Glycosylation, one of the most fundamental post-translational modifications, is altered in cancer and is subject in part, to epigenetic regulation. As there are many epigenetic-targeted therapies currently in clinical trials for the treatment of a variety of cancers, it is important to understand the impact epi-therapeutics have on glycosylation.N-glycans from chemo-sensitive/non-metastatic cell lines following treatment with 5-AZA-dC. These changes correlated with increased mRNA expression levels in MGAT5 and ST3GAL4 transcripts in ovarian cancer cell lines. Using siRNA transient knock down of GATA2 and GATA3 transcription factors, we show that these regulate the glycosyltransferases ST3GAL4 and MGAT5, respectively. Moreover, 5-AZA-dC-treated cells displayed an increase in migration, with a greater effect seen in chemo-sensitive cell lines. Western blots showed an increase in apoptotic and senescence (p21) markers in all 5-AZA-dC-treated cells. The alterations seen in N-glycans from secreted glycoproteins in 5-AZA-dC-treated breast and ovarian cancer cells were similar to the N-glycans previously known to potentiate tumour cell survival.Ovarian and triple negative breast cancer cells were treated with the DNA methyltransferase inhibitor, 5-AZA-2-deoxycytidine (5-AZA-dC). Branching and sialylation were increased on secreted While the FDA has approved epi-therapeutics for some cancer treatments, their global effect is still not fully understood. This study gives insight into the effects that epigenetic alterations have on cancer cell glycosylation, and how this potentially impacts on the overall fate of those cells. Global t2/home) . Promote. [N-glycans from secreted glycoproteins from the ovarian cancer cell line OVCAR3 post-5-AZA-dC treatment [-depth look at the effects of 5-AZA-dC on glycosylation patterns in various tumour tissues, and on tumours of varying grade and stage [Glycosylation is the most common posttranslational modification of proteins and is altered in cancer and other pathologies such as chronic inflammatory diseases exemplified by rheumatoid arthritis . In canc.  demonstrreatment . Similarreatment . Specifireatment . Both pand stage .. [N-linked protein glycosylation, using resveratrol, caused an accumulation of unfolded proteins in the endoplasmic reticulum (ER) of ovarian cancer cells and induced ER stress-mediated apoptosis [The effects on cellular processes and end points when epigenetic alterations occur are well documented , 11. How.  have shopoptosis . In anotpoptosis . It is t. The impact of this treatment on apoptosis, senescence and epithelial to mesenchymal transition (EMT) is described. Survival analyses on online data repositories (kmplot.com) were undertaken to access the effect of glycan alterations in progression of ovarian and triple negative breast cancer (TNBC).The current study follows up on the published work by Saldova et al. . Our objp = 0.033) , and two TNBC (MDA-MB-231 and MDA-MB-436) cell lines were chosen for this study, to determine what effect 5-AZA-dC treatment has on the secreted glycome of chemo-sensitive/chemo-resistant cancer cell lines. The inclusion of two TNBC cell lines (MDA-MB-231 and MDA-MB-436) allowed for comparison with another tumour type. Briefly, A2780 and A2780cis cell lines were treated with 1 μM 5-AZA-dC and the remaining cell lines were treated with 0.1 μM 5-AZA-dC. Following 5-AZA-dC treatment, cells were harvested for flow cytometry dot plot analyses  , whereas the chemo-resistant A2780cis cells showed a decrease in cell numbers (p = 0.004). This suggests that 5-AZA-dC treatment increased resistance to cisplatin in the A2780 cell line while enhancing sensitivity to cisplatin in the A2780cis cell line  and PEO4 compared to PEO1 (p ≤ 0.05), and an increase in GP14 (A2G2S2 isoforms) in PEO4 compared to PEO1 (p ≤ 0.005). There were no significant differences in cell glycans when comparing A2780 and A2780cis cell lines.When comparing chemo-sensitive with chemo-resistant cell lines, some differences in secreted glycans were observed  (p ≤ 0.05) and GP24, 26, 29 (A3G3S3 isoforms) (p ≤ 0.005) (up to 0.51fold) . Chemo-resistant cells (A2780cis and PEO4) and TNBC cells (MDA-MB-231 and MDA-MB-436), show a decrease in these highly branched and sialylated glycans. Significant changes  were observed in the TNBC cell line MDA-MB-436, with a significant increase in the core fucosylated glycan (CoreF), a decrease in tetragalactosylated glycans (G4), and a decrease in tri and tetra sialylated glycans (S3 and S4). Although not statistically significant, only the chemo-sensitive cell lines (A2780 and PEO1) showed decreases (in the blue colour) in core fucosylated glycans and glycans with polylactosamine extentions with outer arm fucose as well as increases (in the red colour) in trisialylated glycans (changes varied from 0.69fold decrease up to 1.75fold increase).In the secreted glycans, the variation in glycans between 5-AZA-dC treated and untreated samples appeared to be cell line specific Fig. b. Specifp ≤ 0.05). Although not significant, chemo-sensitive cell lines showed increases in branching, galactosylation and sialylation, whereas the chemo-resistant ovarian (A2780cis and PEO4) and TNBC cells showed the opposite trend, specifically, a decrease in branching, galactosylation and sialylation)  decrease in galactosylation 3), GP39 4) and tetrasialylated glycans (S4) were significantly decreased in treated A2780 cells  and PEO1 had a trend decrease in E-cadherin (p = 0.051) post-5-AZA-dC treatment compared to untreated controls. This indicated an increase in EMT in these chemo-sensitive cell lines , suggesting a decrease in EMT  post-5-AZA-dC treatment  compared to the slow growing cells (5 × 104 cells) . Additionally, A2780cis cells, having a relatively high proportion of cells in S-phase  compared to untreated controls. For Rb, there was a significant decrease in expression in PEO1 and PEO4 cells compared to untreated controls. In relation to apoptosis, there was a significant increase in PARP cleavage in the MDA-MB-231 (p ≤ 0.05) post-5-AZA-dC treatment. There were no significant differences found for p16 across all cell lines and treatments  and p21 for cellular senescence. 5-AZA-dC treatment induced a significant increase in p21 expression in all cell lines  and ST3GAL4 is not subject to promotor DNA methylation (Supporting data1). Briefly, using data from the ENCODE project, we identified transcription factors (TFs) with binding sites in the proximal promoter regions of both MGAT5 and ST3GAL4. While GATA1-3 were not the only TFs with binding sites in the promoters of both MGAT5 and ST3GAL4, the finding fact that all three were present in both promoters suggested to us that these TFs may play an important role MGAT5 and ST3GAL4 transcription and therefore warranted further investigation [p = 0.005), and increased GATA3 levels correlated with increased MGAT5 levels (coef = 0.886) p ≤ 0.05) . 5-AZA-dC-treated A2780cis cells showed no successful GATA2 knockdown, and a significant increase in MGAT5 and ST3GAL4 was observed (p ≤ 0.05)    Fig. a. Follow05) Fig. a. For 5-05) Fig. a.At the protein level, MGAT5 and ST3GAL4 were significantly decreased following siRNA knock down of GATA2 and 3, but only in untreated cells Fig. b.ρ = 0.483–0.911) (p ≤ 0.05)    Fig. a (kmplot01) Fig. b. ST33GAN-glycosylation of TNBC and ovarian cancer cell lines. DNA methylation was decreased in all cells following pharmacological demethylation, more in the chemo-resistant cells from the chemo-resistant/chemo-sensitive pair. 5-AZA-dC increased resistance to cisplatin in chemo-sensitive cells while enhancing sensitivity in chemo-resistant cell lines.The epigenetic link between glycosylation and cancer is a relatively new concept . This stThere were two glycans, namely hybrid pentamanosylated bisected outer arm fucosylated biantennary digalactosylated monosialylated glycan with polylactosamine extension and biantennary digalactosylated disialylated glycan, increased on secreted glycoproteins in chemo-resistant compared to chemo-sensitive cell lines.highly branched, galactosylated and sialylated glycans on the secreted glycoproteins, in the chemo-sensitive ovarian cancer cell lines A2780 and PEO1 consistent with the findings of Saldova et al. [. [metastasis and EMT [N-linked glycans, when overexpressed in a gastric cancer cell line, induced an error in the trafficking of the E-cadherin protein, resulting in its mis/re-location of E-cadherin from the cell membrane to the cytoplasm, resulting in reduction in cell to cell adhesion [N-linked glycans on E-cadherin results in tumours with increased mobility and metastatic potential [Although not reaching significance, there was an observed increase in a et al.  and Saldt al. [.  and conf and EMT –20. The  and EMT , and N-c and EMT . This co and EMT . MGAT5, otential .apoptosis and senescence were examined. Firstly, PARP cleavage, a marker of apoptosis, showed an increase in all 6 cell lines post-5-AZA-dC treatment. 5-AZA-dC causes DNA damage inducing strand breaks and this recruits PARP, which functions in the DNA damage response and regulates DNA repair, which forms covalent adducts that induce apoptosis [P21 inhibits G1 phase [Differential markers which are well established with other cellular events such as poptosis . DNA metpoptosis , therefoG1 phase , consistG1 phase .P16 is inactivated in tumours and is highly expressed in senescent cells [Rb [BRCA [Another marker of senescence, p16, showed no significant variation in expression. nt cells , Rb is ant cells , preventnt cells  and p16 nt cells . Therefont cells . This prnt cells . DNA metells [Rb ; howeverells [Rb , and theRb [BRCA . InteresRb [BRCA .Expression of glycosyltransferases responsible for altered glycosylation was investigated. We have looked at the glycosyltransferases which were previously found altered and responsible for changes on secreted N-glycans in ovarian cancer [MGAT5 and ST3GAL4 correlated with increases in highly branched and sialylated structures on secreted glycans in the chemo-sensitive ovarian cancer cell lines. Our in silico survival analysis indicated the poor prognostic significance of increased ST3GAL4 expression in ovarian and lymph node positive TNBC patients, in agreement with the fact, that increase in ST3GAL4 and associated sialylation is associated with cancer progression and chemo-resistance [N-glycans post-5-AZA-dC treatment in HepG2 cell lines [in-silico analysis identified potential TFs with binding sites in the proximal promoter regions of both MGAT5 and ST3GAL4, GATA1-3, which may regulate the expression of these glycosyltransferases. While GATA3 expression is required for normal development of the mammary gland, it has recently been reported to be overexpressed in many metastatic breast cancers [GATA2 or GATA3, resulted in a significant decrease in MGAT5, ST3GAL4, or both, on gene expression and protein levels, as confirmed by RT-qPCR and western blots. Interestingly, following 5-AZA-dC treatment, there was only successful knockdown of GATA2 in the A2780 cell line with an associated decrease in MGAT5. In the A2780cis and PEO1 cell lines, the expression of the ST3GAL4 and MGAT5 was significantly increased after 5-AZA-dC treatment. Demethylation of GATA TFs may cause their significant increase following 5-AZA-dC, and therefore, this effect could mask the effect of the siRNA silencing. Decreases in the mRNA and protein levels of GATA2/GATA3 correlated with decreases in mRNA and protein levels of ST3GAL4/MGAT5. The GATA family of TFs contain six proteins, GATA1-6. It has been reported that aberrant DNA methylation on GATA2 affects GATA6 expression levels in gastric cancer progression [n cancer  and founsistance , 23, 36.ll lines , could b cancers . Increas cancers , 40. Bot cancers , 42. Knogression . 