Commercial pesticide formulations contain co-formulants, which are generally considered as having no toxic effects in mammals. This study aims to compare the toxicity of 8 major herbicide active ingredients–namely glyphosate, dicamba, 2,4-D, fluroxypyr, quizalofop-p-ethyl, pendimethalin, propyzamide and metazachlor–with a typical commercial formulation of each active ingredient. Cytotoxicity and oxidative stress capability was assessed in human hepatoma HepG2 cells. Using an MTT assay, formulations of glyphosate (Roundup Probio), fluroxypyr (Hurler), quizalofop-p-ethyl (Targa Super) and dicamba (Hunter) were more toxic than the active ingredient alone. Metazachlor and its formulation Sultan had similar cytotoxicity profiles. Cytotoxicity profiles were comparable in immortalised human fibroblasts. Toxilight necrosis assays showed the formulation of metazachlor (Sultan50C) resulted in significant membrane disruption compared to the active ingredient. Generation of reactive oxygen species was detected for glyphosate, fluroxypyr, pendimethalin, quizalofop-p-ethyl, the formulation of 2,4-D (Anti-Liserons), and dicamba and its formulation Hunter. Further testing of quizalofop-p-ethyl and its formulation Targa Super in the ToxTracker assay system revealed that both products induced oxidative stress and an unfolded protein response. In conclusion, these results show that most herbicide formulations tested in this study are more toxic than their active ingredients in human tissue culture cell model systems. The results add to a growing body of evidence, which implies that commercial herbicide formulations and not just their active ingredients should be evaluated in regulatory risk assessment of pesticides. Pesticides include thousands of chemicals, which are sold to kill insects, nematodes, plants or microorganisms considered as a source of nuisance. Exposure to pesticides has been implicated in the development of human diseases causing cancer ,2, ParkiPesticides are used worldwide as mixtures known as formulations. Commercial pesticide formulations not only contain the declared pesticidal active ingredient but also ‘co-formulants’. Co-formulants serve multiple purposes including increasing solubility of the active ingredient, acting as a surfactant to allow good adherence and coverage over the target organism and allowing penetration of the active ingredient through plant cell walls in the case of herbicides. The composition of co-formulants is frequently kept confidential by manufacturers as they deem them not to have innate pesticidal action and not toxic to non-target species, including humans. Crucially, for regulatory purposes, pesticide long-term toxicity studies in laboratory animals are only conducted with the active ingredients as declared by manufacturers and not with commercial formulations ,13. HoweBufo gargarizans) as a result of exposure to the herbicides acetochlor, butachlor, paraquat and methyl methanesulfonate  solution at 1 mg/mL in phosphate-buffered saline (PBS). The resulting formazan precipitate was then dissolved by addition of 100 µL DMSO and quantified spectrophotometrically at 560 nm using a GloMax Multi Microplate Multimode Reader . Cell viability was expressed as a percentage relative to the negative control, untreated cell samples consisting of cell culture medium only.HepG2 hepatoma cells were seeded into black 96-well plates  at a density of 50,000 cells per well and incubated for 24 h. Following this, cells were treated with active ingredients or formulations at concentrations that produced a lethal concentration 50 (LC50) value of 50%, LC50/2 and (LC50/2)/2. If LC50 values could not be calculated, the highest three test concentrations from the MTT cell proliferation assays were used. Following incubation, the ToxiLight bioluminescent assay  was used according to the manufacturer’s instructions to assess cell membrane rupture and consequent necrosis. Briefly, a 50 μL aliquot of the adenylate kinase (AK) reagent was added to each well and after 5 min the plates were read using the GloMax Multi Microplate Multimode plate reader with excitation 485 nM and emission 520 nM settings. The background luminescence from wells with tissue culture medium alone was subtracted, and luminescence compared relative to the negative control, untreated cell samples consisting of cell culture medium only. Triton X100 (0.05%) was used as a cell membrane and necrosis-inducing positive control agent. HepG2 cells were seeded at 10,000 cells/well in 80 μL medium in 96 well white-walled plates, incubated for 24 h and then treated with the test substances at the desired concentrations. The positive control, reactive oxygen species (ROS)-inducing agent treatment was 50 The ToxTracker assays  were undThe experiments were repeated a minimum of 3 times in different weeks (n = 9). Dose response on HepG2 and immortalised fibroblast cells was used to determine cytotoxicity thresholds using nonlinear dose-relationships. We determined the LC50, the concentration at which 50% of the cells are viable using best-fitted value of a nonlinear regression using sigmoid (5 parameter) equation in the GraphPad Prism 9 software package. Statistical differences of necrosis and oxidative stress were calculated using nonparametrc Mann–Whitney test also using GraphPad Prism 9 software.Primary ToxTracker data were produced as flow cytometry (.fcs) files. Mean GFP expression and viable cell concentration after treatment were exported as text files (.csv) and imported into Microsoft excel for calculation of reporter gene induction and cytotoxicity. A positive response from ToxTracker is reported when a compound induces at least a 2-fold increase in GFP reporter gene expression in any of the six cell assay systems. Subsequent ToxTracker analysis was undertaken at concentrations that do not cause more than 75% cytotoxicity.The toxicity of eight herbicide active ingredients and a corresponding typical commercial formulation on two human cell lines, HepG2 hepatocytes and immortalised fibroblasts, was studied using an MTT cell viability assay, which measures mitochondrial respiration via succinate dehydrogenase activity . All paiWe next undertook a ToxiLight cell membrane damage assay in an effort to gain a deeper understanding into the mechanisms of toxicity of active ingredients and their commercial formulations. The results obtained  showed t2O2 was next investigated as an indicator of oxidative stress, to further explore mechanisms of toxicity. The positive control compound menadione resulted in a significant 6-fold increase in H2O2 production when compared to the negative control untreated samples (p < 0.01) at concentrations of 250 µg/mL and 500 µg/mL (p < 0.01), 50 µg/mL and 100 µg/mL (p < 0.001) induced a significant positive oxidative stress response (p < 0.05), 250 µg/mL (p < 0.01), and 500 µg/mL (p < 0.0001) (p < 0.05) (p < 0.01) and its formulation Hunter at 155 µg/mL (p < 0.01), 310 µg/mL and 620 µg/mL (p < 0.001)  G. Signif < 0.05) E. For di< 0.001) H.Of the active ingredients evaluated, quizalofop-p-ethyl was particularly potent at inducing oxidative stress at all concentrations tested including at the lowest concentration (10 mg/mL) of any active ingredient F suggestThe aim of this study was to investigate the differential toxicity of active ingredients and representative commercial formulations of the eight most extensively used herbicides in the UK by comparing their effect on viability and biochemical parameters indicative of carcinogenesis in two human cell lines. Our results show that the commercial formulations of glyphosate (Roundup Probio), fluroxypyr (Hurler), quizalofop-p-ethyl (Targa Super) and dicamba (Hunter) brought about a much greater decrease in cell viability compared to the active ingredient alone . The actMost of the herbicide formulations evaluated in this study have not previously been tested for toxicity in human cell culture systems. Thus, our investigation is the first to compare the effects on cell viability of pendimethalin and any pendimethalin commercial formulation. Investigations of pendimethalin alone have shown a notable increase in oxidative damage and decrease in viability of human pancreas epithelial PANC-1 tumour cells , and a dNumerous studies have compared pesticide active ingredients and respective commercial formulations in various cell lines. Gasnier and colleagues compared three glyphosate formulations namely Roundup Express, Bioforce and Grands Travaux plus and found all formulations tested resulted in a much larger decrease in cell viability than glyphosate alone in HepG2 hepatocytes . SimilarIt is not clear from this study whether the pesticide co-formulants have their own toxic effects or if they amplify the toxicity of the active ingredients. However, studies have shown that the GBH co-formulant polyethoxylated tallowamine (POE-15), is highly toxic to human cells through negatively effecting cellular respiration and membrane integrity . In addiWe previously demonstrated that the quizalofop-p-ethyl-based commercial formulation Targa Super caused a much greater decrease in cell viability than just the active ingredient after 8 days of differentiation of murine 3T3-L1 cells to adipocytes . This is2O2 production and reporter gene expression, with the latter likely to be more sensitive at detecting an oxidative stress response.We have shown that 2,4-D and to a lesser degree dicamba elicit an oxidative stress response in the ToxTracker cell assay system, and with the former also giving rise to an unfolded protein reaction . In the Given our previous investigation and thus interest in quizalofop-p-ethyl toxicity  and thatAlthough the herbicides tested in this study are the most frequently applied in the UK, it is not clear if these are also the pesticides to which humans at large are most exposed . More biThe evaluation of the contribution of the co-formulants to the toxicity of formulations is a first step. However, it is important to bear in mind that human populations are exposed to mixtures of pesticides. Numerous studies stretching back many years have demonstrated that mixtures of pesticides with each at regulatory permitted doses can nevertheless be toxic ,48,49. Thus, pesticide risk assessment also needs to include mixture effects of active ingredients in combination with co-formulants present in their commercial formulations. Mechanisms of action other than those evaluated in this study can also contribute to carcinogenic outcomes. Endocrine disrupting pesticides such as organochlorine insecticides and the herbicide atrazine can cause carcinogenic effects by altering steroid hormone signalling in tissues, including the breast . RecentlIn conclusion, the results presented in this study show that the most frequently applied commercial herbicide formulations used in the UK and in other countries are more toxic than their corresponding active ingredients. Our data add to the increasing body of evidence, which questions the validity of health risk assessments of herbicide exposure when they are established based tests of active ingredients alone.


