In order to be effective models to identify biomarkers of chemotherapy response, cancer cell lines require thorough characterization. In this study, we characterised the widely used high grade serous ovarian cancer (HGSOC) cell line NIH-OVCAR3 using bioinformatics, cytotoxicity assays and molecular/functional analyses of DNA damage response (DDR) pathways in comparison to an ovarian cancer cell line panel. Bioinformatic analysis confirmed the HGSOC-like features of NIH-OVCAR3, including low mutation frequency, TP53 loss and high copy number alteration frequency similar to 201 HGSOCs analysed (TCGA). Cytotoxicity assays were performed for the standard of care chemotherapy, carboplatin, and DDR targeting drugs: rucaparib (a PARP inhibitor) and VE-821 (an ATR inhibitor). Interestingly, NIH-OVCAR3 cells showed sensitivity to carboplatin and rucaparib which was explained by functional loss of homologous recombination repair (HRR) identified by plasmid re-joining assay, despite the ability to form RAD51 foci and absence of mutations in HRR genes. NIH-OVCAR3 cells also showed high non-homologous end joining activity, which may contribute to HRR loss and along with genomic amplification in ATR and TOPBP1, could explain the resistance to VE-821. In summary, NIH-OVCAR3 cells highlight the complexity of HGSOCs and that genomic or functional characterization alone might not be enough to predict/explain chemotherapy response. Genomic instability, a key enabling characteristic of cancer , is ofteHigh grade serous ovarian cancer (HGSOC) is the most common histological subtype of epithelial ovarian cancer (EOC), and is characterised by p53 loss in ~96% of cases accompanied by a high frequency of copy number alterations (CNAs) . DespiteStudies are underway to identify determinants of sensitivity to existing chemotherapies , and smahttps://dtp.cancer.gov/discovery_development/nci-60/), as well as large scale genomic and drug sensitivity studies including Cancer Cell Line Encyclopedia (CCLE) led by the Broad Institute , and the Genomics of Drug Sensitivity in Cancer (GDSC) study led by the UK Wellcome Sanger Institute (https://www.cancerrxgene.org/).In this study, we investigated one of the most widely studied ovarian cancer cell line, NIH-OVCAR3, as a model of HGSOC, established in 1982 from ascites from a progressive ovarian adenocarcinoma that was resistant to cyclophosphamide, cisplatin and doxorubicin . MoleculBRCA mutations. NIH-OVCAR3 showed no functionally inactivating mutations in HRR genes and were competent in forming RAD51 foci in response to DNA damage induction (an accepted biomarker of HRR function). However, further analysis confirmed functional loss of HRR, likely downstream of RAD51, and concomitant activation of the non-homologous end joining (NHEJ) pathway of double strand break (DSB) repair. Genomic analysis identified amplifications in several DDR genes including ATR and TOPBP1 which, along with high NHEJ activity, may explain the relative resistance to VE-821. Additionally, when DDR genes with alterations (genomic and protein level) in NIH-OVCAR3 cells were analysed within the HGSOCs in the TCGA study, similar alterations in genes including ATR, TOPBP1 and XRCC6 (Ku70) were frequently observed in HGSOCs and are hence likely to be clinically relevant biomarkers of response. In summary, analysis of NIH-OVCAR3 highlights the complexity of the molecular profile of ovarian cancer and the difficulties in predicting sensitivity using a single or even multiple molecular determinants.Our study confirmed NIH-OVCAR3 cells to be highly representative of HGSOCs with functional loss of p53 and a very high number of CNAs using published genomic data (CCLE). However, among a panel of ovarian cancer cell lines analysed, the NIH-OVCAR3 cells exhibited an unexpected drug response profile, which was explored further using genomic, proteomic and functional approaches. NIH-OVCAR3 cells were found to be resistant to the ATR inhibitor (ATRi), VE-821, but as sensitive to the PARPi, rucaparib, and carboplatin as cell lines with known Analysis of the genomic profile of NIH-OVCAR3 cells (CCLE database) confirmed the previous characterisation as being representative of HGSOC  with a lr-value = 0.84) between the two datasets  highlight conflicting results. We found NIH-OVCAR3 cells were 5-fold more sensitive to carboplatin than the 12 ovarian cancer cell lines (50 0.18 µM) compared to 2 representative ovarian cancer cell lines (IGROV1 and CAOV3) in the panel (mean LC50 0.70 µM)   confirmiBRCA mutant cells, their sensitivity to the PARPi, rucaparib was also investigated (BRCA1 mutant HRR defective (HRD) cell lines (COV362 and UWB1.289) [Because of the similar carboplatin sensitivity of NIH-OVCAR3 cells to that of the stigated C. NIH-OVWB1.289) , suggestWB1.289) . CytotoxLoss of p53 function has been considered one of the major determinants of sensitivity to ATR inhibition, however, the NIH-OVCAR3 cells were found to be resistant to VE-821 despite functionally inactivating homozygous mutation in TP53 together with deep deletion. Therefore, we sought to determine if p53 function had been restored in these cells. Western blot analysis revealed that despite deep deletion reported in TP53, the NIH-OVCAR3 cells still expressed p53 protein, confirming our speculation that genomic deletions are unlikely to cause complete loss of protein expression. However, when treated with Nutlin-3, an MDM2 antagonist that inhibits MDM2-p53 interaction resulting in p53 activation, there was no induction of either MDM2 or p21 (indicators of p53 activation) in NIH-OVCAR3 compared to a p53 wild type (WT) cell line, confirming functional loss of p53 caused by the mutations .Ser345, a specific marker of ATR activity [50 and the percentage ATR inhibition at 1 µM VE-821 for the NIH-OVCAR3 cells and nine other cell lines analysed was found to be similar   and its activity . The IC5 similar A indicatNext, we investigated expression of 11 DDR proteins that have previously been identified as determinants of sensitivity to ATR inhibition . The norBRCA1 function, thus confirming HRR deficiency  were analysed as likely determinants of response to ATRi.Genomic/transcriptomic analysis of genes with genomic amplifications in NIH-OVCAR3 in the TCGA dataset identified that all of the 12 genes are also frequently amplified in HGSOCs, which again emphasizes the similarity of the cell line with HGSOCs. The most amplified DDR genes were TOPBP1 (24%) and ATR (21%). However, analysis of the 19 DDR genes with deep deletions in NIH-OVCAR3 cells, showed that deep deletions in these genes are relatively infrequent clinically in HGSOCs and are more often found to show altered (increased/decreased) expression. Hence, deep deletions in these genes are unlikely to be suitable markers of therapy response in HGSOCs .Analysis of DDR protein levels C had idePlatinum sensitivity data was available for 148/201 HGSOCs. We attempted to see if there was any difference in mRNA expression of these genes between the platinum-sensitive and -resistant groups. However, none of the genes showed a statistically significant difference between the two groups , except Cell lines derived from human cancers are extensively used models of diseases in preclinical research. Recent advances in the genomic and transcriptomic characterisation of cell lines has greatly expanded their applicability in exploring drug response. Although often representative of the corresponding cancer type, like individual tumours, cell lines can demonstrate variable drug response profiles due to the complex genomic characteristics and functional activity of important cellular pathways. While being representative of HGSOCs, with functional loss of TP53 and high frequency of CNAs, we found the NIH-OVCAR3 cell line to be an outlier with a complex drug sensitivity profile. The cells were found to be resistant to ATRi, and very sensitive to platinum agents (carboplatin and cisplatin) and PARPi, rucaparib, which prompted exploration of likely alterations in DDR pathways. However, this response could not be predicted by genomic or proteomic characterisation of DDR pathways alone, highlighting the need for functional characterisation in combination with genomics to better predict drug response.Inactivation of p53 is one of the most well studied determinants of sensitivity to ATR inhibition. However, despite the loss of p53 function, the NIH-OVCAR3 cells were resistant to VE-821 and this resistance was not due to inadequate ATR inhibition, which was consistent with that observed in other ovarian cancer cell lines. This highlights that functional loss of p53 may not always cause sensitivity to cell cycle checkpoint inhibition by targeting ATR. This rationale further gains strength from conflicting reports of response to ATRi in different p53 mutant models. In previous reports the ATRi, AZD6738, has been found to be selectively cytotoxic towards p53 mutant CLL cells . HoweverInterestingly, NIH OVCAR3 cells had amplifications in ATR and TOPBP1 genes. Downregulation of these genes has previously been reported to confer sensitivity to VE-821 ,38 and hhttps://www.cancerrxgene.org/). This, however, can be driven by differences in the methodologies used to assess the sensitivity, which in turn can be dependent on the doubling time of the cell line. In this study, we used the colony formation assay which is expected to be free from interpretation bias resulting from differences in cell doubling time and found the cells to be particularly sensitive to platinum agents and PARPi.As opposed to the relative resistance to ATRi, the NIH-OVCAR3 cells showed strong sensitivity to carboplatin and PARPi, rucaparib, similar to cell lines with established HRR defects. In the published literature, sensitivity of NIH-OVCAR3 to platinum agents have been reported with conflicting findings, with Beaufort et al. finding the cells to be relatively sensitive to carboplatin  whilst tBRCA) may have value when looking at larger cohorts, it can limit therapeutic options for some individuals.HRR defects are a known determinant of sensitivity to both platinum  and PARPGiven the loss of HRR, it was unsurprising that the NIH-OVCAR3 cells were found to have a high level of NHEJ activity . However2. The growth conditions of additional 12 ovarian cancer cell lines used for comparison, including UWB1.289 and UWB1.289 + BRCA1 cell lines used as positive and negative controls for HRR assay, are listed in The NIH-OVCAR3 cell line (ATCC HTB-161) was cultured in RPMI supplemented with 10% FBS and incubated at 37 °C in humidified atmosphere containing 5% COThe chemotherapy drugs used for the cytotoxicity assay include Carboplatin , rucaparib and VE-821 . Drugs used for western blot analysis include Nutlin-3  and 4NQO (Sigma Aldrich).v/v) and stained with 0.4% crystal violet. Colonies were counted and the % survival for each treatment was calculated from the relative plating efficiency of treated versus untreated controls.Cells were plated into 6-well tissue culture dishes at low densities and allowed to adhere for 24 h before being treated for 24 h with carboplatin (0–50 µM) or rucaparib (0–30 µM), or 48 h with VE-821 (0–30 µM). DMSO concentration was kept constant at 0.5%, including no-drug control, for drugs dissolved in DMSO. Following treatment, the medium was removed and replaced with fresh drug-free medium to allow colony formation. The cells were fixed in methanol: acetic acid  antibody  at 1:1000 and rabbit monoclonal anti RAD51 antibody  at 1:500. Secondary antibodies used were Alexa 488 conjugated goat anti-rabbit and Alexa 546 conjugated goat anti-mouse , both at 1:1000. The nuclei were stained with DAPI. The number of γH2AX and RAD51 foci in each cell was quantified using ImageJ software and data was plotted using GraphPad Prism . A >2-fold increase in γH2AX foci formation was used as an indicator of DNA damage induction following rucaparib treatment and a >2-fold increase in RAD51 foci formation was indicative of functional HRR.pDRGFP  and pCBASceI  plasmids used for the HRR assay were a kind gift from Maria Jasin ,47. EquipCBASceI (Addgene plasmid # 26477) and pimEJ5GFP  