This approach highlighted a subgroup of genes, which value as promising candidate therapeutic targets was further confirmed by protein expression analyses. The in vitro activity of 15 inhibitor drugs against either these genes or their pathways was then analyzed, in order to identify the most active ones in terms of inherent activity and ability to overcome cisplatin resistance. NSC130813  and triptolide, both targeting NER factors, proved to be the two most active agents, without evidence of cross-resistance with cisplatin. Combined in vitro treatments showed that NSC130813 and triptolide, when administered together with cisplatin, were able to improve its efficacy in both drug-sensitive and resistant osteosarcoma cells. This evidence may indicate an interesting therapeutic future option for treatment of osteosarcoma patients who present reduced responsiveness to cisplatin, even if possible effects of additive collateral toxicities must be carefully considered. Moreover, our study also showed that targeting protein kinases belonging to the mitogen-activated protein kinase (MAPK) or fibroblast growth factor receptor (FGFR) pathways might indicate new promising therapeutic perspectives in osteosarcoma, demanding for additional investigation.Treatment of high-grade osteosarcoma, the most common malignant tumor of bone, is largely based on administration of cisplatin and other DNA damaging drugs. Altered DNA repair mechanisms may thus significantly impact on either response or resistance to chemotherapy. In this study, by using a panel of human osteosarcoma cell lines, either sensitive or resistant to cisplatin, we assessed the value as candidate therapeutic targets of DNA repair-related factors belonging to the nucleotide excision repair (NER) or base excision repair (BER) pathways, as well as of a group of 18 kinases, which expression was higher in cisplatin-resistant variants compared to their parental cell lines and may be indirectly involved in DNA repair. The causal involvement of these factors in cisplatin resistance of human osteosarcoma cells was validated through gene silencing approaches and  Osteosarcoma (OS) is the most common malignant tumor of bone, which accounts for about 5% of childhood and adolescence neoplasms. High-grade OS is usually treated with neoadjuvant chemotherapy protocols based on cisplatin (CDDP), doxorubicin, methotrexate, and ifosfamide. However, despite this aggressive approach, 35–45% of patients still recur and experience an unfavorable outcome –5.Three out of the four conventional drugs, which are most commonly used in first-line chemotherapy for high-grade OS, induce DNA damages either directly (CDDP and ifosfamide) or indirectly (doxorubicin). Therefore, resistance mechanisms related to DNA damage response can significantly impact on OS chemotherapy unresponsiveness. Among these drugs, CDDP is the agent which has most extensively been studied in relation to DNA repair. A consistent body of evidence is showing that the onset of clinical unresponsiveness to CDDP usually creates further therapeutic complications, because patients can also become cross-resistant to the other DNA damaging chemotherapeutic drugs used in first- or rescue treatment protocols , 6.ERCC excision repair 1 (ERCC1) negatively impacts on the clinical responsiveness to CDDP-based treatments and on patients' outcome  , 7, 8. W outcome . HoweverIn addition to NER, other DNA repair pathways, first of all the base excision repair (BER), have been indicated or proved to be implicated in CDDP resistance of several human tumors –12, but Cellular response to CDDP-induced DNA damage is also mediated by downstream effects on cell cycle and mitosis regulation , 11. TheBased on our previously (unpublished) gene expression analyses, we observed that CDDP-resistant human OS cell lines showed increased expression of several kinases in comparison with their corresponding parental cells. Among these kinases, 18 can be targeted by inhibitor drugs of which some have already entered clinical trials or have shown promising preclinical activities in human cancers different from OS.In this study, we first confirmed the expression level of these 18 kinases in human OS CDDP-resistant variants in comparison with their parental cell lines.Moreover, the role of genes belonging to NER or BER pathways and of the aforementioned 18 kinases for CDDP resistance in human OS cells was estimated, in order to indicate new candidate markers, which may be considered to overcome resistance to CDDP in OS patients.in vitro efficacy of drugs targeting the most significantly emerged genes or pathways has been assessed.Finally, the in vitro studies were performed on the U-2OS and Saos-2 human OS cell lines and a panel of variants resistant to CDDP .The The U-2OS and Saos-2 cell lines were purchased from the American Type Culture Collection . Variants resistant to CDDP were established by exposing the drug-sensitive U-2OS and Saos-2 parental cell lines to stepwise increasing concentrations of CDDP and characterized as previously described .DNA fingerprint analyses of 17 polymorphic short tandem repeat sequences were performed for all cell lines, confirming their identity.2 atmosphere. Drug resistant variants were continuously cultured in presence of CDDP at the concentration used for their selection.All cell lines were cultured in Iscove's modified Dulbecco's medium (IMDM), supplemented with penicillin (20 U/ml)/streptomycin (20 U/ml)  and 10% heat-inactivated fetal bovine serum , and maintained at 37°C in a humidified 5% COAnalyses focused on genes belonging to the NER and BER pathways, which are known to play key roles for CDDP resistance in several human cancers, and on the 18 druggable protein kinases selected on the basis of our previous observations, which indicated their increased expression in U-2OS- and/or Saos-2-derived CDDP-resistant variants in comparison with their parental cells . ExpressIn a first set of experiments, each gene was silenced by transfecting cells with three different siRNAs specific for different regions of the same gene  for 24 h, whereas controls were cultured in presence of scrambled siRNAs. Transfection was performed by using each siRNA at a final concentration of 5 nM and 0.3 or 1.25 μl lipofectamin RNAiMAX  per well in a 96-well or 24-well plate according to the manufacturer's protocol. After 24 h, medium was changed and cells were maintained in siRNA-free medium for additional 48 h. The extent of gene silencing was estimated at 72 h for each siRNA by qRT-PCR on the ViiA 7 instrument (Thermo Fisher Scientific) in order to identify the siRNA with the strongest effect on mRNA down-regulation. Gene expression analysis was performed using the TaqMan® Gene Expression Cells-to-CT™ Kit  and appropriate TaqMan® Gene Expression Assays (Applied Biosystems) listed in in vitro sensitivity to CDDP was estimated on the basis of drug dosage response curves, assessed by using the 3--2,5-dephenyltetrazolium bromide (MTT) assay kit . For all cell lines, the IC50 value (CDDP concentration inducing 50% growth inhibition) was determined inside each experimental condition. To quantify the extent of the increased CDDP sensitivity after gene knock-down, ratios between the IC50 values of cells incubated with scrambled siRNAs and those of silenced cells were calculated.The siRNAs producing the highest mRNA down-regulation were then selected to verify whether the inhibition of a specific gene expression was related to a corresponding increase in CDDP sensitivity. For this second set of experiments, 48 h after seeding and transfection, the cells were incubated with different dosages of CDDP for additional 48 h. Controls were incubated with scrambled siRNAs. The Cells were cultured in petri dishes until confluence, harvested by scraping and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific) and Benzonase . Protein concentrations were determined by the Bradford Protein Assay . Equal amounts of cell lysates (80 μg) were separated by SDS-PAGE on 4–20% gradient gels (Thermo Fisher Scientific) and then transferred onto nitrocellulose membranes . Then, membranes were blocked in 5% BSA in 1 X TBS containing 0.1% Tween-20  and incubated in primary antibodies  overnighProtein loading was assessed by coomassie R-250 staining (Bio-Rad). Fold changes in protein expression level were determined by densitometric analysis of western blots and autoradiographs using the publicly