Background: Associating paclitaxel (PTX) to doxorubicin (DXR) is one of the main chemotherapy strategies for breast cancer (BC) management. Protocols currently available consist in administering both drugs on their maximum tolerated dose, not taking into account the possible differences in efficacy due to their combination ratio. In the present study, the short and long-term cytotoxic effects as well as migratory effects of PTX, DXR, and its combinations at 10:1; 1:1 and 1:10 PTX:DXR molar ratios either free or co-encapsulated in liposomes were evaluated against three human BC cell lines . Method: The MTT assay was used to screen for synergy or antagonism between PTX and DXR and the combination index value was calculated using the CalcuSyn software. Nuclear morphological alterations were evaluated by staining the cells with Hoescht 33342. The investigation of senescence and clonogenicity of BC cell lines exposed to different treatments was also studied. In addition, the ability of these cells to migrate was assessed. Results: Taken together, the results presented herein allow us to suggest that there is no benefit in enhancing the PTX concentration above that of DXR in the combination for any of the three cell lines tested. Conclusion: The developed liposomes co-encapsulating PTX and DXR in different molar ratios retained the biological properties of the mixture of free drugs and are valuable for planning new therapeutic strategies. Breast cancer (BC) is the most frequent cancer among women in both more and less developed regions. It accounts for the most frequent cause of cancer death in women in less developed regions and in more developed regions it comes after lung cancer . The tre®, a liposomal formulation of doxorubicin (DXR) and Abraxane®, albumin bound paclitaxel (PTX) nanoparticles, are already widely a successfully used for clinical treatment of BC [Despite the efficacy, the chemotherapy schemes now available lead not only to severe side effects during the treatment, but also after its completion. Of particular importance are the potential long-term sequelae of the cardiotoxicity of anthracycline-based therapy for survivors of breast cancer, which might appear more than 10 years after administration of the regimens. This highlights the need of searching for new therapeutic strategies ,7. Nanomnt of BC . A new ant of BC . Therefont of BC .Liposomal formulations designed according to this strategy have shown to be able to maintain drug ratios in the plasma after injection as well as deliver to the formulated drug ratio directly to the tumor tissue. This ensures that the effect observed in vitro is translated into in vivo, and chemotherapy is optimized ,16,17,18We recently reported the results of antitumor activity and toxicity of long-circulating and fusogenic liposomes co-encapsulating PTX and DXR (LCFL-PTX/DXR) at molar ratio of 1:10, respectively, in mice bearing the 4T1 mammary carcinoma. The LCFL-PTX/DXR presented superior efficacy compared to treatments with free PTX or free DXR, and improved cardiac toxicity profile compared to treatment with the free combination of these drugs, enabling its co-administration .Here we evaluate the short and long-term effects of the exposure of MDA-MB-231, MCF-7, and SKBR-3 human breast cancer cell lines to different ratios of free or liposomal co-encapsulated PTX and DXR. sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000 (DSPE-PEG2000) were supplied by Lipoid GmbH . Cholesterol hemisuccinate (CHEMS), doxorubicin (DXR), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) sodium salt, sodium chloride, sodium hydroxide, and Cremophor EL were obtained from Sigma-Aldrich Co. . Paclitaxel (PTX) was supplied by Quiral Quimica do Brasil S.A .The human breast adenocarcinoma cell lines MDA-MB-231, MCF-7 and SKBR-3 were purchased from the American Type Culture Collection . The different culture media , fetal bovine serum, penicillin/streptomycin and 3--2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Co. . All other chemicals used in this study were of analytical grade. 1,2-Dioleoyl-−1 total lipid concentration, mixed with and PTX (0.25 mg/mL) on a round bottom flask and submitted to evaporation under reduced pressure in a 50 °C water bath until a thin lipid film had been obtained. The round bottom flask containing the lipid film was then maintained for 1 h under a chloroform atmosphere for better dispersion of PTX in the lipids, according to the technique known as annealing [LCFL-PTX/DXR were prepared by the lipid film hydration technique, as described elsewhere . Brieflynnealing . A solutThe diameter of the vesicles and the polydispersity index (PI) were determined by dynamic light scattering (DLS). The measurements were performed at a temperature of 25 °C, using a 90° laser incidence angle. The zeta potential (ζ) of the vesicles was determined by DLS associated with electrophoretic mobility. To perform both measurements, the liposomes were diluted in HBS, pH 7 and evaluated on the Zetasizer Nano ZS90 equipment .v/v). The elution time was of 7 min, the ACE® C8 25 cm × 4.6 mm, 5 μm column  was employed. The column was kept at room temperature, the flow rate was set at 1.0 mL/min and the detection was performed with a fluorescence detector model 2475  with excitation and emission wavelengths of 470 nm and 555 nm, respectively. The PTX and DXR encapsulation percentage (EP) were calculated according to the following equation:PTX and DXR levels were determined by high-performance liquid chromatography . For determination of PTX levels, the mobile phase was composed of 55% acetonitrile in water. The elution time was of 8 min, the Hibar 250-4 LiChrospher 100RP-18, 25 cm × 4 mm, 5 μm column  was used. The column was kept at room temperature, the flow rate was set at 1.2 mL/min and the detection wavelength was 227 nm. For determination of the DXR concentration, the mobile phase was composed of methanol:phosphate buffer 0.01 M , with negative results. The human breast adenocarcinoma cell lines MDA-MB-231, MCF-7 and SKBR-3 were cultivated in DMEM, MEM supplemented with 0.01 mg/mL insulin and McCoy media, respectively, all supplemented with 10% fetal bovine serum (FBS). All cell lines were cultivated in the presence of penicillin (100 IU/mL) and streptomicin (100 μg/mL) and maintained at 37 °C and 5% CO3 cells for MDA-MB-231 cell line; 1 × 104 cells for MCF-7 cell line and 5 × 103 cells for SKBR-3 cell line) per well in 96 well plates, and kept in the incubator for around 24 h prior to exposition to the treatments. To determine the inhibitory concentration of 50% (IC50) of each free or liposome-encapsulated drug, cells were exposed to a minimum of 8 different concentrations of PTX, DXR, LCFL-PTX, or LCFL-DXR for 48 h. To screen for synergy or antagonism between the drugs, cells were exposed to a minimum of 6 concentrations of different fixed molar ratios  of a mixture of free PTX:DXR or LCFL-PTX/DXR (co-encapsulating the different fixed molar ratios) for 48 h. After the incubation time, treatments were removed and 100 mL of MTT (0.5 mg/mL) were added to the wells. The plates were incubated for 2 h at 37 °C. After incubation time, media containing MTT were removed from the wells and 100 mL of DMSO was added to solubilize the formazan crystals. The absorbance of the wells was determined using a bench spectrophotometer model Benchmark Plus  at 570 nm. All experiments were performed in quadruplicate of wells per concentration and triplicate of plates.The MTT assay was chosen as the means to screen for synergy or antagonism between PTX and DXR in the different cell lines studied. For that, cells were plated in different densities (6 × 10fa) was subsequently determined for each concentration of treatment and the data sets were analyzed by the median-effect analysis using the CalcuSyn software . It uses the combination index (CI) value as a quantitative indicator of the degree of synergy or antagonism. Using this analysis method, CI value around 1.0 indicates additive activity, CI value >1 indicates antagonism, and a CI value <1.0 indicates synergism [The test data were converted to mean fraction of cell survival relative to untreated cells (control group). The fraction of affected cells (ynergism .Two controls were performed for the MTT assay. The first consisted in verifying the intrinsic biologic activity of the long-circulating and fusogenic liposomes without anticancer drugs (LCFL-blank) and cremophor against the tested cell lines ,25,26. T5 cells/well in 6-well plates and incubated at 37 °C for 24 h. After incubation time, cells were treated for 48 h with 2 mL of different treatments  all at a total concentration of 70 nM. Control wells received 2 mL of fresh media. After incubation, cells were fixed with formaldehyde 4% for 10 min. Fixed cells were stained with a Hoescht 33342 (0.2 μg/mL) solution for 10 min at room temperature in the dark. Nuclei fluorescence images were captured using a microscope AxioVert 25 with a fluorescence module Fluo HBO 50 connected to the Axio Cam MRC camera . A total of a hundred nuclei per treatment were analyzed using the Software Image J 1.50i  and the plugin “NII_Plugin” available at http://www.ufrgs.br/labsinal/NMA/.To evaluate nuclear morphological alterations after treatments, the different cell lines were plated at a density of 2.0 × 104 cells) were seeded in 24-well plate and incubated at 37 °C for 24 h. After incubation time, cells were treated for 48 h with 500 μL of different treatments . All treatments were added at a total concentration of 70 nM. Control wells received 500 μL of fresh media. After treatment, cells were washed with PBS and fixed in 2% formaldehyde (v/v) and 0.2% glutaraldehyde (v/v) in PBS buffer for 5 min. Fixative was removed by washing cells with PBS and then cells were stained for 12 hours with X-gal staining solution .The staining procedure has been performed as described by Debacq-Chainiaux and coworkers . Briefly5 cells/well in 6-well plates and incubated at 37 °C for 24 h. After incubation time, cells were treated for 48 h with 2 mL of different treatments  all at a total concentration of 70 nM. Control wells received 2 mL of fresh media. Then, cells were harvested and counted. Different densities of viable cells (300 cells for MDA-MB-231 or MCF-7 cell lines and 900 cells for SKBR-3 cell line) were re-plated in 6 well plates to evaluate the ability of the remaining cells to form colonies. Twenty-one days after re-plating, colonies were fixed with formaldehyde 4% for 10 min and stained with 2 mL of crystal violet for 1 hour. Colonies, consisting of a group of at least 50 cells, were counted using an AxioVert 25  microscope, and the percentage of colony formation relative to the control was calculated for each group.Clonogenic assay was performed based on one of the protocols described by Franken and coworkers (2006) . MDA-MB-5 cells/well in 12-well plates and incubated at 37 °C for 24 h. Then, a straight wound was made in individual wells with a 10 μL pipette tip. This point was considered the “0 h,” and the “zero wound” was photographed using a microscope AxioVert 25 with a connected Axio Cam MRC camera . After obtaining the wounds, control wells received 1 mL of fresh media and the other wells received 1 mL of media containing the different treatments  all at a total concentration of 70 nM. The plates were incubated at 37 °C for 24 h. On these experiments, from the platting moment until the end of the assays cells were kept on starvation, meaning the different media contained only 1% FBS. After incubation cells were fixed with formaldehyde 4% for 10 min. Images along the wounds of control and treated groups were obtained in phase contrast. Wound areas were obtained using the MRI Wound Healing Tool plugin for the free version of Image J 1.45 software . The wound healing percentage was calculating according to the following equation:To study the bi-dimensional migration of the different cell lines they were plated at a density of 2.0 × 10P values were <0.05. GraphPad Prism 5.04 Software  was used to calculate all data.The results were expressed as mean ± SD of three independent experiments. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post test. Prior to ANOVA analyses, data were transformed as indicated on the tables to fit the normality and homocedasticity requirements, which were evaluated by Kolmogorov-Smirnov and Levene tests, respectively. Differences were considered statistically significant when P > 0.05). The mean diameter of the different formulations ranged from 226.4 to 249.8 nm. Graphical representations of the intensity particle size distribution for the different formulations are provided in Size measurements of the different formulations demonstrated that the encapsulation of PTX, DXR or co-encapsulation of these drugs into LCFP did not affect significantly the size of the vesicles compared to LCFP-blank (P < 0.05), as summarized in P > 0.05), while a reduction on cytotoxicity was observed for SKBR-3 cell line (P < 0.05). For all three cell lines, encapsulation of DXR in liposomes did not affect its cytotoxicity (P > 0.05). Similarly to the free drugs, LCFL-PTX were more cytotoxic than LCFL-DXR against MCF-7 and SKBR-3 cell lines (P < 0.05). No difference in cytotoxicity between LCFL-PTX and LCFL-DXR was observed for liposome-encapsulated drugs against the MDA-MB-231 cell line (P > 0.05). When evaluated for short-term cytotoxicity, all three cell lines tested were more sensitive to PTX treatment compared to DXR treatment in free form  was observed for the treatment of MDA-MB-231 and MCF-7 cell lines only with PTX:DXR at molar ratio of 1:10, while for the SKBR-3 cell line all molar ratios of combined free drugs led to antagonism (CI > 1.0). Different studies have already reported that combination effect profiles might change upon co-encapsulation of the studied drugs [It is well known that the combination effect  can be affected by the ratio of the drugs in the combination . For thiP > 0.05) after incubation with MTT. This finding proved that in the present study, the reduction of MTT to formazan occurred only in the presence of cells.Control experiments revealed that LCFL-blank and cremophor have no interference in the metabolic activities of the different cell lines as they were assessed on a maximum of 110% and a minimum of 80% compared to that of the control in the evaluated concentration ranges. The absorbances in the cell-free wells exposed to 100 μM of PTX solubilized in cremophor (0.23 ± 0.02), 100 μM of DXR (0.22 ± 0.00) or to LCFL-blank in equivalent lipid concentration to the LCFL-PTX treatment at 100 μM (0.22 ± 0.01) did not vary significantly from that observed for the control group .For the MDA-MB-231 and MCF-7 cell lines, it was observed that the increase of the DXR concentration in the combinations allowed for an enhancement of the cytotoxicity compared to the PTX:DXR combination at 10:1 molar ratio. Considering combinations of PTX:DXR on the free form, the 1:1 and 1:10 molar ratios were 4.6-fold and 5.4-fold more cytotoxic, respectively, than the 10:1 molar ratio against the MDA-MB-231 cell line. For the MCF-7 cell line, the 1:1 and 1:10 molar ratios were 5.1-fold and 5.7-fold more cytotoxic, respectively, than the 10:1 molar ratio. Considering the co-encapsulated PTX:DXR combinations, the 1:1 and 1:10 molar ratios were 1.3-fold and 2.1-fold more cytotoxic, respectively, than the 10:1 molar ratio against the MDA-MB-231 cell line. For the MCF-7 cell line, the 1:1 and 1:10 molar ratios of the co-encapsulated drugs were 4.1-fold and 6.5-fold more cytotoxic, respectively, than the 10:1 molar ratio. No difference in cytotocicity was observed between 1:1 and 1:10 molar ratios combinations of the free drugs or between 1:1 and 1:10 molar ratios combinations of co-encapsulated drugs against MDA-MB-231 and MCF-7 cell lines (P < 0.05).The SKBR-3 cell line presented a different profile as the PTX:DXR combinations at 1:1 and 1:10 molar ratios of the free drugs were 3.0-fold and 39.0-fold less cytotoxic, respectively, than the 10:1 molar ratio. When PTX:DXR were co-encapsulated in liposomes at 1:1 and 1:10 molar ratios, these combinations were 1.3-fold and 5.9-fold, respectively, less cytotoxic than the PTX:DXR combination at 10:1 molar ratio against the SKBR-3 cell line. The combination of PTX:DXR at 1:1 molar ratio presented higher cytotoxicity compared to the 1:10 molar ratio either in free or co-encapsulated in liposomes against the SKBR-3 cell line  tool, developed by Filippi-Chiela and coworkers (2012), divides nuclei into six groups according to their morphological appearances, each having a putative biological meaning: normal (N), irregular , small regular , small , small irregular , large regular  and large irregular  .The morphometric analysis of the nuclei size and irregularity showed that all three cells lines responded in a similar manner to the different treatments, which resulted predominantly in an increase of the percentage of LR nuclei, characteristic of senescence, as shown in The different treatments also resulted in nuclear irregularities, characteristic of mitotic catastrophe or another nuclear damaging event, in a similar extension for the three cell lines. The percentage of LI + I nuclei for the MDA-MB-231 cell line exposed to the different treatments ranged from 5 to 27%, while that for MCF-7 and SKBR-3 cell lines ranged from 6 to 25% and 8 to 24%, respectively. For MDA-MB-231 and MCF-7 cell lines, the higher percentage of LI + I nuclei was observed after treatment with PTX while for SKBR-3 cell line when the cells were exposed to PTX:DXR at 10:1 molar ratio. A higher proportion of nuclear irregularities for treatments containing higher concentrations of microtubule-hyperpolymerizing agents, such as PTX, is expected since these drugs result in damage further leading to mitotic catastrophe ,37,38. TSenescent cells express SA-β-gal activity that is detectable at pH 6.0, distinct from the acidic β-galactosidase activity, present in all cells and detectable at pH 4.0. SA-βgal can be detected using chromogenic substrate X-gal, which yields an insoluble blue compound when cleaved by β-galactosidase .Usually the technique consists in SA-β-gal staining followed by the visual identification of the positive cells. This process is considered to be much more subjective and time consuming than using the NMA tool, previously demonstrated to be useful in predicting the senescent status of a cell population . TherefoFor all three cell lines, cells of the control group were not stained by X-gal, as expected. Blue-stained cells were present in cell populations exposed to all the different treatments , confirming the occurrence of senescence. The clonogenic assay essentially tests every cell of the population for its ability to undergo ‘‘unlimited’’ division. It consists in a method to evaluate the long-term effectiveness of cytotoxic agents . The abiP < 0.05). This lower percentage of colony formation was also observed for MDA-MB-231 and SKBR-3 cell lines exposed to LCFL-DXR compared to LCFL-PTX (P < 0.05), while no difference between these treatments was observed for MCF-7 cell line (P > 0.05).As shown in P < 0.05).In general, the encapsulation of the different treatments did not affect significantly the percentage of colony formation for the different cell lines. Exceptions consisted in the encapsulation of PTX for the MCF-7 cell line, and the encapsulation of PTX:DXR at 1:1 molar for the SKBR-3 cell line, both of which led to a significant reduction of colony formation on these cell lines (P < 0.05), while no difference was observed between these ratios in the MCF-7 cell line (P > 0.05). Considering the co-encapsulated combinations of PTX:DXR, the 1:1 and 1:10 molar ratios reduced 1.6-fold and 4.2-fold, respectively, the percentage of colony formation compared to the 10:1 molar ratio against the MDA-MB-231 cell line. For the MCF-7 cell line, the 1:1 and 1:10 molar ratios reduced on 2.1-fold and 2.5-fold, respectively, the percentage of colony formation compared to the 10:1 molar ratio. This reduction in percentage of colony formation was of 11.2-fold and 20.2-fold for the 1:1 and 1:10 molar ratios, respectively, compared to the 10:1 molar ratio in the case of the SKBR-3 cell line. When comparing the 1:1 and 1:10 ratio combinations of the co-encapsulated drugs, the 1:10 ratio allowed for a significant reduction of colony formation compared to the 1:1 ratio for the MDA-MB-231 cell line (P < 0.05), while no difference was observed for these ratios on the MCF-7 and SKBR-3 cell lines (P > 0.05). Despite the different response profiles observed for the short-term cytotoxicity analyses, all three cell lines behaved similarly in the clonogenic assay. This assay revealed that despite the higher cytotoxicity observed for PTX treatment compared to DXR treatment (either in free form or encapsulated in liposomes) when evaluated for its short-term effect, the cells that remained after 48 hours of treatment with this drug retained a better ability to proliferate compared to the cells treated with DXR. In these experiments, higher concentrations of DXR in the combinations led to the lower percentages of colony formation for the three different cell lines. Considering combinations of PTX:DXR on the free form at 1:1 and 1:10 molar ratios, we observed a reduction level of the percentage of colony formation equal to 3.7-fold and 10.7-fold, respectively, compared to the 10:1 molar ratio against the MDA-MB-231 cell line. For the MCF-7 cell line, the 1:1 and 1:10 molar ratios of PTX:DXR reduced 1.9-fold and 3.4-fold, respectively, the percentage of colony formation compared to the 10:1 molar ratio. In the case of the SKBR-3 cell line, this reduction in percentage of colony formation was of 5.7-fold and 32.8-fold for the 1:1 and 1:10 molar ratios respectively, compared to the 10:1 molar ratio. When comparing the combinations of the free drugs at 1:1 and 1:10 molar ratios, the 1:10 ratio allowed for a significant reduction of colony formation compared to the 1:1 ratio for the MDA-MB-231 and SKBR-3 cell lines  cell migration in confluent, monolayer cell cultures . When peP > 0.05). All treatments containing PTX reduced significantly the percentage of cell migration compared to the control group for both cell lines (P < 0.05), which was expected as microtubule-affecting drugs are known to present significant anti-migration properties [As summarized in operties .P < 0.05). For MCF-7 cell line, all molar ratios of free drug combinations reduced migration percentage to the same extent (P > 0.05). When co-encapsulated in liposomes, all different molar ratios of PTX and DXR reduced migration percentage to the same extent for both cell lines (P > 0.05).Considering combinations of free PTX and DXR in different molar ratios, difference was observed in themigration percentage only between PTX:DXR treatments at molar ratio equal to 1:1 and 1:10 using MDA-MB-231 cell line (P < 0.05). For the MCF-7 cell line, cell migration percentages after exposure to free PTX or LCFL-PTXPTX at 6.4 nM concentration were 74.8 ± 3.1% and 72.6 ± 1.5%, respectively, and differed significantly from those observed for the combined 1:10 treatments (P < 0.05). Thus, these