plasmids used for the NHEJ assay were a kind gifts from Maria Jasin and Jeremy Stark . Equimolw/v) in TBS-T for at least 1 hr before primary antibodies were added as indicated for each western below, diluted in either 5% milk (w/v) or 5% BSA (w/v) in TBS-T. Clarity Western enhanced chemiluminescence substrate (Bio-Rad) was used to visualize the bands following the manufacturer’s protocol, and chemiluminescence measured using G-box gel documentation system .Whole cell lysates were prepared from exponentially growing cells in Phosphosafe extraction reagent buffer  containing 1:100 protease inhibitor cocktail . The protein content of all lysates was determined by Pierce BCA assay (Thermo Fisher Scientific) following manufacturers protocol. Lysates were diluted to equal concentrations between 0.5–1 mg/mL in 2 × Laemmli buffer  for TP53 functional analysis, or XT sample buffer and XT reducing agent (BioRad) for VE-821 inhibition and baseline protein expression analysis, and boiled for 5 min at 95 °C. Gel electrophoresis was performed using 4–20% criterion TGX polyacrylamide gels for the TP53 functional analysis, and 3–8% XT Tris-Acetate gels for the VE-821 inhibition and baseline protein expression for optimum separation based on the molecular weights of the proteins analysed. Proteins were transferred to nitrocellulose Hybond™ C membranes  and blocked with 5% milk , mouse anti-p53  and mouse anti-α-tubulin  as a loading control , mouse anti-p21 control .Cells were treated with 4NQO ± VE-821 at the indicated doses for 1 h before cell lysates were harvested. Primary antibodies: goat anti-ATR , rabbit anti-CHK1 (Ser345)  and rabbit anti-vinculin .Cells were left to reach 70–80% confluence before cell lysates were harvested. Primary antibodies: rabbit anti-ATM , goat anti-ATR , mouse anti-CHK1 , mouse anti-Cyclin E , rabbit anti-DNA-PKcs , mouse anti-Ku70 , rabbit anti-Ku80 , rabbit anti-PARP-1 , rabbit anti-RAD51 , rabbit anti-XRCC1 , rabbit anti-ARID1A . Ponceau-S staining was used to ensure equal loading of the protein.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150942).Cell pellets containing ~40,000 cells were collected of the 13 ovarian cancer cell lines  includinhttps://portals.broadinstitute.org/ccle/data). To confirm the authenticity of the cell line used in this study, RNA-Seq read counts for 2544 genes were extracted and the genes with no data availability or no expression were excluded. The gene expression data of 2426 genes was used to calculate the Pearson’s correlation co-efficient and corresponding p-value between the two datasets. The copy number alteration and mutation data for NIH-OVCAR3 cell line was also retrieved from the CCLE database. The mutations were categorised as homozygous or heterozygous mutation using a variant allele frequency cut-off of 0.8, with mutations with variant allele frequency >0.8 were considered as homozygous mutations. The copy number alterations were categorised using their GISTIC scores with −2 for deep deletion and 2 for amplification. The mutation and copy number alteration data of the CCLE dataset was accessed using cBioportal web tool  [RNA-Seq data (read counts) for NIH-OVCAR3 cell line was downloaded from the CCLE database   AdditionAll statistical analysis was performed using GraphPad Prism 8.BRCA mutational status and platinum sensitivity are good indicators of likely PARPi response they are by no means 100% predictive of response [In conclusion, the data presented here highlight the difficulties in predicting sensitivity to emerging therapeutics. Even with extensive genomic characterisation of cell lines, determinants of sensitivity to therapeutics investigated in isolation in isogenic matched cell lines often fail to translate to the context of cell lines with complex molecular characteristics that mirror individual tumours. The loss of p53 and decreased expression of ATM, XRCC1 and XRCC6 (Ku70) would predict sensitivity to ATRi VE-821. However, the NIH-OVCAR3 cells were resistant to VE-821. Loss of p53 and ATM, as determinants of ATRi response, are also being investigated in numerous clinical trials as outcome measures , and will be interesting in relation to the observations made here in NIH-OVCAR3 cells. Furthermore, mutational characterisation did not predict the profound sensitivity of NIH-OVCAR3 cells to carboplatin and rucaparib and even functional analysis using RAD51 foci indicated its limitations. These findings have parallels in the clinic where although Helleday, T.; Curtin NJ. Therapeutic Compounds (PARP inhibitors in homologous repair/BRCA defective cancer) WO 2005/012305 A2.Boritzki, T.J.; Calvert, A.H.; Curtin, N.J.; Dewji, M.R.; Hostomsky, Z.; Jones, C.; Kaufman, R.; Klamerus, K.J.; Newell, D.R.; Plummer, E.R.; Reich, S.D.; Steinfeldt, H.M.; Stratford, I.J.; Thomas, H.R.; Williams, K.J. Therapeutic Combinations Comprising PARP inhibitor WO/2006/033006.Falcon, S.; Reaper, P.; Pollard, J.; Curtin, N.J.; Middleton, F.K.; Chen, T. Method for measuring ATR inhibition mediated increases in DNA damage. WO2014055756A1.


We demonstrate the power of our improved functional annotation by evaluating the 3D landscape of a transgene integration site and two phenotypically different cell lines. Our work opens up further novel genome engineering targets, has the potential to inform vital improvements for industrial biotherapeutic production, and represents a significant advancement for CHO cell line development.Chinese hamster ovary (CHO) cell lines are the pillars of a multibillion‐dollar biopharmaceutical industry producing recombinant therapeutic proteins. The effects of local chromatin organization and epigenetic repression within these cell lines result in unpredictable and unstable transgene expression following random integration. Limited knowledge of the CHO genome and its higher order chromatin organization has thus far impeded functional genomics approaches required to tackle these issues. Here, we present an integrative three‐dimensional (3D) map of genome organization within the CHOK1SV® 10E9 cell line in conjunction with an improved, less fragmented CHOK1SV 10E9 genome assembly. Using our high‐resolution chromatin conformation datasets, we have assigned ≈90% of sequence to a chromosome‐scale genome assembly. Our genome‐wide 3D map identifies higher order chromatin structures such as topologically associated domains, incorporates our chromatin accessibility data to enhance the identification of active  In this work Bevan and colleagues generate a comprehensive 3D genome map of a site‐specific integration (SSI) CHO host cell line using a combination of ATAC‐Seq, Hi‐C, and Promoter‐Capture Hi‐C. These datasets are used to identify higher order chromatin structures such as topologically‐associated domains (TADs), and puts them in the context of a genome‐wide 3D promoter interactome. The combination of such datasets may be used to design cell engineering approaches for improving CHO performance in the bioprocessing environment.  These cell lines have become the pillars of the biopharmaceutical industry, primarily due to their ability to produce large amounts of human‐compatible transgenic protein. Previous publications of iterative Chinese hamster and CHO cell line reference sequences . Each new subculture was seeded at an initial cell concentration of 0.2 × 106 cells/ml on a 3 day/4 day subculture routine. All samples were taken from a 4‐day culture.CHOK1SV 10E9 cells  and aligned using Bowtie2  of the total CHOK1SV sequence Table  and in l3.2cis‐interactions, indicative of the increased frequency of ligation between juxtaposing regions of the linear genome, was prevalent across the diagonal of a genome‐wide Hi‐C heatmap , each CHOK1SV 10E9 LACHESIS assembly scaffold group aligned, contiguously in large sections, to a particular combination of CHO‐K1GS_HDv1 scaffolds Figures  and S1. The integration of data, such as long‐read PacBio sequencing, along with a comparative analysis to the PICRH Chinese hamster assembly  and inactive (“B”) genomic compartments within our chromosome‐scale assembly Figure . On aver3.4We next used ATAC‐Seq , we found that 43.5% of them overlap accessible (ATAC‐Seq positive) enhancer regions. Across these regions, signal from ATAC‐Seq positive enhancer states (2 and 3) dominate the central regions whereby the ATAC‐Seq negative enhancer states (6 and 7) correspond to signal generated by flanking nucleosomes . A similar pattern is uncovered at promoter regions where the ATAC‐Seq positive promoter‐associated signal (State 4) predominantly overlaps the sequences immediately upstream of transcription start sites .3.5We subsequently employed PCHi‐C  over those accessible, active enhancer regions not identified as promoter interacting in this study. This is consistent with the concept that CTCF is involved in the formation and maintenance of chromatin loop interactions between functional regulatory elements .In the context of cell line engineering, the identification of these functional contacts could be important in fine‐tuning the expression of genes whose reduced or increased expression can have a positive effect on product titer and/or product quality. Candidate enhancers identified by this study to interact with the gene encoding the pioneer transcription factor 3.6Fer1l4 locus created using FLP/FRT recombinase‐mediated cassette exchange .In comparison to the map of chromatin organization previously defined for the CHOK1SV 10E9 cell line, the vast majority of accessible sites were consistent between the two experimental cell lines; however, 383 regions exhibited differential accessibility (Fer1l4 genomic locus resides within an active compartment approximately 70 kb from the periphery of a TAD spanning 215 kb and makes contacts with a number of expressed gene promoters within the flanking 400 kb (Figure The identification of CHO 3D genome organization will help to form a comprehensive platform for the improved identification of novel genome‐engineering target sites and to benchmark the regulatory landscape of known genome‐engineering sites. With relevance to this, we find that the b Figure . As it ib Figure , it is r4As the requirements placed upon the biopharmaceutical industry continue to grow, a deeper understanding of CHO biology and in particular, its effect on cell line performance has become even more critical. Given the level of flexibility required to produce the wide range of complex biotherapeutics now industrially produced in CHO cells, it is highly desirable to develop the tools and increase the capability for rational cell line and vector engineering. Recombinant CHO cell lines used by the biopharmaceutical industry are hampered by issues such as unpredictable expression levels and transgene silencing. There is a currently unmet need within the biotechnology sector to identify endogenous integration sites that confer consistent, predictable, and stable expression of integrated payloads. With the gene‐editing tools now available, an understanding of the endogenous 3D chromatin conformation is essential to reduce the theoretical blank canvas of genome‐engineering targets to a shortlist of desirable candidates. Facilitated by a significantly less fragmented CHOK1SV 10E9 genome assembly, our identification of higher order chromatin structures, spatially segregating the CHOK1SV 10E9 genome based on transcriptional activity, immediately aids in this quest. Importantly, regions in close proximity to TAD boundaries are known to be enriched in expressed housekeeping genes and in factors linked to active transcription (Dixon et al., Aside from the requirements for transgene integration sites, flexibility in terms of recombinant cell line phenotypes, for example, cell lines with an increased capacity for protein translation, increased bioreactor cell viability, or tailored protein glycosylation patterns, would also be highly beneficial for the biopharmaceutical sector. Recent exemplar research has uncovered the positive effects of ACTC1 (Pourcel et al., We acknowledge that the variability in the endogenous chromatin architecture of clonal CHO cell lines, pertaining to general chromatin dynamics and the