EGFR, HER1) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). After initial promising results with EGFR‐targeted therapies such as cetuximab, therapeutic resistance has become a major clinical problem, and new treatment options are therefore necessary. Moreover, the relationship between HER receptors, anti‐EGFR therapies, and the human papillomavirus (HPV) status in HNSCC is not fully understood. In contrast to first‐generation EGFR inhibitors, afatinib irreversibly inhibits multiple HER receptors simultaneously. Therefore, treatment with afatinib might result in a more pronounced therapeutic benefit, even in patients experiencing cetuximab resistance. In this study, the cytotoxic effect of afatinib as single agent and in combination with cisplatin was investigated in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines with different HPV status under normoxia and hypoxia. Furthermore, the influence of cetuximab resistance, HPV, and hypoxia on the expression of HER receptors was investigated. Our results demonstrated that afatinib was able to establish cytotoxicity in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines, independent of the HPV status. However, cross‐resistance between cetuximab and afatinib might be possible. Treatment with afatinib caused a G0/G1 cell cycle arrest as well as induction of apoptotic cell death. Additive to antagonistic interactions between afatinib and cisplatin could be observed. Neither cetuximab resistance nor HPV status significantly influenced the expression of HER receptors in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under hypoxia. Oxygen deficiency is a common characteristic of HNSCC tumors, and these hypoxic tumor regions often contain cells that are more resistant to treatment. However, we observed that afatinib maintained its cytotoxic effect under hypoxia. In conclusion, our preclinical data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance.The epidermal growth factor receptor ( AnnVannexin V‐FITCCIcombination indexEGFRepidermal growth factor receptorHIFhypoxia‐inducible factorHNSCChead and neck squamous cell carcinomaHPVhuman papillomavirusmAbmonoclonal antibodyMFImean fluorescence intensityPIpropidium iodideR/Mrecurrent/metastaticSRBsulforhodamine BTKItyrosine kinase inhibitor1et al., Targeted therapies are at the forefront of personalized medicine in cancer treatment. Thanks to our rapidly expanding understanding of the molecular biology of cancer, an increasing number of patients are currently considered as candidates for treatment with molecular targeted pharmaceuticals. Interestingly, the epidermal growth factor receptor  plays an integral role in the tumorigenesis and is richly expressed in a wide range of malignancies, including head and neck squamous cell carcinoma (HNSCC). Increased or sustained activation of the EGFR signaling pathway can convert a normal cell to a malignant cell by providing sustained signals for cell proliferation, anti‐apoptotic signaling, angiogenesis, and metastasis), thus making it a compelling drug target  inhibiting cell proliferation, cell repopulation, DNA repair, and tumor oxygenation; (b) modulating cell cycle perturbation and cell accumulation in radiosensitive phases; and (c) inducing apoptosis and necrosis  or with platinum‐based drugs plus 5‐fluorouracil in the recurrent/metastatic (R/M) disease setting   is a diagnostic marker for a separate entity of HNSCC with enhanced overall and disease‐free survival, but its use as a predictive marker has not been proven yet . Cells were grown as monolayers and maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37 °C. All cell lines were confirmed free of mycoplasma infection through regular testing .Eleven human HNSCC cancer cell lines with different cetuximab sensitivity and HPV status were included in this study. Cal‐27 and UM‐SCC‐104 were obtained from American Type Culture Collection  and Merck Millipore , respectively. SC263 and SQD9 were kindly provided by Sandra Nuyts , and LICR‐HN1 and SCC22b were kindly provided by Olivier De Wever . 93‐VU147‐T was provided by Josephine Dorsman . All cell lines were HPV negative, with the exception of 93‐VU147‐T and UM‐SCC‐104. All cell lines were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% 2.22) was achieved in a Bactron IV anaerobic chamber , as described previously , afatinib , or afatinib in combination with cisplatin . Two sequential combination schedules were tested:Cell survival was assessed using the colorimetric sulforhodamine B (SRB) assay, as previously described  were calculated using winnonlin software . Possible synergism between afatinib and cisplatin was determined by calculation of the combination index (CI) using the additive model as described by others  and cisplatin , were diluted in sterile PBS. Afatinib  was diluted in DMSO (Merck Millipore SA/NV), and further dilutions were made in cell culture medium. IC2.4et al., 50 concentration of cetuximab. In parallel, controlled parental cells were exposed to the vehicle control (suffix PBS). After 10 dose doublings, dose–response studies were determined for each resistant cell line (suffix R). To examine whether acquired resistance was a transient or permanent effect, dose–response curves of cetuximab were re‐assessed in the resistant cell lines after 6 weeks in culture without cetuximab.Generation of resistant cell clones was performed as described previously . These receptor‐specific antibodies recognize and bind to the extracellular domain of the receptor. Corresponding isotype controls  were included for all samples and served as negative controls. Dead cells were excluded from the analysis by staining with Live/Dead Fixable Far‐Red Dead Cell Stain Kit . All samples were measured on a FACScan flow cytometer . Flow cytometric data were analyzed using flowjo v10.1 . The percentage of EGFR‐, HER2‐, HER3‐ and HER4‐positive cells (overton) was determined in comparison with the corresponding isotype control. Furthermore, the signal for aspecific binding was subtracted from the mean fluorescence intensities (=ΔMFI). This parameter indicates the amount of cellular membrane expression of EGFR, HER2, HER3, and HER4 on individual cells.The cellular membrane expression level of EGFR, HER2, HER3, and HER4 under both normoxia and hypoxia was assessed using flow cytometry. Cells were fixed in 4% formaldehyde (10 min) under normoxia or hypoxia after EGFR, HER2, HER3, and HER4 PE‐conjugated antibody incubation . Induction of apoptotic cell death was investigated flow cytometrically using the annexin V‐FITC (AnnV)/propidium iodide (PI) assay (BD Biosciences). Both assays were performed on a FACScan flow cytometer and analyzed with flowjo v10.1.After overnight incubation, cells were treated for 72 h with afatinib. As the sensitivity to afatinib strongly varied between the cell lines, afatinib concentrations were based on the outcome of the monotherapy experiments and corresponded with the ICm. This caspase‐3/7 apoptosis reagent is cleaved by activated caspase‐3/7, which results in the release of a DNA dye and green fluorescent staining of nuclear DNA. Images were taken every 2 h from the start of treatment. Kinetic activation of caspase‐3/7 was monitored morphologically using live‐cell imaging and quantified using the IncuCyte basic analyzer (Essen BioScience).Alternatively, the induction of apoptotic cell death was examined by real‐time measurements of caspase‐3/7 activity using the IncuCyte ZOOM live‐cell analysis instrument . After overnight incubation, cells were treated with afatinib. The IncuCyte caspase‐3/7 green apoptosis reagent (Essen BioScience) was added at the start of treatment at a final concentration of 2.5 μ2.7post hoc analysis with Tukey's correction for multiple testing was performed.We performed all experiments at least three times independently, unless otherwise stated. In cytotoxicity experiments and IncuCyte Caspase‐3/7 Green Apoptosis Assays, each condition was tested in triplicate in each of the three experiments. Flow cytometry experiments were independently performed three times with one sample for each condition. Results are presented as mean ± standard deviation. The effects of various conditions and treatments were studied using linear regression or linear mixed models in case of nonindependent observations. All models were fitted using a stepwise backward strategy, starting from a model with all fixed effects and their interaction. If the interaction term was not significant, a model with only the main was fitted. If one of these terms was significant, effect sizes were estimated. If the treatment effect was significant, a 0/G1 cells as well as AnnV+/PI− and AnnV+/PI+ cells. Regarding the combination experiments, differences in IC50 values were tested for significance with the Mann–Whitney U‐test.Effects of oxygen and resistance status on the expression of HER family members and afatinib's cytotoxic effect were modeled using a linear mixed model with oxygen status, resistance status, and their interaction as fixed effects. A random intercept for cell line was added to account for the dependence between observations within the same cell line. Furthermore, a separate analysis was performed to test for differences in HER status between HPV‐positive and HPV‐negative HNSCC cell lines. Linear regression models were fitted to study the effect of treatment, oxygen condition, and their interaction on the percentage of Ggraphpad prism 7  