available ImageJ software .in vitro efficacy, are listed in the The drugs targeting the prioritized genes/pathways, which have been tested for their In vitro drug efficacy was assessed in terms of in vitro growth inhibition activity estimated with the MTT assay (as described above) on the two parental cell lines (U-2OS and Saos-2) and their CDDP-resistant variants with, respectively, the lowest and the highest resistance level . For three drugs, X80, quercetin and SSR128129E, the CellTiter-FluorTM Cell Viability assay  was used according to the manufacturer's protocol. For each cell line, the drug IC50 value was determined, in order to highlight the possible presence of cross-resistance due to the mechanisms present in CDDP-resistant variants.in vitro association of each prioritized drug with CDDP was determined after 96 h of combined treatment with the IC50 dosage of each drug. In drug sequence experiments, cell lines were sequentially exposed for 48 h to their corresponding IC50 dosage of CDDP and then to the IC50 dosage of each prioritized drug for additional 48 h. These combinations were then repeated with the opposite sequence. The type of interaction in terms of synergism, antagonism or additivity, was defined on the basis of the combination index (CI) of each two-drugs combination, which was calculated with the equation of Chou-Talalay by using the CalcuSyn software . By following the CalcuSyn software indications, the drug–drug interaction was classified as synergistic (SYN) when CI was lower than 0.90, additive (ADD) when CI ranged between 0.90 and 1.10, or antagonistic (ANT) when CI was higher than 1.10.The two most active drugs which emerged from these analyses were prioritized for further evaluations. The efficacy of the CDK3, FLT4, MAP2K3, MAP2K5, MAPK1, MAPK3, PIK3C3.Expression levels of genes listed in RNA interference was used to determine the causal involvement in CDDP resistance of the genes listed in in vitro CDDP sensitivity. Cell lines silenced with the siRNAs listed in the ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, and XPA increased CDDP sensitivity in the U-2OS/CDDP300 and U-2OS/CDDP1 μg resistant variants. Silencing of ERCC1, ERCC2/XPD, and ERCC4/XPF increased CDDP sensitivity also in U-2OS parental cellsSilencing of FGFR1 silencing increased CDDP sensitivity in the U-2OS/CDDP1 μg and in parental cellsMAP2K3, MAPK3, and PIK3CB was associated with an increase of CDDP sensitivity in the U-2OS/CDDP4 μg resistant variant.Silencing of Additional reversal activity of CDDP resistance, with IC50 ratios close to the 2.0 cut-off value, was observed for the knock-down of these and other genes , even ifERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, XPA, MAP2K3, MAPK3, and PIK3CB. FGFR1 was also selected based on its ability to reverse CDDP resistance also if its expression was found to be moderately higher than in parental cells. All these genes showed evidence of increased expression in CDDP resistant variants and their knock-down proved to be associated with an enhancement of CDDP sensitivity.By coupling the results derived from the assessment of gene expression level in association with CDDP resistance  and the To further validate these genes as candidate therapeutic targets to overcome CDDP-resistance, their expression at protein level was assessed by western blot in both U-2OS and Saos-2 parental cell lines and all their CDDP-resistant variants. All proteins encoded by these prioritized genes proved to be expressed in all cell lines. In the U-2OS series , there wWhen considered together, these results further supported the indication of all these genes as candidate drug targets.in vitro activity of drugs listed in The As shown in In U-2OS variants , higher By considering together the findings obtained in both U-2OS and Saos-2 cell line series, the most active DNA repair-targeting agents, without evidence of cross-resistance, proved to be NSC130813  and triptolide (targeting ERCC3/XPB).The most active kinase-targeting drugs, without evidence of cross-resistance with CDDP, were ulixertinib , hypotemycin (targeting the MAP2K pathway), PD173074 and FIIN-2 (both targeting FGFR1).In order to verify whether treatment with each inhibitor drug was able to increase sensitivity to CDDP, the same group of cell lines were incubated with increasing CDDP concentrations in the absence (control) or presence of the IC20 dosage of each inhibitor drug. A ratio ≥ 2.0 (meaning a decrease of at least 2-fold of CDDP-IC50 in presence of the inhibitor drug) was considered as indication of a drug-induced CDDP sensitization.NSC130813  and triptolide proved to be the two drugs with the most relevant activity, being able to increase CDDP sensitivity for more than 2-fold in all the U-2OS and Saos-2 cell lines .For all these reasons, NSC130813  and triptolide were prioritized for evaluation in combination experiments with CDDP.NSC130813  and triptolide were tested in combination with CDDP, in order to verify whether these treatments lead to positive interactions. As shown in Sequential drug exposure experiments  mainly rMany chemotherapeutic drugs, including several agents used in first-line and rescue chemotherapy protocols for OS exert their activity by directly or indirectly damaging DNA. Consequently, the ability of tumor cells to repair drug-induced DNA damages significantly impacts upon efficacy of these compounds , 10, 11.Our study focused on a group of DNA repair genes and kinases, which we found to be upregulated in our panel of CDDP-resistant human OS cell lines, in order to verify whether they could be considered as new candidate therapeutic targets. In particular, analyses focused on genes belonging to the NER and BER pathways and on 18 druggable protein kinases, which resulted to be overexpressed in association with the development of CDDP resistance.in vitro CDDP sensitivity, confirming its involvement in reduced sensitivity to this drug. The genes that emerged to be most strictly related to CDDP unresponsiveness, were prioritized as candidate targets for the second phase of the study, in which their increased protein expression was confirmed in CDDP-resistant cells, justifying the subsequent in vitro studies of drugs interacting with these markers or their pathways.After a screening to identify the most effective siRNA to knock-down each prioritized gene, drug-sensitive and resistant cell lines were silenced and treated with CDDP, in order to identify those genes which down-regulation produced a corresponding increase of the The impact of these genes for DNA repair activity in our experimental models was further confirmed by functional analyses, in which the cells' capability to repair CDDP-induced DNA damages was assessed after silencing of each prioritized gene by the COMET assay . This evin vitro activity in CDDP-resistant variants compared to parental cell lines, suggesting the presence of cross-resistance mechanisms. The possible reasons for this apparent cross-resistance were not further explored because they were beyond the aims of this study. However, it can be hypothesized that cross-resistance might be due either to differential expression of transporters that recognize these drugs as substrate and efflux them out of the cells or to detoxification processes that are more active in CDDP resistant cells and inactivate these agents. Other reasons may be the activation of alternative or redundant pathways, which replace the function of the targeted pathway in CDDP resistant cells, which consequently become less sensitive to these drugs.Drugs targeting the prioritized targets or pathways were selected on the basis of their reported promising activity in other experimental models and/or their use in clinical trials for human tumors. Among the 15 evaluated agents, TGX221, AZD6482, FR180204, AZD4547, GDC0994 and, at a lower extent, quercetin showed a reduced NSC130813  and triptolide (targeting ERCC3/XPB) proved to be the two agents with the most relevant activity on both CDDP-sensitive and -resistant cell lines. Moreover, these two drugs did not show evidence of cross-resistance with CDDP and proved to reverse CDDP resistance in all drug-sensitive and -resistant cell lines. For these reasons, they were further tested in combined treatments  with CDDP, in order to verify whether these combinations may lead to positive interactions.When considered together, results obtained by the combined treatments indicated