inherent genetic instability of immortalized CHO cell lines, will be an important factor to consider when relating this data to other strains and/or variants. Taken together, however, our in‐depth integrative map of 3D genome organization and chromatin accessibility within an industrially relevant clonal CHO host cell line provides a further important advancement for CHO cell line development and acts as a tool for the improved identification and predictive assessment of genome engineering targets. We predict that the scope of potential genome engineering targets informed by a much greater understanding of the endogenous 3D chromatin architecture will translate into the development of an improved, more streamlined recombinant protein production system within industry, leading to a quicker transition of novel therapeutics into the market. In addition, as the field of CHO cell line development continues to evolve, the knowledge and resources provided here will continue to be highly beneficial and relevant for biotechnological applications for the foreseeable future.Peter Fraser and Stefan Schoenfelder and are cofounders of Enhanc3D Genomics Ltd. The other authors declare that there are no conflict of interests.Peter M. O'Callaghan and Peter Fraser conceived the project and the main conceptual ideas. Stephen Bevan, Peter M. O'Callaghan, Peter Fraser, Robert J. Young, Lin Zhang, and Stefan Schoenfelder contributed to experimental design. Stephen Bevan performed Hi‐C, PCHi‐C, and ATAC‐Seq experiments and analysis. Stefan Schoenfelder provided training and assistance with Hi‐C and PCHi‐C experiments. Simon Andrews designed the PCHi‐C RNA bait library and provided bioinformatics support, tools, and assistance. Peter M. O'Callaghan, Peter Fraser, Robert J. Young, Lin Zhang, and Stefan Schoenfelder supervised the research. Stephen Bevan drafted the manuscript with input from all authors. All authors provided critical feedback and contributed to the analysis and interpretation of the results.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


The experimental determination of the relative biological effectiveness of thermal neutron factors is fundamental in Boron Neutron Capture Therapy. The present values have been obtained while using mixed beams that consist of both neutrons and photons of various energies. A common weighting factor has been used for both thermal and fast neutron doses, although such an approach has been questioned. At the nuclear reactor of the Institut Laue-Langevin a pure low-energy neutron beam has been used to determine thermal neutron relative biological effectiveness factors. Different cancer cell lines, which correspond to glioblastoma, melanoma, and head and neck squamous cell carcinoma, and non-tumor cell lines (lung fibroblast and embryonic kidney), have been irradiated while using an experimental arrangement designed to minimize neutron-induced secondary gamma radiation. Additionally, the cells were irradiated with photons at a medical linear accelerator, providing reference data for comparison with that from neutron irradiation. The survival and proliferation were studied after irradiation, yielding the Relative Biological Effectiveness that corresponds to the damage of thermal neutrons for the different tissue types. Boron Neutron Capture Therapy (BNCT) is currently undergoing a renaissance that may bring this therapy closer to hospital practice . This isnE < 0.5 eV), epithermal (0.5 eV ≤ nE ≤ 10 keV), and fast (nE > 10 keV). Treatment planning is one line of improvement. In a clinical study that was performed at Helsinki University Central Hospital , the res14N14C reaction (625.87 keV). Thus, in practice, a low energy neutron spectrum does not affect the energy of these products. Therefore, the RBE of thermal neutrons is a key factor in BNCT, because it does not depend on the facility. A source of information of the response of cells to neutrons are in-vitro irradiation experiments, where different cell cultures are irradiated in a neutron beam at different doses and the biological end-point considered  is the fraction of cells that has the ability to form clones a few days after the irradiation (clonogenic assay). From the comparison of these survival curves from neutron irradiation with those that were obtained from a reference photon irradiation, the relative biological effectiveness of neutrons as compared to photons can be obtained. Some of these experiments can be found in Refs. ,11,12,13The aim of this work was to measure the biological response of different cell lines  under a pure low energy neutron beam, with almost no contamination of fast neutrons and gamma radiation, and then compare it with the response of the same cell lines under a reference photon irradiation from a hospital LINAC. Dedicated setups were designed for both kinds of irradiations. Two different biological end points have been studied for the determination of the neutron response, but only the common one (clonogenic ability) has been chosen for the thermal neutron RBE calculations.9 nthermal/(cm2s). Neutron irradiations were carried out at the PF1B cold neutron beam line at the Institut Laue-Langevin (ILL) . The arrThe experimental arrangement that is shown in Photon irradiations were carried out at the medical linear accelerator (LINAC) of Virgen de las Nieves Hospital (HVN) in Granada. The Elekta Versa HD™ accelerator delivered a flattened 6 MV high energy photon beam . The irrMeasurements of neutron flux were performed using gold foil activation data. Neutron doses at the cells were estimated with the MCNPX simulation code for the transport of the neutrons and the generated photons . The geoPhoton doses at HVN medical LINAC were known from a computed tomography scan image and the clinically used Pinnacle treatment planning system  , which uAt thermal energies, the neutron capture on nitrogen dominates the local dose deposition among all possible reactions in the tissue. Therefore, the nitrogen content of the cells is essential for the dose estimation. CHNS elemental analysis was performed with a THERMO SCIENTIFIC Flash 2000  analyzer. CHNS elemental analyzers provide a means for the rapid determination of carbon, hydrogen, nitrogen, and sulphur in organic matrices and other types of materials. It is based on the dynamic combustion of a sample. The resultant gases are separated and detected by a thermal conductivity detector (TCD). This technique can determine the quantity of carbon, nitrogen, hydrogen, and sulphur with an error less than 3%. 6 cells was prepared and 2 μg of the pellet were used for the CHNS elemental analysis.The nitrogen content of cells was measured by this method at the Centro de Instrumentación Científica (CIC) from the University of Granada. To this end, a dry pellet of 10 × 10The six irradiated cell lines were of human origin, four from cancer tissue and two from healthy tissue. The A375 cells are from malignant melanoma; Cal33 and SQ20 from head and neck squamous cell carcinoma; U87 from glioblastoma; HEK293 are from embryonic kidney; and, MRC5 are fibroblast from fetal lung tissue. The cell lines were kindly provided from Institute of Advanced Biosciences, Grenoble, except for HEK293, which was commercially acquired (Thermo Fisher). The selected cancer cell lines correspond to the type of tumors that have been typically treated with BNCT. All of them are adherent cells, which is also a requirement in our set-up. 2 95% air incubator. The cells were sub-cultured at a ratio of 1:3 and they were grown to 90% confluence. The medium was replaced every 2–3 days. The cells were cultured in DMEM medium  that contained 10% fetal bovine serum , 1 μM L-glutamine (Gibco), 100 IU/mL penicillin, and 100 IU/mL streptomycin  (complete medium) at 37 °C in a humidified CO5 and 2 × 105 cells in 200 μL culture medium) for neutron irradiation or T25 flasks (between 0.7 × 106 and 1.4 × 106 cells) for photon irradiation. The medium was replaced with fresh complete medium before irradiating the cells.Twenty-four hours before irradiations, cells were cultured in complete medium at 70% confluence in either quartz cuvettes (between 1.5 × 102 and 8 × 103 cells/well), based on the irradiation dose and the growing characteristics of the cell line, to form colonies of more than 50 cells in around two weeks. Every four days, the medium was exchanged with fresh complete medium. Colonies were fixed with 90% ethanol, stained with crystal violet, and then counted using the open access automatic counter program by Nghia Ho [The irradiated cells were detached with 1% trypsin-EDTA (Sigma-Aldrich) and then prepared for clonogenic assays. Cells were seeded in triplicate on six-well plates at appropriate numbers (between 2 × 10Nghia Ho .3 cells/well and incubated for four days. Proliferative ability was determined by a 5-bromo-2-deoxyuridine (BrdU) ELISA kit , following the manufacturer’s instruction.After irradiation, the cells were cultured in triplicate in 96-well plates at a density of 1 × 10For ILL neutron irradiation studies, with nitrogen concentration in the cell lines ranging from 1.0 to 2.3%, the simulated neutron thermal doses rates varied from 0.005 to 0.026 Gy/min., depending on the tissue type. The elemental analyzer method is validated, as the nitrogen content of HEK293 cell line measured is in good agreement with the reference in ICRU report 46 for fetus kidney . Table 1Gamma doses, mainly from neutron interaction with the experimental system, are tissue-independent and, therefore, are the same for all cell lines. In most of the cases, the thermal dose component remained higher than the gamma dose component see . The maiThe biological effect of the secondary gamma radiation would be expected to be less than in higher dose rate LINAC irradiations due to the low gamma dose rate. Indeed, this could be expected in long irradiation, because of repair effects. Nevertheless, the RBE of the gamma dose is set equal to 1 due to the lack of data for the studied cell lines at the present dose rate. Additionally, conclusive data for the possible synergies between the high-LET and low-LET radiations were not found. Thus, a cross term for describing this effect in the survival curves was not considered. Two end-points were studied for the neutron irradiations: survival, by means of clonogenic assay, and proliferation, by using BrdU incorporation assay, around two weeks and four days, respectively, after the irradiation. Both end-points showed different results when data were fitted following a linear quadratic equation, as shown in Because a correlation between the two different end-points could not be established, probably because of the time after irradiation at which they are measured, the comparison with photon irradiation, and the calculations of RBE factors are limited to the most common end-point used, which is the long-term survival by clonogenic assay.S, as studied by clonogenic assays, were fitted using the linear quadratic formula [D is the total absorbed dose and α and β are the parameters that describe the behavior of the survival. These parameters are constants that depend on the tissue/cell line and end-point. Survival data after irradiation,  formula :(1)−lnS=γD, while the survival following irradiations at ILL is due to a dose combination of low-energy neutrons and photons, ILLD. Thus, the total absorbed dose at ILL beam corresponds to the sum of neutron and gamma dose, ILLD = nD + γD. For irradiations at the medical LINAC, the total dose will correspond to that for the photons only, ILLS and γS, the survival that corresponds to the neutron effect alone, nS, can be calculated as:From the two fitted survival data, nD. The data can be fitted to a quadratic function, but the parameter β is assumed to be zero given the characteristics of high-LET radiation and the better results found with a linear fitting. This survival describes the effect if the dose is due to low-energy neutrons alone, ILLS) and the survival after photon irradiations at HVN medical LINAC (γS). As a result of the neutron-induced gamma production in the experimental arrangement, he survival (nS) that was associated with the neutron