Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets.Antiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose–response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen.The selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment.Our results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens. Although many patients benefit from the treatment, hallenge ,2. Follohallenge . Howeverhallenge ,3,5-7.in vitro cell culture models based on the ER-positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D [Cell model systems are valuable tools to study the molecular mechanisms for endocrine resistant breast cancer. We have developed and T47D -11. In land T47D -18. HER2and T47D -21. We hTumor expansion depends on continued growth of tumor cells through mitotic cell division. A key mitotic regulator is the chromosomal passenger complex (CPC), composed of the catalytic component Aurora kinase B and the three regulatory and targeting components; inner centromere protein (INCENP), survivin and borealin. CPC is important for chromosome condensation, correction of erroneous kinetochore-microtubule attachments, activation of the spindle-assembly checkpoint and cytokinesis . The funTo explore the molecular mechanisms driving antiestrogen resistant cell growth, we have utilized a large kinase inhibitor library comprising 195 kinase inhibitors on parental and fulvestrant resistant T47D breast cancer cell lines. We identified Aurora kinase B as a putative novel therapeutic target in fulvestrant and tamoxifen resistant breast cancer cells, and further explored its role in signaling and growth of fulvestrant resistant T47D cell lines by using the selective Aurora kinase B inhibitors, barasertib and hesperadin. Moreover, we investigated the clinical relevance of Aurora kinase B expression in primary tumors from breast cancer patients receiving tamoxifen as first-line adjuvant endocrine therapy.R-1 (182R-1) and T47D/182R-2 (182R-2) were established from T47D grown with 5% fetal calf serum (FCS) and long term treated with 100 nM fulvestrant  as described in [5 U penicillin and 31.25 μg/l streptomycin  were added to the growth medium. Barasertib was purchased from Selleck Chemicals . Stock solutions of 10−3 M fulvestrant were dissolved in 96% ethanol, whereas stock solutions of 10 mM barasertib were dissolved in dimethyl sulfoxide (DMSO).The T47D cell line was originally obtained from the Human Cell Culture Bank  and maintained as previously described . The fulribed in . To enabribed in . The fulThe kinase inhibitor library comprising 195 different kinase inhibitors was purchased from Selleck Chemicals and the experiment was performed as previously described . In brieDose–response growth experiments were performed in 96-well plates. Cells were seeded in their standard growth medium and allowed to adhere for 2 days before 5 days treatment with barasertib or JNJ-7706621  at indicated concentrations. Cell number was determined using a crystal violet colorimetric assay as described previously . All expTo investigate the effect of barasertib on protein expression and phosphorylation of Aurora kinase A, Aurora kinase B and INCENP, as well as PARP cleavage, parental and fulvestrant resistant T47D cell lines were treated for 4–96 hours with 50 nM barasertib . Cell lysis and western blot analyses were performed as previously described . AntibodFor cell cycle analysis, cells were fixed in 70% ethanol and incubated for 30 min with 20 μg/ml propidium iodide  and 40 μg/ml RNase A  . BrieflyR-1 and 182R-2 cells were seeded in SlideFlask Chambers and treated with 0.1% DMSO (control) or 50 nM barasertib. The cells were fixed in 4% formaldehyde, permeabilized by 0.2% Triton X-100, stained with Hoechst 33342  and mounted using Fluorescence mounting medium . Pictures were captured using Zeiss AX10 Imager A2 microscope .T47D, 182The cohort included 268 high-risk ER-positive postmenopausal breast cancer patients diagnosed between 1989 and 2001. The patients had received tamoxifen as first-line adjuvant endocrine treatment according to the guidelines from the Danish Breast Cancer Cooperative Group (DBCG) . The staIHC was conducted on tissue microarrays (TMAs) using a standard immunoperoxidase procedure . In briet-test was performed on triplicate values comparing the growth inhibitory effect in parental and resistant cell lines. In the remaining experiments, group comparisons were done using a two-tailed t-test with Bonferroni adjusted p-values for multiple testing. In the biomarker study, uni- and multivariate analyses were performed. The multivariate analysis included tumor grade, size, nodal status and age as standard covariates. Kaplan-Meier life tables with log-rank testing were generated to assess the association between the percentage of Aurora kinase B positive tumor cells, and disease-free and overall survival. The statistical analysis on the clinical data was performed in R version 3.0.1, with the R package “rms”. For all experiments, P < 0.05 were considered statistically significant.In the kinase inhibitor screen, one-tailed Student’s R-1 and 182R-2) T47D cell lines were subjected to a kinase inhibitor library comprising 195 inhibitors each targeting one or more different protein kinases. The results from the screen are shown in a volcano plot displaying, for each of the kinase inhibitors, statistical significance (P < 0.05) versus fold change in relative growth inhibition between fulvestrant resistant and parental cell lines , resulting in a statistical significant 70% growth inhibition of the resistant cells from 10 nM compared to only 30-40% growth inhibition of the parental T47D cells  for 4 hours resulted in undetectable level of phosphorylated Aurora kinase B in the resistant cells, but did not have any effect on the level of phosphorylated Aurora kinase A. Only very low levels of phosphorylated Aurora kinase A and B were seen in the parental cells, and no effect of treatment with barasertib was observed Figure A. AdditiR-1 and 182R-2 cells, respectively, upon 96 hours treatment with barasertib. This is presumably due to degradation of Aurora kinase B as described by Gully et al. [R-1 and 182R-2, respectively  for 4–96 hours. As seen in Figure y et al. . Comparey Figure , whereasy Figure .Figure 3Aurora kinase B is important for correct cell cycle progression and plays a key role in the maintenance of normal ploidy level during cell division . To inveR-1 and 182R-2  compared with parental T47D cells (4.8%) , cleaved PARP (85 kDa) was seen in the resistant cells, whereas parental T47D cells only expressed full length PARP (116 kDa)  Figure B. Only aTo investigate if barasertib also inhibited growth of tamoxifen resistant cell lines, dose–response growth experiments with increasing concentrations of barasertib (5–50 nM) were conducted on the recently established tamoxifen resistant T47D breast cancer cell lines, TR-1 and TR-2 . As seenTo investigate the association between Aurora kinase B protein expression and antiestrogen resistance, the expression of Aurora kinase B was evaluated in tumors from 261 high-risk ER-positive breast cancer patients, who had received tamoxifen as first-line adjuvant endocrine treatment. Aurora kinase B expression was confined to the nucleus of breast cancer cells and scored as percentage positive tumor cells Figure A-D. Auro50 for barasertib is 0.37 nM and for hesperadin 250 nM (see www.selleckchem.com). Aurora kinase B is a key regulator of mitosis and essential for cell proliferation. It is the catalytic component of the chromosomal passenger complex (CPC), composed of Aurora kinase B, survivin, INCENP and borealin [Although endocrine therapy targeting estrogen signaling through ER has clearly improved survival of breast cancer patients, treatment resistance is complex and a major clinical challenge. To explore the molecular mechanisms behind antiestrogen resistance, we have developed cell lines resistant to the antiestrogens fulvestrant and tamoxifen based on the estrogen responsive T47D breast cancer cell line, and utilized a kinase inhibitor screen to identify kinases involved in growth of the fulvestrant resistant T47D cell lines. We found that the Aurora kinase B specific inhibitor barasertib preferentially inhibited growth of the fulvestrant resistant T47D breast cancer cell lines compared with growth of the parental fulvestrant sensitive T47D cells. To verify the role of Aurora kinase B for fulvestrant resistant cell growth, siRNA-mediated knock-down experiments were performed with several siRNA constructs. However, we did not obtain significant knock-down with any of the constructs, including the construct which in our MCF-7 cells reduced Aurora kinase B expression . Therefoborealin . The comborealin . We showborealin . In addiborealin ,40. AlthIn this study, barasertib preferentially inhibited fulvestrant resistant cell growth and phosphorylation of both Aurora kinase B and its targets INCENP and mitosis specific histone H3 in the resistant cells. Moreover, barasertib obstructed proper chromosome segregation and induced arrest of the fulvestrant resistant cells in the G2-phase. The increased proportion of cells in SubG1 together with PARP cleavage in the fulvestrant resistant cell lines indicates that barasertib-induced cell death is mediated by the apoptotic death pathway, as previously described in a panel of human myeloma cell lines . Parentawww.clinicaltrials.gov). Barasertib was also well tolerated in patients with acute myeloid leukemia enrolled in clinical phase I and I/II studies. These trials showed an overall response rate of 19-25% [Administration of barasertib has been shown to potently inhibit growth of colon, lung, hematologic and breast tumor xenografts as well as a panel of human breast cancer cell lines ,42,43. If 19-25% ,47. Collin vitro studies with the antiestrogen resistant T47D breast cancer cell lines, analysis of the association between Aurora kinase B and survival of breast cancer patients treated with fulvestrant would also be of great interest. Archival breast cancer tissue from such patients is unfortunately not available for us. Our previous findings of the importance of Aurora kinase A and ER for growth of tamoxifen and aromatase inhibitor resistant breast cancer cell lines [At present, we do not know the mechanisms whereby the activity of Aurora kinase B is up regulated in our fulvestrant resistant cell lines or how Aurora kinase B is involved in resistant cell growth. The findings in this study suggest that the protein harbors key functions needed for the resistant cell lines to survive upon development of resistance. Although we do not find over expression of Aurora kinase B in the fulvestrant resistant T47D cell lines, over expression of Aurora kinase B may be important for tumor cell growth. Noteworthy, over expression of Aurora kinase B in Chinese hamster embryonic diploid fibroblasts results in aneuploidy cells capable of forming aggressive tumors in nude mice  and Auroll lines ,38 and fll lines , indicatIn this study we found that Aurora kinase B is an important kinase for growth and signaling in the ER-negative fulvestrant resistant T47D breast cancer cell lines and that Aurora kinase B also plays a role for growth of ER-positive tamoxifen resistant T47D cell lines. Aurora kinase B was identified as a biomarker for reduced benefit of tamoxifen treatment. Therefore, our results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.


Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity. Antitumor virotherapy using oncolytic viruses is a developing strategy to treat cancer . Among oSchwarz and Edmonston attenuated strains of MV use the CD46 molecule as the major receptor to infect human cells, unlike the pathogenic strains that mainly use the CD150 molecule –9. The mThe overexpression of CD46 is probably not the only factor that conditions the ability of MV to preferentially replicate in and kill tumor cells. In fact, there is now evidence that host-cell translational activity upon viral replication  and defeWe have previously shown that the Schwarz attenuated strain of MV induces immunogenic cell death of malignant pleural mesothelioma (MPM) cells , 22. In TM assay that is based on their metabolic activity. We also filmed by time-lapse microscopy some of the MPM cell lines exposed to MV encoding the enhanced green fluorescent protein (MV-eGFP) at an MOI = 1 . By measuring fluorescence at 610 nm, we followed daily MV-ch replication in twenty-two MPM cell lines exposed to different multiplicities of viral infection (MOI) , bronchial epithelial cells (CEB), pulmonary endothelial cells (HMVEC-L) and lung fibroblasts (CCD-19Lu) Figure . We obseWe measured the expression of known MV receptors  on the surface of MPM and healthy primary cells. The majority of MPM cells expressed higher levels of CD46 than healthy primary cells Figure . HoweverTo determine whether CD46 plays a role in MPM cell infection, we exposed eight MPM cell lines to MV-ch in the presence of anti-CD46 mAb or isotype control mAb Figure . On MesoSince the sensitivity of MPM tumor cell lines to MV replication did not correlate with the CD46 expression level Figure , we sougDDX58 gene that encodes the retinoic acid-inducible gene-1 protein (RIG-I) and the IFIH1 gene that encodes melanoma differentiation-associated protein 5 (MDA5). These two proteins are intracytoplasmic sensors of viral ssRNA and dsRNA, able to induce type I IFN response against MV [We first analyzed the expression of two helicase genes: the ainst MV . We obseIFNA1 and IFNB1 that encode IFN-α and IFN-β, respectively  and was increased in the presence of MV in four of these . In the nine other sensitive tumor cell lines, we never detected IFNA1 expression, either in the presence or absence of MV. Regarding IFNB1, a weak constitutive expression in the absence of the virus was detected only in the insensitive Meso52 cell line , whereas the eight other sensitive tumor cell lines did not. We also measured the expression of the IFNL1 gene that encodes the type III IFN, IFN-λ1  and a strong constitutive expression only for one (Meso34). In the presence of MV, we observed a significant increase of MX1 expression only for Meso36. The MX1 expression did not change for all the other sensitive cell lines.Finally, we measured the expression of the L Figure . In the IFNA1, IFNB1 and MX1 and are sensitive to MV replication. These results also signify that the capacity to achieve a complete type I IFN response is defective in numerous MPM cell lines.These results indicate that cells that are able to develop a complete type I IFN response, whether they are healthy primary or tumor cells, are not sensitive to MV infection. On the contrary, tumor cell lines that are unable to develop a type I IFN response are sensitive to MV infection, with the four exceptions, Meso36, 37, 34 and 122, which express We then sought to confirm these results by measuring IFN-α and IFN-β secretion by ELISA in the culture supernatants Figure . RegardiWe also measured the cytoplasmic expression of Mx1 protein by flow cytometry in tumor cell lines and healthy primary cells Figure . We founOverall, these results show that tumor cells that constitutively express Mx1 are not sensitive to MV replication, with the exception of Meso34, 36, 37 and 122. They also demonstrate that the cells that fail to develop a complete type I IFN response, illustrated by the absence of IFN-α, IFN-β and Mx1 expression, are sensitive to MV replication with the same exceptions: Meso34, 36, 37 and 122.In the last set of experiments we tested the effect of type I IFN (IFN-α and IFN-β) on MV replication in sensitive tumor cell lines. We first measured the expression of IFNAR1 and IFNAR2 in all MPM cells lines. A low expression was found on the surface of all tumor cell lines (data not shown). We then exposed the fifteen MPM cell lines sensitive to MV to increasing amounts of IFN-α and IFN-β and measured MV replication using MV-ch Figure . MV replIn this study, we measured the sensitivity of a large panel of human MPM tumor cell lines and of different types of healthy cells to the oncolytic activity of the Schwarz vaccine strain of MV. We found that fifteen out of twenty-two MPM tumor cell lines are sensitive to MV replication and cytopathic effect, whereas replication was limited or absent in the four types of healthy cells  as well as in the seven remaining MPM tumor cell lines. Our data also show that the sensitivity of tumor cell lines is not correlated to overexpression of the MV cell receptor CD46, contrary to what has been described in the literature , 7. NeveWe observed that exposure of insensitive tumor cell lines or healthy cells to high titers of MV (MOI = 10) results in viability loss for the majority of them. This is likely due to the induction of the innate antiviral immune response that is known to affect viability, notably by limiting host translational activity or by inducing apoptosis . This isIFIT1, they found that it was expressed in the three insensitive cell lines and only in one out of the five sensitive cell lines. This last result matches our results that were obtained on a larger series of tumor cell lines and also on some healthy primary cells. The same group confirmed this result in an additional study where they analyzed type I IFN response in five MV-insensitive tumor cell lines compared to one sensitive tumor cell line [Two other groups recently studied the implication of the intracellular antiviral response on the sensitivity of tumor cell lines to MV oncolytic activity –18. Bercell line . They foell line . They alWe observed that some tumor cell lines that appear to develop a type I IFN response are nevertheless sensitive to MV oncolytic activity, as also observed in other studies –18. Thisin vivo with experiments on human MPM tumors engrafted in immunodeficient mice as we previously did for colon and lung adenocarcinoma [in vivo.We thought to confirm these results arcinoma . HoweverMV is currently being evaluated in clinical trials for the treatment of different types of cancers  and the 2 atmosphere.Human MPM cell lines (from Meso4 to Meso225) were established in our laboratory from pleural effusions collected by thoracocentesis, and genetically characterized . All patMycoplasma contamination using the PlasmoTestTM from InvivoGen.Normal peritoneal mesothelial cells MES-F were purchased from Tebu-bio, pulmonary fibroblasts CCD-19Lu from the ATCC-LGC Standards, and pulmonary endothelial cells HMVEC-L from Lonza. These cells were cultured in their specific media according to the manufacturers' recommendations. The bronchial epithelial cells were obtained and cultured as previously described . Cells wLive-attenuated Schwarz vaccine strain of measles virus (MV), MV recombinant for the enhanced green fluorescent protein (MV-eGFP) and MV recombinant for the cherry protein (MV-ch) were produced and purified as previously described . InfectiA day before infection, cells were seeded in 96-well plates, at a density of 5,000 cells/well for the MPM cell lines, and at the recommended density for each healthy cell type. Different multiplicities of infection (MOI) were used for infection with MV-ch . Fluorescence at 610nm was analyzed every day during 10 days using a ChemiDoc™ MP imaging system (Bio-Rad). Quantification was performed using Image Lab 4.1 Software (Bio-Rad) with the relative fluorescence corresponding to the ratio between the fluorescence measured in infected cells and the non-infected cells. The medium was replaced every 3 days to match the conditions of the cell viability assay. For the CD46 blocking assay, we used a CD46-specific mAb  or a mouse IgG1 isotype as a control  at a final concentration of 10μg/mL. These antibodies were added 30 minutes before adding the MV-ch at an MOI = 1. To test the inhibition of MV replication with type I IFN, we added rhIFN-alpha-2a and rhIFN-beta-1a (ImmunoTools) at concentrations ranging from 10IU/mL to 10,000IU/mL, 4 hours before the infection with MV-ch at MOI = 1, and the viral inoculum was not replaced for this assay.Cells were seeded in 96-well plates and infected with MV as described in the previous paragraph. Cell viability was measured using Uptiblue reagent (Interchim), an oxidation-reduction sensor that indicates cell metabolic activity. At days 3, 6 and 9 after the infection, the Uptiblue reagent was added  into the culture medium for 2 hours at 37°C. Fluorescence was then measured at 590nm using a ChemiDoc™ MP imaging system (Bio-Rad). Quantification was performed using Image Lab 4.1 Software (Bio-Rad) and the viability was expressed as a percentage compared to non-infected cell viability. Culture medium containing Uptiblue was then replaced by fresh medium to continue the kinetic experiment.5cells/well for the MPM cell lines, and at the recommended density for healthy cells. Cells were infected with MV-eGFP (MOI = 1). The time-lapse video microscopy was performed using a Leica DMI6000B microscope with a 10x objective. Images were acquired every 15 or 30 minutes for 3 to 4.5 days. We used MetaMorph® Microscopy Automation & Image Analysis Software (version 7.8) and Fiji Software for acquisition and analysis.A day before infection, cells were seeded in 24-well plates, at a density of 10TM488-conjugated anti-mouse IgG antibody  was used as secondary antibody. The results are expressed as RMFI.To measure CD46 expression on the cell surface, we stained the cells with a FITC-conjugated anti-CD46 mAb . Fluorescence was measured on FACS Calibur (BD Biosciences) and analyzed using FlowJo software. The results are expressed as relative mean of fluorescence intensity (RMFI). To measure Mx1 in the cytoplasm 72 hours after infection with MV at MOI = 1, cells were fixed with PBS containing 4% paraformaldehyde for 10 minutes at room temperature. Cell washes and mAb dilutions were performed with PBS containing 0.1% BSA  and 0.1% saponin. Unconjugated anti-Mx1 mAb  and, as a control, an unconjugated mouse IgG2a isotype  were used. A DyLight6cells/well and infected with MV at MOI = 1. 72 hours after infection, total cell RNA was extracted using the Nucleospin® RNA II kit (Macherey-Nagel) and 0.5μg total RNA was reverse transcribed using MMLV reverse transcriptase (Invitrogen). PCR reactions were conducted using QuantiTect primer assays (Qiagen) and Maxima SYBR Green/ROX qPCR Master Mix (Fisher Scientific) according to the manufacturer's instructions. Gene expression was analyzed in non-infected and infected cells using QuantiTect primers pairs for IFNA1 (coding for IFN-α), IFNB1 (IFN-β), IFNL1 (IFN-λ1), Mx1 (Mx1), DDX58 (RIG-I) and IFIH1 (MDA5). The gene expression was expressed as relative expression compared to the expression of a housekeeping gene that encodes human large ribosomal protein (RPLPO).MPM cell lines and healthy cells were seeded in 6-well plates at a density of 0.5×10IFN-α and IFN-β production were measured by ELISA , according to the manufacturer's instructions. Supernatants of MPM cell lines and healthy cells were collected 72 hours after infection with MV at MOI = 1 and used directly for ELISA without freezing.p < 0.05, **p < 0.01, ***p < 0.001.Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software Inc.). To compare two groups, a nonparametric, one-sided, unpaired Mann-Whitney comparison test was used. For statistical analysis comparing more than two groups, nonparametric one-way ANOVA  was used, with Dunn's post-test. All data are presented as mean±SEM. P values less than 0.05 were considered to be statistically significant. *