BAX/BAK and caspase activity. Superior antitumor activity of CC‐115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM.Early data suggested that CC‐115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNA‐dependent protein kinase (DNA‐PK) may have additional targets beyond TORK and DNA‐PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense‐mediated mRNA decay (NMD), as an additional target of CC‐115. CC‐115 treatment showed a dose‐dependent increase of SMG1‐mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress‐inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC‐115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of  In this study, SMG1, a key regulator of the RNA degradation mechanism nonsense‐mediated mRNA decay (NMD), was discovered as an additional target of the clinical compound CC‐115. CC‐115 treatment showed a dose‐dependent increase of NMD transcripts, unfolded protein response (UPR) transcripts, and cell death via SMG1 inhibition in a subset of cell lines, primary multiple myeloma cells, and xenograft mouse models. AMLacute myeloid leukemiaATCCAmerican Type Culture CollectionATRataxia‐ and Rad3‐relatedATMataxia‐telangiectasia mutatedBorbortezomibBLBurkitt's lymphomaCLLchronic lymphocytic leukemiaDSMZDeutsche Sammlung von Mikroorganismen und ZellkulturenDiOC6Dihexyloxacarbocyanine IodideDMSOdimethyl sulfoxideDDRDNA damage responseDNA‐PKDNA‐dependent protein kinaseERendoplasmic reticulumKOknockoutLC–MSliquid chromatography–mass spectrometryMCLmantle cell lymphomaMMmultiple myelomaNMDnonsense‐mediated mRNA decayPIKKphosphoinositide 3‐kinase‐related kinasePTCspremature termination codonsPIpropidium iodideQVDquinoline‐val‐asp‐difluorophenoxymethylketoneSserinesgRNAssingle‐guide RNAsSMG1iSMG1 kinase inhibitorSMG1suppressor of morphogenesis in genitalia 1TORKtarget of rapamycin kinaseTGthapsigarginTthreonineTRRAPtransformation/transcription domain‐associated proteinUPRunfolded protein responseUPF1UP‐frameshift protein 11Nonsense‐mediated mRNA decay (NMD) selectively degrades mRNAs containing premature termination codons (PTCs) and controls the expression of a subset of normal RNAs . NMD is Multiple myeloma (MM) is a heterogeneous and still fatal malignancy of plasma cells. Despite a variety of recent therapeutical developments, including immunomodulatory drugs, the development of resistance to proteasome inhibitors and other drugs in MM remains prevalent, and novel therapeutic targets and strategies are still warranted , 11.CC‐115 was identified as an inhibitor of mammalian Target of Rapamycin Kinase (TORK) and DNA‐dependent protein kinase (DNA‐PK) , 13, 14.Following up on early data suggesting that CC‐115 may have additional targets beyond TORK and DNA‐PK, we characterized CC‐115 activity in a panel of cell lines and then profiled the binding activity of CC‐115 to > 200 kinases. In this way, we identified SMG1 as an additional target of CC‐115. The NMD process is highly complex and also variable, and not all mechanistic details are elucidated [22.1−/− (#HD R02‐049) cell lines]. Cells were cultured according to the vendor's recommendation.This study was conducted in accordance with the Declaration of Helsinki and approved by the AMC Medical Committee on Human Experimentation. Primary MM cell samples were obtained upon informed consent from patients diagnosed at the Academic Medical Center, Amsterdam, The Netherlands. Cell lines were purchased from American Type Culture Collection , the Deutsche Sammlung von Mikroorganismen und Zellkulturen , or Horizon Discovery Ltd  − [% cell death in medium control])/[% viable cells medium control] × 100.Cells were seeded in 96‐well plates (40 000 cells per well) and incubated in the presence of different compounds for indicated time points. Viability was measured by 0.01 μ2.10ad libitum and maintained on a 12‐h light/dark cycle. All animals in the study were tagged with stainless steel metal ear tags. Animals were acclimatized to the animal housing facility for a period of 7 days prior to the beginning of the experiment.All animal studies were performed under protocols approved by Institutional Animal Care and Use Committees. Animals were housed and xenograft studies were performed as previously published . All aniSuspensions of CC‐223 were prepared in aqueous 0.5% carboxymethyl cellulose and 0.25% Tween‐80. The formulations were homogenized using a Teflon pestle and mortar (Potter‐Elvehjem tissue grinder). For multiday studies, the compound was freshly formulated every third day. Between doses, the formulated compound was stored under constant stirring using a magnetic stirrer at 4 °C in the dark. The test article and vehicle were administered by oral gavage.6 ANBL‐6 cells. When tumors reached approximately 75–150 mm3, mice were randomized into various treatment groups and administered PO with CC‐223 or CC‐115 once daily at a dose volume of 5 mL·kg−1. Tumor volumes were determined prior to the initiation of treatment and were considered as the starting volumes. Tumors were measured twice a week for the duration of the study. The long and short axes of each tumor were measured using a digital caliper in millimeters. The tumor volumes were calculated using the formula: width2 × length/2. The tumor volumes were expressed in cubic millimeters (mm3).SCID mice were inoculated subcutaneously with 10 × 10graphpad prism. A one‐way analysis of variance (ANOVA) was performed for tumor volume measurements. Post‐hoc analysis was performed using Dunnett's test where all treatment groups are compared with the vehicle control.Xenograft data are expressed as mean ± SEM. Statistical analyses were performed using −1 for CC‐223 and 5 mg kg−1 for CC‐115 were selected to give similar plasma exposure to both agents [3 and continued until day 36.The study was designed to compare the antitumor activity of CC‐223 and CC‐115. Doses of 10 mg·kgh agents , 13, 24.2.11post‐hoc test was applied to analyze differences between more than two groups. Mann–Whitney U‐test was used to analyze differences between the two groups. A P‐value < 0.05 was considered statistically significant.One‐way ANOVA, with the Greenhouse–Geisser correction and Dunnett's 33.1−/− cell lines  and DNA‐PK, , 13, 14 3.2−/− cells  were treated with CC‐115, CC‐223, and SMG1i. In these cell lines, SMG1i blocked proliferation and survival to the same extent as CC‐115  or threonine (T) residues in the context of the SQ/TQ motif , 26. SQ/lls Fig. . SMG1i hlls Fig.  or TORK lls Fig. . As expells Fig. . Both CClls Fig. . To conf115 Fig. .n = 7) were also highly sensitive to CC‐115 and the more specific SMG1 inhibitor  . Recent ATF4, ATF3, and CHOP, but not so much HSPA5 or sXBP1 across the MM cell line panel. These effects were due to SMG1 inhibitory activity, as ATF4, ATF3, and CHOP were upregulated by CC‐115, but not by the TORK inhibitor CC214‐1 .Cell death via CC‐115 was inhibited by the pan‐caspase inhibitor QVD Fig. , indicatOXA Fig. . An incrOXA Fig. . Variousect Fig. . In contect Fig. .In conclusion, MM cells are particularly sensitive to SMG1 inhibition by CC‐115 and SMG1i, which causes an integrated stress response and apoptosis.3.4in vivo models, using CC‐115 as it has already been tested in humans in advanced solid and hematologic malignancies [in vivo. The ability of CC‐115 to inhibit SMG1 and thus NMD in vivo was confirmed in an early stage of the project by qPCR analysis of HCT 116 xenograft tumors after treatment of mice with CC‐115 , close to their starting tumor volume of 75–150 mm3. In contrast, treatment with the TORKi, CC‐223, decreased tumor size ~2× relative to vehicle‐treated mice .Fig. S7. Bodyweight monitoring in in vivo mouse experiments, related to Fig. 5.Click here for additional data file.


Urothelial carcinoma (UC) of the urinary bladder is a prevalent cancer worldwide. Because histone deacetylases (HDACs) are important factors in cancer, targeting these epigenetic regulators is considered an attractive strategy to develop novel anticancer drugs. Whereas HDAC1 and HDAC2 promote UC, HDAC5 is often downregulated and only weakly expressed in UC cell lines, suggesting a tumor-suppressive function. We studied the effect of stable lentiviral-mediated HDAC5 overexpression in four UC cell lines with different phenotypes . In particular, comprehensive proteomics and RNA-seq transcriptomics analyses were performed on the four cell line pairs, which are described here. For comparison, the immortalized benign urothelial cell line HBLAK was included. These datasets will be a useful resource for researchers studying UC, and especially the influence of HDAC5 on epithelial-mesenchymal transition (EMT). Moreover, these data will inform studies on HDAC5 as a less studied member of the HDAC family in other cell types and diseases, especially fibrosis.  HDACs 1–3 are broadly expressed and support proliferation and survival of many cell types including cancer cells8. In contrast, HDAC5 expression is more variable between tissues10. Its function and its mechanisms of action are less well studied, but the enzyme is implicated in the differentiation and function of neurons, among others13. In cancers, this enzyme can act in a tumor-promoting or tumor-inhibitory fashion depending on the tissue13. It may also have a role in fibrotic diseases14.In humans, the classical histone deacetylase (HDAC) family comprises 11 members. They all remove acetyl groups from histones or non-histone proteins, but differ in their substrates in detail, their cellular localization, their cell-type distribution and accordingly, their overall physiological functions and involvement in diseaseHDAC inhibitors (HDACi) are used and further developed as drugs to treat cancer, especially hematological malignancies, and other diseases. Some of these inhibitors target a broad spectrum of HDACs (pan-HDACi), whereas others are more specific for particular isoenzymes. Since the relevance of each isoenzyme varies between tissues, diseases, and cancer types, HDACi would ideally be tailored to inhibit a specific subset of HDACs in each medical application.16. As few treatment options are available for advanced stage UC, our group and others have explored the usefulness of HDACi to treat this disease. In brief, previous work has confirmed the importance of HDAC1 and HDAC2 for UC cell proliferation and survival17. Notably, specific inhibitors of this enzyme pair are considerably more efficacious than pan-HDACi, suggesting that inhibition of other enzymes like HDAC5 may actually be counterproductive11. In fact, we found very low expression of HDAC5 in UC cell lines, hinting at a tumor-suppressive function18. Therefore, we studied the effect of HDAC5 overexpression in a range of UC cell lines with different phenotypes that cover the range of the disease. Endogenous protein levels of HDAC5 in these cell lines were all low. The cellular effects of HDAC5 overexpression reported previously10 included indeed decreased cell proliferation but also promotion of epithelial-mesenchymal transition (EMT).Urothelial carcinoma (UC) is the most common histological subtype of urinary bladder cancerTo gain deeper insights into the cellular and molecular mechanisms underlying the effects of HDAC5, we performed comprehensive