was used for data comparison and artwork. All statistical analyses were performed in r version 3.3.2 . P‐values below 0.050 were considered significant.33.1et al., et al., 50 values, five of seven HNSCC cell lines  were considered as intrinsically resistant to cetuximab as the percentage of viable cells in these cell lines did not decrease below 50%. SC263 and SCC22b were identified as cetuximab sensitive .We previously identified intrinsic resistance to cetuximab in several HNSCC cell lines such as LICR‐HN1 and Cal‐27  and the ΔMFI of receptor‐positive cells (P ≥ 0.170) were observed between cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. In addition, no significant effect of HPV status was detected for the percentage of EGFR‐, HER2‐, HER3‐, and HER4‐positive cells (P ≥ 0.302) as well as ΔMFI of receptor‐positive cells (P ≥ 0.110). Hence, the percentage EGFR‐, HER2‐, HER3‐, and HER4‐positive cells and ΔMFI of receptor‐positive cells seems to be cell line specific and independent of HPV status.No significant differences in the percentages of EGFR‐, HER2‐, HER3‐, and HER4‐positive cells (P = 0.006) and ΔMFI of EGFR‐positive cells (P < 0.001) under hypoxic conditions in all HNSCC cell lines (Table P = 0.016)   (Table P = 0.024) . This significant increase under hypoxia was mainly noticed in intrinsically cetuximab‐resistant as well as acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines (P ≤ 0.050)  (Table P = 0.425). However, taking the difference in cetuximab resistance status between cell lines into account, a significant decrease in ΔMFI under hypoxia was observed for HER3 in acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines (P ≤ 0.010)  (data not shown).Next, the effect of reduced oxygen levels on the expression of EGFR, HER2, HER3, and HER4 was evaluated. No significant interaction between oxygen condition and cetuximab resistance status on EGFR, HER2, HER3, and HER4 expression was found. When looking at the effect of oxygen availability, there was a significant increase in the percentage of EGFR‐positive cells  after 72 h of treatment was observed in all HNSCC cell lines  nor the HPV status (P = 0.800) seems to influence the inhibitory potential of afatinib. However, certain intrinsically cetuximab‐resistant and acquired cetuximab‐resistant HNSCC cell lines demonstrated higher IC50 values compared to other intrinsically cetuximab‐resistant and acquired cetuximab‐resistant HNSCC cell lines used in this study. For instance, the intrinsically cetuximab‐resistant HNSCC cell lines Cal27 and UM‐SCC‐104 demonstrated considerably lower IC50 values compared to LICR‐HN1 and 93‐VU‐147T. Despite that statistical analysis did not reveal a significant influence of cetuximab resistance on the cytotoxicity of afatinib, these results indicate the possibility of cross‐resistance between cetuximab and afatinib.The cytotoxic effect of the irreversible HER family blocker afatinib was studied in cetuximab‐sensitive, intrinsically cetuximab‐resistant as well as acquired cetuximab‐resistant and PBS‐treated control HNSCC cell lines under both normoxia and hypoxia. A clear concentration‐dependent cytotoxic effect of afatinib  and HPV status (P = 0.630) were not associated with afatinib's cytotoxicity under hypoxic conditions. However, statistical analysis showed a significant interaction between resistance status and oxygen condition (P < 0.001). This implies that the difference in IC50 value for afatinib between the normoxia and hypoxia group varies across the different resistance statuses. As such, we analyzed the difference in IC50 value of afatinib between normoxia and hypoxia for cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. The estimated effect sizes, showing the difference in mean log(IC50) between normoxia and hypoxia, are displayed in Fig. 50) values were observed under normoxia compared to log(IC50) values under hypoxia in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines (P < 0.001). This means that afatinib demonstrated an increased cytotoxicity under hypoxic conditions. However, this effect of hypoxia on afatinib's cytotoxic activity was less pronounced in intrinsically cetuximab‐resistant cell lines.The cytotoxic effect of afatinib was maintained under reduced oxygen conditions Table . CetuximOverall, afatinib showed a clear concentration‐dependent cytotoxic effect in cetuximab‐sensitive, intrinsically cetuximab‐resistant, and acquired cetuximab‐resistant HNSCC cell lines. Furthermore, our results demonstrate that therapeutic resistance to afatinib is not associated with HPV infection or prolonged exposure to hypoxia in our panel of HNSCC cell lines with different sensitivities to cetuximab.3.43.4.10/G1 cells, accompanied by a decrease in the percentage of cells in the S and G2/M phases, in the majority of the HNSCC cell lines, irrespective of their sensitivity to cetuximab.As presented in Fig. 0/G1 cells were analyzed using linear regression analyses  or late (AnnV+/PI+) phases of apoptosis. The majority of intrinsically cetuximab‐resistant HNSCC cell lines demonstrated a dose‐dependent increase in AnnV+/PI− and AnnV+/PI+ cells as well as a corresponding decrease in viable AnnV−/PI− cells after 72 h of treatment with afatinib under normal and reduced oxygen conditions . In contrast, treatment with cisplatin followed by afatinib resulted generally in a significant increase in IC50 compared to the IC50 of cisplatin monotherapy (0.050 ≤ P ≤ 0.825). Furthermore, CI ranged from 1.01 ± 0.06 to 1.96 ± 0.40. Thus, sequential exposure to afatinib followed by cisplatin or the inverse regimen  revealed additive or subadditive to antagonistic, yet no synergistic interactions.To investigate the potential interaction between afatinib and cisplatin, tumor cells were incubated with fixed doses of afatinib for 72 h combined with sequential treatment of 0–10 μ4Therapeutic resistance to EGFR‐targeted therapies remains a major clinical problem. In order to overcome resistance to these EGFR‐targeted therapies, new treatment options are urgently needed. Due to extensive crosstalk among HER receptors, blockade of one HER receptor can be partially compensated by other HER family members, which therefore must be targeted by new therapeutic regimens. In contrast to the first‐generation EGFR inhibitors, afatinib blocks irreversibly EGFR, HER2, and HER4. As a result, we hypothesized that treatment with afatinib might result in a distinct and more pronounced therapeutic benefit. To test this hypothesis, we investigated the cytotoxicity of afatinib in a panel of two HPV‐positive and nine HPV‐negative HNSCC cell lines that are either sensitive or intrinsically/acquired resistant to cetuximab.et al., et al., et al., We observed that the majority of HNSCC cell lines used in this study demonstrated high expression of EGFR, HER2, and HER3, but rather low HER4 expression under both normal and reduced oxygen conditions. It has already been established that EGFR is a key survival factor under hypoxic conditions, as EGFR stimulates HIF signaling to improve cellular survival . Furthermore, afatinib displayed a radiosensitizing effect in preclinical studies (Huguet 50/G1 cell cycle arrest and induces apoptotic cell death. Sequential combinations of afatinib with cisplatin demonstrated additive to antagonistic effects. In contrast to cetuximab, afatinib seems not to be the ideal combination partner with platinum‐based drugs for the treatment of HNSCC. In this study, neither cetuximab resistance nor HPV status significantly influenced the expression of HER family members in HNSCC cell lines. In contrast, the expression of EGFR, HER2, and HER3 was significantly altered under reduced oxygen conditions. Furthermore, the cytotoxic effect of afatinib was increased under hypoxia. Overall, these data support the hypothesis that afatinib might be a promising therapeutic strategy to treat patients with HNSCC experiencing intrinsic or acquired cetuximab resistance. Nevertheless, further identification of predictive biomarkers is necessary.Our results suggest that afatinib has the potential to overcome intrinsic and acquired cetuximab resistance in both HPV‐positive and HPV‐negative HNSCC tumors, as it was able to establish a cytotoxic effect in HNSCC cell lines with different cetuximab sensitivity and HPV status. However, cross‐resistance between cetuximab and afatinib might be possible. Therefore, further research is required to identify predictive biomarkers to optimize patient selection. Treatment with afatinib causes a Gin vitro experiments, performed statistical analysis, and drafted the manuscript. FL obtained funding for the study, participated in the design, and helped to draft the manuscript. JVB assisted in the in vitro experiments, participated in its design, and contributed to draft the manuscript. HB assisted in the in vitro experiments toward his Master thesis in Biomedical Sciences. EF performed statistical analysis. VD and PP participated in the design of the study. MP obtained funding for the study. JBV participated in the design of the study and helped to draft the manuscript. AW conceived of the study, participated in its design and coordination, and assisted to draft the manuscript. All authors read and approved the final manuscript.IDP participated in the design of the study, carried out the JBV participated in advisory boards of Boehringer‐Ingelheim. The other authors declare that they have no conflict of interest.


BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify  Two patients with the same disease who receive the same treatment may respond in different ways. This variation often arises from differences in each patient’s genetic code. Genes encode proteins, and proteins are the targets of most medical drugs and thus determine the patient’s response to treatment.st century is that doctors recognise that patients can respond differently to the same treatment and now try to predict which patients will respond best to which drug – an approach known as personalised medicine. Cancer treatment has been at the forefront of personalised medicine because mutations in different genes underlie each different cancer. By analysing which mutations are present in a cancer, doctors can thus predict which drug (or combination of drugs) will be most effective. This approach has been used successfully in several cancers, including breast and lung cancer, leading to fewer patients being exposed to ineffective treatments and their associated side effects and costs.A major advance in the 21Mesothelioma is a cancer of the lining of the lung that is associated with exposure to the mineral asbestos. Current treatment options for mesothelioma are unfortunately limited and not very effective. No personalised treatments are currently in use and new treatment approaches are desperately needed.Kolluri, Alifrangis, Kumar, Ishii et al. set out to determine if any of the mutations commonly seen in mesothelioma affected how the cancer would respond to 94 anticancer drugs that are either in use or in development. In the laboratory, mesothelioma cells that have mutations in the gene that codes for a protein known as BRCA associated protein-1 (or BAP1 for short) were killed much more effectively by a drug known as TNF-related apoptosis-inducing ligand (TRAIL). The same link was seen in experiments with tumours of mesothelioma cells that had been transplanted into mice, and for fragments of mesothelioma tumours taken from patients. When Kolluri et al. studied why these tumours might be killed more effectively with TRAIL, they found that mutations in the gene for BAP1 result in a change in the levels of proteins that transmit the signal from the receptors targeted by the TRAIL drug.These findings may one day result in a new approach to treating patients with mesothelioma. But first, the next step would be to conduct a clinical trial of TRAIL in patients with mesothelioma and assess if those with tumours that have mutations in the gene for BAP1 do indeed respond better. If this proves to be the case, this would result in a new personalised treatment option for patients that suffer from this disease. BCR-ABL fusion product in chronic myeloid leukaemia (CML) and the receptor products of HER2 mutations in breast cancer have transformed the prognosis of these cancers  developed against the  cancers . Maligna cancers . A pleth mutated  and ther mutated .BAP1) that demonstrated heightened sensitivity to the death receptor agonist recombinant tumour necrosis factor (TNF)-related apoptosis-inducing ligand (rTRAIL). We validated this finding using in vitro, in vivo and ex vivo models supporting the use of BAP1 as a genomic biomarker to identify TRAIL-sensitive MM tumours and a novel stratified approach to treat MM.We aimed to determine if the mutational status of these tumour driver genes could predict response to a range of existing anti-cancer compounds with a view to identifying genomic biomarkers for responsive subsets of MM. We have previously reported on the ability of such unbiased high-throughput chemical screens in cancer cell lines to identify drug-sensitising mutations in other cancer types . To thisEGFR mutations and EGFR TKIs  and treatment with recombinant TRAIL  (BAP1 wild-type (wt BAP1) lines when treated with rTRAIL. We subsequently confirmed this association in a larger panel of MM cell lines  harbouring wild-type BAP1 were resistant. BAP1 LOF mutations correlated with a loss of BAP1 protein expression in the majority of cell lines  . No signll lines . Strikinll lines . No sensnt of MM . A margiMEDI3039 . Thus, wBAP1 expression in four wt BAP1 MM cell lines using a lentiviral shRNA construct. Knockdown of BAP1 resulted in increased cell death following rTRAIL treatment compared with empty vector (EV) control shRNA and the parental cell line in all four MM cell lines (BAP1 (http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/). These include CCRC, bladder and breast cancer lines. Treatment of cancer cell lines harbouring nonsense mutations in BAP1 with rTRAIL resulted in markedly reduced cell viability compared with cancer cell lines harbouring missense mutations  , a wild-type BAP1 expression vector or one of these five mutant BAP1 expression vectors. rTRAIL sensitivity of the parental BAP1-null H226 MM line was significantly diminished following expression of wild-type BAP1 and each of the mutant constructs except those with an inactive deubiquitinating or ASXL protein-binding site (BAP1 is a nuclear deubiquitinase that forms multi-protein complexes with transcription factors to regulate gene transcription . To elucic site) , ΔHBM (dic site) , T493A (ic site) , ΔASXL (ic site)  and ΔCTDic site) . H226 MMing site , implicaing site . Transduing site .ASXL1, but not ASXL2, induced sensitivity to MEDI3039 and rTRAIL in the BAP1/ASXL1/ASXL2-wild-type MM line MPP-89  ((http://www.genome.jp/dbget-bin/www_bget?path:map04210). Among those pathways significantly altered when comparing wild-type versus C91A BAP1 (FDR < 0.2) was that of apoptosis. In particular, there was altered mRNA expression of components of the extrinsic death pathway  and vehicle-treated BAP1-mutant tumours (p=0.019)  as assessed by longitudinal bioluminescence intensity (To test the  of mice . On day p=0.019) . BAP1-wintensity .Malignant mesothelioma remains a devastating disease with limited systemic treatment options . BiomarkBAP1 mutation rates in the order of 20–30% .The genomic data is available in the COSMIC database    . The reshttp://www.sanger.ac.uk/genetics/CGP/Software/PICNIC/). All genes were mapped onto this segmentation data to give a gene level copy number analysis. For genes to be classified as amplified the complete coding footprint of the gene had to map onto segment(s) present in eight or more copies. For genes to be classed as homozygously deleted a minimum of 1 bp of coding sequence had to be present within a segment of copy number ‘0’.Genome-wide copy number data were obtained for the cell lines using the Affymetrix SNP6 microarray analysed using the ‘PICNIC’ algorithm, which segments the genome into integer value copy number segments  . Adherent cell lines in the screens were plated 1 day prior to treatment with each compound using liquid handling robotics and assayed after 6 days of treatment with either the single agent or in combination with rTRAIL. Cells were fixed in 4% formaldehyde for 30 min and then stained with 1 μM of the fluorescent nucleic acid stain Syto 60  for 1 hr. Quantitation of fluorescent signal intensity was performed using a fluorescent plate reader at excitation and emission wavelengths of 630/695 nm. The sensitivity of each cell line to various concentrations of compound was calculated as the fraction of viable cells relative to DMSO-treated cells following a 6 day exposure. All screening plates were subjected to stringent quality control measures and a Z-factor score comparing negative and positive control wells was calculated across all screening plates .We derived the area under the curve (AUC) parameter from the 6 day cell line viability data to identify cell lines that are sensitive to a specific compound, with decreasing AUC associated with increasing sensitivity. The AUCs were computed using a trapezoid integration below the five measured viability of the dose-response curve and scaled so that a constant viability of 1 gives AUC of 1.BAP1, TAOK1, NF2, TP53 and CDKN2A). We defined groups of cell lines based on mutations and copy number alterations (homozygous deletions or amplifications) in these genes. This resulted in a set of input features of 4 genes altered in at least 2 of the cell lines . For the association of gene mutations with sensitivity to each compound we restricted the set of drugs to test to those with ≥2 cell lines with AUC <0.7. This resulted in 45 drugs being suitable for analysis .We used 15 mesothelioma cell lines with molecular and drug response data: H2369, H2373, H2461, H2591, H2722, H2731, H2803, H2804, H2810, H2818, H2869, H513, MPP-89, NCI-H2452 and NCI-H28. We selected five genes for inclusion in the analysis  and Sequenom profiling of a panel of 92 single nucleotide polymorphisms for each cell line to ensure non-synonymous cell lines were not used. As a cell line classified as mesothelioma, H513 (on the list of commonly misidentified cell lines) was included in the drug screen of 15 mesothelioma cell lines conducted. Use of this cell line however was not carried forward to further experiments in the paper. The 25 early passage MM cultures were purchased from MesobanK . All cel2.Cell lines were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium and nutrient mix 12 medium (DMEM:F12) supplemented with 10% fetal bovine serum (FBS), penicillin/streptavidin and sodium pyruvate. Early passage human mesothelioma cultures were cultured in RPMI-1640 medium supplemented with 5% FBS, 25 mM HEPES, penicillin/streptavidin and sodium pyruvate. 