dose alone was obtained by the deduction of the gamma effect, as indicated in Equation (2). The U87 cell line was not photon irradiated and the results from Bayart et al. , obtaine14N14C, where the emitted charged particles (protons and 14C) damage the tissue, as mentioned previously. The RBE of thermal neutrons are calculated from a reference irradiation dose, Drefγ,, and the dose corresponding to only neutrons, nD, as described by nα and nβ:The RBE factors corresponding to thermal neutrons mainly arise from the neutron induced proton emission from nitrogen, γα and γβ previously calculated. For different survival, the corresponding doses calculated with parameters in The irradiations at the medical LINAC serve as reference irradiation, as described with the A new formalism for biological dose estimation has been proposed . It avoiAgain, the high errors on the fitting parameters of the irradiations in some of the cell lines lead to high uncertainties, mainly due to the error of the photon response, which can be reduced with more irradiation experiments. For example, in the case of Cal33 and SQ20, the error is due to the low effect that was observed after LINAC irradiations, where the fitting parameters have an error that is comparable to the value. More irradiations at LINAC may improve this result.Data showing the biological response of low-energy neutron irradiation show marked variability for the six cell lines studied. Therefore, it is crucial to characterize and understand the weighting factors for specific tissues and avoid having to assume such values. The proliferation and survival curves obtained following neutron irradiation seem to be different in most cell lines. Although this fact could suggest contradictory results depending on the end-point selected, the proliferative ability of the viable cells was assessed four days after irradiation while the clonogenic assay estimated the long-term cell survival. Moreover, proliferative and clonogenic potential do not have to necessarily correlate, as different factors may influence attachment, proliferation, and growth, such as cell density and cell-cell contact. Overall, our results suggest that it should be interesting to analyze different parameters, as they could provide more information about the possible behavior and response of cells to irradiation in vivo.We have also shown how differences in the nitrogen content between different cell lines have a strong impact on the dose received, emphasizing the importance of the accurate estimation of the elemental composition of each tissue under the neutron field for a better BNCT treatment planning. As we have demonstrated by determining the nitrogen content of HEK293 and comparing with the literature, the CHNS elemental analyzer seems to provide a simple and useful method for measuring it.The healthy tissue cell line MRC5 has the lowest amount of nitrogen, but high RBE values after low-neutron irradiation, which indicates an intense radio-sensibility, since less nitrogen captures took place, but a low survival was observed. The survival data suggest that Cal33 and SQ20, which are representative of a similar cancer type, behave in a similar way and, therefore, have similar RBE values. They both showed a strong effect after neutron irradiation (as compared to photons) and the data were markedly different from the other cell lines with similar nitrogen content, such as A375 or U87. The results of the A375 study suggest that this cell line appears to be more radio-resistant to neutrons than the others. The numerical values from previous work  are updatW, [The results from the U87 cell line can be compared with a similar in vitro irradiation of rat gliosarcoma cells at BMRR , where tThe accuracy of the photon irradiation data is important when used as a reference radiation in the comparison with neutrons for the RBE estimation. A common type of reference photon radiation, at a specific dose rate, would be desirable. Irradiations at the medical linear accelerator at dose rates of few Gy/min. are a good option in acquiring data for the reference photon dose, providing a comparison with a standard irradiation field for which there is a lot of clinical experience. Because the U87 and SQ20 cells were not available for photon irradiations, the reference irradiation used is not from the same series of irradiation at the LINAC, like the other cell lines. This implies that these RBE results may not be directly comparable with the ones from A375, Cal33, MRC5, and HEK293 until the irradiation in the same type of photon beam is possible. However, the main results from this work are the neutron response and the radiobiological parameters A very important contribution to the especially large uncertainties for the MRC5 cell line is due to the uncertainty of the β value, which makes this value compatible to zero, as it can be seen in αn for thermal neutrons can be obtained with higher precision than has been described previously. Here, they have been obtained with an uncertainty of less than 15% for all cancer and normal cell line.It has been shown how with an appropriate experimental arrangement and a clean cold neutron beam, such as the one at ILL, the radiobiological coefficients The obtained data provide very interesting information on the cell types that may be more radio-sensitive to neutron irradiation, and the difference between the tissues. This could be of relevance for BNCT treatment planning when healthy tissues with different neutron sensitivity need to be considered. It would be interesting to study other non-tumor cells of different origins. The importance of the RBE data that were provided by a pure thermal neutron beam has been pointed out. It is stressed that thermal RBE is a universal value that does not depend on the neutron beam and neutron facility. In order to provide very accurate data, the determination of the fast and epithermal neutron RBE in each facility is extremely recommended due to the different characteristic in their spectrum. Further research work of this type on other cell lines and in vivo studies are desirable for improving the understanding of the biological response to neutrons and for the improvement for BNCT treatment planning.


The response to treatment with calcitriol and calcidiol alone was specific for each cell line. Nevertheless, we observed an enhanced effect of combined treatment with ATRA and calcitriol in the majority of the cell lines. Although the levels of respective nuclear receptors did not correlate with the sensitivity of cells to these drugs, vitamin D receptor (VDR) upregulation induced by ATRA was found in cell lines that were the most sensitive to the combined treatment. In addition, all these cell lines showed high endogenous levels of retinoic acid receptor α (RARα). Our study confirmed that the combination of calcitriol and ATRA can achieve enhanced antiproliferative effects in human osteosarcoma cell lines in vitro. Moreover, we provide the first evidence that ATRA is able to upregulate VDR expression in human osteosarcoma cells. According to our results, the endogenous levels of RARα and VDR could be used as a predictor of possible synergy between ATRA and calcitriol in osteosarcoma cells.The main objective of this study was to analyze changes in the antiproliferative effect of vitamin D3, in the form of calcitriol and calcidiol, via its combined application with all- Osteosarcoma is a high-grade primary mesenchymal tumor characterized by spindle cells depositing an immature osteoid matrix . To dateInduced differentiation of transformed cells into mature phenotypes has proven to be an effective strategy in the treatment of several types of human malignancies ,14, and trans retinoic acid (ATRA) with several natural compounds, kinase inhibitors, chemotherapeutics, and proteasome inhibitors have demonstrated additive or synergistic effects [Despite the many benefits of retinoids as anticancer compounds, their usage in clinical protocols is still limited because of their short intracellular availability, clinically significant toxicity, and the occurrence of resistance . Therefo effects . Our res effects ,23,24,25 effects ,27,28.2 vitamin D3) is the most biologically active form of vitamin D3 [3) could act as chemopreventive agents [Calcitriol tamin D3 . It is mtamin D3 ,31. In atamin D3 . The rece agents . The core agents , prostate agents , breast e agents  and melae agents  have alse agents ,41,42,43e agents .In the present study, we focused on the possible effects of calcitriol and calcidiol alone or in combination with ATRA in patient-derived osteosarcoma cell lines, with special regard to the mechanism of interaction between calcitriol and ATRA.First, we investigated the possible effects of calcidiol and calcitriol—either alone or in combination with ATRA—on the Saos-2 established cell line. Using the MTT assay, an analysis of cell viability was performed on days 3 A and 7 B of the The combined treatment of Saos-2 cells with ATRA and calcitriol enhanced synergistically the inhibitory effect of ATRA alone . The detBGLAP and SPP1 genes, which encode the markers of osteogenic differentiation osteocalcin and osteopontin, respectively, was evaluated using RT-PCR. The changes in the expression of these genes during nine days of treatment are shown in BGLAP expression, whereas calcitriol was able to upregulate BGLAP expression from the first day of treatment , retinoic acid receptor (RARα), and retinoid X receptor (RXRα), which is a dimerization partner for both RARα and VDR.VDR mRNA were found in OSA-02 and OSA-08 cell lines, with high levels found in the Saos-2 reference cell line, as well as in the OSA-09 and OSA-13 cell lines  and to their combinations with the known differentiation inducer ATRA. The Saos-2 established cell line and six patient-derived cell lines were used for experiments.In the present study, we described the responsiveness of seven human osteosarcoma cell lines to two forms of vitamin DAccording to the results from the MTT assay, the Saos-2 established cell line showed only minimal sensitivity to the treatment with calcitriol or calcidiol alone. Although the antiproliferative effect of calcitriol in Saos-2 cell line has already been reported ,46, anotBGLAP, an osteogenic differentiation marker, was highest after combined treatment with calcitriol and ATRA during the entire analyzed period.In the combination treatments, only calcitriol was able to significantly enhance the inhibitory effect of ATRA. Similarly, we observed that the mRNA level of The sensitivity of the patient-derived cell lines to differentiation inducers was indeed specific to each cell line. We realized that the increased sensitivity to all differentiation inducers, including calcidiol, in the OSA-13 cell line could be caused by the low differentiation stage of those cells. OSA-13 was previously described as a tumorigenic cell line with elevated expression of the transcriptional factor SOX-2 .Variability in the responsiveness of cell lines could also be related to differences in endogenous levels of respective nuclear receptors for calcitriol and ATRA, as both compounds function as ligands for the respective receptors and subsequently change gene expression ,49. We hVDR mRNA expression in the THP-1 human monocytic leukemia cell line [Subsequently, we focused on the evaluation of nuclear receptor expression after 24 h of differentiation inducer treatment and observed that 1 µM ATRA was able to regulate VDR expression. This phenomenon has already been observed in mouse and rat osteosarcoma cell lines ,51,52,53ell line . In thisell line .VDR mRNA levels and VDR protein levels in selected osteosarcoma cell lines. Upregulation or downregulation of VDR depended on the cell line. It was described that there is no RARE in the VDR promoter, which suggested that ATRA could not regulate VDR directly [VDR transcription indirectly using regulatory elements that cooperate with the VDR promoter. Moreover, a study on myeloid leukemia cell lines showed that the most important isoform of RAR involved in the regulation of VDR transcription is RARα. In the absence of ligands, RARα led