The application of patient-derived xenografts (PDX) in drug screening and testing is a costly and time-consuming endeavor. While cell lines permit extensive mechanistic studies, many human breast cancer cell lines lack patient characteristics and clinical treatment information. Establishing cell lines that retain patient’s genetic and drug response information would enable greater drug screening and mechanistic studies. Therefore, we utilized breast cancer PDX from the Mayo Breast Cancer Genome Guided Therapy Study (BEAUTY) to establish two immortalized, genomically unique breast cancer cell lines. Through extensive genetic and therapeutic testing, the cell lines were found to retain the same clinical subtype, major somatic alterations, and drug response phenotypes as their corresponding PDX and patient tumor. Our findings demonstrate PDX can be utilized to develop immortalized breast cancer cell lines and provide a valuable tool for understanding the molecular mechanism of drug resistance and exploring novel treatment strategies. The major advantages of using cultured cell lines are their prolonged conservation of genetic and molecular features and can be easily managed and maintained using standard culture conditions to provide enough material for cancer research in a short time period2. However, most commercially available cell lines were established decades ago, with the first human breast cancer cell line created in 19583, and have limited patient-related clinical and treatment information. Establishing cell lines that retain this information will better assist researchers in understanding the molecular biology of cancer.Immortalized cell lines have been widely used for studying cancer biology and treatment response4. We previously established multiple breast cancer PDX models from primary breast cancer patient tumors recruited in a prospective trial, BEAUTY6. Utilizing these PDX models has enabled us to identify novel pharmacogenomic biomarkers associated with various therapies7. Furthermore, we have successfully generated PDX-derived organoids by dissociating PDX tumors into single-cell suspensions and culturing them in vitro and demonstrated their importance in understanding cancer pathology and drug resistance mechanisms7. However, the costs associated with the maintenance and long turnaround time to generate PDX limit their widespread use in laboratory practice. On the other hand, the establishment of immortalized primary breast cancer cell lines from PDX tumors could provide valuable tools for mechanistic studies and elucidating alternative therapies in a relatively cost-effective way.Ex vivo culture of fresh patient tumor tissue or establishing patient-derived xenografts (PDX) in mice, provide models closely representing the heterogeneity and drug response of patient tumors9. The ability of cancer cells to grow on plastic tissue culture plates varies based on the cancer cell histology, tumor grade, presence of specific genetic aberrations as well as appropriate nutritional support4. The human breast cancer cell lines extensively studied by investigators  were established from metastatic lesions, making them less likely to accurately recapitulate the genetic composition or biological behavior of primary breast tumors10. While others have successfully established cell lines from tumors12, only biopsies were obtained from BEAUTY patients, limiting tumor availability for in vitro sub-culture. Previously, Cavotelli et al. and Matossian et al. demonstrated the advantage of utilizing in vivo systems to generate immortalized cell lines15. They suggested sub-culturing tumor biopsies in vivo not only expands the number of tumor cells but enables tumors to adapt to their surrounding microenvironment, ensuring proliferative growth and easy passage-traits essential to the immortalization of the tumor cells15. As a result, when PDX tumors were plated in a single-cell suspension in cell culture, immortalized cell lines were established. These examples provide us with the rationale to examine whether it is possible to derive immortalized cell lines from our primary breast cancer PDX tumors.The process to acquire immortalized cancer cell lines is complex with unpredictable resultsHere we demonstrate not only the feasibility of establishing unique HER2+ and triple-negative immortalized primary breast cancer cell lines from their respective PDX tumors, but also draw genetic and drug