5-AZA-dStudy limitations and future perspectives To further investigate cell-line specific patterns of glycan changes, future investigations would include whole genome methylation and transcriptome analysis post-5-AZA-dC treatment. To better characterise the roles of GATA2 and GATA3 in MGAT5 and ST3GAL4 expression, chromatin immunoprecipitation could confirm that both GATA2 and GATA3 bind to MGAT5 and ST3GAL4 genes thereby regulating their expression. The possibility that 5-AZA-dC treatment results in the hypomethylation these GATA TFs could be confirmed by bisulfite pyrosequencing. Additionally, binding of GATA may be methylation-sensitive. GATA1 TF binds to CGATA elements only if cytosine is unmethylated [thylated .Based on our results, the 5-AZA-dC treatment, currently in the clinical trials, could be potentially beneficial for patients with chemo-resistant cancer but potentially harmful for chemo-sensitive cancer presentations; therefore, patients should be carefully selected for this treatment. Glycomics changes in glycosylation on serum proteins have been altered with cancer treatment and prognosis , 36, 45 N-glycome from secreted glycoproteins in chemo-sensitive cell lines to increase migration post-5-AZA-dC treatment. This was directly related to the altered transcription of the glycosyltransferases ST3GAL4 and MGAT5, regulated in part by GATA2 and GATA3 TFs. Increased expression of ST3GAL4 was associated poor recurrence free survival in ovarian and lymph node positive TNBC patients. While more investigation is required, there appears to be a direct and novel link between the GATA TFs and these glycosyltransferases. 5-AZA-dC also triggers a therapeutic-induced senescence (TIS) and an EMT phenotype in the chemo-sensitive ovarian cancer cell lines A2780 and PEO1, with an associated increase in cellular migration. Based on our results, the 5-AZA-dC treatment could be potentially beneficial for patients with chemo-resistant cancer but harmful for chemo-sensitive patients. Therefore, our study is important for patient selection for this treatment.Our results show that the alterations produced by 5-AZA-dC treatment are cell line specific. This study is the first to connect increases in branching and sialylation on 2 for 3 days and cells were counted and compared.The ovarian cancer cell lines A2780, PEO1, PEO4, and the TNBC cell lines MDA-MB-231, MDA-MB-436 were obtained and cultured as described in Greville et al. . The celFlow cytometry to assess DNA methylation status was performed as described in Greville et al. .Supernatants from the cultured cells were collected and concentrated using an Amicon Ultra-15 10 K ultrafiltration (Millipore) to a final volume of ~ 200 μL. Proteins were precipitated with a half volume of 50:50 TCA: acetone (w/v) on ice. The mixture was then incubated for 45 min on ice and centrifuged at 13,000 rpm for 5 min. The resultant pellet was washed with cold acetone and centrifuged again at 13,000 rpm for 5 min.Cultured cells were harvested and resuspended in 250μL sample buffer  and left on ice for 20 min, pipetted again and left on ice further 20 min. The whole content was then drawn up and down 10times through a 21 gauge needle and centrifuged at 13,400 rpm at 4 °C for 20 min. The resultant supernatant was removed, and the pellets were dried.These final pellets of secreted and cell glycoproteins were dried and resuspended in sample buffer for subsequent glycan analysis.N-glycan analyses including glycan release, labelling, HILIC-UPLC and exoglycosidase digestions were performed as described in Greville et al. [N-glycans were released from glycoproteins in samples by in situ digestion with Peptide N-glycosidaseF  in-gel blocks, and fluorescently labelled with 2-aminobenzamide (2AB) by reductive amination. HILIC-UPLC was carried out on a BEH Glycan 1.7 μM 2.1 × 150 mm column (Waters) on an Acquity UPLC H-Class (Waters) coupled with an Acquity fluorescence detector using 30 min method and calibrated using a dextran ladder. The 2AB-labelled oligosaccharides were digested using arrays of the following exoglycosidase enzymes: Arthrobacter ureafaciens sialidase (ABS), Streptococcus pneumoniae sialidase (NAN1), bovine testes β-galactosidase (BTG), bovine kidney α-fucosidase (BKF), β-N-acetylglucosaminidase cloned from Streptococcus pneumonia, expressed in E. coli (GUH), and jack bean α-mannosidase (JBM), almond meal α-fucosidase (AMF).e et al. . BrieflyGlycan peaks were pooled based on similar structural or compositional features of the peak glycan members. Features pertaining to a peak were determined based on the major glycan members of that peak  were separated by SDS-PAGE using a 4–15% precast TGX gels (BioRad) and transferred onto PVDF membranes using the Trans-Blot®Turbo™ system (Biorad). Blots were blocked and incubated with rabbit monoclonal antibodies targeting PARP, p21, p16, N-Cadherin, Vimentin, GATA2 and GATA3; rabbit polyclonal antibody targeting LC3, mouse monoclonal antibodies targeting E-Cadherin, retinoblastoma (Rb), ST3GAL4 and MGAT5 as described in Greville et al. . The memMGAT5), β-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), GDP-mannose-4,6-dehydratase (GMDS), mannose phosphate isomerase (MPI), tissue specific transplantation antigen (TSTA3) GATA1, GATA2 and GATA3 as described in Greville et al. [Eight gene transcripts were analysed comprising of glycosyltransferases mannosyl--glycoprotein β1,6-N-acetyl-glucosaminyltransferase  or 5 × 104 cells/well/100 μL  were added into stopper-loaded wells to normalise for the effect of proliferation. Proliferation controls were additionally treated with 1% SDS in water to solubilise the colour and incubated 1 hr at RT on a rocking plate. A reading of each well was taken at 595 nm.The Oris™ Cell Migration Assay (Platypus Technologies) was used to assess migration in all untreated and treated cell lines as described in Greville et al. . 2.5 × 1Cells were transiently transfected with 100 nM of either siRNA targeting GATA2 , or GATA3  (Dharmacon) transcripts as described in Greville et al. .T test was used. For RT-qPCR, a 2-way ANOVA was used with a post-hoc T test. p values were adjusted for multiple testing using the Benjamini–Hochberg method. The criterion for significance was set at *p value ≤ 0.05 or **p value ≤ 0.005.All data are expressed as the means ± the standard deviation (SD). Statistical analyses were performed using SPSS statistical software for Windows . For significances in glycan data, the HILIC-UPLC data were logit transformed and then used MANOVA and Tukey test. For Western blot densitometry, proliferation and migration assays, parametric p value. Univariate analysis was carried out using Cox proportional hazard models, and hazard ratios (HR) and 95% confidence intervals (CI) were calculated.The prognostic significance of ST3GAL4 and MGAT5 was evaluated using the Kaplan–Meier method (kmplotter.com). Progression free survival was chosen as the outcome of interest for ovarian cancer, while recurrence free survival was chosen as the outcome of patients with lymph node positive triple negative breast cancer. Expression levels were divided above and below the median into two cohorts; high vs low expression. Differences in survival was determined by the log rank test and expressed as a logrank Additional file 1: Supporting data: Glycosylation gene promoter CpG island analysisAdditional file 2: Table S1. N-glycans and features from secreted glycoproteins from all cell lines.Additional file 3: Table S2. Detailed assignments of N-glycans from cell glycoproteins of A2780 cell line using UPLC and exoglycosidase digestions.Additional file 4: Table S3. P-values for siRNA knock down experiments . Figure S2: Glycosylation changes of cell glycans on A2780 and A2780cis chemo-sensitive/chemo-resistant pair. (A) Representative UPLC chromatograms produced from secreted N-glycans of ovarian chemo-sensitive- chemo-resistant pair  and their separation into 39 peaks. (B) Plotted peak areas from the cell N-glycans of these cell lines. The glycans in each peak (GP1-GP39) and features are listed in Table S2. Significant changes (p < 0.05) are starred: * = P-value ≤ 0.05 or ** = P-value ≤ 0.005. (MANOVA). Heatmap histograms indicating fold changes in 5-AZA-dC treated compared to untreated cells were created using Hierarchial Clustering Explorer HCE 3.5 software. Blue indicates decreases, and red indicates increases. The shade of colour corresponds to amounts of the decreases/increases. Figure S3: Secreted glycans differ in chemo-resistant comparing to chemo-sensitive cell lines. Plotted peak areas of GP13 and 14 from the secreted N-glycans of chemo-sensitive and chemo-resistant ovarian cancer cell lines. Significant changes (p < 0.05) are starred: * = P-value ≤ 0.05 or ** = P-value ≤ 0.005. (MANOVA). Figure S4. Representative Western blots of EMT markers in the 4 ovarian cancer cell lines and 2 TNBC cell lines post-5-AZA-dC treatment (T) compared to non-treated (UT) controls. Figure S5: Migration results are not attributable to proliferation. Migration (A) and proliferation (B) or the representative 1 uM 5-AZA-dC treated relative to the untreated A2780cis cells. Figure S6. Western blot analyses of the senescence markers p16, p21 and Rb and of cellular apoptosis (PARP cleavage) markers, post-5-AZA-dC treatment. Representative Western blots of the senescence associated proteins p21, p16, Rb and the apoptosis marker PARP of 4 ovarian cancer cell lines and 2 TNBC cell lines in 5-AZA-dC treated (T) compared to untreated (UT) controls. Figure S7. SiRNA knockdown of GATA2/3, impacts on the protein expression levels of MGAT5 and ST3GAL4. Western blot analyses of siRNA knockdown of GATA2/3 in 4 ovarian cancer cell lines . GATA2 was knocked down in the A2780 and A2780cis cell lines and GATA3 in the PEO1 and PEO4 cell lines. Results are shown comparing (i) untreated controls (UT) with siRNA GATA knockdown UT (siG2/3) (left) with (ii) 5-AZA-dC treated (T) with siRNA GATA knockdown treated T (siG + T). All results are in triplicates .


Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability  and reproducibility  for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line. At a cost of approximately $3 billion per approved drug, over a decade may have passed from target discovery to drug approval (long development time) and tens of thousands of drug candidates would have likely been dropped due to drug safety and/or efficacy issues4. Cell-based pharmacogenomics screens are commonly used during the preclinical drug screening process to identify druggable targets by characterizing the biological effects associated with drug response and toxicity. However, low interlaboratory reproducibility of cell-based pharmacogenomics screens, lack of robust disease models that recapitulate the natural progression of human cancers, and drug-associated toxicity issues contribute to the high drug attrition rates in oncology13. Biomedical researchers and the pharmaceutical industry are, therefore, developing strategies to improve early-phase drug screening, e.g. novel preclinical models of disease and drug repurposing14.Cancer drug candidates currently have the lowest overall success rates and are 23% less likely to succeed in phase III clinical trials compared with other therapeutic arease.g. MTT assay) performed in two-dimensional (2D) cell culture are typically used to evaluate drug efficacy and potency in cells exposed to a drug for up to 72 hours17. However, it has been challenging to develop robust drug sensitivity assays that produce consistent results within a single lab and/or across multiple laboratories because similarly designed drug screening studies have shown varying results16. Although several recent studies have attempted to address this issue by describing biological  and technical factors  contributing to data replicability  and data reproducibility , few studies have proposed a strategy to identify and correct for sources of variability in drug response19. Regardless of the method used to determine cellular response to drug treatment or whether automated or manual lab work is employed, assay robustness testing is recommended. Ideally, a robust assay should be stable over replicate experiments and between labs. Therefore, minor variations in assay parameters  should not drastically affect the results19.Drug-dose response assays , Z-factor (Z), Signal window (SW) and coefficient of variation (CV)) in single wells or on a plate level, followed by implementation of the optimized experimental parameters using a well-established drug and cell line21. Thereafter, the optimized assay should be validated and the results reported using suitable drug response metrics , Emax , AUC (area under the dose-response curve), GR50 (growth rate inhibition concentration at GR(c) = 0.5), GRmax , GRAOC (area over the curve), DSS (drug sensitivity scoring), and NDR ) to evaluate drug potency , efficiency , and drug response . Due to differences in cellular division rates, metrics for growth inhibition  have been shown to produce more consistent interlaboratory results than conventional metrics 25.Newly developed drug sensitivity assays should first be optimized to establish signal dynamic range using quality control metrics  that can affect data replicability, data reproducibility, and cell viability26. In addition, we show that suboptimal assay conditions can be improved, and together with suitable dose-response metrics can result in consistent data between laboratories.Here, we propose a strategy to identify potential confounders in a 2D 4 cells per 96-well in 100 µl HuMEC basal serum-free medium. Previous studies have shown that complete growth medium supplemented with FBS reduces the effect of proteasome inhibitor bortezomib on proteasome activity27, thereby warranting the use of HuMEC medium for all drug treatments. Due to poor aqueous solubility, the pharmaceutical drugs were dissolved in DMSO  according to the manufacturer’s instructions, further diluted with 1xPBS to the desired working concentration (1–10000 nM bortezomib and 2–1024 µM cisplatin/carboplatin), and stored at 4 °C for up to one week in Falcon TC-treated flat-bottom culture microplates (VWR). However, use of these experimental parameters  resulted in intra- and interexperimental inconsistencies, including differences between replicates and dose-response curves with viability estimations higher than 100% at the start of the experiment  and pharmaceutical agents  in cancer research and drug screening were utilized. To investigate the effect of these chemotherapeutic agents on cell viability in MCF7 and HCC38 cells, we initially performed the resazurin reduction assay using standard experimental protocols and calculated dose-response curves and the half-maximal inhibitory concentration (IC50) after 24-hour drug exposure. Cells were plated at a density of 1.0 × 10e.g. storage of the pharmaceutical drugs, cell culture conditions, and resazurin assay conditions; Table 2 environment. After as little as 48 hours storage at 4 °C or −20 °C, evaporation and eventual concentration of the diluted drugs had a significant effect on cell viability  and the area under the dose-response curve (AUC) values, we tested several experimental parameters that may affect replicability and reproducibility  that could potentially have a detrimental effect on the performance of the resazurin assay or cell viability  resazurin solution incubated for four hours) and the tested drugs in growth medium containing 10% FBS.We then evaluated experimental conditions  as covariates. As expected, the vast majority (approximately 90%) of the variation in cell viability was associated with the choice of pharmaceutical drugs  and cell line . All experiments were performed at least three times in triplicate. Intra- and interexperimental variation in resazurin-based absorbance values were also examined using assay QCM , coefficient of variation (CV)) with thresholds set to Z-factor > 0.4, SW > 2, and CV < 20%. An ideal assay would have a large signal dynamic range and/or small signal variation (standard deviation (SD) or CV), with a Z-factor close to or equal to 128. In addition, the signal window is recommended to be at least 2 SD the largest detected assay signal to differentiate biologically active and inactive compounds29. Here, we show that the optimized assay performed well in all four cell lines using bortezomib and cisplatin. All data points were within the recommended ranges, with the majority of data points for Z-factor > 0.75, SW > 10, and CV < 5%  is conventionally used to determine drug potency with cell-based cytotoxicity tests. According to the IC50 values, HCC38 cells were more sensitive to bortezomib than MCF7, MCF-10A, and MDA-MB-436 cells 24 hours after treatment Fig. . In linereatment . As expereatment .Figure 6The pharmacoDB database was then used to compare the IC50 values for MCF7, HCC38, MCF-10A, and MDA-MB-436 cells treated with bortezomib and cisplatin  were used to assess drug potency and efficiency after adjusting for cell growth rates. Using these metrics, we found that cisplatin was comparably cytotoxic to HCC38 , MCF7 , and MDA-MB-436  than MCF-10A cells . In addition, HCC38 cells  were shown to be more sensitive to bortezomib than the other three cell lines , with differing GRmax values indicating variable cytotoxic response to bortezomib  was in agreement with those determined in the NCI-60 cancer cell line panel (26 hours)mib Fig. . Intereslls Fig. .Figure 832. The optimization strategy used here can be adapted to other cell-based and animal model systems by first identifying confounding factors in the experimental setup, followed by optimization of critical experimental parameters and assessment of data quality. However, it is recommended that each lab evaluates whether the proposed optimal conditions are suitable for the cells (e.g. derived from other tissue types) and assay  used in their experiments. Here, we first optimized the resazurin viability assay using two breast cancer cell lines (HCC38 and MCF7) and then implemented the optimized assay on two additional cell lines derived from breast tissue (MCF-10A and MDA-MB-436). We used the resazurin substrate as a marker of cell viability because it is considered to be a relatively quick, non-toxic, cost-effective, and flexible method that can be utilized with a variety of different culture conditions34. To be able to compare drug potency estimates between different studies, long-established breast cancer cell lines  and pharmaceutical agents  in cancer research and drug screening were utilized37.In the current study, we describe a number of biological  and technical  factors that should be taken into consideration to achieve more reliable and reproducible drug-dose sensitivity screens in 2D models. Although many of these factors are known to affect drug response, few studies have proposed strategies to quantify and correct sources of experimental variability in cell-based drug screens using QCM13. Despite differences in experimental protocols, two large-scale HTS pharmacogenomics studies (Cancer Genome Project (CGP) and the Cancer Cell Line Encyclopedia (CCLE)) were shown to generate highly concordant transcriptional profiles, but discordant drug response. These findings indicate a need to standardize protocols for cell-based drug sensitivity screens by e.g. establishing a consensus on appropriate positive controls , cell culture conditions, etc8. To address this issue, use of long-established breast cancer cell lines and pharmaceutical agents were used in the present study. However, HTS are frequently performed using automated liquid handling robots. Although robotic systems are time effective and can facilitate high precision pipetting, challenges associated with this method include e.g. the inability to adjust drug doses for different cell types, evaporation (edge effect), clogging, air bubbles, use of small volumes and pipetting of viscous liquids38. In order to develop common standards for drug screening, it is also important to investigate the effect of confounding factors on cell viability and identify drugs that have a cytotoxic effect by evaluating assay suitability with QCM28.There has been considerable controversy whether high-throughput screens (HTS) produce consistent results between different centerse.g. cell type, drug, drug dose) to the variance of a dependent variable (e.g. cell viability). In agreement with previous studies, we demonstrated that cell line, drug type, dose and treatment time had a substantial effect on cell viability16. Though we used cell lines derived from different breast