293 T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. All cells were maintained in a humidified environment at 37°C and 5% COAB_626723), caspase 8  Cat# 9746, RRID:AB_2275120), c-FLIP  Cat# ALX-804-961-0100 RRID:AB_2713915), cIAP1 , cIAP2 , FADD , XIAP , survivin , α-tubulin , H2AK119Ub , H2A , anti-mouse HRP  and anti-rabbit HRP . To detect the ubiquitination status of the histones, the cells were lysed with TBS buffer containing 1% SDS, protease and phosphatase inhibitors. The cell extract was denatured by heating up at 95°C for 10 min and centrifuged at 13000 rpm for 10 min. The supernatant was collected and immunoblotted as described above.Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer  with protease inhibitors  on ice to extract protein. 20 μg of protein samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with specific primary antibodies, washed, incubated with secondary antibodies and visualised using an ImageQuant LAS 4000 imaging system . Antibodies used include BAP1  Cat# sc-28383, RRID:Cells were seeded in 96-well plates in 100 μl media per well at a density of 40,000 cells/ml 1 day prior to treatment with soluble recombinant TRAIL  or MEDI3039 . XTT  or MTT  reagent was added on day 3. The absorbance was measured with a spectrophotometer at a wavelength of 490 nm or 560 nm for XTT or MTT respectively. Relative cell viability was calculated as a fraction of viable cells relative to untreated cells.BAP1 cDNA was amplified by PCR from pCMV6-AC BAP1 plasmid  and cloned into the lentiviral plasmid pCCL-CMV-flT vector previously described (BAP1 vector designated pCCL-CMV-BAP1. Vectors expressing mutant BAP1 constructs were generated by site-directed mutagenesis (New England Biolabs) of the pCCL-CMV-BAP1 vector. The primers used are listed below. All mutations were confirmed by sequencing.Full-length escribed  in placeBAP1-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGABAP1-R GTCGGTCGACTCACTGGCGCTTGGCCTTGTAC91A-F ATACCCAACTCTGCTGCAACTCATGCCTTGCTGC91A-R CAGCTGGTGGGCAAAGAACATGTTATTCACAATATCATCHBM-F CGCTGCTGCCAAGTCCCCCATGCAGGAGGAHBM-R GCAGCGTCTAGAAAGGCCGGCAGCCGCTCTD-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGACTD-R GTCGTTCGAATCAGTCAGGCTTCCGCTGCTTGTGGT493A-F GCAGACACGGCCTCTGAGATCGGCAGTGCTT493A-R ACTCTCATTGCTGGGGGTGGGTGAASXL-F AACTACGATGAGTTCATCTGCACCTASXL-R CTGGTCATCAATCTTGAACTTCTTCCTCThe ZS-green luciferase plasmid, pHIV-Luc-ZsGreen  was used for generating ZS-Green luciferase-expressing lentivirus to transduce the H226 cells used in animal experiments.Short hairpin RNAs (shRNAs) were expressed as part of a mir30-based GIPZ lentiviral vector . The clones used in this study include BAP1 (V2LHS_41473), ASXL1 (V2LHS_78829), ASXL2 (V3LHS_313940) and the empty GIPZ control vector.Lentiviral vectors were produced by co-transfection of 293 T cells with construct plasmids together with the packaging plasmids pCMV-dR8.74 and pMD2.G  in the presence of a DNA transfection reagent jetPEI (Source Bioscience UK Ltd). Lentiviruses were concentrated by ultracentrifugation at 17,000 rpm  for 2 hr at 4°C. To determine the titres of prepared lentiviruses 293 T cells were transduced with serial dilutions of viruses in the presence of 8 μg/ml Polybrene (Sigma-Aldrich) and BAP1 expression was assessed by flow cytometry. shRNA- and luciferase-expressing vectors were assessed by analysis of GFP expression. Cell lines were transduced in the presence of 8 μg/ml Polybrene at a range of MOIs and transduction efficacy was assessed by flow cytometry for BAP1 expression.BAP1 construct. SPIA pathway analysis as described in Tarca et al  method . We discca et al  was perfAB_626723; 1:50) and then with an AlexaFluor 488-conjugated anti-mouse antibody . For analysis of apoptosis and cell death all floating and adherent cells were harvested and stained with an Annexin V AlexaFluor 647-conjugated antibody  and 4’, 6-diamidino-2-phenylindole . For analysis of DR4 and DR5 expression on cell surface cells were stained with PE-conjugated antibody  Cat# 307205, RRID:AB_314669, DR5 - BioLegend Cat# 307405, RRID:AB_314677, Isotype control - Biolegend #400112; 1:100). FlowJo software was used to analyse all data.All flow cytometry analysis was performed on a LSR Fortessa analyser . For analysis of BAP1 expression cells were stained with primary antibody to BAP1  primary antibody  was incubated overnight at 4°C, before incubating for 1 hr at room temperature with Alexafluor 488-conjugated anti-rabbit secondary antibody. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific Cat# 62249). Images were acquired (n = 3) with a BioTek Cytation3 Multimode reader. Using a 10x objective 4 fields of view were acquired per well (n = 3) and the level of nuclear ubiquityl-histone H2A intensity was determined within the primary nuclear mask and normalised to total cell number.H226 cells were seeded at 2.5 × 10AB_626723; 1:150) for 15 min at room temperature. Epitope retrieval was completed using HIER using Leica Bond ER2 (high pH) for 30 min and a Leica Bond Polymer Refine with DAB chromogen detections system used.BAP1 immunohistochemistry of human early passage cell lines was conducted on sections of cell pellets mounted on slides. Automated staining on a Leica Bond III staining platform was used. Slides were incubated with BAP1 primary antibody , EGF (20 ng/ml) and FGF (10 ng/ml). After 24 hr the explants were treated with rTRAIL  for a further 24 hr, following which explants were either fixed for PARP immunohistochemistry. The explants were fixed in 10% neutral-buffered formalin (NBF) for 24 hr and then transferred into 70% ethanol followed by paraffin embedding. Subsequently, 5 μm sections were used for immunohistochemistry, as previously described . Informed consent to conduct research on samples collected and to publish results was obtained from patients. The diagnosis of mesothelioma was confirmed histologically for all patients prior to consent and surgery. Patients underwent pleurectomy, following which primary pleural tissue was sectioned into fragments measuring approximately 2 mmescribed .AB_777102) was used at a 1:6000 dilution and the rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam) was used for immunohistochemistry, according to the manufacturer’s instructions. Sections were counterstained with haematoxylin and mounted using Vectamount permanent mounting media . Images were taken at 40x magnification on a Hamamatsu Nanozoomer Digital slide scanner. Cleaved PARP-positive cells were scored as the percentage of cells with nuclear staining.Cleaved PARP primary antibody  Act 1986 . Mice were purchased from Charles River, kept in individually ventilated cages under specific pathogen-free conditions and had access to sterile irradiated food and autoclaved water IMSR_CRL:394) were injected with 1 × 106 H226 cells transduced with a plasmid containing wild-type BAP1 and luciferase on the right flank and with a plasmid containing a catalytically inactive BAP1-mutant (C91A) and luciferase on the left flank in a 1:1 mixture of Matrigel  and medium. Tumour size was assessed by bioluminescence in vivo imaging system  15 min following intraperitoneal injection of 0.2 ml (2 mg) luciferin. Tumours were allowed to establish for 2 weeks prior to baseline assessment of size at day 13. Mice were then divided into two groups each of which received either 600 μg TRAIL or vehicle 6 days a week from day 14 until day 40. Bioluminescence was measured on days 0, 13, 19, 26 and 41. Mice were sacrificed on day 42 and tumours harvested for measurement. TRAIL used in the mouse experiment was made in Henning Walczak’s laboratory as per the established protocol (12 8 week old NOD.CB17-Prkdcscid/NcrCrl (NOD SCID) mice . t-test was used to analyse differences between two groups whilst the analysis of variance (ANOVA) test with a Tukey post-hoc analysis was used to analyse differences between three groups. For multiple groups measured over multiple time points repeated measures ANOVA was used. All  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Charles Sawyers as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andrew Thorburn (Reviewer #1); Dan Longley (Reviewer #3).Thank you for submitting your article "Loss of functional BAP1 is a biomarker for TRAIL sensitivity in cancer" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Kolluri et.al. present convincing data arising from a drug screen in a panel of mesothelioma cell lines that the nuclear deubiquitinase BAP1 regulates TRAIL sensitivity in mesothelioma cells (and some other tumor types too.) Loss of BAP1 expression, which occurs in some cancers, could potentially serve as a biomarker for sensitivity to agonists that target TRAIL receptors (TRAIL R). As noted by the authors TRAIL R-targeted therapeutics have been quite widely tested and usually shown to be safe, however the lack of an ability to identify patients who are likely to benefit from these treatments has stymied development of this class of drugs. Therefore, the area that the authors are addressing is potentially quite important. Reviewers found the data presented to be quite convincing and to support the authors' case that BAP1 can affect TRAIL sensitivity most likely through epigenetic regulation of gene expression by a mechanism involving the AXLS1 protein. However, a number of concerns were raised about the ultimate power of BAP1 status to serve as a biomarker in the clinic, as well as the minimal mechanistic insight provided in the report.After thorough discussion, the reviewers agreed to invite a resubmission of a revised manuscript addressing the following major points:1) A main concern with the paper in its current form is that it doesn't represent a major advance for the field, in the sense that there are many genes and processes that have been shown to confer TRAIL sensitivity and proposed as potential biomarkers, yet none of them so far have worked out to be useful. Reviewers agreed that more evidence is needed in this regard. First, it's not clear whether the effects are really specific to TRAIL. Many