phenotype comparisons between their respective organoid, PDX, and original patient primary tumor. We hope that our findings will provide researchers with additional knowledge and tools to better elucidate the mechanisms behind drug resistance and identify novel therapies for breast cancer patients in the era of precision medicine.Immortalized cell lines, MC-BR-BTY-0019 and MC-BR-BTY-0006, were established from HER2+ and triple-negative primary breast cancer PDX models maintained in NSG mice, respectively. Cells were isolated, cultured, and trypsin-passaged in optimized culture medium and conditions as described in the “Methods” section for ~2 months until being able to be passaged continuously without an apparent sign of senescence; at which point the cells were considered to be immortalized. MC-BR-BTY-0019 cells were round-spindle shape and grew as a monolayer with tight adherence to the plate, while MC-BR-BTY-0006 cells were round shape and had loose adherence to the plates Fig. . MC-BR-BNext, we performed short tandem repeat (STR) profiling to confirm the unique genetic identity of our cell lines. Based on testing results obtained from the analysis of 15 autosomal STR loci and the gender identity locus amelogenin, the profile of MC-BR-BTY-0019 and MC-BR-BTY-0006 did not match any existing cell lines documented by the ATCC, DSMZ, and JCRB human cell line STR profile databases Fig. ; suggestKaryotype images of the two cells, MC-BR-BTY-0019 and MC-BR-BTY-0006 are shown in Fig. Similar to MC-BR-BTY-0019, MC-BR-BTY-0006 exhibited a very complex hyper-diploid karyotype which included numerous clonal structural rearrangements including an isochromosome 3q, a doubled version of an unbalanced, whole arm rearrangement involving chromosomes 17 and 19, resulting in loss of 17p and doubled versions of structural abnormalities of 18q and 21p, as well as numerous other unbalanced abnormalities Fig. .16. The two established cell lines MC-BR-BTY-0019 and MC-BR-BTY-0006 were originally derived from HER2+ and triple-negative breast cancer, respectively. To characterize the molecular subtype of the established cell lines, western blot was performed to examine the level of HER2 and EGFR. We compared the protein levels between the two newly established cell lines with their corresponding PDX-derived organoid, PDX tumor tissues and two commonly studied breast cancer cell lines , ErbB2 receptor tyrosine kinase 2 (HER2), and epidermal growth factor receptor (EGFR)nes Fig. .Fig. 3CeThe cell line MC-BR-BTY-0019, and its PDX-derived organoid expressed HER2 at similar levels in comparison with well studied HER2+ cell line, SKBR3, while slightly decreased in the PDX tumor. MC-BR-BTY-0006 was HER2-negative across all three culture types . The relative abundance of EGFR in MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines was comparable to SKBR3 and MDA-MB-231, respectively, but significantly higher than the corresponding PDX-derived organoid. For MC-BR-BTY-0006, EGFR levels were lowest in its derived PDX tumor tissue, but highest for MC-BR-BTY-0019. Immunofluorescent staining in Fig. 17. Paclitaxel is a standard chemotherapy drug targeting microtubule formation thus inhibiting cancer cell mitosis, and has been clinically used to treat all breast cancer subtypes, including triple-negative breast cancer18. In order to examine whether the response to treatment was similar between cell line and PDX-derived organoid in vitro, the cell lines, as well as the organoids, were treated with specific targeted drugs, lapatinib in HER2+ MC-BR-BTY-0019 and paclitaxel in triple-negative MC-BR-BTY-0006. MC-BR-BTY-0019 cell line was sensitive to lapatinib treatment, consistent with PDX-derived organoid response with an IC50 of 438.531 and 979.49 nM, respectively, for cell line and organoids , forming tumors within weeks19. However, even with commonly used breast cancer cell lines, not all of them can be engrafted and grown into tumor20. When our two newly developed cell lines, MC-BR-BTY-0019 and MC-BR-BTY-0006 were injected into the right flank of the NSG mice, tumors formed after about 2 and 3 weeks, respectively ), BT20 (70 h)25, BT549 (53.9 h (NCI-DTP)), and Hs578T (53.8 h (NCI-DTP)). STR profiling for the two cell lines demonstrated they were unique cell lines without any similarity to existing breast cancer cell lines ), MDA-MB-453 (50–60 h (DSMZ)), BT474 (100 h (DSMZ)), and MDA-MB-361 (103.2 h)nes Fig. , and