whole-cell proteome and transcriptome analyses of the UC cell lines RT112, VM-Cub-1, SW1710, and UM-UC-3 engineered to stably overexpress HDAC5 or transduced with vector only. For comparison, we analyzed a benign urothelial control cell line, HBLAK as a “vector-only” form, as this cell line does not tolerate HDAC5 overexpression  and the HBLAK cell line were authenticated by STR profiling.2. As a benign urothelial control, we used the HBLAK cell line , which were cultured in CnT-Prime Epithelial Culture Medium . All cell lines were authenticated by DNA fingerprint analysis. Normal urothelial cells (UP) were cultured as described19 with informed consent of the donors and approval by the Ethics Committee of the Medical Faculty of the Heinrich-Heine-University, study number 1788.The urothelial cancer cell lines (UCCs) VM-Cub1, RT112, SW1710, and UM-UC-3 were provided by Dr. M. A. Knowles , Dr. J. Fogh  and Dr. B. Grossmann  or by the DSMZ . They were cultured in DMEM GlutaMAX-I  supplemented with 10% fetal calf serum  at 37 °C in a humidified atmosphere of 5% CO2. The HDAC5 open reading frame with a C-terminal FLAG tag was subcloned into the lentiviral transfer vector puc2CL12IPwo using primers forward 5′-CATCTCGAGGCCACCATGCCCAGTTCCATGGG and reverse 5′-ATCGCTAGCTTACTTGTCATCGTCGTCCTTGTAGTCTCCTCCCAGGGCAGGCTCCTGC. The construct was verified by Sanger sequencing. Lentivirus production and cell transduction were performed as previously described21. Briefly, HEK-293T cells were transfected with helper plasmid expression construct pCD/NL-BH, envelope vector (pczVSV-G) and either the vector plasmids puc2CL12IPwo or puc2CL12IPwo-HDAC5-FLAG. Replication-deficient lentiviral particles were harvested 48 h after transfection and used to transduce RT112, VM-Cub-1, SW1710, and UM-UC-3 cells using 8 µg/ml polybrene (Sigma-Aldrich). Twenty-four hours after transduction, the supernatant containing viral particles was removed and the transduced cells were selected and maintained with 1 µg/ml puromycin . Stable expression of HDAC5 was confirmed by immunoblot analysis.The plasmid pcDNA3.1 + HDAC5-FLAG was a gift from Eric Verdin (Addgene plasmid #13822)22.Immunoblot analysis of whole cell extracts was performed as described in detail elsewhere23. Briefly, proteins were stacked in an acrylamide gel (about 4 mm running distance), subjected to silver staining, de-stained, reduced and alkylated and digested with trypsin. Resulting peptides were extracted from the gel and 500 ng peptides prepared in 0.1% trifluoroacetic acid for liquid chromatography and mass spectrometric analysis.To study the effect of HDAC5 overexpression on selected cell lines, quadruplicates from individual culture dishes were prepared from RT112, VM-Cub1, SW1710, and UM-UC3 cells expressing HDAC5 as well as corresponding and HBLAK vector-only cells. Cells were harvested and protein lysates prepared in an aqueous urea-containing buffer  3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 30 mM Tris-HCl, pH 8.0) and prepared for mass spectrometric analysis, as described elsewhere23.Here, first peptides were separated by liquid chromatography an Ultimate 3000 Rapid Separation liquid chromatography system was used for peptide separation over a two-hour gradient before analyzing peptides with a QExactive plus mass spectrometer in data dependent top ten mode as essentially as describedHomo sapiens reference proteome . Further search parameters and parameters for peptide and protein acceptance and quantification were essentially as described previously23. In brief, standard search parameters were applied with a few exceptions, including enabled label-free quantification as well as “match between runs”. Cysteine-carbamidomethylation was considered as fixed whereas protein N-terminal and lysine acetylation and methionine oxidation were considered as variable modifications.For spectra identification and precursor ion intensity-based quantification, the MaxQuant environment  was used with standard parameters. Spectra were matched against sequence data from the Cells were harvested with Trizol (manufacturer) and lysates were stored at −80 °C. Total RNA was then isolated by the Qiagen RNeasy Mini Kit  with DNase treatment. RNA quality was checked by spectrophotometry.Library preparation for RNA-Seq was performed according to the manufacturer’s protocol using the ‘TruSeq Stranded mRNA Library Prep Kit’ from Illumina®. Briefly, 250 ng total RNA were used for mRNA capturing, fragmentation, the synthesis of cDNA, adapter ligation and library amplification. Bead-purified libraries were normalized and finally sequenced on the HiSeq. 3000/4000 system  with a read setup of 1 × 150 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing.http://proteomecentral.proteomexchange.org) via the PRIDE partner repository24 with the dataset identifier PXD01444825. Mass spectrometric.raw files as well as the MaxQuant search result files (txt folder) have been uploaded.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (26 (http://www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-841827.The RNA-seq transcriptomics data (raw FASTQ files) have been deposited to the ArrayExpress repositoryA summary of samples, data collection, experimentation, and accession numbers can be found in Table Proteomic analysis of quantitative data was carried out with Perseus  and within the R environment . Here, only proteins showing at least two different peptides were considered and proteins showing at least three valid quantitative values in at least one group in the respective comparison. For the identification of HDAC5 affected proteins and differences between the cell lines, a two-way ANOVA followed by a Benjamini-Hochberg correction and by Tukey’s honest significance tests was carried out on log2 label-free quantification intensities after missing values were filled in with random values from a normal distribution . Additionally, Student’s t-test was calculated for pairwise comparisons of HDAC5-transduced and vector-only cells and cutoffs were determined by the significance analysis of microarrays method .et al.10. The protein abundance of KRT5, KRT17, and VIM measured by quantitative mass spectrometry strongly supports the cellular phenotypes and detection of KRT5 and VIM by immunoblotting .To further validate the mass spectrometry-based observations, we performed immunoblot analysis. Protein expression levels of proteins of interest (HDAC5 and other EMT markers), normalized to tubulin levels were confirmed by immunodetection as previously described in Jaguva Vasudevan Total RNA of samples used for transcriptome analyses were quantified  and the quality and integrity were measured by capillary electrophoresis using the Fragment Analyzer and the Total RNA Standard Sensitivity Assay . All samples in this study showed high quality RNA Quality Numbers   genome sequence. After grouping of samples  according to their respective experimental condition, multi-group comparisons were made and statistically determined using the Empirical Analysis of DGE . The resulting P values were corrected for multiple testing by FDR and Bonferroni-correction. A P value of ≤ 0.05 was considered significant . The reads of all probes were adapter-trimmed (Illumina TruSeq) and quality-trimmed . Mapping was done against the 10, the greatest variation in gene expression was observed between the VM-Cub-1 variants transduced with vector-only and HDAC5, respectively  of the proteome and RNA-seq datasets Fig. . As showely Fig. . FurtherHDAC5 mRNA, in transduced cell lines RT112, VM-Cub1, SW1710, and UM-UC3 cells, we detected the HDAC5 mRNA expression ranging from 76, 186, 195, and 205-fold higher, respectively. Indeed, this is an average value derived from polyclonal cell populations, in fact, the expression levels in individual cells might vary, and could be even more elevated. A direct comparison of this to physiological upregulated HDAC5 levels observed in HDAC5 expressing tumors appears a lot higher. Here, mRNA levels of 2 to 8-fold higher as in corresponding benign tissues were observed  as well as in urothelial cancer cells  and, most importantly, HDAC5 protein expression is diminished in most urothelial cancer cell lines10. In particular, the last point suggests that in addition to the RNA expression levels, posttranscriptional regulation and RNA turnover mechanisms are likely to contribute and responsible for an overall low expression level of HDAC5 in UC. Therefore, we would like to emphasize that the expression of relatively higher HDAC5 transcripts in the tested cell lines may not be reflected in excessive HDAC5 expression.Of note, compared to parental UC cell lines that displayed low or undetectable levels of 10, where we reported the effect of HDAC5 expression on cellular phenotypes such as proliferation, clonogenic potency, and migration. Intriguingly, in VM-Cub-1, HDAC5 expression dramatically triggered an epithelial-mesenchymal transition (EMT)10. Our proteome and transcriptome data backed and detailed the molecular changes. Specifically, they hinted at the involvement of TGFβ. However, we have not systematically analyzed additional differences among these bladder cancer cell lines. For instance, the HBLAK cells seem to rely more on the pentose phosphate pathway, whereas the other cell lines used more oxidative phosphorylation. Thus, our omics datasets from five urothelial bladder cancer cell lines could be utilized, among other prospects, (a) to identify and validate novel target genes or proteins associated with UC; (b) to uncover new metabolic pathway(s) and signaling network(s) in the direction of identifying potential target for cancer therapy, (c) to study epigenetic regulation, protein modification, and cellular consequences as a result of HDAC5 expression.Analyses of parts of these datasets have been published beforeSupplementary File 1. RNA-seq data overviewSupplementary File 2. QC report of RNA samplesSupplementary File 3. QC report of librarySupplementary File 4. QC report of RNA samples