findings show that the cellular migration inhibition observed for the 1:10 molar ratio combination is not due to an effect of the PTX alone, suggesting a potentiation effect when the treatment was performed with PTX and DXR together.It is interesting to note that even the addition of a small proportion of PTX, as in the 1:10 PTX:DXR molar ratio combination, allowed for a significant reduction in the migration percentage of both cell lines when compared to the treatment with DXR alone, either in free form or co-encapsulated in liposomes. In the 1:10 PTX:DXR molar ratio combination the concentrations of PTX and DXR are 6.36 and 63.63 nM, respectively. In order to verify if a PTX concentration of 6.4 nM was sufficient to inhibit cell migration to the same extent as observed for the 1:10 combinations, we tested this dose either free or encapsulated in liposomes against the two cell lines. For the MDA-MB-231 cell line, cell migration percentages after exposure to free PTX or LCFL-PTX at a 6.4 nM concentration were 98.1 ± 0.1% and 76.4 ± 6.9%, respectively, differing significantly from those observed for the combined 1:10 treatments  that such cells are derived from TIS cells that circumvented senescence . TIS wasIt has been suggested, but not confirmed, that the source of recurrent disease in the clinics might be an initially senescent tumor cell population . The occAnother important hallmark evaluated was cell migration. Although the metastasis event involves multiple processes, it is strictly connected to the initial cell migration and tissue invasion from the primary tumor site . The migIn our previous study with Balb/c mice bearing 4T1 murine mammary carcinoma we report that the addition of small amounts of PTX to DXR (1:10 molar ratio) is enough to significantly enhance treatment activity  and that the LCFL-PTX/DXR improves the cardiac toxicity profile compared to treatment with the free combination of these drugs . These pChemotherapy regimens containing PTX and DXR are pivotal in the management of BC ,4. Nowad


BRAF V600E mutation, TERT promoter mutations, and homozygous deletions of CDKN2A/2B. Emerging evidence support the effectiveness of targeted therapies for brain tumors with BRAF V600E mutation. Here we describe a dramatic radiographical response to combined therapy with BRAF and MEK inhibitors in a patient with epithelioid GBM harboring BRAF V600E mutation, characterized by thick spinal dissemination. From relapsed tumor procured at autopsy, we established a cell line retaining the BRAF V600E mutation, TERT promoter mutation and CDKN2A/2B loss. Intracranial implantation of these cells into mice resulted in tumors closely resembling the original, characterized by epithelioid tumor cells and dissemination, and invasion into the perivascular spaces. We then confirmed the efficacy of treatment with BRAF and MEK inhibitor both in vitro and in vivo. Epithelioid GBM with BRAF V600E mutation can be considered a good treatment indication for precision medicine, and this patient-derived cell line should be useful for prediction of the tumor response and clarification of its biological characteristics.Epithelioid glioblastoma is a rare aggressive variant of glioblastoma (GBM) characterized by a dismal prognosis of about 6 months and frequent leptomeningeal dissemination. A recent study has revealed that 50% of epithelioid GBMs harbor three genetic alterations – The online version of this article (10.1186/s40478-019-0774-7) contains supplementary material, which is available to authorized users. BRAF V600E (50–93%) [TERT promoter mutations (70%), and homozygous deletions of CDKN2A/2B (79%) [Epithelioid glioblastoma is a rare aggressive variant of glioblastoma (GBM), newly proposed in the 2016 WHO classification, characterized by frequent leptomeningeal dissemination  and poor(50–93%) , 8 and Tutations 0%, and hutations 0%, and hBRAF V600E-mutant brain tumors such as ganglioglioma [BRAF V600E-mutant cancers demonstrated that efficacy varied by histologic subtypes in patients with BRAF V600E-mutant brain tumors [BRAF V600E-mutant tumors have multiple concurrent drivers. It is still unclear which patients would benefit from precision medicine. Thorough genetic studies and establishment of patient-derived cell lines would support decision making in relation to targeted treatment.Progress in genetic studies and targeted therapies has vastly improved the treatment of some cancers , 10. Sevioglioma , 12, pleioglioma , 14 and ioglioma , 16. Then tumors , suggestBRAF V600E mutation that showed a dramatic radiographical response to combined therapy with BRAF and MEK inhibitor after spinal dissemination. From a recurrent tumor sample obtained at autopsy, we established a cell line and used it to confirm both in vitro and in vivo that combination treatment was effective. Our findings suggest that targeted therapy would be beneficial for patients with epithelioid glioblastoma harboring BRAF V600E mutation, and that establishment of cell lines and xenografts would be useful for predicting the effectiveness and overcoming the resistance mechanisms of precision-based treatments.Here we report a case of epithelioid GBM harboring Informed consents for collection of samples during surgery and autopsy and their subsequent use for genetic analysis and other research purposes were obtained from the patient’s family.The surgical and autopsy specimens were fixed with 20% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-μm-thick sections stained with hematoxylin and eosin (HE), and the Klüver-Barrera method. The pathological diagnosis was made on the basis of the WHO classification of tumors of the central nervous system (CNS) by an experienced pathologist (AK). Immunohistochemistry (IHC) was performed as described previously using primary antibodies against Ki-67 , BRAF V600E  , 19 and Surgically obtained brain tissue and orthotopic brain tumor tissue were also subjected to electron microscopy. Glutaraldehyde-fixed small tissue blocks were post-fixed with 1% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in Epon 812. Ultrathin sections were then cut and stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7100 electron microscope at 75 kV.The NGT41 cell line was established from a disseminated lesion of the cervical spinal cord at autopsy  and the American Type Culture Collection (ATCC), respectively. Control BRAF-wildtype glioma cell lines, U87MG and T98G, were purchased from ATCC. All cell lines were grown in a humidified incubator at 37 °C under 5% CO2. NGT41, AM38, U87MG, and T98G cells were grown as adherent monolayer cultures in 10% FBS DMEM, and DBTRG-05MG cells were grown in 10% FBS RPMI1640  with Glutamax . Dabrafenib (GSK2118436) was purchased from Selleck , and trametinib (GSK1120212) from AdooQ Bioscience .BRAF V600E and the TERT promoter was performed as reported previously [BRAF V600E and the TERT promoter. The primer sequences were: BRAF V600E; forward 5′-TCATAATGCTTGCTCTGATAGGA-3′ and reverse 5′-GGCCAAAAATTTAATCAGTGGA-3′, TERT promoter; forward 5′-GTCCTGCCCCTTCACCTT-3′ and reverse 5′-CAGCGCTGCCTGAAACTC-3′. The PCR products were then sequenced on a 3130xl Genetic Analyzer  with a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) in accordance with the manufacturer’s instructions. Multiplex ligation-dependent probe amplification (MLPA) was selected to assess the CDKN2A/2B copy number. The SALSA MLPA probe mix P088-C2  was used. In accordance with the manufacturer’s instructions, 250 ng of the DNA was denatured, hybridized, ligated, and subjected to PCR. The carboxyfluorescein (FAM)-labeled MLPA PCR products were separated by capillary electrophoresis on an ABI 3730xl DNA Analyzer (Applied Biosystems). The fragment lengths of the data were analyzed using Peak Scanner v2.0 (Thermo Fisher Scientific). Probe ratios below 0.4 were regarded as homozygous, and those from 0.4 to 0.7 were regarded as heterozygous deletions [The CANCERPLEX cancer genome panel  of 435 cancer-related genes . A 20-μL aliquot of PCR mix, composed of 10 μL of 2× ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 1 μL ddPCR Mutation Assay (Bio-Rad), and 9 μL tumor DNA was loaded into each sample well of an 8-channel disposable droplet generator cartridge (Bio-Rad). An additional 70 μL of droplet generation oil (Bio-Rad) was loaded into the oil well for each channel. After droplet generation, droplets were transferred to a 96-well PCR plate and subjected to thermal cycling. Amplification of the 20 μL reaction mixture was carried out on a QX200 Droplet digital PCR system (Bio-Rad). After PCR, the 96-well PCR plate was transferred to a QX-200 droplet reader (Bio-Rad), and the data were analyzed using QuantaSoft analysis software (Bio-Rad). BRAF V600E mutation-specific signals were generated in the FAM channel, whereas BRAF V600E wildtype signals were generated in the HEX channel.Droplet digital polymerase chain reaction (ddPCR) reagents and Primer/probe mix for Fifteen hundred cells per 100 μL medium were seeded into 96-well plates for one day and 10 μL of each drug was applied for 72 h. As a background control (0% control), 0.2% Triton X-100 was added to the control wells. To each well was then added 10 μL of WST-8 , and the cells were incubated for 4 h. Absorbance at 450 nm was measured in each well using ELx808 . Cell viability was quantified as: cell viability (%) = (Asample - a0%) / (a100% - a0%) × 100, where Asample is the sample absorbance, a0% is the average 0% control absorbance, and a100% is the average 100% control absorbance.50 was calculated using the Online GR Calculator .The normalized growth rate inhibition (GR) analysis was performed to minimize cell line-dependent differences in rate of cell division . FifteenApoptosis and the cell cycle were assessed using a Muse™ Cell Analyzer  with Annexin V and Dead Cell Assay and Cell Cycle analysis kits following the manufacturer’s instructions, as described previously . In brieProteins were extracted using cell lysis buffer, and Western blotting was performed as described previously . The bloFour-to-five-week-old male BALB-c nu/nu mice were purchased from Charles River Laboratories Inc.  and housed under aseptic conditions, which included filtered air and sterilized food, water, bedding, and cages. The mice were anesthetized with medetomidine hydrochloride (0.3 mg/kg), midazolam (4.0 mg/kg) and butorphanol tartrate (5.0 mg/kg) by intraperitoneal injection.6 NGT41 cells were suspended in 50 μL of Neurobasal Medium (Gibco) and injected subcutaneously into the hip. To investigate the efficacy of treatment, these mice received vehicle control ) or combined treatment (20 mg/kg dabrafenib and 2 mg/kg trametinib solubilized in 20% DMSO) by oral gavage for 10 days. The tumors were measured daily with calipers, and tumor volume was calculated using the formula: Tumor volume (mm3) = Length (mm) × Width (mm)2/2 [For the subcutaneous tumor model, 1 × 10 (mm)2/2 . Two add5 cells in 2 μL of Neurobasal Medium) was stereotactically injected into the right caudate putamen at 1.0 mm to the right of the midline, just behind the bregma, and at 3.0 mm depth. The mice were monitored daily and euthanized if body weight loss exceeded 20%, or if they developed neurological symptoms indicative of tumor burden. Brain tissue was subsequently resected and subjected to formalin fixation and paraffin embedding.For the intracranial xenograft model, a NGT41 cell suspension  unless otherwise specified. Error bars represent standard error of the mean. Kaplan-Meier survival curves for intracranial xenografts were assessed by Log-rank test, and p < 0.05 was considered statistically