N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the  Colorectal cancer (CRC)N-glycans, O-glycans, and glycosphingolipid glycans have been reported and recently reviewed  high-mannose-type glycans, and glycans containing sialylated Lewis type epitopes, while showing a decrease of bisection as compared with glycans from nontumor colorectal tissue of the same individuals  and pauci-mannosidic N-glycans  was recently found to be correlated with poor prognosis in (advanced) colon carcinomas and N-glycomic profiling was successfully applied to distinguish colorectal adenomas from carcinomas -MS. The method was applied to characterize the N-glycome of 25 different colorectal cell lines in a fast and robust manner, including biological and technical replicates for all the cell lines. We obtained the comprehensive N-glycan profiles of 21 cell lines derived from primary tumors, two from lymph node metastases, one from a lung metastasis, and one from ascites fluid to assess their potential as glycobiological tumor model systems. Cancer cell line glycosylation features were then correlated with cancer cell markers and phenotypes as well as glycosyltransferase expressions. This study provides new insights into colon-cancer-associated glycan changes and sets a basis for studies into the functions of N-glycans in CRC with cell lines as model systems.Here, we developed and optimized a new analytical method for the more sensitive and higher throughput membrane  startingic acids , followem guanidine hydrochloride (GuHCl), 1-hydroxybenzotriazole hydrate (HOBt), 50% sodium hydroxide, super DHB matrix , and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich . HPLC SupraGradient acetonitrile (ACN) was obtained from Biosolve . Dithiothreitol (DTT), ethanol, sodium bicarbonate (NaHCO3), and glacial acetic acid were from Merck  and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) from Fluorochem . Peptide N-glycosidase F (PNGase F) was purchased from Roche Diagnostics . Recombinant PNGase F from Flavobacterium meningosepticum was a kind gift from Rick R. Drake  and Anand S. Mehta . It was expressed and purified as described previously , penicillin and streptomycin from MP Biomedicals , Lonza, 10x trypsin/EDTA solution (5.0 g/l porcine trypsin and 2.0 g/l EDTA●4Na in 0.9% sodium chloride) from PAA Laboratories GmbH , fetal calf serum (FCS) from PAA Laboratories and Biowest , and T75 cell culture flasks from Greiner-Bio One B.V. . All buffers were prepared using Milli-Q water (mQ) generated from a Q-Gard 2 system (Millipore). Control Visucon-F plasma pool  was obtained from Affinity Biologicals . The mRNA Capture kit was obtained from Roche and the reverse transcription system kit from Promega .Ammonium formiate, 2-aminobenzoic acid (AA), 2-picoline borane, dimethylsulfoxid (DMSO), 8 eviously  and is cSupplemental Table S1) were obtained from the Department of Surgery of the Leiden University Medical Center (LUMC), Leiden, The Netherlands, as well as the Department of Pathology of the VU University Medical Center (VUmc), Amsterdam, The Netherlands. Cells were cultured in Hepes-buffered RPMI 1640 culture medium containing l-glutamine and supplemented with penicillin (5000 IU/ml), streptomycin (5 mg/ml), and 10% (v/v) fetal calf serum (FCS) at the LUMC or in DMEM medium, supplemented with 10% (v/v) FCS and antibiotics, except for the KM12 cell line, which was grown in RPMI/10% FCS/antibiotics and l-glutamine at the VUmc. Cells were incubated at 37 °C with 5% CO2 in humidified air and cell culturing was performed up to a confluence of 80% under sterile conditions. For harvesting of the cells, medium was removed and adherent cells were washed twice with 1x PBS and trypsinized using 1x trypsin/EDTA solution in 1x PBS. To stop trypsin activity, medium in a ratio of 2:5  was added and cells were pelleted at 300 × g for 5 min. Cells were then resuspended in 3 ml 1x PBS and counted using the CountessTM Automated Cell Counter  based on tryptan blue staining. Cells were aliquoted to 2.0 × 106 cells per ml of 1x PBS, and washed twice with 1 ml 1x PBS for 3 min at 1000 × g. The supernatant was removed and pellets stored at −20 °C until further use.Human colorectal cancer cell lines (see 6 cells) from two or three biological replicates of each colorectal cancer cell line  were suspended in 100 μl mQ and sonicated in a water bath for 30 min. As control, 20 μl of human control Visucon-F plasma was used that was brought to 100 μl with mQ. Glycans were released using a PVDF-membrane based protocol adapted from Burnina et al. (6 cells/well or 25 μl/well diluted human plasma in denaturation buffer (5.8 m GuHCl and 5 mm DTT), were loaded in quadruplicate onto preconditioned HTS 96-well plates with hydrophobic Immobilon-P PVDF membrane and incubated for 30 min at 60 °C in a moistured, sealed box as an incubation chamber within an oven. Plates were shaken for 5 min on a horizontal shaker prior to centrifugation . The wells were washed twice with 200 μl mQ with 2 min incubation steps on a horizontal shaker prior to centrifugation and once with 200 μl 100 mm NaHCO3 . For N-glycan release, 50 μl 100 mm NaHCO3 and 1 mU PNGase F (Roche) were added per well. Plates were placed into the incubation device and incubated for 3 h at 37 °C. Glycans were recovered into 96-well collection plates by centrifugation ; eventual residual solution was collected from the membrane.Cell pellets  were added and samples incubated for 30 min at 37 °C and 250 rpm in an Innova 43 incubator shaker . Afterwards, samples were centrifuged 15 min at 500 × g and the supernatant containing the released glycans was collected. Supernatant and residual pellet were stored separately at −20 °C until further use. N-glycans from residual pellets were then released using the PVDF-membrane based protocol as described, but with 1 million cells per well and overnight PNGase F incubation.A subset of CRC cell lines were used to obtain the cell surface a et al. . CRC celN-glycans were derivatized by ethyl esterification adapted from Reiding et al. . Briefly, 20 μl of released N-glycans from the total and residual cell pellets as well as the control samples were added to 100 μl of ethyl esterification reagent . For the cell surface glycans, the supernatant was concentrated in a vacuum centrifuge to a volume of 20–30 μl, and 100 μl ethyl esterification reagent was added. Samples were incubated for 1 h at 37 °C. Subsequently, 100 μl ACN were added and the mixture was incubated at −20 °C for 15 min. Samples were brought to room temperature prior to glycan purification by hydrophilic interaction liquid chromatography (HILIC) solid phase extraction modified from a protocol described previously , equilibrated by pipetting 3 × 20 μl mQ water, followed by 3 × 20 μl 85% ACN. Samples were loaded by carefully pipetting up and down for 50 times. The tips were washed by pipetting 3 × 20 μl 85% ACN/1% TFA and 3 × 20 μl 85% ACN. N-glycans were eluted in 10 μl mQ water. For cell surface glycans, pipette tips of 200 μl volume were packed with cotton wool (ca 1000 μg) to prevent clogging through salts. The cotton HILIC purification was performed as described but with 150 μl pipetting volume, while elution was kept at 10 μl mQ water.Released g et al.  allowingeviously . For purN-glycans were spotted onto an anchor chip MALDI target plate  and cocrystallized with 1 μl of 1 mg/ml superDHB in ACN/mQ  containing 1 mm NaOH. Samples were allowed to dry at room temperature and were then recrystallized with 0.5 μl 5 mg/ml superDHB in ACN/mQ  containing 1 mm NaOH. MALDI-TOF-MS spectra were acquired using an UltrafleXtremeTM mass spectrometer in the positive-ion reflector mode, controlled by FlexControl 3.4 software Build 119 . The instrument was calibrated using a Bruker peptide calibration kit. Spectra were obtained over a mass window of m/z 1000–5000 with ion suppression below m/z 900 for a total of 10,000 shots . Tandem mass spectrometry (MALDI-TOF-MS/MS) was performed for structural elucidation via fragmentation in gas-off TOF/TOF mode.For mass spectrometric analysis, 5 μl of derivatized http://docs.python.org/py3k/reference/index.html). The average spectrum was internally recalibrated using glycan peaks of known composition , smoothed , and baseline-corrected  using FlexAnalysis Software . Peaks of signal-to-noise > 2 were picked, manually revised, and analyzed in GlycoWorkbench 2.1 stable build 146 (http://www.eurocarbdb.org/) using the Glyco-Peakfinder tool (http://www.eurocarbdb.org/ms-tools/) for generation of a glycan compositions list. Using MassyTools version 0.1.5.1, which is a novel in-house software developed for automated data processing, the resulting glycan peak list generated based on the average spectrum was used for targeted data extraction of the area under the curve for each of the CRC cell line N-glycomic profiles  total intensity > 1 × 105, (ii) fraction of analyte area with signal noise > 6 is more than 80%, (iii) fraction of glycan analyte intensity in total spectrum intensity > 40%. The area-under-the-curve values were rescaled to a total relative intensity of 100% for each spectrum.A mean average spectrum over all the total cell line sample spectra was generated using an in-house developed script in Python 2.7.3 . Preprocessed, selected, and rescaled total cell N-glycome data was imported into SIMCA software Version 13.0 , and a principle component analysis (PCA) was performed to reveal outliers and batch effects and to study the effects of possible confounders. A PCA displays the variation in the data on new vectors (principle components) and makes it possible to visualize the differences in the data in a two-dimensional way. The PCA is an unsupervised model that finds “natural” variation in the data without over-fitting. The observations (= cell line samples) are displayed in the score plots, while the variables  are displayed in the loading plots. The glycans located at the outskirts of the loading plot are those contributing most to the displayed principle components. The samples located on the outskirts of the score plot are those showing a particularly large deviation (variation) from the other samples. In addition, the particular locations of data points in score and loading plots are associated. For example, samples with particularly high intensities of glycans located in the top left of the loading plot will locate in the top left of the corresponding score plot. Furthermore, we classified the glycans in traits such as levels of fucosylation and sialylation, for which later also relative abundances were calculated (see below). We used these traits to color the variables in the loading plots to find possible contributions of glycan traits to principle components.Averages, standard deviations, and relative standard deviations were calculated for all biological replicates of the cell lines . Data were then rescaled to 100% excluding high-mannose-type glycans to prevent influences of possible high-mannose-type intracellular precursors. Then, additional glycan-derived traits for complex- and hybrid-type glycans were calculated  and compared . Mann–Whitney and Kruskal–Wallis with Tukey's posttest with significance level α = 0.05 were performed in GraphPad Prism Version 5.04  to explore differences of glycan traits with cell line characteristics such as stage of the original tumor, tumor site (primary versus metastasis), and differentiation.To validate observed trends in PCA, averages per cell type  were used to calculate glycan traits  for 2 h at 60 °C . Analysis on AA-labeled glycans was exclusively used for structural elucidation  and targeted search for indicative fragment ions of blood group antigens .The LC-system was coupled to a CaptiveSpray nanoBooster  for mass spectrometric MS/MS analysis on an AmazonSpeed ion trap  using a captive spray for ionization of samples in the positive ion mode. For the electrospray (1300 V), fused-silica capillaries with an internal diameter of 20 μm were used. Solvent evaporation was achieved at 220 °C with an ACN enriched nitrogen nebulizer gas at a pressure of 0.2 bar. Tandem MS was performed automated on the seven highest precursors per MS with ion detection from g, pelleted, and lysed in 500 μl of lysis buffer. Lysated cells were stored at −80 °C until mRNA was specifically isolated by capture of poly(A+) RNA in streptavidin-coated tubes using a mRNA Capture kit. cDNA was synthesized using a Reverse Transcription System kit following the manufacturer's guidelines. Lysates were incubated with biotin-labeled oligo(dT)20 for 5 min at 37 °C and then 50 μl of the mix was transferred to streptavidin-coated tubes and incubated for 5 min at 37 °C. After washing three times with 250 μl of washing buffer, 30 μl of the reverse transcription mix  were added to the tubes and incubated for 10 min at room temperature followed by 45 min at 42 °C. To inactivate avian myeloblastosis virus (AMV) reverse transcriptase and separate mRNA from the streptavidin-biotin complex, samples were heated at 99 °C for 5 min, transferred to microcentrifuge tubes, and incubated in ice for 5 min, diluted 1:2 (v/v) in nuclease-free water, and stored at −20 °C until analysis.For mRNA analysis, 1 million of the cultured cells were transferred to RNase-Free Eppendorf tubes, centrifuged 5 min at 300 × m/μl of both oligonucleotides and 2 μl of a cDNA solution corresponding to 1:100 (v/v) of the cDNA synthesis product. The thermal profile for all the reactions was 2 min at 50 °C, followed by 10 min at 95 °C and then 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as endogenous reference (Oligonucleotides for 17 glycosyltransferases (GTs) were designed by using the computer software Primer Express 2.0 , synthesized by Invitrogen Life Technologies , and are published elsewhere . PCRs weeference .Supplemental Table S7). In order to improve comparability, mRNA data was as well LOG2 transformed. To assess the quality and discriminative power, GT mRNA data were imported into SIMCA software and a PCA model was built as described above. The nine technical replicates were used as individual cross-validation groups. The median across the included cell lines was calculated and GT mRNA data was compared with relative abundances of corresponding glycan traits based on the total N-glycome. Both derived traits based on MS data as well as GT gene expression data were imported into GraphPad Prism (GraphPad Software), and a linear regression model was calculated.To calculate the relative abundance of the genes, the formula 100 × 2  was used, where Ct is the cycle threshold. In this formula, the Ct value is defined as the number of PCR cycles at which the SYBR-green fluorescent signal exceeds the threshold of 0.2 relative units . AverageSupplemental Table S8) via the database http://www.cbioportal.org/  proteins were solubilized in chaotropic agents and applied to PVDF-membranes in a 96-well format . This novel workflow showed high sensitivity and allowed the robust acquisition of high-quality N-glycan profiles from 250,000 cells. The total cell N-glycan profiling workflow was applied to analyze 25 CRC cell lines in multiple biological and technical replicates per cell line, in order to be able to discern biological and technical variation. Applying an unsupervised principle component analysis (PCA) revealed only little variation between replicates  of each cell line as seen by close clustering of scores . The dataset was further analyzed for possible batch effects. PCA scores were colored according to batches , and technical replicates distributed on different plates as well as different sample preparations were compared. Based on this comparison, batch effects were found to be minor and no correction was applied. Relative intensities for all glycans in the biological replicates of the cell lines with standard deviation and relative standard deviation are given in Supplemental Table S4. Relative standard deviations were below 20% for peaks with relative intensities above 5%, with exception for cell line C10, showing slightly higher variation. Although this method already provided a robust data acquisition, glycan traits were calculated by summing up relative intensities of glycan peaks corresponding to a certain glycan class . The only exception was the cell line HCT116 which also showed a high abundance of complex type glycans (58.9%). Complex type glycans could be detected up to m/z 4500 and their abundance varied considerably among the different cell types . The high abundance of high-mannose-type glycans may reflect a vast contribution of intracellular precursors, especially indicated by the glycan Man9HexNAc2Glc1 (m/z 2067.69), which led to the decision to exclude high-mannose-type glycans to calculate the derived glycosylation traits related to complex type N-glycans . To prove, however, that high-mannose-type glycans are indeed also present on the cell surface, N-glycans were shaved of the plasma membrane in a 30-min incubation step of living cells in the presence of high concentrations of recombinant PNGase F. The relative abundance of high-mannose-type N-glycans on the cell surface with respect to the residual pellet is given in Fig. 2B, showing that high-mannose-type N-glycans are expressed on plasma membrane proteins but to a lower extent as compared with their presence as intracellular precursor. The cell line C10 is an exception and shows similar levels of high-mannose-type glycans on the cell surface and the residual pellet. An exemplary MS spectrum comparing the cell surface N-glycome of CaCo2 cells with the residual pellet containing intracellular precursors is shown in Supplemental Fig. S4.This sensitive and robust 2Xcum) of the data with 13 principal components (PCs) featuring a good prediction power of 65.4% (Q2cum). Coloring the scores according to cell lines showed clear clustering based on the cell type as shown in Fig. 4A, indicating robust glycosylation features shared by biological and technical replicates. Cell line samples clustered within the Hotelling's T 95% with exception of HCT116, which seems to differ vastly from other tested cell lines. Coloring the loading plots, which represent each glycan variable, according to glycan classes helped to exploit which differences in glycosylation drove the separation in the PCA model. Trends observed in PCA models were then compared with relative abundancies of glycan classes by derived trait calculations based on the total cellular N-glycome.For further data analysis, a PCA model based on biological replicates  was generated using SIMCA software. This resulted in a model explaining 84.4% (RN-acetylhexosamine (HexNAc) ratio (B). Equal amounts of HexNAc and Hex indicate the presence of terminal N-acetylglucosamine (GlcNAc) or bisecting GlcNAc, while higher amounts of HexNAc than Hex may additionally point to the presence of terminal N-acetylgalactosamines  in e.g. LacdiNAc structures . PC3 (12.3%) appeared to likewise separate on Hex>HexNAc versus Hex<HexNAc, while PC4 (7.2%) refined the separation into terminal/bisecting HexNAc  and glycans with potential LacdiNAc epitopes . The presence of LacdiNAc epitopes was supported by the detection, albeit with low intensity, of a fragment ion at m/z 429 (HexNAc2 [M+Na]+) in MALDI-TOF/TOF-MS/MS spectra of some glycans such as for instance Hex3HexNAc5dHex1 with m/z 1688.61 [M+Na]+ . To further confirm the presence of LacdiNAc epitopes, glycan samples were labeled by 2-AA and analyzed by RP-LC-ESI-MS/MS. As shown in Fig. 3B and described in Supplemental Table S3, the detection of the fragment ion at m/z 407 (HexNAc2 [M+H]+) and its fucosylated variant at m/z 553 (HexNAc2dHex1 [M+H]+) in the fragmentation spectrum of the glycan Hex3HexNAc6dHex3-AA unambiguously demonstrated that this glycan carries a LacdiNAc motif. Additionally, the sialylated counterpart at m/z 698 (HexNAc2NeuAc1 [M+H]+) was found in some glycans . With 20.0% to 22.7%, SW480, SW620, SW48, Colo205_VUmc, and WiDr showed highest levels of N-glycans with HexNAc>Hex, while CRC cell lines SW1463, DLD-1, HCT15_VUMC, and HCT8 (the latter three share DNA profile) contained low levels . In CaCo2 and SW116 cells, 53.2% and 51.2% of the glycans, respectively, contained the same amount of Hex and HexNAc, which potentially reflected the presence of glycans with terminal or bisecting HexNAc . For SW1116 and CaCo2, the presence of bisection was confirmed