BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression.The anti-apoptotic protein B cell lymphoma/leukaemia 2 ( L), BCL-2-like protein 2 (BCL-W) and myeloid cell leukaemia sequence 1 (MCL-1)] and pro-apoptotic members  [Numerous cancer types have been associated with aberrations in genes encoding B cell lymphoma/leukaemia 2 (BCL-2) family proteins . The BCLt (BID)] –6. Incret (BID)] –13.BCL-2 [BCL-2 inhibition using RNA interference caused an apoptotic response in cell lines with moderate-to-high BCL-2 levels. These findings could be confirmed with the small-molecule BCL-2 family inhibitor ABT263, which inhibits the anti-apoptotic activity of BCL-2, BCL-XL, BCL-W and MCL-1 with inhibition constant (Ki) values of 0.044, 0.055, 7 and 224 nmol/L, respectively [BCL-2 levels responded better to ABT263 treatment than low BCL-2-expressing cell lines. ABT263 furthermore delayed the onset of tumor formation in mice injected with high BCL-2-expressing neuroblastoma cells [BCL-2-expressing neuroblastoma tumors. Unfortunately, the administration of ABT263 in phase I/II clinical studies for adult cancers was associated with dose-limiting thrombocytopenia due to concomitant inhibition of anti-apoptotic BCL-XL, a key survival factor for circulating platelets [Neuroblastoma is the most commonly diagnosed extracranial solid cancer in children, accounting for approximately 15% of all pediatric cancer deaths . A largeBCL-2 . Previouectively . Neuroblma cells . These olatelets –22. Therlatelets , 23, 24.L (Ki of 48 nmol/L), BCL-W (Ki of 245 nmol/L) and MCL-1 (Ki of >444 nmol/L), while maintaining its activity against BCL-2 (Ki of <0.01 nmol/L) [Compared with ABT263, ABT199 displays less activity against BCL-X nmol/L) , 25. ABT-W Ki of 5 nmol/L  nmol/L) . In the 50 and LC50 values of ABT199 were established for 21 classical neuroblastoma cell lines and 3 tumor-initiating cell lines (TIC)  versus micromolar IC50 and LC50 values  for the other cell lines tested. Evaluation of the BCL-2, MCL-1, BCL-XL, BCL-W and BIM protein levels . As ABT199 acts by displacing pro-apoptotic BCL-2 family members including BIM from BCL-2 [MYCN status. MYCN-amplified neuroblastoma cell lines responded more potently to ABT199 than MYCN single copy cell lines, with IC50 values ranging from 10 nmol/L-20 μmol/L versus 10-18 μmol/L, respectively , 13% (KCNR) and 5% (SJNB12) for the high BCL-2-expressing cell lines versus 0% (SKNAS) and 1% (SHEP2) for the low BCL-2-expressing cell lines. Effects on sub-G1 were dose-dependent, with maximum increases observed after treatment with 10 μmol/L ABT199 . Next, in vitro effects of ABT199 on the activation of the intrinsic apoptotic pathway were studied. As ABT199 inhibits the activity of BCL-2 by displacement of pro-apoptotic proteins, we first studied the effects of ABT199 on BIM displacement from BCL-2. Treatment of the high BCL-2-expressing neuroblastoma cell lines with 62.5 nmol/L ABT199 was already sufficient for almost complete displacement of BIM from BCL-2  shows that the effects of ABT199 obtained in CHP126, KCNR and SJNB12 are indeed caused by BLC-2 inhibition rather than off-target effects.In addition, overexpression of BCL-2 in the low BCL-2-expressing neuroblastoma cell line SY5Y resulted in a strong increase in sensitivity to SY5Y . The remin vivo in mice with high BCL-2-expressing KCNR neuroblastoma xenografts. Once-daily oral treatment with 100 mg/kg ABT199 for three consecutive weeks resulted in significant tumor growth inhibition relative to the vehicle treated controls . In vitro efficacy studies furthermore showed that BCL-2 protein and the BIM/BCL-2 complex levels are better predictive biomarkers for ABT199 sensitivity than BCL-2 mRNA levels. Although less strong, MYCN amplification status was also observed to be a predictive biomarker for sensitivity to ABT199.Based on previously performed preclinical studies , the BCLin vivo comparison of ABT199 with ABT263 for solid cancer treatment. Although ABT199 induced a strong apoptotic response, comparison of the efficacy of equal doses of ABT199 and ABT263 in mice with KCNR neuroblastoma xenografts expressing high levels of BCL-2 showed superior antitumor activity for ABT263. As maximum BIM displacement from BCL-2 was observed after ABT199 treatment, the most plausible explanation for the superior efficacy of ABT263 is its additional activity against anti-apoptotic proteins BCL-XL and BCL-W. Results indicate that the simultaneous inhibition of multiple anti-apoptotic proteins is more effective for treatment of BCL-2-dependent neuroblastoma than inhibition of BCL-2 alone.The current manuscript describes the first side-by-side in vitro and in vivo treatment with ABT199 resulted in upregulation of the anti-apoptotic protein MCL-1 and sequestration of released BIM by MCL-1. Upregulation of non-targeted anti-apoptotic BCL-2 family proteins taking over the function of BCL-2 has earlier been described as one of the key mechanisms of tumor resistance to BCL-2 inhibitors including ABT199 [L that sequestered BIM [L were observed after ABT199 treatment, indicating that MCL-1 plays a more important role in neuroblastoma resistance to ABT199 than BCL-XL.The importance of inhibiting multiple anti-apoptotic proteins is strengthened by the observations that g ABT199 –32, 34. ered BIM . No chanin vitro and in vivo treatment with ABT199, which has not been reported for BCL-2 inhibitors before. Since complexation of BIM with MCL-1 stabilizes MCL-1 independent of Noxa [MCL-1 upregulation after ABT199 treatment could be explained by Noxa downregulation and MCL-1 stabilization by BIM. Binding of Noxa to MCL-1 triggers Mule-mediated ubiquitination and proteasomal degradation of the anti-apoptotic protein, resulting in decreased MCL-1 levels , 35–40.  of Noxa , BIM disMCL-1 knockdown with ABT199 treatment resulted in synergistic cell growth inhibition. Potentiating effects of MCL-1 inhibition were confirmed with the small molecule MCL-1 inhibitor A-1210477, but combined effects of ABT199 and A-1210477 were additive rather than synergistic. The less potentiating effects obtained with A-1210477 might be due to the incomplete displacement of BIM from MCL-1, which has been described before. In addition, MCL-1 might have other functions independent of its role in apoptosis such as maintenance of mitochondrial integrity and deregulation of mitochondrial integrity by MCL-1 knockdown in combination with BCL-2 inhibitors might have a more lethal effect on neuroblastoma cells than inhibition of the pro-apoptotic function of MCL-1 alone. Of interest is the observation that several compounds in use or development for cancer treatment also cause MCL-1 inhibition  400/60% phosal 50 propylene glycol (PG) (v/v/v) in final concentrations of 10 mg/mL.ABT199, ABT263 and A-1210477 were purchased from Selleck Chemicals while QVD-OPH was purchased from Sigmaaldrich. For Classical human neuroblastoma cell lines and neuroblastoma tumor-initiating cell (TIC) lines were cultured as previously described , 42. Cel50) and half lethal concentration (LC50) values were derived from dose-response curves. IC50 values at 72 hours were calculated by determining the ABT199 concentrations needed to achieve a 50% reduction in cell viability observed for DMSO-treated cells at 72 hours (set at 100%). LC50 values at 72 hours were calculated by establishing the ABT199 concentrations needed to attain a 50% reduction in the cell viability compared to time point 0.Neuroblastoma cell lines were seeded in triplicate in 96-well  or 48-well (TIC lines) plates using the most optimal confluency for each cell line . Cells w1 fraction. See Cells were treated with 0.1% DMSO (control) or ABT199 using concentration ranges of 7.8 nmol/L to 10 μmol/L. After 72-hour treatment, floating and adherent cells were harvested for FACS analysis to determine the cell-cycle distribution and the apoptotic sub-GCHP126, KCNR, SJNB12, SKNAS and SHEP2 were 24-hour treated with 0.04% DMSO (control) or 62.5 nmol/L to 10 μmol/L ABT199. Floating and adherent cells were harvested to determine cytochrome c in the cytosolic and organelle fractions. See L (clone 54H6) monoclonal antibody ; rabbit anti-human MCL-1 monoclonal antibody ; rabbit anti-human BCL-W (Clone 31H4) monoclonal antibody ; rabbit anti-human Noxa (clone EPR9735B) monoclonal antibody ; rabbit anti-human BIM ; mouse anti-human cytochrome c (clone 7H8.2C12) monoclonal antibody ; rabbit anti-human PARP (Clone 9542S) monoclonal antibody ; rabbit anti-human cleaved caspase 3 (Clone 5AE17) monoclonal antibody ; mouse anti-human α-tubulin (clone DM1A) monoclonal antibody  and horseradish peroxidase (HRP)-conjugated goat anti-rabbit (clone NA9340V) and goat anti-mouse (clone NXA931) secondary antibodies . See The following antibodies were used: rabbit anti-human BCL-2 (clone D55G8) monoclonal antibody ; rabbit anti-human BCL-XL, cells were treated with 0.25% DMSO (control) or 62.5 nmol/L or 1.25 μmol/L ABT199 at 24 hours after seeding and harvested after 24-hour treatment. Co-immunoprecipitation studies have been performed as described in detail in the Cell lines were seeded onto 14-cm culture dishes. For the detection of basal BIM/BCL-2 complex levels, untreated cells were harvested after 72 hours incubation at normal culture conditions. For the detection of BIM displacement from BCL-2 and BIM complexation with MCL-1 and BCL-X5 cells in 4 mL culture medium) and incubated overnight. Next, cells were transfected with non-targeting shRNA  or MCL-1 shRNA (TRCN0000199070 and TRCN0000005518) for the BCL-2 high-expressing cell lines using the PLenti VI system according to the manufacturers protocol (Sigma Aldrich), while SY5Y was transfected with either a luciferase 2 control or a