In this study we prepared a novel formulation of liposomal doxorubicin (L-DOX). The drug dose was optimized by analyses of cellular uptake and cell viability ofosteosarcoma (OS) cell lines upon exposure to nanoliposomes that contained varyingDOX concentrations. We intended to reduce the cytotoxicity of DOX and improve characteristics of the nanosystems.in vitro transfection efficacy. In this experimental study, we prepared liposomes by the pHgradient hydration method. Various characterization tests that included dynamic light scattering (DLS),cryogenic transmission electron microscopy (Cryo-TEM) imaging, and UV-Vis spectrophotometry were employed to evaluate the quality of the nanocarriers. In addition,the CyQUANT® assay and fluorescence microscope imaging were used on variousOS cell lines  and Human primary osteoblasts cells,as novel methods to determine cell viability and  We observed an entrapment efficiency of 84% for DOX within the optimizedliposomal formulation (L-DOX) that had a liposomal diameter of 96 nm. Less than 37%of DOX released after 48 hours and L-DOX could be stored stably for 14 days. L-DOXincreased DOX toxicity by 1.8-4.6 times for the OS cell lines and only 1.3 times for Humanprimary osteoblasts cells compared to free DOX, which confirmed a higher sensitivity ofthe OS cell lines versus Human primary osteoblasts cells for L-DOX. We deduced that L-DOX passed more freely through the cell membrane compared to free DOX.We successfully synthesized a stealth L-DOX that contained natural phospholipid by the pH gradient method, which could encapsulate DOX with 84% efficiency.The resulting nanoparticles were round, with a suitable particle size, and stable for 14days. These nanoparticles allowed for adequately controlled DOX release, increased cellpermeability compared to free DOX, and increased tumor cell death. L-DOX provided anovel, more effective therapy for OS treatment. Cancer is the cause of a majority of serious health problems and mortality worldwide. Osteosarcoma (OS), the most common primary malignant tumor of the bone, has a worldwide incidence of approximately 1 to 3 cases annually per million, and comprises almost 60% of all bone sarcomas . CurrentIn this research, we attempted to enhance DOX importation into several OS cell lines by developing a novel PEGylated DOX- loaded liposome to heighten the intracellular nanosystem uptake. We analyzed this nanosystem in an attempt to improve treatment efficacy and, in particular, reduce toxicity. We also employed this novel method to decrease the toxicity of DOX against normal cells by dose optimization of DOX. DOX hydrochloride (DOX-HCl) was obtained from Ebewe Pharma (Austria). Egg phosphatidylcholine (EPC), and derivatized distearyl phosphatidylethanolamine (mPEG2000- DSPE) were obtained from Lipoid GmbH . Cholesterol (CHOL), fluorescent label (Dil), and phosphate-buffered saline  were purchased from Sigma- Aldrich Co. . Dialysis bags (MWCO 12000-14000) were supplied by Jingkehongda Biotechnology Co., Ltd. . Chloroform, ethanol and other chemicals used in this study were analytical grade. We used the pH gradient hydration method to prepare the liposomes. EPC, CHOL, and DSPE- mPEG (67.9:29.1:3 molar ratios) were dissolved in chloroform, and the mixture was warmed to 55˚C. We added Dil to the lipid phase at 0.1 mol% for lipid staining in order to evaluate cellular uptake. The solvent was evaporated under vacuum in a rotary evaporator until a thin-layered film formed. Once prepared, the film was hydrated with 250 mM of ammonium sulfate. The hydrated film was extruded 5 times through 0.2 μm pore size polycarbonate membranes, and 5 times through 0.1 μm pore size polycarbonate membranes using a mini-extruder in order to decrease liposomal particle size. Various doses of DOX  were loaded into the liposomes. Afterwards, free DOX (unloaded) was separated from liposomal DOX (L-DOX) by dialysis bags that had a cut-off of 12 kDa. Figure 1 shows a schematic diagram of the experimental set-up. The particle size distribution and polydispersity index (PDI) of the liposome particles was determined at 25˚C by dynamic light scattering (DLS) using a ZetaPALS zeta potential and particle size analyzer . Each parameter was measured three times, after which we calculated average values and standard deviations. L-DOX morphology was investigated by cryogenic transmission electron microscopy . The L-DOX particles were lysed with Triton X-100 to analyze DOX concentrations. The DOX encapsulation efficiency of the L-DOX collected fractions was determined by measuring absorption at 480 nm with a UV-Vis spectrophotometer . In order to determine the encapsulation efficiency, we used the following formula: Encapsulation efficiency %= ( Loaded drug in liposome (mg))/)×100We evaluated stability of the L-DOX suspension by particle size analysis and drug encapsulation efficiency of the samples after storage at 4˚C for 14 days. We used dialysis bags (MWCO 12000) to monitor the amount of drug released from the liposomes against PBS  for 48 hours at 37˚C and pH=7.0. Samples of medium were taken at different times and replaced with the same volume of PBS to evaluate the DOX release rate from the liposomes. Samples were analyzed via UV-Vis spectrophotometry at 480 nm. UV absorption showed a linear response in the range of 0.5-50 µg/ml. For each sample we calculated the total concentration of drug loaded in a liposome formulation and percentage of released drug. The nanoparticle bilayer structure and round shape was examined by Cryo-TEM  at 200 kV. 2 in a humidified incubator. LM7 was cultured in E-MEM (Lonza) supplemented with 10% FCS, 1 mg/mL Pen-Strep, 1% non- essential amino acids, 1% sodium pyruvate, 2 nM L-glutamine, and 2% MEM vitamin solution  at 37˚C and 5% CO2 in a humidified incubator. DOX was diluted in RPMI-1640 to the desired concentrations prior to use. RPMI-1640 cell culture medium was purchased from Gibco, Invitrogen . 4´,6-diamidino-2-phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were provided by Thermo Fisher Scientific  and Sigma-Aldrich . The MG-63 cell line was supplied by Pasteur Institute (Iran). The SaOS-2 cells were kindly provided by Dr. F. van Valen , and U2-OS by Dr. S. Lens . SaOS-LM7 cells were kindly provided by Dr. E.S. Kleinerman . Human primary (short-term culture: passage<10) osteoblasts Human 54 were obtained from healthy patients undergoing total knee replacement after they provided informed consent. All cells, with the exception of SaOS-LM7, were cultured in RPMI-1640 medium  supplemented with 10% fetal calf serum (FCS), and penicillin/streptomycin  at 37˚C and 5% COHuman primary osteoblasts cells and the OS cell lines  were seeded on 96-well plates for one day to allow for cell attachment. We replaced the media with media that contained a dilution series of: i. Blank liposomes, ii. DOX, or iii. L-DOX. DOX concentrations varied from 0.1 to 10 mg/mL in (ii) and (iii). Cells were further incubated for 72 hours. Then, the cells were washed with PBS. After removal of PBS, we added 300 μl/well of CyQUANT® lysis buffer supplied with the CyQUANT® kit, and samples were subsequently stored at -20˚C until further analysis. We performed 3 freeze and thaw cycles for the samples. Then, total DNA of duplicate samples was measured with the CyQUANT® cell proliferation assay kit according to the specifications provided by the manufacturer . After exposure, fluorescence measurements were determined using a SynergyTMHT multi-mode microplate reader . 5 SaOS-2 and MG-63 cells to grow in six-well plates in a monolayer for 24 hours. Cells were washed with RPMI-1640 medium and incubated with L-DOX (10 μg/ml) for transfection for 3 hours at 37˚C. The cells were rinsed three times with PBS and fixed with a 4% paraformaldehyde solution . For nuclear counterstaining, we used DAPI (0.125 µg/mL) for 15 minutes. Cellular uptake of DOX was evaluated with a fluorescence microscope . We allowed 5×10High encapsulation efficiency of a low dose therapeutic agent is an important parameter for enhancement efficiency of large-scale production of the nanodrug, and in terms of reducing toxicity against normal cells. Table 1 shows the effects of various DOX doses as a model of a hydrophilic drug. The encapsulation efficiency increased with decreasing DOX doses. Decreasing the drug dose slightly increased the zeta potential. The drug dose did not affect agglomeration of the vesicle. The vesicle size decreased with decreasing drug doses. We selected the 0.5 mg/ml dose of DOX for further analysis. in vitro release studies, we observed 37% release of DOX after 48 hours. The drug release profile increased with a gentle slope  in diameter size (103 nm) with no morphological changes for L-DOX, which confirmed the stability of the presented nanoformulation. There was an 84.32% encapsulation efficiency of DOX into the liposomal vesicle, which slightly reduced (less than 2%) to 83.09% after storage. In the le slope . Figure 3 shows the Cryo-TEM micrograph of the liposome vesicles. The biliary structure (hydrophobic and hydrophilic spaces) of liposomes was clear. DOX was entrapped inside the bilayer membrane, restricted to the hydrophilic part of the liposomal vesicle. Cryo- TEM analysis confirmed the low diameter size and round vesicle shape. 