in RP-LC-ESI-MS/MS on AA-labeled glycans with the indicative fragment ion at m/z 1057 [M+H]+ . For CaCo2 cells, additionally a specific epitope was found in RP-LC-ESI-MS/MS analysis, consisting of NeuAc[GalNAc]Gal-GlcNAc-R . The characteristic fragment ion m/z 860 [M+H]+ was confirmed by MS3  and pointed to the presence of the Sda antigen.PC1 (19.8%) appeared to reflect glycans differing in their hexose (Hex)/c) ratio B. Equal CSupplemental Figs. S5 and 5D). Poly-antennarity with more than four LacNAc units indicates the presence of LacNAc repeats . However, LacNAc repeats can also be present on di-, tri-, and tetra-antennary glycans, which could not be reflected in the trait calculations. MS data revealed the abundance of poly-antennary glycans in cell lines SW480 (21.1%), SW1116 (20.1%), SW48 (19.3%), and SW620 (18.6%) , which locate together with DLD-1, HCT116, and T84 on the left side of the score plot . The highest percentage of tri/tetra-antennary glycans in MS data showed cell line Caco2 (67.2%), followed by SW1398_VUmc (65.6%), Lovo (63.8%), SW48 (62.9%), and WiDr , which cluster all around the intersection of PC1 according to the tri-/tetra-antennary glycans in the loading plot . The lowest abundance of tri/tetra-antennary glycans was found in SW1463 (45.9%) and Co115 . In accordance with their location in the PCA plots, SW1463 (30.8%), Co115 (29.0%), HCT116_VUmc (28.3%), and Colo320_VUmc (26.6%) exhibit the highest abundances of di-antennary glycans, while the highest levels of mono-antennary N-glycans were found in HCT15_VUmc (4.9%), SW948_VUmc (4.4%), HCT116_VUmc (4.3%), RKO_VUmc (4.3%), Colo205_VUmc (4.1%), and SW480_VUmc (3.8%) . In contrast, CaCo2 is as well located on the right of the PCA score plot but is characterized by high levels of tri-/tetra-antennary glycans.The glycan antennarity is reflected in PC5 (6.7%) showing separation of cell lines expressing higher antennary glycans clustering on the left and lower antennary glycans on the right (C). Accordingly, score plots of PC1 versus PC2  show HCT116 and DLD-1 located apart from the other cell lines in the lower half, in line with the low relative quantities of fucosylated glycans in these cell lines (E). Relative quantification of complex type glycans reveals only 24.4%,29.1%, and 41.2% fucosylation in HCT116, DLD-1, and HCT116_VUmc, respectively; whereas all other investigated CRC cell lines contain 74.4% to 86.4% fucosylated N-glycans . Cell line HCT116 has a mutation in the fucosylation pathway and is therefore expected to have low fucosylation. Splitting the total fucosylation in mono-fucosylation (one fucose) and multi-fucosylation  revealed mono-fucosylation to be most abundant in Colo320_VUmc cells (72.4%), followed by SW1463 (67.3%), C10 (66.8%), and Co115 (59.4%). MS/MS spectra give evidence of core-fucosylation in many of the N-glycans, with the loss of the reducing end GlcNAc only occurring after the loss of fucose . Nevertheless, mono-fucosylation can also occur on antenna and is not a reliable measure of core-fucosylated glycans. LS180 cells showed highest multi-fucosylation, based on relative quantification of MS data (53.7%), followed by cell lines T84 (53.4%), HCT8 (51.8%), LS174T (50.7%), and SW1116 . The presence of antenna fucosylation is indicated by MS/MS data . Furthermore, antenna fucosylation with indications of the presence of blood group antigens was found  which is in accordance with literature information of the expression of blood group antigens in some of the cell lines . In cell line SW48 (blood type AB), fragment ions indicative of the presence of blood group A antigen -GlcNAc-) at m/z 308.88 [Hex1dHex1+H]+, m/z 715.28 [Hex1HexNAc2dHex1]+, and m/z 861.31 [Hex1HexNAc2dHex2]+ as well as blood group antigen B -GlcNAc-) at m/z 674.20 [Hex2HexNAc1dHex1]+ were found in RP-LC-ESI-MS/MS analysis of AA-labeled N-glycans . The presence of blood group A antigens further contributes to the trait Hex/HexNAc ratio. However, abundances of blood group antigens were rather low on N-glycans and expression of these antigens might be higher on other glycan and glycoconjugates such as O-glycans and glycosphingolipids.While PC1, 3, and 4 appeared to be linked to antennarity and terminal HexNAc residues, PC2 (13.5%) was found to reflect fucosylation, separating fucosylated  from non-fucosylated glycans . Relative abundances of α2,6-NeuAc containing N-glycans (pure and mixed) based on MS data showed the highest levels with 50.5% in Colo320_VUmc, 49.0% in C10, 48.7% in LS-411N, 45.2% in T84, and 42.9% in LS-180, while levels are lowest in HCT8 with 8.5% and SW48 with 8.8%, which is in accordance to their location in the PCA score plot  . The presence of α2,6-NeuAc is indicated by +319.13 Da in the profile spectra and was confirmed by tandem MS via marker ions of m/z 341.64 [M+Na]+  and m/z 706.71 [M+Na]+ , as shown for the N-glycan Hex5HexNAc4α2,6NeuAc1 with m/z 1982.71 [M+Na]+ (E).PC6 (5.5%) showed separation according to sialylation with α2,6-sialylated glycans clustering in the upper half and α2,3-sialylated glycans in the lower half, respectively . Abundance of α2,3-sialylated glycans (pure and mixed) is highest in Co115 . Also, SW1463 cells showed a high percentage of α2,3-linked sialic acid containing glycans (49.5%), while the lowest levels detected by MS were in HCT116 (8.6%) and CaCo2 . In some cell lines, as HT29, WiDr, SW48, T84, and Lovo, high expression of α2,3-sialylation (16.8% to 20.4%) was accompanied by high levels of multi-fucosylation (42.4% to 53.4%), which is indicative for the expected expression of antennary sialyl Lewis antigens (E and 2F). The presence of sialyl Lewis antigens was confirmed by RP-ESI-LC-MS/MS analysis on AA-labeled N-glycans, in which the diagnostic fragment ion at m/z 803.29 [Hex1HexNAc1NeuAc1dHex1]+ was found in several cell lines .The presence of lactonized α2,3-NeuAc is characterized by +273.08 Da in the profiling spectra as well as by the tandem MS marker ion antigens E and 2F.Overall, observations in the PCA plots could majorly be confirmed by relative quantification using derived traits, showing a vast difference between the cell lines. Furthermore, the coloring of loadings according to glycan classes was found to be a promising way to explore the data and to find the discriminators contributing to each principal component.N-glycan profiles derived from CRC cell lines  with the data obtained for CRC-tumor and control tissues derived N-glycans by Balog et al. . Profiles were further compared with recently published data on colorectal adenoma and carcinoma tissues  but in lower relative abundance than in the total cell line profiles determined in this study.The aim of this study was to further evaluate the potential of the studied CRC cell lines as model system. We therefore compared the g et al.  , indicating differences in fucosylation as well as the ratio of Hex/HexNAc based on corresponding loading plots (B and 4C). However, statistical evaluation by a Kruskal–Wallis nonparametric test did not reveal significant differences in fucosylation nor Hex/HexNAc ratio between colorectal cancer stages (data not shown). Interestingly, each disease stage group contained two subgroups that were identified as CDX1/villin-positive and CDX1/villin-negative cell groups (data not shown). The homeobox gene CDX1 is an intestine-specific transcription factor associated with differentiation and villin expression and therefore was used as a marker for the differentiation state of the cell lines (B), and statistical analysis using Mann–Whitney test revealed CDX1/villin-expression to associate with a terminal HexNAc  as well as multi-fucosylation . Coexpression data from the Cancer Cell Line Encyclopedia supported this association partly and showed CDX1 positively correlated with FUT3 . Furthermore, glycosyltransferase B3GNT8, involved in the generation of LacNAc repeats was positively correlated with CDX1 . We observed a trend toward higher poly-antennarity, reflecting more than four LacNAc repeats, in CDX1/villin positive cells but no statistically significance (data not shown). However, the cell line SW48 appeared as an outlier not following this association and expressing a CDX1/villin+ glycan phenotype while being CDX1/villin-.To gain more insight in the biology of the stage IV A, indicang plots B and 4C.ll lines , 39. Colll lines B, and stSupplemental Table S7). The PCA resulted in five principle components explaining 84.7% (R2Xcum) of the data with a good prediction power of 56.1% (Q2cum). The cell lines clustered per cell type, while technical replicates clustered closely together. Notably, taking the GT gene expression data, the location of the cell lines differs from MS-based data and cell lines HCT116 and DLD-1 do not cluster distinctly apart from the other cell lines . In contrast, C10, HCT8, WiDr, and SW1116 seem to be different from the other compared cell lines taking the measured GT into account. The corresponding loading plot  suggests high branching of N-glycans  as well as LacNAc repeats  and α2,3-sialylation  for these latter cell lines, which is in accordance to obtained high gene expression levels . Cell lines SW1116 and WiDr, furthermore, have high levels of fucosylation in both the loading plot as well as mRNA data. CaCo2 localizes also apart from the majority of cell lines and shows in the loading plot only MGAT4A and partly ST3GAL6 associated . The mRNA data support this observation since only MGAT4A and ST3GAL6 genes are highly expressed in CaCo2, while all other GT gene expressions are below the average of the tested cell lines .In order to obtain information on enzyme expression, mRNA of 17 glycosyltransferases (GTs) were obtained by real-time PCR for a subset of cell lines , involved in the synthesis of tri- and multi-antennary glycans and the relative abundance of the corresponding glycan class , as well as for fucosyltransferase (FUT) 4, the enzyme responsible for Lewis X epitopes on LacNAc repeats, with the trait multi-fucosylation . Furthermore, β-galactoside-α2,3-sialyltransferase 4 (ST3GAL4), involved in the production of sialyl Lewis X, showed significant correlation  with the relative abundance of α2,3-linked sialic acid containing N-glycans based on MS data . Overall, the mRNA data showed more overlap for GTs specifically involved in the N-glycan biosynthesis rather than with enzymes contributing to the biosynthesis (elongation/decoration) of more glycan classes, i.e. N-glycans, O-linked glycans and/or glycosphingolipids. However, FUT8, the enzyme responsible for α1,6-fucosylation of the N-glycan core, showed no correlation with the derived trait mono-fucosylation and instead showed the highest levels for SW48, SW480, SW620, and WiDr. This may indicate that, although MS/MS data suggested mainly core-fucosylated structures, mono-fucosylation is not