pLenti 6/V5-DEST vector constitutively overexpressing the BCL-2 protein. After 72 hours, cells were transferred into 96-well plates for in vitro MTT assays and synergy studies (see below).CHP126, KCNR, SJNB12 and SY5Y were seeded in 6-cm culture dishes . Effects on cell viability were studied after 72-hour treatment with ABT199, using the MTT cell proliferation assay.CHP126, KCNR and SJNB12 transiently transfected with non-targeting shRNA or For synergy studies between ABT199 and the MCL-1 inhibitor A-1210477, non-transfected cells in 96-well plates were co-treated with ten-fold serial dilutions of ABT199 (0-10 μmol/L) and five-fold serial dilution of A-1210477 (0-50 μmol/L).nu−/nu− mice  were obtained from Harlan) and experiments were performed with permission from and according to the standards of the Dutch animal ethics committee . NMRI nu−/nu− mice with KCNR neuroblastoma xenografts of approximately 268 mm3 were orally treated with 100 mg/kg/d ABT199 (n = 5), 100 mg/kg/d ABT263 (n = 5), or vehicle (n = 6) for 21 days. Tumor sizes were measured by an external caliper. See Female NMRI in vitro Western blotting.Per mouse sample, 10 tumor sections of 50 μm were homogenized in 2% Chaps buffer as previously described. Western blot detection of protein levels of BCL-2 like family members was carried out as described for Sections of treated and untreated KCNR tumors harvested at 4 hours after administration of the last dose were homogenized using the Ultra Turrax T25 tissue homogenizer (Janke & Kunkel) and lysed (overnight at 4°C) in 2% Chaps buffer. Co-immunoprecipitation was carried out as described above.The following antibodies were used: rabbit anti-human Ki-67 (clone SP6) monoclonal antibody , rabbit anti-human cleaved caspase 3 (Asp175) polyclonal antibody  and BrightVision horseradish peroxidase-conjugated goat anti-rabbit polyclonal secondary antibody . See http://r2.amc.nl).RNA was extracted from tumors with TRIzol  following the manufacturers protocols. RNA concentration and quality were determined using the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Fragmentation of cRNA, hybridization to hg-u133 plus 2.0, microarrays and scanning were carried out according to the manufacturers protocol . The mRNA gene expression data were normalized with the MAS5.0 algorithm within the GCOS program of Affymetrix Inc. Target intensity was set to 100. All data were analyzed using the R2 genomic analysis and visualization platform (


STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5.in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing’s sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice.An in vitro were not mediated through STAT3 inhibition.LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified  Approximately 30–40% of children with osteosarcoma (OS) still die due to metastatic disease, and survival rates for children with metastatic rhabdomyosarcoma (RMS) and Ewing’s sarcoma (ES) are similarly dismal and have not improved in over 15 years . Dysreguin vitro , and mouin vitro –10.The predominant oncogenic role of STAT3 in many solid tumors has generated interest in exploring STAT3 as a relevant target for therapeutic intervention. However, finding a strategy for specific and effective STAT3 inhibition has proven challenging . STAT3 cin silico site-directed fragment-based drug design, we developed a derivative of LLL12, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5) [in vivo models of breast and colon cancer as well as in vitro models of medulloblastoma alone and in combination with chemotherapy or radiation [in vitro [We previously investigated the allosteric small molecule STAT3 inhibitor, LLL12, demonstrating that it inhibits proliferation and induces apoptosis in childhood sarcoma cell lines ,16, althde (LY5) . LY5 hasde (LY5) . LY5 hasadiation –19. STATin vitro . In RMS,in vitro .in vitro, and evaluate the pharmacokinetic (PK) and anti-tumor properties of LY5 in vivo. Our data showed that LY5 was efficacious in vitro in inhibiting phosphorylation of STAT3 in RMS, OS, and ES cell lines at micromolar concentrations, but knockdown of STAT3 did not confer resistance to LY5. While LY5 demonstrated good PK parameters and oral bioavailability in vivo, inhibition of the tumor growth of patient-derived xenograft (PDX) models or metastatic disease in lung metastasis models was not observed. These data suggest that while LY5 is capable of inhibiting STAT3 phosphorylation, the anti-cancer effects of LY5 are likely due to as yet undefined off-target effects.The purpose of this body of work was to characterize the ability of LY5 to inhibit STAT3 phosphorylation in a variety of sarcoma cell lines 2.The human RMS cell lines  and human OS cell line SJSA were purchased from American Type Cell Culture Collection . The human OS cell line (OS-17), human OS PDX model (OS-1), human RMS cell line (RH30), human RMS PDX models (RH10 and RH36), and human ES cell lines  were generously provided by the Pediatric Preclinical Testing Program (PPTP). The human RMS JR-1 cell line was established at St. Jude Children’s Research Hospital, and characteristics have been described . Human Oin silico site-directed fragment-based drug design was used to develop a novel compound, LY5, synthesized by Chenglong Li’s Laboratory  and purified as previously described [in vitro treatment of sarcoma cell lines, LY5 powder was dissolved in sterile dimethylsulfoxide (DMSO) and the stock solution was stored at -20° C. The LY5 dosing solution for in vivo administration in mice consisted of DMSO/Solutol/20% hydroxypropyl beta-cyclodextrin (v/v/v: 5/10/85). The dosing solution was freshly prepared immediately prior to the first dose and pipetted to mix prior to each injection. A 1 mg/ml dosing solution of LY5 was prepared in a 5% DMSO/30% hydroxypropyl beta-cyclodextrin formulation and sterile-filtered for dosing of normal beagle dogs. Lastly, capsules containing 25 or 50 mg LY5 powder were prepared for oral administration to normal hound dogs.An escribed . For in Cell lysis, protein isolation and immunoblotting were performed as described previously . Twenty Sarcoma cell lines were seeded in 24-well plates in 1X RPMI medium containing 10% fetal bovine serum. Cells were then incubated with DMSO (control) or concentrations of LY5 ranging from 10 nM to 10 μM for 96 hrs. At the end of the incubation period, Alamar Blue  was added to cells at one-tenth the volume of the cell culture medium and incubated for 3 hrs at 37° C in the dark. Fluorescence was then measured at an excitation/emissions wavelength of 570/590 nm and emission wavelength of 590 nm.5’-CAGCCTCTCTGCAGAATTCAA-3’)  using Lipofectamine RNAiMAX  according to the manufacturer’s instructions. After 72 hrs of transfection, cells were harvested and lysed for immunoblotting. For the combined STAT3 knockdown–LY5 cell viability assay, negative control siRNA or STAT3 siRNA #7 was transfected into cell lines. After 48 hrs, cells were treated with 0.5 μM or 1 μM of LY5 for another 48 hrs. Cell viability was then analyzed using Alamar Blue as described above.Cell lines were transfected with 10 nM of AllStars negative control siRNA or human STAT3-targeting siRNA #7  were divided into 3 groups to be dosed with 5 mg/kg LY5 (2 mg/ml) by oral (PO), intraperitoneal (IP) or intravenous (IV) routes to establish oral bioavailability. Animals were euthanized and blood was collected at allocated time points  after LY5 administration, plasma was separated by centrifugation and frozen immediately on dry ice. Samples were stored at -80° C prior to analysis.LY5 was evaluated in two normal purpose-bred beagle dogs  receiving a single dose of 1mg/kg LY5 IV then orally by gavage 7 days later to establish the PK profile and assess the spectrum of clinical toxicities associated with LY5 administration. The oral dosing was repeated in the fed and fasted state. The starting dose was determined based on allometric scaling from the maximal tolerated dose studies performed in mice. Dogs were washed out for 30 days, and then given a dose of 25 mg in a capsule orally, followed by a 50 mg capsule dose 14 days later. Full PK sampling was performed over 24 hours following each dose of LY5. The dogs received LY5 and were used for the PK analysis but were not euthanized upon conclusion of the study.The liquid chromatography-mass spectrometry system utilized for these analyses consisted of a Dionex Ultimate 3000 RSLCnano High Performance Liquid Chromatography system and a Thermo Finnigan Quantum Ultras EMR mass spectrometer with an electrospray ionization source. Positive ionization mode was used for both LY5 and the internal standard, hesperetin. Plasma samples were collected and analyzed by the OSU James Comprehensive Cancer Center Pharmacoanalytical Shared Resource. Pharsight Phoenix WinNonlin software using non-compartmental analysis was used to generate PK parameters including maximum plasma concentration (Cmax), amount of time that drug is present at Cmax (Tmax), and total area under the curve (AUC). AUC all and AUC last were calculated by linear trapezoidal method of concentration-time curve up to the last measured point. AUC inf-obs was calculated by log-linear extrapolation of the terminal phase beyond the last collection time. Bioavailability (F) was calculated for IP and PO administration routes. Plasma stability and liver microsomal stability of LY5 were also assessed through this shared resource.scid -/- female mice  received IV inoculation with 106 OS-17-luc cells expressing a luciferase reporter gene. OS-17-luc cells were generated by infecting OS-17 wild type cells with lentivirus containing the firefly luciferase under a CMV promoter  and selected with 0.5 ug/mL puromycin . Beginning 24 hrs after inoculation, mice received vehicle (n = 10) or 20 mg/kg LY5 once daily via oral gavage . LY5 was prepared by diluting a 40 mg/mL solution/slurry with a 50% propylene glycol/1% Tween 20 in water vehicle immediately before administration. Mice were treated with LY5 for 30 days, and bioluminescent imaging (after IP luciferin administration) was performed to determine pulmonary metastatic disease load. To investigate the pharmacodynamics (PD) effects of LY5, pSTAT3  and total STAT3  were evaluated in pulmonary metastatic lesions using IHC in lung samples from mice euthanized 24 hrs after their last dose.The ability of LY5 to inhibit pulmonary metastases was evaluated using a murine model of OS pulmonary metastasis. 