to transcriptional repression of the VDR gene in this cell type [Our results are consistent with the findings described above. Twenty-four hours of treatment with 1 µM ATRA caused changes in directly . Therefoell type .In accordance with these studies, we focused on the RARα isoform and its agonist ATRA. For better interpretation, we compared the two most different osteosarcoma cell lines—the Saos-2 reference cell line, which had the lowest level of RARα and the highest level of VDR, and the OSA-08 cell line, which had the highest level of RARα and the lowest level of VDR. In the Saos-2 cell line, downregulated expression of VDR was observed after RARα activation by ATRA at both the mRNA and protein levels. In contrast, upregulated expression of VDR was detected in the OSA-08 cell line after ATRA treatment.VDR [VDR by RARα. After the repression is overcome, cells start to express higher levels of VDR and calcitriol, thus inducing a stronger response. This response even enhances the antineoplastic effect of ATRA, so the combination is more effective than the effect of each drug alone. According to this hypothesis, we expected to see high sensitivity to calcitriol in cell lines with low endogenous expression of RARα, but our experimental data on cell proliferation did not confirm this idea: Saos-2, i.e., the cell line with the lowest endogenous level of RARα, did not respond to calcitriol at any of the used concentrations. In this case, we must take into account that another mechanism of resistance to vitamin D3, i.e., an overexpression of VDRE-BP, could be activated in Saos-2 cells [Moreover, the combined effect of ATRA and calcitriol was the most effective in the OSA-08 and OSA-13 cell lines, which had high levels of RARα. These data correlate with the hypothesis that unbound RARα acts as a transcriptional repressor of VDR . We assu-2 cells .To summarize, our results proved that combination treatment with calcitriol and ATRA showed an enhanced antiproliferative effect compared with the effect of those drugs alone in the majority of tested cell lines. Furthermore, this study provides the first evidence that ATRA treatment influences VDR expression in human osteosarcoma cells in vitro. More specifically, ATRA upregulated VDR expression at the mRNA and protein levels in cell lines with high endogenous levels of RARα and low endogenous levels of VDR; only these cell lines were the most sensitive to the combination treatment. In general, the results suggest that the levels of RARα and VDR in osteosarcoma cells could potentially be used as predictors of possible synergy between calcitriol and ATRA.The Saos-2 established cell line (No. HTB-85) was purchased from the American Type Culture Collection . Other cell lines were derived from tumor samples obtained during diagnostic biopsies from patients suffering from osteosarcomas. The samples were processed in our laboratory as previously described . The OSA2. Patient-derived cell lines at passages 10–25 were used for the experiments (Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (Saos-2 cells) or 20%  fetal bovine serum, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 2 mM glutamine . Cell culture was performed under standard conditions at 37 °C in a humidified atmosphere containing 5% COeriments .Calcitriol  and calcidiol (Sigma-Aldrich) were prepared as stock solutions at a concentration of 1 mM in absolute ethanol  and stored at −20 °C. ATRA (Sigma-Aldrich) was prepared as a stock solution at a concentration of 100 mM in DMSO (Sigma-Aldrich) and stored at −20 °C under light-free conditions. All three stock solutions were freshly diluted in cell culture medium for each use.3 cells per well (Saos-2 cells) or 2 × 103 cells per well  in 200 μL of complete DMEM. Cells were allowed to adhere overnight. Subsequently, the medium was removed, and fresh medium containing the appropriate concentrations of drugs alone or in combination was added. Cells were treated with five concentrations of calcitriol , five concentrations of calcidiol , and one concentration of ATRA (1 μM). The plates were incubated under standard conditions for 3 or 7 days.For proliferation tests, 96-well plates were seeded with 5 × 10To prepare samples for immunoblotting and PCR analyses, cells were seeded onto Petri dishes and allowed to adhere overnight. The medium was removed and replaced with fresh medium containing 10 nM calcitriol, 100 nM calcidiol, and/or 1 μM ATRA. For immunoblotting and qPCR, cells were harvested after 24 h of treatment, and for semiquantitative RT-PCR, cells were harvested after 1, 3, 5, 7, and 9 days of treatment.In all experiments, untreated cells were used as controls. In addition, we compared the proliferation activity of untreated cells and cells treated with vehicle (DMSO/ethanol) only and found no significant difference.Cell viability was evaluated using the MTT assay, which was performed as previously described . BrieflyHSP90AB1 expression. Each experiment was performed in triplicate.The expression of osteogenic differentiation markers was evaluated using semiquantitative RT-PCR. The protocol included standard procedures that were previously described . The pri® FAST qPCR Kit  and analyzed using the 7500 Fast Real-Time PCR System and 7500 Software v. 2.0.6 . Changes in the transcript levels were calculated using Cq values standardized to a housekeeping gene (GAPDH) used as an endogenous reference gene control. The established Saos-2 cell line served as the arbitrary calibrator. The primers used for genes of interest are provided in The relative expression levels of selected nuclear receptors were studied using RT-qPCR. Total RNA was extracted and reverse transcribed into cDNA in the same manner as described previously . RT-qPCRCells were lysed in LB1 buffer , and the total protein amount was subsequently measured by the DC Protein Arrays Reagents Package  according to the manufacturer’s instructions. Total proteins (10 μg) were loaded onto 10% polyacrylamide gels, electrophoresed, and blotted on a polyvinylidene difluoride membrane (Bio-Rad Laboratories). The membranes were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20 (Sigma-Aldrich) and incubated with primary antibodies overnight. The next day, membranes were incubated with secondary antibodies at room temperature (RT) for 1 hour. All antibodies used for immunoblotting are listed in p < 0.05 and ** p < 0.001 were considered statistically significant. Analysis of possible interactions of compounds included in this study was performed using the Bliss independence model [t-test (two-tailed): * p < 0.05 was considered statistically significant.Quantitative data were statistically evaluated using SPSS Statistics software . Data obtained in the MTT assay were analyzed by one-way ANOVA, followed by the Scheffé post hoc test: * ce model . Data ob


Drug testing is often performed using 2D monolayer cultures or 3D spheroid models. Here, we investigate the impact that these different in vitro models have on the carboplatin response of four EOC cell lines, and in particular how different 3D models (polydimethylsiloxane-based microfluidic chips and ultra low attachment plates) influence drug sensitivity within the same cell line. Our results show that carboplatin responses were observed in both the 3D spheroid models tested using apoptosis/cell death markers by flow cytometry. Contrary to previously reported observations, these were not associated with a significant decrease in spheroid size. For the majority of the EOC cell lines (3 out of 4) a similar carboplatin response was observed when comparing both spheroid methods. Interestingly, two cell lines classified as resistant to carboplatin in 2D cultures became sensitive in the 3D models, and one sensitive cell line in 2D culture showed resistance in 3D spheroids. Our results highlight the challenges of choosing the appropriate pre-clinical models for drug testing.Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America, underscoring the need for the development of new therapeutic strategies for the management of this disease. Although many drugs are pre-clinically tested every year, only a few are selected to be evaluated in clinical trials, and only a small number of these are successfully incorporated into standard care. Inaccuracies with the initial  These c50’s for carboplatin, as determined by clonogenic survival assay, have previously been reported for TOV3041G, TOV112D, and OV866(2) . 50 (by clonogenic assay) of the most resistant cell lines OV90 and OV866(2)  see . Then, t6(2) see . However6(2) see . For sph6(2) see  inserts.The carboplatin sensitivity of cell lines in 2D culture was originally obtained using the clonogenic assay , where cWhen comparing the percentage of live cells by flow cytometry  in the three methods tested, i.e. monolayer, spheroids from PDMS-based microfluidic devices and Matrigel-assisted spheroids, we observed that the spheroids of TOV3041G were significantly more resistant to carboplatin than in monolayer culture, and that the spheroids of OV90 and OV866(2) were significantly more sensitive than their respective monolayer cultures. This was independent of the spheroid formation method used . HoweverIn recent years, 3D cell culture models such as multi-cellular spheroids have become more widely used as pre-clinical models and are expected to bridge the gap between 2D and animal models. In the particular case of ovarian cancer, 3D spheroids are clinically relevant models since in advanced disease EOC cells can spread to the peritoneum as aggregates/spheroids , 53. In We evaluated the carboplatin response of EOC cell lines grown as spheroids using these two methods and compared these to the respective monolayer cultures. Spheroid size is widely used as a measure of drug response in several studies, including those with EOC cells , 45, 51,vs 3D [In a recent report , a compavs 3D , 46. BasWhen comparing the two spheroid models, we generally obtained similar carboplatin responses, except for the TOV112D cell line. Spheroids of this cell line were more resistant than the monolayer when assayed using the PDMS-based microfluidic device, but more sensitive in the Matrigel-assisted ULA plates. It is possible that this cell line is more affected by the Matrigel presence than the others. It is known that ECM components, like Matrigel, can affect drug responses , performAlthough descriptive, the present work aims to emit a clear warning that there is no strong tendency of EOC cells to become more resistant in 3D than in 2D, and that spheroid size is an inefficient predictor of cell response as size variation can be both a change of cell number or a change in tissue compactness.in vitro pre-clinical model can have a major influence on correctly predicting drug response in human clinical trials. Here, we evaluated carboplatin sensitivity of four epithelial ovarian cancer cell lines using both 2D and 3D in vitro models and tested different spheroid forming methods. Channel-based spheroid forming chips held several advantages over commercially available ultra-low attachment plates or the hanging droplet method. Irrespective of the spheroid forming method used, some striking carboplatin sensitivity inversions were noted within a given cell line when 2D and 3D results were compared. Overall, the present study highlights the challenges of in vitro models and discusses the different facets of 2D and 3D in vitro drug testing.In drug discovery, the choice of an appropriate S1 FigGraphs represent an example of flow cytometry analysis of spheroids treated with 300 μM carboplatin and its respective control. After excluding cell debris, viable cells were selected based on the absence of 7-AAD and/or Annexin-V markers (red square). Apoptotic and/or dead cells are shown in the other quadrants.(TIF)Click here for additional data file.S2 Fig(A) Bright field images at day 4. (B) Bright field images at day 6. Scale bar, 500 μm.(TIF)Click here for additional data file.S3 FigBright field images at day 2. Note that TOV112D cells in these conditions form multiple spheroids of smaller size. Scale bar, 300 μm.(TIF)Click here for additional data file.


Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens. Culicoides are vectors of a variety of pathogens of veterinary or medical importance, including viruses, protozoa and helminths [Culicoides midges transmit bluetongue virus (BTV) and Schmallenberg virus (SBV) that cause severe, economically important diseases in ruminants [Culicoides were also recognised over 40 years ago as being implicated in the debilitating skin condition “sweet itch” in horses [Biting midges of the genus elminths ,2,3,4,5.uminants ,9,10,11.uminants . Culicoin horses , and caun horses .Culicoides nubeculosus (Meigen 1830), which is found across the Western Palearctic region from Spain and the United Kingdom in the west to Poland and Turkey in the east [C. nubeculosus transmits the filarial worm Onchocerca cervicalis [O. cervicalis were found to increase susceptibility to BTV in a small proportion of midges [C. nubeculosus is one of very few species of the Ceratopogonidae that has been maintained continuously in colonies, although only one primary line, that was established from a field population in 1969 [Monoculicoides, to which C. nubeculosus belongs, is notable for larvae that possess heavily sclerotised mouthparts that enable an omnivorous diet and this may facilitate colonisation, as two other species within this subgenus, Culicoides sonorensis Wirth and Jones and Culicoides riethi Kieffer, have been colonised and maintained successfully for several years [Culicoides in Europe including Culicoides imicola Kieffer, the major sub-Saharan vector of BTV and AHSV, and Culicoides impunctatus Goetghebuer, the species inflicting the most severe biting nuisance on humans, have been more challenging to maintain in the laboratory, often refusing to mate and with slow developmental times and high rates of mortality across life stages [The European midge species the east ,16,17,18the east ,11. C. nrvicalis , and coif midges . C. nubeal years . Other se stages .C. sonorensis, previously known as Culicoides variipennis [Culicoides antiviral immunity [Wolbachia [Culicoides species, and the existing C. sonorensis cell lines may have limited applicability to research on arboviruses and other microorganisms prevalent in the European environment.Continuous cell lines derived from arthropods such as mosquitoes and ticks are essential laboratory research tools for isolation and propagation of vector-borne pathogens and investigation of host-vector-pathogen interactions . Biting iipennis ,23,24, hiipennis ,26,27,28immunity  and abilolbachia . One of C. nubeculosus. The ability of one of the cell lines was tested to support the replication of strains of two serotypes of BTV, BTV-1 known to be transmitted by midges and BTV-26 with no known arthropod vector.Here we report the establishment and partial morphological and molecular characterisation of two continuous cell lines from embryos of the European midge species C. nubeculosus eggs used in the study were produced at The Pirbright Institute as described previously [C. nubeculosus midges collected in Hertfordshire, UK [eviously , except hire, UK  and has C. sonorensis eggs [g for 2 min, the supernate was removed and the eggs resuspended in 70% ethanol. After 5 min the egg suspension was centrifuged again, the ethanol replaced with Hanks balanced salt solution (HBSS), and the egg suspension centrifuged again. Finally, the eggs were resuspended in 0.5 mL of HBSS or complete culture medium, transferred to a 35 mm sterile plastic petri dish and crushed with the flattened end of a sterile glass rod. The resultant suspension of midge tissues, eggshells and some uncrushed eggs was diluted with additional HBSS or complete medium in a centrifuge tube and allowed to settle for about 2 min. The topmost layer of settled material was collected in about 0.5 mL and transferred to a flat-sided culture tube  to which was added a further 1.5 mL of complete culture medium. All medium components were obtained from Invitrogen  or Sigma  unless otherwise indicated. The complete media used were: L-15 (Leibovitz) supplemented with 10% tryptose phosphate broth (TPB), 20% foetal bovine serum (FBS), 2mM L-glutamine (L-glut), 100 units/mL penicillin and 100 µg/mL streptomycin (pen/strep) (L-15); HBSS supplemented with 0.5% lactalbumin hydrolysate, 20% FBS, L-glut and pen/strep (H-Lac); L-15B [Drosophila medium  supplemented with 10% FBS, L-glut and pen/strep (Schneider’s). Cultures were incubated at 28 °C in sealed containers in ambient air in a dry incubator. The medium was changed weekly by removal and replacement of 1.0–1.5 ml medium. Cultures were examined weekly with an inverted microscope for signs of tissue viability and cell growth.Primary cultures were prepared following the method used originally with sis eggs  with som); L-15B  supplemeWhen inverted microscope examination revealed the occurrence of significant levels of cell multiplication, characterised by the formation of floating clumps of multicellular vesicles and patches of attached small elongated, spindle-shaped or epithelial-like cells, subculture was carried out. After the medium change, an equal volume of fresh medium was added to a primary culture, the attached tissue clumps and cells were pipetted off, mixed gently and half of the cell suspension was transferred to a new flat-sided tube, leaving the remainder in the parent tube. When the daughter tube showed evidence of further cell growth, the procedure was repeated.g for 5 min, the supernate was discarded, the cell pellet was resuspended in 1.5–2 mL of ice-cold complete medium with 20% FCS, an equal volume of ice-cold complete medium with 20% FCS and 20% dimethyl sulphoxide (DMSO) was added and the cell suspension was immediately divided in 1 mL aliquots into ice-cold cryovials. The cryovials were frozen rapidly in dry ice and transferred within 30 min to the vapour phase of a liquid nitrogen refrigerator. Cryopreserved cells were resuscitated by rapidly thawing the cryovial in a 37 °C water bath, immediately transferring the contents to 9 mL complete medium with 20% FCS at room temperature, centrifuging at 400× g for 5 min, resuspending the pellet in 2.2 mL of medium with 20% FCS, transferring to a sealed flat-sided tube and incubating at 28 °C. When sufficient numbers of daughter tubes were available, the contents of 3–4 tubes were pooled and centrifuged at 400× cytochrome oxidase 1 (cox1) gene was amplified following a previously published protocol [Mycoplasma using the Myco-Alert kit  and the Mycoplasma PCR ELISA kit , following the manufacturers’ instructions.Cytocentrifuge smears were prepared from both cell lines using a Cytospin 3 , air-dried, fixed for 3 min in methanol and stained for 20 min in 10% Giemsa stain. DNA was extracted from both cell lines using a DNeasy Mini Kit  following the manufacturer’s instructions for cultured cells. A 710 bp fragment of the mitochondrial protocol . A pan-bprotocol  was usedg for 5 min and the pellet re-suspended in 5 mL 0.75% sodium citrate and incubated for 35 min at 37 °C. The cell lysate was centrifuged as before, the pellet was re-suspended in 5 mL ice-cold acetic alcohol  and held on ice for 5 min. This process was repeated, and finally, the cell lysate was centrifuged as before and the pellet was gently resuspended in an equal volume of ice-cold acetic alcohol. Single drops were dripped onto ice-cold, wet, clean microscope slides from a height of approximately 90 cm. The slides were air-dried and then stained with 3% Giemsa for 1 h. The slides were examined under a light microscope at a magnification of × 250 to count the chromosomes in at least 100 spreads per cell line.Chromosome spreads were prepared from both cell lines as follows. Cells were seeded into flat-sided tubes and incubated at 28 °C for 44 h; 625 μL of colcemid  was added to arrest cell development at metaphase, and the cells were incubated for a further 18 h. Cells were then harvested, centrifuged at 400× g for 5 min, washed once in PBS and fixed in 2.5% glutaraldehyde (w/v) in 0.1 M phosphate buffer  in a Pelco Biowave . The cells were then washed three times in 0.1M phosphate buffer before being embedded in 3% agarose, set on ice and cut into small cubes. The cubes were then post-fixed and stained with 2% aqueous osmium tetroxide in the Pelco Biowave, followed by 2% aqueous uranyl acetate overnight at 4 °C. To prevent precipitation artefacts, the cubes were washed for a minimum of 5 × 3 min with double-distilled water (ddH2O) between each staining step. The cubes were then washed in ddH2O before dehydration in a graded series of acetone at 30%, 50%, 70% and 90% in ddH2O for 5 min each, followed by 2 × 5 min in 100% acetone. Samples were then infiltrated with medium Premix resin  at 30%, 50% and 75% resin in acetone for 30 min each, followed by 3 × 100% resin steps for 30 min each. Fresh 100% resin was used to embed pellets in silicone moulds before being cured for 48 h at 60 °C. Ultrathin serial sections (70–75 nm) were cut on a UC6 ultramicrotome  and collected on formvar coated copper grids. Grids were post-stained with 4% uranyl acetate and lead citrate, before viewing at 120KV in an FEI Tecnai G2 Spirit transmission electron microscope . Images were taken with a RIO16 camera  using GMS3 software.Cell line CNE/LULS44 was prepared for transmission electron microscopy (TEM) as follows. Cells were harvested, centrifuged at 1000× 5 cells per well and incubated for 48 h at 28 °C inside a humidified container. C. sonorensis-derived KC cells [C. nubeculosus cells, were used as controls. Triplicate wells were infected with ~4 × 104 plaque-forming units (PFU) per well of BTV-1 ) as described previously [2 flasks of BSR cells [g for 10 min. The supernate was discarded and the pellet was suspended in 5 mL of Glasgow’s modified Eagle’s medium (GMEM), treated with an equal volume of Vertrel XF  as previously described [2 = 0.982). The number of PFU-equivalents for BTV-1 were calculated using the formula: y = −1.916ln(x) + 40.776, where y is the Ct value determined by real-time RT-PCR assay and x is the number of PFU-equivalents/mL. For BTV-1, the value of x was x = e(y−40.776)/(−1.916), where e stands