matnes Fig. . While iThe further characterization of these two cell lines revealed significantly abnormal karyotypes. The data we have obtained from the karyotypes indicated these two cell lines had clonal variation within the established cell lines. For MC-BR-BTY-0019, 11 among the 20 examined metaphases represented a tetraploid subclone and MC-BR-BTY-0006 presented clonal structural rearrangements  female mice and maintained as PDX as described in a previous study6. No human subject was involved in this particular study. All protocols for animal studies have been approved by the Institutional Animal Care and Use Committee (IACUC) of Mayo Clinic. PDX tumors were collected when the tumor grew to ~10–20 mm diameter. Primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described7. A single-cell suspension of primary breast cancer cells was initially cultured in mouse embryonic fibroblast (MEF) media  supplemented with glutamax, MEM NEAA, sodium pyruvate, and 5 μM Y-27632-inhibitor (Tocris)). Once the primary cancer cells were established after two passages, cells were maintained in DMEM with 10% FBS supplemented with Anti–Anti (Gibco).Breast tumor tissue obtained from a percutaneous biopsy of patients with primary non-metastatic breast cancer recruited on a prospective trial, BEAUTYMC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were plated onto 16-well CultureWell™ chambered coverglass (Molecular Probe C37000) with 20,000 cells per well. Cells were fixed with 4% formaldehyde the next day and treated with or without 0.1% Triton X-100. The cells were then blocked with 3% BSA for 30 min and incubated with either HER2 or EGFR antibodies overnight at 4 °C. Cells were incubated with appropriate Alexa Fluor antibody for one hour at room temperature and stained with DAPI before visualization by Zeiss LSM 780 confocal microscope at ×100 magnification. Antibodies against HER2  and EGFR  were purchased from Cell Signaling. Secondary Antibody, Alexa Fluor 488  was purchased from Invitrogen.® Turbo™ RTA Mini PVDF Transfer Kit (BioRad #1704272). The membrane was then incubated with the appropriate antibody in TBS-T (1× TBS with 0.1% Tween-20) containing 5% non-fat dry milk overnight at 4 °C followed by incubating with the horseradish peroxidase (HRP)-conjugated secondary antibody for one hour. Proteins were detected using the SuperSignal™ West Dura Extended Duration Substrate (BioRad Cat#34075). Primary antibodies against beta-Actin , HER2/ErbB2  and EGFR  were purchased from Cell Signaling. Secondary horseradish peroxidase-linked anti-mouse  and anti-rabbit  IgG antibodies were purchased from Cell Signaling.MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were lysed by Laemmli sample buffer. 300 mg of PDX tumor was placed into microcentrifuge tubes containing 300 μL NETN lysis buffer  containing phosphatase and proteasome cocktail inhibitors and 0.5 mm RNase-free zirconium oxide beads (Next Advance). Tumors were lysed by placing tubes into a bullet blender for 10 minutes at 4 °C. The concentration of cell lysates was then quantified via Bradford assay and mixed with Laemmli sample buffer containing 10% 2-ME and boiled for 10 min. Equal amounts of protein of each cell line were separated on 4–20% SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes using a semi-dry Bio-Rad Trans-blot apparatus with Trans-Blot7. A single-cell suspension of primary breast cancer cells was initially cultured in MEF media ). ROCK inhibitor was removed from media by changing media one week before drug treatment. Cells were treated with various concentrations of lapatinib or paclitaxel for 5 days. 3D cell TiterGlo kit (Promega) was used to measure 3D culture viability according to the manufacturer’s instructions to obtain dose–response curves.MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines were seeded in DMEM-10% FBS in a 96-well cell culture plate with 10,000 cells per well. After cell attachment, various concentrations of lapatinib (Selleckchem) and paclitaxel (Selleckchem) were used to treat the cells. Following 48-h treatments, MTS (Sigma) was added to the wells according to the manufacturer’s instructions to obtain dose–response curve. MC-BR-BTY-0019 and MC-BR-BTY-0006 organoids were grown from corresponding PDX tumors freshly harvested from mice and cultured in MEF media with 10,000 cells per well in nanoculture plates. Briefly, PDX tumors were collected when the tumor grew