Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs.We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers  and polycomb-repressive complexes . Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT.Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs.The online version contains supplementary material available at 10.1186/s13148-021-01223-1. According to the World Health Organization (WHO) classification system, germ cell tumors (GCT) of the testis can be subdivided into two main groups . One groEven though cure rates are up to 90%, mostly due to cisplatin based therapy, the incidences keep rising . AdditioCompared to most other cancer types, GCTs harbor a very low mutational burden , 8. HencPreviously, we described the cytotoxic effects of the histone deacetylase (HDAC) inhibitor Romidepsin , 18 and bona fide epigenetic targets in GCTs, we re-analyzed microarray expression data of different epigenetic writers, erasers and readers in the GCT entities GCNIS, SE, EC and teratoma  among the different GCT entities  at the BRD4 promoter, indicative of an active DNA demethylation process (ADD) [6-methyladenosine (m6A)-marked enhancer RNAs (eRNAs) have been identified to recruit the nuclear m6A reader YTHDC1, resulting in the formation of m6A-eRNA/YTHDC1 condensates, which promote BRD4/BRD4 co-activator formation [BRD4 was further regulated by miRNA binding to the 3′ UTR of BRD4, as well as post-translationally by acetylation, phosphorylation, ubiquitination and proline hydroxylation [Of note, mRNA levels of ss (ADD) . Moreoveormation . Furtherxylation . Additioxylation , 38. In 50 values  in a nanomolar range, while GSK343, PBIT, PRT4165 and LP99 showed EC50 concentrations up to 50 µM. Additionally, VZ185, a recently published PROTAC [50 > 50 µM), Selisistat, Nicotinamide, GSK2879552, Bizine, BI-9564 and Aurintricarboxylic Acid were excluded from further analyses. Generally, all drugs were less effective in targeting JAR cells (CC) as well as in the MPAF fibroblast cells  , while JAR and JEG-3 were less responsive to the epi-drugs  , JEG-3, BeWo (CC) and GCT72, 1411H (YST), their cisplatin-resistant sublines (-R), as well as another fibroblast cell line (HVHF2) and Sertoli cells FS1   and HDAC1 (Quisinostat target) were observed in 17% and 13% of the samples, respectively. All target genes were expressed in seminomatous and non-seminomatous GCT (with lowest mRNA levels of SUV39H1 in YST), further corroborating the potential use of the selected drugs  were analyzed by screening the 'Testicular Germ Cell Tumors' cohort of ‘The Cancer Genome Atlas’ TCGA , 42, 43), 4342, 4EZH2 (GSK343), which was marginally expressed in PCa and RCC. Based on these data, the three most potent drugs Quisinostat, JIB-04 and Chaetocin were tested in UC, PCa and RCC cell lines . Similar to the observations found in GCTs, JIB-04 and Chaetocin were highly effective in these urological entities, thereby highlighting the pan-urologic applicability of these epi-drugs.In addition, other urological malignancies , prostate cancer (PCa) and renal cell carcinoma (RCC)) were screened for the expression of the target molecules of Quisinostat, JIB-04, PBIT, Chaetocin, MZ-1, LP99, PRT4165 and GSK343 ) induced apoptosis in the majority of the tested GCT cell lines, with TCam-2 and 2102EP being affected the most , PBIT was excluded from further molecular analysis due to its higher ECTo screen for the effects of each epi-drug on the epigenetic landscape, mass spectrometry analyses of histone modifications were performed in the EC cell line 2102EP after treatment with Quisinostat, JIB-04, Chaetocin, MZ-1, LP99, PRT4165 or GSK343, allowing to not only confirm known changes in the epigenetic landscape caused by each epi-drug, but also to identify epigenetic effects beyond the postulated modes of action  analysis was performed in 2102EP EC cells treated with Quisinostat, JIB-04, Chaetocin, MZ-1, LP99, PRT4165 or GSK343  and DDIT4 (DNA damage-inducible transcript 4) and downregulation of EIF3CL (eukaryotic translation initiation factor 3 subunit C-like)  as well as ‘cell differentiation regulators’, while downregulated genes were involved in ‘regulation of respiratory gas’ and ‘response to organic substance’  and ‘negative regulation of transcription’    and the ‘extracellular matrix,’ while 7 downregulated genes were found to be related to ‘cytoplasm’. Furthermore, the STRING algorithm  predicted interaction between the  development- and pluripotency-associated genes DPPA3 and DPPA5   in cell lines of different GCT subtypes   showed similar deregulations in gene expression as found in the RNA seq analysis .A qRT-PCR analysis was performed to validate selected deregulations in gene expression after inhibitor treatment (EC72) Fig. B. In theA proteome profiler phospho-kinase array was used to detect phosphorylation of well-known kinase targets Fig. C. Thus, 22) [PRKCB, which is targetable by Enzastaurin  , which is targetable by kb NB 142–70 , upregulation of PRKCB (Quisinostat), PRKD1 (Chaetocin), HDAC9 and CSF1R (GSK343) was observed  . Quisinoal) Fig. A. Chaeto–70 Fig. A. For GS124 Fig. A. GW-258The combination of Quisinostat and Enzastaurin as well as Chaetocin and kb NB 142–70 significantly reduced cell viability in JAR (30% and 5%) and GCT72 (10% and 10%) cells compared to the mono treatments, while having little to no effect on TCam-2 and 2102EP cells. The two combinations (Panobinostat and Sunitinib) with the PRC2i GSK343 showed promising results in all cell lines, significantly reducing cell viability compared to the mono treatments of the drugs. The combinatorial effect for GSK343 was strongest in TCam-2 (30%) and 2102EP (13%) upon treatment with 20 nM Panobinostat. The combination of GSK343 with Sunitinib was also effective in these two cell lines, since the mono treatments did not reduce cell viability. he combination led to a reduction of cell viability by 60% (TCam-2) and 80% (2102EP).In this study, 16 different and selective epi-drugs were analyzed for their potential to treat (cisplatin-resistant) GCTs. Of these, seven were selected for further analyses due to their cytotoxicity at low nano-  to micromolar  concentrations  upregulated by the epi-drugs . Thus, drugs targeting these genes are putative combination partners for co-application with the corresponding epi-drug. Indeed, our analyses demonstrated that a combinatory application  reduced the viability considerably compared to mono application in a cell line-dependent manner, suggesting that these drugs are promising combination partners for related epi-drugs. Nevertheless, the suitability as a therapeutic option for GCTs and other urological malignancies needs to be extensively analyzed in future studies.We identified four druggable genes . Regarding H2AK119ub after PRT4165 treatment , Ismail et al.  observedmiR-497-5p and its target gene SALL4. Specifically, DNA hypermethylation of the miR-497-5p coding sequence resulted in silencing of its expression, eventually leading to enhanced SALL4 transcription in the CC cell line JEG-3 [SALL4-overexpressing JEG-3 CC cells [Most of the tested epi-drugs in this study were less sensitive in CC cell lines. CC is not only a cancer of the testis, but is also one of the most aggressive gestational trophoblastic neoplasia due to its propensity to invade the vascular system . Specifine JEG-3 . SubsequCC cells . Though,How can we explain the detected off-target effects on the epigenetic landscape? Expression of alternative target molecules of the same enzyme class as the primary target of each epi-drug is quite low in GCT tissues and cell lines  –70. To aIn addition to the changes in histone modifications, epi-drug treatment (except HDACi Quisinostat) affected the DNA-methylation status, i.e. decreased 5mC. Passive DNA-demethylation occurs over several cycles of replication; therefore, the 5mC decrease after 24 h is more likely due to active DNA demethylation (ADD). We observed a tendency for  decreased 5mC, which was accompanied by 5hmC accumulation. 5hmC formation is the first step of ADD and mediated by the TET enzymes (TET1—3) . Thus, tFurther analyses are necessary to validate ADD via the oxidation pathway, which is preferentially utilized in GCTs .CACNA1A/B/C/G; Additional file 2+ uptake through calcium channels being linked to membrane depolarization and cell death in adrenal chromaffin cells [KCN genes), which could also result in pro-apoptotic release of potassium ions. Chaetocin treated cells showed downregulation of potassium and sodium transporters (ATP1A2), eventually leading to a reduction of K+ and accumulation of Na+, which have been associated with apoptosis in lymphoma cells [Quisinostat treatment caused deregulations in expression of genes associated with ion-transport and -binding factors. Downregulation of transporters regulating the ion uptake and upregulation of channels mediating gradient-dependent flow of potassium and chloride ions have been shown to be pro-apoptotic via inducing water loss of the cells . In turnin cells . In addima cells .DDIT4,DNA damage-inducible transcript 4 protein) was also upregulated in H358  [ HOX genes, also showing a tendency towards downregulation in our study , thereby inducing apoptosis and/or cell cycle arrest. Cell lines from different pan-urologic cancer entities were highly sensitive toward the epi-drugs within a therapeutic window, mostly ruling out effects on healthy cells, e.g. fibroblasts. Additionally, we provide a detailed analysis of drug related (off-target) effects on the histone modification landscape. Further, we gained mechanistical insights by characterizing each epi-drug on transcriptional level and analyzing related signaling pathways. Our data provide a valuable source of information on tested epi-drugs, which might be of benefit for drug combination attempts and