only representative for core-fucosylation but also antenna-fucosylation. The discrepancy between GT gene expression and MS data shows that the value of GT expression data for predicting actual glycan structures is rather limited and that a better understanding of factors influencing protein glycosylation may be needed to improve this prediction.Correlation analysis between derived glycan traits based on MS-data and corresponding GT mRNA levels showed significant correlation for mannosyl--glycoprotein-β-1,4-N-glycome of 25 CRC cell lines including 21 cell lines derived from primary tumors and four from metastases revealing vast glycosylation differences between the cell lines. By applying a PVDF-membrane-based N-glycan release in combination with a new derivatization technique for linkage-specific stabilization of sialic acids, we achieved a sensitive high-throughput MALDI-TOF-MS analysis of N-glycans from small numbers of cells. The method proved to be robust for data acquisition while the calculated, derived glycan traits showed an even more pronounced robustness than most individual glycans. Moreover, the differentiation between α2,3- and α2,6-linked N-acetylneuraminic acids by linkage-specific derivatization adds major biological relevance to the results as it allows detection of the presence of sialyl Lewis antigens that are highly associated with cancer  Culturing conditions may influence the cellular phenotype. (ii) Tumor heterogeneity is not represented. (iii) The influence of the tumor environment is not given , which is in accordance with recently reported data by the group of N. Packer (Ø 55.0% high-mannose N-glycans)  gives evidence to the presence of intracellular precursors in the analyzed total cell homogenate glycan pools. Nevertheless, lectin-binding data showed strong interaction of several CRC cell lines with ConA (data not shown) suggesting the presence of high-mannose-type glycans. Of note, this lectin is not entirely specific for high-mannose N-glycans and can also bind to hybrid and di-antennary N-glycans, albeit with lower affinities   but whic glycans . Similar glycans , and als-glycans . From a on times . Especiafinities . Investifinities . This ceIn this study, we identified the presence of Sda antigen  in Caco2 cell lines based on MS data, while mRNA data on the corresponding GT, B4GALNACT2 showed elevated levels for WiDr and SW1116 cells and not for CaCo2. The latter result is in contrast to previous findings by Dall'Olio and coworkers who detected B4GALNACT2 mRNA exclusively expressed in CaCo2 when tested with eight other CRC cell lines (not WiDr and SW1116) . They deN-glycome of membrane proteins from various CRC cell lines as well as CRC tissues using porous graphitized carbon LC-ESI-MS/MS  in SW620 (38.4%), a cell line derived from lymph node metastasis. This is in contrast to literature, often describing the role of bisecting GlcNAc in suppressing metastasis , whereas no or weak staining was observed in healthy tissues —were applied. In addition, different culture conditions may play a role since it has been shown that the protein glycosylation is influenced by cellular, medium, and process effects, which is frequently addressed for the production of glycoproteins in the pharmaceutical industry (N-glycosylation profile between cell lines cultured in different laboratories and the impact of various factors on the glycan profile during the cell culture needs to be further investigated—not only for pharmaceutical productions but also for in vitro experiments with cancer cell lines in order to prevent misleading interpretation of results.Regarding sialylation, we observed a higher level of α2,3-sialylation and a decreased percentage of α2,6-sialylation in CDX1-positive cells that correlate with cell differentiation. In contrast, Sethi and collaborators reported on the presence of α2,3-sialylation exclusively in the poorly differentiated cell line LIM2405 . In geneindustry . Also inet al. (N-glycans in a CRC cell line SW1116 that are expressed specifically on CD26/dipeptidyl peptidase IV and serve as ligands for mannan-binding protein, a C-type lectin involved in host defense and tumor growth inhibition in CRC cells. In this study, we found that SW1116, a well-differentiated cell line derived from a primary stage I tumor, is characterized by an overall enhanced presence of polylactosamine repeats as well as high levels of multi-fucosylation. Supporting the finding of Kawasaki et al., Chen et al. (N-glycan phenotype. Clearly, further glycomic studies on more CRC cell lines as well as CDX1-knockdown cell lines and cells with forced CDX1 expression are needed to evaluate whether CDX1 acts as an inducer of the associated glycomic profiles, and assays on aggressiveness/invasiveness of corresponding cell lines are required to show the biological relevance.Interestingly, we observed a correlation between multi-fucosylation and expression of CDX1 and/or villin. CDX1 is an intestine-specific transcription factor associated with differentiation and villin expression , 39. Invet al.  identifin et al.  reportedN-glycome provided new insight in their glycosylation features that are lacking behind genetics and helps to evaluate cell lines as glycobiological model system. However, in order to choose an optimal model system, a larger-scale study comparing cell line glycosylation with tissue-derived glycan profiles is needed. Also, we observed major differences between the cell lines with regard to their N-glycosylation, demonstrating that the study of a single CRC cell line model can lead to the wrong generalization of glycobiological findings. Moreover, the similarity with regard to other glycan classes needs to be further studied, and the first results on O-glycosylation (in vitro might aid to mimic the tumor. Collecting detailed glycomic data on tumor subpopulations in a database can facilitate this approach by matching the characteristics to available cell line glycomic data and therefore improve the potential of cell lines as model systems.The analysis of the CRC cell lines with regard to their sylation  showed m


Genetic diversity provides the capacity for plants to meet changing environments. It is fundamentally important in crop improvement. Fifty-nine local maize lines developed at INERA and 41 exotic  inbred lines were characterized using 1057 SNP markers to (1) analyse the genetic diversity in a diverse set of maize inbred lines; (2) determine the level of genetic diversity in INERA inbred lines and patterns of relationships of these inbred lines developed from two sources; and (3) examine the genetic differences between local and exotic germplasms.Roger’s genetic distance for about 64% of the pairs of lines fell between 0.300 and 0.400. Sixty one per cent of the pairs of lines also showed relative kinship values of zero. Model-based population structure analysis and principal component analysis revealed the presence of 5 groups that agree, to some extent, with the origin of the germplasm. There was genetic diversity among INERA inbred lines, which were genetically less closely related and showed a low level of heterozygosity. These lines could be divided into 3 major distinct groups and a mixed group consistent with the source population of the lines. Pairwise comparisons between local and exotic germplasms showed that the temperate and some IITA lines were differentiated from INERA lines. There appeared to be substantial levels of genetic variation between local and exotic germplasms as revealed by missing and unique alleles.Allelic frequency differences observed between the germplasms, together with unique alleles identified within each germplasm, shows the potential for a mutual improvement between the sets of germplasm. The results from this study will be useful to breeders in designing inbred-hybrid breeding programs, association mapping population studies and marker assisted breeding.The online version of this article (doi:10.1186/s12863-014-0127-2) contains supplementary material, which is available to authorized users. Genetic diversity in agricultural populations provides the capacity to meet changing environments and market requirements . In cropet al. [Molecular marker analyses provide an important approach for estimating genetic relationships. Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeats (SSR) and Single Nucleotide Polymorphism (SNP) markers have been used to study genetic diversity in maize. Warburton et al.  characteet al. , and to et al.  and mid-et al.  inbred let al.  and 450 et al.  have beeA very large number of SNP markers are now available in maize, many of which have been developed from the DNA sequence of known genes. For this reason, SNP markers are now the assay of choice for a variety of tasks in maize improvement including genetic diversity analysis .per se and in top-cross combination of the newly developed inbred lines but they have not yet been characterized at the molecular level. Knowledge of molecular genetic diversity among the maize inbred lines developed for the national breeding program will provide guidance on how to use the local germplasm more efficiently. A better understanding of how the local lines are related to lines from different sources may be useful for decisions on the incorporation of exotic germplam in existing breeding program.CIMMYT and IITA  are the source of maize breeding materials for a significant portion of Africa. CIMMYT and IITA inbred lines and OPVs are bred to contain considerable diversity and are then taken by National Agriculture Research Programs and selected for further adaptation in their own particular environment(s). Maize germplasm at the Institute of Environment and Agricultural Research (INERA) in Burkina Faso includes different materials from CIMMYT and IITA. The maize breeding program at INERA was established in the late 1980s but little progress was made because a lot of effort was devoted to Open Pollinated Varieties (OPVs) development, which were more suitable to the majority of farmers. However, INERA initiated a new inbred-hybrid breeding program in 1991. Inbred lines are essentially extracted from Open Pollinated Varieties (OPVs), which have the advantage of being both environmentally adapted and adopted by farmers. Several studies have addressed the relative performance The objectives of this study were to (1) analyze the genetic diversity in a diverse set of maize inbred lines; (2) determine the level of genetic diversity in INERA inbred lines and patterns of relationships of these inbred lines developed from two sources; and (3) explore the genetic differences between local and exotic germplasms.Striga hermonthica resistant early yellow population, TZE COMP5-Y. The two other IITA testers (TZEI 10 and TZEI 23) which belong to heterotic group B and 7 inbred lines including