significant unless otherwise specified. All statistical tests were performed using the GraphPad Prism 6 software package .Differences between three or more groups were assessed using two-way analysis of variance (ANOVA) with the A 57-year-old man presented with headaches and slight dysphasia. Post-contrast MR images revealed a well circumscribed mass lesion measuring 6 cm × 5 cm at the left frontal lobe, adjacent to the lateral ventricle. Subependymal enhancement was noted inside the left lateral ventricle Fig. a. The inBRAF V600E and TERT promoter (C250T) mutations, and CDKN2A/2B loss were identified using the CANCERPLEX comprehensive genomic panel. No other genetic mutations were found. These genetic changes were confirmed by Sanger sequencing and MLPA analysis  mutation, and heterozygous deletion of CDKN2A/2B (probe ratios were between 0.4 and 0.7), observed in the tissues obtained at surgery and autopsy  or trametinib , or with both  for 72 h. All BRAF V600E-mutant cell lines were sensitive to both drugs. In NGT41, addition of trametinib did not significantly reduce cell viability compared to dabrafenib alone. Among BRAF V600E wildtype cell lines, U87MG showed a response to trametinib, consistent with a previous report  or each drug  for 1 h. Treatment with dabrafenib reduced the expression of both pMEK and pERK in tus Fig. b. In NGTBRAF V600E-mutant cell lines, but not BRAF wildtype cell lines, treated with dabrafenib and trametinib in combination exhibited significantly greater apoptosis   vs 61.24 ± 9.36 p = 0.0299, Log Rank test) , 100% had biallelic inactivation of CDKN2A, and 47% had TERT promoter mutations [Ink4a/Arflox/lox mice has been reported [Ink4a, an important tumor suppressor, produces the same effect as CDKN2A loss (which encodes p16INK4a) in gliomas. Induction of BRAF V600E alone was not tumorigenic; Ink4a/Arf loss in combination produced well-demarcated gliomas showing evidence of growth into the subarachnoid space, recapitulating the characteristics of epithelioid GBM. Thus, the epithelioid GBM model with BRAF V600E mutation in combination with CDKN2A loss we established recapitulates the characteristics of previously published models.Interestingly, a recent series of 15 anaplastic PXAs reported that 100% had RAF alterations  showed that BRAF mutations were significantly correlated with CDKN2A alterations (p = 0.025)   database included in R2: Genomics Analysis and Visualization Platform Additional file 2:Figure S1. Genetic profiles of surgical tissue and the NGT41 cell line. BRAF V600E and TERT promotor (C250T) mutation was confirmed by Sanger sequencing (A), and heterozygous loss of CDKN2A/2B was identified by the multiplex ligation-dependent probe amplification method (B). Figure S2. Evaluation of BRAF V600E using ddPCR. Tumor DNA was extracted from the area of vivid tumor cells in the FFPE tissue by laser microdissection (A). Fractional abundance (FA) of mutated BRAF V600E was calculated as copies of mutated DNA/(copies of mutated DNA + wildtype DNA) (B). Scale bar A: 200 μm. Figure S3. Calculation of growth rate value in NGT41 and U87MG after combination treatment. Dose response curves on relative cell count showed marked response to BRAF and/or MEK inhibitor treatment in NGT41 (A), but minimal reduction in U87MG (B). Figure S4. BRAF and MEK inhibitor induced greater apoptosis and G0/G1 arrest in the NGT41 cell line. In BRAF V600E-mutant cell lines, each treatment significantly increased the number of apoptotic cells  (A). G0/G1 arrest was induced by each treatment in BRAF V600E mutant-cell lines, whereas no response was observed in BRAF-wildtype cell lines (n = 3) (B). Figure S5. Effect of BRAF and MEK inhibitor in the intracranial model. pMEK and pERK were markedly suppressed in the treatment group (A). Serial body weight calculations in the treatment group were virtually the same as in the control group (B). Histological appearance of intracranial tumor in the treatment group at endpoint was similar to that of the control group (C). Scale bar C: 50 μm. Figure S6. Analysis of the TCGA database included in R2: Genomics Analysis and Visualization Platform showed that BRAF mutations were significantly correlated with CDKN2A alterations (p = 0.025) (A) and TERT promoter mutations (p = 7.03e-03) (B). (ZIP 8066 kb)


In the original publication of this article Fig. 2A is incorrectly published. The correct version of Fig. Under the section “The rationale for the investigation of CAR IS” the line starting with “the surface of an tumour cell or infected target cell” is incorrect, it should be read as “the surface of a tumour cell or infected target cell”.Under the section of “COMPLIANCE WITH ETHICS GUIDELINES” the line starting with “This article does not contain any studies” is incorrect, it should be read as “This article does not contain any studies with human or animal subjects performed by any of the authors”.Under the section “Development of “off-the-shelf” NK cell products” the line starting with “The CAR-modified NK92 cell line may” is incorrect, it should be read as “The CAR-modified NK92 cell line may serve as a future “off-the-shelf”.Under the section “The background of IS” the line starting with “between peripheral blood NK cells in the YTS cell line and various transfectants” is incorrect, it should be read as “between peripheral blood NK cells and various transfectants”.


Camellia oleifera seeds through IR, NMR, HR-ESI-MS and GC-MS spectroscopic methods, namely oleiferasaponin A3, oleiferasaponin A1, camelliasaponin B1, and camelliasaponin B2. The structure of oleiferasaponin A3 was elucidated as 16α-hydroxy-21β-O-angeloyl-22α-O-cinnamoyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3β-O-[β-d-galactopyranosyl-(1→2)]-[β-d-xylopyranosyl-(1→2)-β-d-galactopyranosyl-(1→3)]-β-d-gluco-pyranosiduronic acid. Camelliasaponin B1 and camelliasaponin B2 exhibited potent cytotoxic activity on three human tumour cell lines (human lung tumour cells (A549), human liver tumour cells (HepG2), cervical tumour cells (Hela)). The hypoglycemic activity of oleiferasaponin A1 was testified by protecting pancreatic β-cell lines from high-glucose damage.One new and three known triterpenoid saponins were isolated and identified from  Camellia oleifera was named for its seeds with plentiful edible oil. Tea seed pomace—the byproduct of oil manufacture—contains about 8% saponins, which have historically been wasted without full use -[β-d-xylopyranosyl-(1→2)-β-d-galactopyranosyl-(1→3)]-β-d-gluco-pyranosiduronic acid. The other three known compounds were oleiferasaponin A1 ][β-d-glucopyranosyl (1→2)-α-l-arabinopyranosyl (1→3)]-β-d-gluco-pyranosiduronic acid) [1 ][β-d-glucopyranosyl (1→2)-α-l-arabinopyranosyl (1→3)]-β-d-gluco-pyranosiduronic acid) and camelliasaponin B2 ][β-d-glucopyranosyl (1→2)-α-l-arabinopyranosyl (1→3)]-β-d-gluco-pyranosiduronic acid) [The NOESY spectrum showed the cross peaks between H-22 at se (Xyl) ,20. Syntic acid) , camelli1, oleiferasaponin A3, camelliasaponin B1 and camelliasaponin B2 obtained from Camellia oleifera seed pomace were tested against three human tumour cell lines  using cell proliferation bioassay (SRB). Camelliasaponin B1 and camelliasaponin B2 at the concentration of 20 μM exhibited effective anti-proliferative activity on the human tumour cell lines tested (1 and camelliasaponin B2 at the concentration of 10 μM significantly inhibited the proliferation of human lung cancer cells (A549) (1 and camelliasaponin B2 possessed potent cytotoxic activity. There are some reports about the structure–activity relationships of triterpenoid saponins [Oleiferasaponin As tested —the inhis (A549) —the inhisaponins ,22.1 and camelliasaponin B2 are similar, except for the orientation of C-22 angeloyl. Compared to previously reported results [1 and oleiferasaponin A3 did not show cytotoxic activity.The structures of camelliasaponin B results ,12,13, i1 and oleiferasaponin A3 did not exhibit cytotoxic activity on three human tumour cell lines , so we carried out a hypoglycemic activity study for further exploration of structure–activity relationship. Oleiferasaponin A1 and oleiferasaponin A3 were tested for their protective effect on RIN-m5f (islet-β cells) injured by high glucose. The insulin content of RIN-m5f cells upon treatment under 16.7 mmol/L glucose are shown below  was enhanced, which indicates that oleiferasaponin A1 has potential hypoglycemic activity against the damage induced by high glucose, and oleiferasaponin A1 may be a therapeutic agent for hyperglycemia treatment. Regarding the oleiferasaponin A3 group, no improvement effect on insulin levels was found in RIN-m5f (islet-β cells) injured by high glucose, even at a concentration of 100 μM. The difference of bioactivity between oleiferasaponin A1 and oleiferasaponin A3 is due to the different structure, including aglycone and sugar moieties  is the third most prevalent disease globally, and manifests as a disorder of blood glucose caused by metabolic disorder, which can induce cardiovascular system diseases and cancer, then threatening human health and life. Many studies regarding the cytotoxic activity of triterpenoid saponins have been reported ,12,13, wwn below . With himoieties . Compare4  as solvent. HR-ESI-MS were determined on an electrostatic field orbital trap mass spectrometer  using an ESI source.HPLC was run on Agilent 1260 HPLC . IR (infrared) spectra was recorded on Nicolet iN10  with KBr pellets. NMR spectra was measured on an AVANCE III (600 MHz) spectrometer  using methanol-dCamellia oleifera) was collected from a factory in Shucheng, Anhui province, China. The plant material was identified by one of the authors (Associate Prof. X.F. Zhang), and was deposited in State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University.Tea seed pomace  to furnish two saponin mixtures . The first faction was further purified by HPLC  to afford oleiferasaponin A1 (8.7 mg) and oleiferasaponin A3 (3.9 mg). The second faction was further purified by HPLC  to camelliasaponin B1 (11.6 mg) and camelliasaponin B2 (9.9 mg).The samples were extracted and isolated according to Zhang et al. . The tea3 was dissolved in 1 M HCI  (1 mL) for 3 h at 90 °C, then extracted with chloroform . The aqueous phase was evaporated under N2 flow. The residue was dissolved in 0.2 mL pyridine  containing L-cysteine methyl ester hydrochloride (10 mg/mL) and reacted at 70 °C for 1 h, then evaporated under N2 flow again. After concentrated, 0.2 mL trimethylsilylimidazole  was added for derivatization reaction, and reacted at 70 °C for another 1 h. The reaction mixture was partitioned between n-hexane and water. The organic phase was analysed by GC-MS  . The standard sugar samples were subjected to the same reaction and GC-MS conditions.Oleiferasaponin A−1 penicillin, and 100 μg·mL−1 streptomycin at 37 °C in a 5% CO2 humidified atmosphere. The culture medium was refreshed every other day. After 80% of the cells were fused, cells were kept in logarithmic phase by trypsinization and subculturing.Human lung tumour cell (A549) lines, human liver tumour cell (HepG2) lines, and cervical tumour cell (Hela) lines were obtained from Qingdao Marine Biomedical Research Institute Limited by Share Ltd Testing Center . Cells were cultured in DMEM complete medium supplemented with 10 % fetal bovine serum, 2 mM l-glutamine, 100 U·mL3 cells per well (180 μL per well), and incubated for 24 h. After 24 h, negative control without additions; solvent control was supplied with 0.1% DMSO; positive control with 1 μM adriamycin; 20 μM, 10 μM saponins were added to trial group, all incubated for 72 h. Then, 50% (m/v) ice-cold trichloroacetic acid was added to the medium for fixed cells. After staining by sulforhodamine B, tris solution (150 μL per well) was added to culture medium. Absorbance values were measured at 540 nm using an enzyme-linked immunosorbent reader . The inhibition rate of cell proliferation was calculated as:Human tumour cell lines in logarithmic phase were seeded in a 96-well plate at 4 × 104 cells per well). Cells were set in four groups: normal group with 5.5 mmol L−1 glucose; injured group with 16.7 mmol L−1 glucose; trial group with 16.7 mmol L−1 glucose and different concentrations  of oleiferasaponin A1; positive control group with 16.7 mmol/L glucose and 100 μM ZnSO4. Each group was set three parallels and incubated for 48 h. Next, the medium was removed, cleaning twice with polybutylene succinate (PBS). The cells were incubated in medium with 5.5 mmol L−1 glucose for 1 h. Then, the medium was replaced by medium with 33.3 mmol L−1 glucose and incubated for 2 h. The supernatant was collected for insulin content detection using ELISA kit .Pancreatic β-cell lines (RIN-m5f) were obtained from Qingdao Marine Biomedical Research Institute Limited by Share Ltd. Testing Center . Cells were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin and 1% glutamine. Then, pancreatic β-cell lines (RIN-m5f) were seeded in a 96-well plate . Oleiferasaponin A1 possessed potential hypoglycemic activity protecting pancreatic β-cell lines from high-glucose damage.Four triterpenoid saponins were isolated from