cancer subtypes with differing biological features, the impact of cell type on drug response was likely underestimated due to the use of cell lines from the same tissue. Seeding density, medium type and volume, and resazurin assay time were also shown to have an impact on cell viability, though to a lesser extent8. In the present study, a narrow range of seeding densities (5000 to 10000 cells/well) was used. However, Hafner et al. showed that seeding density can have a major effect on GR values, especially when using a broad range of seeding numbers6. In contrast, the effect of medium type and volume is less studied. Interestingly, we show that different FBS concentrations (0–20% FBS) have a minimal effect on drug sensitivity after 24 hours exposure. As FBS is expected to have an effect on cell proliferation, it would be interesting to assess the impact of FBS concentration on cell viability over time40.Drug screening assays with a low CV , high SW  and a Z-factor close to or equal to 1 should be able to identify biologically active pharmaceutical compounds. Here, CV, SW, and Z-factor were within recommended ranges  in the three independent experiments, with CV below 5%, SW exceeding 10 SD and Z-factor close to 1. Furthermore, variance component analysis can potentially identify confounders in an assay by estimating the contribution of covariates  for each cell line, thereby improving replicability of the drug-dose response curves. Pharmaceutical drugs are typically dissolved in various solvents  for use in in vitro studies. However, solvents such as DMSO can have a profound effect on cell viability, even at concentrations as low as 0.33%. By using a single DMSO vehicle control, we not only observed both over- and underestimation of viability, but also dose-response curves starting at levels above 100% viability. In contrast, matched DMSO concentration controls are highly recommended as they reduce the risk of dose curves starting at >100% viability. Due to the risk of evaporation, matched controls and drugs should be plated in the same location on the 96-well plate. Subsequent use of matched concentration controls is more time consuming because e.g. a dilution series of the solvent needs to be prepared, which takes up more space on the 96-well plate and drives up costs. In the edge effect experiments, we concluded that evaporation was higher around the perimeter of the 96-well plates, thereby affecting the concentration of added components and cell viability measurements. To minimize the edge effect, we excluded perimeter wells and instead filled these wells with PBS. Since the cell viability assay is usually performed over several days, each 96-well should be seeded with an appropriate cell number so that the cell population will be in the exponential phase during the course of the experiment and not affected by high confluency (stationary phase) that in turn could affect drug efficiency41. Lastly, an evaluation of evaporation on plates containing diluted drugs (stored at −20 °C) demonstrated that 96-well flat-bottom plates sealed with parafilm had higher evaporation than 96-well PCR plates sealed with aluminum tape. Consequently, evaporation of diluted drugs and medium components can lead to fluctuations in drug response, thereby making it difficult to estimate accurate IC50, GR50 and GRmax values and achieve reproducible results.Interexperimental variability was reduced by optimizing critical experimental parameters , drug response was relatively comparable. With the exception of MCF-10A cells (treated with bortezomib and cisplatin) and MCF7/MDA-MB-436 cells treated with cisplatin, we show similar GR values as Hafner and colleagues. Drug treatment time differs frequently between studies, ranging from minutes to days. Here, we conducted drug sensitivity screening for 24, 48, 72 hours and observed similar IC50 values between different cell lines after 48 hours despite differences in population doubling time. In addition, medium and drug replacement every 24 hours had no effect on cell viability, indicating that treatment was most crucial during the first 24 hours. In the case of bortezomib, proteasome activity was inhibited after a 2 hour treatment time and G2/M phase arrest was induced within 24 hours. Consequently, the half-life of the tested drugs ranges from 25 minutes to about 20–30 hours in patients (depending on the drug type)44.To evaluate the reproducibility of our results, we compared our data for four breast cancer cell lines  treated with two anti-cancer drugs (bortezomib and cisplatin) with data from pharmacoDB and Hafner e.g. 2D monolayers, 3D spheroids or in vivo models) used to evaluate drug sensitivity45. We identified new critical experimental parameters (e.g. matched solvent concentration controls and drug storage) that need to be optimized to develop high precision, robust and reproducible cell viability assays. IC50 is commonly used by researchers to determine the potency of a drug on a certain cell line. To be able to compare drug potency estimates between different studies, we recommend using both IC50 and GR50. Human error or instrument inconsistencies can result in variations in data measurements that need to be overcome to identify biologically active hits (potent drugs)28. However, using QCM metrics increases the chance to identify hits within experiments. Ultimately, careful consideration to assay optimization and estimation of drug potency during the preclinical drug screening process may help to improve the success rates of cancer drug candidates that reach clinical trials.In summary, optimization is crucial for data reliability regardless of the model type (2 environment in RPMI 1640 (HCC38) supplemented with 2 mM L-glutamine, 2 g/L D-glucose, and 10% fetal bovine serum ; RPMI 1640 (MCF-10A) supplemented with 2 mM L-glutamine, 2 g/L D-glucose, hydrocortisone, epidermal growth factor, cholera toxin, insulin, and 10% fetal bovine serum  or Dulbecco Modified Eagle’s Medium  supplemented with 2 mM L-glutamine, 4 g/L D-glucose, and 10% FBS (Gibco ThermoFisher). Cell growth rates were determined by non-linear regression curve analysis. Authentication of each cell line was performed using the ATCC short tandem repeat (STR) profiling service.Human breast cancer cell lines  were purchased from the American Type Culture Collection  and cultured at 37 °C in a humidified 5% CO3 cells per well in 100 µl culture medium ).Stock solutions for platinum-based agents (10 mM cisplatin and carboplatin) and a proteasome inhibitor (1 mM bortezomib) were prepared using DMSO , further diluted in 1xPBS to the appropriate concentration, and plated in 96-well PCR plates . The pharmaceutical compounds were screened at nine concentrations  using a 2-fold dilution series with matched DMSO concentration vehicle controls. The pharmaceutical compounds were at room temperature (18-25 °C) when added to cells. Proteasome activity was assessed using the Proteasome-Glo Chymotrypsin-like assay (Promega) with bortezomib-treated cells seeded in 96-well clear, flat-bottom microplates (Corning Life Sciences) at a density of 7.5 × 103 cells per well in 100 µl culture medium supplemented with 10% FBS and cultured for 24 hours. Cell viability was assessed after drug treatment for 24, 48 or 72 hours using 0.2 mg/ml resazurin solutions prepared from resazurin sodium salt (Fisher Scientific) dissolved in sterile 1xPBS (ThermoFisher Scientific). In brief, resazurin was at room temperature before adding to the cells in each well. The cells were incubated with 10 µl resazurin solution (10% of cell culture volume) for four hours at 37 °C. The absorbance was measured with a 560 nm excitation filter and a 615 nm emission filter in a Wallac 1420 VICTOR2 microplate reader (Perkin Elmer). For five days, the metabolic activity of each cell line (untreated cells) was measured daily to monitor potential resorufin (fluorescent pink) conversion to dihydroresorufin (non-fluorescent and colorless). Optimization of the resazurin assay parameters is detailed in the Cells were plated in 96-well clear, flat-bottom microplates (Corning Life Sciences), at a density of 7.5 × 1046 in R/Bioconductor (version 3.3.2).Percentage cell viability was calculated as 100% × (absorbance of treated cells – absorbance of background controls) / (absorbance of matched DMSO concentration controls – absorbance of background controls). Normalized growth rate inhibition (GR) was assessed using the absorbance of untreated cells at the time of treatment . The half-maximal inhibitory concentration (IC50), drug potency (GR50), and drug efficiency (GRmax) were determined for each compound using the GRmetrics (version 1.0.0) package3 cells per well in 100 µl culture medium supplemented with 10% FBS and cultured for five days. The resazurin assay was performed daily to determine the cell population doubling time (Td) during the exponential growth phase. The doubling time was calculated in hours using linear regression analysis.HCC38 and MCF7 cells were plated in 96-well flat-bottom plates (Corning Life Science) at a density of 7.5 × 10HCC38 cells were cultured in 10-cm dishes with either different drug concentrations or DMSO (vehicle control) for 24 hours. Cell cycle distribution analyses were performed using harvested cells, fixed with 70% ethanol and stained with propidium iodide/RNase staining solution . Apoptosis was assessed using the sub-G1 cell cycle fraction. Data analysis for cell cycle distribution was performed using the FACScalibur system (BD Biosciences) and Flowing software (version 2.5.1).e.g. cell line, drug, drug dose) in the resazurin-based cell viability assay, analysis of variance (ANOVA), Z-factor (Z), signal window (SW), and coefficient of variation (CV) were calculated as described elsewhere47, with cutoffs set at Z > 0.4, SW > 2, and CV < 20%. In brief, one-way ANOVA (base stats package in R/Bioconductor version 3.3.2) was performed using cell viability as the dependent variable and all other variables  as covariates. The ANOVA percentage of variation explained (η²) was calculated by dividing the sum of squares (Sum Sq) between groups by the Total Sum Sq  was used. The Shapiro-Wilk normality test was used in R to determine whether the data was normally distributed, where the parametric T-test was used if P > 0.05  or the non-parametric Wilcoxon test was used if P < 0.05 . Bar plots were constructed using the ggpubr (version 0.2.1.999)48 and rstatix (version 0.1.1.999)49 R packages to compare different groups with T-test or Wilcoxon test and Benjamini-Hochberg adjusted p-values, as appropriate.A variance component analysis was used to determine the influence of experimental factors (Supplementary Information.Supplementary Table S2