patient/therapy risk assessment.2, while bladder, prostate and renal carcinoma cell lines were grown at 37 °C and 5% CO2. Short tandem repeat (STR) profiles of all cell lines are checked on a regular basis and are available upon reasonable request. All cell lines are scrutinized for mycoplasma contamination on a regular basis using PCR strategy. Briefly, medium from cultured cells was boiled at 95 °C for 5 minutes (min) and 2 µl used in the PCR. The primers were used as a mix of ten primers. Sequences are given in Additional file All GCT cell lines were grown as described previously , 91. Cel3 cells  per 96-well were seeded and different drug concentrations  assays were performed as described previously . In summ50 concentrations for each cell line. For Annexin V/PI staining, cells were washed with Annexin V Binding Buffer before being resuspended in 70 µl Annexin V Binding Buffer and 2.5 μl Annexin V FITC-conjugate  and 15 µg PI  and incubated in the dark at room temperature for 15 min. 500 µl of Annexin V Binding Buffer was added before measurement. For the cell cycle analysis, cells were washed with PBS , fixed by ice cold 70% ethanol and stained with 2 µg/ml PI and 200 µg/ml RNaseA  in PBS shortly before every measurement. At least 5 × 104 cells were counted and analyzed using the MACSQuant software v 2.13.0.Flow cytometry analyses of apoptosis rates and cell cycle distribution was performed by Annexin V/propidium iodide (PI) or PI staining, respectively, using MACSQuant Analyzer 10  after 16 h of treatment with the corresponding ECDNA was isolated using the QIAamp DNA Mini Kit  according to the manual. RNA from cell lines was isolated using the RNAeasy Mini Kit  according to the manual. Proteins were extracted using RIPA buffer  containing protease inhibitor tablets . Histones were extracted using the Histone Extraction Kit according to the manufacturer protocol . Protein concentrations were assessed by the BCA Protein Assay Reagent Kit . RNA concentrations as well as 260/280 nm, 260/230 nm purity ratios were determined by NanoDrop measurement .2O to 200 ng, 100 ng and 50 ng in 200 µl per slot. Slot-Blotter device  was assembled as according to the manual. DNA was blotted onto a positively charged nylon membrane  using vacuum application. Three identical membranes were prepared. The membranes were air-dried for 15—20 min and UV-crosslinked (20 s (s), 1200 mJ/cm2). One membrane was stained with methylene blue  in 0.3 M sodium acetate (pH 5.2)) to validate DNA loading across the samples. The remaining two membranes were blocked in 5% milk powder in PBST for 1 h at RT. Membranes were incubated with 5mC or 5hmC antibody overnight at 4 °C. After washing, membranes were incubated with secondary antibody for 2 h at RT. Signals were detected using the ChemiDoc Imaging Systems . See Additional file Purity of DNA was verified using NanoDrop 260/280 ratio and agarose gel electrophoresis. DNA was diluted in HWestern Blots were performed as described previously . In totaFor the detection of phosphorylated kinase targets, the Proteome Profiler Human Phospho-Kinase Array Kit  was used according to the manufacturer’s instructions. The ChemiDoc Imaging System  was used for detection. 2102EP cells were harvested after 16 h of treatment with the epi-drugs. Pixel density was analyzed using Fiji ImageJ 1.53c .Quantitative RT-PCR (qRT-PCR) was performed as described previously . A total50 concentrations for Quisinostat, JIB-04, Chaetocin, MZ-1, LP99, PRT4165, GSK343 and DMSO as solvent control. The cells were harvested via trypsinization after 16 h. The mass spectrometric analysis of histone modifications was outsourced to the Mod Spec service by Active Motif . Histone extraction and mass spectrometric analysis was performed as described previously [2102EP cells were treated with the ECeviously . Raw dathttps://www.ncbi.nlm.nih.gov/geo/, GSE189472) [p values were corrected for multiple testing by false discovery rate (FDR). Accompanying PCA and heatmap were generated using Qlucore Omics Explorer 3.7 . Values of solvent controls wereaveraged from the studies GSE189472, GSE190792 and GSE190022 [Transcriptome analysis has been performed as previously described at the core facility ‘Genomics & Transcriptomics’  and has been publicly shared via GEO (E189472) , 95. RNAE189472) , 95. Stan = 4), GCNIS (n = 3), SE (n = 4), EC (n = 3), TE (n = 3) and mixed non-seminomas (n = 4). All tumors were classified according to the WHO classification of tumors based on their histology and assessment of tumor or GCNIS amount. GCT cell line datasets included TCam-2 (n = 5), 2102EP (n = 5), NCCIT (n = 4) and JAR (n = 2). Data are available via GEO . Expression intensities < 10 (Affymetrix) and < 8 (Illumina) were considered as not expressed based on expression intensities of SOX2 and SOX17 in SE  and EC  cell lines or tissues.Experimental setup and microarray data of GCT tissues (Affymetrix expression array) and cell lines (Illumina HT-12v4 expression microarray) have already been published , 90, 96.https://david.ncifcrf.gov/) for functional annotations [https://string-db.org) [https://www.cbioportal.org/) [http://www.interactivenn.net/) [http://mkweb.bcgsc.ca/tableviewer/) [https://www.wordclouds.com/) by Zygomatic . PINAv3.0 (https://omics.bjcancer.org/pina/) was used to identify drugs suitable to target molecules induced by epi-drugs [https://app.biorender.com/) .Analysis of RNA seq data was performed using the online tool DAVID Bioinformatics Resources 6.8  , 100. Thal.org/) , 45. Thenn.net/)  and the viewer/) . The funpi-drugs . Proposet test after confirming equality of two variances according to the F test has been performed to analyze differences between groups .A two-tailed Student’s Additional file 1: Figure S1. Identification of 16 potential epigenetic targets for GCT therapy. A Re-analysis of gene expression of epigenetic modifiers in GCT tissue samples , SE (n = 4), EC (n = 3), TE (n = 3) and mixed non-seminomas (n = 4)). B, C A Venn diagramof commonly expressed epigenetic modifiers in GCT tissues (B) and cell lines (C) highlighted 80 commonly expressed genes in GCT tissues and 33 in GCT cell lines . D Re-analysis of the 80 commonly expressed epigenetic modifiers of GCT tissues in GCT cell lines , 2102EP (n = 5), NCCIT (n = 4) and JAR (n = 2)). E Final list of 35 target genes (including BRD4 and EZH2) as well as corresponding drugs. F Western blot analysis verifying protein levels of BRD4, VHL and EZH2 in GCT cell lines. β-Actin was used as loading control. Expression microarray data were re-analyzed in context of this study [is study , 90, 96.Additional file 2: Figure S2. XTT data for 16 epigenetic inhibitors in three GCT cell lines and MPAF fibroblast cells (n = 4). Cells were treated once with indicated epi-drugs. Cell viability was detected after 24, 48, 72 and 96 h. The red line indicates 50% cell viability. Asterisks indicate significant changes between treatment and solvent control  .Additional file 3: Figure S3. XTT data for eight epigenetic inhibitors in 14 GCT cell lines, fibroblast cells MPAF and HVHF2, and Sertoli cells FS1 (n = 4). Cells were treated once with indicated epi-drugs. Cell viability was detected after 24, 48, 72 and 96 h. The red line indicates 50% cell viability. Asterisks indicate significant changes between treatment and solvent control  .Additional file 4: Figure S4. Epi-drugs decrease cell viability in GCTs and other urological entities. A EC50 values for each GCT cell line, fibroblasts and Sertoli cells and epi-drug treatment were calculated from data shown in Fig. S3. B Analysis of the mutational and gene expression alteration spectrum of the top eight target genes using the cBioPortal tool on the 'Testicular Germ Cell Tumors' cohort of TCGA. C–E XTT data for Quisinostat, JIB-04 and Chaetocin treatment in three cell lines each for UC (C), PCa (D) and RCC (E). The red line indicates 50% cell viability. Asterisks indicate significant changes between treatment and solvent control  . F EC50 values from (C), (D) and (E) calculated using GraphPad Prism v8. EC50: half-maximal effective concentration where the cell viability has 50% compared to the solvent control. SE, seminoma; EC, embryonal carcinoma; YST, yolk-sac tumor; CC, choriocarcinoma; SC, Sertoli cells; UC, urothelial carcinoma; PCa, prostate cancer; RCC, renal cell carcinoma.Additional file 5: Figure S5. Raw data of flow cytometry analysis of Annexin V/PI (apoptosis) or PI staining only (cell cycle) of indicated cell lines (columns) after 16 h of treatment with indicated epi-drugs (rows).Additional file 6: Figure S6. Mass spectrometry analysis of histone modifications arranged by frequency. A–C Circos diagrams are illustrating the difference in peptide frequency of given histone modifications of treated 2102EP cells compared to the solvent control. Histone modifications with a general peptide frequency of 10–100% (A), 1–10% (B) and 0–1% (C) are depicted separately for better comparison. Only differences in modification abundancy > 1 (10–100%), > 0.1 (1–10%) or > 0.01 (0.1–1%) were considered. D Depiction of the information given by the circos diagram rings. E Western blot analysis of histone extracts from PRT4165 or solvent control DMSO treated 2102EP cells after 24 h and 48 h. Coomassie staining is shown as loading control. F Western blot analysis of selected histone modifications in GCT cell lines. Cells were treated with indicated epi-drugs for 16 h. Total H3/H4 was used as loading control. ↑: upregulation, ↓: downregulation.Additional file 7: Figure S7. A Unsupervised clustering heatmap of RNA seq data . 