50) of DOX on all cell lines was 5-10 μg/ml (free DOX) and 0.1-1 μg/ml (L-DOX). Figure 5 provides a comparison of cell viability of DOX  on various cell lines. The cell viability of free and L-DOX decreased in the following order for the 1 µg/ ml concentration of DOX: SaOS-2 cells>SaOS- LM7 cells>U2-OS cells>MG-63 cells>Human primary osteoblasts cells. We observed a similar trend for the other DOX concentrations. As expected, DOX had fewer side effects on Human primary osteoblasts cells. We analyzed the viability of free DOX and L-DOX after 72 hours by the CyQUANT® assay on various cell lines . Empty lin vitro transfection image of free DOX and L-DOX on SaOS-2 and MG-63 cell lines monitored by fluorescence microscope. Qualitatively, cells (for both cell lines) treated with L-DOX had greater purple intensity compared to those treated with free DOX. This was particularly observed for the MG-63 cell line. According to Figure 6A and B, greater numbers of cells contained L-DOX. L-DOX transfection showed more DOX distribution in the nuclear region compared to the cytoplasm. Figure 6C confirms the successful cellular uptake of L-DOX for both OS cell lines. Liposome vesicles stained with Dil was accumulated around the nucleus. According to the literature, DOX acts as an apoptosis inducer by down-regulation of topoisomerase II  and mutual repelling force between nanoparticles in the suspension system. in vivo stability. Garbuzenko et al.  particles, increasing the drugdose to 0.5 mg/ml would generate a strong drugconcentration gradient, which supported drugentrapment. Surprisingly, in the case of high drugdosages (1 or 1.5 mg/ml), we observed reducedencapsulation efficiency. We have postulated twopossibilities for the decrease in encapsulationefficiency when the liposome membrane isfaced with an over-capacity of drug. The drugconcentration gradient reverses direction and ananti-encapsulation effect occurs. The amount ofdrug loaded is the same value for all doses but thedenominator (equation 1) has increased. Thus, theencapsulation efficiency deceased. Increased zetapotentials by increased drug dosages might beexplained by approaching to the slipping plane.The liposome formulation synthesized by Eggphosphatidylcholine, CHOL and DSPE-mPEG asthe main components, appeared to be nontoxic inthe cytotoxicity assays. However when DOX wasentrapped, the L-DOX nanoparticles accomplisheda more effective tumor cell kill compared to thefree form for all OS cell lines. Compared to the OScells, Human primary osteoblasts cells experiencedless toxic effects, as hoped. Apart from differentialsensitivity between Human primary osteoblastscells and OS cells, the controlled release of DOXand targeting towards cells were consideredimportant features for L-DOX formulations thatminimized DOX adverse effects. Targeting L-DOXparticles appeared possible by the use of naturalphospholipid (EPC) due to its high similarity withthose present in the cell membrane. Thus, thedevelopment of improved L-DOX synthesized byincorporation of egg phospholipid for the treatmentof OS appeared useful.We selected the SaOS-2 and MG-63 cell lines for thecellular uptake assay as models of sensitive, resistantOS cells. According to the cell viability assay, theSaOS-2 cell line showed the most sensitivity for DOXcompared to the other OS cell lines, with the MG-63cell line as the most resistant. Fluoroscopic imagingof particle uptake confirmed this result. There wasmore intense, widespread purple signal observed inSaOS-2 cells versus MG-63 cells. Interestingly, themorphology of both the SaOS-2 and MG-63 cellschanged upon DOX treatment.There was a slow rate of DOX release from theliposome vesicles (less than 2% within 3 hours).Depicts cells treated with L-DOX within 3 hours forthe cellular uptake assay. In comparison with drugrelease profile results, most of the red ﬂuorescencedetected by fluorescence microscopy might berelated to L-DOX, but not to DOX already releasedfrom liposomes. The negative value for the zetapotential showed that an electrostatic force did nothave any influence on the enhancement of cellularuptake due to the negatively charged cytoplasm.We successfully synthesized a stealth L-DOXthat contained natural phospholipid (EPC) by thepH gradient method, which could encapsulateDOX (0.5 mg/ml) with 84% efficiency. Theresulting nanoparticles had a round shape, suitableparticle size of 96 nm, 14-day stability, adequatelycontrolled DOX release (37% DOX after 48 hours),increased cell permeability compared to free DOX,and increased tumor cell kill. Hence, they couldprovide a novel, more effective therapeutic for OStreatment.


Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts . We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis.Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene,  Cutaneous melanoma is a highly aggressive malignancy that originates from melanocytes and accounts for 75% of skin cancer related deaths . AlthougBRAF and NRAS) and other accompanying non-pathogenic mutations (passenger mutations) have been identified [Remarkable progress has been made over the last two decades in understanding the genetic basis of melanoma tumorigenesis. Genetic mutations , as well as between tumors from different patients (inter-patient). As DNA methylation profiles are highly tissue-specific, and can vary extensively from cell to cell in a tissue, this heterogeneity contributes to the complexity in identifying epigenetic factors involved in tumor progression, and in identifying novel epigenetic prognostic or therapeutic biomarkers . In ordeAlthough several mutational studies have been performed in matched primary and metastatic cancers and cell lines, very few genome-scale DNA methylation sequencing studies of similarly matched samples have been carried out. More importantly, methylation changes identified in the context of metastasis have frequently lacked functional studies, an important link between epigenetic changes and phenotype. In the present study we sought to identify epigenetic changes associated with metastasis, so as to identify putative “epigenetic drivers” of metastasis. To this end, we used sequencing based genome-wide reduced representation bisulfite sequencing (RRBS) DNA methylation analysis of metastatic melanoma cell lines, together with paired primary melanoma cell lines derived from the same patients, to identify metastasis-associated DNA methylation changes, which were then followed up by functional analysis of observed changes at the mRNA level.To identify epigenetic changes occurring in metastatic melanoma, compared to the primary tumor of origin, we profiled whole genome methylation in three primary cutaneous melanoma and three matched metastatic melanoma cell lines. The primary melanoma cell lines were WM115, Hs688(A).T, and WM75, and the matching metastatic melanoma cell lines were WM266-4, Hs688(B).T, and WM373, respectively. In addition, Mel-ST was included as a normal melanocyte cell line . To asser = 0.96) and WM-115 and WM-115-2 (r = 0.98) replicate libraries  as the unit of analysis rather than individual CpG sites or a tiled window approach, as we have described previously [It is common for DNA methylation studies to collectively compare differences in DNA methylation between two groups of samples (such as primary and metastatic tumors). However, because the 3 pairs of cell lines each contained distinct epigenomes Figure , we perfeviously –21. FragIn all three cases, we found primary melanoma cell lines were overall hypomethylated compared to normal melanocytes . We alsoCBX8, EXOC3L2, HES5, and POU3F2) as representative examples to demonstrate that the results from RRBS could be validated in mass spectrometry-based Sequenom analysis (EpiTYPER) [r = 0.88, two-tailed test, P-value = 0.0001, Figure r = 0.98, Figure P-value < 0.05, Figure We chose four genes with their associated DMFs (piTYPER) . These rShared methylation changes between primary and metastatic cell lines were identified from the lists of DMFs. The percentage of overlap of DMFs between any two cell-line pairs varied from 10.2% to 28.2% –S10. OveOf the 249 shared metastatic DMFs, 65 were hypomethylated (associated with 59 genes) and 10 were hypermethylated (associated with 9 genes) in each metastatic cell line versus the matched primary melanoma cell line  and S2. (GABP) and TATA box binding factor (TAF1) sites were enriched in hypermethylated DMFs  in all metastatic cell lines. Suppressor of glucose autophagy associated 2 (CCDC165 or SOGA2) contained two adjacent hypermethylated DMFs in the first intron (within 1kb from the TSS). Both EBF3 and SOGA2 DMFs were situated within core CpG islands. Functional enrichment analysis of the genes that contained hypermethylated DMFs (in promoters or gene bodies) indicates that they were mainly involved in cellular organisation, intracellular signalling and transcriptional regulation (HMGA1), Kv Channel Interacting Protein 3 (KCNIP3) and Apolipoprotein B (APOB) shared promoter hypomethylation in all metastatic melanoma cell lines. HOXD11 was consistently hypomethylated, while ROBO2 shared four intronic hypomethylated DMFs in all metastatic cell lines , was observed for early B cell factor 3  in TCGA. On the other hand, three genes  were significantly hypomethylated in metastatic patients -SKCM (melanoma) dataset, which contains 450K-DNA methylation microarray data for 458 patients (99 primary and 359 metastatic tumors). The CpG sites in the 450K-microarray platform are unevenly distributed in the genome, being mainly contained within promoter regions. For a comparable analysis, we analysed individual CpGs from TCGA 450K data that directly overlapped with, or were adjacent to our identified DMFs (i.e. CpGs within 500 bp upstream or downstream of the DMF) in primary and metastatic melanoma patients. For 26 (i.e. for 5 hypermethylated DMFs and for 21 hypomethylated DMFs) out of the 75 common DMFs identified in RRBS, we found a comparable CpG site that directly overlapped or was adjacent in the TCGA dataset. Similar to the RRBS analysis, significant hypermethylation in metastatic patients was confirmed for three genes . The methylation profiles of the CpGs in commonly analysed DMFs, located in the EBF3 promoter in metastatic melanoma cell lines, we performed locus specific bisulfite cloning and sequencing in four cell lines .T). The results confirmed the EBF3 promoter DMF hypermethylation in the metastatic melanoma cell lines, and we observed excellent concordance for the DMF methylation between RRBS and the bisulfite sequencing  was significantly greater than promoter methylation of primary melanomas or normal melanocytes .T, and WM266-4, Hs688(B).T). Unfortunately, the third cell line pair (WM75 and WM373) was extremely slow growing, and as a consequence no further analysis (including RNA extraction) could be carried out using this cell line pair. Surprisingly, EBF3 mRNA levels were significantly higher in the two metastatic versus the primary cell lines .T versus Hs688(A).T was 29.5-fold higher, P-value = 8.5e–16). The observation that the WM266-4 promoter was relatively more highly methylated compared to the other cell lines . Taken together, these results suggest that high EBF3 promoter methylation is associated with higher levels of EBF3 expression in metastatic melanomas.To determine whether s Figure . , which inhibits DNA methylation in a replication-dependent manner. Following decitabine treatment, we confirmed demethylation of the DMF region in the EBF3 promoter .T cell lines , the WM115 cell line also showed a reduction (37.1%) in mRNA level .T, WM266-4, Hs688(B).T) and in four additional NZM metastatic melanoma cell lines , which were included to facilitate the investigations of EBF3 function. The metastatic NZM melanoma cell lines were comprised of both invasive and non-invasive cell lines, which we have characterised previously [EBF3 resulted in 75% to 90% knockdown of EBF3 mRNA, except in NZM6 and NZM40 where the knockdown efficiencies were 42% and 60% respectively  treated cells ranged from 86.5% to 95.2% as determined by trypan blue staining 72 h following EBF3 knockdown, while the mean viability for control siRNA treated cells (siNeg) ranged from 88.5% to 95%, and viability continued to be high at 96 h following knockdown. Cell