Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets.Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study.  Most patients present with locally advanced or metastatic disease, or develop recurrent disease following curative surgery2. While many systemic treatment options are available, the prognosis of advanced gastroesophageal cancer remains poor, with median survival of less than 1 year3. Greater than 90% of gastric and gastroesophageal junction cancers are adenocarcinomas, with gastrointestinal stroma tumours (GIST), lymphomas, and neuroendocrine tumours found in a small minority of cases4.Gastroesophageal cancers are among the most common and lethal cancers worldwide3. There is an increasing recognition of the limitations of using primary tumour features to guide systemic cancer treatment, due to tumour heterogeneity and the frequent disparity observed between primary and metastatic sites5. Metastatic biopsies are rarely undertaken however, due to both inaccessibility of metastatic sites and procedure morbidity. Circulating tumour cells (CTCs) are the likely intermediates of metastasis dissemination of cancer, and as such, can be expected to include the subpopulations which are responsible for disease progression6. While CTC enumeration has an established prognostic role, the true promise of CTCs is to provide a ‘real time’ view of the cancer using only peripheral blood samples, avoiding the need of repeat invasive biopsies7.Most patients with gastroesophageal cancer will require systemic treatment at some point in their treatment8.Moreover, while most cancer deaths are due to the haematological spread of metastases, research into the mechanism of metastasis initiation, formation, and propagation has been hampered by limited access to cancer cells within the various stages of the metastatic cascade. CTCs additionally provide a unique window into the biology of cancer as it spreads through the blood stream. The rarity of CTCs compared to normal blood cells has provided significant technical challenges in developing isolation methods which are sufficiently sensitive and specific10, and long-term primary CTC cultures provide an ideal laboratory tool for the investigation of the biology of metastasis formation11. However, establishment of long-term primary CTC cell cultures has proved to be challenging. To date, despite intensive efforts, only several long-term CTCs cultures have been reported worldwide, including in colorectal12, breast13, and prostate14 cancer, all established with modest success rates, with 1–16% of blood samples producing stable cultures. To improve culture rates, initial expansion of the CTC population using xenotransplantation into immunodeficient mice prior to in vitro culture has been trialled15.CTC culture provides an expanded cell population for expression analysis, functional assays, and drug sensitivity testingIn this current work we describe establishment of two novel and distinct CTC cell lines derived from patients with metastatic gastroesophageal cancer.A total of 41 blood samples were processed for CTC enumeration, with 23 samples processed for culture using the optimised protocol (15 ml blood sample with negative selection using the CTC Enrichment Cocktail). CTCs were detected in 38/40 samples (93%) by EpCAM  based capture using the IsoFlux system (one specimen clotted and was not processed for CTC enumeration), with ≥10 CTCs found in 22 (54%) of the samples. Numbers of CTCs detected ranged from 0–150, with the mean number of CTCs 27.3  who had a high CTC count of 109. This patient presented with diffuse bone and peritoneal metastasis. Endoscopy demonstrated a large ulcerated gastric mass confirmed on biopsy to be a gastric adenocarcinoma. A matched culture was established simultaneously from the ascitic fluid sample from the same patient (UWG02ASC). Patient 41 also progressed rapidly prior to receiving any anti-neoplastic treatments and passed away.in vitro growth characteristics. UWG01CTC is adherent and requires trypsinisation for passaging, although a loose adherent spheroid phenotype is inducible in a hypoxic environment and serum free media , reaching tumour size endpoints (>10 × 10 mm) within 4 weeks for UWG01CTC Fig.  and 3 weDetailed DNA analyses were limited by poor DNA quality from both source patients’ formalin fixed paraffin embedded (FFPE) samples. However, by STR analysis, 24/28 (85.8%) alleles of UWG02ASC and UWG02CTC were identical to those of the primary tumour from patient 41, confirming the source of the cell lines  stain Fig.  with a hThe primary tumour of patient 41 and UWG02CTC both demonstrated high grade appearances by H + E stain Fig.  with a h20 by IHC  did not change the phenotype of any cell line as detected by IHC (data not shown). Neither CTC cell line expressed mesenchymal markers (vimentin or N-cadherin) or urokinase plasminogen activator receptor (uPAR), a key receptor for the initiation of proteolytic cascadesIHC Fig. . UWG02CTIHC Fig. . No cellIHC Fig. .in vitro culture. These include neuroendocrine markers in UWG01CTC such as chromogranin A, CD56, and synaptophysin  growth was observed on NanoString gene expression profiles using unsupervised hierarchical clustering, though monolayer (2D) cultures grouped together and away from spheroids (3D) and xenografts, which grouped together  relative to UWG01CTC (−log2 9.4 fold). Interestingly, UWG02CTC when compared to UWG01CTC, had a higher expression of genes known to be involved in Helicobater pylori mediated carcinogenesis such as AKT24, ETS225 and MYC26  and PLA2GA, an important regular of metastases in gastric cancer and expressed with constitutively active Wnt29. UWG02CTC also showed higher expression than UWG01CTC of targetable pathways including EGFR, FGFR2, HER-2 (ERBB2), and MET, as well as key genes in the JAK/STAT pathway, genes which overexpression are frequently reported in gastric adenocarcinomas but not gastrointestinal neuroendocrine cancers31.The IHC and STR findings, showing two highly distinct CTC lines, were further explored by NanoString analyses. As expected the sample cohorts derived from patient 20 versus patient 41 grouped together by unsupervised hierarchical clustering  genes in UWG02CTC compared to UWG02ASC and overall these two Patient 41 lines demonstrated very similar gene expression profiles.in vitro sensitivity of both CTC and UWG02ASC cell lines to commonly used DNA-damaging chemotherapeutics with varying mechanisms of action was evaluated. UWG01CTC and UWG02CTC varied in their sensitivities to these drugs as demonstrated by the dose response curves shown in Fig. 50 value for etoposide at 0.036 µM is ~3-fold lower than the minimum value reported for other gastroesophageal cancer cell lines (0.136 µM)32, suggesting that patient 41 may have benefited from treatment with this drug even though etoposide is more commonly used for neuroendocrine tumours. The IC50 values for the other compounds otherwise fall within the ranges reported for other cancer cell lines32. Expectedly, UWG02CTC and UWG02ASC cell lines were very similar in their drug sensitivities  significantly enhanced the radiosensitivity of cells resulting in decreased mean surviving fractions compared with cells exposed to radiation alone, especially after 1 Gy  gastroesophageal cancer cell lines derived from CTCs. These two cell lines are genotypically and phenotypically distinct, reflective of differing tumour biology in the donor patients. Similar to other reported CTC lines, UWG02CTC displayed an epithelial phenotype, with strong expression of EpCAM and pan-cytokeratin. In contrast, UWG01CTC, isolated from a patient with neuroendocrine tumour, showed variable and weak cytokeratin (Cam5.2) immunostaining, with strong expression of neuroendocrine markers . UWG01CTC had no detectable EpCAM, mesenchymal , or cancer stem cell marker . This phenotype makes UWG01CTC a unique EpCAM negative CTC cell line.in vitro and in vivo growth with a high grade histological appearance supportive of a stem cell (CSC) phenotype. It was interesting to detect differences in CD44, ALDH1 protein and STAT3 gene expression between the UWG02CTC and UWG02ASC, which probably reflects the different pathways of tumour cell dissemination. Of note, higher expression of CSC markers and key stem pathways was also found on UWG02CTC versus UWG01CTC, despite ALDH1, CD44, and CD133 reported as CSC markers in both adenocarcinomas and high grade neuroendocrine tumours34.Both CTC derived cell lines recapitulated the phenotype of the source patient’s tumour, highlighting that CTCs are a representative tumour source. The RNA expression profiling confirmed these data, with each cell line’s expression clustered with the source patient’s tumour and the corresponding mouse xenograft. Both CTC derived cell lines demonstrated rapid The preserved overall RNA expression profile and IHC staining pattern seen in tumours excised from the mice, and the cell cultures  derived from these tumours, highlight that these procedures, at least in short term cultures, do not grossly alter the original tumour characteristics, making CTC derived cell lines an excellent model to study tumour biology. Together with the confirmed tumourigenicity of the cell lines by the rapid development of tumours (within 4 weeks of inoculation) in all xenografted mice, and presence of key mutated and amplified genes that are characteristic of gastric adenocarcinomas or high grade neuroendocrine tumours, confirms the establishment of two novel CTC derived immortalised cell lines.35. All cell lines were very sensitive to radiotherapy, with a synergistic effect of carboplatin seen in UWG01CTC. This is consistent with the clinical experience of the radiosensitivity displayed by high grade neuroendocrine cancers, driven by low expression of key DNA damage response enzymes, such as ATM and ATR38. We also explored potential synergistic effects of drug combinations as this may allow clinical dose reduction, which in turn reduces toxicity while retaining the therapeutic efficacy.We also undertook cytotoxic and radiotherapy assays of the CTC cell lines based on standard cytotoxic agents used for these cancers. We found drug sensitivities were generally similar to those previously reported for gastroesophageal cell linesEGFR, FGFR2, ERBB2, and JAK/STAT pathway genes, suggesting potential sensitivity to treatments directed at these validated targets, while UWG01CTC had high expression levels of the DLL3 gene, which is proposed as a potentially druggable target39. While these data show that CTC derived cell lines can be used to define personalised drug sensitivities for gastric cancer patients, it is important to note the rapid disease progression and clinical decline of the patients that yielded CTC cultures. The test results in our study would have been too late to guide management, and faster protocols are needed to inform personalised therapy in a clinically meaningful way. Nevertheless, more comprehensive drug sensitivity testing will be performed on the established CTC lines, and may help to define better drug screening libraries for faster future protocols.Multiple other potential therapeutic targets were identified by RNA expression profiling. UWG02CTC had high expression of 12. While we agree with this requirement, this issue is complicated by the inherent biases when selecting the isolation method and epithelial marker based CTC definition used for enumeration. We employed the standard EpCAM based isolation and cytokeratin based CTC identification in the matched blood sample used for CTC enumeration. Due to the biological differences discussed above, our successful cultures derived from samples with high (109) and low (3) CTC counts, subsequently developing EpCAM positive and negative cell lines, respectively. This finding highlights a key issue in the CTC field. While patient 20 has a low EpCAM+ CTC count, which is within the range of background cells found to express CTC markers  detected from healthy donor blood samples with the IsoFlux system (data not shown), the successful establishment of a CTC cell line argues that this patient had a high number of EpCAM− CTCs with the ability to establish metastatic deposits. This hypothesis is also supported by the clinical picture. CTC numbers are known to increase with disease burden40, and at the time of blood draw patient 20 had high volume and rapidly progressive disease. It is reasonable to suggest this patient had a high number of biologically relevant CTCs which were missed using standard CTC isolation techniques. The negative selection used for CTC culture (CD45 depletion), rather than the