2102EP were treated with indicated epi-drugs for 16 h. B STRING analysis of upregulated genes in Quisinostat treated (16 h) 2102EP cells.Additional file 8: Figure S8. STRING analysis of deregulated genes after 16 h of indicated epi-drug treatments in 2102EP cells . A LP99 upregulated genes, B LP99 downregulated genes, C PRT4165 upregulated genes, D PRT4165 downregulated genes, E GSK343 upregulated genes, F GSK343 downregulated genes.Additional file 9: Figure S9. A Proteome Profiler Human Phospho-Kinase Array raw data. 2102EP were treated with indicated epi-drugs for 16 h (n = 2). B Membrane layout of kinase array with corresponding spotted antibodies given in C. Detectable dots (marked in red) were used for quantification with ImageJ as shown in D. Gray bar indicates cut-off value of 2500.Additional file 10: Figure S10. A XTT cell viability raw data of co-treatments. Cells were pretreated with the 0.5 × EC50 concentration of each epi-drug for 24 h, followed by treatment with different concentrations of the co-drug for 24 h and 48 h. B, C Epi-drug side target gene expression in GCT tissues (B) , GCNIS (n = 3), SE (n = 4), EC (n = 3), TE (n = 3) and mixed non-seminomas (n = 4) and cell lines (C) (TCam-2 (n = 5), 2102EP (n = 5), NCCIT (n = 4) and JAR (n = 2). Expression microarray data were re-analyzed in context of this study [50 values from published data for Quisinostat [50, half-maximal inhibitory concentration; HDAC, histone deacetylase; BET, bromodomain and extra-terminal motif; PRC, poly repressive complex.is study , 90, 96.sinostat , JIB-04 sinostat , Chaetocsinostat , MZ-1 [1sinostat , LP99 [3sinostat , PRT4165sinostat  and GSK3sinostat . GCT, geAdditional file 11: Table S1. A Raw data of mass spectrometry analysis of histone modifications given as peptide frequency (n = 3). B Corresponding bar charts to (A) scaled to histone peptide frequency of 100%, 10% and 1%. C–I RNA seq data given as fold change compared to solvent control (log2) as well as DAVID functional annotation analysis for C Quisinostat, D JIB-04, E Chaetocin, F MZ-1, G LP99, H PRT4165 and I GSK343.Additional file 12: Table S2. A Studied cell lines including appropriate culture conditions. B Sequences of used oligonucleotides. C Epi-drug solvents and suppliers. D Used antibodies.


Tongue cancer is one of the most aggressive forms of oral squamous cell carcinoma which needs more investigations. Herein, we aimed to establish and characterize a tongue cancer cell line. Tumor tissue was obtained from a 70-year-old woman with tongue cancer. The established cell line named as EZB-ICR and characterized for doubling time, expression of specific markers, HPV corporation and migration status using flow cytometry, immunofluorescence staining, multiplex PCR, and migration assay. EZB-ICR was negative for expression of mesenchymal specific markers, cytokeratin19, pan-cytokeratin, vimentin and EPCAM, but was positive for S100 and Nestin. No appearance of human papilloma virus DNA was seen. The doubling time of EZB-ICR was 31 hours and migration assay confirmed its metastatic nature. To the best of our knowledge, EZB-ICR is the first tongue human cancer cell line established in Iran, and its features make it appropriate for cancer-based in vitro studies and contribute to more studies on tongue cancer. Head and neck cancer is the world’s fifth most common type of cancer worldwide. Oral squamous cell carcinoma (OSCC) is a prevalent cancer consisted of more than 90% of oral malignancies and in spite of recent advancements in diagnostic approaches and treatments, 5 year survival showed no difference in recent years . The most common oral squamous cell carcinoma is found in tongue and mouth floor . The age range of these patients is 20–44 years old and consuming tobacco and alcohol are among the most important risk factors. Furthermore, genetic, environmental or viral factors may lead to the pathogenesis of tongue cancer .The feature of tongue cancer is completely different from malignancies of other parts of oral cavity because tongue has a significant lymphatic structure and as a result shows aggressive metastatic pattern . Based on the American Cancer Society, 5-year relative survival rate for tongue cancer is as follows: 81% for localized (no sign the cancer has spread outside), 68% for regional (spread to nearby structures or lymph nodes), and 39% for distant (spread to distant parts of the body) type of tongue cancer . Although tongue cancer is increasing throughout the world, no effective treatment is exist and common therapies such as surgery, radiation therapy, chemotherapy and combination therapies are not enough . Thus more in vitro and in vivo investigations using tongue cancer cell lines are undoubtedly needed to establish new therapeutic intervention for tongue cancer. Here, we established a new tongue cancer cell line named EZB-ICR and characterized it for the expression of various markers, growth condition, HPV status and metastatic pattern.Cell line establishment2. The media was changed every 48 hours. In this experimental study, tumor tissue sample was obtained from a 70 years old woman patient with tongue cancer. The fragments were washed with sterile phosphate buffered saline (PBS) containing 1% pencillin/streptomycin. Next, the segments were cultured as a transplantation method in complete DMEM  and 1% penicillin/streptomycin) at 37°C in a humidified atmosphere containing 5% COGrowth curve and proliferation assay4 cells were seeded on six well tissue culture plates and the number of the cells was calculated each day for 7 days; this experiment was repeated three times. Growth curve and doubling time were measured by online doubling time calculating software (http://www.doubling-time.com).For evaluation of doubling time and growth rate of the cell line established, 2.5 × 10Flow cytometryFor phenotypical characterization, the cells were trypsinized and washed with PBS. Then the cells were stained with the appropriate flourchrome labeled monoclonal antibodies against mesenchymal lineage markers such as anti-CD34, CD19, CD166, CD105, CD44, CD10, CD146, CD29, CD45, CD73, CD90, CD24, CD69 and also some chemokine/chemokine receptors like MCP1, IP-10, RANTES, EPCAM and CXCR4, CCR5. The cells were fixed by 1% cell fix for 10 minutes and then washed with PBS. Next, 500ml of 0.2% Saponin was added, and incubated for 15 minutes and washed again. Consequently, cells were stained with above mentioned antibodies  and finally were evaluated using FACSculibure flow cytometer and analyzed by FlowJo software version 10.Immunofluorescent assay4 cells were seeded on chamber slides for 24 hours. Then, the slides were washed by PBS and fixed with cold methanol at room temperature, and blocked with 1% bovine serum albumin (BSA) in PBS containing 1% Triton X-100 (PBST). Subsequently, slides were incubated with primary monoclonal antibodies overnight at 4°C in 1.5% BSA in PBST. Then, slides were washed with PBST and incubated with secondary fluorescent antibodies  for 2 hours at room temperature. After that, the slides were mounted by solution containing propidium iodide (PI) and observed by fluorescent microscope. Our negative controls were the slides without antibodies and those treated with secondary antibody. Nestin, cytokeratin 19, vimentin, pancytokeratin and S100 were assessed with immunofluorescent assay using appropriate antibodies . 3 × 10Migration assay3 cells were cultured in the upper 8 μm transwell cell culture chambers in the FBS deficient condition media and the lower wells was filled out by 650 μl of DMEM media containing 10% FBS. The cells were incubated at 37 °C in 5% CO2 humidified chamber for 24 hours. Then, the non-migratory cells were eliminated from the upper side by cotton swab and the cells which were transferred from upper to lower chamber were fixed and stained with 10% ethanol and 0.5% Crystal Violet. At the end, the migrated cells were counted by phase contrast microscope. This assay was performed in triplicate for each cell line including EZB-ICR, KB  and MDA-MB-231 (as an invasive cell line). The motility of cells was investigated by migration assay via transwell inserts. 5×10Colony formation unit assay (CFU)th and 10th days of culture, cells were washed with PBS and stained by 0.5% Crystal Violet in methanol for 15 minutes at room temperature, finally flasks were washed by PBS and colonies were calculated by following formulation: colony number/cells of seeding ×100.The EZB-ICR cells were harvested with trypsin–EDTA and around 1,000 single cells were seeded in 25mm tissue culture flask in DMEM containing 10% FBS and 1% penicillin/streptomycin. On the 5Human Papilloma Virus (HPV) molecular genotyping (32 genotypes)Total DNA of EZB-ICR cells was extracted by fully automated instrument, part of SPF10 region of the HPV genome was amplified using Multiplex PCR method and the genotype of HPV was determined by various specific probes using reverse hybridization.Establishing and characterizing tongue cancer cell lineEZB-ICR cell line was generated from a 70-year-old woman patient that used waterpipe (hubble-bubble) sometimes. Examination diagnosed aggressive squamous cell carcinoma in right oral tongue lesion in 2016. The tumor mass size was 2.7x1.8x 0.7 cm at right lateroinferior site with ulcerative surface and also, base, apex and medial margins grossly not involved and inferior margin grossly seems to be involved. The patient underwent a hemiglossectomy surgery. The cells were firstly extracted as explants of chopped tumor mass and the cells were grown out after 3 weeks. EZB-ICR cell line was passaged frequently until 72 passages .Doubling timeDoubling time of EZB-ICR cell line was assessed at passage 25 in the course of the exponential growth phase of the cell line by calculating the number of cells for 7 days. Accordingly, the doubling time of EZB-ICR was about 31 hours .Flow cytometryFlow cytometry examination indicated that EZB-ICR cell line was negative for mesenchymal stem cell markers such as CD166, CD44, CD90,CD146 and also markers such as