viability data were also confirmed by caspase-3 assays (data not shown). Overall, these data suggest that reduction in MTT values following EBF3 knockdown were not due to cell death, but rather were due to inhibition of total cell mass, for example through inhibiting proliferation  at 48 h  at 48 h following EBF3 depletion  were positively correlated with the fold-reduction in migration or invasion  by real-time PCR in the melanoma cell lines following EBF3 knockdown  and poor clinical outcome [In a similar approach, using 450K-DNA methylation arrays to analyse multiple cancer types , shared “driver” epigenetic differences were identified between 3 pairs of metastatic and primary cancer cell lines. In metastatic tumors, site-specific hypomethylation was associated with reactivation of a cryptic transcript of TBC1 Domain Family Member 16  comparing the frequency of commonly shared DNA methylation alterations of genes/regions with other genes/regions in multiple samples, and (ii) measuring effect sizes or degree of epigenetic changes to determine the functional role of each epigenetic change. Similar to frequency-based approaches used to identify cancer driver mutations , we hypoDNMT, TET family genes, or EZH2, lead to epigenomic and trancriptomic alterations that differ from tumors without these mutations [RRBS analysis greatly expands the characterization of global methylation patterns in melanoma, as compared to array-based platforms. In the latter, predominantly promoter methylation changes are characterized , 14, 15,utations .EBF3 was hypermethylated in all three metastatic melanoma cell lines. An adjacent EBF3 promoter region, approximately 200–300 bp upstream from the region identified by RRBS, was investigated in TCGA melanoma data, and was also found to exhibit high levels of DNA methylation. Promoter-specific hypermethylation is associated with tumor suppressor gene silencing in cancer [The promoter of n cancer , and hasn cancer , 28, 29.EBF3 promoter hypermethylation in the metastatic melanoma cell lines was unexpectedly associated with elevated (rather than decreased) EBF3 expression, and this was also observed in melanoma patients in the TCGA database. Analysis of TCGA data suggested that EBF3 was relatively highly expressed in a subgroup of metastatic melanomas as compared to primary melanomas. EBF3 plays an important role in cell lineage commitment, migration and differentiation of several cell types, including B-lymphocytes and neurons [EBF3 promoter (at a region between +700 and +1030 downstream from the transcription start) and down-regulation of EBF3 expression in contrast to up-regulation, as observed in our study, has been reported in several cancers [EBF3 has previously been postulated to be a tumor suppressor gene [EBF3 may have oncogenic tumor promoting properties associated with melanoma metastasis. An oncogenic role for EBF3 has been reported in several other cancer types, including elevated expression of EBF3 in phaeochromocytoma. In addition, forced expression of EBF3 in medulloblastoma cancer stem cells, or in HepG2 hepatocellular carcinoma cells was associated with enhanced tumorigenesis, or increased cell cycling, respectively [ neurons , 31. Hyp cancers –34. Therectively –37.EBF3 gene expression was associated with demethylation of the EBF3 promoter DMF, supporting the notion that reducing methylation of the EBF3 promoter causes reduction of the corresponding EBF3 mRNA levels.When melanoma cell lines were treated with a demethylating agent, decitabine, reduction in EBF3 knockdown in our functional studies was associated with reduced MTT values, migration and/or invasion in half (4 of the 8) of the metastatic melanoma cell lines investigated . In the remainder of the melanoma cell lines this effect was not observed, although in some cases the melanoma cell lines did have a relatively high initial level of EBF3. Moreover, in the cell lines where the knockdown led to a reduction in migration or invasion, EBF3 knockdown was also associated with reduced MTT values , a transcriptional repressor, which is known to bind to the 5′ promoter region of EBF3 at approximately 1,870 nucleotides upstream of the EBF3 transcription start. The binding of ARX in neuronal cells was sufficient to repress endogenous expression of EBF3 [EBF3 expression. We hypothesize that transcriptional repression is abrogated by the EBF3 promoter methylation, leading to elevated EBF3 expression in metastatic melanoma.Known transcription factor binding sites near the differentially methylated  of EBF3 , 39. TheEBF3 was identified as a candidate oncogenic metastasis epi-driver. A number of other common significant differential methylation changes between primary and metastatic melanomas were also identified, and will need further investigation to determine whether they may play a role in melanoma metastasis.This study demonstrates a possible approach to identify candidate epigenetic drivers. Additional studies involving greater numbers of paired primary/metastatic cancer samples will be required to identify further candidate epigenetic drivers of melanoma metastasis. From the present RRBS analysis of paired primary and metastatic melanoma cell lines, ® CRL-1675™), WM266-4 (ATCC® CRL-1676™), Hs688(A).T (ATCC® CRL-7425™), and Hs688(B).T ATCC® CRL-7426™). These cells were received in October 2013, together with ATCC certificates of analysis and authentication. Low passage number cells were used at all stages of this project. These cells were cultured in Minimum Essential Medium (MEM-α) (Invitrogen) supplemented with 1% penicillin-streptomycin  and 10% FBS. Four additional melanoma cell lines were generously provided by B. Baguley (University of Auckland) in May 2015; NZM6, NZM9 NZM11 and NZM40, and were cultured in MEM-α media supplemented with 1% penicillin-streptomycin, 5% FBS and 0.1% Insulin-transferrin-selenium (Roche). These cell lines have very recently been tested and authenticated. All cells were grown under standard conditions , except WM115, which were cultured at 35°C. One of the primary/metastasis cell line pairs (WM75 and WM373), which was included in our studies, was extremely slow growing. We received only DNA for these cell lines, which was generously provided to us by Patricia Brafford and Dr Meenhard Herlyn . As a consequence, performing functional experiments with WM75 and WM373 was not feasible, and only DNA level data was reported for this cell line pair.Mel-ST cells  were generously donated in January 2007 by Professor Robert Weinberg , and cultured in DMEM plus 10% fetal bovine serum (FBS). This cell line has not since been tested for authentication. Four melanoma cell lines used in this study were obtained from America Type Culture Collection : WM115 , bisulfite converted and PCR amplified (16–18 cycles). Following purification, libraries were quantified and the quality was assessed. Single-ended RRBS libraries with 100 bp read length were sequenced on an Illumina HiSeq2500.RRBS libraries for the cell lines were prepared according to previously published methods –43. GenoSequenced reads were assessed using FastQC. Quality trimming and adaptor trimming were performed using the fastq_quality_trimmer and our in-house cleanadaptors program , respectFollowing alignment, fragment-based methylomes were generated using DMAP . Correlahttp://www.epidesigner.com/). The sequences of the primers are given in Primers were designed using the EpiTYPER assay designer  as previously described [EBF3 siRNA (siEBF3)  was used to knockdown EBF3 mRNA levels. Sequences were 5′-GCAGGCAACCCUCGAGAUA-3′. 5′-CAUCAUAAUUGGCGACAAC-3′, 5′-UCUUUGAUCUGUUUCGUUA-3′, 5′-CAACCAUAGAUUACGGCUU-3′. The ON-TARGET-plus Nontargeting Pool (siNeg)  was used as a negative control. 2 μl of Lipofectamine RNAiMAX and 2–10 nmol/L of siEBF3 were used for each well of a 6-well-plate, in 500 μl of OPTI MEM I media and 1500 μl of cells (3 × 105/mL) suspended in the appropriate culturing medium, without the addition of antibiotics (penicillin-streptomycin), as this can interfere with the reverse transfection. Cells were transfected for 48 hours. Real-time PCR was used to determine the knockdown efficiency at 48 h, 72 h and 96 h post-transfection (in Hs688(A).T and WM115 cell lines). For WM115 the knockdown was maintained until after 96 h. For Hs688(A).T the knockdown was maintained until after 72 hours, but was lost at 96 hours post-transfection. Efficient knockdown was observed at 48 h and 72 h, but not 96 h post-transfection. Cell viability following EBF3 knockdown was assessed by trypan blue exclusion assays, as described previously [siEBF3 treated cells ranged from 86.5% to 95.2%, while the mean viability for siControl (siNeg) treated cells ranged from 88.5% to 95%.siRNA-mediated knockdown of escribed . ON-TARGEBF3 expression levels were analysed using the qBase software and the delta-delta-Cq method [EBF3 expression were: F: 5′- AGATCTTGCTCTGTTCTGACTC-3′, and R: 5′- GCTTTTGTGGACTTTGTGGAG- 3′. Expression of EBF3 was normalised to house keeping genes RPL27 and SPR14. Primers for RPL27 were: F: 5′- TGGCTGGAATTGACCGCTA- 3′, and R: 5′-CCTTGTGGGCATTAGGTGATTG-3′ and for SRP14 were: F: 5′- ACGGAGCTGACCAGACTTTTC -3′ and R: 5′- TGGTTCGACCGTCATACTTCTT -3′. Details of primers used for real time analysis of the six EMT related genes are given in Expression levels of mRNAs following knockdown were determined by (RT-QPCR), as previously described  using SYq method . qPCR prEBF3 gene as described previously [Demethylation experiments: Demethylation in the described cell lines was induced with 5-Aza-2′-deoxycytidine . First, we performed a viability assay (based on trypan blue staining) at different concentrations of the drug and chose 5 μM as the optimal concentration to induce demethylation. Cells were treated at 5 μM for 72 h and DNA and RNA were extracted following this treatment. The experiments were performed in triplicate. RT-PCR experiments were performed for the eviously .EBF3 promoter (993 bp from the TSS); EBF_993_F: 5’-GGAGTTAATTGTTTTAAAAATTAA G- 3′ and EBF _993_R: 5′-ATTTTCTCTTAACAAAAAAATACCTAAAAC-3′. Amplified bisulfite genomic DNA products were cloned and individual cloned plasmids were sequenced using Sanger sequencing to confirm demethylation of the EBF3 promoter, as described previously [Primers for bisulfite genomic sequencing were designed using the MethPrimer program  to inclueviously .4 cells were seeded in 96 well plates, and each assay was performed with at least 5 replicates. The MTT assay was normalized by cell counting, where the same number of cells were used for both EBF3 knockdown and negative control assays.MTT assays following knockdown were performed as previously described , except 4 cells were seeded into transwell inserts with 8μm micropore filters either without or with matrigel coating. Media containing 10% FBS was added to the lower chamber as the chemoattractant. Five random fields of view were captured per transwell insert to count the number of migrating cells.Boyden chamber invasion assays were carried out as previously described , with th


Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2.Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process  N-terminal methionine excision (NME) is an essential cotranslational pathway and is the first modification to which proteins are subjected, whilst still bound to the ribosome and before their synthesis has even been completed Entamoebag humans . It has in vivo and the molecular consequences of MetAP2 inhibition in mammalian cells. We confirmed the selectivity of the inhibitory effects of fumagillin on endothelial cells and several new sets of tumor lines. Large-scale N-terminal proteomic characterization in cell lines responsive and unresponsive to fumagillin treatment highlighted the in vivo requirement of both MetAPs for M[VT]X-targets and, potentially, for lower-level MGX targets. Interestingly, glutathione redox homeostasis was altered by MetAP2 inhibition in fumagillin-sensitive cells, but not in fumagillin-insensitive cells. Moreover, the identification of the MetAP2 protein and its quantification by targeted selected reaction monitoring (SRM) mass spectrometry (MS) revealed that the protein accumulated at extremely low levels, but that these levels were slightly higher in fumagillin-insensitive than in fumagillin-sensitive cells. Consistent with this finding, transcripts analysis showed that MetAP levels were strongly correlated with the inhibitory activity of fumagillin in cells, particularly in terms of MetAP1 accumulation within the cell. We suggest that MetAP1 expression could be routinely checked in several types of tumor, as a prognostic marker for predicting the efficacy of any treatments targeting MetAP2 activity and for the fine-tuning of therapeutic strategies.Here, we carried out a comprehensive proteomic analysis with an array of cell lines, to investigate the NME We set out to determine the precise selectivity of the MetAP2 inhibition phenotype in large panoply of cell lines. To this end we used a very-well characterized molecule, fumagillin, which has been shown to have potent anti-cellular proliferation activity on endothelial cell lines at very low concentration and specifically inhibits MetAP2 by covalent binding the active pocket of the enzyme . We perfAs MetAP2 inhibition leads to cell growth arrest, we monitored the different phases of the cell cycle in HUVEC, in the presence and absence of fumagillin. Between 24 h and 72 h, the treated HUVEC were arrested in the G0/G1 phase of the cell cycle Figure . These rClinical trials with the fumagillin analog TNP-470 revealed that this molecule had a short half-life in the blood (~7 min), possibly due to its rapid metabolism by epoxide hydrolase . In our in vivo substrate specificity of MetAP1 and MetAP2 and the cellular impact of MetAP2 inhibition in sensitive and insensitive cell lines. MetAPs target the N-termini of newly synthesized proteins. We therefore investigated the status of the protein N-termini (the N-terminome), by characterizing the protein N-termini and their modifications by strong cation exchange (SCX) N-terminus enrichment followed by MS/MS analysis , in twoWe further investigated the effect of fumagillin on proteomes, by classifying the proteins for each cell line and set of conditions according to their N-terminal status, focusing on the removal or retention of the iMet . MetAP2 inhibition did not change the number of identified proteins common to cell lines for the iMet- and iMet+ classes . Howeverin vivo substrate specificity of MetAP1 and MetAP2, with the predictions obtained with TermiNator3, one of the most reliable predictors of N-terminal protein modifications, including NME (https://bioweb.i2bc.paris-saclay.fr/terminator3/). The experimental data were found overall to be well correlated with the predictions for NME efficiency   . GiSaccharomyces cerevisiae and Arabidopsis thaliana  substrates, and also, to a less extent, for M[G] substrates. This result is consistent with previous reports showing that cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, a thioredoxin , an SH3-us study . Indeed,us study . We alsous study . Indeed,us study , 47. More arrest . InteresArabidopsis thaliana that decreases in NME levels affect glutathione redox homeostasis. A link between NME inhibition and glutathione has also been established in yeast and Archeae [We discovered that one of the first alterations induced by MetAP2 inhibition was a redox homeostasis defect. Indeed, our redox analyses and proteomic data indicated that GSH redox homeostasis played a role in the phenotype of MetAP2-inhibited cells. In both sensitive and non-sensitive cells the total glutathione pool increased, indicating a clear challenge to the cellular redox system in both situations Figure . However Archeae . MetAP2  Archeae , 30, 48. Archeae , 41. Thu Archeae . In addi Archeae , 44, 45. Archeae . Given t Archeae . Targeti Archeae . Hence, In conclusion, MetAP1 level is a key determinant of the MetAP2 inhibition phenotype. Since we showed here that cell sensitivity to fumagillin is correlated with mRNA levels, and with MetAP1 mRNA levels in particular, we therefore suggest that mRNA MetAP1 levels could be routinely checked in several type of tumors and used to predict whether treatment targeting MetAP2 is likely to be effective, thereby guiding the choice of the appropriate treatment. We discovered a clear link between MetAP2 inhibition sensitivity and redox homeostasis which could be utilize for therapeutic strategy improvement. We think that determinations of MetAP1 levels and of imbalances in redox homeostasis are potentially useful new tools that may foster the development of new personalized anticancer strategies.15N- and 13C-labeled arginine residues were purchased in crude (PEPotec SRM Peptides) and purified form (AQUA peptides) for LC-SRM assays from Thermo Fisher Scientific . All other reagents and chemicals were purchased from Sigma Aldrich .Modified porcine trypsin was obtained from Promega ; synthetic stable isotope-labeled peptides with C-terminal 6 sensitive and insensitive cells were plated in petri dishes and cultured as described in the Supplementary Information. After 24 h medium is removed and changed with fresh medium containing or not 5μM fumagillin. Cells were cultured for additional 24h before collecting them for further analysis. Cell viability was assessed with the Promega CellTiter-Blue™ reagent, according to the manufacturer's instructions. After 24 h of culture, the cell medium was supplemented with 50 μL of the test compound dissolved in DMSO (<0.1% in each preparation). After incubation for 72 h, we added 20 μL resazurin and incubated the mixture for a further 2 h before recording fluorescence  in a Victor microtiter plate fluorimeter . Experiments were performed in triplicate.Cancer cell lines were obtained from the American Type Culture Collection . Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics  and were cultured as described in the Supplementary Information and according to the supplier's instructions. For the proteomics and transcriptomics analysis 6.2×10in vivo substrate specificity of MetAPs in different conditions.In this studies we compared fumagillin-sensitive and –insensitive lines. Especially for proteomic analysis, to have a broader significance, two cell lines of each class were analyzed. The selection of these lines is described in the first result section. The quality of the methodology used allowed us to characterize more than 1000 unique N-terminal peptides per cell lines  allowing to decipher the p-Value is shown as follow: * pVal<005, ** pVal<0.01, *** pVal<0.001, ns non-significant. For mass spectrometry analysis, the overall pipeline is given in Except otherwise stated, all results were produced from at least 3 independent biological repeats (n) and when the mean is computed the associated standard error of the mean is given. The statistical significance is assessed by running a two-sided t-test (GraphPad Software) and the associated Cell pellets were washed with DPBS. RNA was extracted with the NucleoSpin RNA kit (Macherey-Nagel), according to the manufacturer's instructions. The RNA was quantified and its quality was assessed using the Agilent 2100 Bioanalyzer with the eukaryote total RNA 6000 Nano assay. 1 μg of total RNA was reverse-transcribed in a 20 μL final reaction volume using the High Capacity cDNA Reverse Transcription Kit (Life Technologies) with RNase inhibitor according to the manufacturer's instructions. Quantitative PCR for MetAP1, MetAP2 and reference genes was performed using 10 ng cDNA with TaqMan Real-Time PCR assays (Life Technologies). 10 reference genes have been tested and the two most stable genes (18S and PPIA) selected by GeneX software (GeneX Gene Expression Database) were used to normalize the data. The TaqMan Real-Time PCR assays used are: MetAP1-Hs00299385_m1, MetAP2-Hs00199152_m1, 18S-Hs99999901_s1 and PPIA-Hs99999904_m1.Proteomic analyses were performed essentially as previously described . FurtherThree human cell lines  were studied. Cells were lysed and proteins were extracted, reduced, alkylated, and precipitated. Proteins were digested with trypsin, in solution, and the resulting tryptic peptides were desalted by SPE and analyzed by microLC-SRM. A first experiment was performed with six crude synthetic peptides specific to MetAP2 and MetAP1, for the relative quantification of MetAP2 in three cell lines. We then carried out a second experiment with one highly purified and precisely quantified AQUA peptide, for the absolute quantification of MetAP2. Further details are provided in the Supplementary Information.et al. [−1 propidium iodide for 15 minutes. Cell cycle profiles were acquired with an FC500 cytometer (Beckman-Coulter).Glutathione determinations were carried out essentially as described by Rahman et al. . Cells w