to ~10–20 mm diameter. Primary breast cancer cells were isolated after dissociation of tumor tissue from mouse cells, as previously described3, mice bearing MC-BR-BTY-0019 were randomized into two groups and treated with either vehicle control or lapatinib at a dose of 50 mg/kg (i.p. 5 days/week) for 3 weeks. Mice bearing MC-BR-BTY-0006 were randomized into two groups and treated with paclitaxel at a dose of 12.5 mg/kg (i.p. 2 times/week) for 3 weeks. The two PDX tumors were implanted into 30, 6–8-week-old female NSG mice, respectively, as previously described5 followed by the same treatments as the cell line re-grafted mice. Mice in which did not take tumor were excluded. Study was not blinded. Tumor size was monitored twice every week and mice were euthanized when they met the euthanization criterion. Error bars represent SEM.Two million cells of MC-BR-BTY-0019 or five million cells of MC-BR-BTY-0006 were injected into the right flank region of 20, 6–8-week-old female NSG mice, and tumors were monitored weekly for the first two weeks and every other day thereafter for tumor growth. MC-BR-BTY-0019 developed detectable tumors in 2 weeks, and MC-BR-BTY-0006 developed detectable tumors in 3–4 weeks. When the tumor volume reached ~50–100 mmImages of established cell lines (MC-BR-BTY-0019 and MC-BR-BTY-0006) were taken using Invitrogen microscopy at ×20 at passage 5. The doubling time of MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines at passage 5 was determined by counting cells every 24 h for 5 days after plating 30,000 cells and 85,000 cells in 24-well plates for MC-BR-BTY-0019 and MC-BR-BTY-0006, respectively. Cells were trypsinized and counted using trypan blue staining the live cells every 24 h. Growth curves of each cell line were plotted after normalizing to day 1, and the doubling time was calculated using exponential regression of the cell growth curve with the software, Prism Graphpad. Error bars represent SEM.Karyotyping of two cells of each cell line, MC-BR-BTY-0019 and MC-BR-BTY-0006, were performed after G-band staining, and analysis was done by a certified cytogenetist at Mayo Clinic. Briefly, 1 million cells were cultured in a T75 flask, and Colcemid (1 µg/mL) was added to the medium for 14–16 h. Cells were detached with TrypLE Express for 5–10 min and pelleted down after centrifugation. 5 mL of 50/50 mixture of 0.8 M sodium citrate hypotonic solution and 0.075 M potassium chloride hypotonic solution was added to the cell pellets, followed by adding 2 mL of 3:1 methanol to glacial acetic acid fixative. Cells were pelleted down and fixed with 10 mL of 3:1 methanol to glacial acetic acid. Slides were incubated in a Thermotron slide drying chamber at 25 °C and 65% relative humidity. Cells were then processed for G-band staining. 20 cells were analyzed with 2 cells fully Karyotyped.STR profiling was performed by LabCorp Genetica using genomic DNA obtained from MC-BR-BTY-0019 and MC-BR-BTY-0006 cell lines and PDX. 15 autosomal STR loci and the gender identity locus amelogenin were utilized to determine the uniqueness of cell lines when compared to ATCC, DSMZ, JCRB databases. Mouse loci were utilized to detect mouse cell contamination.Early passage (within passage 10) PDX tumor samples were collected after euthanizing the tumor-bearing mice. Half of the tumor was fixed in formalin for pathology. The other half was used for tumor cell line establishment. Morphologic review of hematoxylin and eosin-stained sections from each PDX tumor was performed along with a review of immunohistochemically stained slides to ensure that the PDX tumor retained features of the original patient tumor. After cell lines were established, they were reseeded in 150 mm dishes for 3 days. Cells were trypsinized after washing with 1× PBS, counted and resuspended into DMEM with 10% FBS. CDX tumors were collected and fixed in formalin for pathology. Sections from each tumor were stained in the Pathology Research core using antibodies against ER , PR , Ki67 , and EGFR , and in the clinical IHC laboratory at Mayo Clinic against HER2 . ER and PR were scored as follows: negative (<1% reactive cells), focally positive (1–10% reactive cells), and positive (>10% reactive cells). HER2 was scored following CAP/ASCO guidelines for clinical testing. Ki67 was scored approximating the percentage of positive nuclei to the nearest 10%. EGFR was scored as negative, weak, moderate, or strong membranous