CD10, CD45, CD34, CD105, CD106, CD14,CD24, CD29, CCR5, CD69, CXCR4, IP10, MCP-1, RANTES and EPCAM as depicted in ImmunostainingAccording to immunofluorescent staining assay, EZB-ICR cell line was strongly positive for the expression of S100 and nestin but it was negative for cytokeratin19, pan-cytokeratin and vimentin .HPV molecular genotypingEZB-ICR cell line was negative for 32 genotypes of HPV. These genotypes were divided to 4 subgroups which included high risk , partially high risk , Low risk  and finally unclassified HPV genotypes . Migration assayMetastatic ability of EZB-ICR cell line was evaluated by migration assay and compared with MDA-MB-231 breast cancer cell line as an invasive cell line and KB cell line which originally from epidermal carcinoma of the mouth. EZB-ICR showed higher capability of migration compared to MDA-MB-231 and in particular KB cell lines .Colony formation assayThe colony formation assay was applied to recognize the ability of the EZB -ICR cell line to grow into a colony forming shape. In this regard the number of colonies after 5 and 10 days were respectively 63 and 45% .Establishment of cell line is a profitable option for further understanding the pathways which contribute to the development of models for cancer metastasis and invasion. In addition, cell lines are required for evaluating the effects of numerous novel drugs and finding better treatments . Several studies identified that the wide variety of patients who suffered from tongue cancer were smokers and most cell lines were determined from them, whereas few cell lines recognized from tumors of nonsmokers .In the present study, we established and characterized the tumorigenic and metastatic EZB-ICR tongue cell line. EZB-ICR cell line did not express any surface mesenchymal markers. Also, it was not positive for the expression of cytokeratin19, pan-cytokeratin and vimentin. In contrast, this cell line was positive for the expression of S100 as well as nestin.In vivo and in vitro experimental studies showed the over-expression of one subgroup of S100, S100A4, related to potential metastatic and poor clinical outcomes in different kinds of cancers . Experimental studies demonstrated the expression of nestin in several tumor cells like gastrointestinal stromal tumors, malignant melanoma, OSCC, pancreatic, prostate and breast cancers . High expression of nestin correlates with consistent growth and poor treatment prognosis in most solid tumors . As our established cell line expresses both S100 and nestin, these markers may be considered as tongue tumor markers for diagnostic and therapeutic options.It has been recently demonstrated that S100 plays significant roles in biological processes such as proliferation, apoptosis and cell survival associated with both normal development and tumorigenesis . Wanga et al., (2017) established a tongue cancer cell line from a nonsmoker 44-year-old woman (UCSF-OT-1109). Experimental studies demonstrated that never-smoking patients had aggressive patterns and their survival were even less than smoker patients . EZB-ICR and UCSF-OT-1109 both were isolated from nonsmoker patients and showed similar morphology. Although the connection of HPV infection to OSCC has been hypothesized, it did not emerge in neither EZB-ICR nor UCSF-OT-1109 . In contrast to EZB-ICR that did not express CD44 and EpCAM, UCSF-OT-1109 was positive for both markers .Migration index showed that EZB-ICR has strong motility in comparison with MDA-MB-231 and KB cell lines. So, this behavior could answer important questions regarding the metastatic pattern of the EZB-ICR cell line.To the best of our knowledge, EZB -ICR is the first human tongue cancer cell line that registered in the Cell Bank of Pasteur Institute of Iran. Furthermore, the EZB-ICR has enabled us to figure out more about the characteristics of tongue cancer, consequently, enhance our information in regard to finding novel therapeutic possibilities. The key important limitation in this study was that the number of tongue cancer cell lines in Iran is rare so we did not have a chance to compare EZB-ICR with other tongue cancer cell lines.


Synovial sarcoma is a soft-tissue sarcoma that lacks effective systemic therapy and carries poor prognosis due to frequent late local recurrence and metastases. The cancer is known to be driven in part by increased expression of the pro-survival protein BCL-2. Surprisingly, synovial sarcoma proved resistant to BCL-2 inhibitors in pre-clinical trials. We identified increased activity of a second pro-survival protein, MCL-1, as responsible for this resistance. We showed that co-targeting both BCL-2 and MCL-1 proves to be an effective therapeutic approach both in cell culture and animal models of synovial sarcoma, supporting translation into clinical trials.Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy. Synovial sarcoma (SS) is a rare but deadly soft-tissue sarcoma (STS) with no targeted therapy options. SS accounts for only about 10% of STS, but despite its relative rarity and predominant incidence in pediatric and young adults, 4000 cases are fatal each year . StandarL, and MCL-1), BH3-only pro-death , and terminal-effector molecules (BAK and BAX), the latter of which creates mitochondrial pores upon activation and oligomerization [L, and MCL-1 via BH3 mimetic drugs, which free BH3-activator proteins to initiate apoptosis, has been a major goal in pharmaceutical development [The BCL-2 family is a highly conserved family of proteins that functions primarily in and around the mitochondria, involving pro-survival , AMG 397 (NCT03465540), AZD-5991, and an inhibitor closely related to S63845 . The relative tolerability of venetoclax makes it an attractive agent to include in combination therapies, and clinical trials exploring concomitant dosing of MCL-1 inhibitors plus venetoclax are underway in several hematologic malignancies ,19. SS.PDX and SYO.1 cell lines were cultured in DMEM  with 10% FBS  and 1 µg/mL penicillin and streptomycin. SW982, ASKA, HS-SY-II, and Yamato cells were grown in RPMI1640  and 1 µg/mL penicillin and streptomycin. Routine mycoplasma testing was performed on all cell lines. The SS.PDX cell line was established ex vivo from the SS patient-derived xenograft model. SYO.1 and SW982 cell lines were kindly provided by Cyril Benes and the Center for Molecular Therapeutics at Massachusetts General Hospital . ASKA, HS-SY-II, and Yamato cell lines were obtained from Riken Bioresource Center . http://hgserver1.amc.nl, accessed on September 2019) [RNA expression data for sarcoma samples were obtained and analyzed through the R2: Genomic Analysis and Visualization Platform. (er 2019) . L (2764), BIM (2933), NOXA (14766), and SS18 (70929) from Cell Signaling Technology . Secondary antibodies used were anti-mouse IgG  and anti-rabbit IgG . S63845 was from Abmole , and venetoclax (ABT-199) was kindly provided by AbbVie Inc . Primary antibodies used for western blotting were as follows: GAPDH (sc-3233) and MCL-1 (s-19) from Santa Cruz Biotechnology; cleaved PARP1 (5625), cleaved Caspase III (9579), BCL-2 (4223), BCL-X6 cells were injected s.c. into each flank of 6–8-week-old NOD/SCID gamma (NSG) mice. The patient-derived xenograft model was acquired from Jackson Laboratories (J000104314). For the SYO.1 xenograft model, 5.0 × 106 cells were injected s.c. into each flank of 6–8-week-old female NOD/SCID gamma (NSG) mice. Treatment began when tumors reached approximately 150 to 200 mm3, as calculated using the L*W2 *.52 method. Mice were randomized into treatment cohorts, and tumor size was measured at least three times per week for the duration of treatment. Venetoclax was dissolved in 60% Phosal 50 PG, 30% PEG400, and 10% ethanol for a final dosage of 100 mg/kg body weight and administered via oral gavage five days a week [For the SS patient-derived xenograft model, 5.0 × 10s a week ,22. Nexts a week ,15. Then3 cells/well in 180 µL media. After 24 h, cells were treated with increasing concentrations of ABT-199 and S63845, alone or in combination, for 72 h, at which point 25 µL CTG reagent was added to each well, and plates were read using a Synergy H1 Hybrid microplate reader . These experiments were each repeated in triplicate over the course of several weeks. For crystal violet assays, 5.0 × 104 cells were seeded in 6-well plates for 24 h and then treated with ABT-199 and S63845, alone and in combination. Media was changed every other day until the untreated well was fully confluent, at which point cells were stained with 0.1% crystal violet . For FACS measurements of apoptosis, 2.0 × 105 cells were seeded in 6-well plates and after 24 h, were treated with ABT-199 and S63845 as indicated. After 24 h, cells were collected per BD Biosciences Annexin V/PI staining protocol and then assayed on a Millipore Guava flow cytometer. FACS experiments were conducted in biological triplicate. For CellTiter-Glo (CTG)  assays, cells were seeded in quadruplicate in 96-well plates at a concentration of 1.5 × 10NOXA overexpressing SS.PDX and SYO.1 cell lines were generated as previously described . In briet-test comparisons were performed using R2: Genomics Analysis and Visualization Platform . For the in vivo experiments, Unpaired two-tailed t-test comparing tumor volumes, and paired two-tailed t-test in comparing mouse weights, were performed using GraphPad Prism version 8.4.2 for macOS . For CTG experiments, the Bliss additivity method was used to calculate synergy scores [For RNA expression data, one-way ANOVA followed by two-tailed y scores .L) or pro-apoptotic proteins  can alter sensitivity to BCL-2 inhibition [L (BCL2L1) expression trended lower in the SS tumors, suggesting it is not a critical factor in venetoclax resistance [Despite high BCL-2 expression being diagnostic for SS, BCL-2 targeting has proven to