positive selection used for CTC enumeration (EpCAM capture) was critical to detect these cells. The optimal isolation method of CTCs continues to evolve in the face of these issues, and while more emphasis has been given to epithelial to mesenchymal transition (EMT) phenotype CTCs in recent years41, the data presented here highlight the persistent challenges identifying the optimal markers to isolate CTCs.Other studies have suggested a high CTC count (>300) is necessary for successful culture12. It is unclear whether this finding is indicative that a high proliferation rate is a requirement for successful cultures, or is a result of selection pressures for aggressive clones in the culturing process.Both CTC cell lines displayed a highly aggressive phenotype, with rapid growth, high grade histological appearance, and high Ki67 expression. In both cases, this phenotype mirrored the rapid clinical progression seen in the corresponding patient. Similar results have been seen in other CTC cell lines28, while UWG02ASC was negative for CD44. This is consistent with other results showing that CTC cultures exhibit a stem cell phenotype, and further supports the notion that CTC cultures develop from the CTC population which are able to establish metastases42.The matched culture derived from ascitic fluid (UWG02ASC) simultaneously established from patient 41 as UWG02CTC demonstrated an identical phenotype, with the exception of cancer stem cell marker expression. UWG02CTC was strongly positive for CD44, a key CSC marker in gastric cancerIn conclusion, we report the first two long-term CTC cell lines developed from patients with metastatic gastroesophageal cancer, providing a high fidelity platform for high-throughput gene expression, drug response profiling and functional genomics studies. The two CTC cell lines displayed distinct profiles which faithfully recapitulate the source patient’s tumour. Our results support the developing role of CTC culture as an essential laboratory resource for the understanding of the biology of metastases and importantly undertake personalised screening for therapeutic strategies.Peripheral venous blood samples were collected from patients with metastatic gastroesophageal cancer prior to treatment. Patients had histologically confirmed gastric or gastroesophageal cancer treated at the Illawarra Cancer Centre, Wollongong Hospital, NSW Australia. Informed consent was obtained from each patient prior to enrolment, and the study was approved by South Western Sydney Local Health District Human Research Ethics Committee (Project Number 15/072). All experiments were performed in accordance with relevant guidelines and regulations.43. Enumerated CTCs were defined by the standard EpCAM/Cytokeratin/DAPI positive and CD45 negative phenotype40.Initially 7.5 ml of blood was collected in EDTA tubes. This was increased to 15 ml after seventeen patients were enrolled for higher CTC capture to improve culture success rates. Blood samples were transported immediately at room temperature for CTC isolation. A separate matched 7.5 ml EDTA blood sample was collected at the same blood draw for EpCAM based capture for CTC enumeration using the IsoFlux system (Fluxion Biosciences), and processed as per manufacturer instructions44, in both cell lines successful cultures were established in both media formulations, and we did not find any phenotypic differences between media conditions (data not shown). All media was supplemented with antibiotics  prior to a density gradient separation with Leucosep tubes (Stemcell Technologies) to isolate a peripheral blood mononuclear cell (PBMC) layer. We initially found excessive lymphocyte contamination prohibiting CTC growth with the RosetteSep Human CD45 Depletion Cocktail alone. This improved with changing to the CTC Enrichment Cocktail. The PBMC layer was washed twice, then immediately plated into 24-well ultra-low attachment plates (Corning) with serum free Advanced DMEM/F12  supplemented with epidermal growth factor , fibroblast growth factor  and N2 supplement (Life Technologies), or ADMEM/F12 with 10% foetal calf serum (FCS) in hypoxic conditions. Separate CTC cultures were maintained in 10% FCS or serum free media. While serum free media is reported to support a cancer stem cell phenotypePatient 41 had marked peritoneal disease and gross ascites from gastric adenocarcinoma. At the same time as the blood draw for CTC isolation, 200 ml of ascitic fluid were collected from a peritoneal catheter. This sample was transferred immediately to the laboratory, centrifuged to collect a cell pellet, washed twice in ADMEM/F12 with 1% antibiotic/antimycotic (Sigma Aldrich), and cultured as per conditions listed above. Cultures derived from the patient’s CTC and ascites samples were maintained independently under identical conditions. Once established as cell lines, for all subsequent routine cell culture and experimental assays, cells were maintained in ADMEM/F12 with 10% FCS supplemented with EGF.6 cells from early passages of each cell line were injected subcutaneously into the flank of NOD scid gamma (NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ) mice. Mice were monitored for tumour growth and sacrificed when the tumour grew greater than 10 × 10 mm, or the animal demonstrated signs of stress (such as >15% weight loss) or evidence of impedance of tumour on movement. Tumours were collected from the sacrificed mice for subsequent culture, RNA and DNA extraction, and histological analysis. For culture from xenografts, tumour tissues were cut into approximately 1 mm pieces and then incubated with tumour dissociation enzymes  in ADMEM/F12, after which the tumour homogenate was centrifuged, the pellet resuspended, and plated in serum-free media and hypoxic conditions as above. All procedures were carried out in accordance to the Australian Code for the Care and Use of Animal for Scientific Purposes 8th edition 2013, and approved by the University of Wollongong’s Animal Ethics Committee (study AE15/17).For confirmation of tumourigenicity, 1–2 × 10Expression of key proteins on the cell lines, mouse xenografts, and representative sections from the matching patient’s tumour specimen were compared using immunohistochemistry (IHC). For cell lines, cells were collected, centrifuged with supernatant removed, then clotted with plasma and commercially prepared thrombin (Fibri-Prest Automate from Stago) to prepare a cell block. All samples were fixed in 10% formalin and paraffin embedded, with 4 µm sections cut for staining. Antigen retrieval and development was performed on the fully automated Bond system according to manufacturer’s instructions  or AllPrep DNA/RNA/Protein Mini Kit , respectively, according to the manufacturer’s instructions. All samples were quantified using the NanoDrop . DNA/RNA samples had A260/280 ratios between 1.8 and 2.1.45. As recommended, genes with expression levels at or below the level of the negative controls were removed from analysis. Differential expression of key transcriptomic pathways were compared between cell lines, with fold change and P values calculated using nSolver default settings. With the remaining list of genes, a filter cutoff of fold change ≥ ±2 and P value < 0.05 were used to identify the significant gene expression changes46.Total RNA extracted from fresh-frozen (25 ng) and FFPE (150 ng) specimens were run on the NanoString nCounter Sprint system using the 770 gene PanCancer Pathways panel with additional cancer stem cell and proteolytic genes as per the manufacturer’s instructions (NanoString Technologies). RNA extracted from patient 20 FFPE specimen had low RNA integrity and did not meet the threshold for further analysis. Results were analysed using the NanoString nSolver 4.0 and Advanced Analysis Module, which normalizes gene expression to a set of positive and negative control genes built into the platformSTR profiles for the cell lines and their matching patient FFPE tumour tissue were verified by the PowerPlex 18D System, using the following 18 markers (seventeen STR loci and Amelogenin): D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, vWA, D8S1179, TPOX, FGA, D19S433 and D2S1338. As per standard practice, cell lines were considered to match if profiles are more than 80% identical to source patient sample.DNA of the cell lines and xenografts were sequenced using the Oncomine Comprehensive v3 Assay on an Ion S5 Sequencer (Thermo Fisher Scientific). The assay detects specific hotspots as well as coding sequences in particular genes. STR and Oncomine assays were performed by KCCG Sequencing Laboratory & Cancer Diagnostics, Garvan Institute of Medical Research, Sydney Australia.50 . Data is presented as a mean ± standard error of the mean (SEM) from ≥2 independent experiments.Approximately 10,000 cells were seeded per well in triplicate into a 96-well plate 48–72 h prior to drug treatment. Cells were incubated with serial dilutions of each drug for 72 h with drug vehicle  and the dilution kept constant across all drug concentrations and controls . The viability of cells were assayed using CellTitre 96 Aqueous One Solution Cell Proliferation Assay  using a SpectroMax 250 UV plate reader using SoftMax Pro software . Cell viability of treated cells was normalized against vehicle controls. This data was analysed using a logarithmic sigmoidal dose–response curve using the variable slope parameter to determine IC50 values determined as monotherapies and ranged from 0.125×, 0.5×, 1×, 2× and 4× the IC50 of each drug, as optimal for the Chou-Talalay median-effect equation47. To determine synergism/antagonism, the combination index (CI) and dose response index (DRI) was calculated using CalcuSyn , with Monte-Carlo simulation to assign confidence. By the Chou-Talalay method, a CI = 1 represents an additive effect, a CI value > 1 represents antagonism and a CI value < 1 represents synergism.For combination drug assays cells were seeded as above and then treated with the drug combinations specified. The drug concentrations were kept at a constant ratio based on their approximate IC48. UWG02CTC and UWG02ASC were not suitable for this assay as they formed loose and small colonies. Briefly, cells were first acclimated for at least a week in normoxic conditions, then plated into 12.5 cm2 tissue culture flasks in regular cell culture media (as above) in order to reach ~60% confluency 3 days later. Cells were then pre-sensitized with 0, 1, 2 and 5 µM carboplatin for 48 h prior to exposure to 1 or 2 Gy X-Ray radiation delivered in a single fraction at room temperature. Cells were then seeded into triplicate tissue-culture petri dishes  in 10 mL of ADMEM/F12 with 10% FCS, pencillin/streptomycin, EGF and L-glutamine, at different cell densities per dish (ranging from 1000 to 20000 cells per dish). After approximately 15 doubling times, petri dishes were washed with PBS and adherent cell colonies fixed and stained with a 1:3 crystal violet:ethanol solution. Colonies (≥50 cells/colony) were manually counted and presented as Mean Plating Efficiency  and Surviving Fraction  as previously described49. Unirradiated control samples (with and without drugs) were handled under the same conditions as the irradiated samples.The sensitivity of the UWG01CTC cells to 10MV x-ray irradiation with drug pre-treatment was investigated using clonogenic survival as the radiobiological endpoint as previously described50. Cells were irradiated in T12.5 cm3 flasks using a Varian 2100iX linear accelerator. Prior to irradiation flasks were completely filled with medium. Flasks were located at midpoint in a 23 cm thick Solid Water phantom . Flasks were surrounded with Super-Flex bolus  to ensure electronic equilibrium. Doses of 1 or 2 Gy were delivered using parallel opposed beam geometry, in a single fraction at room temperature. Briefly, cells (2.0 × 104) were stained with PI master mix  for 30 min at 37 °C. DNA content was then measured using a Becton Dickinson BD LSR II FACSort flow cytometer  and the proportion of cells in G0/G1, S and G2/M phases of cell cycle were calculated on the basis of DNA distribution histograms using FlowJo software as previous described 51.Measurements of cell cycle distribution after irradiation were performed essentially as previously describedt test, Fisher’s exact test, one-way analysis of variance test, and Dunnett’s test were performed. P values < 0.05 were considered statistically significant.Apart from the NanoString analyses, all the experimental data were expressed as means ± SEM and statistically analyzed using GraphPad Prism . For comparisons, two-tailed Student’s Supplementary Information.


R), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1’(R), and 6’(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1( Acute myeloid leukemia (AML) is one of the main causes of death related to leukemia, with 70% of patients either not responding to treatment or eventually relapsing to secondary chemoresistant AML. Standard therapy has been almost unchanged for the last 40 years, and new drugs or drug combinations targeting the resistant fraction of patients are actively researched .www.fda.gov), and is under evaluation for AML treatment [B-cell lymphoma (Bcl)-2 and myeloid cell leukemia (Mcl)-1 are two major anti-apoptotic proteins, which are frequently deregulated in cancer. The Bcl-2 family of proteins is the target of recently developed synthetic BH3 mimetics, namely ABT-263 (Navitoclax) and ABT-199 (Venetoclax) or natural compounds [reatment . Howeverreatment . Recentlreatment .Aplysina aerophoba to act as a cytostatic drug against a panel of cancer cell lines and sensitizes lymphoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [Pseudovibrio denitrificans Ab134 isolated from the sponge Arenosclera brasiliensis [Marine organisms and sponges in particular constitute a precious source of biologically active compounds, which provide potential agents against cancer ,13,14,15 (TRAIL) . Fistula (TRAIL) ,25,26,27iliensis , which e1H and 13C NMR spectra of fistularin stereoisomers [R), 6(S) for C1 and C6, the absolute configuration at the two secondary carbinols C11 and C17 is rather complicated. This high molecular weight molecule presents four secondary alcohols that hampered the co-determination of their absolute configuration by chemical modification and NMR studies. Suitable crystals for X-ray studies have never been obtained for one of the stereoisomers of fistularin-3. Moreover, the carbon configuration also seems to vary depending on sponge species, and even mixtures can occur [Aplysina oroides, which was recognized as a 11R isomer of fistularin-3 extracted from Aplysina cauliformis, attributing to this latter the 11S configuration [Due to the similarities of oisomers  confusioan occur . The namguration . FinallyR), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3 -11(R), 17(S)-fistularin-3, and abbreviated here as RS-F3), isolated from the sponge Suberea clavata. We characterized the complete absolute configuration of RS-F3, revealing that this compound is in fact the (+)-11(R), 17(S)-fistularin-3 isomer. We confirmed the potential of this isomer to interfere with DNA methyltransferase (DNMT) 1 and assessed its anticancer potential on a panel of AML cell lines and show that it induces differential proliferation arrest and cytotoxicity. We also found that RS-F3 induces an early decrease of Mcl-1 protein levels in sensitive AML cell lines, which was independent of Noxa, but could be rescued by proteasome inhibition. Moreover, combination treatments with ABT-199 resulted in strong induction of apoptosis in AML cell lines resistant to Bcl-2 inhibition, which was not observed in healthy human progenitor/stem CD34+ cells or platelets.Here, we report the activity of the stereoisomer (+)-1(Suberea clavata (200 g) was extracted at room temperature with methanol/dichloromethane (MeOH/DCM) (1/1) to give 57.8 g of dried extract. The crude extract was submitted to n-butanol/H2O partition to obtain a desalted butanol extract (30 g). A portion (5 g) of the later extract was submitted to filtration on Phenomenex Sepra C18  silica gel and eluted with a gradient H2O/MeOH (100/0 to 0/100) and MeOH/DCM (100/0 to 0/100) to give 6 fractions (f1 to f6). The fraction f3 (1.3 g) was submitted to reversed phase (Sunfire C18) HPLC chromatography with H2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH: 85/15 to 0/100 in 40 min and yield 13 subfractions (F1 to F13). The fraction F6  was pure (+)-11(R), 17(S)-fistularin-3 (1H and 13C data of the compound (+)-11(R), 17(S)-fistularin-3   E.To investigate involvement of molecular markers well-known to contribute to AML cell survival in the effect of RS-F3, we assessed the expression of the anti-apoptotic protein Mcl-1, associated with resistance to chemotherapy in AML ,30. KineWe previously showed that isofistularin-3 induced endoplasmic reticulum (ER) stress in RAJI Burkitt lymphoma cells  and ER s50 above 1 µM [Since combination treatments targeting Mcl-1 and Bcl-2 are considered a promising anticancer strategy against AML ,7,34, weove 1 µM ,35. We pInterestingly, in the presence of the caspase inhibitor, U-937 cells shifted to a caspase-independent apoptotic-like cell death, whereas apoptosis was fully prevented, and a small fraction of necrotic cells appeared in the case of OCIAML-3 cells . Under t+ cells from healthy donors, a reduction of cell viability of about 30% was observed, which is less than a half of the cell death occurring in the Bcl-2-positive AML cell lines tested  6(S), 1’(R), and 6’(S) configuration (R)-C17(S) exhibited lower global energy score than other stereoisomers (R) 6(S), 1’(R), 6’(S) configuration, RS-F3 showed the lowest global energy score (−44.34) when DNMT1  [Four C11-C17 diastereoisomers for fistularin-3 can exist in theory, but not all the diastereoisomers have been discovered in Nature so far. To gain structural insights of these fistularin-3 stereoisomers on DNMT1 activity, we implemented simulated docking experiments on DNMT1 with four C11-C17 diastereoisomers with established 1  was usedS-adenosyl methionine (methyl donor) concentration did not affect the inhibition of isofistularin-3 against DNMT1 activity, the methyl donor binding site would not be an isofistularin-3 binding site [To further support the predicted binding modes of RS-F3 on DNMT1, we used a different docking program, AutoDock 4.2 , which iing site . Taken aIn the case of most favorable docking pose, RS-F3 penetrated the negatively charged cleft that is surrounded by CXXC and RFTS domains and consolidated the interaction between CXXC and RFTS domains. Furthermore, RS-F3 stretched to the DNA binding loop on CXXC domain, SKQ, that could influence to the interaction between CXXC domain and DNA . Given tWe then assessed the ability of RS-F3 to inhibit DNMT1 activity in vitro. We used epigallocatechin-3-gallate (EGCG), a well-known in vitro DNMT1 inhibitor, as a positive control. Results showed that 50 µM RS-F3 reduced the activity of purified DNMT1 by about 25% C.Finding new effective treatments against AML is one major challenge of current anticancer research. Several marine compounds have already shown promising activity against AML cells ,44 and cR, 17S-fistularin-3, briefly RS-F3. RS-F3 was tested for its anticancer effect on a panel of AML cell lines. Three AML cell lines decreased proliferation and viability at 5–15 µM. These results are similar to those obtained previously with the compound isofistularin-3 from Aplysina aerophoba, a stereoisomer of fistularin-3 with a non-specified configuration at C17 [We report here an interesting cytostatic and cytotoxic effect against AML cell lines of a brominated sponge-derived compound of the fistularin family, named (+)-11n at C17 . In thisWe thus investigated the anti-proliferative molecular mechanism of RS-F3 by assessing levels of Mcl-1. We showed that sub-toxic doses of RS-F3 reduced Mcl-1 levels in AML cells lines at early time points. However, in HEL cells, which continued to proliferate and survive to RS-F3 treatment, we detected no Mcl-1 decrease. The JAK2 V617 mutation status of this cell line could be implicated in this differential behavior; however, this hypothesis remains to be tested. Overall, these data suggest an important role for Mcl-1 in the anti-proliferative effect of RS-F3. Mcl-1 downregulation could result from transcriptional inhibition, increased degradation or from the combination of these two phenomena. When U-937 cells were treated with two different proteasome inhibitors together with RS-F3, Mcl-1 levels were rescued. Our data suggest a contribution of proteasomal degradation to RS-F3-induced Mcl-1 decrease.+ cells), to sub-micromolar concentrations of ABT-199. These data attest for a promising sensitizing activity of this marine compound towards Bcl-2 inhibitors, which could be exploited in the treatment of AML with high Mcl-1 expression and resistance to BH3 mimetics.Importantly, in this study we found that combination treatments of RS-F3 with the Bcl-2 inhibitor ABT-199 sensitized U-937 and OCIAML-3 cell lines, but not Bcl-2 negative TF-1 cells or healthy platelets , 6(S), 1’(R), 6’(S) fistularin-3 stereoisomers and to bind to the interface between CXXC and RFTS domains. In addition, RS-F3 exhibited an inhibitory effect on the in vitro DNMT1 activity. Altogether, these data support the increasing knowledge about the potential of marine bromotyrosines to modulate epigenetic enzymes and lead cancer cells to proliferation arrest or to death.Bromotyrosine derivatives comprise several biologically active molecules ,24,27,59Suberea clavata , which was collected by hand using SCUBA diving in Solomon Islands, Russel Group, off Lologhan Island on the 30/06/2004 between 6 m and 12 m deep. A voucher sample is deposited at the Queensland Museum under the accessing number G322641 and was identified by Dr. J.N.A. Hooper as Suberea clavata . The sponge was deep frozen on board until work up.Compound RS-F3 was extracted from the freeze-dried sponge 2O partition to obtain a desalted butanol extract. A portion of the extract was submitted to filtration on Phenomenex Sepra C18 silica gel and eluted with a gradient H2O/MeOH (100/0 to 0/100) and MeOH/DCM (100/0 to 0/100) to give 6 fractions. The fraction f3 was submitted to reversed phase (Sunfire C18) HPLC chromatography with H2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH: 85/15 to 0/100 in 40 min and yield 13 subfractions (F1 to F13). The fraction F6 was pure (+)-11(R), 17(S)-fistularin-3.The lyophilized sponge was extracted at room temperature with methanol/dichloromethane (MeOH/DCM) (1/1). The crude extract was submitted to n-butanol/HR), 17(S)-fistularin-3 was established using the Mosher method and intensive purification by RP-HPLC, LCMS and NMR analysis .The absolute configuration of (+)-11; bortezomib, Venetoclax (ABT-199), and ABT-263 were purchased from Selleckchem . All drugs were dissolved in DMSO.+ progenitor/stem cells were isolated from cord blood of healthy donors and cultured as previously described [2.The AML cell lines U-937, HL-60, HEL, THP-1, and TF-1 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) were cultured in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal calf serum  and 1% antibiotic–antimycotic (Lonza); TF-1 cells were also supplemented with 5 ng/mL granulocyte–macrophage colony-stimulating factor; PeproTech, London, UK). The AML cell line OCIAML-3 was cultured in MEM-ALPHA (Lonza) supplemented with 15% FCS and 1% antibiotic–antimycotic. PBMCs and platelets  from human healthy donors were isolated and cultured as previously described . Human pescribed . All celCell number and viability were determined using a semi-automated image-based Cedex cell counter  hinged on the Trypan Blue (Lonza) exclusion assay. Colony formation assays were performed as previously described , with a Lymphocyte subpopulation from PBMCs was identified by flow cytometry  based on cell size and granularity. Data were recorded statistically (10.000 events/sample) using the CellQuest 4.0.2 software  and analyzed using Flow-Jo 8.8.7 software .Platelet viability was assessed by measuring ATP levels with the Cell Titer-Glo Luminescent Assay , according to the manufacturer’s instructions. Data were recorded using an Orion Microplate Luminometer . In parallel, viability was evaluated by measuring phosphatidylserine exposure by FACS analysis of cells stained with FITC Annexin V . The platelet-toxic compound ABT-263 and a challenge with 50% DMSO for 1 min were used as a positive control for platelet mortality.The modality of cell death was assessed performing nuclear morphology analyses by fluorescence microscopy , after staining with Hoechst 33342 and propidium iodide.Enzymatic activity of caspases-3/7 was determined using the Caspase-Glo 3/7 Assay . The assay was performed according to the manufacturer’s instructions and luminescence was measured using an Orion Microplate Luminometer .