8-week-old C.B-17 Murine K7M2 and human MG63.3 OS cells were treated with increasing concentrations of LY5 for 2 hrs or 7 hrs. Western blotting was performed for pSTAT3 and STAT3 as indicated above to confirm inhibition of STAT3 phosphorylation in the cell lines prior to initiation of the PuMA assay. In addition, a sulforhodamine B colorimetric assay was performed to assess the cytotoxicity of LY5 in K7M2 and MG63.3 cells as previously described .5 MG63.3; 5 x 105 K7M2) were administered via tail vein injection into mice. Following humane euthanasia, the lungs were insufflated with a culture medium/agarose mixture to a constant hydrostatic pressure of 20 cm H2O and prepared for ex vivo culture as previously described [GFP-positive K7M2 and MG63.3 cells , were used to propagate subcutaneously implanted tumors. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee at University of Texas Health Science Center at San Antonio. Ten mice were used in each control or treatment group. Tumor volumes (cm3) were determined and responses were determined using three activity measures as previously described [C.B-17SC escribed .in vivo xenograft models, an event was defined as four times the tumor volume at baseline similar to the PPTP criteria [The data were analyzed by using GraphPad Prism software . Data are presented as mean ± standard deviation. Data were analyzed by one-way analysis of variance (ANOVA), and Tukey’s post-test was used to assess differences between groups. Differences were considered statistically significant at p < 0.05. For criteria . The procriteria .Murine and canine experiments were conducted in accordance with the institutional animal care and use committee of the Research Institute at Nationwide Children’s Hospital and the Ohio State University approved protocols (IACUC# 2009A0196-R1) and University of Texas Health Science Center at San Antonio (IACUC# 15015x) for patient-derived xenograft models, designed to minimize the numbers of mice used and to minimize any pain or distress. The institutional review boards/ethics committees at the respective institutions specifically approved this study.KINOMESCAN, www.kinomescan.com). LY5 exhibited no binding to any of the kinases at 1 μM and binding only to polo-like kinase 3 (PLK3) at 5 μM . ES and OS cells were also sensitive to LY5 administration . These data supported our further study of LY5 in vivo.To determine the relevance of decreased STAT3 phosphorylation in sarcoma cells, we analyzed with LY5 . LY5 decWe used both murine and canine models to evaluate the PK profile of LY5. In a pilot study to gain an initial understanding of plasma PK, mice were dosed with 5 mg/kg LY5 via IV, IP, or PO routes , and animals were euthanized at various time points between 5 minutes and 24 hours to collect plasma for analytical analysis . LY5 shoWe subsequently examined systemic bioavailability of LY5 in normal beagle dogs with IV bolus and oral gavage administration at a dose of 1 mg/kg. The administration of 1 mg/kg LY5 in normal dogs was well tolerated without apparent acute clinical toxicities during the 24 hour PK collection timeframe. The overlaid plasma concentration and time profiles of LY5 with IV bolus administration are shown in in vivo murine model of metastatic OS. Mice received 1 x 106 OS-17 cells stably expressing luciferase by tail vein injection and were then treated with 20 mg/kg LY5 daily for 30 days. Consistent with the in vitro study, LY5 inhibited STAT3 phosphorylation in pulmonary metastatic lesions as demonstrated by immunohistochemistry  using Western blotting and an ex vivo pulmonary metastasis assay (PuMA) [in vitro as determined by cell viability assays . Upon treatment of LY5 at various doses for 2 hrs, LY5 decreased STAT3 phosphorylation at a concentration of 1 μM in both cell lines  . Both lill lines . STAT3 pll lines . Using tis assay . Higher 0 μM LY5 , which w0 μM LY5 . TherefoTo further examine the efficacy of LY5, we administered LY5 using subcutaneous PDX models of RMS (RH10 and RH36) and OS (OS-17 and OS-1). RH10, RH36 and OS-1 were derived from tissue explants, therefore no corresponding cell lines exist. Tumors were harvested 2 hrs after final drug administration. Similar to the lung metastasis growth in To address whether LY5 might have anticancer effects independent of STAT3, we transfected RMS, ES, and OS cells with siRNA targeting STAT3  and thenin vitro but poor PK properties. Previous studies had described LY5 to have potential anti-tumor activity [in vitro and in vivo had not yet been characterized. The present study was performed to generate preclinical data in potential support of the continued development of LY5 as a novel cancer therapeutic. Our data showed that LY5 has excellent target specificity for STAT3, anti-proliferative activity at nanomolar drug concentrations against several sarcoma cell lines, and promising oral bioavailability in mice and dogs. However, drug concentrations needed to achieve tumor growth inhibition in both ex vivo and in vivo assays were discordant with those necessary to achieve target inhibition in vitro, suggesting that the anti-tumor effects of LY5 are not due to inhibition of STAT3 phosphorylation. The mechanism responsible for this observed discrepancy is currently unknown, however our results indicated that the anti-cancer activity observed in sarcoma cells exposed to LY5 was likely due to an off-target effect(s). Because LY5 demonstrated a lack of in vivo activity further investigation of these potential mechanisms is currently being pursued.STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction and its activation in tumor cells promotes metastasis and resistance to chemotherapy. LY5 was developed as a small molecule allosteric inhibitor of STAT3 building off of a prior analog LLL12 that had good activity activity –21 but iin vitro versus in vivo. Indeed, our unpublished data suggest that STAT3 may be more important for tumor cell-microenvironment interactions rather than for direct cell survival.Experimental evidence has demonstrated that inhibition of JAK/STAT3 signaling impairs proliferation and survival in sarcoma cell lines, supporting the notion that STAT3 is a relevant therapeutic target ,33–35. Iin vivo combined with data generated from siRNA studies downregulating STAT3 expression in sarcoma cell lines strongly suggested that its effects in vitro were not tied to inhibition of STAT3 phosphorylation.Clinical application of STAT3 inhibitors has been challenging. While a variety of small molecule and nucleic acid antisense inhibitors have been evaluated in the preclinical setting and in early-phase clinical trials, none have yet attained FDA approval. As previously discussed, we had developed another small molecule inhibitor of STAT3 (LLL12) that reduced proliferation, and induced apoptosis and cell cycle arrest of a variety of tumor cell lines ,16,36. Hin vitro and in vivo, few STAT3 inhibitors have been thoroughly studied for in vivo efficacy and pharmacology [in vivo activity that could be tied to a defined PK/PD relationship. Reporting results such as these may help combat the language of failure and improve outflow from the drug pharmaceutical pipeline.While investment in pharmaceutical research and development has increased substantially in the past two decades, productivity has declined . One of macology . Since Pmacology , we examin vitro and exhibited favorable PK properties in both mice and dogs including good oral bioavailability. However, the concentrations of LY5 necessary to observe an anti-tumor effect in vivo were discordant with the concentrations of LY5 necessary for inhibition of STAT3 phosphorylation. Further, LY5 anti-proliferative activity was similar in control cells and cells where STAT3 had been depleted, indicating that the anti-tumor effects of LY5 are not due to STAT3 inhibition.The novel small molecule inhibitor LY5, inhibited STAT3 phosphorylation and tumor cell viability S1 FigLY5 exhibits no interactions sterically or allosterically to the active sites of 96 protein or lipid kinases at 1 μM LY5 (A), mapped S-Score (35) = 0. At 5 μM LY5 (B), only interaction with PLK3 was identified, mapped S-score (35) = 0.01.(PDF)Click here for additional data file.S2 Fig(A) ES cell line ES-3 was transfected with either negative control siRNA (NC siRNA) or STAT3-targeting siRNA #7. After 72 hrs of transfection, cells were harvested and analyzed for total (T) STAT3 and GAPDH protein expression by immunoblotting. (B) RH30, ES-3, ES-7, and EW-8 cells were transfected with NC siRNA or siRNA targeting STAT3 for 48 hrs, followed by incubation with 0.5 μM or 1 μM LY5 for another 48 hrs. Cell viability was evaluated using Alamar Blue staining. Samples were set up as triplicates.(PDF)Click here for additional data file.S1 TableComparison of maximum drug concentration (Cmax), time at Cmax (Tmax) drug exposure (AUC), and oral bioavailability (F) of LY5 in mice  (A) and two study dogs (B and C).(PDF)Click here for additional data file.