be an ineffective strategy in preclinical SS models . As the hibition ,25,26,27hibition . As expesistance . In contL and MCL-1 were largely unchanged across cell lines with either construct. We next analyzed changes in BCL-2 family members after endogenous expression of SS18-SSX in a normal human fibroblast line, CRL-7250 [The SS18-SSX fusion protein can both activate and repress target gene transcription . The fusCRL-7250 . ConsistCRL-7250 ,31,32,33As low NOXA expression has been demonstrated to mediate venetoclax resistance in hematologic cancers ,34, we hAfter observing that NOXA expression sensitized SS cell lines to venetoclax and S63845 similarly sensitized SS cell lines, we chose to expand our investigation into the efficacy of venetoclax in combination with S63845 as a therapy for SS. We exposed a panel of SS cell lines to venetoclax and S63845, both alone and in combination. After 72 h of treatment, cell viability was assessed using CTG assay. Each drug showed limited efficacy as a single agent, yet strong synergistic cell killing in combination across all five cell lines expressing the SS18-SSX fusion gene A. In conWe also conducted seven-day treatments to assess SS response to the combined treatment B. BecausBCL-2 and MCL-1 are critical anti-apoptotic proteins and BH3 mimetics against these two molecules that induce apoptosis ,35. We fThese data prompted further investigation into how venetoclax and S63845 interacted to increase the amount of apoptosis. BIM is a pro-apoptotic protein that mediates venetoclax-mediated cell killing ,21. AnalWe next assessed the anti-tumor effect of venetoclax and S63845 in a patient-derived xenograft (PDX) model of SS. Mice were treated with 100 mg/kg of venetoclax  delivereSS accounts for ~10% of pediatric STS and, due to its metastatic potential and the very limited efficacy of chemotherapies, is often fatal . The knoVenetoclax is effective in the clinic as a monotherapy against chronic lymphocytic leukemia (CLL), which expresses BCL-2 at high levels , as wellMCL-1 is among the most well-characterized resistance factors for venetoclax. As a structurally related anti-apoptotic BCL-2 family member, MCL-1 can bind to pro-apoptotic factors released from BCL-2, such as BIM and BAK, thus preventing venetoclax-mediated apoptosis ,43,44. NSS has long been known to characteristically overexpress BCL-2 ; SS18-SSMultiple MCL-1 inhibitors are now being evaluated in early clinical trials . In addiDespite high levels of BCL-2 in SS, the activity of venetoclax in preclinical models of SS has been disappointing. This study demonstrates that suppressed NOXA expression leads to an MCL-1-mediated resistance to venetoclax in SS, thus revealing an Achilles’ heel that can be exploited by concomitant treatment with an MCL-1 inhibitor. Simultaneous disruption of both BCL-2 and MCL-1 via small-molecule inhibitors induces robust regression of tumors in a SS PDX model and cell-line xenograft model as well as multiple cell lines derived from both primary and metastatic disease. Given the lack of therapeutic options for SS, these data provide the rationale for clinical evaluation of this combination.


Clinical trials have evidenced that several natural compounds, belonging to the phytochemical classes of alkaloids, terpenes, phenols and flavonoids, are effective for the management of various types of cancer. Latest research has proven that natural products and their semisynthetic variants may serve as a starting point for new drug candidates with a diversity of biological and pharmacological activities, designed to improve bioavailability, overcome cellular resistance, and enhance therapeutic efficacy. This review was designed to bring an update regarding the anticancer potential of betulonic acid and its semisynthetic derivatives. Chemical derivative structures of betulonic acid including amide, thiol, and piperidine groups, exert an amplification of the in vitro anticancer potential of betulonic acid. With the need for more mechanistic and in vivo data, some derivatives of betulonic acids may represent promising anticancer agents. The extensive research over the past decades has identified an increased number of natural compounds from various medicinal plants that elicit chemopreventive potential. These phytochemicals represent an important potential source of anticancer molecules as such or after various physico-chemical modulations, which can be successfully used in different therapeutic protocols ,2. The aCancer is one of the largest causes of mortality in the world and due to its prevalence, the discovery of novel anticancer drugs has great importance. Plants, microorganisms, and marine organisms are considered valuable sources for the discovery of anticancer drugs ,5.Throughout history, natural products and their derivatives have proved to be useful for the development of chemotherapeutics, presenting a great structural diversity as well as molecular and pharmacological properties that favor such development .Vinca rosea L. (Apocynaceae), were the first agents to advance into clinical use. At low doses, they interrupt microtubular activity, whereas, at higher doses, they cause cell cycle arrest and apoptosis  in betulin)  = 4.2–32.0 µg/mL), as shown in 2)n in the amide residue did not affect the inhibitory activity, however, the introduction of the second amide residue increased the inhibition of the tumor cell growth (I (D)). The apoptosis-inducing activity of betulonic acid amides was higher than that of betulonic acid for MT-4 and MOLT-4 cell lines. Thus, the introduction of amide function at the position C28 of PT led to the formation of effective inducers of apoptosis methyl}-3-oxolup- 20(29)en-28-oate (XXI) were also examined in terms of pro-apoptotic activity; they induced DNA fragmentation in colon cancer cell line SW480, thereby providing strong signs of apoptosis. In addition, acridine orange/ethidium bromide staining of A549 lung carcinoma cells treated with these derivatives also showed induced apoptosis [The pro-apoptotic and cytotoxic activity of betulonic acid derivatives was reported by Csuk et al. The research groups have synthesized C(2)-methylene derivatives either by Mannich reactions, accompanied by poptosis .Kazakova et al. have reported the biological activity of C-28-imidazolides of betulonic acid, containing 3-oxo-, 3-hydroxyimino-, and 2-cyano-2,3-seco-4(23)-ene fragments in cycle A. Firstly, they assessed the cytotoxicity of the imidazolides (cycle A) using different human tumor cell lines , dyed with sulforhodamine B, and treated with 10 μM of the derivatives for 48 h. The results have shown that the imidazolide of 3-hydroxyimino-lup-20(29)-en-28-carboxylic acid (cycle A) (XXII) was the most efficient in inhibiting the cell growth of all six lung cancer cell lines, all seven large intestine cancer cell lines, all six leukemia cell lines, all six melanoma cell lines, all four breast cancer cell lines, two nervous system cancer cell lines, one prostate cancer cell line, three ovarian cancer cell lines, and five renal cancer cell lines. The cell survival rates are depicted in Owing to the hydrophobic properties of betulonic acid, its in vivo anti-cancer effectiveness has not been intensively studied . In a reBased on the remarkable results obtained for the imidazolide of 3-hydroxyimino-lup-20(29)-en-28-carboxylic acid (XXII), Kazakova et al. have studied its antineoplastic activity in vivo on two ascites tumors (Ehrlich tumor and P388 lympholeukemia) and three solid tumors  inoculated in BDF1 hybrids (DBA2 × C57B1/6J), BALB/C, and DBA mice. This chemical derivative injected intraperitoneally, five times daily at a dosage of 70 mg/kg, had no antineoplastic effect neither on the Ehrlich ascites tumor nor P388 lympholeukemia (at a dose of 30 and 50 mg/kg), respectively. Interestingly, the administration of 50 mg/kg through the same procedure suppressed the development of breast adenocarcinoma (Ca755) by 56–77%. The result was maintained without an improvement in life duration until day eight of the study. In mice with large intestinal adenocarcinoma treated by the same procedure, a 53% decrease in tumor growth was seen only after 5 days of therapy without any change in the mice’s lifespan .The research study conducted by Zhukova et al. examined the effects of betulonic acid and its methyl esters derivatives on the pathological modifications in the kidneys of the C57BL/6 mice, presenting Lewis pulmonary adenocarcinoma. Compounds were administered intraperitoneally in a dosage of 50 mg/kg for 8 days. After 8 days, mice were decapitated under ethereal anesthesia and sections of the treated kidneys were analyzed using periodic acid–Schiff reaction-hematoxylin and eosin-orange G dyeing. These chemical derivatives had a beneficial impact on the course of paraneoplastic nephropathy; they decreased the volume density of epithelial cell necrosis at 56%. On average, the number of cells with edematic cytoplasm, vesicular lipid infiltrate, and disrupted brush lymbus fell by 8–13% in comparison to untreated tumoral cells .Data presented in this work show that following structural modifications, which involved the introduction of amide, thiol, and piperidine groups, amplification of the in vitro anticancer potential of betulonic acid can be achieved. Studies published in the state of the art literature revealed that betulonic acid derivatives possess an important in vitro cytotoxic and pro-apoptotic activity. However, the number of in vivo studies is limited. The screening of scientific literature on this topic indicates a demand for in-depth in vivo data regarding the effect and mechanism of action of the betulonic acid derivatives in order to benefit from these structures in different therapeutic protocols designated for the management of various types of cancer.