®  supplemented by 1× protease inhibitor cocktail  according to manufacturer’s instructions. Western blots were carried out, as previously described [Whole cell extracts were prepared using M-PERescribed , using tCHOP gene expression levels were evaluated by Q-RT-PCR, as previously described . XBP1 spDNMT1 activity was measured using the in vitro DNMT activity/inhibition assay  according to manufacturer’s instructions. The methylation reaction was performed by incubating 25 ng of purified DNMT1 with compounds for 2 h at 37 °C in the presence of 0.01% Triton X-100. The methylated DNA was then recognized by the His-tagged methyl-CpG binding domain protein 2b. The addition of a poly-histidine antibody conjugated to horseradish peroxidase then provided a colorimetric readout quantified with a spectrophotometer  at the wavelength of 450 nm.p-values below 0.05. All histograms represent the mean ± SD of at least 3 independent experiments.Statistical analyses were carried out using the GraphPad Prism 7.0 software . One-way or two-ways ANOVA, followed by the Holm-Sidak multiple comparison tests were used for statistical comparisons. Statistical significances were evaluated at R), 17(S)-fistularin-3 (RS-F3). We showed RS-F3 to induce an interesting anticancer effect against AML cell lines. Our data suggests an important role for Mcl-1 in mediating the anti-proliferative activity of the drug. Importantly, we show that this fistularin-3 stereoisomer synergizes with the Bcl-2 inhibitor ABT-199 in killing Bcl-2-positive AML cell lines. Moreover, our docking and in vitro studies support the increasing knowledge about the potential of marine bromotyrosines to modulate DNMT1 activity.We determined the absolute configuration of the fistularin-3 stereoisomer (+)-11(


Carnosic acid was used as starting material to synthesize royleanone derivatives featured C11–C14 para quinone. The importance of C-20 group of royleanone derivatives was verified by the cytotoxicity assay of royleanonic acid, miltionone I and deoxyneocrptotanshinone. Following our synthetic route, 15 amide derivatives were synthesized and 8 compounds exhibited moderate cytotoxic activities against three human cancer lines in vitro.The online version of this article (10.1007/s13659-018-0173-y) contains supplementary material, which is available to authorized users. Royleanones, possessing a characteristic 11,14-para benzoquinone 12-hydroxy abietane skeleton, were first isolated by Handa’s group in 1945 from the roots of Inula royleana as yellow pigment [ pigment –5. These pigment .para benzoquinone moiety in royleanones could not act as Michael acceptor, the cytotoxic activity of royleanones would be weak. In fact, royleanones has IC50 of 32.5 µM to MIAPaCa-2 cell line and deoxyneocryptotanshinone was inactive (IC50 > 100 µM) to the same cell line [para benzoquinone derivatives might lead to more potent analogues than columbaridione.Our research interests are largely related to the antitumor molecules, especially tricyclic terpenoids derivatives –12. Tansell line . Royleanell line . The difpara benzoquinone. As a result, 15 new amide derivatives were synthesized , intramolecular esterification occurred to afford lactone 2 with DCC. The 12-hydroxy group was then protected as MOMO ether. After hydrolysis, the resulting free phenol 10 was oxidized into para benzoquinone 11. Liberation of MOMO ether by HCl afforded the desired royleanonic acid.To verify the hypothesis of the importance of C-20, we synthesized royleanonic acid as indicated in Scheme p-TsOH in MeOH, Deoxyneocrptanshinone was obtained in 70% yield  (Table 7 had a significant cytotoxicity toward Hep G2 and MCF 7 human cancer lines in vitro (IC50 1.02–2.03 μM).All the synthesized compounds were evaluated for cytotoxicity against three human cancer cell lines . Eight royleanone derivatives showed potent cytotoxicity against three human cancer cell lines IC50 < 0 μM . These results may attribute to mechanism other than Michael addition of DNA, RNA etc. [7 gave the best result. All derivatives are active towards HpeG2 cell line. These findings may serve as new clues for discovering antitumor molecules with new structure scaffold.Our experiments demonstrated that, for the first time, modification of orthoquinone into RNA etc. . The exiSupplementary materials such as experimental procedures, MS and NMR spectra of the synthesized compounds are available in the online version of this article free of charge. Supplementary material 1 (DOC 5423 kb)Below is the link to the electronic supplementary material.


These hyperploid cell lines form xenografts in mice and show metabolically active and necrotic areas on fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. WES of ITOC cell lines recapitulate the genomic tumor profile of ICGC GB-SCC database. We further identified smokeless tobacco associated genetic alterations  and oncogenic PIK3CA mutation in GB-SCC cell lines. Transcriptome profiling identified deregulation of pathways commonly altered in cancer and down-regulation of arachidonic acid metabolism pathway, implying its possible role in GB-SCC. Clinical application of high throughput sequencing data depends on relevant cell line models to validate potential targets. Extensively characterized, these oral SCC cell lines are particularly suited for mechanistic studies and pre-clinical drug development for smokeless tobacco associated oral cancer.Smokeless tobacco associated Gingivobuccal squamous cell carcinoma (GB-SCC) is a major public health problem but available oral cancer cell lines are mostly from smoking associated tongue SCC raising the need for pertinent GB-SCC cell line models. As part of the International Cancer Genome Consortium (ICGC) Project, 4 novel cell lines, namely, Indian Tata Memorial Centre Oral Cancer (ITOC) −01 to −04 were established and characterized with conventional methods, karyotyping, ultrastructure,  Smokeless tobacco is consumed in various forms with areca nut, lime or betel leaves as quid, pan, masheri or gutka4. Most oral cancers are squamous cell carcinomas (SCC) and arise from the lip, tongue, gingivobuccal mucosa, retromolar trigone, floor of mouth or the hard palate. The form of tobacco use not only influences the site of cancer within the oral cavity but also its biology and mutational profile. In western countries where smoking is the predominant form of tobacco use, tongue cancer is the most common oral cancer while in south Asian and east Asian countries where tobacco chewing is more prevalent, Gingivobuccal SCC (GB-SCC) is the most common oral cancer. GB-SCC differs from the tongue SCC in its clinical behaviour5 as well as molecular8 and immunoproteome profile9, yet the treatment modalities are identical. Previous studies have revealed that as compared to tobacco smoking, the smokeless tobacco associated cancers have predominance of specific transversions or transitions in TP53 and the genome12.Advanced stage oral cancer with poor clinical outcome is a major public health problem in south Asian and east Asian countries where smokeless tobacco use is prevalent6 and The Cancer Genome Atlas (TCGA) project in USA for diverse HNSCC13. Large number of novel and recurrently mutated genes and deregulated pathways in HNSCC, with some being unique to GB-SCC were identified. Mechanistic studies on the role of an increasing number of genetic variants being identified in different cancers and their translational exploitation requires cell lines from same organ and histology with full molecular genetic characterization.Comprehensive molecular genetic characterization using whole genome, exome and transcriptome analysis has been reported in over 400 Head and Neck SCC (HNSCC) tumours. These include GB-SCC by our group as part of the International Cancer Genome Consortium (ICGC) India Projectin vitro models due to the ease of performing large scale experiments, genetic manipulation and reproducibility14. Cell lines maintained using appropriate conditions retain parent tumor features and phenotype, making them preferred tumor representative models in laboratory studies16. The dearth of commercial cell lines for tumours which are uncommon in large parts of the world necessitates the need to establish and characterize novel cell lines from primary tumor17.Primary cultures and immortalized cell lines are indispensible source for conducting translational cancer research. Tumor-derived cell lines are widely used 20 and none from the Indian subcontinent. As part of our systematic efforts to establish relevant cell lines in the ICGC India oral cancer project, we have established and performed comprehensive molecular genetic characterization of 4 oral squamous cell carcinoma (OSCC) cell lines from tumour tissues obtained from tobacco chewing Indian patients. These cell lines are named ITOC-01, 02, 03 and 04, after the ethnic origin, institute and cancer site - Indian Tata Memorial Centre Oral Cancer (ITOC). These cell line models established from smokeless tobacco consuming patients provide a platform to carry out wide spectrum of assays spanning from functional genomics to drug response studies. To the best of our knowledge this is the first study from Indian subcontinent characterizing smokeless tobacco GB-SCC derived cell lines.Of the several hundred HNSCC cell lines reported worldwide, very few are from GB-SCC in patients with smokeless tobacco useDetails of the patients from whom these cell lines were derived are provided in Table Classical epithelial morphology and characteristic SCC features were seen in all four parent tumors Fig. . Detaile21.Genotyping confirmed the human origin and unique genotype of each cell line  with GeneJuice transfection reagent. EGFP expression observed after 48 hours of transfection revealed low transfection efficiency in ITOC-01 (1%) and ITOC-02 (6%) but high efficiency in ITOC-03 (30%) and ITOC-04 cell lines (20%)  and Bonferroni post-tests showed high significance between control and time points of 24 and 48 hours  mice in 3, 2 and 2 weeks respectively Fig. . GrosslyTP53, FAT1, CASP8, NOTCH1, KMT2B, PCLO, UNC13C, SMG1, FAT3, EP300, KMT2D, SYNE2, TRPM3, PIK3CA and NSD1), 12 were mutated in one or more of the ITOC cell lines  and inflammatory pathways , implying their role in cancer progression29. Mutational signature in the 4 cell lines were similar with C > T transitions (44%) being most common, followed by T > C (20%), C > A (21%) and C > G (15%) transversions  was done at a mean depth of 155x, 112x, 128x and 113x for ITOC-01, -02, -03 and -04 cell line respectively with mean mapping quality of 58.12. A total of 24150, 21921, 22576 and 22018 exonic variants were identified in the ITOC-01, 02, 03 and 04 cell lines respectively, of which, 369, 265, 343 and 312 somatic variants were considered deleterious. Each cell line had a mutational profile matching with the