Cognitive decline is among the most feared aspects of ageing. We have generated induced pluripotent stem cells (iPSCs) from 24 people from the Lothian Birth Cohort 1936, whose cognitive ability was tested in childhood and in older age. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating oriP/EBNA1 backbone plasmids expressing six iPSC reprogramming factors (OCT3/4 (POU5F1), SOX2, KLF4, L-Myc, shp53, Lin28, SV40LT). All lines demonstrated STR matched karyotype and pluripotency was validated by multiple methods. These iPSC lines are a valuable resource to study molecular mechanisms underlying individual differences in cognitive ageing and resilience to age-related neurodegenerative diseases. Human iPSC lines from the Lothian Birth Cohort 1936 comprise individuals with rich life-course cognitive performance data , affordi1.2Human peripheral blood mononuclear cells (PBMCs) were obtained from 24 unrelated members of the Lothian Birth Cohort 1936. Demographic parameters are 50% female (n = 12), 100% white Scottish . Line doPBMCs were reprogrammed to generate induced pluripotent stem cells (iPSCs) using episomal plasmids encoding human OCT3/4 (POU5F1), SOX2, KLF4, L-Myc, shp53, Lin28, SV40LT. All lines were reprogrammed and stored within 22 months of each other. EBNA-related gene analysis demonstrated that iPSCs were EBNA transgene-free (and therefore exogenous reprogramming factors were no longer present) by passage 17–21 (depending on line). Qualitative tests for parental cell type by TCR-αβ and TCR-γδ T-cell clonality assay revealed that 83% (n = 20) of lines were non-T cell-derived, 17% (n = 4) were T-cell derived. T-cell derived lines are: EDi021-A, EDi025-A, EDi026-A, and EDi035-A. All lines have been confirmed mycoplasma negative .TM pluripotency result, the PluriTest novelty score was borderline (1.688) (All lines demonstrated stem cell-like morphology  and expr (1.688) . Further (1.688) . At 14 d (1.688) , EDi035- (1.688) , and EDi (1.688) , as asseAll lines showed a normal karyotype  between All lines were confirmed to be of human origin and iPSCs matched the profile of parent PBMCs by Short Tandem Repeat (STR) analysis. Parent line data was not available for EDi026-A and EDi028-A. Genetic profiles for these lines were compared to the cell line genetic profiles available in the DSMZ STR database and did not match any other reported profiles in the DSMZ database. These profiles were found to be unique and did not match to any previously submitted profiles from the iPSC Core. The genetic profiles established here can be used for future comparisons for these cell lines. Whole genome sequence data for all 24 lines has been deposited at the European Genome-phenome Archive (EGA), which is hosted by the EBI and the CRG, under accession number EGAS00001003819.An overview of iPSC line characterisation can be found in 22.1Blood samples were collected with NHS Lothian Research Ethics Committee Approval (10/S1103/10). Blood samples were collected in Sodium Citrate BD Vacutainer CPT tubes  (three tubes per participant). For samples EDi021-A, EDi025-A, EDi028-A, EDi030-A, EDi031-A, EDi032-A, EDi033-A, EDi034-A, and EDi035-A PBMC isolation was performed by Roslin Cells. For all other lines, PBMC isolation was performed by the Edinburgh Clinical Research Facility (ECRF).2.2Generation of human iPSCs lines from PBMCs was performed using nucleofection of episomal plasmids containing POU5F1, SOX2, KLF4, LIN28, L-MYC, TP53shRNA, and SV40LT.6 cells per nucleofection of PBMCs were nucleofected with the Amaxa Human T-cell Nucleofector® Kit  and a 5p plasmid mixture using program V-024 on a Amaxa Nucleofector 2D Device . Each transfection contained the following seven factors: OCT4, SOX2, KLF4, LMYC, LIN28, SV40LT and p53 shRNA. These were delivered on the following plasmids from Addgene, together with an EBNA1 plasmid for episomal plasmid maintenance: pEP4 E02S ET2K (Cat. 20927), pCXLE-hOCT3/4-shp53-F (Cat. 27077), pCXLEhUL (Cat. 27080), pCXLE-hSK (Cat. 27078), and pCXWB-EBNA1 (Cat. 37624). Each transfection used 0.5 µg of plasmid pCXWB-EBNA1 and 0.83 µg of each of the remaining four plasmids. After nucleofection, cells were immediately plated in either αβ T-cell medium  or non T-cell medium  onto mitomycin treated mouse embryonic fibroblasts (MEF) and placed in a 37 °C incubator with 20% O2 and 5% CO2.Briefly, ~5 × 102, and 5% CO2 throughout the reprogramming process.Two days after nucleofection, 2 mL/well of Primate ESC medium  containing 5 ng/ml bFGF (for MEF condition) was added to the wells without aspirating the previous medium. Beginning on day four, the medium was gently aspirated from each well and 2 mL of the appropriate fresh reprogramming media was added to each well. Medium was replaced every other day. At approximately day 18 post nucleofection, individual colonies were observed in all wells of each condition. Individual PBMC-iPSC colonies with ES/iPSC-like morphology appeared between day 25–32 and those with best morphology were mechanically isolated, transferred onto 12-well plates with fresh Matrigel™ Matrix , and maintained in mTeSR®1 medium . The iPSC clones were further expanded and scaled up for further analysis. All cultures were maintained at 37 °C, 20% O2.32, 5% CO2 incubator. Briefly, 70–90% confluent human iPSC colonies were passaged every 7 days chemically  or mechanically by StemPro® EZPassage™ Disposable Stem Cell Passaging Tool  and re-plated at a 1:6 or 1:9 ratio depending on the cell line. The iPSCs were passaged every 5–7 days. The iPSCs were expanded for 6–22 passages during which period various characterization assays were performed. The iPSCs were cryopreserved using CryoStor CS10  and an isopropanol freezing vessel at −80 °C overnight. The cryopreserved vials were subsequently stored in liquid nitrogen tanks for long-term storage. Working Cell Banks (WCB) of iPSCs were cryopreserved at passage 9–14 and then Distribution Cells Banks (DCB) were created between passages 18–22.Human iPSCs were cultured in mTeSR®1 medium  on growth factor-reduced Matrigel™ Matrix  -coated plates at 37 °C in a 20% O2.4The absence of mycoplasma contamination in the iPSC lines were confirmed monthly using the MycoAlert Detection Kit, a selective biochemical test .2.5250 ng of genomic DNA was extracted using the KingFisherTM DUO Prime purification system (Thermo Fisher Scientific) and the MagMAXTM DNA Multi-Sample Ultra 2.0 Kit . An embryonic stem cell line (H9) was included alongside LBC lines a negative control. DNA Amplification was conducted using TaKaRa Ex Taq® DNA Polymerase  and a Bio Rad 1000 Touch Thermal Cycler. Primers that recognize EBNA1 along with housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was used as a housekeeping gene, were included in this study . PCR was2.6TCRB and TCRG T-Cell Clonality testing was conducted using Gene Rearrangement and Translocation assays from Invivoscribe Technologies, Inc. Genomic DNA was harvested from all iPSC lines using the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit (Cat. A36570) from Applied Biosystems and it was re-suspended to a final concentration of 100–400 μg per ml in dilution buffer. Three Clonal Control DNA and one Polyclonal Control DNA provided with the kit were used. PCR was carried out as per the manufacturer’s protocol. PCR products were analysed using 6% TBE gel electrophoresis with gel red staining.2.7Human PBMC-iPSCs were incubated in Colcemid  for 30 min at 37 °C and then dissociated using TrypLE for 5 min. They were then washed in phosphate buffered saline (PBS) and incubated at 37 °C in 5 mL hypotonic solution  for 30 min. The cells were centrifuged for 2.5 min at 1500RPM and re-suspended in fixative  at room temperature for 5 min. This was repeated twice, and finally cells were re-suspended in 500 μl of fixative solution and submitted to the Cedars-Sinai Clinical Cytogenetics Core for G-band karyotyping. Karyotyping of each iPSC line was conducted at early and late passage, between passages 6–22. Approximately 20 metaphase spreads were counted per line.2.8iPSCs were plated on Matrigel™  -coated glass coverslips or optical-bottom 96-well plates  and subsequently fixed in 4% paraformaldehyde ). The blocking buffer used was 5% goat serum  and 5% donkey serum  with 0.15% Triton X-100 in PBS, except for SSEA4 and OCT4 staining, for which 5% goat serum with 0.15% Triton X-100 in PBS was used as the block. All cells were blocked for one hour at RT, then incubated with primary antibodies  for eith2.9Alkaline phosphatase staining was performed using the Alkaline Phosphatase Staining Kit II  according to the manufacturer's instructions.2.10PluriTest was used to assess the pluripotency of undifferentiated iPSCs . Cell pe2.11Applied Biosystems TaqMan®hPSC Scorecard™ Panel (Thermo Fisher Scientific) was used as an additional technique to assess pluripotency and tri-lineage differentiation potential of iPSC lines using real-time qPCR assays . Total R2.12IPSC lines were allowed to differentiate by EB formation. Briefly, iPSCs were lifted from 3 wells of a 6 well plate using a cell scraper and seeded in a T25 flask treated with poly-HEMA to prevent cell attachment in EB media containing: IMDM basal media (Cat. 12440061), 17% KnockOut Serum Replacement , 1% non-essential amino acids (Cat. 11140050), 1% Antibiotic-Antimycotic (Cat. 15240062) and 110 µM β-Mercaptoethanol (Cat. 21985023), all from Thermo Fisher. EBs were allowed to form by self-aggregation, grow and differentiate for 14 days in EB culture media replacing it twice a week. Differentiation to endoderm, mesoderm and ectoderm was assessed by TaqMan® hPSC Scorecard™ Assay .3Short Tandem Repeat (STR) Analysis is conducted to confirm iPSC genetic identity. For that, a frozen vial of the parent PBMCs and a frozen vial of the reprogramed iPSC line at late passage  are sent to IDEXX BioResearch. STR profile and interspecies contamination testing is analysed. iPSC line human authentication was conducted at IDEXX BioResearch by Cell Check™. Profiling included using a nine marker STR profile  and interspecies contamination check for human, mouse, rat, African green monkey and Chinese hamster cells. Comparative analysis was conducted between parent PBMCs and reprogrammed iPSC lines.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: US patent US 10,221,395 B2 has been granted describing some of the methods to reprogram to iPSCs. Apart from this issued patent filing the authors have declared that no other competing financial interests exist.