perturbations that make tumor cells more sensitive to apoptosis will appear to confer sensitivity on a canonical apoptosis inducer like TRAIL. Therefore, reviewers request a series of experiments addressing this issue, to define whether or not BAP1 mutation/loss is a general sensitizer to death ligands and/or general apoptotic stimuli. Reviewers suggest testing a different death ligand, such as a Fas agonist (e.g. CH-11 antibody) or, alternatively, a TNF/IAP antagonist combination. For the general apoptotic stimulus, the reviewers propose disease-relevant genotoxic agents such as pemetrexed or cisplatin.2) Second, reviewers agreed that more work should be performed to establish the notion of BAP1 status as a biomarker for TRAIL efficacy. Although the pattern presented is very strong, many BAP1 positive cells are sensitive to TRAIL and vice versa, indicating that other factors in addition to BAP1 define sensitivity to TRAIL. The report will be significantly strengthened by performing cause-effect relationship experiments in a much larger number of cell lines. More specifically, reviewers would like to see the BAP1 depletion experiments done in a larger panel of cell lines. The report currently shows the effect of BAP1 knockdown in only two cell lines: H226 and MDA-MB-231. All other cause-effect experiments involve artifact-prone overexpression experiments of BAP1 (wt or various mutants). Given the ready access to cell lines and proven shRNAs for BAP1, the conclusions would be significantly strengthened by testing the effect of BAP1 depletion in two additional MM cell lines that are BAP1 positive and TRAIL-resistant. Additionally, BAP1 mutations are most frequent among kidney clear cell carcinomas (CRCC) estimated at 10% by tumorportal.org. The impact of the report would be significantly increased by measuring TRAIL sensitivity upon BAP1 depletion in two CCRC cell lines expressing wild type BAP1.3) Third, the clinical relevance would be increased by testing a large number of patient samples in the ex vivo analyses in 4) Another major issue was the minimal mechanistic insight as to how BAP1 status regulates sensitivity to TRAIL. What is the impact of BAP1 on known regulators and effectors of the TRAIL receptor pathway? Some data and discussion on the relationship between BAP1 mutation and gene expression of TRAIL-R1/2, FADD, FLIP, caspase-8, etc. is needed. What happens to these genes when WT versus mymutant BAP1 is overexpressed in BAP1 negative cell line?5) Additionally, insight into how the ASXL1 connection translates into increased cell death should be provided, such as additional measures of key cell death signaling nodes as described in #4 above in experiments where ASXL1 activity/expression has been manipulated.6) In the absence of true validation of BAP1 status as a biomarker in the clinical setting, the title was deemed overambitious, and reviewers request a more accurate title and some attenuation of some of the conclusions describing BAP1 as a biomarker. […] After thorough discussion, the reviewers agreed to invite a resubmission of a revised manuscript addressing the following major points:1) A main concern with the paper in its current form is that it doesn't represent a major advance for the field, in the sense that there are many genes and processes that have been shown to confer TRAIL sensitivity and proposed as potential biomarkers, yet none of them so far have worked out to be useful. Reviewers agreed that more evidence is needed in this regard. First, it's not clear whether the effects are really specific to TRAIL. Many perturbations that make tumor cells more sensitive to apoptosis will appear to confer sensitivity on a canonical apoptosis inducer like TRAIL. Therefore, reviewers request a series of experiments addressing this issue, to define whether or not BAP1 mutation/loss is a general sensitizer to death ligands and/or general apoptotic stimuli. Reviewers suggest testing a different death ligand, such as a Fas agonist (e.g. CH-11 antibody) or, alternatively, a TNF/IAP antagonist combination. For the general apoptotic stimulus, the reviewers propose disease-relevant genotoxic agents such as pemetrexed or cisplatin.We agree and highlight in our manuscript that to date a key limiting factor of TRAIL therapeutics has been the lack of a validated biomarker for sensitivity. However, no approach thus far has included the search for a sensitising mutation using an unbiased drug screen in sequenced cell lines. Importantly the retrospective identification of drug sensitising mutations has re-directed the use of other anticancer therapies with initially disappointing results in unselected populations.BAP1 as a sensitising mutation to TRAIL, and other death receptor agonists, we have treated a panel of BAP1 mutant and wild-type MM lines with additional apoptotic stimuli as suggested. No sensitising association with BAP1 was observed for pemetrexed or cisplatin, current first line agents for the treatment of MM  Second, reviewers agreed that more work should be performed to establish the notion of BAP1 status as a biomarker for TRAIL efficacy. Although the pattern presented is very strong, many BAP1 positive cells are sensitive to TRAIL and vice versa, indicating that other factors in addition to BAP1 define sensitivity to TRAIL. The report will be significantly strengthened by performing cause-effect relationship experiments in a much larger number of cell lines. More specifically, reviewers would like to see the BAP1 depletion experiments done in a larger panel of cell lines. The report currently shows the effect of BAP1 knockdown in only two cell lines: H226 and MDA-MB-231. All other cause-effect experiments involve artifact-prone overexpression experiments of BAP1 (wt or various mutants). Given the ready access to cell lines and proven shRNAs for BAP1, the conclusions would be significantly strengthened by testing the effect of BAP1 depletion in two additional MM cell lines that are BAP1 positive and TRAIL-resistant. Additionally, BAP1 mutations are most frequent among kidney clear cell carcinomas (CRCC) estimated at 10% by tumorportal.org. The impact of the report would be significantly increased by measuring TRAIL sensitivity upon BAP1 depletion in two CCRC cell lines expressing wild type BAP1.BAP1 can affect TRAIL sensitivity as evidenced in our panel of MM lines  Third, the clinical relevance would be increased by testing a large number of patient samples in the ex vivo analyses in Since review we have obtained 7 video assisted thoracoscopic surgery (VATS) pleural biopsy samples from treatment naïve patients with a suspected diagnosis of MM. Four of these proved to comprise benign tissue only. We attempted to assess apoptosis in response to MEDI3039 treatment in explants from the remaining three. However, as these were VATS rather than pleurectomy biopsies, the tissue volume was extremely small precluding appropriate immunohistochemical analysis. Successful explant data is ultimately subject to the frequency of pleurectomy cases performed at our local surgical centre and within the suggested time period for resubmission there were none. We agree however that an expanded cohort would strengthen the clinical relevance and continue with our explant programme to this end.4) Another major issue was the minimal mechanistic insight as to how BAP1 status regulates sensitivity to TRAIL. What is the impact of BAP1 on known regulators and effectors of the TRAIL receptor pathway? Some data and discussion on the relationship between BAP1 mutation and gene expression of TRAIL-R1/2, FADD, FLIP, caspase-8, etc. is needed. What happens to these genes when WT versus mymutant BAP1 is overexpressed in BAP1 negative cell line?BAP1 null MM line with overexpression of BAP1 wild-type or the catalytically inactive BAP1 C91A. Signaling pathway impact analysis highlighted apoptosis as being significantly altered by BAP1 function. Thus we proceeded as suggested to specifically assess the impact of BAP1 function on the death receptor pathway at a gene expression and protein level. This data supports a role for BAP1 in the modulation of transcription of death receptor pathway proteins.We have extended our mechanistic data as suggested. We have compared gene expression data from a We have added the following text to the Results section:BAP1-null H226 cells transduced with the C91A BAP1 mutant or with wild-type BAP1, and carried out a signalling pathway impact analysis SPIA  Additionally, insight into how the ASXL1 connection translates into increased cell death should be provided, such as additional measures of key cell death signaling nodes as described in #4 above in experiments where ASXL1 activity/expression has been manipulated.BAP1 null MM cells transduced with the ASXL1/2 binding mutant ΔASXL BAP1. The changes observed in this analysis are consistent with that of those cells transduced with the catalytically inactive C91A BAP1 mutant. This supports the role of both functions, deubiquitinase activity and ASXL binding, in the regulation of expression of components of the death receptor pathway.We have conducted immunoblot analysis of the death receptor pathway on These changes to the manuscript are detailed above in point 4.6) In the absence of true validation of BAP1 status as a biomarker in the clinical setting, the title was deemed overambitious, and reviewers request a more accurate title and some attenuation of some of the conclusions describing BAP1 as a biomarker.We have amended the title to:“Loss of functional BAP1 augments TRAIL sensitivity in cancer cells.”We have minimised the reference of BAP1 as a biomarker replacing such instances with reference to its use as a ‘genomic stratification tool’ or ‘potential biomarker’.