FGFR3) occur in 10–20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents.Mutations and fusions in Fibroblast Growth Factor Receptor 3  and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis.Acquired resistance to FGFR inhibitors was generated in two FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation.Long term treatment of FGFR3-fusion harbouring bladder cancers.We demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for The online version contains supplementary material available at 10.1186/s12885-022-09478-4. FGFR3 aberrations (activating mutations or aberrant gene fusions) in these tumours, and their preclinical sensitivity to FGFR-targeted therapy [Urothelial bladder cancer is responsible for approximately 150,000 deaths per year worldwide, and the median survival of patients with metastatic disease is approximately 18 months . FibroblFGFR3 aberrations lead to oncogenic signalling through the MAPK and PI3K pathways. Activating mutations in FGFR3 occur in 10–20% of muscle-invasive bladder cancers [FGFR3 gene, with 5 mutations, R248C, S249C, G372C, Y375C, and K652E accounting for greater than 90% of all mutations [ cancers –9 and a utations , 12. Theutations –15.FGFR3 chromosomal translocations which generate oncogenic FGFR3 fusion proteins [FGFR3 (which include the kinase domain) fused in-frame to either transforming acid coiled-coil 3 (TACC3) or BAI-Associated Protein 2-Like-1 (BAIAP2L1) [FGFR3 component of the fusion gene is identical, with a conserved breakpoint lacking only the final exon (exon 19). Expression of these fusion proteins in normal human urothelial cells has been shown to induce mitogenic activation of the MAPK pathway [A smaller proportion of bladder cancers (3–6%) have proteins , 16, 17.AIAP2L1) , and for pathway .FGFR3 mutations and fusions are sensitive to FGFR inhibitors such as BGJ398 , PD173074 (Pfizer) and erdafitinib  [FGFR2/3 aberrant bladder cancers in 2019 [FGFR3-fusion harbouring bladder cancer lines have so far been limited to a single cell line, RT-112, which harbours a FGFR3-TACC3 fusion as well as an amplification [FGFR3-fusion driven bladder cancer cell lines is unknown.Preclinical studies have demonstrated that human bladder cancer cell lines with Janssen) –22, ther in 2019 . Despite in 2019 , 24. Sevfication –27. Mechfication , activatfication , ERBB2, fication , and incFGFR3-fusions, RT4 (FGFR3-TACC3 fusion) and SW780 (FGFR3-BAIP2L1 fusion). Through continuous culture in the presence of the FGFR inhibitor BGJ398, we derived lines highly resistant to BGJ398. These cell lines were also found to be cross-resistant to erdafitinib  and TAS-120 futibatinib, an irreversible FGFR inhibitor that is currently undergoing phase II clinical testing [FGFR3-fusion driven bladder cancers.To address this, we undertook this preclinical study to investigate potential resistance mechanisms to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring  testing . ProfiliFGFR3-BAIP2L1 fusion) and RT4 (FGFR3-TACC3 fusion) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 1% GlutaMax, 1% HEPES, 1% Penicillin/Streptomycin, and 10% Fetal Bovine Serum (Invitrogen), and incubated at 37 °C in 5% Carbon Dioxide. BGJ398 was obtained from Novartis  or from Selleck Chemicals . AZD8931, erdafitinib and TAS-120 were purchased from Selleck Chemicals.The urothelial carcinoma cell lines SW780 (Resistance to BGJ398 in SW780 and RT4 bladder cancer cell lines was established by 1) sustained exposure to 1 µM BGJ398 for 3 months (labelled SW780 RS and RT4 RS), with fresh drug added each time the cells were passaged, and (2) gradual increase in dose exposure to BGJ398 over 3 months, commencing at a low dose (3 nM) and doubling the dose each week until reaching 1 µM at which dose cells were subsequently maintained (labelled SW780 RD and RT4 RD). Parental cell lines were passaged in parallel in equivalent concentrations of DMSO. Parental and resistant cells were regularly assessed for Mycoplasma contamination and confirmed to be negative, and the authenticity of the cell lines verified using the Promega StemElite ID System (Supplementary Table  sustaineCell viability was measured using the CellTitre-Glo (CTG) luminescent cell viability assay . Cells were seeded in white 96 well flat bottom plates at a density of 1500–5000 cells per well, then treated the following day with drug for 72 h. Luminescence was measured using a SpectraMax L Microplate Reader  and compared to DMSO treated cells. BGJ398, erdafitinib, TAS-120, Trametinib and AZD8931 were all purchased from Selleck Chemicals and dissolved in DMSO.Changes in cell cycle kinetics and apoptosis were assessed following 24 h of drug treatment using Propidium Iodide staining as described previously , followeRT4-RS and SW780-RS cells were cultured in fresh media without drug for 24 h before collection to negate effects induced by acute drug exposure. Control and resistant lines were then lysed in Radio immunoprecipitation assay (RIPA) buffer  containing complete Protease Inhibitor Cocktail Tablets  and PhosSTOP (Roche). A total of 200 µg of lysate was then incubated with human Phospho-RTK Arrays  as per manufacturer’s instructions, and blots were read using the ChemiDoc X Imaging System .N-morpholino) ethanesulfonic acid) buffer (ThermoFisher). Proteins were transferred using the iBlot® Dry Blotting System (Invitrogen) and signal detected using the Li-Cor® Odyssey Infrared Imager . The following antibodies were obtained from Cell Signaling Technologies : pERK p44/42 MAPK T202/Y204 (9106); t-ERK p44/42 MAPK (9107), p-ERBB3 Tyr1289 (2842S), ERBB3 (4754S), p-ERBB2 Tyr 1248 (2247), ERBB2 (2242), pEGFR Tyr1068 (D7A5) and EGFR (2232). Anti-β-tubulin (ab6046) was obtained from Abcam  and anti-β-actin (A5316) from Sigma.Protein lysates were prepared as above, denatured using 10 × NuPage Sample Reducing Agent , and run on NuPAGE Novex 4–12% Bis–Tris precast gels (Invitrogen) in MES . Data shown is mean ± SEM from 3 technical replicates from a representative experiment unless stated otherwise. Biological replicates were performed for the majority of experiments and are stated in the figure legends. Groups were compared using parametric unpaired Student’s t-test with Welch’s correction. max of BGJ398 [FGFR2/3 driven bladder cancers, and the covalently binding irreversible FGFR inhibitor TAS-120 (futibatinib) [SW780 and RT4 cell lines with acquired resistance to the FGFR inhibitor BGJ398 were generated by continuous culture in 1 µM BGJ398 for 3 months, a dose which is approximately twofold higher than the clinically reported Cf BGJ398 . The celbatinib)  which isTo investigate the mechanistic basis for acquired resistance to FGFR inhibition, the phosphorylation status of 49 receptor tyrosine kinases (RTKs) was compared between parental and SW780-RS and RT4-RS cells using phospho-RTK arrays. This screen identified an increase in pERBB3 in both SW780-RS and RT4-RS cell lines Fig. A, with mBased on the consistent increase in pERBB3 across the resistant lines, we examined the effect of combining BGJ398 with the pan-ERBB family inhibitor AZD8931 in FGFR inhibitor-resistant cell lines . CombinaWhile these analyses identified mechanisms of acquired resistance associated with long-term FGFR inhibition, it is now evident that several tumours, including bladder cancers, can also rapidly adapt to targeted therapies and reactivate signalling through various mechanisms , 33, 34.We therefore investigated whether the rapid reactivation of MAPK signalling in FGFR-fusion harbouring bladder cancer cells could be attenuated by combined treatment with an FGFR inhibitor and the pan-ERBB inhibitor, AZD8931, and whether this could enhance the growth inhibitory effect of FGFR inhibitors. Indeed, combined treatment of both RT4 and SW780 parental cells with BGJ398 and AZD8931 significantly attenuated the induction of pERBB3, and further suppressed pERK levels compared to the effect of either agent alone Fig. A, E. FurFGFR3-fusion harboring cell lines was also induced when the FGFR inhibitors erdafitinib and TAS-120 were combined with AZD8931. Indeed, combining either erdafitinib or TAS-120 with AZD8931 induced synergistic suppression of proliferation of SW780 and RT4 parental cells, demonstrating this is a highly effective combination regimen that can enhance the activity of multiple FGFR inhibitors  and RT4 (FGFR3-TACC3 fusion).FGFR inhibitors such as BGJ398 and the clinically approved agent erdafitinib induce objective responses in ~ 25–40% of patients with metastatic urothelial cancer harbouring erations , 24, 35.erations –38. To bFGFR3 altered cell lines remain dependent on FGFR for growth in vitro, with some cell lines switching their dependence to ERBB family members [FGFR3-fusion harbouring cell lines investigated in the current study were sensitive to FGFR inhibition, we did note considerable variability in response in the SW780 cell line with different serum batches and serum concentrations (data not shown), indicating this is an important consideration in these studies.Interestingly, a prior study reported that not all  members . While t members . NotablyFGFR3-fusions that had not been previously characterized in terms of response to FGFR inhibition. While profiling of changes in receptor tyrosine kinases in cell lines with acquired resistance identified increased expression of several candidates, only expression of pERBB3 was consistently increased in both cell line models and was independent of the mode of resistance induction (RS vs RD). Importantly, combination treatment with the pan-ERBB inhibitor AZD8931 profoundly re-sensitised these lines to FGFR inhibition, indicating a direct role for pERBB3 in conferring resistance. This finding is consistent with observations previously reported in RT-112 cells [In this study, we examined resistance mechanisms to FGFR inhibition in two contexts – acquired resistance following long term exposure, and rapidly following 4–72 h treatment – in two bladder cancer cell lines harbouring 12 cells , 39, sugFGFR3-fusion harbouring bladder cancer cells to rapidly reactivate MAPK signalling. We demonstrated that this is associated with increased pERBB3 levels, and that the increase in pERBB3 and reactivation of pERK could be attenuated by combined treatment with the pan-ERBB inhibitor, AZD8931. In parallel, combined FGFR/ERBB inhibition further inhibited cell growth of FGFR3-fusion driven bladder cancer lines. These findings are consistent with those previously reported in RT-112 cells, where pERBB2 and pERBB3 were both rapidly elevated within 24 h of BGJ398 treatment [FGFR2-amplified gastric cancer cells [FGFR3-fusion driven bladder cancers following FGFR inhibition, our findings in the SW780 and RT4 cell lines adds to the previous findings in RT-112 cells to demonstrate compensatory activation of ERBB3-driven signalling as a consistent mechanism of rapid adaptive resistance to FGFR inhibitors in these tumours.An important finding of the current study is the capacity of reatment . Furtherreatment . While wreatment , it is per cells . A furthFGFR3-fusion harbouring bladder cancer, and our demonstration that dual targeting of FGFR and ERBB family receptors synergistically inhibits growth, suggests that dual blockade of FGFR and ERBB-driven signalling may represent a more effective treatment strategy for these tumours. Additionally, ERBB3-targeting antibodies are now entering clinical trial for other indications (e.g. seribantumab for NRG1-fusion harboring tumours) [Critical to preventing the emergence of resistance is the more efficient targeting and elimination of FGFR-driven tumours from the onset of treatment. In this regard, the finding that compensatory activation of ERBB3-driven signalling occurs rapidly in response to FGFR inhibition in multiple models of tumours)  (NCT0438Finally, while these studies establish a role of increased ERBB3 phosphorylation in driving resistance to FGFR inhibition, additional studies are needed to understand the specific mechanisms driving this event. Possibilities include the upregulation of ERBB3 ligands  which caFGFR3-fusion driven bladder cancer cell lines. Our findings suggest that upfront combination treatment with FGFR and ERBB3 inhibitor warrants further investigation for FGFR3-fusion driven bladder cancers.In summary, we identify consistent increased activation of pERBB3 as a resistance mechanism to FGFR inhibition in Additional file 1.