Apoptosis was assessed via the Caspase-Glo 3/7 assay system. Western blotting was used to assess changes in expression of downstream effectors. TNBC patient-derived xenograft (PDX) models were treated with AZD1775, capecitabine, or the combination and assessed for tumor growth inhibition. From the initial PDX screen, two of the four TNBC PDX models demonstrated a better response in the combination treatment than either of the single agents. As confirmation, two PDX models were expanded for statistical comparison. Both PDX models demonstrated a significant growth inhibition in the combination versus either of the single agents.  An enhanced anti-proliferative effect was observed in the adavosertib/5FU combination treatment as measured by live cell analysis. An increase in apoptosis was observed in two of the four cell lines in the combination when compared to single-agent treatment. Treatment with adavosertib as a single agent resulted in a decrease in p-CDC2 in a dose-dependent manner that was also observed in the combination treatment. An increase in γH2AX in two of the four cell lines tested was also observed. No significant changes were observed in Bcl-xL following treatment in any of the cell lines. The combination of adavosertib and capecitabine/5FU demonstrated enhanced combination effects both in vitro and in vivo in preclinical models of TNBC. These results support the clinical investigation of this combination in patients with TNBC, including those with brain metastasis given the CNS penetration of both agents.Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. Adavosertib (AZD1775) is a potent, small-molecule, ATP-competitive inhibitor of the Wee1 kinase that potentiates the activity of many DNA-damaging chemotherapeutics and is currently in clinical development for multiple indications. The purpose of this study was to investigate the combination of AZD1775 and capecitabine/5FU in preclinical TNBC models. TNBC cell lines were treated with AZD1775 and 5FU and cellular proliferation was assessed in real-time using IncuCyte Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by a lack of hormone receptor expression and non-amplified HER2 . TNBC acKd 3.2 and 3.9 nM, respectively) [2 cell cycle checkpoint [TP53-deficient cells with impaired G1 checkpoint function can be sensitized to DNA-damaging chemotherapeutics, with the addition of AZD1775 leading to abrogation of the G2 checkpoint [TP53-deficiency, possibly due to baseline replicative stress or compromised DNA repair proficiency [TP53-deficient colon cancer cell lines and the 5FU prodrug capecitabine in xenograft models [2 DNA damage checkpoint induced by 5FU, causing premature entry of mitosis. Adavosertib AZD1775) is a potent, small-molecule, ATP-competitive inhibitor of the Wee1 and 2 kinases (ctively)  that pot is a poteckpoint ,7. In reeckpoint . TP53-deeckpoint . AZD1775ficiency . AZD1775Capecitabine is an oral antimetabolic fluoropyrimidine deoxynucleoside carbamate drug that is converted to 5-fluorouracil in patients by thymidine phosphorylase, which then concentrates in the tumor tissue . CapecitThe purpose of this study was to evaluate rational combination partners for AZD1775 in preclinical models of TNBC using FDA-approved chemotherapies  and other targeted agents available through the National Cancer Institute’s Cancer Therapy Evaluation Program (CTEP) . Following the initial screen evaluating these combinations in TNBC PDX models, larger efficacy and mechanistic studies were performed for the combination of 5-FU/capecitabine and AZD1775.In an effort to minimize the number of animals required to evaluate multiple combinations with AZD1775, we performed an initial screen using four TNBC PDX models treated with AZD1775, capecitabine, paclitaxel, gemcitabine, doxorubicin, navitoclax (BCL-2 inhibitor), VX970 (ATR inhibitor), and romidepsin (HDAC inhibitor) or combinations of these agents with AZD1775. Each group consisted of three mice with two tumors each. As demonstrated in The combination of AZD1775 and capecitabine was identified for further evaluation based on an enhanced combination effect observed in two models  and the fact that both drugs cross the blood–brain barrier. Of note, in TNBC013, capecitabine as a single agent resulted in a very high TGI of approximately 75%, which may have limited the detection of a combination effect. To confirm potentiation of the activity of AZD1775 with the addition of capecitabine in TNBC, we performed further in vivo studies using 2 TNBC PDX models (TNBC012 and TNBC013) with 5 animals (10 tumors) in each group. These models were selected for confirmation based on known p53 mutations and these tumors were isolated from brain metastasis in patients, which is relevant in TNBC given the high incidence of brain metastasis and CNS penetration of both agents. The dose of capecitabine was lowered in these experiments for TNBC013 based on the TGI observed with a higher dose in Following confirmation of combination activity in vivo, we performed in vitro experiments to further characterize the anti-proliferative activity of the combination using live cell imaging and the sulforhodamine B (SRB) assay. We selected 5FU for use in vitro based on the required activation steps for the conversion of capecitabine to 5FU in vivo. We observed a statistically significant decrease in proliferation with the combination as compared to either AZD1775 and 5FU alone as assessed by live cell imaging in two of the four TNBC cell lines (MDA-MB-231 and CAL-51) A,D. In cTo determine the effect of AZD1775 and 5FU on apoptosis, the Caspase 3/7 assay was utilized. At 24 h, we observed no significant apoptosis in the MDA-MB-231 or HCC1937 cell lines with single agent or combination treatment A,C. Howep < 0.01). Additionally, there was also a statistically significant increase in S-phase arrest in the combination, when compared to AZD1775 as a single agent (p < 0.05)   C. Similactively) D.Immunoblotting was performed to elucidate the mechanism of the combination effects of AZD1775 with 5FU in the four TNBC cell lines. Following 48 h exposure to the drugs, two of the four cell lines (MDA-MB-468 and HCC1937) demonstrated a decrease in p-CDC2 in a dose-dependent manner with AZD1775 as a single agent, and the decrease was only maintained in the combinations for the HCC1937 cell line. An increase in in γ-H2AX was observed in the AZD1775 single agent at both dose levels in the MDA-MB-231 and the HCC1937 and was enhanced in the 125 nM combo for the HCC1937. In the MDA-MB-48 and CAL51, no increase in γ-H2AX was observed in either AZD1775 as a single agent. However, there was an enhanced increase in the 250 nM combination. A decrease in Bcl-xL was observed with the 5FU and combination treatment in the MDA-MB-468 and CAL-51 cell lines, indicating an increase in apoptosis .Approximately 15% of breast cancers in the United States are classified as TNBC and, despite recent advances in local and systemic therapies, patients with TNBC continue to be at increased risk of metastatic recurrence and have inferior outcomes compared to other breast cancer subtypes . TherefoOur study demonstrates that the addition of AZD1775 to capecitabine enhanced the anti-tumor effects in additional TNBC PDX models and TNBC cell lines. We observed an increase in apoptosis in several cell lines with the addition of AZD1775 to either agent. AZD1775 treatment led to a decrease in p-CDC2 and an increase γ-H2AX that was either maintained or enhanced in the combination with capecitabine demonstrating a DNA damage response. This is similar to what was observed in pancreatic cancer cell lines where gemcitabine, when combined with AZD1775 increased the amount of γ-H2AX staining by flow cytometry . In factIt is thought that the addition of AZD1775 to DNA-damaging agents like chemotherapies may be a viable treatment strategy for various tumor types. Hirai et al. demonstrated that combining AZD1775 with gemcitabine, 5FU, capecitabine, and irinotecan enhanced the effects of each of these chemotherapies in colon and breast cancer models . SimilarBRCA1 mutations and no difference in response between patients with p53 mutations and wild-type p53 was observed. In this trial, post treatment tumor biopsies demonstrated a decrease in p-CDC2 and an increase in γ-H2AX which is consistent with our observations preclinically [Several clinical trials have been conducted or are currently ongoing evaluating AZD1775 as a single agent and in combination with other cancer therapies. In a phase I trial of AZD1775 in patients with refractory solid tumors, the compound was tolerable with myelosuppression as the main dose-limiting toxicity. Partial responses were observed in two patients with inically . In a phinically . Stable TP53 mutations and brain metastasis [Further adding to the promise of AZD1775 as a cancer therapeutic is documented CNS penetration, which is particularly interesting in TNBC with its high incidence of tastasis ,26. CNS tastasis . Penetratastasis . Capecittastasis ,28,29.3, and were randomized into treatment groups as previously described [n = 3/group) were treated with AZD1775 alone or in combination with capecitabine, paclitaxel, gemcitabine, doxorubicin, navitoclax, VX970, and romidepsin. In the expanded cohorts, mice (n = 10/group) were treated with AZD1775 , or capecitabine , as single agents or in combination as described previously [2) × 0.52. Percent tumor growth inhibition (TGI) values were calculated using the following formula: ((MTVvehicle − MTVtreated)/MTVvehicle) × 100.Patient-derived tumors were acquired from the University of Colorado Hospital  following consent and in accordance with protocols approved by the Colorado Multiple Institutional Review Board. Female athymic nude mice (4–8 weeks of age) were purchased from Envigo . All animal studies were approved by the Institutional Animal Care and Use Committee (00021). Tumor specimens were implanted into the hind flanks of mice and maintained for several generations as described previously . For treescribed ,32,33. Feviously ,35. MiceAZD1775 was provided by AstraZeneca or purchased from MolPool . Capecitabine was purchased from Active Biochem . The 5-fluorouracil was purchased from the University of Colorado Hospital Pharmacy . HCC1937, CAL-51, MDA-MB-231, and MDA-MB-468 were purchased from ATCC and maintained in DMEM (Corning) containing 10% FBS  with 1% Pen/Strep and 1% non-essential amino acids . Cells were routinely tested for mycoplasma and were authenticated at the Barbara Davis Center for Diabetes Molecular Biology Service Center . The anti-proliferative effects of AZD1775 in combination with 5FU were evaluated on HCC1937, CAL-51, MDA-MB-231, and MDA-MB-468 using the IncuCyte Zoom live cell imager . All cell lines were plated in 96-well plates at optimal density and allowed to adhere for 24 h. After 24 h, the cells were treated with AZD1775 (125 or 250 nM) and 5-fluorouracil (5FU) (7.5 µM), as single agents or in combination. Doses of each compound were based on previously published data ,37. PlatThe effect of AZD1775 in combination with 5FU on cell cycle was evaluated in HCC1937, CAL-51, MDA-MB-231, and MDA-MB-468 cell lines. All cell lines were plated in 6-well plates at optimal density and allowed to adhere for 24 h. After 24 h, the cells were treated with AZD1775 (125 or 250 nM) and 5FU (7.5 µM), as single agents or in combination for an additional 24 h. Cells were then trypsinized, washed with PBS and resuspended in Krishan’s stain and stored at 4 °C overnight. Cell cycle distribution was analyzed by flow cytometry in the University of Colorado Cancer Center Flow Cytometry Core . g at 4 °C. The supernatant was removed and transferred to a new tube and protein concentration was determined using a Pierce BCA Protein Assay Kit . A total of 40 µg of protein was electrophoresed on a precast 4–12% BisTris gel . Proteins were then transferred onto a nitrocellulose membrane using Invitrogen Power Blotter (Thermo Fisher). Membranes were blocked for 1 h at room temperature using 3% casein. After blocking, the membranes were probed with the following primary antibodies (1:1000) overnight at 4 °C with rocking: CDC2, p-CDC2, H2AX, Bcl-xL, and Actin. Following washing three times with TBST, the membranes were incubated for 1 h at room temperature with anti-rabbit or -mouse IgG  conjugated sary antibody at a dilution of 1:15,000. Images were captured using the Odyssey Infrared Imaging System .  or 5-fluorouracil (5FU) (7.5µM), as single agents or in combination for 24 h. Following treatment, lysis buffer containing protease and phosphatase inhibitors were added to the wells and the cells were scraped on ice. The lysed proteins were added to a microcentrifuge tube and sonicated for 30 s. Following sonication, the lysates were centrifuged for 10 minutes at 16,000× t-test with Welch’s correction GraphPad Prism 8.0, .We compared combination treatment groups to each of the single agents and performed an unpaired The combination of AZD1775 with capecitabine leads to enhanced anti-proliferative effects in vitro and in vivo. Enhanced apoptosis and cell cycle arrest was also observed. Additionally, an increase in the DNA damage response was observed by immunoblotting in the AZD1775 single agent and this was maintained in the combination. This work supports the future investigation of the combination of AZD1775 and capecitabine in patients with metastatic TNBC.