that NSC130813  and triptolide have to be administered together with CDDP, in order to improve its efficacy in both drug-sensitive and resistant OS cells. If transferred to a clinical setting, this association has to be considered regarding possible effects of additive collateral toxicities.NSC130813 , also known as [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl]], is a compound which was shown to act synergistically with CDDP and mitomycin C by interfering DNA repair through the disruption of the interaction between ERCC1 and ERCC4/XPF . TargetiTriptolide is a diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities . In the It has been demonstrated that triptolide covalently binds to human ERCC3/XPB, inhibiting its DNA repair-related activity , 24, 25.On the basis of this body of evidence, we have explored whether in OS cells triptolide-mediated inhibition of NER may improve CDDP activity. Our findings indicated that inhibiting DNA repair through the simultaneous administration of CDDP and triptolide may be a new interesting treatment avenue to overcome CDDP resistance in OS.ClinicalTrials.gov Identifier: NCT03117920), acute myeloid leukemia , and different advanced solid tumors , but results of these regimens are presently not available. Moreover, other triptolide derivatives and analogs have been used in clinical studies aimed to test their efficacy and safety  and PD173074 (targeting FGFR1) showed some promising activity, without evidence of cross-resistance with CDDP. Although these drugs were not further analyzed in combined treatments with CDDP, the obtained findings suggest that targeting protein kinases that influence DNA repair activities may indicate new promising therapeutic perspectives in OS, demanding for additional investigation. This perspective is particularly interesting because there are many protein kinase inhibitors in various stages of clinical development worldwide and the majority of them are used for cancer treatment  models may further support their clinical use. All these activities are presently planned and will focus on the drugs screened and highlighted by this study.Our results can also be the basis for further In high-grade OS, when patients fail to respond to first-line treatment and relapse, therapeutic options and drugs effective for rescue chemotherapy protocols are scarce, also because resistance mechanisms developed against first-line chemotherapeutic drugs can also be responsible for reduced responsiveness to the agents used in the subsequent regimens.Inhibition of DNA repair can be considered as a promising treatment strategy to enhance the efficacy of currently available DNA damaging drugs.There are several genes and proteins involved in modulating the cellular response to DNA damage, each one may serve as target to enhance the efficacy of conventional therapeutic modalities. The current efforts in the development and deployment of several classes of DNA repair targeting compounds justify the hope to achieve new tailored treatment approaches through the use of these inhibitor drugs, which may ultimately drive toward innovative regimens aimed to improve patient outcomes.The evidence emerged within this study about the possibility of successfully combining CDDP with drugs targeting DNA repair factors or protein kinases involved in these processes may indeed indicate new therapeutic options for specific OS patient cohorts, who have reduced cure probabilities.Datasets generated and analyzed in this study will be made available by the authors to qualified researchers, upon justified request.in vitro drug testing. ET, MF, SV, and AF-R performed the gene silencing screening. ET and MF carried out the DNA damage evaluations. ET, MF, CH, and SL took part in the molecular biology and western blot studies. ET, MF, CH, MP, and MS performed the statistical analyses. All authors have made a substantial, direct, and intellectual contribution to the work.ET, MF, CH, and MS designed the study. MS coordinated the study and drafted the manuscript, which was revised by all authors, who approved it for publication. ET, MF, AF-R, MP, and FM carried out the cell culture-based experiments and the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. CH is Associate Editor of the section Pharmacogenetics and Pharmacogenomics of Frontiers in Pharmacology.


CPT2, LDHA, MCM2, and SKP2, in both BrCa and PCa cell lines. Hence, [C16Pyr][Amp] was shown to be a promising anticancer therapeutic agent for BrCa and PCa cell lines.Breast (BrCa) and prostate (PCa) cancers are the most common malignancies in women and men, respectively. The available therapeutic options for these tumors are still not curative and have severe side effects. Therefore, there is an urgent need for more effective antineoplastic agents. Herein, BrCa, PCa, and benign cell lines were treated with two ionic liquids and two quinoxalines and functional experiments were performed—namely cell viability, apoptosis, cytotoxicity, and colony formation assays. At the molecular level, an array of gene expressions encompassing several molecular pathways were used to explore the impact of treatment on gene expression. Although both quinoxalines and the ionic liquid [C2OHMIM][Amp] did not show any effect on the BrCa and PCa cell lines, [C16Pyr][Amp] significantly decreased cell viability and colony formation ability, while it increased the apoptosis levels of all cell lines. Importantly, [C16Pyr][Amp] was found to be more selective for cancer cells and less toxic than cisplatin. At the molecular level, this ionic liquid was also associated with reduced expression levels of  Cancer is one of the leading causes of disease-related death worldwide. In particular, breast (BrCa) and prostate (PCa) cancer are the first and second most common cancers in women and men, respectively . ConcernIonic liquids (ILs) and quinoxalines have emerged has potential anticancer drugs ,13. ILs Quinoxalines are chemical compounds whose structures are similar to quinolone antibiotics. Quinoxaline derivates are synthetic heterocyclic compounds formed by the fusion of two aromatic rings, i.e., benzene and pyrazine, with a nitrogen atom replacing carbons in the naphthalene ring . AlthougConsidering the promising applications of both ILs and quinoxalines in biomedical research and the pharmaceutical industry, we aimed to investigate the effect of these synthetic compounds—in particular, two ILs, one with imidazolium ([C2OHMIM][Amp]) and the other with pyridinium ([C16Pyr][Amp]) cations, as well as quinoxaline-1,4-dioxide and 2-methylquinoxaline-1,4-dioxide—in BrCa and PCa cell lines with a malignant phenotype. These four compounds were chosen as they have shown promising results in previous publications ,22.Ten immortalized epithelial cell lines from the breast and prostate were used to assess the impact of four compounds—namely two quinoxalines  and two ILs based on ampicillin . The BrCa cell lines used were HTB22 (MCF-7), HTB133 (T-47D), HCC1937, and MDA-MB-231, while 22Rv1, LNCaP, Du145, and PC-3 were the PCa cell lines chosen. The remaining two cell lines were benign and used as controls: MCF-10A (breast) and RWPE-1 (prostate).50) of each cell line and the dose–response curves were evaluated with 3--2,5-diphenyltetrazolium-bromide (MTT) assay. The IC50 of the compounds was determined by exposing each cell line to different concentrations of the compounds . Considering these results, phenotypic assays were only performed with the IL [C16Pyr][Amp], at both the IC50 and at the double IC50 concentrations for each cell line.For each compound, the half-maximal inhibitory concentration  in both BrCa and PCa cell lines  than in tumor cells, indicating that cisplatin is non-specific for tumor cells and induces toxicity to normal cells [Amp] presented the highest SI value for MDA-MB-231 (SI = 11.00). This value is 2.5-fold the SI value found for cisplatin, which is a promising result since MDA-MB-231 is an aggressive cell line derived from a metastatic site of pleural effusion. Similarly, although to a lower extent, the SI values of [C16Pyr][Amp] for HTB22 and HCC1937 were also remarkable. Compared to cisplatin, the SI value of [C16Pyr][Amp] was 8-fold higher in HTB22. Concerning the PCa cell lines, [C16Pyr][Amp] exhibited the greatest SI value in LNCaP (SI = 7.16), which is approximately 11-fold the value obtained for cisplatin. Importantly, in PC-3, this compound also presented an