Single crystal X-ray diffraction analysis of 1–3 and a dimethylsulfoxide (DMSO) solvate of 2 (2-DMSO), has identified the presence of a range of intra- and intermolecular interactions including N-H⋯N, N-H⋯O=C and N-H⋯O=S(CH3)2 hydrogen-bonding interactions, C-H⋯π interactions and off-set, face-to-face stacking π-π interactions that support the variety of slipped stack, herringbone and cofacial crystal packing arrangements observed in 1–3. Additionally, the cytotoxicity of this series of aromatic oligoamides was assessed against two human ovarian (A2780 and A2780cisR), two human breast (MCF-7 and MDA-MB-231) cancerous cell lines and one non-malignant human epithelial cell line (PNT-2), to investigate the influence of the terminal functionality of these aromatic oligoamides on their biological activity. The chemosensitivity results highlight that modification of the terminal group from Ac to Boc in 1 and 2 leads to a 3-fold increase in antiproliferative activity against the cisplatin-sensitive ovarian carcinoma cell line, A2780. The presence of the amine termini in 3 gave the only member of the series to display activity against the cisplatin-resistance ovarian carcinoma cell line, A2780cisR. Compound 2 is the lead candidate of this series, displaying high selectivity towards A2780 cancer cells when compared to non-malignant PNT-2 cells, with a selectivity index value >4.2. Importantly, this compound is more selective towards A2780 (cf. PNT-2) than the clinical platinum drugs oxaliplatin by > 2.6-fold and carboplatin by > 1.6-fold.The synthesis and characterization of three aromatic oligoamides, constructed from the same pyridyl carboxamide core but incorporating distinct end groups of acetyl (Ac)  The rise of cancer cell resistance towards clinical anticancer drugs, combined with the poor selectivity they can demonstrate for cancers over non-malignant tissue and the occurrence of adverse side-effects, has driven the search for new compounds with increased antiproliferative activity and selectivity. . Whilst 50 values (1.1–4.3 μM) against the human colon carcinoma (DLD-1), mesothelioma (I45), lung carcinoma (A549), and human non-small cell lung carcinoma (H1299).Systematic solid-state studies of aromatic oligoamides have identified that small structural variations in these molecules can have a profound influence on their conformational behavior and such studies can to help deepen our understanding of their structure-activity relationships (SARs). A crystallographic study of aromatic oligoamides by Nissinen and co-workers  showed t1, tert-butyloxycarbonyl (Boc) 2 and amine 3 , carboplatin (CARB) and oxaplatin (OXA), which all demonstrate high cytotoxicity.Gaining an understanding of the influence of the structure of an aromatic oligoamide on its biological activity is central to the development of new molecules within this class that have the potential to demonstrate improved cytotoxicity towards cancerous cells. To probe the influence of the terminal group on the solid-state structure and antiproliferative activity of these aromatic oligoamides, we undertook the synthesis, crystallographic analysis and cytotoxicity studies of three aromatic oligoamides based on the same pyridyl carboxamide core but including different end groups; acetyl (Ac)  amine 3 . We repo1–3, which all have the same pyridyl carboxamide core, but incorporate different terminal groups of Ac 1, Boc 2 and NH23 . Electronspray ionization mass spectrometry identified the molecular ion peaks at m/z 432.1670 [M + H]+1, 548.2503 [M + H]+2 and 348.1453 [M + H]+3.Aromatic oligoamides, nd NH23  have beend NH23  . 1–3 and 2-DMSO . X-ray diffraction analysis identified the nature of the non-covalent interactions present in the solid state for each of the studied aromatic oligoamides.Single crystals suitable for X-ray diffraction were obtained for 1 were grown by vapor diffusion of diethyl ether into a dimethylformamide solution at ambient temperature. 1 crystallizes in a monoclinic crystal system and solution refinement was performed in the P21/c space group -H (2/4)⋯N (1) (2.6583(17)-3.2310(16) Å, 1 displays a slipped stack crystal packing arrangement,  solvate, 2-DMSO, were grown from two different crystallization conditions at ambient temperature, firstly, through the slow evaporation of chloroform to give 2 and secondly, through the slow evaporation of a 9:1 chloroform:DMSO solvent mixture to generate 2-DMSO. In the former conditions, 2 crystallizes in an orthorhombic crystal system and solution refinement was performed in the P212121 space group -H (2/4A)⋯N (1) 2.34(4)-2.36(4) Å, The molecular structures of 2 adopts a herringbone crystal packing arrangement -H (3)⋯O (4) = C and N (5)-H (5)⋯O (1) = C hydrogen bonding interactions, (2.03(4)-2.231(4) Å, tert-butylcarboxyaminophenyl rings on neighboring molecules -H (3)⋯O (4) = C, (2.012 (18) Å, 3)2 hydrogen-bonding interaction present which involves one of the NH’s of a terminal group moiety and the O atom of a DMSO solvent molecule (N (5)-H (5)⋯O (7) = S(CH3)2, (2.007 (19) Å, In the crystal packing of 3 were grown through the slow evaporation of chloroform at ambient temperature. 3 crystallizes in a monoclinic space group and solution refinement was performed in the P21/c space group -H (3B)⋯O (2) 2.15 (7) Å (3 (angement  orientat (c axis  and this15 (7) Å , N (8)-H15 (7) Å  and N (115 (7) Å . The sec15 (7) Å . Additio(7) Å (3 . (Egli e(7) Å (3 .CDDP), carboplatin (CARB) and oxaliplatin (OXA) and compounds 1–3 were screened for their cytotoxicity against human cell lines: cisplatin-sensitive ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780cisR) and breast adenocarcinomas (MCF-7 and MDA-MB-231). The IC50 values were obtained via the MTT assay after a 96 h incubation period of each compound with the cells at 37°C and 5% CO2 . The amine-terminated compound 3 in non-toxic towards the breast adenocarcinomas cell lines (MCF-7 and MDA-MB-231), with IC50 values greater than the tested threshold (>100 μM). Notably, 3 is non-toxic against the cisplatin-sensitive ovarian carcinoma A2780 but is the only one in the library which displays any level of antiproliferative activity against the cisplatin-resistant ovarian carcinoma cell line, A2780cisR, with a moderate IC50 value of 61 ± 1 μM.Cisplatin  and breast adenocarcinomas (MCF-7 and MDA-MB-231). Results highlight the Boc-terminated compound CDDP, OXA, and CARB and compounds 1–3 were also screened against the non-malignant prostate cell line , PNT-2, to determine any cancer cell selectivity. The results for CDDP, OXA, and CARB show that these clinical platinum drugs have high to moderately cytotoxicity towards PNT-2, with IC50 values of 1.3 ± 0.2 μM (OXA), 8.5 ± 0.4 μM (CDDP) and 27 ± 2 μM (CARB). Unlike the clinical platinum drugs, compounds 1–3 are non-toxic towards PNT-2  The selectivity index (SI) values were calculated for all the compounds, using the IC50 values obtained against PNT-2 and dividing by the IC50 value against the cancer cell line in parenthesis in 1 shows only slight increases in selectivity, with an SI > 1.3*  for this compound against the triple negative breast cancer (TNBC) cell line, MDA-MB-231, is higher than those observed for the clinical platinum anticancer drugs OXA and CARB (of 0.5 and 0.8 respectively).1 Compound 2 displays a notable SI > 4.2* (p < 0.05) against A2780 and a very moderate increase in selectivity towards MCF-7  and MDA-MB-231 (SI > 1.4*).3, is the only compound to display increased selectivity for A2780cisR when compared to PNT-2, with a SI > 1.6* (p < 0.05), which, albeit is very modest, is higher than the SI values observed for CDDP (0.6), OXA (0.6) and CARB (0.3*). Overall, these results highlight that small structural changes to the terminal groups of these aromatic oligoamides can have a marked effect on their biological activity against the tested ovarian and breast cancer cell lines. Herein, it is shown that modification of the terminal groups from Ac to Boc leads to a notable increase in the SI against A2780 but similar SI values are observed against MDA-MD-231, whilst variation of the terminal group to NH2 leads to a change in the SI of the aromatic oligoamide against A2780cisR with a slight increase in SI  being observed by this amine-terminated compound.1, tert-butyloxycarbonyl (Boc) 2 and amine 3. Single crystal X-ray diffraction analysis of 1–3 and 2-DMSO has identified the presence of an array of non-covalent interactions including N-H⋯N and N-H⋯O=C hydrogen-bonding interactions, a series of C-H⋯π and π-π stacking interactions that support the diverse crystal packing arrangements present in these aromatic oligoamides including slipped stack (1), herringbone (2) and cofacial/slipped stacked (3). The crystal packing of 3 also reveals the presence of hydrogen-bonded dimer formed by the presence of reciprocal intermolecular N-H⋯O=C hydrogen bonding interactions formed between the NH of the terminal amine groups and the O atom on the carbonyl group in the amide group of an adjacent molecule.In conclusion, we have synthesized and characterized a series of aromatic oligoamides based on a common pyridyl carboxamide core but incorporating distinct end groups: acetyl (Ac) 1–3  were obtained via a 96 h MTT assay, and screening against human ovarian carcinomas (A2780 and A2780cisR), human breast adenocarcinomas (MCF-7 and MDA-MB-231) and non-malignant prostrate cell line (PNT-2). Generally, compounds 1–3 display either moderate cytotoxicity or are non-toxic against A2780cisR, MCF-7 and MDA-MB-231 cancer cell lines. The Boc-terminated compound, 2, is the lead candidate of the tested aromatic oligoamides displaying an IC50 value of 24 ± 0.9 μM against A2780. Unlike the tested clinical platinum anticancer drugs, compound 2 is non-toxic towards PNT-2 (IC50 > 100 µM), meaning it displays an SI value >4.2*-fold towards A2780 (cf. PNT-2), making it more selective towards ovarian cancer than the platinum drugs CDDP and OXA , 6.5 (CARB); 2.6 (OXA)). The insights gained from this study, regarding the importance of small structural modifications on influencing the biological activity of aromatic oligoamides, will facilitate the future design of related compounds with improved cytotoxicity against ovarian and breast cancer cell lines.To understand SARs, the cytotoxicity of the compound