GB-SCC or HNSCC mutational profile reported in the ICGC GB-SCC and TCGA HNSCC database , cell lines at late passage  and xenograft tumors was performed by evaluating RNA-seq of three normal buccal mucosa tissues and four OSCC cell lines generated ~65 million reads each. Compared to the 3 normal buccal mucosa tissues, 661, 811, 885, 907 genes were upregulated and 1627, 1997, 2343, 1950 genes down-regulated in ITOC-01, -02, -03 and -04 cell lines respectively  and down-regulation of Vimentin validates the epithelial origin of these cell line and also corroborates with the earlier result of expression of keratin 8 in OSCC cell lines  were down-regulated in ITOC cell lines.RNA-seq data was validated by quantitative polymerase chain reaction (qPCR) for 30 genes which are known to be deregulated in cancer and also in these cell lines Fig. . Gene exnes Fig. . The AAM18. The Expert protein analysis system (ExPASy) – Cellosaurus database has reported over 150 SCC cell lines derived from tongue cancer but very few from buccal mucosa32 or gingiva35. Surprisingly, no GB-SCC cell lines are reported in literature or databases from the Indian subcontinent where it is the most common oral cancer. One possible reason may be the difficulty in culturing and establishing cell lines from heavily infected locally advanced gingivobuccal cancers.Human cancer cell lines are indispensable to functionally characterize new or previously uncharacterized genetic alterations identified by large scale genomic studies. In the precision medicine era, the utility of a cell line for identifying clinically relevant targets requires these cell lines to closely recapitulate the molecular genetic profile of specific human cancers. Despite the common SCC histology, prior tobacco exposure and several common genetic alterations, the carcinomas arising at different primary sites within the Head and Neck region have several distinct featuresTP53 mutations across parent tumor, cell line at early and late passage and xenograft tumors affirms the genomic characteristics of primary tumor being preserved throughout the cell line cultivation and further in the xenograft model as well. The genomic and histopathological analyses assure the OSCC cell lines are representative models of the parent tumors. Through exome and transcriptome analysis we confirm that ITOC cell lines recapitulate the mutational and gene expression profile of GB-SCC and HNSCC in the ICGC and TCGA studies13. Of the 15 most recurrently mutated HNSCC or GB-SCC genes, 8 genes  were mutated in >10% of smokeless tobacco associated GB-SCC tumours of ICGC6 but not in the cigarette smoking associated HNSCC of TCGA study13 and may be considered as smokeless tobacco associated genes  had deleterious mutation in GB-SCC ITOC cell lines  but not in the Tongue-SCC ITOC-02 cell line. This highlights the need for establishing, characterizing and using cell lines from tumours of the same subsite within HNSCC that have developed after similar form of tobacco exposure. Interestingly, the tongue SCC ITOC-02 cell line had truncating mutations in a few genes which are rarely associated with some cancers, PYROXD2, PCMTD1 reported to be mutated in hepatocellular cancer37 and PABCP1 reported to be upregulated in gastric carcinoma and targeting miR-34c38.We have successfully established and characterized 4 OSCC cell lines including 3 from GB-SCC derived from tobacco chewers. We demonstrate these cell lines to be of human origin with characteristic features of SCC. Identification of identical nes Fig. . Three oPIK3CA mutation (H1047R). It would be particularly useful to study PIK3CA inhibitors like Alpelisib (BYL719) which has been evaluated in only 2 PIK3CA mutated HNSCC cell lines39. Encouraging results from first in human use of Alpelesib in diverse tumours with oncogenic PIK3CA mutations has been reported40 and merits further evaluation in HNSCC including GB-SCC.Moreover, ITOC-01 is the only GB-SCC cell line harbouring an oncogenic 10, however, this was not observed in any of the 4 ITOC cell lines. Similar to our finding, Fadlullah et al.20 had also not observed any correlation between the patient habits and mutation signatures. It is important to note that smokeless tobacco is used in many forms with or without lime, areca nut and other potentially carcinogenic ingredients. Hence there is a need to re-examine signature 29 and its association with smokeless tobacco and forms of smokeless tobacco use.Earlier studies have proposed genetic signature 29 with abundance of C > A transversion for smokeless tobacco associated cancers41. For the ITOC cell lines we used stringent bioinformatic methods to deduce somatic mutations. This seems to be a valid approach since the known mutational profile of HNSCC and GB-SCC is recapitulated in the ITOC cell lines. Since gene expression is tissue dependent, to interpret deregulated pathways in specific cancers, it is important to use representative normal tissues. In the TCGA HNSCC study with 16 GB-SCC tumours, their head neck region normal tissue dataset did not have any normal buccal mucosa sample for gene expression analysis. Similarly, the oral cancer cell line panel study used cultured keratinocytes in lieu of normal oral mucosa tissue20. Pure normal cell population in culture may be used as a convenient surrogate for normal tissues. However it is well known that isolation of specific cell populations, immortalization of normal or tumour cells, and the culturing conditions could all result in major alterations in gene expression as compared to their counterpart normal tissue or primary tumor42. This may result in misidentification of altered genes and pathways leading to false discovery. Possibly as a result of using normal buccal mucosa for determining differential gene expression, the deregulated genes in ITOC cell lines show excellent correlation with the qPCR data 43, epigenetic markers ; ECM receptor interaction molecules (laminin - LAMC2) and IL23A. A recent study has shown up-regulation of LAMC2 as a marker of cancer invasion in oral leukoplakia44. We propose a similar role could be observed in OSCC-GB. ITOC cell lines also show up-regulation of ECM receptor interaction and focal adhesion pathway, similar to previous large scale transcriptomic analysis of 326 oral cancer tissues45. Previous report by ICGC India team has shown loss-of-function somatic mutations in AAM pathway leading to better patient survival46. Considering the identification of somatic mutations and down-regulation of gene expression in AAM pathway components of these novel cell lines it would be interesting to further elucidate the role of AAM pathway in OSCC.The ITOC cell lines show up-regulation of inducers of epithelial to mesenchymal transition  (Gibco) supplemented with antibiotic] at 37 °C in a humidified atmosphere containing 5% CO2. Culture medium was replaced every 48 hours and the epithelial pool of cells was enriched by differential trypsinization and subsequent removal of fibroblasts. Cells at 80% confluency were split in the ratio of 1:2 twice a week. Four cell lines were established – 3 from GB-SCC  and 1 from tongue SCC (ITOC-02).This study was approved by Tata Memorial Centre (TMC) – Advanced Centre for Treatment, Research & Education in Cancer (ACTREC) Institutional Ethics Committee III (Project No. 3). This study was conducted at TMC, ACTREC and all experiments involving human tissue were performed in accordance with relevant guidelines and regulations. Tumor tissues were collected at the time of surgery after obtaining informed consent from patients with smokeless tobacco associated oral SCC. Tumor tissues were transported in 5 mL Plain Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco) with double strength antibiotic mixture of Penicillin 400 U/mL, Streptomycin 40 µg/mL, Gentamycin 50 µg/mL and Mycostatin 10 U/mL (PSGM). Tissues were processed within half an hour of resection and were rinsed twice with sterile 1X phosphate buffered saline (PBS) followed by 10% Povidone-iodine (Wockhardt) and PBS wash. Tumor tissues were minced finely to small pieces of approximately 1–2 mmMorphological characteristics of the cell lines were studied using Olympus IX51 phase contrast microscope and ultra structure analysis was performed with Transmission Electron Microscope  at 120 kV .Cells were harvested and arrested with colcemide (0.05 µg/mL) for 1 hour at 37 °C. Mitotic cells were centrifuged at 2000 rpm for 5 minutes followed by hypotonic KCl treatment for 30 minutes at 37 °C. Cells were fixed in fresh methanol: glacial acetic acid  for 15 minutes at 4 °C. G-banding was performed by treating the slides with trypsin followed by Giemsa staining (GTG). Metaphases were captured using Olympus BX61 microscope  and analyzed using Applied Spectral Imaging (ASI) GenASIs software version 7.0 .http://strdb.cogcell.org) and ATCC STR database (http://atcc.org/STR_Database) of cell lines using match threshold of ≥80%.Genomic DNA of cell lines was used for STR analysis of 8 autosomal markers  and 1 gender marker, Amelogenin. PCR products were analyzed with 3500 Genetic Analyzer (Applied Biosystems) and compared with COG STR database -2,5-diphenyltetrazolium bromide (MTT) solution was added at a final concentration of 0.5 mg/mL and incubated for 4 hours at 37 °C, 5% CO2. Purple formazan crystals were dissolved by addition of 10% SDS. Absorbance was measured at 540 nm with a spectrophotometer (BMG Labtech). Cell proliferation was assayed at different time intervals - 0, 24, 48, 72 and 96 hours. MTT readings were recorded in triplicates and the overall mean plotted against time. Standard error of the mean (SEM) were calculated for each day and the exponential growth curve plotted. Doubling time calculated using the equation ln(2)/K, where K is the growth rate constant.Cells were seeded at a density of 5 × 10Lymphocytes isolated from fresh blood of a healthy donor were used as control for ploidy studies. Cells fixed in 70% ethanol for 60 minutes were incubated with 100 µg/mL RNase A (Sigma-Aldrich) and stained with 50 µL propidium iodide (Sigma-Aldrich) for 30 minutes at 37 °C. Acquisitions were performed on BD FACSCalibur (Becton Dickinson) and data analyzed using Modfit software (version 2.0).Cells were grown on sterile coverslips for 48 hours, fixed in ice-cold methanol at −20 °C for 10 minutes. Coverslips were rinsed with PBS and permeabilized using 0.3% Triton X and ice-cold methanol at room temperature (RT). Blocking was carried out using 3% bovine serum albumin (BSA) in PBS for 15 minutes at RT. Cells were incubated with primary antibodies for 1 hour at RT; p53 , Cytokeratin 8  followed by 1 hour incubation with fluorescein isothiocyanate (FITC) secondary antibody , Alexa Fluor 568 goat anti-mouse IgG (H + L), Molecular Probes). Coverslips were mounted using Vectashield (Vector Labs) mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) and viewed under confocal microscope .5 in 35 mm cell culture dish were transfected with 1 µg of pEGFP-C1 plasmid using GeneJuice (Merck) transfection reagent as recommended by the manufacturer’s instructions. Cells were incubated at 37 °C for 48 hours and transfection efficiency was evaluated by counting 200 cells from five different fields under a fluorescent microscope.Cells seeded at a density of 3 × 106 in 6-well plate and on reaching 100% confluency a wound was inflicted with a pipette tip and images were captured every 24 hours on Olympus IX51 microscope and analyzed using ImageJ software. The relative cell free area at respective time points was calculated by considering the inflicted