Mutations of histone variant H3.3 are highly recurrent in childhood glioblastoma and in young adults with Giant Cell Tumor of the Bone (GCTB). The heterozygotic representation of the mutations in the tumors, and with potential histone H3 and H3.3 redundancy, suggest that the mutations are gain-of-function by nature. To address common H3.3 point mutations, we have generated data from GCTB patient samples with H3.3 G34W substitutions and engineered human GFP-tagged H3.3-mutated isogenic cell lines for high throughput data comparisons. First, a total of thirty-six patient samples and cell lines were used to acquire gene expression transcriptome data using microarray and RNA-sequencing. The expression data were validated with the orthogonal nCounter assay. Second, to uncover the H3.3-GFP interaction proteomes from the isogenic cell lines, immunoprecipitation of unmutated wild type, K27M, G34R, and G34W substitutions were performed. The RNA-sequencing data and the H3.3 interaction proteome enable potentially important functional insight into the tumorigenic process and should spur further detailed analysis. In contrast, tumors from children and young adults develop from a less complex cytogenetic background, simplifying the process of uncovering the driving forces of tumorigenesis and their transcriptional profiles.Recurrent genomic alterations are a cornerstone in cancer development and reproducible mutational profiles have been observed at defined genomic locations. Whole genome sequencing of tumors from adults and seniors have uncovered genomic landscapes with hundreds of small and large-scale mutations, revealing the complexity of chromosomal rearrangements influencing tumorigenesis2 and Giant Cell Tumor of the Bone (GCTB)3 where very few other genic mutations were discovered after whole-exome sequencings. Yet, the histone mutations affected global epigenetic modifications of important residues, dramatically changing gene expression of targeted genes, seemingly driven by the histone mutations that lead to amino acid substitutions at or very near frequent post-translational modified amino acids; namely the K27M, G34R/V/L/W, and K36M substitutions3.This is exemplified by the recent discovery of recurrent mutations of histone H3 and the replication-independent variant H3.3 in pediatric Glioblastoma5. A similar example is the H3.3 K36M substitution binding the enzymes MMSET and SETD2, leading to global reduction of H3K36 methylation6. These two examples indicate that mutations of the N-terminal tail of histones can acquire novel binding properties and greatly influence transcription. We address the possibility that the cancer-related histone H3.3 substitutions in this study bind yet unknown proteins, allowing them to be purified and characterized by gel separation and mass spectrometry.The power behind these histone mutations can be traced to their role as pre-eminent binding sites for proteins influencing transcription. The first, and perhaps the best example, is the H3.3 K27M substitution leading to binding and catalytic inhibition of the polycomb repressive complex 2 (PRC2), with global loss of H3K27 methylation across the genome as a result7.As presented here, the transcriptome and interactome from GCTB biopsies and isogenic cell lines, respectively, with mutations of H3.3 have been made available . We firs7. These genes are all targeted by the E2F transcription factor family.We there reported on the comparison of unmutated and H3.3 G34W tumors and found a distinct gene expression pattern likely dictated by the single mutation in H3.3. We uncovered several known genes associated with GCTB, e.g. that RANKL is affected via the downregulation of its decoy receptor OPG, and that the entire IGFBP-family of genes appear to be downregulatedWT to indicate an engineered isogenic version of H3.3 to avoid confusion with patient samples. The H3.3 interaction proteome uncovered 493 proteins of which about half (225) were commonly bound by all constructs  were established in HEK293 cells where e.g. H3.3 WT have been denoted isoH3.3nstructs , and aronstructs . While snstructs .Our analysis in the previous study mainly focused on the H3.3 G34W substitution in GCTB, filtered against other H3.3 interaction proteomes to enrich for the once that specifically represented G34W . This st7. While the description of the methods here is very similar to that report, we specifically want to emphasize the samples and the methods used to generate the data, in particular the previously unavailable H3.3 interaction proteome from cancer-related recurrent substitutions.Please note that samples and the generation of data presented in this Data Descriptor have been previously presented in Lim et al.The data in this data descriptor contain information generated from Giant Cell Tumor of the Bone biopsies and established control cell lines. Twenty tumor biopsies were collected from two cohorts, one from Germany and one from South Korea, to establish primary cell lines from the two clinics. Informed consent to analyse tumour tissue and to publish clinical details was obtained from all individuals included in the study. The use of patient samples and the experiments performed in this study was approved by and in accordance with guidelines and regulations by the Ethics Committees of the University of Heidelberg, University of Leipzig, University Medical Center Hamburg-Eppendorf, and the National Cancer Center of Korea (IRB NCC2015-0070).We performed gene expression microarray analysis on 29 samples, of which 9 were German primary cell lines, 12 were Korean biopsies and 6 Korean cell lines, and 2 were control cell lines. Control cell lines were the bone-marrow derived mesenchymal stromal cell line KM1234 and the hFOB1.19 osteoblast cell line. In total there were 19 samples of H3.3 G34W and 10 were H3.3 WT. The samples were run on a HumanHT-12 v4 Expression BeadChip (Illumina). RNA sequencing was performed on 6 Korean primary cell line samples of which 3 were H3.3 G34W and 3 were H3.3 WT. We used the TrueSeq RNA kit for library preparation and paired-end sequencing on an Illumina HiSeq2500 instrument and accumulated about 14 Gbp read bases per sample with a Q30 phred quality score of 91-96%.2, 3 μl [25U/μl] Benzonase nuclease  and Complete protease inhibitors (Roche), Pepstatin A (Roche) and Aprotenin (Roche). Cleared lysates were diluted 1:2.5 in dilution buffer (10 mM Tris-HCl (pH7.5), 150 mM NaCl, 0.5 mM EDTA). Total protein concentrations were determined by BCA protein assays  and PAGE-gel followed by Coomassie (BioRad) staining to determine equal loading prior to immunoprecipitation. The equal loading-adjusted protein lysates were incubated with equilibrated 25 μl GFP-Trap-A bead slurry (Chromotek), washed and recovered with 2x Laemmli buffer (BioRad) supplemented with 10% ß-mercaptoethanol. A new PAGE-gel was run to separate the eluates, and stained with Silver stain kit  for verification. Each lane was divided into three equal portions and subjected to in-gel tryptic digestion and LC-MS/MS separation with the Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer instrumentation (Thermo Fisher Scientific).The GFP-expressing isogenic cell lines were used for immunoprecipitations of the H3.3 construct with interacting proteins from total protein extracts. Isogenic HEK293 cells were grown in DMEM media with 10% FBS in a Ø10 cm dish, harvested at subconfluency by cell scraping, rinsed twice with PBS and lyzed with 200 μl ice-cold lysis buffer, and placed at + 4°C for 30 minutes on rotation. The lysis buffer consisted of 50 mM Tris-HCl (pH7.6), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, supplemented with 2.5 mM MgClThe presented data was analyzed with the following software and applications:8, mapped the reads with TopHat9 to the human genome reference hg19, and assembled the reads with CuffLinks10.For the RNA-sequencing data, we trimmed the reads using trimmomatic0.36Protein scores from the LC-MS/MS analysis were generated and analyzed with default settings using the SEQUEST package (Thermo Fisher Scientific).The gene expression microarray data, generated by the Illumina HT12 array platform using total RNA from Giant Cell Tumor of the Bone biopsies, have been made available by the Gene Expression Omnibus (GEO) in Data Citation 1.The RNA sequencing data, generated by poly-dT selected RNA from primary cell lines isolated as outlined in www.ebi.ac.uk/pride/archive/) in line with the ProteomeXchange (PX) consortium guidelines. The public dataset  is available under Data Citation 3.The liquid chromatography and mass spectrometry (LC-MS/MS) data, generated from GFP immunoprecipitation experiments of isogenic HEK293 cell lines containing H3.3-GFP constructs, have been made available at the PRIDE Archive (https://www.proteinatlas.org/about/download).Data indicating the cellular compartments of individual proteins were provided by the Human Protein Atlas  were validated with deep sequencing technology of poly-dT selected RNA from primary cell lines (Data Citation 2) originating from an independent set of samples, not overlapping with samples used to generate the microarray data .Second, we performed validation of the gene expression data with the RNA hybridization-based nCounter assay (NanoString Technologies). We found a strong correlation between the two methods  of the H3.3-GFP fusion proteins to uncover the H3.3 interactome, with emphasis on proteins that specifically interacted with the mutated forms. Mock IP with parental control did not pull down any proteins after the washing procedures. From the H3.3 interactome, we found hnRNPA1L2 to be the strongest interactor to H3.3G34W and validated the mass spectrometry data with PAGE and Western blot analysis 7. Here, we make all data available to allow further analysis of expression data and the WT and mutant H3.3 interaction proteomes .