The present review aims at compiling the findings of particularly interesting studies that use cancer cell line models for assessment of antioxidant and oxidative stress modulation properties of plant-derived bioactive compounds.Medicinal and aromatic plants (MAPs) are known and have been long in use for a variety of health and cosmetics applications. Potential pharmacological usages that take advantage of bioactive plant-derived compounds' antimicrobial, antifungal, anti-inflammatory, and antioxidant properties are being developed and many new ones explored. Some phytochemicals could trigger ROS-mediated cytotoxicity and apoptosis in cancer cells. A lot of effort has been put into investigating novel active constituents for cancer therapeutics. While other plant-derived compounds might enhance antioxidant defenses by either radical scavenging or stimulation of intracellular antioxidant enzymes, the generation of reactive oxygen species (ROS) leading to oxidative stress is one of the strategies that may show effective in damaging cancer cells. The biochemical pathways involved in plant-derived bioactive compounds' properties are complex, and  Nevertheless, interactions with other cell types are not usually considered in cell culture assays, so that the in vivo environment might not be fully mimicked. As a consequence, experimental design should be done carefully, with appropriate controls included in every assay [Cell culture techniques allow investigators to look ry assay .in vitro assessment of medicinal and aromatic plants (MAPs) regarding their antioxidant and oxidative stress-modulating properties in cancer cell lines is presented.There are several strategies for oxidative stress modulation such as ionizing radiation  and platMedicinal and aromatic plants (MAPs) are vegetal materials that have pharmacological properties and also have aromatic and gastronomic uses . The useIt is estimated that more than 80% of the world's medicinal plants grow in Asia and America , 10. MAPα inhibition and in vitro decrease of nitric oxide generation [Another application of herbal products takes advantage of their high flavonoid content that confers them free radical scavenging properties that may, in turn, reduce cellular oxidative damage and ultimately help in the maintenance of intracellular antioxidant defenses . In addineration . Naturalneration .Since the discovery of the vinca alkaloids in the 1950s and their further application in cancer therapy, the interest in MAPs has increased . The cheA number of plant-based anticancer drugs are currently under clinical studies  see Tab. Amin etIn eukaryotic cells, the most important redox process is aerobic respiration, which is mediated by mitochondrial electron transfer. This leads to the generation, in turn, of most of the endogenous intracellular reactive species (RS). For a proposed classification of RS that are produced within cells, see Generation of RS might be (1) exogenous, from sources such as UV radiation, toxic chemicals, cigarettes, drugs, pollutants, alcohol, physiological changes , and exercise or (2) endogenous, from intracellular sources including peroxisomes, mitochondrial metabolism, and the NADPH oxidase complex and myeloperoxidases, which play a fundamental role in maintaining a balanced cellular metabolism .Oxidative stress (OS) is defined as an imbalance in ROS levels and cellular antioxidant endogenous mechanisms. Although ROS are involved in innate immunity and inflammatory signaling through pathogen elimination , ROS preHigh ROS concentrations have a major negative impact in biomolecules  , with hiIntracellular ROS reduction is regulated by antioxidant enzymes such as glutathione peroxidases (GPx), catalases (CAT), and superoxide dismutases (SOD). However, in some cases, antioxidant defense mechanisms may be not enough to maintain a redox balance and might become easily saturated, causing permanent genome impairment and toxicity .In cancerous cell lines, high levels of ROS are necessary to maintain a fast proliferation rate. Szatrowski and Nathan have reported that some cancer types such as melanoma, neuroblastoma, colon carcinoma, and ovarian carcinoma generate large quantities of hydrogen peroxide . Furtherin vitro effects of plant extracts on cancer cell lines, has increased substantially over the last 10 years, as shown in The number of publications, in a PubMed search for in vitro cell functions. The use of immortalized cell lines facilitates in vitro research, but normal cell lines need to be included as experimental controls [Cell culture techniques provide a relatively easy and affordable way into eukaryotic experimentation. Cell culture is a very useful tool for the study of pathologic/normal mechanisms in cell biology and is considered essential when screening for potential new drugs for complex diseases, such as cancer. The use of a primary culture from certified vendors and proper culture media with adequate supplements and growth factors are crucial to secure consistent and stable controls .Cell line models have been widely used in OS research and also in the study of new candidate molecules for the treatment of OS-related diseases. For instance, procarbazine, an azo derivative, has been part of cancer treatment schemes since its discovery 50 years ago. Procarbazine's mechanism of action as anticancer drug involves oxidation of the molecule and production of intracellular hydrogen peroxide .Regulation of ROS is fundamental for homeostasis and normal functioning of cells. Several studies have addressed this complex series of phenomena. For instance, in a study by Schlenker et al., OS was evaluated using a bile duct epithelial cell (BDE) model to determine the earliest effects of RS in cell injury. The study authors found that OS causes a significant decrease in cell volume and affects multiple cell functions such as ion permeability, activation of ion channels, and apoptosis . Glorieuin vitro screening of MAP-derived antioxidants has been on the rise in the last years. In the next section, we will look at several promising applications of MAPs based on data from in vitro cancer studies.As shown in The methodology used in all the studies presented in this review has been summarized in A study by Yedjou and Tchounwou evaluated the effect of garlic extract in an HL-60 model (human leukemia cells). This extract was found cytotoxic (via apoptosis) and an inductor of oxidative stress on tested cells. An interesting finding of the above study is the potential of malondialdehyde (MDA) as a biomarker of oxidative stress associated with caspase activation and DNA fragmentation. Mechanistic data regarding the observed cell responses to garlic extract is, however, still lacking .in vitro effect of apigenin, a natural polyphenolic compound rich in flavones, on cell lines HT-29  and HCT-15  models, the authors [In a study on the  authors  showed tThe anticancer properties of this herbal mixture used in traditional medicine have been assessed on several cancer cell lines  . In the Chemotherapeutics are usually toxic for both noncancer and cancer cells. Appropriate combinations of available drugs are crucial to effectively kill cancer cells, reduce side effects and improve survival rates. A study by Tyszka-Czochara et al.  evaluateInula helianthus aquatic on HT-29 and HCT 116 colorectal cancer cell lines was studied in a recent work by Li et al. [The effect of this sesquiterpene lactone naturally produced by i et al. . The autIwasaki et al. in a recently published work  studied Curcuma longa L. that has shown many pharmacological activities, such as modulation of the nuclear NF-κB signaling pathway. Hong et al. [Curcumin is a bioactive compound isolated from g et al.  showed tg et al. .The role of natural antioxidants in oxidative stress-related diseases has been studied for several years. MAPs can act as protective agents against OS and thus their potential for use in such diseases might be worth exploring.Fucus spiralis on H₂O₂-treated MCF-7 cells (breast adenocarcinoma) [A study of five antioxidant fractions from seaweed rcinoma)  showed aCannabis sativa, on a hydrogen peroxide ROS-induced murine RAW 264.7 macrophage model. This study identified the selective receptor antagonists needed to elucidate CBG's known antioxidant properties at the mechanistic level. The authors found that activation of CB2 receptors is involved in CBG effects, with receptor inhibition leading to lower oxidation as a cytoprotective effect. CBG was found to downregulate several oxidative markers through inhibition of phosphorylation and modulation of MAPK. In addition, CBG exerted antioxidant activity by inducing superoxide dismutase-1 (SOD-1) overexpression, which causes an imbalance in proapoptotic factors to avoid cell death.In another recent study, Giacoppo et al.  evaluate(60)Co γ-radiation DNA damage in exposed lymphocytes. Furthermore, these natural polyphenols could inhibit lipid peroxidation and activate antioxidant enzymes.Plant compounds such as flavonoids, polysaccharides, and phytohormones could function as radioprotectors, meaning they can protect adjacent normal cells from the harmful effects of radiation therapy. It has been proposed that the above compounds might also offer protection against ionizing occupational and environmental radiation exposure . In a reConversely, plant products could act as radiosensitizers by enhancing the effect of ionizing radiation therapy against radioresistant cells. A study conducted by Wang et al.  showed tin vitro oxidative stress cell model. Hydrogen peroxide is a chemical that is frequently used for oxidative stress induction in cultured cells; however, concentrations and exposure times vary extensible and may require standardization to assure reproducibility and data comparability [Lastly, it is important to mention that many studies currently in place evaluate the protective effect of novel antioxidant plant compounds against oxidative stress. Due to the complexity of biological systems, an interesting approach in antioxidant molecules testing might be the construction of an rability –58.in vitro cell models is critical for a comprehensive understanding of the biological properties of plant-derived compounds in cancer research. According to the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM), there are several in vitro cell models accepted for drug development applications. In the case of oxidative stress, studies on cell models help to integrate multiple metabolic routes and identify novel plant-based drugs or therapeutic targets. For instance, human-induced pluripotent stem cells (hiPSC) might be a valuable model for neurotoxicity studies with