staining. The pathology of parental PDX tumors and CDX tumors were compared to demonstrate their similarities and differences.6 cells were washed in 1× PBS and resuspended into 500 μL 1× PBS. Subsequently, 500 μL of ice-cold better C1 and 1.5 mL of ice-cold distilled water were added, and the lysed cells were centrifuged at 4 °C for 15 min at 1300×g. The supernatant was discarded and the pellet was resuspended in 250 μL of ice-cold buffer C1 and 750 μL of ice-cold distilled water and mixed by vortexing. The nuclear pellet was obtained after centrifuging the mixture again at 4 °C for 15 min at 1300×g and resuspended into 1 ml of Buffer G2 by vortexing for 10–30 s at maximum speed to lyse the nuclei and denature proteins. Finally, 25 μL of Qiagen protease cocktail was added and the mixture was incubated at 50 °C for 60 min to further digest the denatured proteins. All subsequent steps were performed according to the manufacturer’s Genomic-tip protocol. For PDX tissues, genomic DNA was isolated after homogenization using QIAGEN DNeasy Blood & Tissue Kit following the manufacturer’s protocol.Genomic DNA was isolated from cultured cell lines MC-BR-BTY-0019 and MC-BR-BTY-0006 using the QIAGEN Blood and Cell Culture DNA kits. Briefly, 5 × 10DNA-sequencing was performed by Mayo Clinic Core facility using Sure Select XT Whole Exon Capture v5 + UTRs 75 MB and Illumina HiSeq 4000 Paired-End Sequencing. Paired-end libraries were prepared using 1.0 μg of genomic DNA following the manufacturer’s protocol (Agilent) using the Agilent Bravo liquid handler. Whole exon capture was carried out using 750 ng of the prepped library following the protocol for Agilent’s SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products are then amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. The concentration and size distribution of the completed libraries was determined using an Agilent Bioanalyzer DNA 1000 chip  and Qubit fluorometry . Libraries were sequenced at an average coverage of ~250× following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 150 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kits and HCS v3.3.52 collection software. Base-calling is performed using Illumina’s RTA version 2.7.3.6. All of the sequence results used in the subsequent analysis passed quality controls according to the Illumina manufacturer  and also passed the quality control measures of the FASTQC software. To map reads to the human genome, we used the BWA-MEM (v 0.7.10) alignment program within the Mayo Clinic’s DNA sequencing analysis pipeline, GenomeGPS (v4.0.1)33. We were able to map reads to the human reference genome  at consistently high rates, ranging between 98.7% and 99.9% . For each sample, the percentages of the intersection of the v4 and v5 capture regions were 89% (at least 40× coverage), over 80% (at least 50×), and over 60% (at least 100×) , tumor tissue, PDX, and PDX-derived immortalized cell lines for two patients (MC-BR-BTY-0019 and MC-BR-BTY-0006) of the BEAUTY study2ratio, was obtained by PatternCNV (version 2.02.04). The log2ratio was computed by applying the log2 transformation to the ratios of the normalized coverage between a tumor sample and a reference set computed for 10 bp windows. The reference set included germline samples from samples that were whole-exome sequenced using the same exome capture methods.A measure of relative copy number across the genome, log35 was applied to detect heterozygous variants in the BEAUTY blood samples of these two patients. At each of these sites, we computed the allelic frequencies (AF), the ratio of the number of alternate-allele supporting reads to the total number of reads at the variant site in each of the three tumor samples for the patient in which the heterozygous variant was detected in the blood sample. We performed the transformation 0.5–|0.5–AF| to obtain symmetrically reflected allelic frequencies (RAF). In general, higher values of RAF indicate greater heterozygosity, and lower values indicate less heterozygosity. We evaluated the log2ratio and RAF profiles to center the log2ratios to the diploid state.Multi-sample variant calling using GATK’s UnifiedGenotyper2ratio and RAF profiles using circular binary segmentation implemented in the DNACopy package36 with standard-deviation-based segment merging applied at the optimal level as determined by a visual review of the segmentation profiles. We used an in-house method for estimating