This offers a unique opportunity to apply approaches that may provide genetic profiles that define response and resistance, with the potential to apply these as predictors in clinical decisions.Numerous collections of cancer cell lines provide opportunities to characterize genetic signatures that distinguish response and resistance to a variety of drugs. The NCI-60, Cancer Cell Line Encylopedia (CCLE), and the Genomics of Drug Sensitivity in Cancer (GDSC) provide a wealth of information that includes genomic sequences, mutational status, gene expression, as well as response to panels of drugs used in cancer therapies1. The data collection includes DNA sequence, mutation status, and gene expression data that we have used to develop a pipeline of computational approaches that predict response and resistance. Drug response is determined by a 9 step twofold serial dilution of drug concentration and measuring cell viability. From these, two quantitative values are provided: the drug concentration required to reduce viability by 50% (IC50) and the area under the dose-response survival curve (AUSC). Gene expression data is available from the Affymetrix U219 gene array platform.The GDSC is a collection of 1047 cell lines from diverse tumor types that have been tested with 265 drugsBecause expression patterns may vary widely simply based on tissue specificity, we chose to limit our initial analysis to B-cell malignancies, represented by 71 cell lines derived from leukemias, lymphomas, and myelomas. Our approach comprises a series of steps in which we classify response and resistance, develop a differential classification profile of gene expression patterns, identify features by pathway analysis, and validate on cell lines and reported clinical outcomes. We demonstrate this approach to stratify and predict response to the protein tyrosine kinase inhibitor, dasatinib.5. For this report, we develop an approach that identifies a five gene signature distinguishing dasatinib response and resistance.Dasatinib is a multi-target kinase inhibitor that has affinity for about 50 kinases and is most widely used to manage chronic myelogenous leukemiaWe arranged the 71 B-cell lines by response, using both Area Under the Survival Curve (AUSC) and IC50. The distribution of response favored non-response, with only 14 lines showing a strong response to low doses, and 11 lines showing essentially no response. Specifically, we classified Responders as lines that show an AUSC < 0.75, and an IC50 value less than the maximum drug concentration divided by 4 and Non-Responders were defined as having an AUSC > 0.98 and an IC50 greater than maximum dose tested. This resulted in the 14 strong Responder lines vs. 11 highly resistant (Non-Responder) lines, representing the extreme ends of response and resistance. Our rationale was that underlying this wide separation of response may be a common expression signature or pathway(s) that can serve as a predictive biomarker.Differential gene expression between the Responder and Non-Responder lines was performed using Significance Analysis of Microarray see , with a 8. Activation of various oncogenes and tumor suppressor genes gives rise to the malignancies at different stages of B-cell development  pathway has been demonstrated to be active at different stages of B-cell developmentent Fig. . The disz-score was the BCR pathway . Using the log-fold ratio scores of Responders relative to Non-Responders, molecules of the BCR pathway reflected a uniquely activated pattern in the Responders  on the 228 differentially expressed genes and their log-fold ratio data of Responders relative to Non-Responders. Notably, the top canonical pathway with a significant ers Fig. .Fig. 2BCp-value < 0.05) were further analyzed. This resulted in the identification of five genes  and Non-Responders (low expression). The genes from the BCR pathway were analyzed using a Mann–Whitney t-test between Responders and Non-Responders. Genes that were significant  and limited response (AUSC between 0.85–0.98). The five genes displaying the best separations were chosen for use in a predictive scoring system described below. Analysis of variances were performed using all four groupings and indicated 4 of the 5 genes showed highly significant differences between the Responders and Non-Responders, and trends of decreasing expression across the increasing resistance groupings.Eleven cell lines not included in the differential expression analysis had available gene expression data , but not dasatinib response data. These were tested in-house for dasatinib response. The CCLE/GDSC expression platforms were then normalized to one another to obtain comparable values (not shown). During the validation phase, we used the AUSC as the sole metric indicating response defining a Responder (AUSC < 0.8) and Non-Responder (AUSC > 0.8).A simple algorithm was developed based on average expression values of the Responders and Non-Responders for each of the five genes, which we refer to as Response Averages (RA). Lines that had expression lower than the RA of CD19 were immediately binned as a Non-Responder. Lines that had expression higher than that of the CD19 RA were given a score of “1” for each remaining gene  for which the expression exceeded the RA. If a cell line’s score was less than 3, they were binned as Non-Responders. This scoring system accurately predicted the response of 9 of the 11 previously untested lines.As described above, the discriminating genes were originally determined using the extreme Responders vs. the extreme Non-Responders. When applying the scoring system to the intermediate responding lines (that were not included in the gene discrimination modeling), 30 lines were correctly predicted for an accuracy rate of 67%. It should be noted low CD19 does place 23 out of 23 of these cell lines in the Non-Responder category. Thus, low CD19 is an effective discriminator of non-response. Nevertheless, the five gene discrimination was very accurate in distinguishing the highly sensitive from the highly resistant lines. Despite the difficulty of determining the response of the equivocal intermediate group, the sensitivity of the test set, training set, and intermediate group is 78.9% and specificity is 74.6%.MM lines (plasma cells) rarely express CD19, and show a low activation of the BCR pathway. Thus, our BCR gene discrimination model would indicate poor response. Indeed, a recent clinical study (NCT00429949) of relapsed, refractory, or plateau phase MM patients was discontinued after using dasatinib as a single agent in which a partial response occurred in only 1 of 21 enrolled in the study.9. WM primary patient lines exhibited good response to dasatinib in primary patient samples (n = 32)9 supporting our findings that the expression of CD19 and four other molecules of the BCR pathway are associated with dasatinib response.Waldenström’s macroglobulinemia (WM) is also a plasma cell malignancy, but in contrast to multiple myeloma, is CD19+, and has recently been described to express an activated BCR pathway11. Kim et al.12 showed acquired bortezomib resistance (BTZ-R) was accompanied by a re-activation of the BCR pathway. Along with this re-activation, a collateral sensitivity to dasatinib was observed.Mantle cell lymphoma (MCL) is mid-stage B-cell malignancy and may, or may not, express CD1913. The U266-VR line had a 2.8-fold increase in FPKM reads for CD19. In addition, the U266-P line had a dasatinib AUSC value of 0.91, whereas U266-VR had an AUSC of 0.73. These data further support that CD19 and the BCR pathway are consistent biomarkers associated with B-cell lineage cells response to dasatinib.We wondered whether this relationship between bortezomib and dasatinib would also be seen in the U266 multiple myeloma line. U266-P and U266-VR (Velcade resistant) was developed in our laboratory and had RNAseq data availableWe show here that cell line expression and drug response can be interrogated through differential expression and pathway analysis to find meaningful relationships and identify biomarkers of drug response.This is just one example of the use of available data bases to develop response signatures. Similar approaches may provide gene signatures across many other drugs within the GDSC, CCLE, or similar large cell line data bases.Supplementary Methods


The antitumoral effects of different Toll-like receptor (TLRs) agonists is mediated by activating immune responses to suppress tumors growth, although TLR ligands may also have a direct effect on tumoral cells. Given that TLR signaling induces hematopoietic cell differentiations this may serve as a novel differentiation therapeutic approach for AML.® Aqueous One Solution Cell Proliferation Assay (Promega). Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. Differentiation was measured by staining cells with the EuroFlow™ antibody panel for AML and analyzed by flow cytometry. FlowJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test.We investigated the effects of agonists for the ten human TLRs on the proliferation, apoptosis, cell cycle and differentiation of ten different types of myeloid leukemia cell lines . Proliferation was measured using the CellTiter 96The activation of particular TLRs on some cell lines can induce growth inhibition and Imiquimod (a TLR 7 agonist) was the most effective agonist in all leukemic cell lines examined. Imiquimod was able to induce apoptosis, as well as to induce cell cycle alteration and upregulation of myeloid differentiation markers on some of the cell lines tested.Our results, together with the known efficacy of Imiquimod against many tumor entities, suggest that Imiquimod can be a potential alternative therapy to AML. This drug has a direct cytotoxic effect on leukemic cells, has the potential to induce differentiation, and can also stimulate the activation of cellular immune responses anti-AML. Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells and the most common acute leukemia in adults. AML arise from the acquisition and accumulation of genetic and/or epigenetic lesions by progenitor hematopoietic cells which results in disruption of differentiation and increase proliferation, leading to blast-cell accumulation in the bone marrow and other tissues. Both prognosis and treatment choice for AML patients are determined by several aspects, as the presence or absence of specific genetic alterations, which determine AML classification in different risk based-categories. The majority of patients with AML have an intermediate cytogenetic risk; some of these patients do well with chemotherapeutic consolidation, but others have a very poor outcome and will suffer from relapse. Generally the clinical outcome remains problematic, with a poor prognosis and an overall survival rate of only 23.6% at 5 years. The treatment of AML patients has no changed in the last years and novel agents are necessary to improve cure rates and decrease toxicity. Currently, the standard therapy approach for AML is chemotherapy using a combination of cytotoxic agents, aimed at killing highly proliferating cells. Use of differentiation therapy is a very interesting possibility allowing more efficacious and less toxic therapeutic regimen. An example is the treatment of acute promyelocytic leukemia 5–10% of AML) with all-trans retinoic acid (ATRA) as differentiation agent, that lead to the long term survival and presumed cure of well over 85% of patients. However, ATRA does not work for other types of AML, so alternative differentiation therapy need to be identified  × 100.Cells were seeded in 96-well plates  and treated with TLR agonists for 48 or 72 h. Cells were analyzed for proliferation by a colorimetric method for determining the number of viable cells, using the CellTiter 964 cells in 100 µl medium were cultured in 96-well plates for 24, 48 or 72 h in the presence or absence of the TLR ligands. Apoptosis was evaluated by measuring annexin-V and propidium iodide (PI) binding using Annexin-V apoptosis detection kit (Santa Cruz Biotechnology). Viable cells were negative for both annexin-V and PI, early apoptotic cells were positive for annexin-V staining, and late apoptotic cells were positive for both annexin-V and PI staining. Cell cycle analysis was performed using DNA-Prep kit (Beckman Coulter) following the protocol recommended by the manufacturer for cell permeabilization and PI staining. The samples were analyzed by flow cytometry using a FACSCanto II flow cytometer (Becton–Dickinson). Samples were run in duplicate with 10,000 events counted per sample.7.5 × 1020,000 cells in 100 µl medium were cultured in 96-well plates for 72 h in the presence or absence of the TLR agonists. Immunophenotyping was performed by staining cells with the EuroFlow™ antibody panel for AML , and anat test for dual comparisons. Data are expressed as mean ± standard deviation. Significance was accepted at *P < 0.05 and **P < 0.01 levels.Statistical differences were determined using two-tailed Student’s The aim of the current study was to investigate the effects of agonists for the ten human TLRs on the proliferation and differentiation of myeloid leukemia cell lines. To address this question 10 different myeloid leukemia cell lines were used. HL-60 and Kasumi-1 (which are AML of M2 subtype), MOLM-13 (AML of M5a subtype), U-937 (a lymphoblast expressing monocytic like characteristics), K-562 , EOL-1 (from acute eosinophilic leukemia), HEL (an erythroleukemia cell line), KG-1 and the subline KG-1a (established from an erythroleukemia that evolved into AML) and NB4 (from acute promyelocytic leukemia M3).First, the mRNA expression of TLRs in the 10 cell lines was examined by RT-PCR , 87.5% in the presence of Poly (I:C) low molecular weight, and 84% in the presence of LPS. These differences were statistically significant in all cases.To study the effect of the TLR ligands on the proliferation of the cell lines, we measured viable cells in the cultures incubated for 48 or 72 h, in the presence or absence of each ligand. Results showed that Imiquimod (a TLR7 ligand) treatment induced growth inhibition in all cell lines in a time-dependent manner, reaching the higher reduction in cell number at 72 h . These alterations of cell cycle and viability were similar to that induced by Imiquimod in this cell line.We also analyzed apoptosis and cell cycle of cells at 72 h after treatment with R848, and only statistically significant differences were found in HL-60 cell line. In these cultures R848 caused an increase in the percentage of cells in early and late apoptosis, as well as a reduction of cells in the GTogether these data suggest that cell cycle arrest contributes to the reduction of proliferation in Imiquimod treated cultures in some cell lines, while Imiquimod inhibits cell proliferation and activates apoptosis in all tested myeloid leukemia cell lines.To study the effect of the TLR ligands on the differentiation of the cell lines, we performed the immunophenotyping of cells incubated for 72 h in the presence or absence of each ligand, by staining cells with the EuroFlow™ antibody panel for AML . The fluorescence mean intensity (FMI) of each specific monoclonal antibody staining on viable cells  was analyzed; only some differences were found in Imiquimod treated cells  as well as induce differentiation on the remaining viable cell population.TLRs are crucial drug targets due to their significant biomedical impact as they can stimulate immune responses. Generally, immunotherapy based on TLR agonists is very promising for the prevention and/or treatment of several disorders including microbial infection and cancer. Many patent applications, clinical trials and drug validation assays have involved TLR agonists, either individually or in pharmaceutical combinations with other ingredients (for a review see ).The antitumoral effects of different TLR agonists is mediated by activating immune responses to suppress tumors growth by different mechanisms: increasing dendritic cell maturation and presentation, activating NK or T cytotoxic cells or increasing infiltrating immune cells in the tumors. Nevertheless, we hypothesize that TLR agonists may have a direct effect on the tumoral cells in addition to their known immune activating effect. TLR ligation on healthy HSPCs induces proliferation and differentiation towards the myeloid lineage bypassing exogenous growth factors , 5, and Therefore, we tested agonists for the 10 human TLRs on 10 different myeloid leukemia cell lines. None of the ligands used induced proliferation, but some of them were able to induce growth inhibition; Imiquimod (TLR7 ligand) was the most effective agonist in all leukemic cell lines examined. R848 (TLR7/8 ligand) treatment also induce growth inhibition whereas TL8-506 (TLR8 ligand) had no effect, suggesting that the observed growth inhibition could be TLR7 dependent. The growth inhibition induced by Imiquimod seems to be at least partially caused by alteration of the cell cycle in some cell lines and by induction of apoptosis in all of them. In this context, the effect of Imiquimod in inhibiting proliferation and in inducing apoptosis, in all cell lines, was time-dependent.Our results are in agreement with previous reports showing that Imiquimod induces tumor-selective apoptosis and cell cycle arrest of different type of tumor cells, including skin cancer, prostate cancer, and endometrial cancer cells –19. MoreImiquimod is currently used as a topical and noninvasive treatment for superficial basal carcinoma, viral warts and other skin lesions in the clinic . The majAs TLRs are able to induce HSPCs differentiation, then we tested the ability of the agonists to induce differentiation of AML cell lines by measuring the expression level of 12 markers that are currently used to the detection and classification of AMLs . Not obvIt should be noted that most established leukemia cell lines may acquire irreversible changes that make cells resistant to TLR ligand differentiation, therefore, the differentiation detected in some cell lines in response to Imiquimod, although slight may be considered significant, and strongly suggests that the effect of Imiquimod and other TLR agonists in freshly isolated leukemia cells deserves further studies.Our results reinforce the idea of TLR-targeted differentiation as a potential therapy for AML, and suggest that Imiquimod may be a useful treatment option, as this drug has a direct cytotoxic effect on leukemic cells, has the potential to induce differentiation, and can also stimulate the activation of cellular immune responses anti-AML. Further studies are needed to confirm this therapeutic option.