wound at 0 hours as 100%. One-way analysis of variance (ANOVA) and Bonferroni post-tests were performed to analyze effect of wound healing with respect to time.Cells were seeded at a density of 2 × 105 cells/mL were suspended in 0.2% agar in IMDM with 10% FBS and overlaid onto 0.5% basal agar coat in 35 mm plates. The plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 21 days and colonies formed were visualized under an inverted microscope. Images were captured using Axiovert 200 inverted microscope (Carl Zeiss).1 × 107 cells were injected subcutaneously in nude (ITOC-01) and NOD-SCID mice . All experiments were performed in accordance with relevant guidelines and regulations of Institutional Animal Ethics Committee. The mice were housed in sterile pathogen-free environment and observed daily for appearance of tumors. FDG PET studies were performed for the xenografts established from these cell lines in nude and NOD-SCID mice . Tumors from sacrificed mice were stained with hematoxylin and eosin (H&E) and images captured using Axio Imager.Z1 upright microscope (Carl Zeiss).For animal study approved by Institutional Animal Ethics Committee (Project no. 05/2012), 1 × 10Genomic DNA libraries were prepared using Nextera® Rapid Capture Enrichment Kit (Illumina) as per the manufacturer’s instructions and paired-end reads were generated using Illumina HiSeq 1500 platform. Approximately 37 Mb region of human genome comprising of 214,405 exons was captured. Detailed analysis provided in Supplementary Materials and Methods.47. Mutational analysis was carried out by bidirectional Sanger sequencing of PCR products on ABI 3730 capillary sequencer (Applied Biosystems).Genomic DNA was isolated from cells , fresh frozen tissue  and formalin-fixed paraffin-embedded (FFPE) tissue  as per the manufacturer’s instructions. Primer sequences used for amplification of Exon 1–11 of TP53 gene were as described earlierTotal RNA was isolated from the 4 tumor cell lines and 3 normal buccal mucosa tissues using miRNeasy mini kit (Qiagen). RNA libraries were prepared using TruSeq RNA sample preparation kit (Illumina) as per the manufacturer’s instructions and paired-end reads were generated using Illumina HiSeq 2500 platform. Please refer to Supplementary Materials & Methods for detailed RNA-seq analysis. RNA-seq data was validated by qPCR . Primer sequences are specified in Supplementary Table Supplementary InformationDataset 1Dataset 2Dataset 3


RIL/PDLIM4 gene was identified as a tumor suppressor, its expression is frequently altered in various types of malignancies. The product of RIL/PDLIM4 gene is an adapter protein involved in the actin cytoskeleton remolding and assembly of stress fibers crucial for cell motility and epithelial-mesenchymal transition. Although the exact mechanism tethering RIL to cancer development remains unknown some pieces of evidence suggest that RIL may act by suppressing activation of the proto-oncogene tyrosine-protein kinase Src. To further explore this issue we tested how different expression levels of RIL affected the activity of Src in breast cancer cell lines. RIL was ectopically overexpressed in the cell cultures with its relatively low endogenous level, or, otherwise, was downregulated by RNA interference. Whereas we observed no correlation between expression levels of RIL and activity of Src we found that in several cell lines elevated levels of RIL were associated with higher cell migratory activity along with the increased incidence of breast xenograft formation and metastasizing. The obtained data suggest that in some breast cancer models RIL may not act as Src kinase inhibitor, but rather play the role of a potential oncogene that promotes cell motility and contributes to cancer cells spreading. RIL gene  encodes a highly conserved adapter protein, the member of the ALP/Enigma family , 2. The Conversely, a growing body of data suggests that RIL may also act as an oncogene –18. The In order to find a correlation between the level of RIL expression and the activity of Src in the breast cancer model we tested a panel of eight human breast carcinoma cell lines. Expression levels of RIL were measured by qPCR, the proportion of active Src (phosphorylated at Y419) was determined by ELISA .Quantitative evaluation of the relationship between the two estimated parameters was performed by the Pearson bivariate correlation test. The calculated coefficient stood at -0.172 suggesting no evident correlation between the expression level of RIL and the activity of Src.The absence of the expected pattern could be explained by the fact that the activity of Src is controlled by numerous tyrosine kinases and phosphatases varying depending on the cell line. The analysis of the panel of different breast cancer cell lines fails to take into account the individual characteristics of each cell culture, which may affect the obtained results.In order to eliminate the impact of cell cultures heterogeneity a panel of breast cancer cell lines with artificially induced and suppressed level of RIL was established. RIL was upregulated in the relatively RIL-deficient cell lines by the introduction of a lentiviral expression construct. To downregulate the expression of RIL in breast carcinoma cell lines with the relatively high levels of endogenous RIL we used RNA interference mediated by the introduction of lentiviral constructs expressing RIL-specific shRNAs . The traThe created panel of transgenic breast cancer cell lines was used to measure the effect of suppression and overexpression of RIL on the activity of Src .According to the results, the influence of RIL on Src activity varies distinctly between cell lines. While for the T47D and HCC1395 cell cultures the low level of RIL correlates with the considerable inactivation of Src, its activity in BT549 and MDA-MB-468 does not significantly respond to RIL overexpression. Meantime, in MDA-MB-231 and MDA-MB-436 cells the high level of RIL is associated with the increased Src kinase activity, demonstrating the positive correlation between the studied parameters.The observed RIL-dependent activation of proto-oncogene Src may increase the malignant properties of cells, providing evidence for the oncogenic activity of RIL. To test this effect, we compared the phenotypic characteristics of breast cancer cells within the established panel.Migratory activity is considered among critical parameters that determine the aggressiveness of the tumor. The migration level of the RIL (+) and RIL (-) transgenic breast cancer cell lines was evaluated by the Boyden Chamber assay .in vivo could favor a metastatic spread of cells and a poor outcome of the disease.According to the data obtained, in two (MDA-MB-231 and HCC1395) of the six cell lines the expression of RIL demonstrates a statistically significant association with the increased migratory activity. The other two cell cultures (MDA-MB-436 and BT-549) show a similar trend. In these cell lines RIL acts as an oncogene stimulating the invasive behavior, which in vitro, we attempted to confirm the observed effect in vivo by evaluating the impact of RIL on tumor formation in the orthotopic xenograft mouse model. The transgenic cells derived from MDA-MB-231 demonstrating the most pronounced positive correlation of RIL expression with the cell migration rate were used within this experiment  group , 5C. RILin vitro results obtained for the correspondent breast cancer cell culture , but also for HCC1395 cell line  which shThis assumption is consistent with the results of our previous study, which revealed that RIL overexpression correlated with the unfavorable prognostic parameters of tumors, namely an increased CD74 level, reduced expression of E-cadherin, and inactivated intracellular tumor suppressor protein Scribble. Although the RIL-dependent mechanisms of tumor formation remain elusive, RIL may be involved in breast cancer development through the CD74-mediated mechanism typical of malignant cells  where inAlong with the previous evidence of RIL being a tumor suppressor, the present results reveal its more ambiguous role in breast cancer development. As a scaffold protein, RIL could participate in a wide range of protein-protein interactions, whereby its contribution to malignant phenotype could be primarily affected by the cellular context and the spectrum of abnormalities accumulated at the preceding steps of carcinogenesis.Summing up, RIL should not be considered a canonical oncogene or tumor suppressor. The assessment of the role of RIL in cancer development should be carried out taking into consideration the epigenetic features of the tumor or the cell line. Revealing mechanisms by which RIL affects positively or negatively malignant phenotypes of cancer cells could contribute to future personalized approaches to cancer therapy.Breast cancer cell lines were obtained from the American Type Culture Collection  and maintained according to the ATCC’s instructions.The cell lines used for the artificial modulation of RIL expression were selected within a single molecular subtype of breast cancer (triple-negative subtype).To upregulate RIL the selected cell lines were transduced with the lentivirus expression construct pLM-CMV-FLAG-RIL. The recombinant RIL was fused with the Flag epitope and placed under the control of CMV promoter. As a control, the corresponding cell lines were transduced with a similar lentiviral construct in which RIL was replaced with a Red fluorescent protein (pLM-CMV-RFP).To downregulate RIL the pLSLPw-shRIL lentiviral construct was used that contained shRNA expression cassette driven by the H1 promoter within the 3′-LTR. As a negative control we used the pLSLPw-shE6 construct that expressed a shRNA specific for the human papillomavirus E6 protein absent in the human genome.The corresponding genetic constructs were introduced into breast cancer cell lines by lentiviral transduction .To obtain the lentivirus vector particles, HEK293T cells were co-transfected with four plasmids: the lentivirus vector itself, two helper plasmids (pGag1 and pRev2), and pVSV-G. The transfection was performed via the liposome method with the use of LipofectAmine and Plus-reagents (Invitrogen) according to the conditions provided by the manufacturer. The medium containing recombinant virions was collected from the transfected cells and used to infect the target breast cancer cells.Proteins from whole-cell lysates were separated by 10% SDS–PAGE and analyzed by Western blotting for the expression of RIL ; GAPDH  was used as a loading control.Quantitative analysis of RIL expression was carried out by TaqMan probe-based Real-time PCR; GAPDH was used as the housekeeping gene. Primers and TaqMan probes were designed using Primer-BLAST tool; a comparative Ct method was used to determine the relative expression level of target genes.Bipartite Transwell chambers with 8-μm diameter pore  were used to assess cell migration rate in accordance with the Corning Cell Migration Assay. The migratory activity was estimated as the ratio of the number of cells on the lower side of the membrane to the number of non-migrated cells remaining on the upper side of the membrane; the proposed method allowed to avoid the cell count error which occurs when only the migrated cells were taken into account.® Phospho-SRC (Y419) and Total SRC ELISA kit was used to assess the activity level of Src, according to the manufacturer’s recommendations. Fluorometric analysis of breast cancer cell lysates was performed at 450 nm using Microplate Reader . The relative activity of Src was estimated as the ratio of the optical absorption value obtained for the Phospho-SRC (Y419) to the total amount of endogenous Src.RayBioSCID Beige mice (CB17. Cg-PrkdcscidLystbg-J/Crl) were purchased from Charles River Laboratory and kept under conditions of the SPF vivarium at the Institute of Bioorganic Chemistry (IBCH RAS); the experiments were performed in accordance to the guidelines recommended by the Bioethics Commission of IBCH RAS.Eight-week old female mice were injected unilaterally with 1 × 10^6 cells in 50 µL of PBS into the fourth abdominal fat pad by subcutaneous injection at the base of the nipple. Tumor growth was monitored externally using Vernier calipers for up to 30 weeks; the animals were euthanized when the tumors reached 10% of body weight.Lymph nodes, liver, heart, and lungs of the each euthanized mice were dissected, homogenized under cryogenic conditions, and used for the extraction of total RNA. The presence of the transgenic human cells (corresponding to the secondary sites of tumor development) in the analyzed organs was defined by RT-PCR. Puromycin-resistance gene presented in the transplanted cell lines (MDA-MB-231 shRIL and MDA-MB-231 E6) was chosen as a marker of metastases.p-value ≤ 0.05 was considered to be statistically significant.Statistical differences between groups of nominal, independent samples of small size were determined by Fisher’s exact test; the Bivariate (Pearson) correlation test was used as a measure of strength of the association between two continuous independent variables. All data were analyzed using GraphPad Prism, error bars representing standard deviation of the mean. A