The objectives of the present study were to compare changes in muscle excursion, total collagen, and collagen subtypes after tenotomy over time and after delayed tendon repair.Tenotomy on the extensor digitorum tendon of the right second toes of 48 New Zealand White rabbits was performed; toes on the left leg were used as controls. Passive muscle excursion, total collagen content, and type I, III, and IV collagen contents were measured at 1, 2, 4, and 6 weeks after tenotomy. Next, passive muscle excursion and total collagen content were measured at 8 weeks after delayed tendon repair at 1, 2, 4, and 6 weeks after a tenotomy.p < 0.05). After tendon repair, increases in total collagen content after tenotomy were not reversed, despite early repairs at 1 and 2 weeks after tenotomy.Passive muscle excursion decreased sequentially over time after tenotomy. Meanwhile, total collagen increased over time. These changes were significant after 4 weeks of injury. Type I collagen significantly increased, type III collagen significantly decreased, and type IV collagen had no significant change over time. Passive muscle excursion was negatively correlated with total collagen and type I collagen after tenotomy at each time point after tenotomy (Increases in type I collagen were found to be associated with decreased excursion after tendon rupture. The increase in collagen that was observed after tenotomy was not reversed by repair within 8 weeks. Tendon injuries are often overlooked and untreated. Left alone, these injuries eventually reduce active joint motion . ProlongCollagen is an important component of the extracellular matrix (ECM) of connective tissue layers that surround muscle cells . We repoIt is unknown if delayed repair after tenotomy can reverse decreased muscle excursion and increased collagen content over time after tenotomy or when the delayed repair effect disappears after tenotomy. This information is an important clinical decision-making tool when choosing surgical methods, such as direct repair, tendon transfer, or tendon graft, for treatment of a neglected tendon rupture. Accordingly, this study was designed to examine whether collagen subtypes in a tenotomized muscle change over time and whether changes in total collagen content and muscle excursion after tendon rupture are reversed by delayed tendon repair.n = 48 subjects, 96 hind limbs) were obtained from DooYeol Biotech . Rabbits were cared for in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals. Rabbits were single-housed in standard stainless steel cages and allowed free access to standard rabbit chow and water, with 12-h photoperiod , 17–21 °C ambient temperature, and 50 ± 10% relative humidity.All experimental methods were approved by the Institutional Animal Care and Use Committee of Yonsei University. New Zealand White rabbits  was chosen as the experimental muscle because it has a long and large tendon that can be easily manipulated surgically. This muscle is easy to define because all muscle fibers of the muscle of the second toe originate from the medial surface of the tibia. The EDII tendon is also an intrasynovial tendon, as are the human finger flexors, which are commonly injured .Animals were randomly assigned to eight treatment groups (six rabbits in each) at the beginning of the study. For the first experiment, tenotomy of the EDII was performed on the right legs and the intact left legs served as a control. Passive muscle excursion and the content of hydroxyproline, as well as type I, III, and IV collagen, were measured at 1, 2, 4, or 6 weeks after tenotomy. For the second experiment, tenotomy of the EDII was performed on the right legs and the intact left legs served as the control. The tendon was repaired at 1, 2, 4, or 6 weeks after tenotomy. Passive muscle excursion and hydroxyproline content were measured in both legs at 8 weeks after repair.Anesthesia was induced with an intramuscular injection of 15 mg/kg of Zoletil 50  and 5 mg/kg of Rompun  and was maintained with enflurane, which provided approximately 30 min of adequate sedation. The right hind limbs of the animals were shaved and positioned supine on the operating table. The knee was in approximately 90° of flexion and the hip was in flexion and external rotation. Under aseptic conditions, a longitudinal skin incision was made over the medial side of the distal tibia and the tendon of the EDII was exposed. The tendon was transected at the level of the metatarsal. The wound was closed and animals were returned to their cages.At various times after the tenotomy was performed as described above, animals were anesthetized, their incisions were re-opened, and the proximal tendon stump of the muscle was completely released from the adhesions with the surrounding tissues. Because end-to-end repair was impossible due to the decreased muscle excursion, the tendon was sutured to the ankle extensor retinaculum at the point of 50% excursion. After wound closure, animals were allowed to move freely in their cages.The proximal tendon stump of the muscle was completely released from the adhesions with the surrounding tissues before the measurement of muscle excursion. A marking suture was placed in a proximal tendon end in the myotendinous junction, which was selected as a measurement point to avoid tendon length change through tendon stretching. The EDII tendon was pulled with a hemostat by the operating surgeon. Traction force was maximally applied to the EDII tendon to the extent that further plastic deformation did not occur by stretching of the tendon. The location of the marking suture in the retracted position and the maximal manual traction position were marked in the medial tibia bone. Muscle excursion was obtained by measuring the distance between the two bone markers using a digital caliper.Animals were sacrificed with an overdose of pentobarbital (150 mg/kg i.p.). The EDII were harvested from the bilateral lower limbs after measuring muscle excursion. Hydroxyproline, a major component of collagen, was quantified as a measure of collagen content using the Hydroxyproline Colorimetric Assay Kit  according to the manufacturer’s instructions.www.antibodies-online.com; catalog numbers: ABIN628250, ABIN628251, and ABIN628256, respectively) according to the manufacturer’s instructions.Collagen was solubilized with pepsin under acidic conditions and then further digested with pancreatic elastase at neutral pH to convert polymeric collagen to monomeric collagen. Type I, III, and IV collagen were measured using specific enzyme-linked immunosorbent assay (ELISA) kits  was used for all statistical analyses. The muscle excursion, the content of hydroxyproline, and type I, III, and IV collagen content relative to controls were used to account for individual differences between rabbits. The values for these parameters at each time point after tenotomy were compared in a linear mixed model. Muscle excursion and hydroxyproline content relative to control values at 8 weeks after tendon repair were compared with a linear mixed model, according to repair timing. The muscle excursion ratios and the hydroxyproline ratios to the control value after tenotomy and after delayed tendon repair were compared using the Mann-Whitney test according to the period after the tenotomy. Correlations between muscle excursion ratio in relation to the control value and hydroxyproline and type I, III, and IV collagen content ratios to control values at each time point after tenotomy and between the muscle excursion ratio to the control value and hydroxyproline content ratio to the control value at 8 weeks after tendon repair were also analyzed. For all analyses, the level of significance was All eight treatment groups of six rabbits survived without complications until scheduled sacrifice.p < 0.01; Table The mean passive muscle excursions after tenotomy decreased from 3.7 ± 0.5 mm at 1 week to 2.4 ± 0.4 mm at 6 weeks. The mean ratios thereof in relation to control values significantly decreased over the same time period from 94 ± 6 to 60 ± 12% . There was no significant correlation between muscle excursion values and type III collagen content  or type IV collagen content  after tenotomy . The mean ratios of muscle excursion according to timing of delayed repair to control values significantly decreased from 97 ± 9% at 1 week to 58 ± 10% at 6 weeks (p < 0.01; Table p < 0.01; Fig Mean passive muscle excursions after tendon repair according to timing of delayed repair significantly decreased over time (p < 0.01; Table p < 0.01; Fig. r = −0.721, p = 0.00, Fig. Hydroxyproline content in the muscle after tendon repair increased with the timing of the delayed repair. The mean ratios of hydroxyproline in muscle after delayed tendon repair to control values significantly increased over time (To delineate the relationships between changes in muscle excursion and collagen content after tendon rupture and delayed repair, tenotomy on EDII muscles of rabbits was performed on the right legs with or without subsequent repair. Intact left legs served as controls. Muscle excursion, hydroxyproline content, and type I, III, and IV collagen content relative to control were used to account for differences between individuals. Total collagen and type I collagen significantly increased with time after tenotomy, excursion and type III collagen decreased significantly, and there was no significant change in type IV collagen. Muscle excursion ratio was significantly negatively correlated with total collagen and type I collagen after tenotomy. Total collagen content at 8 weeks after tendon repair increased significantly, and muscle excursion decreased significantly with delay in repair, such that excursion was significantly negatively correlated with total collagen. The increase in collagen that was observed after tenotomy was not reversed by repair. Improvement in excursion was noted when repair was delayed by 1 or 2 weeks; however, when it was delayed by 4 or 6 weeks, excursion decreased. Increases in type I collagen were associated with decreased excursion after tendon rupture.In the present study, the ratio of type I to type III collagen at 1 week after tenotomy was 3.6:1. Type I collagen was negatively correlated with passive excursion, while type III was positively correlated with passive excursion. Type I and III collagen are important components of the perimysium and epimysium, and their relative amounts differ between species and between muscle groups within the same species , 11. TheType IV collagen is a non-fibrillar collagen and an important component of the endomysium . It formPassive muscle excursion decreased sequentially with time after tenotomy, becoming prominent at 4 and 6 weeks after tenotomy. The decreased excursion at 4 and 6 weeks after tenotomy did not increase after delayed repair. The hydroxyproline content relative to control increased after repair (Fig. There are several limitations to the present study. First, the applied maximal passive tension may be subjective when measuring passive excursion. Muscle excursion has been measured during full flexion and extension of joints using ultrasonography or magnetic resonance imaging, but in cases of tendon rupture, excursion measurement by joint motion is impossible , 21. In Increases in type I collagen were found to be associated with decreased excursion after tendon rupture. The increase in collagen that was observed after tenotomy was not reversed by repair within 8 weeks.