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Basal/human epidermal growth factor receptor (HER)2-positive (HER2+) breast cancer is resistant to monoclonal antibody (herceptin) treatment. There are currently only three basal/HER2+ breast cancer cell lines available, but they are not from Chinese populations.Three immortalized cell lines  were established from invasive ductal breast carcinoma tissue of two patients treated by surgical resection at our center. The cell lines were characterized in terms of histology, therapeutic response, and biomarker expression. Their tumorigenic potential was evaluated in an athymic nude (BALB/C nu) mouse xenograft model. Cell authentication testing by the techniques of short tandem repeat.ZJU-0327, ZJU-0725, and ZJU-1127 cell lines were maintained for more than 110 passages in vitro. The cells grew as monolayers; showed typical epithelial morphology and ultrastructure; were polyploid; had doubling times of 18, 57.5, and 18 h, respectively; had a near-tetraploid (ZJU-0327 and ZJU-1127) or aneuploid (ZJU-0725) karyotype with structural aberrations and tumor protein 53 mutation; insensitive to chemotherapeutic drugs and/or radiation; show high invasiveness and tumorigenicity in mice; and had no mycoplasma contamination. The cell lines were basal/HER2+, expressed cluster of differentiation, and were associated with poor prognosis. Cell authentication testing by the American Type Culture Collection confirmed the human origin of the cell lines, which did not match those in existing databases.The three novel basal/HER2+ breast cancer cell lines recapitulating the malignant characteristics of the parent tumor’s, and can be useful for clarifying the molecular pathogenesis of basal/HER2+ breast cancer. Breast cancer is the first occurring incidence overall and is the second leading cause of death in the United States in 2017 , althougEstablishing cell lines experimental systems is important for basic and pre-clinical studies as they allow investigation into the molecular mechanisms of carcinogenesis and the testing of therapies . Most inTo this end, we established three new breast cancer cell lines: ZJU-0725 and ZJU-1127 were derived from invasive ductal carcinoma tissue of one female Chinese patient, whereas ZJU-0327 was derived from another patient. Herein, we describe the characterization of these cell lines in terms of cell morphology, ultrastructure, DNA content, population doubling time (PDT), tumorigenicity, invasive potential, therapeutic sensitivity, protein expression, mycoplasma contamination, TP53 mutation, status karyotype, breast cancer biomarker expression and STR authentication.3), and Basal-Like Group (BT-549 and MDA-MB-231) obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences  in early 2016. The cells were authenticated by DNA-fingerprinting in the Cell Bank at a regular basis. HBL-100 was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium  with additive , and 1.5 g/l NaHCO3, 2.5 g/l Glucose, and 0.11 g/l sodium pyruvate. MCF-10A was maintained in Mammary Epithelial Cell Growth Medium (MEGM) . MCF-7 was maintained in Minimal Essential Medium(Invitrogen) with additive, and 1.5 g/l NaHCO3, 0.023 IU/ml bovine insulin, and 0.11 g/l sodium pyruvate. T-47D and Sk-Br3 were maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen), with additive, and 1.5 g/l NaHCO3. BT-549 was maintained in RPMI 1640 medium with additive, 1.5 g/l NaHCO3, 0.11 g/l sodium pyruvate, and 0.023 IU/ml bovine insulin. MDA-MB-231 was maintained in L-15 medium  with additive. Cells were cultured in a humidified 5% CO2 incubator at 37 °C. The medium was renewed when a color change was observed.We used three panels of breast cancer lines as controls—i.e., Normal Phenotype Group (HBL-100 and MCF-10A), Luminal Phenotype Group   to obtain a single-cell suspension. The cell pellet was resuspended RPMI 1640 medium supplemented with additive, and then seeded in 60-mm Petri dishes in a humidified 5% CO2 incubator at 37 °C. After 2 days later, floating cells were washed away when the medium was renewed. Cancer-associated fibroblasts (CAFs) were removed by scraping using a Corning Small Scraper . Cancer cells were harvested by treatment with 0.25% trypsin–EDTA (Invitrogen) and subcultured. The cells were sampled at intervals and stored in liquid nitrogen.Tissue samples were obtained from patients diagnosed with invasive ductal breast carcinoma, who underwent mammary gland excision at Sir Run Run Hospital of Zhejiang University and were processed for primary culture as previously described . BrieflyFor ultrastructural analysis, cells at passage (P)72 were fixed with 1.2% glutaraldehyde and 2.0% glutaraldehyde for 1 h at 4 °C, post-fixed in 1% osmium tetroxide dehydrated in a graded series of alcohol, and embedded in resin. Ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and visualized under a HT7700 microscope .CK-pan is a widely used epithelial maker . ZJU-032Normal human lymphocytes were used as an internal standard for cell cycle analysis. ZJU-0327, ZJU-0725, ZJU-1127, and MCF-7 in exponential growth phase were processed according to a standard cell cycle staining protocol  and then sorted by flow cytometry. The proliferation index (PI) was calculated as (G2/M + S)/(G0/G1 + S + G2/M), and the S-phase fraction (SPF) was calculated as S ÷ (G0/G1 + S + G2/M) to determine the percentages of proliferating and S-phase cells, respectively.3 and 1 × 104 cells/well as six replicates in 0.2 ml culture medium, with medium containing no cells serving as a control. The growth rate was evaluated at exactly 12 h intervals for 72 h by measuring absorbance at 450 nm. Growth curves were plotted, and PDT (population doubling time) was calculated using the formula PDT = 0.693t/ln (Nt/N0), where t is culture time, N0 is initial absorbance, and Nt is absorbance at time t.The growth kinetics of ZJU-0327, ZJU-0725, and ZJU-1127 cells at P40 were examined with the CCK-8 assay . Cells were seeded in 96-well plates at a density of 5 × 10To screen for TP53 mutations, total DNA was extracted from three primary breast cancer cell lines using a DNeasy Blood and Tissue kit  according to the manufacturer’s instructions. Total DNA was amplified polymerase chain reaction (PCR) using a primer set containing exons 1–11 of TP53  according to the manufacturer’s instructions. DNA fragments were visualized under ultraviolet illumination.Proteins samples of Normal Phenotype Group, Luminal Phenotype Group, Basal-Like Group and Established Group cells  were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad) and transferred to a polyvinylidene difluoride membrane  that was blocked in 0.1% Tween-20 in Tris-buffered saline (TBS) containing 5% non-fat dry milk(BD Biosciences) for 2 h at room temperature, and probed overnight with primary antibodies against Ki-67, HER2, progesterone receptor (PR) A/B, estrogen receptor (ER)-α, keratin-18, Forkhead box (FOX)A1/hepatocyte nuclear factor (HNF)3α, anterior gradient homolog (AGR)2, epidermal growth factor receptor (EGFR), secreted protein acidic and rich in cysteine (SPARC), vimentin, caveolin 1 and 2, cluster of differentiation (CD) 44, signal transducer and activator of transcription (STAT)3α/β, octamer-binding protein (OCT)4, (sex-determining region Y)-box (SOX)2, Nanog, CD146, vitamin D3 receptor (VDR), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . After three 5-min times of washes with 0.1% Tween-20 in TBS, the membrane was incubated with diluted horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody , and imaged with a Tanon 5200 Multi scanner  after processing with the EZ-ECL kit .3 cells were exposed to 0, 2, 5, 10, or 15 Gy radiation for 72 h. Cells were seeded at a density of 5 × 103 in 96-well plates, with six replicates per treatment. Cell viability following treatment was evaluated by incubating with CCK-8 reagent for 2.5 h and then measuring the absorbance. Inhibition rate (IR) was calculated with the formula IR = 100 − (mean absorbance of test well/mean absorbance of control well) × 100%.ZJU-0725 and ZJU-1127 cells at P70 were subjected to chemoradiotherapy ; and ZJU-0725 and ZJU-1127 cells at P75 and Sk-Br7) at P50 resuspended in 0.1 ml PBS were subcutaneously injected into the right flank of each mouse (n = 4 per group). Tumor diameter was recorded every 5 days. On the last day, tumor tissue was excised, fixed in 10% formalin, embedded in paraffin, and processed for routine histopathologic examination by hematoxylin and eosin (H&E) staining.Female athymic nude mice   were used for heterotransplantation experiments. Animal were maintained in laminar flow cabinets under specific pathogen-free conditions. ZJU-0327, ZJU-0725, and ZJU-1127 cells  coated with 0.3 g/l Matrigel (BD Biosciences). Cells were added to the upper compartment of the chamber, while the lower compartment was filled with 600 μl culture medium. After 48 h, the cells on the top membrane surface were removed, and those that invaded the Matrigel layer on the lower membrane were fixed in methanol for 15 min, stained with 0.1% Crystal Violet for 15 min, and immersed several times in water, and air dried, and sealed with resin. Cells in 10 randomly selected fields were counted under a microscope.l-lysine-coated slides, dried overnight at 37 °C, deparaffinized in xylene, rehydrated through a graded series of alcohol, and stained with H&E. Immunohistochemical analysis was performed using a standard avidin–biotin technique after a microwave antigen retrieval step in which slides were heated in a pressure cooker containing 10 mM sodium citrate (pH 6.5) for 10 min. Endogenous peroxidase was deactivated by treatment with 3% H2O2 for 5 min, blocked with 10% normal goat serum for 30 min at room temperature, and probed overnight at 4 °C with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, p120, and Ki-67 . The following day, slides were incubated with poly-HRP secondary antibodies for 1 h in the dark at room temperature. Immunodetection was performed using 3,3′-diaminobenzidine substrate kit. Sections were counter-stained with hematoxylin to visualize nuclei.Immunohistochemical analysis was carried out using paraffin-embedded sections of tumor xenograft samples from mice. The specimen were cut into 4-μm-thick sections, that were mounted on poly-Cells were authenticated according to the protocol of the ATCC  based on 17 STR loci plus the gender-determining locus, amelogenin. Samples were processed with an ABI Prism 3500 × 1 Genetic Analyzer, and data were analyzed using GeneMapper IDX v.1.2 software (Applied Biosystems). Appropriate positive and negative controls were used, and the results were confirmed against ATCC and Deutsche Sammlung von Mikroorganismen und Zelkulturen (DSMZ) databases for each sample submitted .Data were analyzed using Prism software  and are presented as mean ± standard deviation of at least three independent experiments. Differences with a P value of < 0.05 were considered statistically significant.The ZJU-0327, ZJU-0725, and ZJU-1127 cell lines were deposited in the China Center for Type Culture Collection (CCTCC) . ZJU-0327 was derived from one female breast infiltrating ductal carcinoma patient aged 63 years old with axillary lymph node metastases (1 +/15), whereas ZJU-0725 and ZJU-1127 were derived from another patient aged 65 years old with the same diagnosis (1 +/19). To establish primary breast cancer cell lines, original cultured cells were subcloned at a ratio of 1:6, and the epithelial cell cluster was marked. Fibroblasts were removed by scraping method. Established cells grew as an adherent monolayer and were spindle shaped with epithelial and malignant characteristics, although ZJU-0725 cells were more narrow than cells in the other two lines  from proline (CCC) to arginine (CGC) at codon 72 of exon 4 and a missense mutation from tyrosine (TAC) to asparagine (AAC) at codon 