Soft tissue sarcomas (STS) are a rare and highly heterogeneous group of solid tumors, originating from various types of connective tissue. Complete removal of STS by surgery is challenging due to the anatomical location of the tumor, which results in tumor recurrence. Additionally, current polychemotherapeutic regimens are highly toxic with no rational survival benefit. Cold atmospheric plasma (CAP) is a novel technology that has demonstrated immense cancer therapeutic potential. Canady Cold Helios Plasma (CHCP) is a device that sprays CAP along the surgical margins to eradicate residual cancer cells after tumor resection. This preliminary study was conducted in vitro prior to in vivo testing in a humanitarian compassionate use case study and an FDA-approved phase 1 clinical trial (IDE G190165). In this study, the authors evaluate the efficacy of CHCP across multiple STS cell lines. CHCP treatment reduced the viability of four different STS cell lines  in a dose-dependent manner by inhibiting proliferation, disrupting cell cycle, and inducing apoptosis-like cell death. Sarcoma is one of the major types of cancer and refers to tumors derived from mesenchymal or neural crest cells . AccountMany STS subtypes are highly malignant and aggressive, more so in adults than in children. For example, adult patients with rhabdomyosarcoma , synovial sarcomas , and fibrosarcoma (originating from tendon and fascia), have substantially lower five-year overall survival rates and higher risk of recurrence or metastasis than pediatric patients ,14,15,16For the past several decades, surgery has been the first-line treatment for STS . To mitiDue to the difficulty of obtaining wide surgical margins near vital organs, positive microscopic margins, defined by residual disease within <1 mm of the surgical margin, are often a consequence of retroperitoneal STS . RetropeAdjuvant radio- and chemotherapies can be effective modalities for ensuring control of residual disease but are rarely beneficial to STS. Radiotherapies often fail to improve local control, making reresection necessary ,24. ChemCold atmospheric plasma (CAP) is a novel and a relatively new technology with various biomedical applications, including cancer therapeutics ,34. CAP The purpose of this study was to evaluate the efficacy of CHCP on sarcomas, specifically STS. We examined the effects of CHCP on the cell viability of four human STS cell lines, each representing a different STS subtype shown in To determine the appropriate eradication dose for each STS subtype, MTT assays were performed 48-h post-CHCP treatment on all four STS cell lines . In Figuf ≤ 0.033 and ** f ≤ 0.002381)  . A serieExpression of Ki67 proliferative marker was investigated 6-, 24-, and 48-h post-CHCP treatment, shown in Cell cycle was continuously monitored for 48 h following CHCP treatment, depicted in To confirm the CHCP-induced apoptosis, cells were stained for FITC Annexin V and Propidium Iodide (PI), and live, early apoptotic, and late apoptotic/dead cell populations were quantified by flow cytometry 24- and 48-h post-CHCP treatment, shown in To the authors knowledge, this is the first study to compare the efficacy of CAP across multiple STS cell lines. Fibrosarcoma had the greatest susceptibility to CHCP, followed by synovial sarcoma, and rhabdomyosarcoma. Differences in CHCP sensitivity were also observed in our previous study which compared the efficacy of CHCP across multiple carcinoma cell lines; renal adenocarcinoma was highly susceptible to CHCP treatment whereas ovarian adenocarcinoma was relatively more resistant and required a higher dose for eradication . FurtherInitially identified in our previous studies on carcinoma cell lines ,43, inhi5 cells/well 24 h prior to CHCP treatment [4 cells/well because CHCP had no effect on 94T778 cell viability at higher seeding densities. This could be because of its unusually large cell size and its proportionately higher amount of histone mRNA, which would require a higher dose of CHCP to degrade. It was also observed that 94T778 grew relatively slower than other cancer cell lines, suggesting that slow-growing cells would be less susceptible to CHCP since histone mRNA would less likely be exposed during S-phase. However, other morphological or molecular differences could also play a role in 94T778 resistance. Determining these would require a stand-alone study of its own.In our previous studies for the establishment of CHCP dosimetry, cancer cells were seeded in concentrations of 1.0 × 10reatment . HoweverMoreover, in vitro follow-up studies will consider differential gene regulation as a mechanism of survival since we recently discovered BCL2A1 to be a key gene for CHCP resistance in breast cancers . We antiFinally, based on our preliminary report, CHCP treatment of 120P for 7 min was determined to be the eradication dose for liposarcoma and would be the standard for future animal and clinical studies for STS.All experiments were performed at the Jerome Canady Research Institute for Advanced Biological and Technological Sciences (JCRI-ABTS), Takoma Park, MD, USA.Cold Plasma Device. CHCP was developed at JCRI-ABTS . The CHCP scalpel generates CAP through a USMI SS-601 MCa high-frequency electrosurgical generator and a USMI Canady Cold Plasma Conversion Unit. In-depth description and schematics of plasma generation by CHCP was detailed in our previous study [us study . To estaus study . Mock anCell Culture. Connective tissue fibrosarcoma (HT-1080), synovial sarcoma (SW-982), rhabdomyosarcoma (RD), and well-differentiated retroperitoneal liposarcoma (94T778) human cell lines were purchased from ATCC . Following ATCC’s protocol, cells were cultured in either Eagle’s Minimum Essential Medium, Dulbecco’s Modified Eagle’s Medium, or Roswell Park Memorial Institute-1640 Medium supplemented with 10% fetal bovine serum  and 1% Pen Strep  in a 37 °C and 5% CO2 humidified incubator (Thermo Fisher Scientific). When cells reached approximately 80% confluence, HT-1080, SW-982, and RD were seeded at a concentration of 5.0 × 103 cells/well into 96-well plates  and were treated with 1 or 3 LPM at 20–120 P for 1–2 min for cell viability assays. Additionally, 94T778 was seeded at a concentration of 2.5 × 104 cells/well into 12-well plates (USA Scientific) and were treated with 3 LPM at 120 P for 1–7 min for cell viability, proliferation, cell cycle, and apoptosis assays.Cell Viability Assay. Thiazolyl blue tetrazolium bromide (MTT) assays were performed on all four cell lines 48-h post-CHCP treatment. Reagents were purchased from Sigma-Aldrich  and assays were performed following manufacturer’s protocol. The absorbance of the dissolved compound was measured by a BioTek Synergy HTX  microplate reader at 570 nm.Cell Proliferation Analysis. Images of immunofluorescent Ki67 expression in CHCP-treated 94T778 cells were obtained to detect changes in cell proliferation 6-, 24-, and 48-h post-CHCP treatment. Cells were seeded onto fibronectin-coated (Sigma-Aldrich) 12 mm diameter round cover glass (Thermo Fisher Scientific) in 12-well plates. Following CHCP treatment (3 LPM at 120 P for 5 min), cells were washed, fixed, and stained with Alexa Fluor 488 conjugated Ki-67 Rabbit mAb  and Isotype Control  in a 1:200 dilution. After overnight incubation in 4 °C, the round cover slides with cells were transferred onto 1″ × 3″ × 1 mm microscope slides. Cells were covered with Antifade Mounting Reagent with DAPI  before finally covered by a 24 × 50 mm cover glass  and cured for 2 nights in 4 °C. Slides were imaged using Confocal LSM 510  with a 63 × lens and 405 and 488 nm laser bands.Cell Cycle Analysis. The IncuCyte S3 Live-Cell Analysis System  continuously monitored cell cycle progression of 94T778 stable cell line every hour and up to 48 h following CHCP treatment (3 LPM at 120 P for 5 or 7 min). The stable cell line was generated using the IncuCyte® Cell Cycle Green/Red Lentivirus Reagent (Sartorius).Apoptosis Assay. Flow cytometry was used to detect apoptosis in 94T778 cells 24- and 48-h post-CHCP treatment. CHCP doses of 3 LPM at 80 P or 120 P for 5 or 7 min were selected. All live attached and dead detached cells were collected, washed, and stained with FITC Annexin V  and Propidium Iodide (Thermo Fisher Scientific) according to the manufacturer’s protocol. Early and late apoptosis was quantified and analyzed using FCS Express .Statistics. MTT assays were repeated 3 times with 2 replicates for each condition. Flow cytometry was repeated 3 times with 1 replicate for each condition. Data was plotted by Microsoft Excel 2016 as the mean ± standard error of the mean. A student t-test or a one-way analysis of variance (ANOVA) was used to check statistical significance where applicable. The differences were considered statistically significant for a p ≤ 0.05 or b p ≤ 0.05. A post-hoc test was used to check statistical significance where applicable. The differences were considered statistically significant for * f ≤ 0.0033 and ** f ≤ 0.002381. Confocal microscopy and IncuCyte experiments were each performed 1 time with 2 replicates for visualization, so statistics were not considered for these experiments.Overall, our study strongly demonstrated CHCP to be a promising adjuvant for STS treatment. The in vitro preliminary data showed that CHCP effectively reduced STS viability by inhibiting proliferation, disrupting cell cycle progression, and inducing apoptosis. The next report will reveal the results of our gene expression studies on ex vivo STS tumor samples from our completed FDA-approved phase 1 clinical trial to elucidate the CHCP mechanism.