N-glycan imprint of four PDAC cells which were previously shown to differ in their galectin-4 expression and metastatic potential in vivo. Next to the sister cell lines Pa-Tu-8988S and Pa-Tu-8988T, which were isolated from the same liver metastasis of a PDAC, this included two primary PDAC cell cultures, PDAC1 and PDAC2. Additionally, we extended the N-glycan profiling to a normal, immortalized pancreatic duct cell line. Our results revealed major differences in the N-glycosylation of the different PDAC cells as well as compared to the control cell line, suggesting changes of the N-glycosylation in PDAC. The N-glycan profiles of the PDAC cells, however, differed vastly as well and demonstrate the diversity of PDAC model systems, which ultimately affects the interpretation of functional studies. The results from this study form the basis for further biological evaluation of the role of protein glycosylation in PDAC and highlight that conclusions from one cell line cannot be generalised, but should be regarded in the context of the corresponding phenotype.To characterise pancreatic cancer cells from different sources which are used as model systems to study the metastatic behaviour in pancreatic ductal adenocarcinoma (PDAC), we compared the  Asymptomatic early stages as well as lack of reliable early diagnosis biomarkers hinder the detection of pancreatic cancer before spreading to other organs, and contribute to its high mortality4. Once metastases have occurred, most pancreatic cancers are resistant to chemotherapy and only 15–20% of the patients benefit from surgery, resulting in a 5-year-survival below 5%5. For pancreatic ductal adenocarcinoma (PDAC), which accounts for about 90% of pancreatic cancers, a very early formation of metastases is typical, hence increasing malignancy and therapy resistance considerably6. For these reasons, understanding the biology and mechanisms underlying PDAC progression and metastasis formation is essential to improve early detection and treatment.Although death rates for many cancers have decreased by more than 20% in the last 30 years, the incidence and mortality for pancreatic cancer is still risingK-ras, and in the tumour-suppressor genes TP53, SMAD4, and CDKN2A7. However, multiple pathogenic pathways leading to PDAC have been proposed, also as a result of the identification of different precursor lesions8.Several studies identified genetic alterations in main signalling pathways with high occurrence of mutations in the oncogene 10. Recently, fucosylated haptoglobin, but also glycosylated isoforms of α-1-acid glycoprotein, α-antitrypsin, and sialylated (Sambucus nigra lectin-responsive) α-1-β-glycoprotein have been explored for early detection of PDAC14. With regard to understanding PDAC biology and metastasis, characterisation of the aberrant glycosylation on cancer cells is a first step and can identify potential biomarkers, while in vitro and in vivo studies as well as manipulations of the glyco-phenotype can provide information on mechanism and therapy targets. However, the cell line model systems need to be well characterised in order to choose the model most reflecting the cancer phenotype and to interpret results correctly.On a glycomic level, several carbohydrate antigens such as CA-19–9, carcinoembryonic antigen, CA242, or combinations of those are used for pancreatic cancer diagnosis or monitoring16. While the two sister cell lines PaTu-S and PaTu-T were derived from the same liver metastasis of a patient with PDAC, thereby having the same genetic background, their metastatic behaviour differed vastly in vitro and in vivo in Danio rerio (zebrafish)16. Since galectin-4 is a glycan binding protein, and differentially binds the two cell lines, we hypothesised that the surface glycosylation would differ between PaTu-S and PaTu-T. Therefore, we characterised the N-glycome of these cell lines in order to (i) investigate if the N-glycan phenotype differs between the tumour-like PaTu-S and the metastatic PaTu-T, and (ii) characterise the N-glycan phenotype for future in vitro and in vivo studies using PaTu-S and PaTu-T as model systems. We expanded the characterisation to two primary cultures (PDAC1 and PDAC2), which as well showed different galectin-4 expression and metastatic behaviour17, and included the comparison to a normal, immortalised pancreatic duct cell line (hTERT-HPNE). Hitherto, only few studies have been performed to comprehensively characterise the glycosylation of cell line model systems using mass spectrometry19 and, importantly, evaluating their potential as model system by comparing cell line glycosylation profiles with those of tissues20. Especially in biopharmaceutical production, the selection of the right production system gained importance21, while for functional studies this awareness is still scarce.We previously described the effect of galectin-4 expression of two closely related PDAC cell lines (the established sister cell lines Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T)) on their metastatic behaviouN-glycome, emphasising the importance of the phenotypic characterisation of cell lines for the interpretation of in vitro or in vivo experiments. Interestingly, the tumour-like PaTu-S revealed the most deviating complex-type N-glycan features as compared to the immortalized normal pancreatic duct cell line, while the metastatic sister cell line PaTu-T showed most similar characteristics to this normal duct cell line.Our results show that the investigated cells differ vastly in their in vitro and in vivo, were characterised with regard to their N-glycan profile and compared to an immortalised, normal pancreatic duct cell line. While comparison of PaTu-S and PaTu-T is of particular interest due to their shared genetic background, the early-passage primary cultures PDAC1 and PDAC2 may better represent the genetic characteristics of the original tumour. As described before, PaTu-S showed a primary, tumour-like behaviour with an epithelial phenotype, whereas PaTu-T revealed a mesenchymal phenotype with high migratory and metastatic capacity in vitro and in vivo in zebrafish16. The primary cell cultures PDAC1 and PDAC2 were isolated from two different patients with PDAC in the same stage based on the pathological tumour-node-metastasis (pTNM) staging system. However, PDAC1 was derived from a male and PDAC2 from a female with a shorter survival time (8.5 months in PDAC2 vs. 21.4 months in PDAC1)22. In culture, PDAC2 revealed a less cohesive pattern of growth, suggesting a more mesenchymal phenotype as compared to PDAC1. In mouse models, PDAC1 showed a significantly lower migratory and invasive potential as compared to PDAC217, which was comparable to the behaviour of PaTu-S and PaTu-T in zebrafish, respectively. In contrast, both PDAC1 and PDAC2 showed a dramatically more aggressive behaviour in the zebrafish model as compared to PaTu-S and PaTu-T. For PDAC1 more than 23% of the fish were dying within 48 h of the experiment and for PDAC2 44% . Furthermore, for both PDAC cells a strong occurrence of brain metastases was observed in zebrafish .Two PDAC cell lines and two primary PDAC cell cultures, which were characterised by different metastatic behaviour N-glycosylation, N-glycans were released from the proteins using a PVDF-membrane-based 96-well plate protocol18. Prior to MALDI-TOF-MS analysis, N-acetylneuraminic acids (NeuAc) were subjected to linkage-specific derivatisation using a recently developed protocol allowing structural stabilisation of the sialic acid residues and the mass spectrometric distinction between α2,3 and α2,6-linked species on the basis of mass shifts induced by ethyl esterification  and lactonisation 23. Compositions of detected glycans were confirmed by fragmentation analysis using MALDI-TOF/TOF-MS/MS as well as additional reversed-phase liquid chromatography (RP-LC)-MS/MS of 2-aminobenzoic acid (AA)-labelled glycan pools. A list of compositions with averaged abundance per sample and fragmentation information is given in Supplemental Table 23.To determine how the pancreatic cancer cells PaTu-S, PaTu-T, PDAC1, and PDAC2 and the normal pancreatic duct cell line hTERT-HPNE differ in their N-glycan profile spectra with the main peaks annotated are shown in Fig. N-glycan profiles of all investigated cells ranged from m/z 1000 to approximately m/z 4000. Profiles were dominated by high-mannose-type N-glycans for all cell lines yet to different extent. The normal pancreatic duct cell line hTERT-HPNE and PaTu-S showed comparable levels of high-mannose type N-glycans (ø 56% and ø 59%), while the more metastatic cells PaTu-T, PDAC1, and PDAC2 showed elevated levels of ø 66–72% high-mannose N-glycans with highest abundance in PaTu-T  model was built on the relative abundance of derived traits resulting in a model with five principal components, covering 85% of variation (R2Xcum) in the data with a very good prediction power (Q2cum) of 76% 1 + Na]+  and confirmed the presence of an ethyl esterified α2,6-sialylated antenna. In contrast, the relative abundance of α2,3-sialylation was highest in normal hTERT-HPNE cells (ø 47%), followed by PaTu-T (ø 37%), PDAC1 (ø 28%), PDAC2 (ø 19%) and was lowest in PaTu-S  was highest in hTERT-HPNE cells (ø 61%), whereas lowest in PDAC2 : Maackia amurensis agglutinin  are summarized in Supplemental Table N-glycan derived traits for sialylation, the relative abundance of α2,3-sialylation based on MAA-binding  one and two showed most pronounced separation between PaTu-S and the control cell line on PC1  at m/z 2142.78 [M + Na]+ is shown in Fig. et al. for a liver metastatic pancreatic cancer cell line24, but also in our control pancreatic cell line fucosylation was very low  the main separation was between the two cell lines PaTu-S and PaTu-T versus the two primary cell cultures PDAC1 and PDAC2 Fig. . InvestiN-glycans carrying additional fucoses , a derived trait representative for sialyl Lewis antigens. Highest abundance from MS-based analysis was observed for PDAC2 (ø 10%), followed by PaTu-S (ø 8%), PaTu-T (ø 5%), and PDAC1 (ø 6%), whereas hTERT-HPNE showed lowest abundance  Lewis antigens were performed  can influence the cells’ characteristics. Feldman et al. reviewed critically the use of in vitro models in pancreatic cancer research and questioned the