126 of exon 5  resulting from unbalanced translocation with materials of known or unknown origin were observed. There were also additions in chromosomes 1, 3–8, 11–15, and 18–21, and loss of chromosomes 9, 16, and 22. ZJU-0725 was also near tetraploid, with a modal number of chromosomes of 78–85. Typsin-Giemsa banding revealed a double-deletion of chromosomes 3 and 5; additional chromosomes 2, 9, 21, and 22; derivative chromosomes of X, 2, 8, and 9; additions in chromosomes 1–8, 11, 12, 15, 16, 18, 20, and 21; and loss of chromosomes 9 and 22. ZJU-1127 was aneuploid with near pentaploid to hexaploid complement, with chromosome number ranging from 128 to 133. Trypsin-Giemsa banding revealed a deletion chromosomes 11; additional chromosomes 2, 19, 21, and 22; derivatives of all chromosomes including X; and no chromosome loss. Unknown derivative marker chromosomes were also observed in the three cell lines .In this study we successfully established three primary Basal/HER2+ breast cancer cell lines , two of which originated from a single patient. The cell lines were derived from poorly differentiated in situ breast invasive ductal carcinoma of untreated female patients from China. In both cases, there was no family history of breast carcinoma, and no other factors that are known to have contributed to the disease.Many studies have described methods for establishing breast cancer cell lines, including differential trypsinization –16, adheThe purity of ZJU-0327, ZJU-0725, and ZJU-1127 cells ranged from about 97%  was present in codon 72 of exon 4, this is common in human cancers and is considered non-pathogenic. Codon 126 of exon 5, which is located in the sequence-specific DNA-banding region, harbored a TAC (Try) to AAC (Asn) missense mutation (MUT_ID:1320) (http://www.p53.iarc.fr) (Fig. Mutations in tumor suppressor genes including TP53 are often detected in several cancers including breast cancer , 28. We Chromosomal abnormalities can lead to the development of diseases including cancer. Our three breast cancer cell lines showed abnormal karyotypes, including increases in chromosome number as well as chromosomal breaks and translocations Fig. b–d. AlthBasal/HER2+ breast cancer shows poorer survival than other phenotypes , 30. We 3 cells (Fig. We investigated the malignant potential of our cell lines in a xenograft model and found that all three cell lines formed tumors in nude mice, with the largest tumors formed by ZJU-0327 and ZJU-1127 cells in 30 days. The different growth patterns observed in vitro and in vivo were likely due to environmental variations. Our histological analysis confirmed that the tumor xenografts were poorly differentiated Fig. a–c. Althlls Fig. d, e.We observed differences in protein expression between tumor xenografts and clinical specimens. The expression patterns of CK5/6, P120, and E-cadherin were similar in tissue sections from xenograft and corresponding original tumors. PR and HER2 expression was comparable between ZJU-0725 and ZJU-1127 tumor xenografts and original tumors, whereas Ki67 was similarly expressed in ZJU-0327 cell-derived tumors and clinical samples. We also observed that compared to the original tumors, Ki67 and ER-α immunoreactivity was increased and decreased, respectively, in ZJU-0725 and ZJU-1127, while HER2 and EGFR immunoreactivity was upregulated and ERα and PR were downregulated in ZJU-0327. The difference may be due to a well-differentiated cell subpopulation and/or the presence of factors that increased the differentiation of ZJU-0327, ZJU-0725, and ZJU-1127 cells during the establishment of these cell lines. Expression of the proto-oncogene and receptor tyrosine kinase EGFR was correlated with cell proliferation, survival, migration, and differentiation, and its over-expression can lead to the development and recurrence of breast cancer . We founWe established three novel basal/HER2+ breast cancer cell lines from female Chinese patients who were diagnosed with invasive breast cancer. The cell lines recapitulate the malignant characteristics of the primary tumor. These three cell lines can serve as new models for investigating the etiology and molecular pathogenesis for basal/HER2+ breast cancer and for testing novel therapeutics for its treatment.


TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients’ samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains  and losses (chr X), with additional more CNVs found in CDDP-resistant cells . In the patients’ samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g.,  NANOG or the oncogene KRAS. Another typical alteration is the KIT/KITL pathway activation, characteristic for early stages and pre-invasive tumors [Testicular germ cell tumors (TGCT) are the most frequent solid tumors in young adult men aged 18–45 years. The incidence of TGCT has increased over the past decades, but the reason for this observation is unknown ,3,4,5. Ae tumors ,13,14,15e tumors ,16. Moree tumors .Based on the characteristic histological patterns, TGCT are classified into seminomas and nonseminomas. These two main TGCT subtypes show distinct clinical characteristics, differing in the course of the disease and treatment response. Introducing platinum-based regimens into the treatment of TGCT dramatically increased the overall cure rate, with over 90% of patients remaining in long-term complete remission ,19. NeveNANOG, OCT3/4/POU5F1), the cytotoxic stress/DNA damage drives these cells via p53 signaling directly to apoptosis, not allowing for any attempts of damage repair. The loss of this specific type of DDR, e.g. accompanying the differentiation of tumor cells, either spontaneous or induced by the applied chemotherapy, may then result in the establishment of the CDDP resistance [TP53 and MDM2 genes [XRCC2, HMGB4) [PIK3CA, IGF1R) [MAD2g) [BMI1) [The principal cause of the development of cisplatin (CDDP) resistance, which almost inevitably leads to the treatment failure and death of the affected TGCT patients, has not been identified, and the molecular basis of cisplatin resistance is poorly understood. In general, the resistance to cisplatin may arise at any phase of its kinetics and action . Howeversistance ,27,28,29M2 genes . In furt, HMGB4) ,32, onco, IGF1R) ,33, cell [MAD2g)  or epige) [BMI1) .In this project, we generated cisplatin-exposed counterparts of TGCT cell lines and investigated their newly acquired features in comparison with the original cells, to uncover particular genes or pathways involved in the acquired cisplatin resistance. In a group of primary tumor samples of TGCT patients with different stages of the disease, we then looked for similar aberrations and for aberrations that could be related to the disease development.50 approximately 10 times higher than the original sensitive cells (referred to as NCCIT) .NCCIT cell line was cultured with increasing doses of CDDP for 20 months (further referred to as NCCIT_CDDP), achieving a significant resistance to CDDP, with ICp = 0.05), which was accompanied by a decreased number of resistant NCCIT_CDDP cells in S phase . Interestingly, the trend in the cell cycle distribution between parental vs. derived Tera-2 cells was rather opposite. Tera-2_CDDP showed a decrease in G1  and an increase in S phase in comparison with parental cell line . The apoptotic rate of the cell lines was analyzed before and after a short exposure to cisplatin. Apoptosis analysis in the absence of CDDP indicated a good and consistent viability of all analyzed cell lines regardless of their different proliferation rate (p = 0.05) and a dramatic drop in the number of alive cells . In Tera-2 cell lines, mild increase in apoptotic response was also observed in the original CDDP-naive cells, and no change was observed in CDDP-exposed cells after incubation with CDDP.There was a notable difference in the proliferation between the two original cell lines, NCCIT showing much faster proliferation rate than Tera-2. The derived counterparts of both cell lines showed a slower proliferation rate compared with that of the parental sensitive cells, which was nonsignificant and only transient in case of NCCIT_CDDP (around day 6), but profound and permanent in case of Tera-2_CDDP a. To furion rate d. After OPN1LW gene (present in 91% of sequencing reads). Among the acquired variants found in NCCIT_CDDP cells we identified genes involved in cellular pathways crucial for survival, proliferation, metastatic dissemination as well as metabolic set up of cancer cells – e.g., genes involved in DNA damage repair (BCOR), chromatin remodeling , major signaling pathways , metabolic pathways  or maintenance of epithelial-mesenchymal balance (KDR). Interestingly, we found three different missense variants in ATRX gene acquired during the development of CDDP resistance  data of both original and derived TGCT cell lines showed a significantly increased total number of acquired gene variants in the cells that developed CDDP resistance , there PAX5, DAB2, SPINT2, TFDP1, histon methyltransferase NSD1, as well as other genes previously described to regulate proliferation (DLK1), cell signaling , metabolism , cell migration and adhesion . Altered expression was also present in PYCARD gene, which is a key mediator in apoptosis and inflammation. We also detected alteration in the expression of two homeobox genes (SIX6 and EN2).From transcriptome sequencing, a set of 50 most differentially expressed genes between CDDP-resistant (CDDP_NCCIT) and sensitive cell lines (NCCIT and both CDDP_Tera-2 and Tera-2) was identified . UnsuperSBF2/RNF141 and EML4/MTA3, resulting from an in-frame deletion and tandem duplication, respectively. Their presence was also confirmed by Sanger sequencing. In the case of the Tera-2 cell line, three in-frame fusion transcripts were found in both the original and derived counterparts. Two of them (SPG7/CDH15 and ZNHIT6/COL24A) originated from tandem duplication in the coding sequence, while in ZNF160/ZNF415 the coding sequence of one gene was translocated to the 5′ untranslated region of the second gene  analysis revealed large-scale gains and losses in all cell lines. Apart from the obligatory 12p gain, characteristic of TGCT, other CNVs could be identified particularly on chromosome 1, 20, 21 and X. Further acquired CNVs changes were observed in the NCCIT_CDDP cell line comparing with the original NCCIT counterpart  .Tumor mutation burden (TMB) was slightly higher in the CDDP-derived cells than in the original cell lines and did not differ between NCCIT and Tera-2 cell lines  or amplicon sequencing with the Comprehensive Cancer panel , together with patients’ germline DNA as a background control. TFAP2C gene, and another clinically interesting mutation with high variant frequency (30%) within TRERF1 gene. Of the other two patients, one harbored a stop/gain mutation in DOHH gene—metalloenzyme involved in lysine metabolism; and the other was detected with 7 acquired missense variants including genes RIN1—member of RAS signaling pathway, FOXG1—member of FOXO signaling pathway, and DMRTA1—transcription factor. There was no overlap in the affected genes among these 3 patients.In 3 out of 4 patients analyzed in detail by WES, significant acquired somatic gene aberrations were found . The patNSD1, encoding histone methyltransferase, that was altered in 9 of 27 (33%) patients. Interestingly, we also found variations in the SETD2 gene, another histone methyltransferase, in 3 of 27 (11%) patients. Variations in 3 other genes—FANCA, IL21R, and JAK1 were present in more than two patients. The genes with the highest variant frequency  included ATRX, ARID2, FANCA, MDM2, PDGFRA, NSD1, SETD2, and