for exponential, the mathematical constant or base of the natural logarithm that equals 2.71828. CNE/LULS44 cells were seeded in 24-well plates at a density of approximately 5 × 10KC cells , maintaiArrrr/01 ) or BTV-eviously . Brieflyeviously . PFU equSR cells  inoculatescribed , then trescribed . RNA wasC. nubeculosus eggs supplied on three occasions between March and June 2018, using one or a combination (1:1) of the four culture media L-15, H-Lac, L-15B and Schneider’s. Of these, five cultures were lost to contamination because the egg batches contained some larvae that had hatched prior to surface sterilisation; larvae that hatched from intact eggs after surface sterilisation did not pose a contamination risk, but did consume embryonic tissues. A further 10 cultures did not contain viable embryonic tissues and were discontinued. Two primary cultures contained small clumps of viable tissues attached to the surface of the tube within 48 h. Some of these clumps contained contractile tissue that twitched or pulsated, and small, spindle-shaped or elongated cells began to migrate out from some of the clumps. This process continued slowly over the subsequent weeks, with a gradual increase in confluence and muscular activity. After 2–8 months, multicellular vesicles began to appear, either attached to the cell layer or free-floating individually or in clusters , and sequences from Turkey  and France . Five of the eight published C. nubeculosus sequences were only 472 bp in length which may explain why BLAST query coverage was lower (70%) for several of the matches to our 616–617 bp sequences. The pan-bacterial 16S rRNA PCR did not amplify any products from either cell line, and both tests for contaminating Mycoplasma gave negative results. Both cell lines were confirmed as C. nubeculosus cell lines comprised diverse populations of the attached round, spindle-shaped and elongated cells, cell clumps and floating multicellular vesicles derived from at least two different cell phenotypes. With time, and in later passages, elongated and clumped cells predominated in CNE/LULS44 (n = 6 [n = 12), while the remaining cells were aneuploid, with 4–21 and 4–24 chromosomes per spread from CNE/LULS 44 and CNE/LULS47 respectively . Following inoculation with BTV-26, no replication was detected by RT-PCR in CNE/LULS44 or KC cells  [Wolbachia [Culicoides species of either New or Old World origin. The two new C. nubeculosus cell lines reported here, CNE/LULS44 and CNE/LULS47, will therefore greatly increase the scope for research into many aspects of midges as vectors of viruses, protozoa and helminths, especially those relevant to Europe. They represent a novel comparative resource that is likely to expand research on Culicoides genetics and genomics and also provide an additional platform for the study of vector-host interactions. An important advantage is that long-term maintenance of both the cell lines and the C. nubeculosus colony from which they were derived is sustainable, ensuring the continued availability of matched resources. The first dge eggs ,23. Sinc and W8) . Two add and W8) . Of thes and W8) ,27,28,29C. sonorensis cell lines [C. nubeculosus eggs. The age of the eggs was important, as the presence of hatched larvae prior to surface-sterilisation invariably resulted in contamination of the cultures; hatching could be successfully delayed up to 5 days by holding eggs at 4 °C but not at 15 °C. A variation of the surface-sterilisation protocol applied to tick eggs [C. sonorensis eggs [C. nubeculosus eggshells with a flattened glass rod was likely to be less damaging to the embryonic tissues than the glass tissue homogeniser used for C. sonorensis eggs [C. nubeculosus cells, while they grew prolifically in media based on L-15 (Leibovitz) medium. Interestingly, L-15 (Leibovitz) medium supplemented with 20% FBS, rather than Schneider’s medium, was used to propagate KC cells for karyotyping [Culicoides species of veterinary or medical importance.In the present study, the techniques applied to the generation of the ll lines ,24 were ick eggs , using 0sis eggs . Crushinsis eggs , but it yotyping . These aC. nubeculosus cell lines are phenotypically heterogeneous, comprising round, spindle-shaped and elongated cells of different sizes, growing as attached individual cells, cells forming attached sheets and clumps, and floating multicellular vesicles. At the time of writing, the proportion of cells forming floating vesicles in CNE/LULS44 has greatly decreased, while the proportions of attached, sheet- and clump-forming cells remain constant, whereas vesicle-forming cells remain numerous in CNE/LULS47, making up at least half of the total biomass in the cultures. The phenotypic diversity is reflected in Giemsa-stained and TEM preparations, which demonstrate multiple cell types. In the TEM preparations, CNE/LULS44 cells displayed prominent mitochondria, some of which appeared to be multilobed or to have bizarre morphology. Little information on Culicoides mitochondria is available from published studies; numerous large mitochondria were reported in sheath cells surrounding mechanoreceptor sensillae of C. nubeculosus [Culicoides punctatus midgut cells [C. sonorensis cell lines was also reported [C. nubeculosus cell lines do not harbour any endogenous viruses; it will be important to apply whole genome sequencing to both cell lines in future to explore their putative viromes [Morphologically, both eculosus , and ovoreported ; the tworeported , does no viromes .Culicoides spp. cells. The majority (68.5%) of C. sonorensis KC cells were reported to have a diploid number of 2n = 6 chromosomes; of the remainder, 30% were tetraploid and 1.5% had 24 chromosomes [C. nubeculosus cell lines in the present study, except that a much smaller proportion of spreads were tetraploid, and 32% and 20% of spreads were aneuploid in CNE/LULS44 and CNE/LULS47, respectively. Aneuploidy has frequently been reported in cell lines derived from other arthropod species including ticks [There is very little published information on chromosome numbers in omosomes . These vng ticks , sand flng ticks .Culicoides, we tested the ability of one of the novel C. nubeculosus cell lines to support replication of two serotypes: BTV-1, a typical serotype that is Culicoides-transmitted, and BTV-26, an atypical serotype that is suspected not to be transmitted by insects and failed to replicate in KC cells or C. sonorensis midges [C. sonorensis. However, in contrast to these results for intracellular viral RNA, preliminary results indicated that extracellular viral RNA was present at lower levels in the supernates of CNE/LULS44 cultures than in those of KC cell cultures, which suggests the possibility that in CNE/LULS44 cells, a large proportion of virus particles remained cell-associated. Further studies using a wider range of midge-transmitted BTV serotypes are required to confirm and investigate this observation. In contrast, BTV-26 failed to replicate in both KC and CNE/LULS44 cells, confirming its status as an atypical bluetongue virus which appears to be unable to replicate in Culicoides midges. As BTV is one of the most economically important and widespread livestock viruses transmitted by s midges . Using aC. nubeculosus for BTV has been reported to be low, based on studies in which viral RNA replication was detected in only a small proportion of tested individuals following infection by membrane feeding on BTV-1-spiked horse blood [C. nubeculosus was a competent experimental vector of BTV-4 following membrane feeding and supported virus replication following feeding on an infected sheep [O. cervicalis microfilariae rendered a small proportion of individuals in a refractory C. nubeculosus population capable of transmitting an unspecified serotype of BTV [C. nubeculosus may be more complex if the apparent low ability of CNE/LULS44 cells in vitro to release mature BTV-1 particles reflects the conditions in intact midges. Despite broad assessments of susceptibility to infection and studies of barriers to arbovirus dissemination in Culicoides [Vector competence of se blood . Earliered sheep , and thae of BTV . The aute of BTV ,53. Howelicoides ,53,54, BC. nubeculosus has been tested experimentally for the ability to support replication of, and/or transmit, other midge-borne arboviruses. Virus replication following intrathoracic inoculation, but not an oral infection, was demonstrated for the orbiviruses epizootic hemorrhagic disease virus, AHSV and Eubenangee virus, and the orthobunyavirus Akabane virus, all of which could be transmitted orally by C. sonorensis [C. nubeculosus with the orthobunyavirus SBV was demonstrated experimentally [C. nubeculosus following oral infection was demonstrated for two other orthobunyaviruses, Main Drain virus [C. nubeculosus cell lines to support replication of other insect-borne orbiviruses, tick-borne orbiviruses, SBV and other orthobunyaviruses, and indeed intracellular bacteria of the genera Wolbachia [Cardinium [Rickettsia [Culicoides biting midges.norensis ,57,58,59in virus  and Shunin virus . It willolbachia , Cardiniardinium ,63 and Rckettsia , represeC. nubeculosus cell lines reported here will greatly expand the scope of research into Culicoides-borne pathogens at the cellular and molecular level. Virus replication and production can now be compared in vitro between cells of midge species with the different vectorial capacity for arboviruses such as BTV, facilitating a better understanding of the infection and release mechanisms underlying vector competence and virus transmission. The study not only provides a baseline for further characterisation of the existing Culicoides spp. cell lines, but will also facilitate the establishment of new cell lines from major arbovirus vector species for which only preliminary studies of colonisation have been carried out, such as the major Afrotropical BTV vector C. imicola [C. nubeculosus cell lines CNE/LULS44 and CNE/LULS47 are deposited in the Tick Cell Biobank at the University of Liverpool and will be available for distribution from December 2020.In conclusion, the two new  imicola . The C.