Curcuma longa (Zingiberaceae family), which possess several pharmacological properties. However, in vivo administration exhibited limited applications in cancer therapies.Curcumin is one of the leading compound extracted from the dry powder of 1–10, acetone 11–17 and cyclopentanone 18–24 series. All the curcuminoids were synthesized by the acid or base catalyzed Claisen Schmidt condenstion reactions, in which β-diketone moiety of curcumin was modified with mono-ketone. These curcuminoids 1–24 were screened against HeLa, K562, MCF-7 (an estrogen-dependent) and MDA-MB-231 (an estrogen-independent) cancer cell lines. Among them, acetone series 11–17 were found to be more selective and potential cytotoxic agents. The compound 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines. Among the cyclohexanone series, the compound 4 exhibited  potential cytotoxicity against four proposed cancer cell lines, respectively. All the curcucminoids were characterized with the detailed 1H NMR, IR, UV–Vis, and mass spectroscopic techniques. The structure of compound 4 was confirmed by using the single X-ray crystallography. Additionally, we are going to report the first time spectral data of -2,6-bis(2-methoxybenzylidene)cyclohexanone (1). Structure–activity relationships revealed that the mono-carbonyl with 2,5-dimethoxy substituted curcuminoids could be an essential for the future drugs against cancer diseases.Twenty-four curcumin derivatives have synthesized, which comprises cyclohexanone 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines.Curcuminoids with diferuloyl(4-hydroxy-3-methoxycinnamoyl) moiety with mono carbonyl exhibiting potential cytotoxic properties. The compound  Curcuma longa  × 100%50 value (concentration of compounds inhibited 50% of cell viability) was determined from the graph of cell viability vs absorbance.IC4 was solved by direct methods and refined by full-matrix least-squares techniques on F2 using SHELXTL software package. Crystallographic data of the reported structures have been deposited at the Cambridge Crystallographic Data Centre with CCDC deposition numbers of 1548735. Copy of available material can be obtained free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).X-ray analysis for all these samples were performed using Bruker APEX II DUO CCD diffractometer, employing MoKα radiation (λ = 0.71073 Å) with φ and ω scans, at room temperature. Data reduction and absorption correction were performed using SAINT and SADABS programs –53. The 14 was exhibited (IC50 = 3.02 ± 1.20 and 1.52 ± 0.60 µg/mL) against MCF-7 and MDA-MB-231 breast cancer cell lines. Structure activity relationship revealed that the role of methoxy groups are important. Curcumin derivatives, 4, 5, 9, 14, 11 and 17 exhibited significant cytotoxic activity  cancer cell lines. Curcuminoids with diferuloyl (4-hydroxy-3-methoxycinnamoyl) moiety with mono carbonyl exhibiting potential cytotoxic properties. The compound


TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations  in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups . The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including  Head and neck cancer is the sixth most common cancer worldwide . Collectin vitro models of OSCC may not accurately reflect the in vivo characteristics of OSCC, given that the majority of these models have not been subjected to detailed genetic analysis and have not been linked to detailed clinical data. The few efforts made to ensure conservation between OSCC tissues and experimental models have been limited to mutational analysis of selected genes or to a probe-based capture techniques ; whereby Vt1 is the tumor volume at time t1 and Vt2 is the tumor volume at time t2. Mice were euthanized on day 80 or earlier (if there were signs of illness or discomfort), and tumors on the flanks for each mouse were retrieved. The resected tumors were fixed in 4% formalin overnight and then transferred to 70% alcohol and processed for paraffin embedding for histopathological evaluation by pathologists . Images of hematoxylin and eosin stained slides were acquired with the OlyVIA imaging software version 2.4 .We examined the tumorigenicity of seven ORL cell lines in the subcutaneous flank model . These ORL cell lines were derived from patients with diverse risk habits and represented cell lines of the 3 different gene expression clusters (described below). A minimum of five NU/NU mice were used for each cell line. The ORL cell lines were resuspended at a concentration of 2 × 105 in DMEM-F12 serum-free medium. Mice were examined twice weekly for tumor development in the tongue under anesthetic condition using isoflurane. Tumor measurement was given visually by the same operator for the duration of the study. Mice were euthanized after 40 days, the neck area of each mouse was carefully dissected to retrieve 4 to 5 cervical lymph nodes, and tongues for each mouse were also retrieved. The resected tissues were fixed in 4% formalin overnight and then transferred to 70% alcohol and processed for paraffin embedding for histopathological evaluation by pathologists (AM and TGK). Hematoxylin and eosin stained slides were acquired with the OlyVIA imaging software version 2.4 .The tumorigenicity of the seven ORL cell lines mentioned above was also examined by the orthotopic tongue model. The orthotopic tongue model was established as reported previously . BrieflyORL cell lines and NOK were harvested by trypsinization at 70–80% confluence and DNA were extracted using the QIAamp DNA Mini kit  according to manufacturer's instructions. Copy number alterations (CNA) analysis was performed on the Genome Wide Human Cytoscan HD array . Data was analyzed with Chromosome Analysis Suite v2.0 (CHAS) and R software. Detailed analysis steps are included in Supplementary Methods.ORL cell lines and NOK were lysed with TRI Reagent  at 70–80% confluence and extracted according to manufacturer's instructions. Libraries were constructed using 1 μg total RNA following Illumina TruSeq RNA Sample Preparation v2 Guide and 100 base pair paired-end sequencing was conducted as previously described [n = 7,053 genes) in the R environment program (R version 3.0.2) using the R package Consensus Cluster Plus [Sequencing reads were mapped to human reference genome (Ensembl release GRCh37) using Tophat2 version 2.0.9 with default parameters . Variantter Plus . The optter Plus  as descrTP53 and CDKN2A mutations were conducted by Sanger sequencing on DNA extracted from donor tissue where available, while expression of EGFR was confirmed by western blotting. Details of these experiments are included in Supplementary Methods.Validation of 2O) was tested at concentrations ranging from 0.1 to 25 μM. Culture medium containing 0.01% (v/v) DMSO was used as vehicle control. Cells were treated for 48 hours and evaluations were done with six independent replicates for each drug. At the end of the treatment period, 20 μl of MTT solution (5 mg/ml) was added to each well and incubated for 4 hours at 37°C. The media was then removed and 100 μl of DMSO was added to solubilize the formazan crystal. The optical density was recorded by Synergy H1M microplate reader  at 570 nm. Five parameter logistic (5-PL) dose response curves were plotted using SoftMax Pro 5.4.5  and the 50 percent inhibitory concentration (IC50) of each drug of was obtained from the curve. Statistical differences were assessed by the Student's t-test. p < 0.05 was considered to be statistically significant.To determine whether gene expression profiles influence response to therapeutic drugs, we treated a sub-set of ORL cell lines from different gene expression clusters with DNA damaging agents that are routinely used in the clinic and cell viability was evaluated using 3--2,5-diphenyltetrazolium bromide (MTT) assay. Four cell lines each from Cluster 1  and cluster 2  were used. Briefly, 3000 cells in 80 μl culture medium per well was seeded in 96-well plates. After 16 hours incubation, the drugs which were diluted in 20 μl of culture medium were added into each well. Irinotecan hydrochloride , cisplatin  and topotecan hydrochloride  were tested at concentrations ranging from 0.4–100 μM, whereas mitomycin C  and examined on an upright Olympus IX71 microscope  with double bandpass filters to detect fluorescent-stained nuclei . Images were captured from 3–10 different fields of each treatment concentration and further analyzed with EBImage [T-test was used to determine statistical significance between the percentages of EdU positive cells in each treatment concentration relative to untreated control cells. P value less than 0.05 was considered statistically significant.Three cell lines each from Cluster 1  and cluster 2  were used to examine the effects of CDK1 inhibitor, RO3306. Cells were seeded on cover slips overnight and treated at concentrations ranging from 0–7 μM, for 24 hours. Following this, cell proliferation was evaluated by 5-ethynyl-2′-deoxyuridine incorporation using Click-iT EdU Cell Proliferation Assay Kit  according to manufacturer's protocol. Briefly, cells were incubated with 10 μM EdU for 2–6 hours prior to fixation with 3.7% formalin. The cells were permeabilized with 0.1% Triton X-100 in phosphate buffer, followed by EdU detection via a copper-catalyzed reaction and nuclei staining by Hoechst 33342 . The cover slips were then mounted on glass slides by using VECTASHIELD