genuinely ‘normal’ character of hTERT-HPNE35 and also Maqsood et al. discuss the advantages and challenges of immortalization of cell lines36, while others described human cells immortalized with hTERT as closest resemblance to primary human cells as in vitro system without turning the cells into tumor cells as seen for immortalization with viral oncogenes37.Notably, the immortalised normal cell line hTERT-HPNE showed highest binding of anti-sialyl Lewis X antibodies  was found higher expressed in PaTu-T compared to PaTu-S and was identified to suppress FUT1/2 expression in pancreatic and colon cancer cell lines, leading to the reduced expression of the resulting epitopes38. Aubert et al. hypothesized that FUT1 is competing with α2,3-sialyltransferases for modification of type 2 chains, thereby skewing the glycan profile to sialyl Lewis epitopes through reduced FUT1 expression during pancreatic cancer progression39. Their results showed reduced α1,2-fucosyltransferase activity in pancreatic cancer cell lines in comparison to a normal pancreatic cell line39. Furthermore, restoration of FUT1 in the pancreatic cancer cell line BxPC-3 led to reduced adhesive and metastatic properties, and was associated with a reduction of sialyl Lewis A and sialyl Lewis X expression39. Conversely, our results showed low expression for Lewis B and Y in the normal cell line, whereas in PDAC2 we observed high levels of Lewis B and Y together with high levels of  Lewis A and X. This is in contrast to reports which identified Lewis Y and B epitopes mainly on normal pancreatic tissues, whereas Lewis X, sialyl Lewis X, and sialyl Lewis A were detected in pancreatic cancer tissues32. Interestingly, in colon cancer cell lines, Lewis Y expression was positively associated with sialyl Lewis A41, which matches our observations in the here tested cell cultures. These somewhat controversial results obtained from different cell lines support the view that conclusions from a single cell line cannot be generalised and should be in the context of the corresponding phenotype. Importantly, the same epitopes can be present on different glycan classes  and play different roles, showing the added value of a glycan class-specific phenotypic characterisation by e.g. mass spectrometry.In line with these results, we previously showed enhanced levels of FUT1 and FUT2 as well as the resulting Lewis Y and blood group antigens in PaTu-S compared to PaTu-TN-glycan feature, which was strikingly different between the investigated cell lines, was the higher or equal number of HexNAc than Hex of various N-glycans. Based on the loading plot of the PCA this feature was most dominant in PaTu-S cells , or attachment of N-acetylgalactosamine  residues in so-called LacdiNAc (LDN) structures as well as blood group A epitopes  or SdA antigen . Accordingly, galactosylation was lowest in PaTu-S (ø 85%), whereas hTERT-HPNE was characterised by almost complete galactosylation of complex type N-glycans  was found  was further confirmed with a monoclonal antibody against this epitope by flow cytometry, demonstrating its expression in PaTu-S cells (ø MFI 124.1), while expression on the other PDAC cells was  absent and only low in hTERT-HPNE  epitopes and their involvement in cancer progression are scarce, and further investigations are necessary to increase our understanding about their functions.Another lls Fig. . Derivedlls Fig. . The hig.78 Fig.  by MALDItra Fig. . A fragmN-glycans, defined as compositions with HexNAc = 4, were lowest expressed in PaTu-S (ø 24%) and increased in the more metastatic PaTu-T (ø 48%), PDAC1 (ø 46%), PDAC2 (ø 50%) as well as in the control pancreatic cell line  Lewis antigens. Clearly, functional studies with more cell lines as well as in-depth studies on the role of the individual glycan motifs in these model systems are needed to elucidate the meaning of these diverse (N-)glycan profiles in the process of metastasis in PDAC.Strikingly, PaTu-S cells, which show the more epithelial tumour-like behaviour, differed most strongly from the normal epithelial-like pancreatic duct cell line with regard to complex-type N-glycomic characterisation of different PDAC cells revealed major differences as compared to the N-glycome of a control normal pancreatic cell line, suggesting changes of the N-glycosylation in PDAC. However, we also found pronounced different N-glycan profiles between the four PDAC cells, which did not directly relate to their functional behaviour. These data suggest not only different mechanisms of cancer progression and metastasis by the model systems studied, but also show the diversity of model systems. The latter raises the concern about choosing the right model system as well as drawing conclusions and generalising results from only one or few cell lines. PDAC2 cells, for example, were characterised by high expression of Lewis B and Y,  Lewis A and sialyl Lewis X and showed high metastatic potential in vivo in zebrafish, whereas the PaTu-S cells shared high levels of sialyl Lewis A and Lewis Y antigens and were least invasive. Sialyl Lewis A is used as diagnostic marker (CA19-9) in PDAC and was lowest expressed in the control cell line, but also the metastatic cell line PaTu-T did not show high expression of this antigen. Furthermore, the normal pancreatic duct cell line shared high levels of sialyl Lewis X with PDAC2, demonstrating that further studies on different glycan-based mechanism involved in PDAC metastasis formation are needed and that different model systems may lead to different conclusions. Moreover, the results of this study challenge the genuinely normal character of the control cell line hTERT-HPNE and raise concerns as to what extent transfections (not only in this case) have unwanted effects on the geno- and phenotype of the cells – making proper characterisations essential.The here presented To improve the quality and reliability of cell lines as  model systems, it is essential to characterise the model system and link it to the investigated disease/cancer phenotype. Mass spectrometry offers a great tool for the glycomic characterisation in an untargeted and glycan-type specific manner, while data from direct binding studies with anti-glycan antibodies and lectins by flow cytometry supported and refined the mass spectrometry-based results. The use of lectins and antibodies gave further an insight in the overall surface glycosylation, while the mass spectrometric characterisation of other glycan classes is still needed and ongoing.Importantly, discrepancies between results obtained from different cell lines demonstrate that findings from one cell line cannot be generalised for the biology of a cancer. Experiments with higher numbers of cell lines as well as functional analysis of individual glycan motifs within these model systems are needed to improve the impact of these studies.3), and glacial acetic acid were from Merck . Ammonium formiate, dimethyl sulfoxide (DMSO), 8 M guanidine hydrochloride (GuHCl), 1-hydroxybenzotriazole (HOBt) hydrate, 2-aminobenzoic acid (AA), 2-picoline borane, 50% sodium hydroxide (NaOH), trifluoroacetic acid (TFA), and super DHB matrix  were obtained from Sigma-Aldrich  and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) from Fluorochem . N-Glycosidase F (PNGase F) was purchased from Roche Diagnostics , and the peptide calibration standard was purchased from Bruker Daltonics . MultiScreen HTS 96 multiwell plates (pore size 0.45μm) with high protein-binding membrane (hydrophobic Immobilon-P PVDF membrane) were purchased from Millipore , 96-well polypropylene 0.8 mL 96-deepwell plate and 96-well PCR plate polypropylene from Greiner Bio . Control Visucon-F plasma pool from 20 healthy human donors (citrated and 0.02 M HEPES buffered) was obtained from Affinity Biologicals . All buffers were prepared using Milli-Q water (mQ) generated from a Q-Gard 2 system (Millipore).HPLC SupraGradient acetonitrile (ACN) was obtained from Biosolve  and dithiothreitol (DTT), ethanol, sodium bicarbonate  was acquired from the American Type Culture Collection . An overview of cell characteristics is given in Table 17 and primary PDAC cells were kept at low passage number (<30).Pa-Tu-8988S and Pa-Tu-8988T pancreatic cell lines were purchased at the DSMZ culture bank . PDAC1 and PDAC2 primary cell cultures were isolated from patients at the University Hospital of Pisa  as described in Avan N-glycans were released from eight biological replicates per cell line (0.5 × 10 E6 cells each in four (batch A) or two (batch B) technical replicates randomly distributed across the plate) using a 96-well plate PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid derivatisation, purification by cotton-HILIC-SPE, and MALDI-TOF-MS analysis as described earlier18. As controls, Visucon pooled human plasma as well as water blanks were used.18. Shortly, a composition list was generated based on smoothed , baseline corrected , and internally re-calibrated average spectra using FlexAnalysis 3.4 (Stable Build 76) and the open-source software mMass (http://www.mmass.org)47. Compositional annotations as well as MS/MS peak annotations were performed in GlycoWorkbench 2.1 stable build 146 (http://www.eurocarbdb.org/) using the Glyco-Peakfinder tool (http://www.eurocarbdb.org/ms-tools/). Calibration . The curated data is available as Supplemental Table Spectra were processed as described earlierhttp://www.r-project.org/) was used to remove batch-effects using the sva library for ComBat Batch correction. Furthermore, derived traits such as galactosylation, fucosylation, sialylation, etc. were calculated in Rstudio by applying in-house developed scripts combined with open-source packages  and labelled using an equal volume of freshly prepared labelling solution  and an incubation for 2 h at 65 °C49. Samples were cooled down to room temperature, brought to 85% ACN and purified by HILIC-SPE as described previously18.Remaining released N-glycans (5 µL sample) were then injected into a nano-RP-LC-ESI-ion trap-MS(/MS) system consisting of an Ultimate 3000 RSLCnano system  coupled to an ESI nano sprayer . Samples were loaded on a trap column  for concentration prior to separation on an Acclaim PepMap RSLC nano-column . A flow rate of 500 nL/min was applied in a multistep linear gradient  = 1%; t = 5 