As a result, the modified LLO (LLO@rGO) cathode exhibits superior electrochemical performance including cycling stability and rate capability compared to the pristine LLO cathode. In particular, the LLO@rGO with a 0.5% rGO content can deliver a high discharge capacity of 166.3 mAh g−1 at a 5C rate. The novel strategy developed here can provide a vital approach to inhibit the undesired side reactions and structural deterioration of Li-rich cathode materials, and should be greatly useful for other cathode materials to improve their electrochemical performance. Layered lithium-rich manganese oxide (LLO) cathode materials have attracted much attention for the development of high-performance lithium-ion batteries. However, they have suffered seriously from disadvantages, such as large irreversible capacity loss during the first cycle, discharge capacity decaying, and poor rate performance. Here, a novel method was developed to coat the surface of 0.4Li Nowadayvehicles . Howevervehicles ,5,6. In vehicles ,8,9, metvehicles ,11,12 anvehicles ,14,15. Avehicles . By far, oxides , fluoridGraphene is a two-dimensional, macromolecule carbon atom sheet with a honeycomb structure. Owing to its excellent electronic conductivity and mechanical properties, graphene can serve as an ideal conductive additive for hybrid nanostructured electrodes ,20,21,222MnO3∙0.6LiNi1/3Co1/3Mn1/3O2) was synthesized by using a simple and feasible carbonate co-precipitation method in our work. Then, the LLO surface was modified with an alkyl-bromide surfactant, which has been successfully applied in other fields [Therefore we developed a novel and robust method to coat thermally-reduced graphene oxide (GO) uniformly on the LLO surface, mainly based on electrostatic forces. Specifically, LLO , shows a far higher positive potential. Obviously, the surfactant greatly changes the surface charge state. Such a phenomenon is due to a bilayer surfactant structure formed on the LLO surface. Previous study showed that the alkyl-bromide surfactant can be adsorbed on a cathode oxide based upon the interaction between the cationic heads of the surfactant, and the surface O of the cathode oxide [To verify the working mechanism of the surfactant, the zeta potentials of pristine LLO, surfactant-modified LLO in aqueous solutions, were measured as shown in de oxide . Thus, ide oxide . Hence, the final charged state of the surfactant-modified LLO will be determined by the outside cationic heads of the out-layer surfactant, and it is highly positive , which c38H80BrN-GO mixture shows a positive potential. This is because the negatively-charged GO sheets are fully covered by the surfactant molecules, and are reversed to a positively-charged state. The results prove the strong interaction between the surfactant and the GO via the electrostatic interaction. No doubt, GO will coat tightly on the surfactant-modified LLO surface when they are mixed. Moreover, after the GO coating, the surface state of the LLO@rGO particles should be dominated by the coating nanosheets, which is negative, as shown in To confirm the strong electrostatic interaction between the GO sheets and the surfactant, their zeta potentials were also measured. The zeta potential of GO sheets is presented in 2 (R3m) phase, a typical α-NaFeO2-type structure. The weak peaks at 20–25° belong to a monoclinic Li2MnO3 (C2/m) phase [The effect of rGO coating on the crystal phase evolution of LLO was first determined. m) phase ,33,34. Bm) phase ,36. Thesm) phase . HoweverNext, the effect of rGO coating on the morphology of LLO were also checked to see if the rGO nanosheets could wrap the LLO particles well, as shown in To further exam the details of the morphology of rGO-coated LLO, transmission electron microscopy (TEM) images of LLO and LLO@rGO are shown in It is obvious that the rGO wraps the LLO particles tightly, owing to the addition of the surfactant. We believed that such kind of structure is beneficial for the improvement of the electrochemical properties of the LLO material.X-ray photoelectron spectroscopy (XPS) was used to monitor the evolution of the chemical states of the particle surface to further ensure the coating of rGO. + ion is extracted from the active LiMO2 phase, since the voltage gradually increases up to 4.50 V from 4.45 V, accompanied by the oxidation of Ni2+ to Ni4+ and Co3+ to Co4+. Then, the voltage maintains a long plateau at 4.50 V, during which Li+ ions are further removed from the Li2MnO3 component (TM layer) [+ ion transport.Electrochemical properties of the modified LLOs and pristine LLO were side-by-side compared to determine the effect of rGO coatings with the aid of the surfactant. The first cycle voltage profiles of LLO and LLO@rGO electrodes obtained at 0.1C are shown in M layer) . Figure −1 at the 5C rate, which is far higher than that of the LLO electrode (122.8 mAh g−1). This high-rate capability is attributed to the highly conductive ultrathin graphene (0.5%) around LLO particles. Moreover, the rGO coatings can also reduce the electrolyte decomposition and serve as a fast pathway for electron transportation during the charge/discharge process [−1 when the discharge rate is back to 0.1 C. Cycling stability is also an important issue for high-performance materials. To further elucidate the changed electrochemical performance of the LLO by the rGO modification, extended rate capability testing was implemented, as presented in  process ,44. MeanIt can be seen that the discharge capacity of the pristine-LLO electrode is going to decay from 40 cycles. Surprisingly, the LLO@rGO electrode manifests a stable capacity retention after 100 cycles. The improved cycling capacity is owed to the physical protection layer provided by a proper amount of rGO wrapping layers. The rGO layer can separate active material and electrolyte away, while suppressing the attack from HF and inhibiting the surface side reaction ,46. s, Rct, Q and W represent the ohmic resistance of the electrode, charge transfer resistance at the electrode/electrolyte interface, double layer capacitance, and Warburg impedance of the Li+ diffusion in the electrode, respectively. It can be seen that the values of Rs of various cells are almost the same. Rct shows big difference in different cells. The LLO@rGO-0.5%-based cell displays the lowest value of Rct compared with those of other cells, as shown in To clarify the kinetic features of pristine-LLO and LLO@rGO-based cells, EIS measurement was also performed. As shown in 4·6H2O, MnSO4·H2O and CoSO4·7H2O (Ni:Co:Mn = 0.6:0.2:0.2) was stirred at room temperature to achieve a precursor solution. Then, an adequate amount of precipitant (NH4HCO3) was added to the above solution, which was kept stirring at room temperature for 2 h. Next, the solution was filtered, washed and dried. The dried powder was mixed with LiOH·H2O, and then calcined at 400 °C for 2 h, 600 °C for two more hours, and 800 °C for a final five hours in air, to obtain the pristine LLO.The layered lithium-rich manganese oxide (LLO) cathode material was synthesized by a carbonated co-precipitation method. An aqueous solution of NiSO38H80BrN) was added into the LLO solution under continuous stirring. Next, different amounts of GO solution (12 mg/mL) were added to the modified-LLO solution at different weight ratios , and were stirred at 60 °C overnight. The mixed solutions were centrifuged and then dried at 60 °C for 12 h. Finally, the as-prepared powders were annealed at 400 °C for 5 h in an Ar atmosphere to obtain the LLO@0.5% rGO, LLO@1.0% rGO and LLO@2.0% rGO.GO was prepared by the Hummer’s method. To wrap reduced graphene-oxide (rGO) onto the LLO surface, 0.15 g LLO powders was first dispersed in 10 mL deionized water. Then, 3 mg dimethyldioctadecylammonium bromide  (ESCALAB 250Xi) . Morphological observations were carried out on a Zeiss Supra 55 field emission scanning electron microscope (FE-SEM) . The transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM) were used for morphology and micro-crystal observation with a JEM-2100 transmission electron microscope . The weight contents of rGO in the LLO@rGO composites were determined using thermo-gravimetric analysis  .X-ray diffraction (XRD) patterns of the samples were obtained by a Rigaku D/max 2500 diffractometer with Cu Kα radiation at V = 4 0 kV and I = 150 mA in a 2θ range of 10–80°, and the scanning rate was 10° minN-methyl-2-pyrrolidone, was prepared and then coated on an aluminum foil to fabricate working electrodes. The cells were assembled in an Ar-filled glove box with water and oxygen contents less than 0.1 ppm, using polypropylene film (Celgard 2400) as the separator. The electrolyte was a solution of 1 M LiPF6 in a 1:1 (v/v) mixture of ethylene carbonate (EC)/dimethyl carbonate (DMC). The cells were galvanostatically charge-discharged at various current rates (1C = 250 mA g−1) between 2.0 and 4.8 V at room temperature. The cyclic voltammetry (CV) tests were conducted between 2.0 V and 4.8 V at a scan rate of 0.1 mV s−1 on an electrochemical workstation. Electrochemical impedance spectroscopy (EIS) was also performed on an electrochemical workstation . The frequency of EIS ranged from 100 kHz to 10 mHz with an amplitude of 5 mV.All the electrochemical tests here were carried out on CR2032-type coin cells with lithium metal as their anodes. The details are described as follows. A slurry, with 75 w% active materials, 15 w% acetylene black, and 10 w% polyvinylidene fluoride dispersed in −1 at 5C rate. The results show that the rGO coating enables a greatly enhanced rate performance as well. Such improvement is due to the enhanced interfacial charge transfer, the reduced side reactions and the inhibited structural deterioration of Li-rich cathode materials. Using a proper surfactant to enhance the coating of graphene derivatives on different surfaces is demonstrated to be very promising to realize outstanding energy storage devices. In conclusion, we have successfully fabricated a high performance rGO-coated LLO cathode material. A surfactant was proved to be beneficial for the robust coating of rGO nanosheets on the LLO powder surface. The rGO-coated LLO cathode material exhibited outstanding rate capability as 166.3 mAh g