MYCL amplification and/or expression. Fourteen SCLC cell lines, including four with MYCL amplification, were examined for the effects of JQ1 on protein and gene expression by Western blot and mRNA microarray analyses. The sensitivity of SCLC cells to JQ1 was assessed by cell growth and apoptosis assays. MYCL was expressed in all the 14 cell lines, whereas MYC/MYCN expression was restricted mostly to cell lines with gene amplification. ASCL1, a transcription factor shown to play a role in SCLC, was also expressed in 11/14 cell lines. All SCLC cell lines were sensitive to JQ1 with GI50 values ≤1.23 μM, with six of them showing GI50 values <0.1 μM. Expression of MYCL as well as MYCN, ASCL1 and other driver oncogenes including CDK6 was reduced by JQ1 treatment, in particular in the cell lines with high expression of the respective genes; however, no association was observed between the sensitivity to JQ1 and the levels of MYCL, MYCN and ASCL1 expression. In contrast, levels of CDK6 expression and its reduction rates by JQ1 were associated with JQ1 sensitivity. Therefore, we concluded that CDK6 is a novel target of JQ1 and predictive marker for JQ1 sensitivity in SCLC cells.We aimed to elucidate the effect of JQ1, a BET inhibitor, on small cell lung cancers (SCLCs) with  MYC family oncogenes, MYC, MYCN or MYCL, is amplified in approximately 20% of SCLCs, with the frequency of MYCL amplification the highest among the MYC family genes in clinical SCLC samples ., 16.MYC MYC family genes together, even though only one of them is amplified in each tumor/cell line . Th. Th50 vaCell cycle arrest and/or apoptosis have been shown to occur in several types of cancer cells by JQ1 treatment , 14. p21MYCL gene is a target of JQ1 for transcriptional silencing in the majority of SCLC cell lines  is a transcription factor necessary for the development of neuroendrocrine cells and the growth of SCLC cells . It was MYC family gene amplification/expression and/or ASCL1 expression are associated with the growth inhibition effect of JQ1 to SCLC cell lines. Since two of the four MYCL amplified cell lines, one of the two MYC amplified cell lines, one of the two MYCN amplified cell lines, and two of the six MYC non-amplified cell lines were highly sensitive to JQ1 (GI50 <0.1 μM)  and the 8 cell lines with low sensitivity to JQ1 (GI50 ≥0.1 μM). None of these proteins showed an association between their levels of expression and the sensitivities to JQ1  Figure , sensitid Figure  and comps to JQ1 .MYC, MYCN and ASCL1, in human cancer cells [MYCL as well as MYC, MYCN and ASCL1 were transcriptionally suppressed by JQ1 in SCLC cells, we further performed mRNA microarray analysis in 12 of the 14 SCLC cell lines, which were collected after 24 hours of culture with and without 1.0 μM of JQ1. As summarized in MYCL as well as MYCN and ASCL1 were transcriptionally suppressed by JQ1, in particular, in cell lines with high expression levels . In contrast, transcriptional suppression was not detected in the MYC gene, as reported previously in SCLC cell lines [MYC amplification and overexpression. The results of mRNA microarray analysis were overall similar to those of Western blot analysis in most of cell lines, with some variations probably because of culture and experimental conditions. The result indicates that MYCL is also a target of JQ1 for transcriptional silencing in SCLC cells. However, even though MYCL was overexpressed in H1963, no reduction was observed by JQ1, consistent with the result of Western blot analysis, supporting that the rearranged/fused MYCL gene in this cell line is not a target of JQ1, likely because of the use of the RLF promoter for transcription.JQ1 is known to induce growth suppression by transcriptional inhibition of numerous genes, including er cells , 20-25. FOSL1, BCL2, CDK6, IL7R, RUNX2 and CDC25B have been shown to be targets of JQ1 for transcriptional silencing in other types of cancers [FOSL1, IL7R and RUNX2 was very low and not markedly changed by JQ1. However, the expression of BCL2, CDK6 and CDC25B was high  in several cell lines, and their levels were decreased by JQ1 treatment in most cell lines, in particular those with high levels of expression. Namely, expression of BCL2 was markedly reduced by JQ1 in H345 and H2107, while expression of CDK6 was reduced by JQ1 in H1618, Lu135, H82, H526 and Lu134. Expression of CDC25B was reduced by JQ1 in 11 of the 12 cell lines. cancers , 26-31. 50 values at 1.0 μM of JQ1. Only CDK6 showed a significant association (p=0.003 by Pearson correlation test), while none of MYCL, MYCN or ASCL1 showed such an association. We also investigated the association between the expression levels in non-treated cells and the GI50 values. BCL2 (p=0.011) and CDK6 (p=0.002) were predictive markers for the effectiveness of JQ1 on growth inhibition, although the association with BCL2 expression was much weaker than that with CDK6 expression. Therefore, we conclude that CDK6 plays a common role for the growth inhibition by JQ1 in most SCLC cells. Thus, the sensitivities to JQ1 for growth inhibition could be in part defined by the levels of CDK6 expression and its reduction rates in SCLC cells.Therefore, we further investigated the association between the reduction rate of expression and the GIhttp://software.broadinstitute.org/gsea/msigdb/collections.jsp). GSEA revealed a significant downregulation of 3 of the 50 hallmark gene sets. HALLMARK_MYC_TARGETS_V2 was the most significantly downregulated pathway, supporting the conclusion that MYC family genes were transcriptionally inhibited by JQ1 in SCLC cells. The second most significantly downregulated pathway was HALLMARK_E2F_TARGETS. Gene sets significantly upregulated by JQ1 included HALLMARK_P53_PATHWAY and HALLMARK_APOPTOSIS. These results support the occurrence of G1 arrest and apoptosis in SCLC cells by JQ1 treatment.To examine whether JQ1 specifically abrogates MYC-dependent transcription and which pathways are affected by JQ1, we further performed gene set enrichment analysis (GSEA) to identify functional gene sets with significant downregulation or upregulation by JQ1 treatment Figure  , H526 from Dr. C. C. Harris , Lu135 and Lu134 from Dr. T. Terasaki , Lu165 from RIKEN BioResource Center , and H2141, H1184 and H209 from the American Type Culture Collection . Cells were cultured in RPMI medium supplemented with 10% FBS with 5% COin vitro cultures. These properties make the use of SCLC cell lines difficult for drug screening due to the following reasons. First, if cell growth is assessed in 2 to 3 days after drug addition, as in other cancer cell lines, the number of non-treated SCLC cells often does not double at the time of assessment, compared to the number of plated cells. Therefore, if a drug has an effect to induce only growth arrest but not cell death, the GI50 (50% growth inhibition) values would be very high [The population doubling times of SCLC cell lines are generally very long, and those in the 14 cell lines used in this study ranged from 3 to 7 days. The cells proliferate as multi-cell floating aggregates in ery high . For thiery high , 37.Lysate preparation and Western blot analyses were performed as described previously . AntibodTotal RNA was extracted using Maxwell® 16 LEV simplyRNA Tissue Kit (Promega) and qualified with a model 2100 Bioanalyzer (Agilent). All samples showed RNA Integrity Numbers higher than 7.9 and were subjected to microarray experiments. Ten nanograms of total RNA were processed and hybridized to GeneChip® Human Transcriptome Array 2.0  using the GeneChip® WT Pico Reagent Kit (P/N 703262 Rev. 2). After hybridization, arrays were washed and stained in the Affymetrix GeneChip Fluidics Station 450 and scanned in the Affymetrix GeneChip Scanner 3000 7G. The data were processed by the MAS5 algorithm, and the mean expression level of a total of 67,528 genes was adjusted to 1,000 for each sample.


However, there is concern regarding the effect of locally applied BMP-2 on tumor recurrence and metastasis. The purpose of this project was to evaluate the effect of exogenous BMP-2 on osteosarcoma migration and invasion across a panel of tumor cell lines in vitro tumor growth and development was assessed across multiple standard and patient-derived xenograft osteosarcoma cell lines. Tumor migration capacity, invasion, and cell proliferation were characterized. In addition, the effect on metastasis was measured using a xenograft model following tail-vein injection. The effect of exogenous BMP-2 on the development of metastases was measured following both single and multiple BMP-2 administrations.The effect of BMP-2 on There was no significant difference in migration capacity, invasion, or cell proliferation between the BMP-2 treated and the untreated osteosarcoma cell lines. There was no significant difference in pulmonary metastases between either the single-dose or multi-dose BMP-2 treated animals and the untreated control animals.In the model systems tested, the addition of BMP-2 does not increase osteosarcoma proliferation, migration, invasion, or metastasis to the lungs. Osteosarcoma is a rare primary bone tumor, making up less than 1% of cancers in the United States. ChemotheHistorically, extremity osteosarcomas were typically removed by performing amputations or disarticulations.  In the mBone Morphogenetic Protein-2 (BMP-2) has been shown to stimulate bone growth. The Food and Drug Administration (FDA) has approved BMP-2 to be used in spinal fusion surgery, fixation of open tibial fractures, oral and maxillofacial surgery, and in the management of recalcitrant non-unions.  However,Standard osteosarcoma cell lines, 143b, HOS, U2OS, Saos-2, and MG63 (ATCC), the metastatic cell line, SaOS-LM7 , and xenograft cell lines, OS17 and OS31,  were groTo further characterize the osteosarcoma lines utilized, we quantified gene expression for BAMBI, SOST, and NOG across 5 standard and 3 xenograft cell lines using quantitative real-time PCR. The experiment was performed both with and without the addition of BMP-2 over a 48 hour period. RNA was extracted using PureLink RNA Mini Kit  and subsequently converted to cDNA using SuperScript III First- Strand Synthesis System (Life Technologies) according to the manufacturers’ instructions. Gene expression quantitation was carried out using a 7,500 Fast Real-Time PCR system and Taqman Gene Expression assay mix . The housekeeping gene GAPDH was used as a control and multiple wells of scrambled control were included as negative controls. Reactions for each sample were performed in triplicate. mRNA levels were quantified using ΔΔCt method as per the manufacturer’s instructions, 7500 Sequence Detection System (Life Technologies). MSCs were used as a calibrator.Random migration motility was measured via a wound-healing assay as described previously.  