the use of the oxidative stress biomarker Nrf2 [in vitro toxicology testing platforms.The use of ker Nrf2 . Such a Evaluation of potential natural drugs for modulation of oxidative stress in cancer might, in our opinion, be addressed efficiently and rigorously using cell line models. The NCI60 human tumor cell line anticancer drug screen panel had been used for testing of natural crude extracts collections since 1989. The panel requires previous individual cell line screening, which has led, for example, to the discovery of bortezomib, the first therapeutic proteasome inhibitor for the treatment of multiple myeloma [Additionally, it is important to consider that a wide range of subtypes of cancer cell lines is currently available through certified vendors and collections. Colorectal cancer cell lines, for instance, show different genomic patterns and mRNA expression profiles  and may in vivo experiments with animal models might be optimized based on in vitro data. Information from in vitro and in vivo studies will, in turn, facilitate and improve further clinical trials [In conclusion, carefully planned experiments on cell culture models allow a throughout assessment of the molecular pathways involved in the mechanism of action of a bioactive compound. The use of these models for evaluation of ROS production and oxidative stress modulation properties of plant-derived biocompounds is well documented and growing. Consequently, l trials .in vivo environment. Three-dimensional (3-D) cultures closely emulate tumor formation processes by synthetic tumor microenvironment mimics (STEMs). Models of human lung epithelial cells, pulmonary vascular endothelial cells, and human bone marrow-derived mesenchymal stem cells have been used for induction of ROS production and evaluation of upregulated efflux transporters [Cell culturing aims at mimicking sporters . Finally


Donor-specific induced pluripotent stem cells (iPSCs) offer opportunities for personalized cell replacement therapeutic approaches due to their unlimited self-renewal potential and ability to differentiate into different somatic cells. A significant progress has been made toward generating iPSC lines that are free of integrating viral vectors, development of xeno-free culture conditions, and differentiation of pluripotent stem cells (PSCs) into functional somatic cell lineages. Since donor-specific iPSC lines are genetically identical to the individual, they are expected to be immunologically matched and these iPSC lines and their cellular derivatives are not expected to be immunologically rejected. However, studies in mouse models, utilizing rejection of teratomas as a model, have claimed that syngenic iPSC lines, especially the iPSC lines derived with integrating viral vectors, could be inherently immunogenic. This manuscript reviews current understanding of inherent immunogenicity of PSC lines, especially that of the human iPSC lines and their cellular derivatives, and strategies to overcome it. Isolation of human embryonic stem cells (hESCs) from early stage embryos  and repr−/− and CD8−/− mice could form teratomas , but 20 × 106 allogenic mESC can form teratomas in all the transplanted animals  (The issue of inherent immunogenicity of human pluripotent stem cell (hPSC) lines was first examined with hESC lines, and they were shown to express low levels of MHC class I molecules but no MHC class II molecules . Drukker process . To testman PBMC  and showman PBMC . Howeveres (CTL) .−/− mice and immune-competent CD1 mice and did not find any sign of immune-reactivity at the transplantation site, evident by lack of granulocytes infiltration in Prk−/− mice and abrogation of granulocyte and lymphocyte infiltration in immune competent mice upon endotoxin administration along with hESC, suggesting that the hESCs exhibit immunosuppressive properties in vivo  ruling out an active immune response against the grafts /chimeric antigen receptor (CAR) engineered antitumor T cells that have produced remarkable clinical outcomes in cancer patients –58. Howevia tumor antigen-specific TCR and CAR engineering approaches, and these T cells have been shown to exhibit antigen-specific effector function (in vitro as well as in vivo (A significant progress has been made toward generating effector T cells, APC, NK cells, and other blood lineages from the hESC and iPSC lines –65. Funcfunction –70. Dono in vivo , 71. Whi in vivo  and the Since the isolation of first hESC lines and especially the derivation of donor-specific iPSC lines, regenerative medicine field has progressed at an exponential pace. Methods have been developed to generate donor-specific iPSC lines that are free of integrating viral vectors from different somatic cell lineages, including terminally differentiated primary immune cells, and different types of functional somatic cells have been derived, some of which have even advanced to the clinical trials stage. However, several challenges, such as the inherent immunogenicity of iPSC lines, have also been identified that need to be addressed. Since iPSC lines derived from different somatic cells and with different iPSC derivation methods can harbor different genetic and epigenetic signatures, it is necessary to fully characterize the mechanism of development and maintenance of pluripotency to generate iPSC lines with stable genomic architecture. This is important given that incomplete reprogramming could generate a neoantigen repertoire that could translate into enhanced immunogenicity of iPSC lines. Furthermore, while inherent immunogenicity of iPSC lines is not desired, iPSC-derived immune cells need to be fully functional. Therefore, according to the somatic cell lineage of choice, appropriate models need to be developed to characterize their molecular, cellular, as well as functional profiles. Addressing these issues is critical for developing safe and effective CRT.AC conceived and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, FR, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Long non-coding RNAs (lncRNAs) constitute non-protein coding transcripts with a size > than 200 nucleotides. They are involved in many cellular processes, such as chromatin remodelling, transcription, and gene expression. They play a role in the development, progression, and invasion of many human cancers, including osteosarcoma. This rare tumor entity predominantly arises in children and young adults. Treatment consists of polychemotherapy and surgical resection, increasing survival rates up to 60%. In the present review, the role of lncRNAs with prognostic, predictive, therapeutic, and diagnostic significance in osteosarcoma is discussed. Moreover, their potential application in clinical practice is highlighted. Osteosarcoma is a rare tumor entity especially affecting children and young adults . TreatmeNon-coding RNAs are a class of non-protein coding transcripts which can be divided by their function or size . Short nThe following review will provide an overview of lncRNAs involved in the pathogenesis, progression, and potential treatment of human osteosarcoma . Maternally expressed gene 3 (MEG3) is under-expressed in several human cancers, including non-small cell lung cancer, colorectal cancer and osteosarcoma [Ewing-sarcoma associated transcript-1 (EWSAT1) and downregulation of MEG3 in the presence of EWSAT1 leads to proliferation, invasion, and cellular migration of osteosarcoma cells, based on gain- and loss-of-function assays [MEG3 in human osteosarcoma tissue is associated with advanced clinical stage (I/II vs. III) and presence of distant metastasis. Moreover, low MEG3-levels correlate with poor overall survival (OS) in osteosarcoma patients [MEG3 could serve as a prognostic tissue biomarker in osteosarcoma, with high levels indicating a good prognosis.The lncRNA osarcoma ,36,37. In assays . Consequpatients . TherefoHighly upregulated in liver cancer (HULC) is upregulated in human osteosarcoma [HULC are associated with advanced clinical stage (IIA vs. IIB/III), presence of distant metastasis and poor in OS. In osteosarcoma cell lines MG-63, U2OS, OS-732, and Saos-2, HULC is significantly overexpressed as compared with the primary osteoblast cell-line hFOB1.19 [HULC via transfection of short-hairpin RNA targeted HULC #2  reduces cellular proliferation, migration and invasion, and induces apoptosis by acting as a sponge for microRNA-122 (miR-122) in MG63 and OS-732 cell lines [HULC may not only serve as a prognostic tissue biomarker in human osteosarcoma as high HULC-levels are associated with advanced clinical stage and presence of metastasis, but also constitutes a potential therapeutic target, since experimental knockdown reduces the migratory potential of osteosarcoma cell lines.Other than MEG3, lncRNA osarcoma . High lehFOB1.19 ,39. Knocll lines . On the Taurine upregulated gene 1 (TUG1) is likewise upregulated in human osteosarcoma tissue as compared to adjacent normal tissue [Taurine upregulated gene 1-overexpression measured via quantitative polymerase chain reaction (qPCR) is significantly associated with a tumor size exceeding 8 cm, advanced clinical stage and administration of adjuvant chemotherapy. Moreover, high levels of TUG1 significantly correlate with poor progression-free and overall-survival. Interestingly, TUG1 was found at significantly lower levels in serum samples of osteosarcoma-patients following surgery in comparison to serum samples taken preoperatively. Moreover, TUG1 plasma-levels correlate with disease status, with elevated levels indicating disease progression or recurrence. Of note, knockdown of TUG1 via using small-interfering RNA targeting TUG1 results in decreased cell proliferation, colony formation, enhanced apoptosis, and induced cell-cycle arrest in the G1/S-phase in U2OS and Saos-2 cell lines [TUG1 acts as an endogenous sponge to directly bind miR-9-5p and thus reduce its expression. Moreover, it diminishes expression of POU class 2 homeobox 1 (POU2F1), thus acting in a TUG1/mir-p-5p/POU2F1-axis in osteosarcoma cells (Saos-2 and U2OS cell lines) [TUG1 may not only serve as a prognostic and therapeutic, but also diagnostic and surveillance-marker in patients with osteosarcoma.The lncRNA l tissue . Taurinell lines . In the l lines) . Taken tActivated by transforming growth factor-beta (lncRNA-ATB) is involved in osteosarcoma pathogenesis via regulation of a miRNA [lncRNA-ATB are not only increased in patients with osteosarcoma, but are also overexpressed in osteosarcoma tissues and cell lines U2OS, SAOS2, MG-63, and HOS. Moreover, lncRNA-ATB targets miR-200s, thus upregulating miR-200s target genes Zinc finger E-box-binding homeobox 1 and 2 (ZEB1/2) [lncRNA-ATB