Background: Systemic treatment options for metastatic renal cell carcinoma (RCC) have significantly expanded in recent years. However, patients refractory to tyrosine kinase and immune checkpoint inhibitors still have limited treatment options and patient-individualized approaches are largely missing. Patients and Methods: In vitro drug screening of tumor-derived short-term cultures obtained from seven patients with clear cell RCC was performed. For one patient, a patient-derived xenograft (PDX) mouse model was established for in vivo validation experiments. Drug effects were further investigated in established RCC cell lines. Results: The proteasome inhibitor carfilzomib was among the top hits identified in three of four patients in which an in vitro drug screening could be performed successfully. Carfilzomib also showed significant acute and long-term cytotoxicity in established RCC cell lines. The in vivo antitumoral activity of carfilzomib was confirmed in a same-patient PDX model. The cytotoxicity of carfilzomib was found to correlate with the level of accumulation of ubiquitinated proteins. Conclusions: In this proof-of-concept study, we show that patient-individualized in vitro drug screening and preclinical validation is feasible. However, the fact that carfilzomib failed to deliver a clinical benefit in RCC patients in a recent phase II trial unrelated to the present study underscores the complexities and limitations of this strategy. Metastatic renal cell carcinoma (RCC) has a poor prognosis with 5-year survival rates between 8% and 12% ,4,5. DesAdvanced RCC is characterized by a high degree of genomic and functional intratumoral heterogeneity ,7. UnderIn the present proof-of-concept study, we demonstrate the feasibility of simultaneous patient-individualized in vitro drug screening and PDX development in metastatic RCC. The potent proteasome inhibitor carfilzomib was identified as the most effective compound in vitro and its antitumoral activity was confirmed in vivo. In light of a negative phase II study of carfilzomib in patients with advanced RCC, published recently , complexl-glutamine . All experimental procedures were approved by the Ethics Committee of the University of Heidelberg School of Medicine (S-453/2010). Written informed consent was obtained from all patients.Short-term tumor cell cultures were established as previously described in . Brieflyhttps://dtp.cancer.gov/organization/dscb/obtaining/available_plates.htm (accessed on 21 November 2013). The compounds of the library included chemotherapeutic agents, small molecule inhibitors, and antihormonal substances. Briefly, tumor cells were seeded into 96-well plates with 10,000 cells per well and allowed to adhere overnight. Drugs were then added to the tumor cells at a final concentration of 1 and 10 μM, respectively. The drug concentrations used for hit discovery, i.e., 1 and 10 μM, were based on the published literature [VHL status [2.Short-term tumor cell cultures were subjected to a medium-throughput functional drug screening procedure using the National Cancer Institute’s (NCI) Developmental Therapeutics Program Approved Oncology Drugs Set IV. A full list of compounds can be found under L status . All celPrimary tumor cells were injected subcutaneously into the flanks of Naval Medical Research Institute (NMRI)-nude mutant mice. The growth of palpable tumors was monitored and followed by randomized treatment with the pharmacologic agents (vehicle control or 3 mg/kg/d carfilzomib i.v.). The treatment group consisted of five animals; the control group consisted of four animals. To determine therapeutic efficacy, tumors were measured twice weekly. The study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Society of Laboratory Animals (GV SOLAS). All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Freiburg regional council  and performed at Charles River Laboratories, Freiburg, Germany.The Proteome Profiler™ Array for human ubiquitinated proteins was used as recommended by the manufacturer .t-test for independent samples was used wherever applicable. p values ≤ 0.05 were considered statistically significant.Student’s two-tailed Short-term tumor cell cultures from seven RCC patients were established and used for functional drug screening with the NCI Approved Oncology Drugs Set IV . For pat50 was reached), and only drug screens with at least one compound producing such a reduction of cell viability were considered successful in our analyses [A drug hit was defined as a ≥0.5-fold change in cell viability by a compound .We were able to further validate the result that carfilzomib is highly effective in RCC001 by testing short-term tumor cell cultures obtained from two distinct anatomic locations c,d. BothTaken together, functional drug screening of tumor-derived short-term cultures and validation in a same-patient PDX model demonstrate antitumoral effects of the proteasome inhibitor carfilzomib in advanced RCC.We next investigated whether carfilzomib has acute antitumoral activity in established RCC cell lines as well as non-cancerous, kidney-derived HEK293 cells. First, we tested the response to carfilzomib in these cell lines and found growth-inhibitory activities in all cell lines, starting at a concentration of 0.01 μM .VHL wildtype) and 786-0 cells (VHL mutated), with HEK293 cells used as controls, and analyzed the response to carfilzomib in comparison to sunitinib, sorafenib and axitinib, which are VEGF receptor targeting receptor tyrosine kinase inhibitors (TKIs) approved for the treatment of metastatic RCC  under normoxic conditions. In case of hypoxia, and when VHL is inactivated, HIFs accumulate and activate signaling pathways that lead, among others, to enhanced angiogenesis via the VEGF/VEGF receptor (VEGFR) axis. This pathway is the basis for established and novel therapeutic interventions in RCC that target VEGF, VEGFR or HIF2α [Unfortunately, in a recent phase II study of nine patients with treatment-refractory metastatic clear cell RCC, there was an absence of antineoplastic activity of carfilzomib . All ninionality . The VHLor HIF2α .In a situation, however, where destabilized pVHL with at least partially retained functionality is expressed, pVHL activity could potentially be rescued by inhibition of the proteasome with a subsequent re-activation of HIF degradation . Given tFirst, it is conceivable that short-term cultures and established cell lines do not adequately reflect the complex microenvironment in a patient tumor .Second, proteasome inhibition has been reported to attenuate immune cell functions . RCC is There are a number of additional points that need to be taken into account when patient-individualized drug screening and validation are considered. The screening set-up may have favored finding drugs like carfilzomib, since it is known for relatively fast apoptosis induction, while, for example, TKIs are more dependent on cell growth and inhibition of signal transduction pathways. In addition, the target structure of TKIs may not be expressed by the cells analyzed. As such, for drugs with different types of kinetics and mechanisms of action, different assays may be appropriate .It may be also possible to exploit the drug screening results for potential combination therapies. For example, in patient RCC006, carfilzomib and the EGFR-targeting TKI erlotinib were both found as hits . A previThe off-label use that almost inevitably follows drug screening efforts has legal ramifications that can prolong or prevent drug procurement. In addition, low engraftment rates and slow tumor growth can delay the generation of PDX models. Moreover, serial passaging has been shown to alter drug sensitivities and evolutionary trajectories away from the primary tumor ,38,39. IVHL mutation testing has not been performed. However, since the vast majority of patients with clear cell RCC harbor inactivated VHL, it is highly likely that this is also the case in our patients. We found a more pronounced accumulation of ubiquitinated proteins in a VHL-deficient RCC cell line pointing to a more general role of pVHL in the elimination of abnormal proteins, as previously suggested [Further limitations of our proof-of concept study are the small sample size and the lack of genetic information of the tumors. Unfortunately, uggested .Taken together, our results show that patient-individualized drug screening and parallel in vivo validation is feasible but at the same time underscore the complexity of this approach. Further studies are warranted to understand the apparent disconnection between preclinical and clinical findings and to optimize tools for drug discovery in advanced RCC.


