of each combination, CI values were generated for each combination point indicating synergy, additivity or antagonism [In combination drug studies, glycolytic inhibitors were assessed in combination with traditional drugs. For these treatments a range of different concentrations of the glycolytic inhibitor were combined with a constant fixed concentration, around the ICtagonism . CI valutagonism .p-value using the Miller-Siegmund minimal P correction.Statistical tests were undertaken using GraphPad Prism software version 6. Student’s t-test was used to compare two groups and ANOVA followed by the Tukey post-test was used to compare more than two groups. For survival analysis, we undertook Kaplan Meier analysis using X-tile  which alTo assess the variation in expression of key components of the glycolytic pathway in ovarian cancers, expression levels of GLUT1, HKII, PKM2 and LDHA were investigated in a series of 380 ovarian tumours by Automated Quantitative Analysis (AQUA). A three label immunofluorescent protocol was used generating a quantitative score for each tumour core. Representative immunofluorescence images illustrating the expression of the four glycolytic targets in TMA cores of ovarian cancers are shown in Fig. P = 0.0011; t-test)  and this was reflected in a difference between HGSOC and clear cell disease   and again this difference was reflected in HGSOC being lower than clear cell   Fig. . SimilarVA) Fig. . In contVA) Fig. . For PKMVA) Fig. .Fig. 2ExP = 0.023; t-test)   than early disease (stages I/II) (st) Fig. . In contVA) Fig.  while noP < 0.01) are drawn as lines that connect pairs of markers. Thickness of connection lines reflects significance and positive significant relationships are displayed in grey colour. The colour of each marker indicates the number of significant connections. High number of significant connections is displayed in yellow colour while low in blue. The correlation values (FDR P < 0.01) are summarised in Additional file S2.A heatmap correlating the expression of the four examined glycolytic enzymes across the dataset is shown in Fig. To assess the growth dependence of ovarian cancer cells on glucose, the proliferation of a small panel of ovarian cancer cell lines was monitored under a range of glucose concentrations after a 5-day incubation period. Growth was compared with controls in medium without glucose. Fig. 50 concentrations are listed in Table PEA1 / PEA2 and PEO1 / PEO4 are two pairs of cancer cell lines established from two individual patients with HGSOC . The fir50 value equal to 280 nM and PEA2 equal to 460 nM. In contrast, the PEO4 platinum-resistant cell line presented greater sensitivity having a threefold lower IC50 value  compared to the platinum sensitive PEO1 cell line (4.8 μΜ). STF31, another GLUT1 inhibitor [50 values of 1.3μΜ and 0.9μΜ respectively. In contrast, the platinum-resistant line PEO4, having an IC50 value of 0.9μΜ, showed increased sensitivity to the inhibitor compared to its paired platinum-sensitive line PEO1, with an IC50 value of 1.5μΜ. 3PO is a recently identified PFKFB3 inhibitor [50 value. Oxamic acid is an established LDH inhibitor [50 value of 16 mM. Regarding the second pair, the PEO4 platinum resistant cell line proved to be more resistant to oxamic acid, having an IC50 value threefold higher than the corresponding value of PEO1 , of the cytotoxic drug; hence in PEA2 cells, 4μΜ of cisplatin was required to produce a similar inhibitory effect in cell number to that of 1μΜ cisplatin on PEA1 cells. Both drugs were delivered at the same time and cancer cell proliferation was examined by the SRB assay after a 3-day treatment period. Combination Index values (CI) were generated for each combination point, using Calcusyn software, providing a quantitative evaluation of the combination efficacy. Concentrations at which synergistic interactions  between the two compounds were identified are indicated by asterisks in Fig. Combinations of the PFKFB3 inhibitor, 3PO, with cisplatin and paclitaxel were next investigated against the paired cell lines. 3PO was able to enhance the effect of cisplatin in both the chemosensitive PEA1 and chemoresistant PEA2 cell lines. A range of different concentrations of 3PO were used in combination with a constant fixed concentration Additional file 2:TMA dataset. Mean AQUA expression values for GLUT1, LDHA, HKII and PKM2 in 380 ovarian cancer samples. Histology and stage are shown for individual tumours. (XLSX 77 kb)Additional file 3:Table S2. Spearman correlation of the expression of four glycolytic enzymes in a cohort of 380 ovarian cancers. Spearman rho correlation values  along with the respective adjusted P value  of statistically significant correlations thresholded at FDR P < 0.01 are summarised. (DOCX 21 kb)Additional file 4:Figure S1. Growth response curves of PE01 and PE04 ovarian cancer cells treated with combinations of glycolysis inhibitors with chemotherapy or metformin. A. 3PO with cisplatin. 3PO concentrations between 0.5-30μΜ alone (blue line) or combined with a constant concentration of cisplatin (red line) were evaluated. In green the effect of 0.5μΜ (PE01) or 1μΜ (PE04) cisplatin on cell viability is presented. B. 3PO with paclitaxel. 3PO concentrations between 0.5-30μΜ alone (blue line) or combined with a constant concentration of paclitaxel (red line) were evaluated. In green the effect of 2μΜ paclitaxel (both PE01 and PE04) on cell viability is presented. C. Oxamic acid with metformin. Concentration response curves of PE01 and PE04 ovarian cancer cells treated with oxamic acid concentrations between 1.56-100mΜ alone (blue line) or combined with 2 mM (PE01) or 0.5 mM (PE04) metformin (red line). In green the effect of 2 mM (PE01) or 0.5 mM (PE04) mM metformin on cell viability is presented. Cell viability was determined by an SRB assay after a 3-day treatment. Mean results of 6 replicates are reported and error bars represent standard deviations. Values are shown as a percentage of control. Asterisks indicate synergistic combination points with * CI value lower than 0.8 and ** CI value lower than 0.3. (PPTX 1444 kb)