min, c(B) = 1%; t = 20 min, c(B) = 25%; t = 25 min, c(B) = 70%; t = 30 min, c(B) = 70%; t = 31 min, c(B) = 1%; t = 55 min, c(B) = 1%) with 0.1% formic acid in water as solvent A and 0.1% formic acid in 95% ACN and 5% water as solvent B.Released, AA-labelled, and purified m/z 400 to m/z 2000 for MS analyses. Selected precursor masses  and solvent evaporation was achieved at 220 °C with a stream of nitrogen at a flow rate of 3 L/min.Mass spectrometry was performed on an AmazonSpeed ion trap  in positive ion mode with a mass window of et al.38. Briefly, pancreatic cancer and normal cells were incubated for 1 h at 37 °C in TSM , biotinylated plant lectins (10 µg/ml) Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA)  or at 4 °C with antibodies (5–10 μg/mL) against the glycan antigens Lewis X (clone P12), Lewis Y (clone F3), Lewis A (clone unknown) and Lewis B (clone T218) , sialyl Lewis A , sialyl (dimeric) Lewis X  and against the antigen LDNF50, kindly provided by Dr. Richard Cummings . Next, cells underwent a 30 min staining with a fluorescent secondary antibody , using cells stained only with these secondary antibodies as background fluorescence negative controls. Binding of lectins and anti-glycan antibodies was measured using a FACSCalibur or a BD LSRFortessa™ flow cytometer . FlowJo v10.3  and Summit software (BD Biosciences) were used to determine cell population mean fluorescence intensity (MFI). Averages of measurements from a minimum of three independent experiments were calculated.Flow cytometry was performed as described in Belo Supplemental Tables S1-S8


In GC cell lines, MCT1, 2, and 4 were up-regulated with different expression levels; MCT1 and MCT4 were more widely expressed in GC cell lines compared with MCT2. Inhibition of MCTs by siRNA or AR-C155858 reduced cell viability and lactate uptake in GC cell lines. The effect of inhibition of MCTs on tumor growth was also confirmed in xenograft models. Furthermore, MCT inhibition in GC cells increased the sensitivity of cells to radiotherapy or chemotherapy. Compared with normal gastric tissue, no significant alterations of expression levels in tumors were identified for MCT1 and MCT2, whereas a significant increase in MCT4 expression was observed. Most importantly, MCT4 was highly overexpressed in malignant cells of acsites and its silencing resulted in reduced tumor cell proliferation and lactate uptake in malignant ascites. Our study suggests that MCT4 is a clinically relevant target in GC with peritoneal carcinomatosis.Monocarboxylate transporters (MCTs) play a major role in up-regulation of glycolysis and adaptation to acidosis. However, the role of MCTs in gastric cancer (GC) is not fully understood. We investigated the potential utilization of a new cancer therapy for GC. We characterized the expression patterns of the MCT isoforms 1, 2, and 4 and investigated the role of MCT in GC through  Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer deaths globally . In advaIn rapidly growing cancer cells, oncogenes and hypoxia stimulate the glycolytic metabolism, which generates increased amounts of lactic and carbonic acids –8. To avin vitro and in vivo . Co. Co47]. Peritoneal carcinomatosis in advanced GC remains an intractable clinical problem despite advances in the treatment of GC. Peritoneal dissemination is the most frequent pattern of gastric cancer recurrence (33-50%) , 52, 53.Our results suggest that MCT4 plays a central role in tumor metabolism in GC with peritoneal carcinomatosis and targeting MCT4 in combination with chemotherapy could be a novel strategy in the treatment of GC.in vivo GC xenograft models and enhance the anti-tumor activity of radiotherapy or conventional chemotherapy. Fourth, for identifying the significant differences in MCT expression between normal tissue and tumor tissue, we compared the levels of MCT expression of normal gastric tissue, primary GC tissue, and PDCs derived from malignant ascites by using real-time PCR.The objective of this study was to assess whether the MCTs could be a therapeutic target for treatment of advanced GC patients. First, we examined the expression levels of MCTs including MCT1, 2, and 4 in human GC cell lines by reverse transcription polymerase chain reaction (RT-PCR) and western blot. Second, to investigate the functional role of MCTs in GC, we explored the effect of knockdown of MCTs on cell proliferation and lactate uptake. Third, we examined whether inhibition of MCTs could translate in This study was approved by the Samsung Medical Center Institutional Review Board  in accordance with the ethical principles of the Declaration of Helsinki and local guidelines. Written informed consent was obtained from all participating patients.We screened 16 established GC cell lines including 6 primary cell lines , 4 metastatic cell lines , and 6 ascite cell lines  in order to identify the cell lines that expressed high levels of MCT1, 2, and 4.Total cellular RNA was extracted using an RNeasy MiniKit (Qiagen) and treated with DNase I. One microgram of RNA was converted to cDNA using an Omniscript RT Kit (Qiagen). Real-time PCR was performed by using a Prism 7900HT Sequence Detection System (PE Applied Biosystems).For western blot analyses, antibodies against MCT1 , MCT2 , MCT4 , and β-actin  were used. The protocols used are described in the Supplementary Materials.Silencing of MCT expression was done with siRNA targeting MCT1, MCT2, and MCT4  using HiPerfect transfection reagents (Qiagen) according to the manufacturer's instructions.5 cells per 6-well plate, Corning) were transfected with siRNAs and incubated for 3 days. Cell proliferation following each treatment was compared with untreated cells. For the clonogenic assay, cells were transfected with siRNAs for 24 hours, irradiated with a 137Cs source , incubated for 14 days, and then colonies were counted. All these experiments are described in detail in the Supplementary Materials.To assess cell numbers, cells (1 × 1014C]-L-lactate (Amersham Biosciences) was expressed as counts per million per milligram of protein. The protocols used are described in the Supplementary Materials.For lactate uptake measurements, siRNA-transfected cells were equilibrated in buffered solution adjusted to pH 6. [in vivo, we implanted mice subcutaneously or intraperitoneally with GC cell lines and assigned the animals to two groups based on their treatment with PBS only or AR-C155858. All mice experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the appropriate Institutional Review Boards at Samsung Medical Center (Agreement- 20141211001). Xenograft models of GC are described in the Supplementary Materials.To determine whether an MCT inhibitor has antitumor effects Forty-five matched pairs of primary GC and normal gastric tissue and PDCs (N = 48) were collected at the Samsung Medical Center. All GC tumors and control tissues were confirmed by the hospital's clinical pathology department. To establish PDCs from metastatic GC patients with malignant effusion, those who were enrolled in the SMC Oncology Biomarker study (NCT#01831609) were screened for the expression of MCTs by western blot. Details of cell lines and cell cultures are provided in the Supplementary Materials.All patients provided written informed consent. This study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Samsung Medical Center.MCT4 expression status in patients enrolled in the ARTIST phase III clinical trial [We examined al trial . All slit-test or one-way ANOVA using GraphPad Prism version 4.01 . For analysis of the MCT4 IHC cohort, SPSS statistical software version 20  was used. All comparisons were examined by the 2χ test or Fisher exact test. The Kaplan-Meier test was used to evaluate the survival rate and survival curves were compared by the log-rank test. A Cox proportional hazard model was applied for multivariate survival analysis. All tests were two-tailed, and P values < 0.05 were considered significant.Preclinical data were evaluated by a two-tailed


There is limited evidence assessing the effects of omega-3 polyunsaturated fatty acids (PUFAs) on oesophageal adenocarcinoma, both in vitro and in vivo. We evaluated the effects of the omega-3 PUFA and oxaliplatin on OE33 and OE19 cells.The two oesophageal cells were treated with Omegaven® (fish oil emulsion), EPA, DHA and oxaliplatin and incubated for up to 144 h.p = 0.001), DHA by 59% (p < 0.000) and Oxaliplatin by 77% (p < 0.000). For OE19 cells, the EPA reduced growth by 1% (p = 0.992), DHA by 26% (p = 0.019) and oxaliplatin by 76% (p < 0.000). For both cells, Omegaven® resulted in reduced cell growth at intermediate concentrations (20-40 μM) and increased cell growth at low (10 μM) and high (50 μM) concentrations. DHA, Omegaven® and oxaliplatin were associated with significant downregulation of VEGF and p53 protein, and upregulation of p21 protein. DHA, Omegaven® and Oxaliplatin also led to significant downregulation of the total ERK1/2 and Akt proteins.The following inhibitory effects were observed on OE33 cells: EPA reduced cell growth by 39%  had greater in vitro anti-cancer effects than EPA alone.The online version of this article (10.1186/s12944-018-0664-1) contains supplementary material, which is available to authorized users. There is an increasing interest in natural therapies with anti-inflammatory effects that exhibit anticancer benefit, including omega-3 PUFAs. The main omega-3 PUFA are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which have anti-inflammatory effects –3. Only Helicobacter pylori (H. pylori) related chronic gastritis and atrophy [H. pylori infection causes the induction of various pro-inflammatory cytokines such as TNF-α, Interleukin (IL) -1 and IL-6 [Several factors in the cancer microenvironment influence the carcinogenesis process, including the delivery of bioactive molecules, such as cytokines and growth factors that are responsible for increased cell proliferation, inhibition of apoptosis and induction of angiogenesis . In many atrophy . It is eand IL-6 . For theWu et al., treated gastric cancer cell lines with EPA and DHA, and found inhibited macrophage activated cell migration by down regulation of the matrix metalloproteinase 10 gene, and subsequent down regulation of extracellular signal-related kinase (ERK) . SlagsvoIn this exploratory