In addition to 2D migration and clonogenic assays, the combination was studied in 3D spheroids and a syngeneic mouse model of HCC. Results: Vandetanib ICin vitro. In vivo, combination therapy significantly reduced cancer growth and improved overall survival, an effect that persisted for the duration of vandetanib treatment. Conclusion: In 2D and 3D studies in vitro and in a syngeneic model in vivo, the combination of vandetanib plus radiotherapy was more efficacious than either treatment alone. This new combination therapy for HCC merits evaluation in clinical trials.Hepatocellular Carcinoma (HCC) is increasing in incidence worldwide and requires new approaches to therapy. The combination of anti-angiogenic drug therapy and radiotherapy is one promising new approach. The anti-angiogenic drug vandetanib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and RET proto-oncogene with radio-enhancement potential. To explore the benefit of combined vandetanib and radiotherapy treatment for HCC, we studied outcomes following combined treatment in pre-clinical models. Methods: Vandetanib and radiation treatment were combined in HCC cell lines grown  The risk of liver cancer has been steadily increasing since the end of the 20th century , now accRadiosensitisers can act via intrinsic methods by directly impairing cell function and ability to survive radiation-induced damage, as is the case with platinum-based DNA chelators ,9,10,11 HCC development is often characterised by expansion of the vascular system, resulting in aberrant tumour vasculature and an immunosuppressive environment ,17,18. Tin vitro with 2D and 3D models and in immunocompetent mouse models.To evaluate vandetanib as a radiosensitiser in HCC, the effect of vandetanib-radiation combination therapy on HCC and cancer-related processes such as migration, invasion, growth and viability was studied in vitro can be narrowed down to intrinsic TKI-mediated disruption of VEGFR-2 activation of the RAS/RAF/MEK/ERK pathway [In the normal tissue micro-environment, expression of VEGFR-2 is thought to be limited mainly to the endothelium and absent in the surrounding epithelial tissue. It is not clear how this expression changes following cellular differentiation into cancer and the possibility exists for cancer cells with VEGFR-2 expression. The expression of VEGFR-2 in human and murine cell lines was measured with western blotting to assess functional VEGFR-2 signalling and susceptibility to vandetanib and similar TKIs, both of which have implications for cancer growth and vandetanib treatment . VEGFR-2 pathway  localise pathway  inside tin vitro, a series of experiments was performed on a range of liver cancer cell lines. Cells were treated with serial dilutions of vandetanib and survival was quantified to determine the Inhibitory Concentration at 50%-maximal  over controls for both drug treated groups.The clonogenic radiosensitivity assay was performed to characterise the adiation  and expein vitro [p ≤ 0.05) and the combined treatment was found to produce an additive reduction in migration (p≤ 0.05), suggesting vandetanib inhibition of migration proceeds by alternate mechanisms to those of radiation-induced inhibition.Vandetanib treatment is known to reduce migration of HCC cell lines in vitro  and we pp≤ 0.05) or 5 p ≤ 0.001). Similarly, when the human hepatoma cell line PLC-PRF-5 was grown in 3-D, significant growth reduction was measured in spheroids treated with combined 5 p ≤ 0.001) and 5 p ≤ 0.001). To study the relationship between the tumour micro-environment and radiosensitivity, spheroid studies were performed with cancer cell lines grown in co-culture with fibroblast or hepatic stellate cell types. In 3D spheroid co-culture of HLF and NIH3T3 fibroblasts receiving combination treatment, invasion was significantly reduced over radiation (p ≤ 0.05) and vandetanib (p ≤ 0.0001) and likewise in 3D spheroid co-culture of JHH7 cancer cells and LX2 hepatic stellate cells, spheroid growth was inhibited in combination treatment compared to 5 p≤ 0.05) and 4 Gy radiation (p ≤ 0.005). JHH7 spheroids co-cultured with NIH3T3 and treated with combined vandetanib and radiation showed significant growth reduction compared to either single treatment (p ≤ 0.01 vs. 5 p ≤ 0.01). Similar results (p≤ 0.05 vs. control) and Hepa1-6 (p≤ 0.05 vs. 4 Gy radiation and control) when grown in spheroids. The murine cell line BNL1ME A.7R.1 was tested to eliminate the possibility of oversensitivity to either treatment as BNL.1ME was also cultured for tumour engraftment in the animal model  size reduction compared to control. When treated with either vandetanib or radiation alone, the tumours remained stationary for seven days, at which point the growths recurred. In combination therapy, the tumours initially shrunk in size compared to the starting measurements and growth remained inhibited longer than the single treatment groups. Tumour growth reduction was significant versus control (p ≤ 0.0001), and regression in size was seen until day 10, concurrent with the end of vandetanib treatment, when the tumours began to exhibit signs of growth recurrence.Past studies have characterised the effect of vandetanib in animal models and shown radiation enhancement ,28,29,30In vitro radiosensitivity assays suggest a prolonged exposure is required for an intrinsic radiosensitisation effect, as evidenced by the results from the clonogenic assay  sub-additive [in vitro results, particularly the clonogenic assay which also found the duration of vandetanib exposure to be an important factor for achieving radiosensitisation, it would appear the radiosensitisation occurs at a stage after the initial binding and inhibition of the receptor. The dynamics of in vivo treatment with vandetanib have been shown to alter the physical properties of the tumour microenvironment, by increasing oxygenation immediately following vandetanib treatment [in vivo.In immunocompetent mouse studies, combination treatment was found to be additive with mean survival for the combination therapy equivalent to the sum total survival reached by both treatments alone C. By tumadditive  effect wadditive ,28,29,30reatment  and the reatment ,48. Intereatment ,50,51 dureatment ,24 vascureatment . Targetiin vitro results vandetanib significantly enhances the effect of radiation on cell viability as seen with the clonogenic assay, migration in the scratch assay, and growth and invasion in the spheroid study. The effects of combined treatment, on not only these various components of the tumour machinery but on the ability of the tumour to develop, are most visible in the animal model where a reduction in size persisted for the duration of treatment and additive effects to overall survival were found. The sum total of these results indicates vandetanib enhances HCC treatment when administered concomitantly with radiation and suggests a potentially significant benefit could be derived from the development of combined vandetanib-radiation therapy for the treatment of HCC patients.It is apparent from the The human cell lines used in this study are shown below in Cell lines were grown under standard cell culture conditions Plasmids containing a pcDNA3-EGFP (Doug Golenbock: Addgene plasmid # 13031) or pQCXI Neo DsRed-LC3-GFP (David Sabatini: Addgene plasmid # 31183 ) retroviin vitro sample irradiation. X-ray emissions at effective energies of 65.2 keV [An XStrahl  Small Animal Radiotherapy Research Platform (SARRP), S/N 525722, was used for 65.2 keV  were del65.2 keV  indicatiDosimetry for the SARRP was measured using a PTW 30012 Farmer ionisation chamber  and a UNIDOS E electrometer  corrected and calibrated to the National Physical Laboratory  primary standard. Reference conditions: Open field; source to surface distance (SSD), 33 cm; phantom thickness, 5.5 cm; detector’s geometric centre at 2 cm depth, 35 cm; and half-value layer, 0.658 mm Cu.HCC cells were seeded in DMEM and serum starved for 24 h prior to harvesting. HUVEC cell lines were serum starved and treated with vandetanib for 24 h, then dosed with 50 ng/mL VEGF for 20 min prior to fixation. Cell culture samples were washed with PBS and frozen at −80 C before being lysed on ice with RIPA Lysis buffer. Protein concentration was measured by Bradford assay against BSA standard, and 30 MD, USA)  Optical TM media containing 10 Cell line ICHCC cells from tissue culture cell lines were trypsinised and counted, and 2000 cells at a concentration of 250 cells/mL were plated on 10 cm dishes (Corning) for 24 h before addition of vandetanib in 0.1% DMSO and/or treatment with irradiation. Drug exposure was maintained for either 24 or 72 h, depending on the experiment, before media was changed to remove the drug from the cellular environment. Drug controls were treated with 0.1% DMSO. Radiation controls received mock irradiation to maintain equivalent cell culture conditions. After ten days, the cells were fixed in methanol and stained with 0.4% Methylene Blue in Methanol or 330 HCC cells were plated to confluence on a 96-well plate and a 2 mm section of confluency was cleared with an Incucyte Woundmaker  across the centre of the monolayer. Cells were treated with vandetanib in 0.1% DMSO or vehicle (0.1% DMSO) and/or irradiation. Migration into the clearance was imaged at regular 4 h intervals with a Nikon Biostation  and quantified with ImageJ software . Signifig for 5 min and allowed to form aggregates under normal cell culture conditions for a period of three days. Following aggregation, the cells were placed on ice, the media was removed and replaced with 100 Spheroids were formed according to a standard spheroid protocol . HCC celsoftware . Signifisoftware  . Animal experiments were conducted with approval from UK Home Office (licence 70/8861) in accordance with the European Commission Directive 2010/63/EU , the UK Home Office (Scientific Procedures) Act (1986), and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with project approval from UCL Biological Services. Animals were housed in a temperature-controlled facility with 12 h on–12 h off light cycle and provided water and laboratory rodent food in vivo passages in BALB/C mouse and allowed to reach 60–80% confluency before harvest under sterile conditions and subcutaneous injection of 5 × 10The BNL.1ME mouse cell line was selected from among three hepatic murine cell lines  due to rin vitro. The effect of the combination therapy was most marked in vivo, where a syngeneic mouse model of HCC found combination-treated tumours to reduce in size significantly more than those receiving single treatment, a reduction that persisted for the duration of vandetanib treatment and led to an additive benefit to overall survival when compared to either treatment alone. Collectively, these findings help to characterise vandetanib as a radiosensitiser for HCC and suggest the need for clinical trial exploration of this treatment combination.In 2D and 3D studies of radiosensitisation in multiple HCC cell lines, combined treatment with vandetanib and radiation was more beneficial than either treatment alone, reducing cell viability, migration, and invasion