The identification of noninvasive biomarkers able to detect renal cell carcinoma (RCC) at an early stage remains an unmet clinical need. The recognition that altered metabolism is a core hallmark of cancer boosted metabolomic studies focused in the search for cancer biomarkers. The present work aims to evaluate the performance of the volatile metabolites present in the extracellular medium to discriminate RCC cell lines with distinct histological subtypes (clear cell and papillary) and metastatic potential from non-tumorigenic renal cells. Hence, volatile organic compounds (VOCs) and volatile carbonyl compounds (VCCs) were extracted by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography–mass spectrometry (GC–MS). Multivariate and univariate analysis unveiled a panel of metabolites responsible for the separation between groups, mostly belonging to ketones, alcohols, alkanes and aldehydes classes. Some metabolites were found similarly altered for all RCC cell lines compared to non-tumorigenic cells, namely 2-ethylhexanol, tetradecane, formaldehyde, acetone (increased) and cyclohexanone and acetaldehyde (decreased). Furthermore, significantly altered levels of cyclohexanol, decanal, decane, dodecane and 4-methylbenzaldehyde were observed in all metastatic RCC cell lines when compared with the non-metastatic ones. Moreover, some alterations in the volatile composition were also observed between RCC histological subtypes. Overall, our results demonstrate the potential of volatile profiling for identification of noninvasive candidate biomarkers for early RCC diagnosis. Renal cell carcinoma (RCC) accounts for more than 90% of all kidney malignancies  and reprRCC has a favorable prognosis when detected at an early stage . HoweverThe use of metabolomics for investigation of novel biomarkers in oncobiological studies has been rising over the years . MetabolVolatile organic compounds (VOCs) are end products of cellular metabolism excreted by cells ,21 and tHence, this study aimed to investigate, for the first time, the performance of VOCs and VCCs in discriminating RCC cell lines  from one non-tumorigenic renal cell line (HK-2). We further investigated which metabolites might discriminate RCC cell lines according to their histological subtype (ccRCC vs. pRCC) and metastatic potential (metastatic vs. non-metastatic RCC). Prior to analysis by gas chromatography-mass spectrometry (GC–MS), VOCs present in the extracellular medium were directly extracted by headspace solid-phase microextraction (HS-SPME), while VCCs were first derivatized before HS-SPME to enhance the detection of aldehydes (CHO-) and ketones (CO-) . KnowledIn this study, the exometabolome volatile profile of several RCC cell lines  and one non-tumor renal cell line (HK-2) was analyzed to investigate whether volatile compounds might be able to discriminate tumorigenic from non-tumorigenic cells. The two most common histological subtypes, ccRCC and pRCC, at different disease stages were selected for this study, thus extending our investigation to the metabolic differences that characterize the histological subtype and tumor stage. The number of peaks detected in the extracellular medium in VOCs and VCCs analyses were 105 and 111, respectively . A very 2 > 0.5). Pairwise multivariate supervised analysis (PLS-DA) was performed between each RCC cell line and the non-tumorigenic cell line for both analytical methodologies, VOCs and VCCs (m/z 58), and the decrease in the levels of acetaldehyde and cyclohexanone. These results suggest the existence of a common regulatory mechanism in ccRCC and pRCC cells.The compounds altered between each tumor cell line versus the non-tumor are shown in The analysis of the volatile profile of blanks enabled the interpretation of dysregulations occurring in RCC cells in terms of excretion or consumption of culture medium nutrients. The heatmap depicted in 2 > 0.7; 2 = 0.211) was observed in VCCs (2 > 0.9) was observed in both analytical methodologies , and decreased levels of acetaldehyde and cyclohexanone. These results suggest that different histological RCC subtypes have, at least in part, a common regulatory mechanism that affects the metabolic pathways in which these compounds are intermediates.The profiling of volatile compounds present in the culture medium (exometabolome) unveiled important alterations associated with the excretion and consumption of nutrients by RCC cells lines when compared with a non-tumor cell line. In addition, different RCC histological subtypes (ccRCC and pRCC), as well as different stages of the disease (metastatic and non-metastatic) disclosed characteristic exometabolome signatures. These metabolic alterations may constitute a panel of potential biomarkers yet to be investigated in urine from RCC patients, compared with cancer-free individuals. Common to all cancer cell lines under study, we found increased levels of 2-ethylhexanol, tetradecane, formaldehyde, acetone and an unknown compound  in the extracellular medium. The main mechanism that affects the production of hydrocarbons is the oxidative stress, since the production of these volatile metabolites is mainly due to the induction of ROS-mediated polyunsaturated fatty acid peroxidation . In factAnother major change observed in this study was a significant increase of some alcohols (namely 2-ethylhexanol) in RCC cell lines when compared to the non-tumorigenic cell line. These results can be explained by a possible up-regulation of cytochrome P450 (CYP450) enzymes, which can hydroxylate several volatiles formed during ROS-mediated lipid peroxidation, leading to the production of the corresponding alcohols . This thIn addition, our metabolomic results also showed that most of the aldehydes  were significantly increased in RCC cell lines. Increased levels of aldehydes may derive from different mechanisms including a direct origin in the reduction of hydroperoxides by CYP450 or from the conversion of some alcohols to their corresponding aldehydes mediated by alcohol dehydrogenases (ADHs) . Since tRemarkably, acetaldehyde, another well-known aldehyde, appears significantly decreased in all RCC cell lines when compared to the non-tumorigenic cell line. This finding is opposite to what would be expected based on aforementioned higher activity of ADH in renal cancer cells. The ability of acetaldehyde to bind to DNA and cellular proteins to form adducts may explain the results verified in the exometabolome . MoreoveKetones were also found altered in the exometabolome of RCC cells compared with the non-tumor cell line, with special emphasis for acetone whose levels are significantly increased. Several studies ,23,41 ham/z 59) were found significantly altered only in this pRCC cell line. Contrary to other RCC histological subtypes, ccRCC and pRCC arise from adjacent parts of the proximal tubule [In contrast, cyclohexanone was found decreased in the exometabolome of all RCC cells. Of note, this finding is not in agreement with the previous study by Wang et al.  where thl tubule , which ml tubule , thus bel tubule ,44, our Regarding the comparison metastatic vs. non-metastatic stage in both the clear cell and the papillary RCC cell lines, the results suggest a variation of the volatile compounds in a stage-dependent manner since statistically significant differences in the levels of several metabolites were found. Curiously, the separation between metastatic and non-metastatic cell lines was more robust in the pRCC subtype, which justifies the markedly altered profile of the pRCC cell line, Caki-2. These results are consistent with the findings reported by Schaeffeler et al. , which dThis study demonstrates for the first time, to our knowledge, the potential of volatile compounds in discriminating RCC cells from nonmalignant cells, according to the histological subtype and tumor stage. However, the metabolic pathways that lead to VOCs and VCCs production are not yet clearly understood, hindering an accurate interpretation of the results. Nevertheless, our findings support the hypothesis that altered energy production in RCC cells enables them to survive under oxidative stress conditions and to migrate to other tissues and form metastases . This stO- hydroxylamine  and thymol (≥ 98.5%) were obtained from Sigma-Aldrich . The antibiotic mixture penicillin/streptomycin (10.000 U/mL/10.000 mg/mL), heat-inactivated fetal bovine serum (FBS) and trypsin-EDTA (0.25%) were purchased from GIBCO Invitrogen . Hydrochloric acid (HCl) and sodium hydrogencarbonate were obtained from Merck .All chemicals were of analytical grade and dissolved in deionized water unless otherwise indicated. RPMI-1640 medium, sodium chloride , 2. All cell lines were routinely tested for Mycoplasma spp. contamination .Five human RCC immortalized cell lines  and one non-tumorigenic renal cell line (HK-2) were purchased from the American Type Culture Collection  and their characteristics are summarized in g, 4 °C). Then, the supernatant was divided into two 2.5 mL aliquots and immediately stored at −80 °C until analysis. A pool of all samples and blanks, called quality control (QCs), was also prepared under the same collection and storage conditions.For each cell line, two different cell stock vials were grown in parallel and the experiments carried out up to 7 consecutive passages in plastic T25 culture flasks, after an adaptation stage of at least three passages for all cell lines. After 24 h, a confluence of 50%–60% was observed, the culture medium was discarded and replaced with 7 mL of fresh culture medium . The culture flasks were maintained in the incubator for another 48 h reaching near confluence, together with blanks (T25 culture flasks with the same culture medium but without cells). After 48 h, the extracellular medium from each culture flask with cells and blanks was collected and centrifuged  hydroxylamine, 40 g/L) and the compounds were extracted using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber with an incubation time of 6 min at 62 °C and extraction using the same temperature during 51 min, under continuous stirring (250 rpm) [Sample preparation was performed as described in previous studies published by our group ,24,28,48m/z at a scan rate of 6 scans/s. VCCs were analyzed using a 436-GC system  coupled to a EVOQ Triple Quadrupole (TQ) mass detector and a Bruker MS workstation software version 8.2. The column, carrier gas and oven temperature conditions were the same described above for VOCs analysis. The MS detector was operated in the electron impact mode (70 eV) at 270 °C, the temperature of transfer line and manifold were 260 °C and 40 °C, respectively. Data acquisition was performed in the full scan mode considering a 50–600 m/z mass range.VOCs detection was accomplished using a 436-GC system  coupled to a SCION Single Quadrupole (SQ) mass detector and a Bruker Daltonics MS workstation software (version 8.2). A fused silica capillary column Rxi-5Sil MS  was used and helium C-60  was the carrier gas (flow rate 1 mL/min). The oven temperature was fixed at 40 °C for 1 min, increasing to 250 °C (rate 5 °C/min), held for 5 min, followed by increasing to 300 °C (rate 5 °C/min) and held for 1 min. The MS detector was operated in electron impact mode (70 eV) at 260 °C, the transfer line temperature was 250 °C and the manifold temperature was 40 °C. Data acquisition was performed in full scan mode with a mass range between 40 and 350 Compounds detected in both VOCs and VCCs analytical methodologies were identified by comparison of the MS fragmentation with the mass spectra present in the National Institute of Standards and Technology (NIST 14) database and by comparing the experimental Kovats retention index (RI) with literature. When possible, the identification was confirmed by comparison of the retention time and MS spectra of the samples with the commercially available standard compounds analyzed using the same conditions.m/z range 50–250; MS data noise level 1 × 104; m/z tolerance 0.3; chromatogram baseline level 5 × 104 and peak duration range 0.05–1 min. For VCCs analysis, the parameters used were RT range 9–45 minutes; m/z range 50–500; MS data noise level 1 × 104; m/z tolerance 0.3; chromatogram baseline level 1 × 104; peak duration range 0.03–0.2 min. GC contaminants from the chromatographic column or fiber (among others) were manually removed from the final data matrix.Before statistical analysis, the data obtained from GC–MS were converted into a manageable format (netcdf) and preprocessed using MZmine 2.53 . The prep-values for multiple comparison using the MetaboAnalyst 4.0 [p-value < 0.05 were considered statistically significant. In addition, the AUC (area under the curve) was computed for each discriminant compound using the same analytical platform. The effect size and the standard error were also computed as described by Berben et al. [The statistical analysis pipeline used for VOCs and VCCs final matrix was the same. First, data were normalized by the total area (TA) of the chromatogram and scaled to Pareto. Then, PCA  was applied to detect trends, outliers and to evaluate the distribution of the QCs, followed by PLS-DA  to discriminate classes and to identify the specific metabolic signature of each cell line, in SIMCA 15.0.2 . A default sevenfold internal cross-validation and permutation testing were used to confirm the robustness of PLS-DA models. All volatile compounds with VIP (variable importance in the projection) higher than one were considered important for group discrimination and were subsequently submitted to the Mann–Whitney test. The false discovery rate (FDR) method was used to adjust lyst 4.0 , and comn et al. . FinallyIn this study, we used an in vitro metabolomics approach to obtain the volatile signatures of RCC cells with distinct histological subtypes and stages. The results showed that alterations in the volatile profile are capable of discriminating RCC from non-tumorigenic renal cells. This unique volatile profile of RCC cells can be useful to unveil promising noninvasive biomarkers for early diagnosis in more complex biological matrices, such as urine. A multi-biomarker panel can provide a more robust and accurate tool for RCC diagnosis than the use of only one metabolite as a biomarker. Indeed, 2-ethylhexanol, tetradecane, formaldehyde, acetone, cyclohexanone and acetaldehyde were able to discriminate RCC cell lines from the non-tumor cell line and may constitute a potential panel of candidate biomarkers for RCC diagnosis. This study also demonstrated that the volatile profile of metastatic cell lines markedly differed from the non-metastatic in each RCC subtype, indicating some promising biomarkers applicable to the categorization of tumor stage, pending further investigation and validation.