study, we evaluated the effect of the four single treatments  and oxaliplatin) on OE33 and OE19 cell growth and expression of the following cytokines: IL-6, TNF-α and VEGF in the cell culture supernatant. In addition, we also evaluated expression of the following proteins p53, p21, Akt, ERK1/2 in the cell lysate.The two oesophageal cancer cell lines used were OE19 and OE33. OE19 is a human oesophageal cancer cell line derived from a 72 year old white male patient with moderately differentiated UICC stage 3 adenocarcinoma. The OE33 cancer cell line is derived from a 73 year old white female with UICC stage 2A lower oesophageal adenocarcinoma arising in a background of known Barrett’s metaplasia. These cell lines were purchased from Public Health England cell collection (The European Collection of Authenticated Cell Cultures).2. Both cell lines were cultured in RPMI 1640 medium  supplemented with 2 mM Glutamine and 10% foetal bovine serum (FBS).Cell lines were cultured as a monolayer at 37 °C and 5% COCell lines were passaged no more than 15 times following resuscitation from liquid nitrogen, to reduce the risk of phenotypic alterations. Passaging was undertaken once cells had reached approximately 80% confluence as follows: Cells were washed with 10 mL pre-warmed (37 °C) PBS once, followed by addition of 5 mL of 1X trypsin for 5 min at 37 °C for cell detachment. The trypsinisation process was halted following addition of an equivalent volume of RPMI media containing 10% FBS. Cells were pelleted at 400 x g, resuspended in fresh medium containing 10% FBS, and aliquoted appropriately into cell culture flasks as per experimental requirements.The treatments tested were EPA, DHA, Oxaliplatin , and Omegaven® . EPA and DHA stocks were prepared as 50 mM stocks dissolved in DMSO and oxaliplatin was prepared as a 50 mM stock dissolved in 5% dextrose. All treatments including the vehicle control, received equivalent volumes of DMSO or 5% dextrose. Omegaven® is a 10% fish oil lipid emulsion containing 1.25 to 2.82 g/100 ml EPA and 1.44 to 3.09 g/100 ml DHA as per the Omegaven® summary of product characteristics. The rationale for selecting Omegaven® was that it was commercially available, the omegaven® emulsion was also investigated over the same time period in a pilot clinical trial in patients with advanced oesophago-gastric cancer and the intention was to mirror the in vitro laboratory work with the clinical trial.OE33 and OE19 cell lines were grown in RPMI 1640 + 2 mM Glutamine + 10% foetal bovine serum (FBS) medium for 24 h, then the media was removed and replaced with medium containing 10 μM, 20 μM, 30 μM, 40 μM and 50 μM of EPA, DHA and Oxaliplatin treatment and in order to equate the Omegaven® emulsion mixture to treatment concentrations using the single agents, the emulsion was diluted in RPMI medium + 10% FCS via serial dilution to make treatments of approximately 10 μM, 20 μM, 30 μM, 40 μM and 50 μM of EPA and DHA. The cell lines were incubated for 72, 96, 120 or 144 h to determine the anti-proliferative effects. The cell culture supernatant was collected at 72 and 144 h and stored at − 80 °C for cytokine analysis. The cells were then harvested and counted.3 cells/well in 1 mL of RPMI 1640 medium. Cells were incubated for 24 h, and then the media was replaced with media containing the relevant treatments. Cells were then incubated for 72, 96, 120 and 144 h before counting; Each well was washed × 1 with 1 mL PBS and 0.5 mL of 1X trypsin added to each well, which was neutralised with 0.5 mL RPMI + 10% FBS once cells had detached. The well contents were transferred to coulter count cups containing 9 mL of isoton solution  and cells were counted using the Z2 particle analyser.Cell proliferation was undertaken using a Z2 particle size analyser  to count raw cell numbers; this was performed in both cell lines for comparison in triplicate. OE19 and OE33 cell lines were seeded into 24-well plates at a density of 2 X 10Cell culture media supernatants were collected at 72 and 144 h after incubation with treatment as described above, and stored at − 80 °C for analysis of IL-6, TNF-α, VEGF using an ELISA assay.5 cells per 75 cm flask and allowed to adhere overnight. Cells were then treated with EPA, DHA, Omegaven® or Oxaliplatin at concentrations of 10 μM and 50 μM and incubated for 72 and 96 h.The cell lines (OE19 and OE33) were seeded at 1.5 X 10After 72 and 96 h, all flasks were removed from the incubator and placed on ice. Cells were gently scraped using a sterile cell scraper, media containing cells was collected and cells pelleted . The supernatant was discarded, and the remaining cell pellet washed with 1 mL PBS. Cells were again pelleted, the PBS carefully aspirated and 100 μL of Roche Complete Lysis M  cell lysis buffer added to the cell pellet. The pellet was mixed by vigorous pipetting and the cells lysed on ice for 10 min. Cell debris was pelleted and the supernatant stored at − 80 °C for later analysis of total Akt, Erk1/2, p53 and p21 proteins.The data was not normally distributed hence all data were transformed to log 10. In order to identify whether there was any significant effect of each treatment, a two tailed t test was used to compare cell counts and all cytokine expression compared to the control.Growth inhibition of both cell lines was observed following treatment with EPA, DHA and oxaliplatin. The growth inhibition occurred in a time-, dose- and cell line dependent manner, with OE33 cells exhibiting more sensitivity to treatment than OE19 cells.p = 0.001), DHA reduced cell growth by 59% (p < 0.000) and Oxaliplatin reduced cell growth by 77% (p < 0.000). For OE19 cells, the EPA reduced growth by 1% (p = 0.992), DHA reduced growth by 26% (p = 0.019) and oxaliplatin reduced cell growth by 76% (p < 0.000). For both cell lines, the effects of Omegaven on cell growth did not follow a linear pattern, but more a “U-shaped” curve, with increased cell growth at low (10 μM) and high (50 μM) concentrations, and decreased cell growth at intermediate (20–40 μM) concentrations, see Additional file The following inhibitory effects were observed on OE33 cells: Compared to control, EPA reduced cell growth by 39% p = 0.00, DHA red® and oxaliplatin treatment was associated with significant down regulation of VEGF expression compared to control. A similar concentration of EPA was associated with an upregulation effect  significantly downregulated Akt expression in both cell lines after 96 h Fig. . All tre0 μM signIt has been reported in four previous studies on gastric adenocarcinoma cell lines that omega-3 PUFAs (mainly EPA and DHA) present in fish oil are associated with anti-proliferative effects , 10–12, We have shown for the first time that EPA, DHA, and oxalipatin have anti-proliferative effects on OE33 and OE19 cell lines. There was a more pronounced dose response effect with DHA and oxaliplatin than with EPA. Oxaliplatin as a single treatment or coupled with EPA has been previously investigated in colorectal cancer cell lines , 16. TheThis study differs from previous studies that investigated omega-3 PUFAs in that we used EPA and DHA as a single treatment and in combination in the form of a fish oil emulsion (Omegaven®) in oesophageal adenocarcinoma cell lines for the first time. At low 10 μM) and high (50 μM) concentrations of Omegaven®, treatment resulted in a significant increase in cell number for both OE19 and OE33 cell lines, this effect was lost at intermediate concentrations of 20 μM and 30 μM as shown in the Additional file 0 μM and Granci et al., investigated the effects of Omegaven®  in combination with oxaliplatin in colorectal cancer HT-29 cells, with this combination associated with further significant reduction of cell viability when compared to oxaliplatin alone .Omegaven® emulsion has various other substances such as preservatives, stabilisers and also other fatty acids including sodium oleate (monounsaturated fatty acid). The latter might theoretically have altered the growth characteristics of the oesophageal cancer cell lines as it had previously reported to increase breast cell line proliferation and decrease apoptosis , 21. In In colonic cancer cells, both EPA and DHA (10–30 μM) were associated with inhibition of VEGF expression . In vivoIn the current series of experiments, the most profound effects on cytokine production were on VEGF. The treatment of OE33 cells with DHA, Omegaven® and oxaliplatin in this experiment, showed significant decreases in VEGF expression after prolonged treatment of 144 h  was examined and we found that EPA treatment was associated with a significant increase in IL-6 and TNF-α expression as early as 96 h of treatment in OE33 cells and 120 h in OE19 cells. DHA, Omegaven® and oxaliplatin led to dose- and time dependent biphasic changes in OE33 cells. This sort of inconsistent low dose vs high dose effect appears to be a common finding in response to a variety of dietary agents and has previously been reported by Lavigne et al. in 2008, while investigating the effects of isoflavones (Genistein) in breast cancer cell lines . Cai H eIt had been previously reported that omega-3 PUFAs inhibited the activation of NF-kB, which normally regulates the Interleukin-6 and TNF-α expression , 27. The® and oxaliplatin treatments in both cells were associated with significant downregulation of p53 and upregulation of p21 protein, but this was not the case for EPA  forms of these proteins. Future work should assess the effects of treatment on protein phosphorylation for both Akt and ERK 1/2, which would give a more comprehensive indication of which up-stream pathways are directly implicated in regulating PUFA efficacy.We have demonstrated that DHA, Omegaven® and oxaliplatin were associated with downregulation of p53 and VEGF in two oesophageal adenocarcinoma cell lines. Of the PUFAs studied, DHA alone or in combination (Omegaven®) had greater in vitro anti-cancer effects than EPA alone. It appeared to exert its anti-cancer effects via multiple different pathways. Further, the magnitude of the anti-proliferative effect observed for DHA was similar to that observed with oxaliplatin in these oesophageal cancer cell lines. We speculate that it could be used as an adjunct to chemotherapy for oesophageal adenocarcinoma to enhance treatment efficacy.