TZEI 158, TZEI 161, TZEI 124, TZEI 148, TZEI 8, TZEI 149 and TZEI 146, were derived from the broad-based Striga hermonthica resistant and drought tolerant early yellow population, TZE-Y Pop DT STR.A total of 100 maize lines representing INERA, CIMMYT, IITA and temperate germplasms were chosen for molecular characterization. CIMMYT and IITA lines were chosen based on their resistance/tolerance to biotic and/or abiotic stresses and INERA lines were chosen to represent a sample of advanced lines selected for morphological characteristics and disease tolerance. The 100 lines included 59 lines from INERA, 16 lines from CIMMYT/Zimbabwe, 15 lines from IITA, and 10 temperate lines obtained from National Institute for Agricultural Research . The temperate lines represented European and U.S inbred lines. The INERA germplasm included 3 OPVs , 34 and 21 lines extracted from FBC6 and ESPOIR, respectively . FBC6 has a mixed genetic background, It was developed from a mixture of 8 varieties /IRAT/Réunion and FBC4, Maka and IRAT217 from INERA. ESPOIR was developed from Population 66 SR of CIMMYT/IITA using different cycles of recurrent selection and FBMS1 was developed from a mixture of different sources of sweet corn. FBML10 was derived from IITA line,TZI35, and selected for more uniform grain colour. The list of the inbred lines together with kernel colour, environmental adaptation and reactions to stresses (where available) are listed in Additional file http://www.lgcgenomics.com/nucleic-acid-extraction/kits/). They were then genotyped by Kbiosciences  using their Kompetitive Allele-Specific PCR (KASP) SNP genotyping system. The KASP assay uses a technique based on allele specific oligo extension and fluorescence resonance energy transfer (FRET) for signal generation. The fluorescent reporting system is comprised of four single-labelled oligonucleotides that hybridize to one another in free solution to form a fluorescent quenched pair which upon introduction of complementary sequences generates a measurable signal. Complete details on the principle and procedure of the assay are available at http://www.lgcgroup.com/products/kaspgenotyping-chemistry/#.VHhVZIvz0SU. SNP markers used in this study were chosen to cover all the 10 maize chromosomes and represented all the CIMMYT SNP markers that GCP  converted to KASP system and made available for maize genotyping. A detailed list of SNPs used can be found in Additional file Genomic DNA for each sample was extracted from seedling leaves (at V3 to V5 stage) using a magnetic bead and Klearcall extraction buffers , unbiased estimation of gene diversity, observed heterozygosity, and polymorphism information content (PIC) value, were calculated using PowerMarker software . The PICet al. , was useet al. . The genet al.  with PowSPAGeDi software  was usedQ values) for each inbred line were estimated to have its memberships in multiple subgroups. Inbred lines with membership probabilities ≥0.60 were assigned to the corresponding subgroup and lines with membership probabilities <0.60 assigned to a mixed subgroup.Three multivariate analysis including cluster analysis, principal component analysis and model-based population structure analysis were employed to subdivide inbred lines into genetic subgroups. A dendrogram was constructed, in cluster analysis, from the roger’s genetic distance matrix using the neighbor-joining algorithm  with PowOf the 1237 SNPs in the KASP assay, 1151 (93%) were successfully called in the 100 lines. SNP markers that were monomorphic (75 SNPs) or had missing data points more than 20% (19 SNPs) in the diversity panel of inbred lines were removed from further analysis. As a result, a total of 1057 SNPs (91.83%) was called successfully with high quality. Of the 100 maize lines, 4 lines were deleted from the next statistical analysis due to missing data called ≥20%. A detail list of these informative SNP loci including chromosome, base change, minor allele frequency (MAF), heterozygosity, gene diversity, and PIC is presented in Additional file Of the 1057 SNPs, 55.91% (591 of 1057) had a MAF >0.2 and were selected as markers with normal allele frequencies. Approximately 25.26% SNPs (267 of 1057) had a MAF ≤0.1, and 10.12% (107 of 1057) had a MAF ≤0.05. In addition, 88 (8.33%) showed almost equal allele frequencies (with MAF close to 0.5) for two alternative alleles  and high PIC values (>0.25). Lu et al.  recommenTo understand the effect of SNP subset and the influence of the two subsets in INERA germplasm on genetic diversity, the parameters of genetic diversity were estimated for each group with the 1057 SNPs and the 580 SNPs. Using all the 1057 informative SNPs and 96 inbred lines, temperate germplasm was found to show the highest average PIC value and gene diversity, followed by INERA germplasm for gene diversity and by CIMMYT germplasm for PIC value, whereas the IITA germplasm showed the lowest PIC value and gene diversity  value of this analysis is shown in Figure Group 1 (named temperate) consisted of 10 temperate lines, 7 CIMMYT lines and 1 IITA line  has been proposed as an alternative to Structure analysis for studying population structure of genotypic data . PrincipPrincipal component analysis (PCA) classified the INERA inbred lines into four distinct groups Figure  which inIn order to gain further insight into genetic differentiation between INERA germplasm and CIMMYT, IITA and temperate germplasms, a principal component analysis was performed on the following sets of inbred lines: set 1 consisted of INERA and temperate lines, set 2 : INERA and CIMMYT lines and set 3 : INERA and IITA lines.PCA on set 1 showed a clear separation of INERA and temperate germplasms Figure a. HoweveRoger’s genetic distance of the 96 lines ranged from 0.0205 to 0.448, with the overall average distance of 0.314. However, the majority (64.2%) of the inbred lines fell between 0.300 and 0.400  was observed for 331 (31.3%) SNPs in INERA versus Temperate, 263 (24.6%) SNPs in INERA versus CIMMYT lines, and 252 (23.8%) SNPs in INERA versus IITA. A distribution of allele frequency difference observed in the three pairwise comparisons, ranging from 0.1% to 71% is shown in Figure The temperate, CIMMYT and IITA inbred lines were included in this study for comparison purpose. They are not representative of all temperate, CIMMYT, and IITA germplasms. Thus, we only identified missing alleles present in INERA lines. In total, there were one hundred missing alleles identified in INERA germplasm which were present in other germplasm. The counterpart allele frequency in temperate, CIMMYT and IITA germplasm ranged from 3% to 70%. There are 28, 7 and 3 of such alleles that were frequent only in temperate, CIMMYT and IITA germplasm, respectively, but completely lacking in INERA germplasm , 55 unique alleles were identified that only existed in that specific germplasm collection but not in others  was homogeneous or contained genetically distinct subgroups. All the different multivariate methods supported the presence of genetically distinct groups. The model-based population structure and principal component analysis showed, to some extent, a separation by origin of the lines with related lines tending to cluster together. It has been reported that the clustering observed in the tropical populations is largely consistent with the pedigree information . TherefoThere are few studies on molecular characterization of maize germplasm from Burkina Faso. Previous studies (unpublished) characterized a collection of landraces and improved varieties using enzymatic and SSR markers and showed the influence of flux of genes in maize diversity of Burkina Faso.Hybrid maize programs were initiated at INERA but little progress has been made. The current hybrid breeding program uses improved OPVs, adapted to local environments and adopted by famers as source populations for extraction of inbred lines. FBC6 and ESPOIR are the main sources from which the available advanced inbred lines were extracted. The present study identified genetic variation and subgroups among INERA inbred lines. Model-based population structure and PCA of the 54 INERA maize lines, extracted from the two different sources, included in this study showed a separation of the lines into three distinct subgroups and a mixed group, which is believed to include recombinant lines since the two parental sources had a common parent. The extraction of inbred lines from FBC6, which was developed by mixing many different varieties, might have resulted in the separation of the lines into genetically distinct subgroups. In contrast, the lines extracted from ESPOIR did not show any major differentiation suggesting that the separation of lines from FBC6 could be explained by the presence in its genetic background of genes from diverse geographic origins. Lines extracted from FBC6 and tested in this study consisted of three groups. Two of the three groups included lines that are closely related in terms of the pedigree. In other studies of tropical maize lines considered extremely diverse, SSR marker variation did not provide any evidence of population structure other than among individuals closely related by pedigree . It has The present study showed that INERA inbred lines are fixed (lower level of heterozygosity) and exhibited an amount of genetic diversity between different lines. This makes them a valuable source for association mapping studies. Allelic frequency differences observed between INERA and; temperate, CIMMYT, and IITA lines, together with unique alleles identified within each germplasm set, suggests that a mutual improvement between INERA and each of temperate, CIMMYT and IITA sets of germplasm is possible.The present study investigated the genetic diversity among maize inbred lines developed at INERA and the relationship between these lines and temperate lines, and CIMMYT and IITA lines. The 1057 informative SNP markers revealed genetic variation among the inbred lines from different sources as well as between the INERA germplasm set. Two subsets of INERA germplasm included in this study were divergent but there was also a mixed group that presumably share a common genetic background. There appeared to be substantial progress in national program inbred line development as revealed by the low level of heterozygosity and the uniqueness of the majority of the lines. Principal component analysis showed a genetic differentiation between INERA and, temperate and IITA lines but not with CIMMYT lines. However, the unique alleles identified within each set of lines suggest that the exotic lines can provide new desirable alleles for local lines. This study has confirmed a set of SNPs previously reported by Lu et al.  and SemaThe data sets supporting the results of this article are included within the article and its additional files.