Glioblastoma multiforme is the most common primary brain tumor. Unfortunately, it is also one of the cancer types that has the worst morbidity and mortality ratios, so new targets and treatments need to be found. The metabolism of glutamine is fundamental for the proliferation of many tumor cells, including glioblastomas. Glutaminase isoenzyme GLS is one of the responsible enzymes for the pro-oncogenic pathways that induce metabolic reprogramming and leads to altered levels of some amino acids and other key intermediary metabolites in glioblastoma. Using the clinically approved GLS inhibitor CB-839 (Telaglenastat), we found significant changes in glutamine metabolism, including both the oxidative and reductive fates of Gln-derived alpha-ketoglutarate in the tricarboxylic acid cycle, in three glioblastoma cell lines. One of them, the T98G glioblastoma cell line, showed the greatest modification of metabolite levels involved in the de novo biosynthetic pathways for nucleotides, as well as a higher content of methylated and acetylated metabolites.GLS and GLS2. In glioblastoma, GLS is commonly overexpressed and considered pro-oncogenic. We studied the metabolic effects of inhibiting GLS activity in T98G, LN229, and U87MG human glioblastoma cell lines by using the inhibitor CB-839. We performed metabolomics and isotope tracing experiments using U-13C-labeled Gln, as well as 15N-labeled Gln in the amide group, to determine the metabolic fates of Gln carbon and nitrogen atoms. In the presence of the inhibitor, the results showed an accumulation of Gln and lower levels of tricarboxylic acid cycle intermediates, and aspartate, along with a decreased oxidative labeling and diminished reductive carboxylation-related labeling of these metabolites. Additionally, CB-839 treatment caused decreased levels of metabolites from pyrimidine biosynthesis and an accumulation of intermediate metabolites in the de novo purine nucleotide biosynthesis pathway. The levels of some acetylated and methylated metabolites were significantly increased, including acetyl-carnitine, trimethyl-lysine, and 5-methylcytosine. In conclusion, we analyzed the metabolic landscape caused by the GLS inhibition of CB-839 in human glioma cells, which might lead to the future development of new combination therapies with CB-839.Most tumor cells can use glutamine (Gln) for energy generation and biosynthetic purposes. Glutaminases (GAs) convert Gln into glutamate and ammonium. In humans, GAs are encoded by two genes:  The reprogramming of cellular metabolism is now considered a hallmark of cancer . The tumEGFR), inactivating mutations and/or deletions in the tumor suppressor phosphatase and tensin homolog (PTEN) and the cyclin-dependent kinase inhibitor A (CDKN2A) genes; all these changes lead to the overactivation of core signaling pathways such as RAS/RAF/MAPK, PI3K/AKT, or cyclin-dependent kinases 4 and 6 (CDK4/6) Gln into citrate , principally in cancer cells . Gln can citrate .13C-Gln tracing experiments showed a consistent decline in relative amounts of m+4 labeled-isotopologues: succinate (m+4), fumarate (m+4), malate (m+4), aspartate (m+4), and citrate (m+4), which indicate the reduced contribution of glutamine oxidation to these metabolites. There was also a reduction in the contribution of reductive-carboxylation-related labeling in the following metabolites: fumarate (m+3), malate (m+3), aspartate (m+3), and citrate (m+5). On average, fractional labeling was reduced five-fold in T98G, which proved to be the most Gln/GA dependent among the GBM cell lines assayed for maintaining TCAC, three-fold in LN229, and two-fold in U87MG, which was found to be the less Gln/GA dependent assayed cell line  were sharply diminished in T98G (0.02 to 0.2-fold) and reduced in LN229 (0.1 to 0.5-fold) and U87MG (0.2 to 0.5-fold), as depicted in Moreover, pyruvate and alanine levels were also depleted upon CB-839 treatment, as shown in Regarding pentose phosphate pathway-related metabolites, we found diminished levels of 6-phosphogluconate and/or 6-phosphoglucono-δ-lactone in CB-839 treated cell lines, as depicted in The N-acetylation of several amino acids  was positively increased following GLS inhibition by CB-839 in the three assayed cell lines . In someA large accumulation (11-fold) was detected for acetyl-carnitine in the treated T98G cells. The levels of acetyl-carnitine doubled for both LN229 and U87MG cell lines treated with CB-839 a. On theThe metabolism of purine nucleotides was significantly modified in all GBM cell lines treated with CB-839. Inosine monophosphate (IMP) and the adenosine monophosphate analog AICAR were augmented in the three assayed GBM cell lines  was lost, and labeled adenosine monophosphate (AMP) (m+2) dropped from 0.25 to 0.01, while labeled guanosine metabolites (m+3) were also sharply diminished for guanosine (from 0.26 to 0.007), guanosine monophosphate (GMP) (from 0.48 to 0.03), and guanosine diphosphate (GDP) (from 0.53 to 0.04)  . As reprThe level of 3-methyl-adenine increased in all the assayed GBM cell lines, as depicted in We wondered if higher levels of methylated metabolites could be attributable to a potential lower activity of αKG-dependent dioxygenases, including histone demethylases and the ten-eleven-translocation (TET) family of 5-methylcytosine hydroxylases. Consequently, we calculated the relative levels of αKG and succinate, the substrate and product, respectively, of αKG-dependent demethylases, and expressed it as the αKG/succinate ratio, which we found to have diminished to 0.13 for T98G, 0.55 for LN229, and 0.55 for U87MG cells, following an inverted pattern compared to the increased levels in methylated metabolites for each treated cell line . GLS inhibition by CB-839 induced a dose-dependent decrease in the cell proliferation of all the assayed GBM cell lines , as expe13C-Gln compared to the oxidative (m+4)-related mark  in adenylosuccinate, AMP, and GMP, among others  reduction to ubiquinol (QH2). CB-839 might be indirectly causing a change in the NADH/NAD+ ratio while affecting flux through the ETC [Of note, CB-839 treatment decreased the succinate biosynthesis derived from glutamine, as noted by the sharp decrease in succinate m+4 labeling for all cell lines. Accordingly, succinate metabolite levels were also down when treated with CB-839. However, as noted by lower αKG/succinate ratios , the dro the ETC . Such a In addition, the results of our study established that GLS inhibition by CB-839 simultaneously provoked TCAC imbalance, as discussed before, causing an asymmetric drop between succinate and other TCAC intermediates, which could originate as a possible malfunction of demethylases. In our models, the levels of the methylated metabolite N,N,N-trimethyl-lysine were significantly increased in all three cell lines analyzed when treated with CB-839. Free N,N,N-trimethyl-lysine is likely to arise from methylated protein degradation, including histones, so it could be interpreted as an indicator of overall protein methylation levels, and this accumulation might also be related to lower levels of L-carnitine found in the three CB-839 treated cell lines  lowering oxidative and reductive anaplerotic activity, (ii) modifying the de novo synthesis of nucleotides, (iii) increasing acetylation and methylation overall levels, and (iv) reshaping urea cycle reactions. All these findings paved the way for further CB-839 or other GA-targeted studies and extended the basis for its future clinical application in GBM, especially pointing to the future development of combination targeted therapies that are designed based on a specific tumor molecular signature.


A new preparation method for extreme fluorescent green emission tryptophan-stabilized silver nanoclusters (Tryp-AgNCs) is presented in this scientific research. The produced silver nanoclusters are dependent on tryptophan amino acid which contributes to normal growth in infants and the sublimation and recovery of human protein, muscles, and enzymes. Herein, we have introduced a green method by using microwave-assisted rapid synthesis. The subsequent silver nanoclusters (AgNCs) have excitation/emission peaks at 408/498 nm and display a considerable selectivity to Fe(III) ions. The tryptophan amino acid molecule was used in the synthesis process as a reducing and stabilizing agent. The Tryp-AgNCs’ properties were investigated in terms of morphology, dispersity, and modification of the synthesized particles using different advanced instruments. The luminescent nanoclusters traced the Fe(III) ions by the luminescence-quenching mechanism of the Tryp-AgNCs luminescence. Therefore, the extreme selectivity of the prepared nanoclusters was exhibited to the Fe(III) ions, permitting the sensitive tracing of ferric ions in the lab and in the real environmental samples. The limit of detection for Fe(III) ions based on Tryp-AgNCs was calculated to be 16.99 nM. The Tryp-AgNCs can be efficiently applied to a paper test strip method. The synthesized nanoclusters were used efficiently to detect the Fe(III) ions in the environmental samples. Moreover, we examined the reactivity of Tryp-AgNCs on various human tumor cell lines. The results show that the Tryp-AgNCs exhibited their activity versus the cancer cells in a dose-dependent routine for the perceived performance versus the greatest-used cancer cell lines. Iron compounds are distinguished by their numerous applications, including in many industries, such as building materials, drinking water, sewage pipes, food additives, water treatment compounds, dyes in paints, and manufacturing in the plastics’ industry. Various quantities of iron ions are eliminated from the environment and water sources. Iron is not only present in the hemoglobin inside the red blood cells, but it is also used to transport oxygen to vital tissues. Therefore, it is necessary to find advanced or straightforward methods to trace the concentration of iron ions inside cells or in the surrounding environment. In comparison to other transition metal ions, iron is a relatively poisonous metal. The World Health Organization (WHO) set the content limitation of iron ions in drinking water to be 0.3 mg/L ,2. MoreoFluorescent chemosensors are deployed in many applied methods, due to their high sensitivity and rapid response in detecting many different significant ions, including biological molecules ,12, drugAgNCs, comprising of accumulation of a limited number of atoms, have a diameter of around 2 nm which is close to the Fermi wavelength. Concerning this considerable small size, the band structures of AgNCs are discontinuous and break down into discrete energy levels, and AgNCs thus give distinctive physical and chemical characteristics. These significant properties involve quantized charging property and tunable fluorescence with high optical stability. The AgNCs are diverse from the larger particle size Ag nanoparticles ,37. TypiIn this work, we used tryptophan amino acid as a momentous chelated ligand as the stabilizing and reducing agents for Tryp-AgNCs synthesis . The usehttp://www.sigmaaldrich.com, accessed on 20 May 2022). The dialysis membrane of 12–14 KD was obtained from the Spectrum Chemical Co.  . All of the used solvents were of the highest grade available.The tryptophan, ethylenediaminetetraacetic acid (EDTA), all-metal ions, and chemical compounds were purchased from Sigma Aldrich Co.  (−1). Lastly, the X-ray Photoelectron Spectroscopy (XPS) analyses were applied using a K-ALPHA  with monochromatic X-ray Al K-alpha radiation −10 to 1350 eV spot size 400 µm at pressure 10−9 mbar with full-spectrum pass energy 200 eV and narrow-spectrum 50 eV.The absorbance spectra of the investigated solutions were conducted in a 1 cm quartz cell using an Evolution™ 200 series  UV-Vis spectrophotometer. The luminescence emissions under a certain excitation wavelength were recorded using a JASCOFP8200 spectrofluorometric instrument. The high-resolution transmission electron microscope (HR-TEM) measurements were collected by JEOL-100S Japan, operative at 200 kV. The Fourier Transform Infrared Spectroscopy (FTIR) was used to characterize the synthesized nanoclusters, utilizing a 4100 Jasco-Japan (4000 to 400 cm3) was mixed with 6 mL of 23.3 mg/mL of tryptophan in water. The prepared mixture was left to stand for three minutes under vigorous stirring, then 0.3 mL 1M NaOH was added to the mixed solution, and the net solution was further stirred for another 3 min. After adjusting the pH to 12.5 using sodium hydroxide solution, the solution was then transferred to a 10 mL microwave sealed tube. After transferring the prepared solution to the microwave tube, the reaction tube was kept in the microwave oven for 20 min  with a constant irradiation power. After 20 min, the microwave-assisted Tryp-AgNCs were dialyzed in ultra-pure water for 24 h to remove all of the excess molecules. After dialysis, the Tryp-AgNCs were kept in the dark at 4 °C. The quantum yield of the Tryp-AgNCs was evaluated by using the integrated fluorescence intensities of the nanomaterials and the fluorescein dye that dissolves in ethanol solution as a reference , R2 = 0.9956, can be fit, providing a substantial association constant (Ksv) of 1.9 × 106 M−1 (Our mechanism depends on the hypothesis that the Fe(III) chelated with the AgNCs’ surface via induction of the metal-complex formation with the active functional groups of tryptophan of the Tryp-AgNCs, causing a considerable fluorescence quenching of the Tryp-AgNCs, probably with an electron transfer mechanism. The obtained data suggest that the Tryp-AgNCs exhibit a great selectivity toward the Fe(III) ions. These characteristic properties can be attributed to the significant electron-accepting capability of the Fe(III), and therefore the electrons were simply captured by the Fe(III). The Fe(III) electronic configuration is 3dcapacity , escorti 106 M−1 b. This pSeveral pieces of research reported the AgNCs that were stabilized with different scaffold molecules. These nanoclusters were utilized to detect the significant metal ions based on the nanoclusters’ aggregation ,58. ThisTo study the selectivity of the Tryp-AgNCs, the influences of several metal ions on the synthesized nanoclusters were investigated under the same experimental conditions a. No con® cellulose paper strip with a high-quality cotton liner that is modified to provide an α-cellulose content of more than 98%. The sensing nanoclusters were embedded by the tryptophan scaffolds, which can be entrapped in the test strip. The free unbind Tryp-AgNCs were washed out with bi-distilled water. The test strips’ sensitivity toward the Fe(III) ions was investigated by immersing the strips in different Fe(III) concentrations (0 to 4 μM), as shown in The Tryp-AgNCs, as a new optical sensor, can be applied to a paper test strip method. The Tryp-AgNCs were spread onto a WhatmanThe tracing of the Fe(III) cations was studied, using the Tryp-AgNCs in real samples in mineral and tap water. The examined samples were utilized straightforwardly without applying any extra treatment. Additionally, the definite Fe(III) cation concentrations in bi-distilled water concentrations were prepared in the environmental samples. In particular, the data are dependent on the fluorescence standard relationship obtained in the lab measurements. The Fe(III) cation concentrations in the mineral and tap water samples were effectively detected. The obtained data are shown in −1 ~5.4 µM) for ferric ions in the drinking water.In summary, the developed green optical sensor Tryp-AgNCs displayed a lower LOD than that of some previously published optical sensors exhibited in 50 values which reflected the effect of the synthesized particles on the cancer cells in the current investigation.The synthesized Tryp-AgNCs compound was investigated for its anticancer activity on a group of human cancer cell lines; human epidermoid carcinoma (A431), colorectal carcinoma (HCT-116), prostate adenocarcinoma (PC3), lung adenocarcinoma (A-549), breast cancer adenocarcinoma (MCF-7), pancreatic carcinoma (PaCa2) and osteosarcoma (HOS) cancer cells. The MTT assay  was applied in the current investigation. The results are shown in It has been revealed that the results of the observed activity, obtained from the tested particle, were generally displayed on the cancer cells in the current investigation, in a dose-dependent manner exhibiting such behavior against the HOS, MCF7, and A431 cancer cell lines. The tested particle, Ag-Tray, exhibited activity from high to moderate against most of the cancers’ cell lines at 100 µM, exhibiting a degree of selectivity at the remaining applied concentrations. The highest activity results were revealed against certain cancer cells, MCF7 and HOS, followed by PC3, A431, and PaCaII. The tested compound showed weak activity against the remaining cancer cell lines, A549 and HCT116. It was found that the MCF7cancer cell was the most susceptible cell at all of the tested concentrations. In addition, the highest inhibition was observed for the MCF7cancer at 100µM, revealing 95.7 ± 0.3 as percentage inhibition. However, although influenced by moderate activity at 100 µM, the PC3 human cancer cell line did not show any observed behavior at the remaining tested concentrations. The same also applies to the PaCaII cancer cells which showed good activity only at 100 µM. Additionally, the most unaffected tested cancer cell at all of the concentrations was HCT116. There is an observed large difference in the displayed activity results on cells such as PC3 and PyCall.The very small effect observed of the tested particle on the RPE1 Human retinal pigment epithelial normal cell line only at 100 µM, while that cell was not influenced at 50, 25 and 12.5 µM, revealed, obviously, the interesting selectivity of the activity towards the cancer cells with no influence on human normal cells.3+ with a noticeably significant linear relationship between the emission of nanoclusters and the concentration of the Fe3+. The activity of the prepared nanoclusters on different human tumor cell lines was studied and the obtained data prove that the Tryp-AgNCs provided their activity against the cancer cells in a dose-dependent way, in terms of the detected behavior against most of the utilized cancer cells lines. Lastly, the results exhibit that the Tryp-AgNCs will be an impactful optical sensor that can detect Fe3+ in real samples and biological analysis.We successfully synthesized the Tryp-AgNCs by using a green, one-step, and straightforward method. The achievement was ascribed to our full knowledge about tryptophan as a reducing and protective agent, making the preparation simple, low-cost, and eco-friendly. The resulting nanoclusters have a green emission under UV excitation. Further, the Tryp-AgNCs effectively provided an optical sensor for detecting Fe


Penile cancer is a rare disease in demand for new therapeutic options. Frequently used combination chemotherapy with 5 fluorouracil (5-FU) and cisplatin (CDDP) in patients with metastatic penile cancer mostly results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance against standard therapy could help to understand molecular mechanisms underlying chemotherapy resistance and to identify candidate treatments for an efficient second line therapy.rCDDP2500, adapted to 2500 ng/ml CDDP; UKF-PEC-1r5-FU500, adapted to 500 ng/ml 5- FU; UKF-PEC1rCDDP2500/r5-FU500, adapted to 2500 ng/ml CDDP and 500 ng/ml 5 -FU). Afterwards cell line pellets were formalin-fixed, paraffin embedded and subject to sequencing as well as testing for homologous recombination deficiency (HRD). Additionally, exemplary immunohistochemical stainings for p53 and gammaH2AX were applied for verification purposes. Finally, UKF-PEC-1rCDDP2500, UKF-PEC-1r5-FU500, UKF-PEC1rCDDP2500/r5-FU500, and UKF-PEC-3  were tested for sensitivity to paclitaxel, docetaxel, olaparib, and rucaparib.We generated a cell line from a humanpapilloma virus (HPV) negative penile squamous cell carcinoma (UKF-PEC-1). This cell line was subject to chronic exposure to chemotherapy with CDDP and / or 5-FU to induce acquired resistance in the newly established chemo-resistant sublines (PEC-1rCDDP2500 was characterized by an increased HRD score, which is supposed to be associated with increased PARP inhibitor and immune checkpoint inhibitor sensitivity in cancer. However, UKF-PEC-1rCDDP2500 did not display sensitivity to PARP inhibitors.The chemo-resistant sublines differed in their mutational landscapes. UKF-PEC-1The online version contains supplementary material available at 10.1186/s12885-022-10432-7. In developed countries penile cancer is a rare tumor. The incidence in Central Europe and the United States is about 1.0 per 100,000 men per year . There aIn metastasized penile carcinoma, one of the frequently used chemotherapy regimens is a combination of cisplatin (CDDP) and 5-fluorouracil (5-FU) –9, but rAn unfixed partial penectomy specimen of a 64-year-old patient with a squamous cell carcinoma of the penis measuring 5.4 cm in largest dimension located primarily on the foreskin was received by the Dr. Senckenberg Institute of Pathology, Frankfurt am Main for intraoperative frozen section diagnosis of the resection margins. The tumor was diagnosed as HPV independent penile squamous cell carcinoma, usual type, according to WHO classification of Tumours 5th Edition, Urinary and Male Genital Tumours. The tumor was excised in toto (R0) and showed infiltration of corpus spongiosum without infiltration of the urethra (pT2). Perineural invasion was noted (Pn1). The tumor was graded G2 on a scale of G1 – G3. The tumor showed usual type morphology and infiltrative borders . An adjaFormalin fixed and paraffin-embedded (FFPE) material as well as unfixed material was taken from the Dr. Senckenberg Institute of Pathology, University Hospital, Frankfurt am Main, Germany. The material was taken after diagnostics had been finished. It was double pseudonymized. Tissue sample and patient data used in this study were provided by the University Cancer Center Frankfurt (UCT). Written informed consent was obtained from the patient and the study was approved by the institutional review board of the UCT and the ethical committee at the University Hospital Frankfurt, according to the declaration of Helsinki (project-number: SUG-02–2017).rCDDP2500 (adapted to 2500 ng/ml CDDP), UKF-PEC-1r5-FU500 (adapted to 500 ng/ml 5- FU), and UKF-PEC1rCDDP2500/r5-FU500 (adapted to 2500 ng/ml CDDP and 500 ng/ml 5 -FU), were established by continuous exposure to increasing drug concentrations, as described previously [For primary tumor cell line generation (UKF-PEC-1), a penile cancer specimen (diameter 5 mm) was removed from the primary tumor and dissected with a scalpel into little pieces and treated twice with trypsin (0.2%) for 30 min afterwards. Trypsin was inactivated by 10% FBS. The tumor material was cultivated in epithelial medium  until cell adhesion. Media was changed at five days intervals. The drug-resistant sublines UKF-PEC-1eviously . The subeviously .In addition, the penile carcinoma cell line UKF-PeC-3 was derived from a HPV DNA subtype 16-positive tumor with a pT3 pN0 L0 G2 R0 .All cell lines were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum , 100 IU/ml penicillin and 100 µg/ml streptomycin as well as 4 mM Glutamin at 37 °C. Mycoplasma testing was performed.2 cells per well were plated into 96-well plates and incubated at 37 °C and 5% CO2. After 0, 1, 4 and 7 days, 100 µl of CellTiter-Glo (Promega) was added to each well (according to manufacturer´s instruction) and luminescence was measured on a plate reader . Population doubling times (in h) were then calculated for time points day 1 to day 4 (72 h) using http://www.doubling-time.com/compute.php, which uses the equation: Doubling time = duration × log(2)/log − log. The growth curves were generated using GraphPad Prism 9.3.1.To determine population doubling times , 4 × 104/100 µL per well in 96-well plates) were incubated in the presence or absence of drug for 120 h. Then, 25 µL of MTT solution (2 mg/mL (w/v) in PBS) were added per well, and. plates were incubated at 37 °C and 