Cells we3D spheroid cell invasion assay was performed using Cultrex® 96-well 3D Spheroid BME Cell Invasion Assay Kit  according to manufacturer’s instruction. Briefly, osteosarcoma cell lines in spheroid formation ECM were seeded 3000 cells per well in 3D culture qualified 96-well spheroid formation plate, followed by incubation at 37oC for 72 hours to promote spheroid formation. Upon completion of spheroid formation, the spheroid is embedded in an invasion matrix composed of basement membrane proteins. After allowing the matrix to form hydrogel network on which invasive cells can travel, 100 μl of cell culture medium containing chemoattractant and BMP-2 were added. Then each day until day 6 the morphology of spheroid of each well was photographed using the 4x objective of a Nikon Inverted Microscope ECLIPSE TE200 attached to a CCD camera . All cell lines were assayed in triplicate and qualitative analysis of the figures obtained was performed. Non-invasive cell line, MCF-7, and invasive cell line, MDA-MB-231, were included as a measure of quality assurance, and wells without the invasion matrix were also included as negative controls.Haptotaxis, defined as cell movement towards an immobilized ECM protein gradient, was measured in the Boyden chamber system and was performed using the Chemicon QCM Quantitative Cell Migration Assay . Cells were serum-starved overnight before being seeded into Boyden chambers. Cells that migrated outside the chamber were stained and extracted in 300 μL of extraction buffer. Absorbance at 562 nm was measured using a microplate reader , which indicates the relative number of cells that migrate out of the upper chamber and into the lower chamber, which contains the chemoattractant. The fibrosarcoma cell line, HT1080, was used as a positive control and the non-invasive cell line 3T3 was used as a negative control.In vitro invasion through matrigel was quantitatively measured using the Biocoat Matrigel Invasion Kit . Serum-starved cells were plated in the invasion chambers with an 8μm pore size polycarbonate membrane, over which a thin layer of matrigel matrix was applied. Invading cells migrate through the matrix layer and become attached to the bottom of the polycarbonate membrane, which is then stained, extracted, and measured using a microplate reader with an absorbance of 562 nm .6 cells in 200μL of media were implanted using a 26-gauge needle. Tumor cells were allowed to grow for one week. Thereafter, 20 animals were randomized to either a treatment group (n = 10) or a control group (n = 10). The treatment group received a single 30μg tail vein injection of E. coli-derived BMP-2  that had been reconstituted in accordance with manufacturer’s recommendations to a concentration of 1mg/ml. The control group received no additional injections. Thereafter, animals were carefully observed for seven weeks, at which time they were euthanized by asphyxiation with 100% CO2. Body weight and harvested lung weight was recorded for each animal. The lungs were stained in Bouin’s solution and photographed. The experiment was repeated using three sequential 30μg BMP-2 tail vein injections, spaced in one-week intervals and compared with a control group that received no treatment.Experiments were performed with the approval of the Albert Einstein College of Medicine Institutional Animal Care and Use Committee (IACUC) and in accordance with the institutional animal welfare policy. Six to eight week-old female CB17 SCID mice were obtained  and housed in a pathogen-free barrier facility at all times. Heterotopic tumor implantation using SaOS-LM7 was effected via tail vein injection using a 26-guage needle. After standard alcohol preparation, approximately 102. Two animals met the criteria for euthanasia prior to the study endpoint and were sacrificed.Basic animal housing and maintenance was managed within the barrier facility by staff members of the animal institute in accordance with all rules and regulation of the facility. Animals were housed in cages with 5 animals per cage. The cages containing tumor bearing animals were clearly marked. All of the mice were observed daily including weekends by the PI’s staff. Body weight was checked twice weekly or as clinically indicated. Water was provided at all times. Feeding was in keeping with routine cycles and timing. A 12 hour light/12 hour dark cycle was utilized. PI’s staff did not enter the mouse room during the dark cycle. Temperatures of 65–75°F with 40–60% humidity are maintained. A warming lamp was available while the mice were anesthetized and through the recovery period. Upon recovery, mice were returned to their cages. Minimal blood loss was expected and anesthesia time was less than 1 minute. Animals were monitored for one hour after anesthesia to confirm normal behavior, then as regularly scheduled. Animals were routinely evaluated daily by both the investigators and the housekeeping staff. Any identification of an unhealthy or ill animal was immediately conveyed to the PI. Mice were assessed daily for ulceration and animal activity and were assigned a body conditioning score daily as well. Animals which became moribund, defined as weight loss exceeding 20% of starting weight and/or a significant decrease in activity, which developed dyspnea or which were assigned a body conditioning score of 2 were euthanized by asphyxiation with COIn vitro proliferation assays, migration assays, and invasion assays were all performed in triplicate for each dose level of BMP. The data was summarized by calculating means and standard deviations for each BMP dose level. Student t-test was utilized assessing the effect of each dose level of BMP on proliferation, migration, and invasion in treated cells compared with untreated cells. P value ≤0.05 was considered statistically significant. In vivo assays assessing the effect of single-dose and multiple-dose BMP on the development of lung metastases was determined utilizing 10 mice in each experimental arm and 10 mice in each control arm. Mean lung weight, mean body weight, and mean lung weight/body weight, as well as standard deviations, were calculated for each experimental and control arm. Percentage was defined as mean lung weight divided by mean body weight. Student t-test was utilized assessing the effect of single-dose and multiple-dose BMP on the development of metastases comparing lung weight and lung weight/body weight in mice in the experimental arms versus mice in the untreated arms. P value ≤0.05 was considered statistically significant.Results following the addition of BMP-2 to eight cell lines  are summarized in Random cell migration was assessed using a wound-healing assay. The addition of BMP-2  did not yield a decrease in the scratch wound width across all 8 osteosarcoma cell lines . Chemotain vitro, regardless of dose used.The addition of BMP-2 to osteosarcoma cell lines does not increase random cell migration or cell migration in response to a chemical stimulus Cell invasion was measured using a Biocoat Matrigel Invasion Chamber. Results of the experiment are summarized in in vivo by measuring pulmonary tumor growth in mice. Animals treated with a single dose of parenterally administered BMP-2 were compared to animals which were untreated. The lung to body weight ratio was used to account for variances in weights between animals. Results are summarized in A xenograft murine model (SaOS-LM7) was used to assess osteosarcoma metastasis in vivo following triple-dose BMP-2 administration, given once per week. These animals were compared to animals which were untreated. Results of the experiment are summarized in A xenograft murine model (SaOS-LM7) was similarly used to assess osteosarcoma metastasis in vivo and in vitro experiments in this study demonstrate that the addition of exogenous BMP-2 does not increase osteosarcoma proliferation, migration, invasion, or metastasis to the lungs. This finding is consistent with our previously reported results, which demonstrated that the local addition of exogenous BMP-2 failed to increase local recurrence rates. [Overall results from the e rates. BMPs belong to the TGF-β superfamily and are involved in a broad array of cellular roles including development, proliferation, and differentiation. Both BMP-2 and BMP-7 demonstrate potent osteogenetic properties, and have been approved by the FDA for use in a number of clinical scenarios to promote bone healing. In theory, the use of these proteins at the time of surgical reconstruction could decrease the non-union rate at the allograft-host bone junction. However, as previously mentioned, the medical community remains exceedingly cautious about using BMP-2 in the context of osteosarcoma. BMPs and BMP receptors are expressed in osteosarcoma, which has lead to the concern that administering exogenous BMP-2 may promote tumor recurrence. , 11 In rConversely, other investigators have characterized BMP-2 as an inhibitor of tumorgenesis in osteosarcoma. Wang and colleagues reported that BMP-2 treated severe combined immunodeficiency (SCID) mice did not develop tumors following heterotopic tumor implantation. They concluded that addition of BMP-2 results in inhibition of tumor-inducing gene expression and an upregulation of osteogenic differentiation markers.  Geng andThere are a number limitations of this study. The use of an immunocompromised xenograft model does not account for any potential confounding host factors associated with the administration of exogenous BMP-2. In addition, measuring lung metastasis by comparing the lung weight in BMP treated and control group mice is not the most sensitive method of analysis, and may not detect very small differences in the treated mice. During data analysis of the metabolic activity of the cell lines, there was a wide range of values. This may be due to cells remaining on the plate, a common effect demonstrated by solid tumors that prefer cell-cell interactions over cell-substratum interactions.Previous experiments have demonstrated that the addition of exogenous BMP-2 does not increase the rate of local tumor recurrence. In this study, we likewise demonstrate that the systemic administration of exogenous BMP-2 does not increase metastasis in a xenograft model of osteosarcoma. We appreciate and agree with the concerns raised to date, which prioritize patient survival over functional outcome. That said, it is hoped that these results will drive future work intended to either demosntrate that BMP-2 is indeed cancer promoting or to demonstrate that BMP-2 can be safefly utilized within the context of osteosarcoma reconstructive surgery. Although we believe these results support the consideration of future clinical studies, we acknowledged that this is a very complex system and that the use of a single BMP is unlikely to fully quell the aformentioned concerns.S1 FigFor all four cell lines included the addition of BMP-2 did not results in increased proliferation of tumor cell lines.(TIF)Click here for additional data file.S2 FigFor all four cell lines included the addition of BMP-2 did not results in increased proliferation of tumor cell lines.(TIF)Click here for additional data file.S3 FigThere was a signficant difference in expression between the experimental group exposed to BMP-2 and the control group that was not exposed to BMP-2.(TIF)Click here for additional data file.S4 FigThere was a signficant difference in expression between the experimental group exposed to BMP-2 and the control group that was not exposed to BMP-2.(TIF)Click here for additional data file.S5 FigThere was a signficant difference in expression between the experimental group exposed to BMP-2 and the control group that was not exposed to BMP-2.(TIF)Click here for additional data file.S6 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.S7 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.S8 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.S9 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.S10 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.S11 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.S12 FigThe cell line without BMP-2 and invasion matrix, with invasion matrix and without BMP-2, and with BMP-2 and the invasion were compared and did not demonstrate a qualitative difference.(TIF)Click here for additional data file.