interesting SI (SI = 2.63), especially because of the metastatic and castration-resistant nature of this cell line. Hence, [C16Pyr][Amp] might be a potential compound for clinical purposes, with the highest selectivity for tumor cells without significantly impairing normal cell lines when compared to cisplatin.Knowing the relevance of this chemotherapeutic agent in the clinic, we used this drug as a control. In our study, [C16Pyr][Amp] presented lower ICll lines . Moreoveal cells ,39,40. IOverall, a time- and dose-dependent reduction in the viability of BrCa and PCa cells was observed upon [C16Pyr][Amp] treatment . The decThese results were paralleled by those obtained in the analysis of the resistance to apoptosis. Indeed, [C16Pyr][Amp] significantly increased apoptotic levels in a dose-dependent manner in all cancer and benign cell lines . The HTBRegarding the cytotoxic effect, [C16Pyr][Amp] promoted a slight lactate dehydrogenase (LDH) increase in the medium (data not shown) comparative to the control condition, which is indicative of necrosis and cytotoxicity due to a damaged plasma membrane . Herein,The in vitro colony formation assay is based on the ability of a single malignant cell to grow and divide, thereby forming colonies, contrarily to healthy cells . All celCPT2, LDHA, MCM2, and SKP2 . Concerning the MCM2 gene, a higher difference was depicted in HTB133  and MDA-MB-231 , although 22Rv1 also presented a statistically significant decrease. Both the LDHA and SKP2 genes also depicted significantly decreased expression in the BrCa cell lines, SKP2 being also significantly downregulated in both PCa cell lines, although to a lower extent. Curiously, the hormone-dependent cell lines (HTB133 and 22Rv1) depicted a more impressive decreased expression of these genes compared to the respective BrCa hormone-independent and PCa castration-resistant (MDA-MB-231 and Du145) cell lines.Considering the antitumor activity of [C16Pyr][Amp] exhibited in the functional assays, the main cellular pathways possibly affected by [C16Pyr][Amp] treatment were assessed through the identification of altered gene expression that could corroborate the anticancer effects shown. For this purpose, the cell lines that best responded to treatment with [C16Pyr][Amp], identified in the previous trials and representative of the wide range of breast and prostate tumors, were chosen. Specifically, two hormone-dependent (HTB133 and 22Rv1) and two hormone-independent BrCa and castration-resistant PCa (MDA-MB-231 and Du145) cells were treated with the highest [C16Pyr][Amp] concentration and further tested via transcript analysis. A commercial custom array panel of cancer research was used to evaluate the expression of several genes involved in cell cycle, apoptosis, DNA repair, cellular metabolism, and mTOR or MAPK/ERK pathways. From the 96 genes analyzed in the array, four were downregulated in the four tested cell lines upon [C16Pyr][Amp] treatment: and SKP2 . The CTPDespite technological and social development, cancer remains an important cause of morbidity and mortality. The global increment in cancer incidence and mortality rates, among several other complex reasons, is a consequence of demographic factors  and socioeconomic development, which alter the prevalence and distribution of cancer risk factors . BrCa an50 determination, demonstrated that this compound has potent antiproliferative activity against five different human cancer cell lines, but with no exploration of the functional and genetic alterations of the cells treated. In that study, [C16Pyr][Amp] proved to be active in doses between 0.005 and 132.700 µM [Amp][Amp] in BrCa and PCa cell lines. These lower concentrations are possibly related to the hydrophobic nature of the [C16Pyr] cation conferred by the alkyl side chain [Amp] and [C16Pyr][Amp], in BrCa and PCa cell lines. These compounds were previously reported to display a growth inhibitory effect in HTB133 and PC-3 cancer cells . AlthougMIM][Amp], in thistabolism  and, conde chain . ILs arede chain . This inde chain . Indeed,de chain ,54. [Amp] formulation in inhibiting or attenuating the aggressive features of cancer cells. Overall, this compound was effective in attenuating the malignant phenotype of cancer cells by reducing cell viability and colony formation ability while inducing apoptosis. Moreover, the SI of [C16Pyr][Amp] revealed selectivity toward the majority of the tested tumor cells, mainly for MDA-MB-231 (SI = 11.00) and LNCaP (SI = 7.16), contrarily to the cisplatin SI values. This is a rather promising result, mainly for MDA-MB-231, since it is an aggressive cell line derived from a metastasis of pleural effusion. Moreover, [C16Pyr][Amp] presented a 2–11-fold augmentation of the SI values compared to cisplatin, a drug commonly used in the clinic, thus suggesting the higher selectivity of [C16Pyr][Amp] than cisplatin. In general, cancer is mainly treated with long-term intensive chemotherapy sessions to shrink the tumor so that it can effectively be removed by surgery, if necessary. However, chemotherapy is a highly debilitating therapeutic approach for patients, with severe side effects due to the lack of selectivity toward tumor cells. Despite its cytotoxicity, cisplatin is a widely known chemotherapeutic agent frequently used in the clinic, as well as docetaxel, doxorubicin, cyclophosphamide, and paclitaxel, already reported in BrCa and PCa cell lines . In ordeThe MTT assay is among the most commonly used methods to evaluate the cytotoxicity of a chemical through the analysis of the capacity of mitochondrial enzymes to reduce the tetrazolium dye MTT to formazan crystals ,56. HoweKaushik et al. reported that ammonium and imidazolium ILs also inhibit the colony formation capability of brain cancer cells in a concentration-dependent manner . Herein,CPT2, LDHA, MCM2, and SKP2 genes upon treatment with [C16Pyr][Amp]. These genes are mainly involved in cellular metabolism and cell division and their decreased expression corroborates the exacerbated effects observed on cell viability and apoptosis after [C16Pyr][Amp] exposure. CPT2 encodes a mitochondrial enzyme involved in lipid metabolism, regulating fatty acid oxidation (FAO) in normal cells [Amp], a gene expression panel of 96 genes was analyzed and revealed downregulation of the al cells . In cancal cells . The stral cells . Indeed,al cells . [Amp] exposure paralleled the reduced cell proliferation found in in vitro assays. Indeed, MCM2 has been proposed to be an alternative proliferation marker to ki67 in the BrCa model [Amp] treatment suggests a role of this IL in DNA replication and cell cycle rate reduction in both BrCa and PCa cell lines.[C16Pyr][Amp] treatment of the BrCa and PCa cell lines also significantly downregulated nwinding ,78. MCM2nwinding ,80. Morenwinding  and prednwinding . Herein,Ca model . Both MC samples , showing kinases ,86. SKP2 kinases ,88,89,90 kinases . Accordiechanism . Hence, ctivated . In the Our study demonstrated that [C16Pyr][Amp] has potential as an anticancer therapeutic agent in BrCa and PCa cell lines with disparate phenotypes. The tumorigenic features of these cell lines were impaired upon [C16Pyr][Amp] treatment, leading to a decreased number of malignant viable cells, accompanied by increased cell death, mainly by apoptosis. The aggressiveness of malignant cells was also quantitatively diminished, herein reflected by a reduction in colony formation capacity. At the molecular level, downregulation of the genes involved in two well-known hallmarks of cancer, namely cellular energetics and sustaining the proliferative signaling in the context of DNA replication and cell cycle regulation, might contribute to the malignant behavior attenuation of cell lines upon [C16Pyr][Amp] treatment in phenotypic assays. The selectivity and lower toxicity of [C16Pyr][Amp] compared to cisplatin render it a promising therapeutic agent that might be administered alone or in combination with the currently used conventional therapies. In vivo studies are needed to further evaluate the antitumor effect of C16, as well as to identify targetable proteins that may serve as surrogate markers of therapy response in BrCa and PCa patients.v/v) was used to culture the PC-3 cell line; Keratinocyte Serum-Free Medium (SFM) was used to culture the RWPE-1 cell line (Gibco). Specifically, MCF-10A was cultured with DMEM/F12  supplemented with 5% horse serum (Gibco), 20 ng/mL of