results in enhanced cell proliferation, migration and invasion in the MG63 cell line. Clinically, high levels of LncRNA-ATB correlate with presence of metastasis, advanced tumor stage and local recurrence [lncRNA-ATB may be used both as a prognostic and diagnostic biomarker in the management of osteosarcoma.As with HULC and TUG1, lncRNA  a miRNA . Serum l(ZEB1/2) . Overexpcurrence . TherefoZEB1 antisense 1 (ZEB1-AS1), which is overexpressed in human osteosarcoma tissues [ZEB1-AS1-levels are associated with advanced tumor stage, a tumor size > 8 cm, presence of metastasis, poor recurrence-free survival, and poor OS. Experimentally, overexpression of ZEB1-AS1 in the osteosarcoma cell line HOS via transfection with pcDNA3.1-ZEB1-AS1 plasmid results in enhanced cellular proliferation and migration, whilst knockdown of ZEB1-AS1 by transfection with short-hairpin (shRNA)-ZEB1-AS1 has the opposite effect. ZEB1-AS1 is the antisense transcript along the ZEB1 locus and leads to expression of ZEB1 mRNA and protein in the HOS cell line. On the other hand, the knockdown of ZEB1-AS1 results in diminished ZEB1-expression in the Saos-2 cell line [ZEB1-AS1. Additionally, miR-200s-levels negatively correlate with ZEB1-AS1 and ZEB, as ZEB1-AS1 relieves the inhibition of ZEB1 exerted by miR-200s [ZEB1-AS1 could therefore serve as a marker to indicate patients’ prognosis.Another lncRNA targeting ZEB1 and miR-200s is  tissues . High ZEell line . Of notemiR-200s . In clinFGFR3 antisense transcript 1 (FGFR3-AS1) [FGFR3-AS1 leads to upregulation of Fibroblast growth factor receptor 3 (FGFR3) expression via antisense pairing with the 3′ untranslated region (3′UTR) of FGFR3. Moreover, the knockdown of FGFR3-AS1 via transfection of MG63 cell line with the FGFR3-AS1 shRNA expression plasmid pGPU6/GFP/Neo inhibits cell cycle progression and proliferation in-vitro. Moreover, tumor growth is likewise reduced in-vivo, using xenograft models implanted with FGFR3-AS1 stably knocked down MG63 cells [FGFR3-AS1 may be used in clinical practice as a molecular tissue marker for predicting prognosis of patients with osteosarcoma.Another lncRNA upregulated in human osteosarcoma tissue is FR3-AS1) . On the 63 cells . Thus, FLung cancer associated transcript 1 (LUCAT1) is specifically overexpressed in osteosarcoma cell-lines resistant to methotrexate (MTX), one of the most effective drugs used in treatment of patients with osteosarcoma [LUCAT1 does not only diminish expression of resistance-related genes MDR1, MRP5, and LRP1 during treatment with MTX, but also reduces the number of invasive cells. The interaction between LUCAT1 and ABCB1 is mediated via miR-200c, which binds to the 3′UTR of ABCB1 and is itself regulated by LUCAT1 [LUCAT1-levels could serve as predictive biomarkers in patients with osteosarcoma regarding MTX-sensitivity, provided that further experiments show that LUCAT1 is also overexpressed in human osteosarcoma tissue samples. The lncRNA osarcoma . Cell liy LUCAT1 . ConsequForkhead box protein C2 antisense 1 (FOXC2-AS1) is overexpressed both in human osteosarcoma tissues and cell-lines MG63 and KH-OS resistant to doxorubicin (DXR), another important chemotherapeutic agent used in osteosarcoma treatment [FOXC2-AS1 overexpression in human osteosarcoma tissues correlates with a poor patient outcome and promotes DXR-resistance in vitro using MG63 and KH-OS cell lines [FOXC2-AS1 improves the sensitivity of osteosarcoma cells to DXR. Moreover, FOXC2 is upregulated in DXR-resistant osteosarcoma cell lines MG63/DXR as well as KH-OS/DXR and human tissues and its levels positively correlate with FOXC2-AS1-expression. As with FOXC2-AS1 knockdown, the inhibition of FOXC2 results in diminished resistance of osteosarcoma cells deriving from the cell lines MG63/DXR and KH-OS/DXR to DXR. At the same time, FOXC2 contributes to the resistance of osteosarcoma by inducing expression of ABCB1. Therefore, FOXC2-AS1 and FOXC2 seem to contribute to DXR-resistance in osteosarcoma cells by both enhancing ABCB1-expression [FOXC2-AS1 may not only serve as a predictive biomarker in osteosarcoma indicating resistance to DXR but also as a therapeutic target to increase sensitivity to DXR.Long non-coding RNA reatment . FOXC2-All lines . On the pression . TherefoMFI2 is overexpressed in osteosarcoma tissue [MFI2 seems to regulate Forkhead box P4 (FOXP4), thus promoting proliferation and migration of osteosarcoma cells [MFI2 may be of value in osteosarcoma in order to reduce tumor progression.The lncRNA a tissue . Its knoma cells . TherefoBreast cancer anti-estrogen resistance 4 (BCAR4) has both prognostic and therapeutic significance in osteosarcoma [BCAR4 in-vitro using cell lines MG63 and U2-OS inhibits osteosarcoma cell proliferation and migration by inhibiting the transcription of GLI family zinc finger 2 (GLI2)-dependent target genes. Depletion of BCAR4 results in reduced transcription of TGF-beta1, IL-6, RPS3, and MUC5AC, all being target genes of GLI2 [BCAR4 could serve as a therapeutic target in the treatment of osteosarcoma, as this would inactivate the GLI2 pathway and GLI2-dependent target genes.The lncRNA osarcoma . On the osarcoma ,41. On t of GLI2 . ConsequMetastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), one of the first lncRNAs identified, is overexpressed in osteosarcoma cell lines Saos-2, U2OS and HOS compared with the osteoblast cell line hFOB1.19 (p < 0.05), and is overexpressed in human osteosarcoma tissues [MALAT-1 results in decreased cell proliferation and migration by inducing cell cycle arrest from the G0/G1 to S-phase and apoptosis in osteosarcoma cell lines U2OS and HOS. Moreover, the knockdown of MALAT-1 reduces the formation of acting stress fibers and vasculogenic mimicry (VM) in three-dimensional cultures, referring to the ability of osteosarcoma cells to form alternative circulatory systems [MALAT-1 seems to exert its oncogenic function via regulating the Ras homolog gene family, member A (RhoA)/Rho-Kinase (ROCK)-pathway, as downregulation of MALAT-1 reduces protein levels of RhoA, ROCK1 and 2 in osteosarcoma cell lines U2OS and HOS. Moreover, the downregulation of MALAT-1 reduces tumorigenesis in mice implanted with osteosarcoma cell lines HOS and U2OS cells transfected with MALAT-1 siRNA in comparison to mice implanted with HOS and USO2 cells transfected with non-specific siRNA. Another effect of MALAT-1-knockdown is the inactivation of the Rac1/JNK-pathway via activation of miR-509 and the downregulation of High-mobility group protein B1 (HMGB1) [MALAT-1 may constitute a therapeutic target in the management of osteosarcoma, in order to inhibit cellular proliferation, migration, and invasion.Long non-coding RNA  tissues . Knockdo systems ,42. MALA (HMGB1) ,44. BothP21-associated ncRNA DNA damage activated (PANDA), which is overexpressed in the osteosarcoma cell-line U2OS [PANDA-depletion reduces the number of viable tumor cells but has no effect on apoptotic cells. Moreover, silencing of PANDA leads to an upregulation of cyclin-dependent kinase-inhibitor p18 mRNA and an accumulation of cells in the G1-phase of the cell-cycle whilst count of cells in the S and G2/M-phase is decreased [PANDA could be used in practice to induce cell-cycle arrest in the G1/S-phase in osteosarcoma cells.Another lncRNA involved in cell-cycle regulation is ine U2OS . It is iecreased . ConsequMALAT-1 and PANDA, lncRNA Cancer susceptibility candidate 2 (CASC2) is significantly downregulated both in human osteosarcoma tissue and osteosarcoma cell lines MG-63, U2OS, SAOS2, and SOSP-9607 . The latter one is positively correlated with expression of CASC2 and consequently expressed at low levels in osteosarcoma. At the same way, experimental overexpression of CASC2 results in reduced infiltration, colony formation, and cellular growth in osteosarcoma cell-lines [CASC2-mimics may be used in clinical practice to diminish tumor growth and progression.Other than ll-lines . Moreovell-lines . TherefoNeuroblastoma associated transcript 1 (NBAT1) is likewise downregulated in osteosarcoma tissues and five osteosarcoma cell lines  [NBAT1-levels correlate with clinical stage, presence of distant metastasis and a poor patient outcome. Experimental overexpression of NBAT1 results in decreased proliferation, migration, and invasion, whilst knockdown leads to enhanced proliferation, invasion, and migration in vitro. In xenograft models, overexpression of NBAT1 is associated with lower tumor volumes and slow growth, whilst NBAT1-silencing results in enhanced tumor growth. Interestingly, NBAT1 upregulates PTEN, PDCD4, PTM1, and RECK via an inactivation of miR-21, whilst overexpression of miR-21 reverses this effect [NBAT1-mimics could serve as therapeutic agents to reduce tumor growth and progression.d MG-63) . Low NBAs effect . In clinNBAT1, MEG3, and CASC2, or be overexpressed, such as MFI2, BCAR4, and HULC. They may serve as predictive  prognostic , therapeutic  and even diagnostic  markers. Especially the latter group is of interest, considering the evolving role of liquid biopsies in the management of osteosarcoma. Liquid biopsies subsume minimally-invasive measures as urine-sampling and blood-tests that are taken to detect and monitor human cancers, with the big advantage for patients of getting by without invasive diagnostic methods as tissue biopsy and explorative surgery. As prognostic biomarkers, lncRNAs can aid prognostication regarding outcome of patients with osteosarcoma, thus eventually guiding treatment decision for or against more aggressive therapeutic approaches. Moreover, predictive lncRNAs may help clinicians to change treatment protocols. Considering that the therapy of patients with osteosarcoma has not significantly changed over the last years, lncRNAs with therapeutic significance may constitute promising targets in the treatment of patients with osteosarcoma. Yet, it has to be borne in mind that far not all lncRNAs can be detected via minimally-invasive methods  and thus still rely on conventional detection methods as tumor biopsy or analysis of the entire tumor specimen. Consequently, further studies are warranted focusing on the applicability of lncRNAs as minimally invasive biomarkers, by detecting them through the blood stream, urine, or even saliva. Moreover, result obtained in experimental studies using osteosarcoma cell lines cannot be simply transferred into the clinical setting. However, these results give a hint towards a potential clinical applicability of lncRNAs, be it as therapeutic targets or prognostic, predictive, and diagnostic biomarkers. Several long non-coding RNAs are involved in the development, progression and invasion of osteosarcoma . They ma