5 % CO2for 4 h. Afterwards, cells were lysed and incubated overnight, using 100 µL of a buffer containing 20% (w/v) sodium dodecylsulfate and 50% (v/v) N,N-dimethylformamide (pH 4.7) at 37°C and 5 % CO2. Absorbance was determined at 560 nm and 620 nm on a plate reader . After subtraction of the background absorption, the results were expressed as the percentage of viability relative to control cultures that received no drug. Drug concentrations that inhibited cell viability by 50% (IC50) were determined using CalcuSyn .Cell viability was tested by the 3--2,5-diphenyltetrazolium bromide (MTT) dye reduction assay after 120 h of incubation modified as described previously . Cells  with the Maxwell® RSC FFPE Plus RNA Kit  and QIAamp DNA FFPE Tissue Kit , respectively. The concentration of DNA and RNA was measured with the Qubit 4 Fluorometer . For the library preparation 20 ng for the OncomineTM Comprehensive Assay v3  were used. A 540 chip was applied.www.ncbi.nlm.nih.gov. To achieve reliable results, only alterations with fulfilled quality criteria were considered, such as allele frequency ≥ 5% and a coverage of at least 500 × for the Ion S5TM. Classification and interpretation of detected filtered and unfiltered variants were evaluated. The variant annotation provided by the respective software was manually reviewed according to the online databases ClinVar [For quality assurance the DNA and RNA were analyzed by DNA and RNA Screen Tape assay with the Agilent 4200 TapeStation . This automated electrophoresis solution is used to determine the integrity, too. The RIN and DIN (RNA and DNA Integrity Number) are software algorithms for determining RNA and DNA quality, respectively. RIN and DIN values range from 1 to 10; 10 being completely intact DNA or RNA, 1 being completely degraded. Next-generation sequencing: OncomineTM Comprehensive Assay v3 . Data analysis was performed using the analysis software platforms provided by ThermoFisher. The primary analysis of the sequencing data was completed by Torrent SuiteTM software. Afterwards data were analyzed with the Ion ReporterTM software (version 5.12.0.0), filter chains Oncomine Variants 5.10 and Oncomine Extended 5.12 were used. Genomic alterations were identified by the alignment on the reference genome hg19 (GRCh37) available at  ClinVar  and Cosm ClinVar . Other dFor DNA isolation from formalin-fixed, paraffin-embedded (FFPE) patient tumor tissue and FFPE-processed primary parental cells and the adapted chemo-resistant sublines were used. Areas with high tumor content were marked on hematoxylin and eosin (H&E)-stained slides with a 2 μm tissue section by experienced pathologists (RW). Afterwards, tissue cores (1.0 mm diameter) from FFPE blocks were taken from the marked area. DNA was isolated using the truXTRAC FFPE total NA Kit  based on focused ultrasonification and column purification according to the manufacturer's instructions. The DNA was eluted in 50 μL nuclease-free water and stored at − 20 °C. The DNA was quantified using the Quantus™ Fluorometer and ONE dsDNA Assay Kit . Additionally, samples have been screened for fragment length using Bioanalyzer 2100  and Agilent DNA 7500 Kit  in order to include samples with fragments larger than 150 bp for subsequent Copy-number variation analysis.Array-based genome-wide copy-number analysis was conducted using OncoScan FFPE Microarrays . For the assay, an input amount of 79.2 ng DNA was used. The OncoScan® assay utilizes the molecular inversion probe technology (MIP), which is optimized for highly degraded FFPE samples, for the identification of copy number (CN) alterations, loss of heterozygosity (LOH) and somatic mutations (SMs). MIP probes in the OncoScan® assay capture the alleles of over 220,000 SNPs within ~ 900 cancer genes. Furthermore, the MIP probes also enable detection of 74 frequently tested somatic mutations in nine genes implicated in cancer . The molecular inversion probe processing was done according to the OncoScan FFPE Assay Kit Protocol . At the end of the hybridization period, arrays were stained and washed using the GeneChip® Fluidics Station 450 and loaded into the GeneChip® Scanner 3000 7G  where array fluorescence intensity was scanned to generate array images (DAT file). Array fluorescence intensity (CEL) files were automatically generated from DAT files by the Affymetrix® GeneChip® Command Console® (AGCC) Software version 4.0. Resulting data files (CEL files) were processed and viewed by using Chromosome Analysis Suite (ChAS) software version 4.2.0.80. Segmentation was performed using ASCAT . With this package, we also derived copy-number profiles of tumor cells and estimates of normal cell contamination and ploidy.HRD score was calculated from output of ASCAT using implementations in R as described by Sztupinszki et al. . EstimatParaffin sections were stained with the antibodies p53 and gammaH2AX. For staining, the DAKO FLEX-Envision Kit  and the fully automated DAKO Omnis staining system or manual procedure  were applied according to manufacturer´s instructions. The antibodies used were Anti- Anti-phospho-Histone H2A.X (gammaH2AX) (Ser139)  applied for 30 min after 95 °C epitope retrieval in pH9 and anti-Human p53 Protein  applied for 20 min after epitope retrieval in pH9. Epitope visualization was performed using DAKO EnVision™ FLEX DAB + Substrate Chromogen System . Nuclear counterstain was done using DAKO hematoxylin solution . Slides were finally digitized using Pannoramic Scan II  and 5-FU-adapted sublines (IC50 UKF-PEC1r5-FU500: 1.707 μg/mL 5-FU ± 0.140 vs. IC50 UKF-PEC1: 0.953 μg/mL 5-FU ± 0.042)  pathway (CDK4 pathway). It promotes G1–S cell-cycle transition and is often defective in cancer and, therefore, dysregulation of CDKs are a hallmark of cancer .RAF1 is part of the MAPK signaling pathway. Copy number alterations were described in penile carcinomas . Rb1, CDNOTCH3 is expressed predominantly in vascular smooth muscle, central nervous system, some thymocyte subsets and regulatory T cells as well as by resting macrophages. NOTCH3 expression and signaling plays a role in pro-inflammatory macrophage activation and in the activation of NF-κB . It is iWe found FGFR4 mutations in all our penile cancer cell lines. FGFR4 belongs to the family of fibroblast growth factor receptors. FGFR4 mutations were described in many tumors, i.e. breast, colon, and head and neck with controversial interpretation of their significance . FGFR4 aSLX4 coordinates three different endonucleases directing cleavage and resolution of damaged DNA and Holliday junctions arising between homologous DNA duplexes , 40. We CDK12 is a well-known gene that is involved in carcinogenesis , 46. IntrCDDP2500/r5-FU500 was adapted with both substances simultaneously. The cell lines UKF-PEC1r5-FU500 and UKF-PEC1rCDDP2500 were adapted individually. If the same mutations were seen in each case, this would require that the cells always react in the same way when adapted to a cytostatic drug. However, the cells do not do this, but can develop different mechanisms as shown before [It was shown in this study, that amplifications, copy number variations and mutations in the cell lines adapted to CDDP and 5-FU are more in number than in the double resistant cell line. The cell line UKF-PEC1n before , 48, 49.r5-FU500 harbored c.574C > T, UKF- UKF-PEC1rCDDP2500 harbored c.524C > A and UKF-PEC1rCDDP2500/r5-FU500 harbored c.524C > A mutations. Consequently, the primary tumor showed a p53 wild type pattern via immunohistochemistry and the cell lines showed strong staining results. The antibody applied here reacts with wild type and mutant type of the p53 protein. Nevertheless, different p53 antibodies are present which can react with specific mutations of the p53 gene.To prove p53 expression in the primary tumor and the cell lines, immunohistochemical staining for p53 was performed. Sequencing results showed low level p53 mutations  in the primary tumor. According to the sequencing results the cell lines harbored the following p53 mutations: UKF-PEC1 harbored c.574C > T, UKF-PEC1Additionally, staining for gammaH2AX was performed. GammaH2AX is a marker which is indicative for the occurrence of double strand breaks and is linked to genomic instability. It was studied in a number of cancer subtypes . Cisplatwww.mycancergenome.org), MLH1 is altered in 3.85% of penile carcinoma patients [Immunohistochemistry for defective mismatch repair (MMR) proteins like MLH1, MSH2, MSH6, and PMS2 was analyzed by Stoehr et al. in 70 patients with penile cancer. They showed a normal expression of the MMR proteins . In contpatients . CDKN2B patients . RICTOR patients .TERT, the telomerase reverse transcriptase gene, is found to be reactivated in many cancer cells and enables cancer cells to evade senescence resulting from telomere shortening . TERT muIt is well known that CDDP alters the expression of many genes involved in several cellular processes. Ryan et al. could show increase of sensitivity to CDDP in neuroblastoma cells by downregulation of NTRK2, a potent oncogene involved in chemotherapy-resistance in neuroblastoma, with mR204 . In our PIK3CA is a known oncogene involved in the PI3K-AKT-mTOR pathway which plays a role in many human cancers with respect to cellular proliferation, survival and angiogenesis. However, Adimonye et al. conclude, that this pathway is not a key driver in the carcinogenesis of penile squamous cell carcinomas . FallahiCREBBP and FGF3 mutations have been frequently described to be associated with tumorigenesis –63. CREBThe MRE11-RAD50-NBS1 protein complex is able to recognize and process DNA double strand breaks and maintains genomic stability in the cell. Mutations in the genes are associated with many cancers, i.e. colon and breast carcinoma . AlblihyThe CDDP-resistant subline showed further amplifications: MRE11, RAD51B and AKT1. MRE11 and RAD51B play a role in the DNA repair reactions through homologous recombination . An assorCDDP2500 cells was not associated with enhanced sensitivity to the taxanes docetaxel and paclitaxel and the PARP inhibitors olaparib and rucaparib, indicating that an increased HRD score is not necessarily associated with elevated PARP inhibitor sensitivity. This may be due to molecular changes associated with resistance formation to chemotherapeutic agents such as increased levels of membrane transporters, e.g. ABCB1, or acquired resistance to apoptosis, which may also provide resistance to PARP inhibitors [The HRD score quantifies the so-called genomic scar evoked by HRD and has become a diagnostic marker . HRD faihibitors , 76. ImmWe performed an OCA panel on one case of penile carcinoma with additional chemo-resistant sublines. Therefore, further samples need investigation to confirm the mutational status. Additionally, and due to the nature of the test, only limited numbers of genes were targeted. The primary tumor paraffin block material aged seven years in the archive prior to investigation whereas the cell lines were paraffin embedded in the same year as the processing procedure took place. Therefore, impaired DNA and RNA quality of the primary tumor can be explained.Additionally, the HRD score of 42 needs validation in bigger cohorts of penile carcinomas to establish an individualized score for this entity.We generated a p16 negative cell line of a squamous cell carcinoma of the penis, and from this parental cell line three chemo-resistant sublines were established: one subline adapted to 500 ng/ml 5-FU, one subline adapted to 2500 ng/ml CDDP and one subline adapted to both drugs. A molecular profile including the HRD score was performed with this panel of cell lines. Acquired chemoresistance to CDDP resulted in an increased HRD score. With the use of PARP inhibitors, an increased sensitivity to these drugs was not shown in our cell line model.Additional file 1: Supplementary Figure 1. Dose-response curves for UKF-PeC1, UKF-PeC3 and their adapted chemoresistant sublines against CDDP and 5-FU.Additional file 2: Supplementary Table 1. Quality parameters for the primary tumor and the cell lines investigated.