Epidermal Growth Factor (EGF) , 0.5 mg/mL of hydrocortisone , 100 ng/mL of cholera toxin (Sigma-Aldrich), 10 µg/mL of insulin (Sigma-Aldrich), and 1% penicillin–streptomycin (Gibco). All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and routinely tested for Mycoplasma spp. contamination .Ten American Type Culture Collection (ATCC)  immortalized epithelial cell lines available at the Instituto Português de Oncologia do Porto laboratory were cultured in the recommended medium supplemented with 10% fetal bovine serum (FBS)  and a 1% antibiotic–antimycotic (Anti–Anti (100×), Gibco, Waltham, MA, USA). The cancer cell lines chosen present distinct features, covering a large spectrum of breast and prostate tumors, while the benign cell lines were used as controls. Briefly, minimum essential media (MEM) was used to culture the HTB22 and Du145 cell lines; Roswell Park Memorial Institute (RPMI)-1640 was used to culture the HTB133, HCC1937, 22Rv1, and LNCaP cell lines; Dulbecco’s Modified Eagle Medium (DMEM) was used to culture the MDA-MB-231 cell line; RPMI-1640/Ham’s F-12 Nutrient Mixture  and two ILs ([C16Pyr][Amp] (C16) and [C2OHMIM][Amp]) based on ampicillin. The quinoxaline derivates were purified by reduced pressure sublimation, and thermal stability was verified by differential scanning calorimetry (DSC), as previously described ,94. The 50 value and the dose–response curves of each compound were determined with an MTT assay. Cells were seeded onto 96-well flat-bottomed culture plates at 2 × 103 (MCF-10A and Du145), 7 × 103 (HTB22), 2.5 × 103 , 4 × 103 (22Rv1), 5 × 103 (LNCaP), and 1.6 × 103 (PC-3) cells per well. The IC50 value was determined using nonlinear regression (curve fit) with all logarithmic absorbance values. For control purposes, cell lines were also exposed to the compounds’ vehicles, i.e., purified water and DMSO for the quinoxalines and ILs, respectively. To assess the toxicity of the compounds, cisplatin was used as a control. All cells were treated 24 h after being cultured (day 0) and were evaluated with 72 h of exposure to the compounds (day 3). The IC50 values of cisplatin were calculated for all cell lines after 72 h with concentrations of 50 nM, 500 nM, 5 µM, 50 µM, and 100 µM, considering the IC50 values previously described in the literature [Amp] and cisplatin was carried out using Wilcoxon matched pair tests and Pearson’s correlations. For RNA expression in the RT2 profiler PCR array, p-values were calculated based on a Student’s t-test of the replicate 2(−ΔΔCT) values for each gene in the control and treatment groups. Analysis was performed with GraphPad Prism 7, and statistical significance was set at p < 0.05.One-way analysis of variance (ANOVA) with the post-hoc Dunnett’s multiple comparison test was used to compare the results obtained in each parameter for the different compounds concentrations and the control/vehicle, when appropriate. Comparison of IC


Canonical Wnt/ β-catenin pathway is one mechanism being activated in platinum-resistant epithelial ovarian cancer (EOC). Detecting potential targets for Wnt pathway modulation as a putative future therapeutic approach was the aim of this study.Biological effects of different Wnt modulators  on the EOC cell lines A2780 and its platinum-resistant clone A2780cis were investigated via multiple functional tests. Immunohistochemistry (IHC) was carried out to compare the expression levels of Wnt marker proteins  in patient specimens and to correlate them with lifetime data.We could show that activated Wnt signaling of the platinum-resistant EOC cell line A2780cis can be reversed by Wnt manipulators through SB216763 or XAV939. All Wnt manipulators tested consecutively decreased cell proliferation and cell viability. Apoptosis of A2780 and A2780cis was enhanced by triptolide in a dose-dependent manner, whereas cell migration was inhibited by SB216763 and triptolide. IHC analyses elucidated significantly different expression patterns for Wnt markers in the serous subtype. Herein, higher plasmatic snail/ slug expression is associated with improved progression-free (PFS) and overall survival (OS).According to the described effects on EOC biology, all three Wnt manipulators seem to have the potential to augment the impact of a platinum-based chemotherapy in EOC. This is promising as a dominance of this pathway was confirmed in serous histology.The online version of this article (10.1007/s00432-020-03317-4) contains supplementary material, which is available to authorized users. Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and the seventh most common cancer in women worldwide  were cultured in Roswell Park Memorial Institute 1640 medium with GlutaMAX  supplemented with 10% fetal bovine serum  at 37 °C in the presence of 5% COXAV939 is a small molecule inhibitor of Wnt signaling pathway. It acts through binding and inhibiting tankyrase's catalytic poly ADP ribose polymerase (PARP) domain, which usually destabilizes axin as part of the destruction complex  and following this, in contrast to XAV939, an activator of the Wnt/ β-catenin pathway  were seeded in 96-well plates overnight and afterwards incubated with different concentrations of SB216763  , XAV939  (Sigma-Aldrich) and triptolide  (Sigma-Aldrich).5 × 10S-phase-dependent synthesis of DNA during the cell cycle and, therefore, cellular proliferation was analyzed with thymidine analog 5-bromo-2′-deoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA) after 72 h. Cell viability was determined using a 3--2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) colorimetric assay after 48 h and 72 h. The procedures of both techniques were already reported by our colleagues , cells treated with 2‰ (for incubation with SB216763) and 5‰ (for incubation with XAV939) dimethyl sulfoxide (DMSO) were carried out. For the evaluation the control without DMSO was set 100%.The M30 CytoDeath apoptosis assay is used for determination of early apoptosis. A specific epitope of cytokeratin 18, which is presented after cleavage by caspases during apoptosis, is detected. OC cells were grown on microscope slides to subconfluency, incubated for 48 h in the presence of triptolide at different concentrations , fixed and stored at − 20 °C. After thawing, washing and incubation with M30 CytoDEATH antibody   according to the manufacturer's protocol. Following induced cell death, mouse monoclonal antibodies bind against cytoplasmic histone-associated DNA-fragments (mono- and oligonucleosomes). In brief, 5 × 104 cells/well were grown overnight in 96-well culture plates and then incubated with 6.25 nM, 12.5 nM, 25 nM and 50 nM of triptolide for 24 h. Details on the further procedure can be found in Geiger et al.  for 5 min and methanol for 5 min, followed by freezing at − 20 °C. Manufactured slides were treated with anti-β-catenin IgG  . Significant differences to controls were determined by one-way ANOVA followed by Dunnett's multiple comparisons test or two-way ANOVA followed by Sidak's multiple comparisons test. A probability of n = 109], endometrioid [n = 21], clear cell [n = 11], mucinous [n = 12]) who underwent radical cytoreductive surgery in our department between 1990 and 2002. Histopathological diagnoses were established by a specialized gynecologic pathologist with staging and grading according to TNM and FIGO (WHO) classification. 75.2% of patients presented with advanced disease (FIGO IIB-IV), while only 24.8% were diagnosed in early disease (FIGO I-IIA). Except for patients in stage FIGO IA with low-grade histology, all patients received adjuvant platinum-based chemotherapy. Lifetime data  from EOC patients were taken from our patient charts, the Munich Cancer Registry and aftercare calendars. Median age at primary diagnosis was 59.0 years with a 95% confidence interval (CI) of 57.0–61.0 years. 