TSHR. The highest number of altered genes (15) was found in the only one patient of this cohort who died of the disease progression. The only gene that was found to be altered in relation to CDDP resistance in TGCT cell lines and was present also in TGCT patients was ATRX gene .In the larger group of patients who underwent amplicon sequencing, a number of gene aberrations were detected, some of the genes being affected in multiple patients . The mosThe comparison with clinical and laboratory data did not prove any relation between the number of detected variants and the histologic subtype (seminoma vs. nonseminoma) but there was a trend towards increasing number of variations in advanced stages of the tumor .The WES-sequenced patient samples were also analyzed for CNV and TMB. Apart from the expected 12p gain, we could observe large scale gains and losses on other chromosomes—e.g., gains on chromosome 17 in 2/4 pts . The oneThe topic of cisplatin resistance is highly relevant as it represents the leading cause of treatment failure in TGCT management. No single major aberration has been so far associated with CDDP resistance in TGCT but increasing number of potential genes, pathways, genomic and epigenetic changes have been emerging from recent experimental and descriptive studies. In order to add further information to the understanding of the basis and conditions leading to cisplatin resistance development, we established two CDDP-exposed cell lines and studied their proliferative and genetic properties. NCCIT and Tera-2 are both non-seminoma TGCT cell lines that contain embryonic carcinoma cells. NCCIT is referred to include also a teratocarcinoma component. Both cell lines showed similar initial sensitivity to CDDP. With the identical approach of intermittent CDDP exposure and resurrection periods, NCCIT developed a significant CDDP resistance  whilIt is well known that cytotoxic chemotherapy is not effective for cancer cells in the G0/G1 phase, and resistant cancer cells are prone to rest at this stage ,39,40. TATRX gene and the alterations of NSD1 gene. ATRX was the gene with the highest frequency of newly acquired mutations found in the CDDP-resistant cell line and the only one mutated also in primary TGCT tumors. This gene is located on chromosome Xq21.1 and encodes an α-thalassemia mental retardation X-linked protein, a member of the SWI/SNF superfamily of chromatin modifiers. It regulates various cellular processes such as replication stress response, transcription or mitotic recombination, and has been shown to play a critical role in sexual differentiation [ATRX sequence or alterations of its expression were found in various cancers including glioma, neuroblastoma, osteosarcoma, colorectal cancer or lung cancer [ATRX mutations, involves apart from thalassemia and mental retardation also gonadal dysgenesis of various degree, in extreme cases even male to female sex reversal [The DNA and RNA sequencing revealed two specific gene aberrations that may have a strong functional relation to TGCT and CDDP resistance development – the mutations in ntiation . Aberratg cancer . They hareversal , and as NSD1 gene were the most common aberrations detected in TGCT patients in this study including one with CDDP-resistant disease. The other CDDP-resistant patient had large scale losses including this gene region on chromosome 5, and downregulation of this gene was also found within the 50 most differentially expressed genes in CDDP resistant cell line comparing to the sensitive cells. NSD1 is located on chromosome 5q35.3, encodes nuclear receptor binding SET domain protein 1, which functions as the methyltransferase H3K36. It has been reported as an oncogene or tumor suppressor depending on cellular context; and its aberrant expression or mutations have been described previously [NSD1 are strongly associated with widespread genome hypomethylation [Mutations of eviously ,45. Altehylation . It is ihylation .ATRX and NSD1 are involved in epigenetic regulations, DNA methylation and chromatin remodeling correlates well with the current understanding to TGCT biological background, where larger genomic and epigenomic aberrations rather than point mutations are supposed to represent the crucial events in TGCT pathogenesis. In accordance with this, repression of H3K27 methylation was associated with acquired CDDP resistance in TGCT in one recent study [The fact that both altered genes nt study .FOXG1 [OPN1LW [EOMES [TFAP2C [DMRTA1 [MDM2, which is the only one gene that have been related to CDDP resistance in TGCT patients repeatedly [Of the other genes found altered in primary TGCT tumors in this study, some have been related to TGCT previously. These include genes related to TGCT occurrence in published genome wide association studies (GWAS) or other genetic association studies, such as FOXG1 , OPN1LW  [OPN1LW , EOMES [W [EOMES ,51, TFAP [TFAP2C , DMRTA1  [DMRTA1 ,53,54; apeatedly ,31,55,56SETD2 and FANCA genes. SETD2 gene, whose variants were found in 10% of patients in our cohort, is another gene with the influence on epigenetic remodeling. It is an important partner of NSD1 gene, acts as a tumor suppressor [NSD1, it has not been described in relation to TGCT, but it seems to be involved in spermatogenesis and its aberrations may be related to male infertility [FANCA gene mutations were also present in 10% of TGCT patients; while this gene is well known for its function in post-replication DNA repair and maintenance of chromosome stability, its high expression levels were found in pachytene spermatocytes (in mice) and its role in the maintenance of reproductive germ cells and in meiotic recombination is suggested [In the primary tumor samples we also identified mutations of several genes that have not been associated with TGCT so far but which role in TGCT could be implied. These include e.g., ppressor ,58,59,60ppressor . Like NSertility . FANCA guggested .MAP2K1, NSD1, TRERF1 and TFAP2C in resistant primary TGCT and several other genes, namely KDR and MAP3K4 in the resistant cell line. TFAP2C gene encodes a sequence-specific DNA-binding transcription factor, which is involved in activation of several developmental genes. It has a significant role in the development of primordial germ cells, and its altered expression was found in some cancer types, including murine germ cell tumors [TRERF1 gene encodes a basal cell cycle regulatory protein that has been shown to play a key role in development of estrogen independence/resistance in solid tumors—e.g., breast cancer [KDR gene encoding a tyrosine kinase protein is necessary for survival, proliferation, and migration of endothelial cells [MAP3K4 gene is an important component of the MAPK pathway, plays a crucial role in epithelial to mesenchymal transition, supports the male gonadal determination, and has been tested as a prognostic factor in other cancer types [CDDP resistance was in our study associated with aberrations in genes l tumors ,65,66,67t cancer . KDR genal cells , testis-al cells , also beal cells . MAP3K4 er types ,73.The signaling pathway analysis revealed an increased number of mutations in the cell cycle control and proteasome pathway in resistant NCCIT_CDDP cells, comparing with the other cell lines. This may underline the previously mentioned role of changes in DDR and cell cycle control, enabling the cell cycle arrest and repair of chemotherapy-induced adducts in the resistant cells.TGCT are tumors with quite low TMB in comparison with other solid tumors of adults. This corresponds with the generally low amount of identified gene mutations among TGCT patients. Interestingly, the CDDP-resistant patient showed an increased TMB, contrary to the remaining patients.In contrast to TMB, large CNVs are expected in TGCT and were also present in our sample cohort. As in the case of low TMB, the abundant CNVs are probably related to the TGCT origin. While the low amount of mutations detected in TGCT may be the result of embryonic DDR leading to apoptosis and not allowing for (aberrant) DNA repair, the germ cell character and abnormalities in cell divisions and mitotic/meiotic switch form strong predispositions for larger genome/chromosome rearrangements. The hallmark of TGCT—the amplification of the short arm of chromosome 12—was clearly present in all analyzed samples, both cell lines and patients. Of other CNV changes, several were present in the majority of samples, while additional ones were found in CDDP-resistant samples only. Of note, many of the newly acquired gains and losses in the resistant cell line and the resistant patient’s sample affected similar chromosomal loci. It again underlines the high functional significance of CNVs and larger genomic changes in TGCT development, only our understanding and interpretation of these very complex alterations is still limited.TGCT samples were obtained after patients’ informed consent and ethical committee  approval . Their characteristics are summarized in Two non-seminoma TGCT cell lines—NCCIT (Lot Number: 59069063) and Tera-2 (Lot Number: 4018167) were purchased from ATCC  , as described in To determine the differences in cell cycle distribution and cell proliferation between original and CDDP-treated cell lines, several staining protocols and measurements were used (details in Nucleic acids (DNA and RNA) from both the cell lines and patients’ frozen samples were isolated with DNeasy Mini Kit and RNeasy Mini Kit . DNA and RNA from FFPE samples were extracted with RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher Scientific) .The sequencing data are available at GEO, accession number GSE136560 , Prism , and R-project .50 values, which were calculated in the R-project [The resistance of the cell lines to cisplatin, as determined by MTS assay, was assessed from the IC-project  using pa-project  and furtAll group comparisons were calculated using non-parametric tests .Principal component analysis was used to determine the genetic relations among the original and derived cell lines.Kegg pathway analysis was made on WES data by counting and visualization of significant variants present in the selected group of pathways that could be assumed to be involved in TGCT development and progression.DMRTA1) or TGCT resistance (MDM2). The ATRX and NSD1 genes, newly depicted in this study, represent strong candidates for the functional role in TGCT and CDDP resistance ethiopathology, though their effect will require further evaluation. Their identification also supports the emerging role of altered epigenetic gene regulation in TGCT biologic background.In summary, our study suggests that the development of CDDP resistance in TGCT is associated with increased number of genetic aberrations on the level of SNVs/TMB as well as CNVs. Some of these aberrations may represent potential driver aberrations, while most of them may be the results of the on-going malignant transformation from a very unique type of tumor with specific embryonic and germ cell features to a malignant solid tumor with more general characteristics and higher genomic instability. Our findings support the hypothesis about the key role of a unique DDR for the high TGCT sensitivity to platinum-based chemotherapy; the acquired CDDP resistance being associated with decreased apoptotic response, changes in the cell cycle control with decreased proliferation and increased cell cycle arrest, allowing the repair of chemotherapy-induced damage. Several genes have been identified to have a potential effect on CDDP resistance development – some of them being previously referred to in relation to TGCT susceptibility (e.g.,