Medulloblastoma (MB) is the most common malignant paediatric brain tumour. Recurrence and progression of disease occurs in 15-20% of standard risk and 30-40% of high risk patients. We analysed whether circumvention of chemoresistance pathways  can restore chemotherapeutic efficacy in a panel of MB cell lines.O6-methylguanine-DNA-methyltransferase (MGMT) and BCL2 family members are differentially expressed  in MB cell lines. Based on these findings, each pathway was then inhibited or circumvented and cell survival assessed using clonogenic assays. Inhibition of ABCB1 using vardenafil or verapamil resulted in a significant increase in sensitivity to etoposide in ABCB1-expressing MB cell lines. Sensitivity to temozolomide (TMZ) was MGMT-dependent, but two novel imidazotetrazine derivatives  demonstrated ≥7 fold and ≥3 fold more potent cytotoxicity respectively compared to TMZ in MGMT-expressing MB cell lines. Activity of the BAD mimetic ABT-737 was BCL2A1 and ABCB1 dependent, whereas the pan-BCL2 inhibitor obatoclax was effective as a single cytotoxic agent irrespective of MCL1, BCL2, BCL2A1, or ABCB1 expression.We demonstrate, by immunohistochemistry in patient tissue microarrays, that ABCB1 is expressed in 43% of tumours and is significantly associated with high-risk. We show that ABCB1, ABCB1 is associated with high-risk MB; hence, inhibition of ABCB1 by vardenafil may represent a valid approach in these patients. Imidazotetrazine analogues of TMZ and the BH3 mimetic obatoclax are promising clinical candidates in drug resistant MB tumours expressing MGMT and BCL2 anti-apoptotic members respectively.The online version of this article (doi:10.1186/2051-5960-2-57) contains supplementary material, which is available to authorized users. Medulloblastoma (MB) is the most frequent primary malignant brain tumour in children . CurrentChemotherapy is now widely accepted as part of standard therapy for MB. The high frequency of recurrence, however, supports the hypothesis that a substantial number of patients have intrinsically drug resistant tumours . Chemothet al. to foetal cerebellum  respectively. The MED5R cell line maintained the mutation in culture whereas the MED6 cell line reverted to wild type  MB cell lines in response to etoposide alone or in combination with the ABCB1 inhibitors VPL and vardenafil. The IC50 concentrations for etoposide reflected the expression levels of ABCB1, with MED1 demonstrating the greatest degree of resistance to etoposide  . Obatoclax caused significantly decreased clonogenic cell survival resulting in less than 2% clonogenic survival at the highest concentration (3 μM) in all 3 cell lines  in MB cells. Thirdly, our data indicate that, despite the expression of several BCL2 anti-apoptotic family members, the BH3 mimetic obatoclax is effective as a single agent in MB.Benefit of adjuvant chemotherapy has been demonstrated in children  particul50 in the ABCB1 positive MED1 cell line was slightly above the concentration used in clinic [50 was below the dose limiting toxicity. The data suggest however that the application of drugs that circumvent ABC transporters could therefore represent a valid addition to current MB chemotherapy regimens, particularly in high risk MB. We have used two inhibitors to overcome ABCB1 resistance in MB cells. VPL  is a nonspecific ABCB1 inhibitor and its low affinity to ABCB1 necessitated use of a high dose to block ABCB1 in clinical trials, resulting in unacceptable cardiac toxicity [in vivo in pre-clinical models.Multidrug transporters serve as a major mechanism of defence in cancer chemotherapy. We hypothesise that the treatment of MB with current chemotherapeutic drugs, although effective as adjuvant therapy, leads to selection of an ABCB1 transporter expressing sub-populations of cells that in time can give rise to a more resistant tumour . Importan clinic  while thtoxicity . Vardenatoxicity , 67. It toxicity , 69. OurL and is currently in phase 1 clinical trial in small cell lung cancer (SCLC) and chronic lymphocytic leukaemia [50 concentration was still not reached and these concentrations would result in significant toxicity in vivo[Many efforts have been made towards development of small molecules able to overcome the anti-apoptotic activity of anti-apoptotic BCL2 family proteins. ABT-737 can overcome BCL2 and BCL-Xeukaemia , 70. Alteukaemia , 71, 72.eukaemia . Additioeukaemia , 76. Oury in vivo, 78. Obay in vivo. Obatocly in vivo. We beli50 concentration in the MGMT negative cell lines (MED1) was below the maximum tolerated dose of TMZ used in clinical trial [O6-guanine which cannot be removed by MGMT and both analogues have been shown to cause double DNA strand breaks leading to death in glioma cells. Overcoming MGMT resistance in MB may be reliant on MB tumours being proficient in mismatch repair. This is potentially not an issue since MMR proficiency was recently demonstrated in 74/74 primary MB tumours in a study by von Bueren et al.[O6-methylguanine, TMZ methylation at N7-methylguanine and N3- methyladenine purines can also be repaired by PARP through BER. Our data demonstrate that sulfoxide and propargyl analogues are cytotoxic to all 4 MB cell lines regardless of MGMT expression and that, unlike TMZ, the efficacy of the propargyl analogue cannot be potentiated by the PARP inhibitor rucaparib. This suggests that putative propargyl modification at N3 and N7 positions may not be recognized by PARP, or cannot be repaired by BER. Stability studies of the 2 analogues have shown that N3-propargyl is more stable than TMZ in plasma (100 minutes versus 30 minutes respectively); however, the N3-sulfoxide analogue is extremely unstable in plasma and hence unsuitable as a clinical candidate. Hence, propargyl could be a good substitute for TMZ in recurrent and progressive MBs and may also be effective in primary tumours.TMZ and Lomustine are alkylating agents commonly used in clinic, however, the DNA damage cause by these drugs can be repaired by MGMT . Notablyal trial . Two noval trial . It is hen et al.. In addiin vivo models but also to use them in combination. We believe that such an approach will help to redefine MB as a more chemo-sensitive disease.We have tested three mechanisms of resistance to chemotherapy mediated by ABCB1, MGMT and anti-apoptotic BCL2 family members in MB cell lines. Targeting these factors either by using an ABCB1inhibitor (vardenafil), a novel agent that can circumvent DNA repair  or a BH3 mimetic to inhibit BCL2 anti-apoptotic proteins (obatoclax) could all be viable therapeutic routes in this malignant tumour in children. Our study of a small subset of MB cell lines, however, indicates that these resistance mechanisms are not mutually exclusive. Hence, the next stage should not only be to test the efficacy of these agents in Additional file 1: Table S1: Clinicopathological characteristic of MB patients included in Nottingham TMA. (DOCX 14 KB)Additional file 2: Table S2: Antibodies used in the IHC analysis. (DOCX 15 KB)Additional file 3: Table S3: Correlation of ABCB1 expression with clinicopathological characteristics of DKFZ + Nottingham TMA cohorts. (DOCX 15 KB)Additional file 4: Table S4: Clinical characteristics of the 5 patients from whom 6 new MB cell lines were derive. (DOC 39 KB)Additional file 5: Figure S1: Patterns of MB marker subtype immunostaining on each patient’s original tissue sample. (JPEG 1 MB)Additional file 6: Figure S2: Molecular sub-classification of the 8 MB cell lines. (PDF 160 KB)MYCC and MYCN copy numbers in MB cell lines. (PDF 323 KB)Additional file 7: Figure S3: Elevated CTNNB1) sequencing in MED5R and MED6 tumours and cell lines. (PDF 32 KB)Additional file 8: Figure S4: β-catenin Additional file 10: Figure S6: MED1 cells show high ABCB1 protein expression. (JPEG 185 KB)Additional file 11: Figure S7: ABCB1 expression is found in a small subpopulation of cells. (PDF 37 KB)Additional file 12: Figure S8: MGMT expression in the original patient tumours. (JPEG 768 KB)Additional file 13: Figure S9: Chemosensitization of TMZ and propargyl by rucaparib in MB cell lines. (PDF 133 KB)