28 relapses and 101 deaths were documented. A summary of patient characteristics can be found in Table Specimens represent a cohort of 153 patients with EOC  using a microtissue arrayer . Every tumor sample was used for three biopsies, resulting in 459 TMAs in total. Afterwards, sections of 5 µm were prepared and transferred to microscope slides. To determine whether there was enough representative tumor tissue left, a haematoxylin and eosin stain was done.IHC was performed using a combination of pressure cooker heating and the ZytoChem-Plus HRP Polymer-Kit with DAB as chromogenic substrate according to a previous publication by our lab . Furthermore, expression-dependent differences in overall survival (OS) (median ± SEM) and progression-free survival (PFS) (median ± SEM) were tested by chi-square statistic of the Log-Rank test (Mantel-Cox) in Kaplan–Meier curves with SPSS Statistics 25 . P-values < 0.05 were considered to be statistically significant for further analyses.Mean values of the three representative IHC scores of every probe were calculated for further analysis. GraphPad Prism 8 was used for the comparison of immunoquantitation between histological subtypes, FIGO stages and platinum-response (< 6 months to primary surgery)/ -sensitive (≥ 6 month to primary surgery) with Mann–Whitney-p < 0.05 or p < 0.001) shift from ß-catenin staining from the nucleus to the membrane was noted following SB216763 (100 µM) and XAV939 (50 µM) treatment in comparison to controls for both cell lines A2780 and A2780cis. The biggest impact presented XAV939 treatment in A2780cis, where the cell proportion with nuclear ß-catenin localization decreased from 99.1% (control with 5‰ DMSO) to 25.2% (p < 0.001)  impairment of proliferation in A2780 (17.1%) and A2780cis (8.9%). While XAV939 (50 µM) showed no influence in both cell lines, SB216763 (100 µM) also significantly (p < 0.05) inhibited proliferation in A2780cis (84.5%) , we compared the highest concentrations of each inhibitor against its control. Controls for triptolide without DMSO were set 100.0%. After 72 h, triptolide (50 nM) displayed a significant (p < 0.01 or p < 0.001) at the selected concentrations of 100 µM SB216763, 50 µM XAV939 and 50 nM triptolide . But in contrast to cell proliferation assay, metabolic activity decreased for all three inhibitors in platinum-sensitive  and -resistant cells  in MTT assay. Except for SB216763 and XAV939 in A2780cis, these findings were highly significant (p < 0.001) in a dose-dependent manner up to 95.4% for A2780 and to 53.1% for A2780cis, respectively. Controls with 0‰ DMSO showed 100.0% M30 CytoDEATH negative cells  increased significantly (p < 0.01 or p < 0.001) dose-dependent rise of absorbance up to 8.9-times in A2780 and 2.7-times in A2780cis at 50 nM   in endometrioid , clear cell  and mucinous  type compared to serous  histology. Moreover, in comparison to serous subtype, expression of plasmatic snail/ slug (8.8 ± 0.2) and membranous E-cadherin (7.6 ± 0.3) was significantly lower in endometrioid  and clear cell  histology. Representative stainings are displayed in Fig. To understand the role of Wnt pathway in the clinical context, immunoquantitative comparisons of Wnt signaling markers among four histological subtypes  were performed. IHC score revealed multiple significant expression differences especially of serous histology compared to other subtypes Table . Membranp < 0.01]), ß-catenin (plasma) (8.8 ± 0.4 to 6.7 ± 0.3 [p < 0.001]) and E-cadherin (plasma) (7.7 ± 0.5 to 6.8 ± 0.3 [p < 0.05]) were noted for FIGO stage I-II compared to FIGO stage III-IV  (9.2 ± 0.4 to 7.9 ± 0.2 [No significant expression differences were identified for Wnt markers in platinum-resistant compared to platinum-sensitive patients (data not shown).p = 0.001]). A similar trend was noted for OS, although this difference did not reach statistical significance (50.4 ± 13.2 to 27.6 ± 3.6 months [p = 0.058])  expression with a cut-off IHC score of seven or beneath  is a key mechanism in cell migration and Wnt signaling is one of EMT's major pathways  after SB216763 or XAV939 treatment in both cell lines. In parallel to the functional tests, both drugs led to an immunohistochemical shift of β-catenin from the nucleus  to the membrane , which finally contrasts the general role of SB216763 as an activator of the pathway Supp. 1 BrdU cell proliferation and MTT cell viability assay for different Wnt signaling manipulators in different concentrations in A2780 and A2780cis after 72h: (a) In the BrdU assay only triptolide displayed a dose-dependent decrease of cell proliferation in A2780 and A2780cis, whereas SB216763 and XAV939 showed no dose-dependency . (b) In the MTT assay, according to the findings in the BrdU assay, only triptolide displayed a dose-dependent decrease of cell metabolic activity in A2780 and A2780cis, whereas SB216763 and XAV939 showed no dose-dependency Below is the link to the electronic supplementary material.


Uveal melanoma is a rare intraocular malignancy. Half of the patients diagnosed will develop metastases within 10 to 30 years, most commonly in the liver. Although there has been a significant development in the treatment of melanoma, no effective treatment to prevent or treat metastases of uveal melanoma is available. Oncolytic viruses are now being studied for various types of cancers and show promising results. Preclinical results show cytolytic activity of enteric cytopathic human orphan virus type 7 (ECHO-7) strain Rigvir in human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma and pancreas adenocarcinoma cell lines. The aim of this study was to test the possible cytolytic activity in human uveal melanoma cell lines.The results suggest cytolytic activity of oncolytic ECHO-7 virus strain Rigvir in MP41, 92-1 and Mel-202 cell lines. While uveal melanoma is a rare disease it is one of the most commonly diagnosed intraocular malignancies, which comprises about 5% of all melanoma cases reported . Most ofBRAF mutations [GNAQ and GNA11 [BAP1 gene shows inactivating mutations in 85% of aggressive tumours and is considered to be a marker of metastatic disease [Cutaneous and uveal melanoma both arise from melanocytes, however, genetically they are different. In cutaneous melanoma most of the mutations concern the mitogen-activated protein kinase pathway (MAPK). This pathway is responsible for cell growth, differentiation and survival. The mutations altering this pathway most commonly are found in the serine/threonine-protein kinase B-Raf (BRAF) kinase; 40–50% of cutaneous melanoma have nd GNA11 . These gnd GNA11 . The tum disease .Due to the distinctive genetic features of uveal and cutaneous melanoma, specific model systems for each should be used in preclinical studies and active substance testing. Until recently, there was a lack of cell models for uveal melanoma research because most of the available cell lines were from cutaneous melanoma. However, recently a couple of uveal melanoma cell lines have been developed . They inCytolytic effect of the present ECHO-7 virus strain has been observed in human melanoma, rhabdomyosarcoma, gastric adenocarcinoma, lung carcinoma and pancreas adenocarcinoma cell lines . CurrentThe oncolytic potential of Rigvir has been observed in several cancer cell lines. Therefore, the experiments were extended to examine the effect on cell viability of oncolytic ECHO-7 virus strain on uveal melanoma cell lines. The following seven cell lines were tested: Mel–202, MP41, MP38, MP65, MP46, MM28 and 92-1.Only 3 of the seven uveal melanoma cell lines could be successfully propagated in the laboratory, Mel-202, MP41 and 92-1, and used in the analysis. The results indicate that Rigvir had cytolytic effect in Mel-202, MP41 and 92-1 uveal melanoma cell lines.2 growth conditions.Cell lines MP38 , MP65 (ATCC CRL-3299), MP41 (ATCC CRL-3297), MP46 (ATCC CRL-3298), MM28 (ATCC CRL-3295) were obtained from ATCC, and 92-1  and Mel-202 (cat.no. CLS3527) were obtained from Sigma-Aldrich. All cell lines are of human origin. The cell lines were cultured in Roswell Park Memorial Institute Medium (RPMI) + 20% foetal bovine serum (FBS) + 1% penicillin/streptomycin under 37 °C, 5% CO6 TCID50/ml) was obtained from the manufacturer.ECHO-7 virus strain  was treated with gamma irradiation; the measured absorbed dose in the samples was 41.6 -43.3 kGy.2 in a 24 well plate. After 24 h cultivation (to ensure complete cell attachment to plate surface) the medium was renewed.10,000 cells for culture 92-1 and MP41, 30,000 cells for Mel-202 were seeded with culture medium (RPMI + 20% FBS + 1% penicillin/streptomycin) per 1 cmTwo concentrations of ECHO-7 virus Rigvir strain were used: The number of viral particles added was multiplicity of infection (MOI) 7 and 70 (1% and 10% (v/v), respectively). Also, two concentrations of inactivated ECHO-7 virus strain were added: MOI of 7 and MOI of 70 as negative control. An equal volume of medium (without virus) was added to the control cells. Each test group was run in triplicate (n = 3).Cell proliferation was monitored for 96 h