Since, phosphatidyl-inositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and fibroblast-growth-factor-receptor-3 (FGFR3) mutations often occur in such tumors, here, we tested targeted therapy directed to such genes in TSCC/BOTSCC cell lines. We also combined the two types of inhibitors with each other, and cisplatin or docetaxel that are used clinically.Human papillomavirus positive  approved drugs, alpelisib (BYL719) and erdafitinib (JNJ-42756493) alone and in combination with cisplatin or docetaxel. Viability, proliferation, apoptosis and cytotoxicity responses were thereafter followed by WST-1 assays and the IncuCyte S3 Live® Cell Analysis System.The HPV+ CU-OP-2 had a pS249C-FGFR3, and like CU-OP-20, a pE545K-PIK3CA mutation, while no other lines had such mutations. Irrespectively, dose dependent responses to all PI3K/FGFR inhibitors were obtained, and upon combining the inhibitors, positive effects were observed. Cisplatin and docetaxel also induced dose dependent responses, and upon combination with the inhibitors, both positive and neutral effects were found.HPVThe data suggest that FDA approved drugs alpelisib and erdafitinib efficiently inhibit TSCC/BOTSCC cell line growth, especially when combined irrespective of presence of corresponding mutations and should be further explored, for use upon recurrent disease. Stock solutions were at 10 mM for all inhibitors, except BEZ235 where a stock solution of 2 mM was recommended. Further dilutions were done in PBS, and were for: AZD4547 5-25 μM; JNJ-42756493 0.01-10 μM; BEZ235 0.25-1 μM; and for BYL719 0.5-20 μM. Cisplatin  was diluted in PBS and used at 2.5-7.5 μM. Docetaxel  was diluted in PBS and used at 0.6-6 nM. All experiments were repeated minimum three times.Viability was quantified using the WST-1 assay  and followed for 72 h according to the instructions of the manufacturer and before , 42.® Cell Analysis System, using the IncuCyte Caspase-3/7 Green Apoptosis Assay Reagent and the Incucyte™ Cytotox Red Reagent , and followed for proliferation, apoptosis and cytotoxicity by images taken every 2 h  decreased viability consistently (p<0.01) , while CU-OP-2 and -3 were more resistant , . For UPCI-SCC-154 and UT-SCC-60A only data on the FDA approved drugs are shown, since data on the others have been published  , for all remaining, at least p<0.05  of BYL719-JNJ-42756493 were calculated for all lines 24-72 h after treatment as done before , 42–44. To summarize, combining PI3K-FGFR inhibitors, generally resulted in positive combinatory effect in most cell lines.All cells lines presented dose dependent responses to single treatments with cisplatin or docetaxel as shown with WST-1 assays in All CU-OP lines presented decreased viability compared to PBS with 2.5-7.5 µM of cisplatin 72 h after treatment, except for CU-OP-20 with 5 µM cisplatin     of BYL719 and/or JNJ-42756493 with cisplatin or docetaxel were calculated for all cell lines 48-72 h after treatment , 38–40, To summarize, mainly positive and neutral effects were obtained upon combining BYL719 and JNJ-42756493 resp. with cisplatin and docetaxel on CU-OP lines, with the best effects observed for CU-OP-2 and CU-OP-17.Effects on proliferation, apoptosis and cytotoxicity by single and combined BYL719 and JNJ-42756493 treatments on the CU-OP lines were followed by the IncuCyte S3 Live−Cell Analysis System. All lines showed complete inhibition of proliferation with all BYL719 doses, and the highest JNJ-42756493 dose, and upon combined exposures excluding the highest doses of both inhibitors, proliferation was inhibited with all combination doses Figure 6Cytotoxicity was observed for all CU-OP lines with the two highest BYL719 (1 and 10 µM) and the highest JNJ-42756493 (10 µM) concentrations. Upon combined exposure, cytotoxicity was observed in most lines, especially those including 1 µM BYL719 .Combining BYL719 (0.5-10 µM) or JNJ-42756493 (0.01-1 µM) with either 7.5 µM cisplatin or 6 nM docetaxel, inhibited proliferation completely (data not shown). Cytotoxicity was observed with the two highest BYL719 (1 and 10 µM) and the highest JNJ-42756493 (10 µM) doses with cisplatin or docetaxel for CU-OP-2 and -3, while CU-OP-20 and CU-OP-17 were generally less sensitive especially when docetaxel was present with JNJ-42756493 (data not shown). Prominent apoptosis was only observed in combinations including the highest JNJ-42756493 dose (10 µM) (data not shown).In this study, the FDA approved drugs alpelisib and erdafitinib induced dose dependent effects with decreased viability and proliferation on HPV+ and HPV- TSCC/BOTSCC cell lines, irrespective of whether the cells had FGFR3 and/or PIK3CA mutations. Furthermore, upon combining these inhibitors mainly positive or neutral effects were disclosed with comparable decreases in viability and proliferation using lower doses of the inhibitors. All tested TSCC/BOTSCC cell lines above also presented dose-dependent effects with diminished viability and proliferation when treated with cisplatin and docetaxel often used clinically, although e.g., CU-OP-2 and CU-OP-3 were relatively more resistant. Subsequently, only the CU-OP lines, were followed for their responses to targeted inhibitor-chemotherapeutic drug combinations, and here also positive and neutral effects were disclosed.The data on the FDA approved drugs alpelisib and erdafitinib paralleled the inhibitory effects on viability and proliferation, as well as the effects on cytotoxicity and apoptosis of recently tested inhibitors BEZ235 and AZD4547 on UPCI-SCC-154 and UT-SCC-60A , 45. TheThat the cell lines above had drug dependent dose decreases in viability and proliferation, to FDA approved inhibitors, and that effects were enhanced upon combining the two was anticipated, since UPCI-SCC-154 and UT-SCC-60A had responded in an analogous way, to corresponding inhibitors BEZ235 and AZD4547  was superior to the included PI3K inhibitors with regard to inducing cytotoxicity , 42, 43.Furthermore, all TSCC/BOTSCC cell lines, with/without FGFR or PI3K mutations, were sensitive to PI3K and FGFR inhibitors and those with such mutations were not necessarily more sensitive or resistant to the inhibitors, similar to that reported for other tumor types , 46–53. The HPV+ and HPV- TSCC/BOTSCC cell lines were also examined for their responses to single cisplatin and docetaxel therapy and also here CU-OP-2 and -3 were the most resistant ones, while the remaining cell lines tended to be more sensitive. This pattern was not reflected upon by the effects of cisplatin and docetaxel on proliferation, which was largely inhibited in a similar way for all cell lines. At the doses used however, neither cisplatin or docetaxel disclosed prominent effects on cytotoxicity or apoptosis, but better effects may have been obtained if higher concentrations or repeated treatments had been used. Nonetheless, here we aimed to use as low doses as possible, in order to disclose combinational effects upon combining the chemotherapeutic agents with the inhibitors.When examining potential combinational effects using cisplatin and docetaxel together with BYL719 or JNJ-42756493, mainly neutral and positive, with occasional negative effects were disclosed. Of note were some positive combinations with BYL719 and cisplatin or docetaxel especially on CU-OP-2, but also with the other cell lines, and the data suggest it could be worthwhile elaborating further on optimizing the effects of such combinations.To our knowledge, the pursuit of potential positive effects of alpesilib and erdafitinib upon combination with cisplatin and docetaxel have not been examined before in TSCC/BOTSCC, and there are only limited studies on other cell lines, comprising some of the drug-inhibitor combinations in this report. One study on nasopharyngeal cancer cell lines, showed neutral or limited positive effects upon combining BYL719 and cisplatin, while another report on a non-small lung cancer cell, showed more positive effects , 54. HowThis report has limitations. Despite six cell lines were included, more could have been examined. Nonetheless, UPCI-SCC-154 and UT-SCC-60A have extensively been used and the CU-OP cell lines, have the benefit of including lines with PIK3CA and FGFR3 mutations –41. IrreTo conclude, this study supports the potential of further exploring the combined use of FDA approved drugs alpelisib and erdafitinib for the treatment of recurrent TSCC/BOTSCC especially when other options e.g., check point inhibitors are not useful.The original contributions presented in the study are included in the article/SH and OK did the majority of the experiments, interpreted the data, calculated the statistics and contributed to the writing of the manuscript. CB performed the CAST-PCR to detect the mutations of the cell lines presented in this study. NW and RU collaborated with SH and OK and performed some experiments contributed together with SH and OK in the graphs of the manuscript. NW and RU initiated the experiments and the interpretation of the initial experiments and contributed to the writing of the material and methods section, all under the supervision of SH. MZ and AN assisted in the analysis of the data and in their clinical interpretation. TD, SH, and OK made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data and have been involved in drafting the manuscript and revising it critically for important intellectual content. TD has also provided the sources of the performance of the experiments. TD, OK, SH, MZ, and RU provided the financial support for conducting the research project. All authors contributed to the article and approved the submitted version.This research was funded by the Swedish Cancer Foundation (grant no. 18 0440), the Stockholm Cancer Society (grant no. 181053), the Swedish Cancer and Allergy Foundation (grant no. 190), the Royal Swedish Academy of Sciences (grant no. 2017-2018), the Stockholm City Council (grant no. 20180037), Karolinska Institutet (grant no. 2018:0007) and Stiftelsen Clas Groschinsky Minnesfond , Lindhes Advokatbyrå , and University of Medicine and Pharmacy Grigore T Popa Iasi (grant no. 6983/21.04.2020).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.