Despite being one of the leading cancer types in the world, the diagnosis of oral cancer and its suitable therapeutic options remain limited. This study aims to investigate the single and chemosensitizing effects of TW-37, a BH3 mimetic in oral cancer, on human oral cancer cell lines.We assessed the single and chemosensitizing effects of TW-37 in vitro using trypan blue exclusion assay, Western blotting, DAPI staining, Annexin V–FITC/PI double staining, and quantitative real-time PCR. Mcl-1 overexpression models were established by transforming vector and transient transfection was performed to test for apoptosis705 and nuclear translocation in human oral cancer cell lines at the early time points. Furthermore, TW-37 potentiated chemosusceptibility of cryptotanshinone in human oral cancer cell lines by suppressing STAT3–Mcl-1 signaling compared with either TW-37 or cryptotanshinone alone, resulting in potent apoptosis.TW-37 enhanced the cytotoxicity of human oral cancer cell lines by inducing caspase-dependent apoptosis, which correlates with the reduction of the myeloid cell leukemia-1 (Mcl-1) expression via transcriptional and post-translational regulation. The ectopic expression of Mcl-1 partially attenuated the apoptosis-inducing capacity of TW-37 in human oral cancer cell lines. Besides, TW-37 decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at TyrThis study not only unravels the single and chemosensitizing effects of TW-37 for treatment of human oral cancer but also highlights the likelihood of TW-37 as a good therapeutic strategy to enhance the prognosis of patients with oral cancer in the future. Globally, head and neck squamous cell carcinoma (HNSCC) is one of the leading cancer types, of which oral squamous cell carcinoma constitutes > 90% of cases . DespiteIn cancer therapy, targeting anti-apoptotic Bcl-2 family proteins is an attractive approach because of their oncogenic potential. The “BH3 mimetics” were designed to mimic the BH3 domain of pro-apoptotic Bcl-2 proteins and to antagonize anti-apoptotic Bcl-2 family proteins, resulting in apoptosis . Of thesBased on the related literature , this st2 incubator. In addition, TW-37 was purchased from ApexBio , whereas cryptotanshinone and cycloheximide (CHX) were purchased from Sigma-Aldrich . ABT-737 and Stattic were supplied from Selleckchem , Z-VAD-FMK was purchased from R&D Systems , and MG132 was provided from Santa Cruz Biotechnology . All chemicals were dissolved in dimethyl sulfoxide and stored at −20 °C.HSC-3, Ca9.22, and HSC-4 cell lines are obtained from Hokkaido University , and the HN22 cell line was provided by Dankook University . All cell lines were maintained in DMEM/F12 medium  supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin) at 37 °C in a 5% COWe seeded all cell lines in six-well plates and treated those with TW-37. Figure All cell lines were extracted with 1X RIPA lysis buffer  using phosphatase inhibitor tablets  and protease inhibitor cocktails . We then performed protein quantification using a DC protein assay kit . After normalization, protein lysates containing nearly 20–50 μg of protein were boiled with a protein sample buffer at 95 °C for 5 min and separated on SDS–PAGE. Next, the proteins were transferred to Immun-Blot PVDF membranes after the electrophoresis and blocked with 5% skimmed milk for 1 h at room temperature (RT). The membranes were incubated with the indicated primary antibodies overnight at 4 °C and were maintained with corresponding horseradish peroxidase (HRP)–conjugated secondary antibodies for 2 h at RT. The antibodies for all the target proteins have been listed in Table After the TW-37 treatment, all cell lines were fixed overnight with 100% ethanol at −20 °C and 100% methanol at RT for 10 min. The fixed cell lines were then deposited on slides and stained with DAPI solution (5 μg/mL). Furthermore, we observed morphological changes of nuclei in apoptotic cell lines using a fluorescence microscope .We measured the induction of apoptosis using the FITC Annexin V Apoptosis Detection Kit  according to the manufacturer’s protocol. Briefly, all floating and adherent cell lines were collected, washed two times with PBS, and pelleted by centrifugation. After that, cell lines were resuspended in annexin V binding buffer containing 3-μL annexin V–FITC and 1-μL PI and incubated for 15 min at RT in the dark. Subsequently, we transferred cell lines to a FACS tube and analyzed by flow cytometry using a FACSCalibur and counted 10,000 events per sample.Mcl-1 gene was normalized to the amount of GAPDH and was evaluated using the 2–ΔΔCt method. The qPCR primers were as follows: Mcl-1 sense, 5′-GTA TCA CAG ACG TTC TCG TAA GG-3′; Mcl-1 antisense, 5′-CCA CCT TCT AGG TCC TCT ACA T-3′; GAPDH sense, 5′-GTG GTC TCC TCT GAC TTC AAC-3′; and GAPDH antisense, 5′-CCT GTT GCT GTA GCC AAA TTC-3′.Using TRIzol Reagent , the total RNA was extracted. Afterward, 1 μg of RNA was reverse transcribed using an AMPIGENE cDNA Synthesis Kit , and the resultant cDNA was subjected to PCR using AMPIGENE qPCR Green Mix Hi-Rox (Enzo Life Sciences). We performed real-time PCR using the StepOnePlus Real-Time PCR System . In this study, the PCR conditions for all genes were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The relative amount of Mcl-1 (NM_021960) gene from cDNA that was synthesized in HSC-3 cell lines using gene-specific primers , followed by cloning into a pGEM® T-easy vector . The Mcl-1 was then established by sequence analysis. Finally, we cloned the Mcl-1 gene into the multi-cloning site of the pcDNA3.1 (+) vector . For the overexpression vector transfection, we transfected cell lines with vector constructs (pcDNA3.1 or pcDNA3.1-Mcl-1) using Lipofectamine 3000 transfection reagent (Life Technologies) according to the manufacturer’s protocol.We amplified the open reading frame of human t-test (two tailed) using SPSS 22 . In this study, P < 0.05 was considered statistically significant.We compared the control and the treated groups through Student’s We first performed a concentration–response test using the trypan blue exclusion assay to assess the potential cytotoxic effect of TW-37 on human oral cancer cell lines. TW-37 exhibited a concentration-dependent cytotoxic effect on HSC-3 cell lines Fig. b. In addAs TW-37 is a potent inhibitor of anti-apoptotic Bcl-2 family proteins, such as Bcl-2, Bcl-xL, and Mcl-1, we assessed whether TW-37-induced apoptosis in human oral cancer cell lines could regulate these proteins. The findings revealed that the Mcl-1 expression notably decreased after the TW-37 treatment, whereas the Bcl-2 and Bcl-xL expressions were inconsistent  and pcDNA3.1-Mcl-1 (to overexpress Mcl-1) to elucidate the biological role of Mcl-1 protein in TW-37-induced apoptosis. The ectopic expression of Mcl-1 modestly attenuated the expression of cleaved PARP after the TW-37 treatment , is a STAT3 inhibitor that binds to the SH2 domain of STAT3 [To date, numerous STAT3-targeted therapies inhibiting the SH2 domain, upstream tyrosine kinase, and DNA-binding domain have been considered potential therapeutic approaches for clinical applications because targeting STAT3 could be a reasonable strategy for the development of chemotherapeutic drugs against cancer . Cryptotof STAT3 . In addiof STAT3 –27. Repoof STAT3 , 29. AccThis study demonstrates that TW-37 alone and in combination with cryptotanshinone exerts a potent apoptotic effect on human oral cancer cell lines by inhibiting STAT3–Mcl-1 signaling. Overall, this study highlights that targeting STAT3 using TW-37 could be an attractive approach in enhancing the prognosis of patients with oral cancer in the future.Additional file 1: Figure S1. TW-37 induces apoptosis in human oral cancer cell lines by suppressing p-STAT3Tyr705. Ca9.22, HSC-4, and HN22 cell lines were treated with 2.5- and 5-μM TW-37 for 48 h. a The cell viability was measured using the trypan blue exclusion assay. Bar graphs represent the mean ± SD values of three independent experiments. *P < 0.05. b Protein levels of cleaved caspase-3 and cleaved PARP were analyzed using Western blotting. Actin was used as a loading control. c All cell lines stained with DAPI were visualized by fluorescence microscopy . d The expressions of p-STAT3 (Y705) and STAT3 were detected using Western blotting. Actin was used as a loading control. Figure S2. TW-37-induced apoptosis depending on caspase activation in human oral cancer cell lines. HSC-3 cell lines were pretreated with 10-μM Z-VAD for 2 h with/without 5-μM TW-37 for 48 h. a Protein levels of cleaved PARP were analyzed using Western blotting analysis. Actin was used as a loading control. b Bar graphs represent the mean ± SD values of three independent experiments. *P < 0.05; #P < 0.05. Figure S3. TW-37 slightly decreases Mcl-1 mRNA levels at 24 and 48 h. The HSC-3 cell lines were treated with 5-μM of TW-37 for 24 or 48 h. Relative mRNA levels of Mcl-1 were measured with qPCR and normalized to GAPDH. Bar graphs represent the mean ± SD values of triplicate experiments. *P < 0.05. Figure S4. STAT3 inactivation is sufficient to induce apoptosis via the inhibition of Mcl-1 expression. The HSC-3 cell lines were treated with 5-μM of cryptotanshinone or stattic for 24 h. a The cell viability was measured using the trypan blue exclusion assay. b Protein levels of p-STAT3 (Y705), STAT3, Mcl-1, and cleaved PARP were analyzed using Western blotting analysis. Actin was used as a loading control. c Relative mRNA levels of Mcl-1 were measured with qPCR and normalized to GAPDH. d The HSC-3 cell lines were transiently transfected with 0.5-μg pcDNA3.1 or pcDNA3.1-Mcl-1 for 6 h. Protein levels of p-STAT3 (Y705) and STAT3 were determined using Western blotting analysis. Actin was used as a loading control. All bar graphs represent the mean ± SD values of three independent experiments. *P < 0.05. Figure S5. TW-37-induced apoptosis is mediated by the suppression of STAT3-Mcl-1 signaling unlike ABT-737. The HSC-3 cell lines were treated with the indicated concentrations of TW-37 or ABT-737 for 48 h. a Protein levels of p-STAT3 (Y705), STAT3, Mcl-1, and cleaved PARP were analyzed using Western blotting analysis. Actin was used as a loading control. b Bar graphs represent the mean ± SD values of three independent experiments. *P < 0.05.