with a live cell imaging system . Phase contrast microscopy images were taken after 0, 3, 6, 12, 24, 48, 72 and 96 h. The live cell imaging system was set to recognize differences in cell population and morphology of different cell growth phases.The inhibitory effect of ECHO-7 virus Rigvir strain was calculated at the end of culturing time (96 h), using the formula: Inhibition (%) = 100-(100xA/B), where A stands for cell number with Rigvir at the end of culturing time, and B is control  the cell number at the end of the culturing time. GraphPad Prism 8 was used for two-way ANOVA statistical analysis. The difference was taken as statistically significantly different when P < 0.05.The results show that MP41, Mel-202 and 92-1 cell line cells detached from the cultivation surface when ECHO-7 virus Rigvir strain was added to the medium. Control samples with PBS and inactivated Rigvir continued to proliferate. This observation indicates a cytolytic/cytotoxic effect of Rigvir in these three cell lines  was 86.4% and by MOI of 70 ECHO-7 viral particles it was 85.9% Fig. a.92-1 cells treated with MOI of 70 Rigvir particles also showed earlier cytotoxic response than for the MOI of 7 treatment group. The maximal inhibition of cell growth by MOI of 7 Rigvir compared to control (PBS) was 92.4, and by MOI of 70 it was 95.5%  was 99.3% and by MOI of 70 it was 99.4%  and Gα11 (encoded by GNA11) having 90% homologous amino acid sequences [The present results suggest that of ECHO-7 virus strain Rigvir has cytolytic activity in 92-1, MP41 and Mel-202 cell lines. These cell lines originate from primary uveal melanoma tumours, possess a loss of heterozygosity of chromosome 3, and show BAP1 protein expression. The MP41 cell line has a mutation in GNA11 (c.626 A > A/T), the 92-1 cell line has mutations in GNAQ (c.626 A > T) and EIF1AX (c.17 G/A). The Mel-202 cell line has mutations in GNAQ (c. 629 G > A) and SF3B1 (c.1793 C > T) . These mequences .The results suggest a cytolytic effect of Rigvir in the MP41, Mel-202 and 92-1 human uveal melanoma cell lines. Currently there is no treatment to prevent or treat metastasis in the uveal melanoma patients and it is crucial that new treatment for this type of cancer are being sought.The results show that Rigvir decreases uveal melanoma cell proliferation under in vitro conditions; further experiments are necessary to elucidate if Rigvir oncolytic virus would also have clinical effect in uveal melanoma patients.This study is limited by the three uveal cell lines that were propagated in the laboratory. In order to use a live cell imaging system and capture the cytolytic effect, a certain cell layer density should be reached. Four of the cell lines have long cell doubling times (up to 96 h) that limited their use in the real time imaging system. Further studies should involve 3D cell models; however, a majority of the commercially available cell lines unfortunately do not form even cell monolayers.


Ovarian clear cell carcinoma (OCCC) is a rare subtype of gynecological cancer for which well-characterized and authenticated model systems are scarce. We provide an extensive characterization of ‘105C’, a cell line generated from an adenocarcinoma of the clear cell histotype using targeted next-generation sequencing, cytogenetic microarrays, along with analyses of AKT/mTOR signaling. We report that that the 105C cell line is a bona fide OCCC cell line, carrying PIK3CA, PTEN, and ARID1A gene mutations, consistent with OCCC, yet maintain a stable genome as reflected by low copy number variation. Unlike KOC-7c, TOV-21G, and RMG-V OCCC lines also mutated for the above genes, the 105C cells do not carry mutations in mismatch repair genes. Importantly, we show that 105C cells exhibit greater resistance to mTOR inhibition and carboplatin treatment compared to 9 other OCCC cell lines in 3D spheroid cultures. This resistance may be attributed to 105C cells remaining dormant in suspension culture which surprisingly, contrasts with several other OCCC lines which continue to proliferate in long-term suspension culture. 105C cells survive xenotransplantation but do not proliferate and metastasize. Collectively, we show that the 105C OCCC cell line exhibits unique properties useful for the pre-clinical investigation of OCCC pathobiology. Ovarian clear cell cancer (OCCC) is a histotype of epithelial ovarian cancer (EOC) that accounts for 5–10% of all ovarian cancers diagnosed . HistoloOCCC patients make up to 26% stage I/II ovarian cancer patients and show a good outcome ,8,9. HowIn this study, we characterize the 105C cell line that was originally derived from the ascites of an ovarian cancer patient and identified as “adenocarcinoma of the ovary with clear cell differentiation” . This hiCell lines were cultured in Dulbecco’s modified Eagle medium/Ham’s F12 (Wisent) and supplemented with 10% FBS (Wisent). The 105C cell line  was obtained from Dr. Hal Hirte . The KOC-7c, RMG-I, SMOV-2, OVMANA, OVTOKO, TU-OC-1, and OVSAYO cell lines were provided by Dr. Hiroaki Itamochi . TOV-21G cell line was provided by Dr. Anne-Marie Mes-Masson . ES-2 cell line was kindly provided by Dr. Barbara Vanderhyden .2 concentration of 5%. For experiments requiring spheroid formation ultra-low attachment plates  were used. At the time of experimentation, cell lines were positively identified using short tandem repeat (STR) analysis performed at the Centre for Applied Genomics (TCAG) in Toronto, Ontario, Canada and cross referenced to STR fingerprints in the CLASTR 1.4.4 database (https://web.expasy.org/cellosaurus-str-search/) (Cells were maintained in at 37 °C and a COsearch/) . Cells ip-value : >0.01 filtered out). In addition to the Sunnybrook Genomics Core Facility Standard Filter, we removed any hits where the number of sequence reads mutated were less than 30% of total reads obtained. Data accession number GSE160571.To determine the mutational profile of the 105C cell line we used the Oncomine Comprehensive v3 assay . Genomic DNA obtained from 105C cell line was sent to the Sunnybrook Research Institute Genomics Core Facility in Toronto, Ontario, Canada where the assay was performed according to the manufacturer’s protocol. The data was analyzed by the Sunnybrook Genomics Core Facility using their standard filter , Single nucleotide variant (SNV), Insertion or deletion (InDel) and Copy number variant (CNV); Variant effects: frameshift insertion, non-frameshift insertion, frame shift deletion, non-frameshift deletion, stopless, missense, nonsense; UCSC Common SNPs: filtered out; 1000 Genomes-Minor Allele Frequency (MAF): >0.01 filtered out; 5000 Exomes Global MAF: >0.01 filtered out; CNV Confidence Range: >10; To compare and contrast the mutational profile of the 105C cell line to other OCCC cell lines we downloaded the cell line specific mutational information from the CCLE at the DepMap repository . Next-gehttp://genie.cbioportal.org/was first used to identified sample IDs that were classified as “Ovarian Cancer” (under the “Cancer Type” header). These IDs were then used to generate a “User-defined Case List” from the GENIE Cohort v6.1-public dataset. A custom Python script was used to extract mutation data from the dataset and limit analysis to genes mutated in the 105C cell line. To compare our data with tumor samples, we obtained data from the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) dataset. To identify samples of interest, the data selector at https://training.galaxyproject.org/training-material/topics/variant-analysis/). Briefly, reads obtained from SRA were mapped to hg19 using the Burrows–Wheeler Aligner (BWA)  ,2917,29]To identify significant gene deletions and amplifications that might contribute to the 105C phenotype and determine the degree of genomic instability describing the 105C cell line relative to other OCCC lines, we performed copy number analysis using the Cytoscan HD platform on 105C, KOC-7c, SMOV-2, TU-OC-1, and OVSAYO lines. This data along with CCLE and GENIE copy-number data allowed us to compare the 105C line to 11 OCCC cell lines as well as patient tumour samples . Our anaIt is well-established that tumors resident in the peritoneal cavity can produce ascites and that tumour cells released into the ascites can subsequently migrate to other surfaces resulting in the formation of secondary tumour sites . TherefoTo determine whether the OCCC cell lines including 105C cells remain in a quiescent state during spheroid