BRAF-mutant (n = 11), NRAS-mutant (n = 10), BRAF/NRAS wild type (n = 10), and GNAQ/GNA11-mutant uveal melanomas (UVMs) (n = 8). Cell surface expression of established IFNγ downstream targets PD-L1, PD-L2, HLA-A, -B, and -C, HLA-DR, and nerve growth factor receptor (NGFR) were analyzed by flow cytometry. Basal cellular expression levels of HLA-A, -B, -C, HLA-DR, NGFR, and PD-L2 predicted the levels of IFNγ-stimulation, whereas PD-L1 induction was independent of basal expression levels. Only 13/39 (33%) of the melanoma cell lines tested responded to IFNγ with potent induction of all targets, indicating that downregulation of IFNγ signaling is common in melanoma. In addition, we identified two well-recognized mechanisms of immunotherapy resistance, the loss of β-2-microglobulin and interferon gamma receptor 1 expression. We also examined the influence of melanoma driver oncogenes on IFNγ signaling and our data suggest that UVM have diminished capacity to respond to IFNγ, with lower induced expression of several targets, consistent with the disappointing response of UVM to immunotherapies. Our results demonstrate that melanoma responses to IFNγ are heterogeneous, frequently downregulated in immune checkpoint inhibitor-naïve melanoma and potentially predictive of response to immunotherapy.Immune checkpoint inhibitors that block the programmed cell death protein 1/PD-L1 pathway have significantly improved the survival of patients with advanced melanoma. Immunotherapies are only effective in 15–40% of melanoma patients and resistance is associated with defects in antigen presentation and interferon signaling pathways. In this study, we examined interferon-γ (IFNγ) responses in a large panel of immune checkpoint inhibitor-naïve melanoma cells with defined genetic drivers;  The identification of checkpoint signaling pathways that dampen anti-cancer immune responses and the subsequent development of checkpoint inhibitors have transformed the treatment of patients with metastatic cancer. Antibodies blocking immune checkpoints such as the cytotoxic T-lymphocyte-associated protein-4, the programmed cell death protein 1 (PD-1), and its ligand PD-L1 induce durable anti-tumor immune responses in many advanced malignancies, including melanoma, non-small-cell lung cancer, and renal cell carcinoma. PD-1 inhibition in melanoma promotes tumor regression and prolonged overall survival in 30–40% of patients with advanced disease (Recent studies have shown that resistance to immune checkpoint blockade involves defects in the interferon-γ (IFNγ) signaling pathway –9. Once B2M) gene, the structural component of MHC class I complexes, is enriched in pre-treatment tumor samples from melanoma patients with innate and acquired resistance to checkpoint inhibitor therapy , HLA-A, -B, -C, and HLA-DR] could serve to assess the integrity of IFNγ signaling in melanoma. We also examined the potential influence of melanoma driver oncogenes on IFNγ signaling activity and found that uveal melanoma (UVM) cells show evidence of diminished IFNγ pathway activity with minimal baseline and IFNγ induction of HLA-DR, NGFR, and PD-L2. Importantly, nearly 70% of melanoma cells included in this study showed incomplete responses to IFNγ stimulation, indicative of pre-existing resistance to immunotherapy. Furthermore, our data confirm that measuring IFNγ output with a select number of targets may be useful for detecting intrinsic defects in the IFNγ/JAK/STAT pathway, including JAK and STAT mutations which are associated with PD-1 inhibitor resistance , 13.A total of 39 cell lines were included in this study. Oncogenic driver mutation status is shown in Table 2. For IFNγ treatment, 7 × 104 melanoma cells per well were plated in complete media in six-well plates. After an overnight incubation, the complete media was replenished, and cells treated for 72 h with 1,000 U/ml IFNγ  or vehicle control [0.1% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline ]. Cells were collected, washed with PBS, and analyzed by flow cytometry.Cell lines were cultured in Dulbecco’s Modified Eagle Medium or Roswell Park Memorial Institute-1640 media supplemented with 10 or 20% heat inactivated fetal bovine serum , 11.25 mM glutamine , and 10 mM HEPES (Gibco) and were maintained at 37°C in 5% CO5) were incubated for 30 min on ice with mouse anti-human antibodies against HLA-ABC (clone W6/32), HLA-DR (clone L243), CD271/NGFR (clone ME20.4), CD273/PD-L2 (clone 24F.10C12) , and CD274/PD-L1  conjugated to phycoerythrin (PE), fluorescein isothiocyanate, PE-cyanine (Cy)7, allophycocyanin, and brilliant violet 421, respectively. All antibodies were titrated prior to experiment to ensure optimal concentrations were used. Fc block (BD Biosciences) was used to prevent non-specific staining due to antibody binding to Fc receptors. Fluorescence minus one controls  were included with each experiment. Prior to acquisition, cell viability was determined by staining cells with either 5 µM DAPI , Zombie Yellow dye (BioLegend), or Live Dead near-IR fixable dye . For the analysis of interferon gamma receptor 1 (IFNGR1) and B2M expression, cells were first stained with a fixable viability dye and either PE-conjugated anti-CD119 (clone GIR-208) or PE-Cy7 conjugated anti-B2M (clone 2M2), both from BioLegend. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm kit and stained intracellularly with the same antibody that was used for cell surface stain.Staining was performed in flow cytometry buffer . Cells (2 × 10Samples were acquired on BD LSRFortessa X20 flow cytometer (BD Biosciences) and the FlowJo software  was used for data analysis. At least 10,000 live events were acquired. General gating strategy included forward and side scatter area to exclude cell debris, time parameter to exclude electronic noise, forward scatter area and height to exclude doublets and gating on viable cells . Relative marker expression levels were calculated by dividing the geometric mean fluorescence intensity (MFI) of the antibody-stained sample by the FMO control MFI  human skin cutaneous melanoma (SKCM) and UVM datasets using single sample gene set enrichment analysis (ssGSEA) . RNA cou package , 18. The package .http://www.ingenuity.com) was used to identify mutations in genes associated with the JAK-STAT (KEGG) signaling pathway  using BWA . V600BRAF-mutant, 10 NRAS-mutant and 10 BRAF/NRAS wild type (BRAF/NRASWT) cutaneous melanoma cell lines, and 8 GNAQ/11-mutant UVM cell lines , and HLA-DR molecules was examined in a panel of 39 human melanoma cell lines with defined oncogenic driver mutations  and the uveal MP46 cells (relative MFI = 2.3) . PD-1 ligands PD-L1 and PD-L2 were expressed at comparably low levels in our panel of melanoma cells  BRAFV600-mutant, 1/10 (10%) NRAS-mutant, and 7/8 (87.5%) uveal cell lines  revealed a broad range of expression for all five markers Figure ; Table 1p < 0.01) and NGFR expression  . The cell surface protein expression patterns of these markers in our melanoma panel did not precisely reflect their transcript expression patterns in the human SKCM dataset of TCGA  and HLA-DR (HLA-DRA) were correlated  and PD-L2 (PDCD1LG2) transcripts were correlated (Spearman’s rank = 0.793 p < 0.01) in the TCGA SKCM dataset, although we did not observe any correlation in their cell surface protein expression  transcript expression was also different between the TCGA uveal and cutaneous datasets, whereas HLA-A transcript expression was indistinguishable between the TCGA uveal and cutaneous tumor groups   because PD-L1 and PD-L2 showed disparate expression at baseline  .Comparison of all five target molecules also showed positive correlation between IFNγ-induced expression of PD-L1, PD-L2, and HLA-DR. In particular, post-stimulation levels of PD-L1 and PD-L2 were correlated  demonstrated diminished response to IFNγ stimulation, usually manifested as no induction  of one or more markers in response to IFNγ stimulation. HLA-ABC expression was absent in the 7% demonsWe also examined the impact of IFNγ treatment on cell cycle progression in our panel of melanoma cells using flow cytometry. Of the 38 melanoma cell lines tested, three showed increasing cell death in response to IFNγ, with greater than 10% increase in sub G1 Table . Of thesvia alternate pathways or downstream elements.Analysis of the IFNγ target proteins, HLA-ABC, HLA-DR, NGFR, PD-L1, and PD-L2, in a panel of 39 melanoma cell lines revealed that IFNγ stimulated cell surface expression of all five markers in only 13 melanoma cell lines tested. The degree of IFNγ-mediated induction was highly variable for all five markers but closely reflected the corresponding basal expression levels for HLA-ABC, HLA-DR, PD-L2, and NGFR. By contrast, PD-L1 expression was frequently absent at baseline (relative MFI < 1.5) but was still induced to high levels after IFNγ treatment in the majority of cell lines. Consequently, although the JAK/STAT/IRF1 pathway is critical for the IFNγ-mediated induction of HLA-ABC, HLA-DR, and the two PD-1 ligands , 23, theHLA-DRA, NGFR, CD274 (PD-L1), and PDCD1LG2 (PD-L2) transcripts, and this was associated with reduced transcript expression of the IFNγ master transcription factors STAT1, STAT3, and IRF1 and with reduced IFNγ transcriptome signatures. It is worth noting that although transcriptome data are derived from high quality tumor samples with at least 60% tumor nuclei, they do contain variable levels of tissue-infiltrating immune and stromal cell populations that may influence the level of transcript expression  was compared. This may reflect disparate baseline expression levels due to IFNγ-independent regulatory influences but also the complexity and redundancy of the IFNγ signaling pathway. For instance, whereas the JAK–STAT1/2/3–IRF1 axis is critical for PD-L1 regulation, the JAK–STAT3–IRF1 node is important for PD-L2 stimulation . We alsopression . NeverthIncomplete responses to IFNγ-stimulation, usually manifested as lack of induction of one or more markers were evident in 26 of 39 (67%) melanoma cell lines. Although it is still not clear whether incomplete IFNγ stimulation in melanoma cells has significant impact on patient responses to immunotherapy, it is evident that this pathway is important for response to PD-1 blockade. In particular, nuclear expression of the IFNγ transcription factor IRF1  is assocIt is worth noting that of the 26 melanoma cell lines displaying incomplete induction of the 5 target proteins, 8 showed cell cycle distribution changes in response to IFNγ treatment. Importantly, 5/7 melanoma cell lines with no IFNγ-mediated PD-L1 induction showed no cell cycle profile changes after treatment with IFNγ. This may reflect the critical role of the STAT1 transcription factor in promoting PD-L1 expression and mediating IFNγ-induced cell cycle effects , 32. FivB2M gene  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Royal Prince Alfred Hospital Human Research ethics committee.SA, ES, and AS performed the experiments. ES, HR, and SL wrote the manuscript. SA, SL, ES, and HR analyzed and interpreted the data. SA, AS, RK, SL, ES, and HR read, revised, and approved the final manuscript.RK served on advisory boards for Roche, Amgen, BMS, Merck, Novartis, and TEVA and has received honoraria from Merck, BMS, and Novartis. The remaining authors declare no conflict of interest.