formation, cells were seeded to 24-well ULA plates and cultured for up to 22 days. Trypan blue exclusion counting was performed at different time points. Interestingly, we found that a subset of OCCC cell lines  continue to proliferate in suspension culture while the majority of the OCCC cell lines including the 105C cells showed significant loss of cell viability when cultured in suspension . The KOCPIK3CA and PTEN) impact signaling we performed western blot analyses on proliferating monolayer cells and cells cultured in suspension, using antibodies targeting various components of the pathway  reduction in cell viability. AZD-8055 caused a large drop in cell viability (64%) with the TOV-21G cell line in suspension culture and much less so when treated at the time of spheroid reattachment. The OVMANA cell line in suspension culture showed a 28% loss in cell viability in the presence of AZD-8055 and a similar (32%) decrease when treated with the IC50 concentration of AZD-8055 for monolayer cells. The 105C cell line in suspension culture was highly sensitive to AZD-8055 dropping cell viability by 55% and when treated at the time of spheroid reattachment, 46% of the cells remained viable at the IC50 determined for spheroid suspension culture. It is also notable that the viability of all cell lines was more adversely affected by AZD-8055 treatment while cells were in suspension culture relative to when treated at the time of spheroid reattachment. This was most evident at the 1uM dose of AZD-8055 used as the positive control for AZD-8055 efficacy. Spheroids are considered metastatic units that eventually will adhere to the peritoneum to continue to invade at secondary sites ,39. To gsticware . In thisIn addition to mTOR inhibition we also treated OCCC cell lines with carboplatin, a therapeutic often used to treat EOC. To determine whether proliferation rate played a role in sensitivity to carboplatin, we selected cells that proliferate while in suspension  and cell lines that are static in suspension culture (105C and OVMANA). Notably cell lines which proliferate when in suspension culture, showed increased sensitivity to carboplatin treatment when cultured as spheroids. The OVMANA and 105C cell lines, which do not proliferate in suspension culture, were however, not affected by carboplatin treatment at either concentration . ClearlyNOD-scid IL2Rgammanull (NSG) female mice. Tumour formation was not observed in any of the mice after intraperitoneal injection. We detected small white nodules at the sites of subcutaneous injection which were excised for cell culture and histology. Nodules from each mouse strain were pooled, digested with collagenase to generate a single cell suspension, and placed in cell culture. Cells dissociated from the nodules grew in culture and the recovered cells were identified by STR analysis , at the time of this writing, showed that a stomach adenocarcinoma (TCGA-FP-8631-01) contained the same ARID1A mutation found in the 105C line.Gene mutational analysis provided the first indication that the 105C cells were derived from an OCCC given the presence of the  in OCCC . While o alleles ,28. An aPTEN resulted in the loss of detectable PTEN by western blot in 105C cell line protein lysates like KOC-7c and TOV-21G cell lines which also carry PTEN mutations. We also performed an extensive analysis of the literature and using the CCLE database in combination with reports providing detailed gene mutational data [ARID1A, PTEN, and PIK3CA [CTNNB1 (42% of samples), KMT2D (31%), KMT2B (19%), and PIK3R1 (19%) [KMT2A, KMT2D, CTNNA1, and CTNNB1 genes [KMT2A, KMT2D [KMT2C and KMT2D genes [The combination of three mutations in nal data ,26,40, wnal data  only thrd PIK3CA . It is ad PIK3CA  whereas d PIK3CA  showed tR1 (19%) . InteresB1 genes ; KOC-7c A, KMT2D , and TOV2D genes ,45 and l2D genes ,42.The epithelial to mesenchymal transition in cells of epithelial origin has been identified as a hallmark of cancer and specifically implicated as promoting dissemination and invasiveness during tumorigenesis . In the The expression HNF1B has been identified as a marker that is expressed preferentially in OCCC and is silenced in the other subtypes ,29,30. WWe also examined HNF1B gene expression using the cBioPortal platform in EOC cell lines and found that the five OCCC cell lines  show the highest HNF1B mRNA levels amongst the 47 EOC cell lines analyzed in the CCLE. However, robust HNF1B expression is not an exclusive property of OCCC lines as indicated by Wiedmann et al., as they showed OCCC cell line HNF1B levels similar to that of the SKOV3 cell line . AdditioPTEN and PIK3CA mutations show the lowest levels of genomic instability. Numerous reports have indicated that loss of PTEN activity is mechanistically linked to loss of genomic stability; therefore, it is surprising that the 105C, KOC-7c, and TOV-21G cell lines show the lowest level of genomic instability relative to the other OCCC cell lines [Previous studies have highlighted that EOC subtypes differ with respect to genomic stability: HGSC exhibit a high level of genomic instability due to mutated DNA repair pathways which results in a large proportion of their genome undergoing copy number variation, whereas the OCCC and endometrioid subtypes are generally much more stable and show far less copy number alteration ,58. We all lines ,61,62,63ll lines . PIK3CA and PTEN was not observed as the TOV-21G and KOC-7c exhibited increased sensitivity while the 105C line showed decreased AZD-8055 sensitivity when maintained in spheroid culture. We also asked whether the efficiency by which OCCC cell line spheroids can reattach to tissue culture plastic changed if only exposed to AZD-8055 while in spheroid culture or when treated just the time of spheroid reattachment to tissue culture plastic. We found that the OCCC cell line spheroids exhibited similar sensitivity to AZD-8055 regardless of when they were treated meaning that an initial exposure while OCCC spheroids where in suspension inhibited their ability to reattach efficiently. The viability of OCCC cell line spheroids treated once they were transferred to tissue culture plastic did not change significantly meaning that inhibition of mTORC1/2 signaling in OCCC cell line spheroids caused cell death in suspension culture and inhibited the ability of OCCC spheroids to attach to an adherent surface. Hence, in our studies, mTORC1/2 activity in OCCC cell lines is not only important to actively proliferating monolayer cells but can also significantly reduce their ability to remain viable in suspension culture. Our data suggests that mTORC1/2 inhibitors could reduce metastatic spread of OCCC as AZD-8055 can effectively kill cells in spheroid form and then reduce their ability to interact with an adherent substratum. Dysregulation of the AKT-mTOR pathway has been implicated in the development and progression of many different types of cancers ,66. GiveThese results suggest that further investigation into the mechanisms governing the suspension proliferation rate of cell lines may be helpful in not only identifying markers that can be informative regarding the extent to which patients might benefit from chemotherapeutic intervention, but also identifying new targets of therapeutic potential.We performed xenografting experiments with the 105C cell line using various strains of immunocompromised murine models which did not result in robust tumour formation. Interestingly, 105C cells were isolated from the site of injection. Hence, 105C cells can take up residence in murine tissue to form small nodules. The mutation profile, genome copy number variation and other properties did not predict the xenograft result given that TOV-21G, RMG-V, and KOC-7c cell lines readily produce xenograft tumors in mice with a similar mutation profile and degree of genome copy number variation to the 105C line ,76,77. WIn conclusion, we have identified and extensively characterized the 105C cell line as derived from OCCC despite the initial tumor pathology suggesting a mixed tumour type. The 105C line is one of the few carrying the prototypical OCCC mutation profile and yet is weakly tumourigenic in a xenograft model. The addition of the readily available 105C line to the compendium of OCCC cell lines provides researchers with a well-characterized model system for preclinical studies focused on the discovery and testing of novel therapeutic agents to treat OCCC.