tumor purity, tumor ploidy, and determining thresholds for the classification of heterozygous variant sites by copy number state. From these classifications, we obtain integrated log2ration/RAF segments. To assess the similarity of copy number alteration profiles across samples by the patient, we calculated empirical probabilities based on copy number at sites of heterozygous variants in the patient blood sample.We segmented the log34 and annotated with variant effect predictor (VEP). Additional criteria were applied to arrive at a filtered set of somatic SNVs and INDELs that were highly likely to be true somatic mutations , JointSNVMix2 (version 0.8b2), and MuTect (version 1.1.7), and somatic indels were called using SomaticIndelDetector (GATK version)37 and that were also identified as cancer genes by membership in four other cancer gene resources: the MSK-IMPACT clinical test38, the FOUNDATION ONE CDx test (Foundation Medicine), Vogelstein et al. (2013)39 and the SANGER Cancer Gene Census40. For genes identified by these resources, we further used OncoKB37, the Cancer Gene Census40, and the COSMIC database41 to identity those for which the particular types of alterations and, for SNV/INDELs, the particular mutations, are known to be relevant for breast cancer. For loss-of-functions alterations to tumor suppressors, we feature those with inferred loss of function for both alleles. We additionally feature RB1 loss of heterozygosity (LOH) as it has been shown to cause functional loss of RB1 in breast cancer42. While CDKN2B was only identified as a cancer gene in 3 of these 5 resources, we also featured it also because the copy number alteration that affected CDKN2A also affected this adjacent cancer gene.As part of our characterization of the tumor samples, we have featured several important alterations Fig. . We seleFurther information on research design is available in the Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary InformationReporting Summary


Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous.Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches.Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells.Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells. Hepatocellular carcinoma (HCC), which ranks sixth among the most common malignancies and fourth in mortality among all kinds of cancers in the world, causes about 841,000 new cases and 782,000 deaths annually Epithelial-mesenchymal transition (EMT), which is mainly involved in the transient de-differentiation of epithelial cells into mesenchymal phenotypes, has been recognized as a major trigger for cancer metastasis and invasion in recent years N-glycan on the cell surface has been proved to be associated with the metastasis and EMT of HCC N-glycans catalyzed by the glycosyltransferase FUT8 Glycosylation is one of the most extensive and important protein post-translational modifications and participates in many essential biological processes including the cell growth, molecular recognition, signal transduction, immune defense, and other biological processes in normal tissue. The alteration of glycosylation has been proved to be associated with the progression and metastasis of many cancers, such as bladder Figure ), and the possible roles of EMT-related glycoproteins and site-specific glycans were further investigated by molecular biological approaches. Using these approaches, we revealed that the cellular uptake capacity of folates can be enhanced by increased core-fucosylation on FOLR1 especially at the glycosite Asn-201 to promote the EMT of HCC, which demonstrated a specific role of protein glycosylation in the EMT progress of HCC.In this study, site-specific glycosylation alterations in the EMT process of HCC were systematically investigated using glycoproteomic methods .In order to systematically investigate the alterations of site-specific glycosylation in the EMT process of HCC, two widely used HCC cell lines SMMC-7721 and HepG2 were treated by HGF to induce their EMT Figure ). Based on the expression of EMT markers, the whole EMT process could be divided into three major stages, including epithelial (E: 0 h), intermediate (I: 6 h and 12 h), and mesenchymal stages  . These results demonstrated that HGF treatment caused up-regulated expression of N-cadherin and down-regulated expression of E‑cadherin in a time-dependent manner in both cell lines, indicating the successful induction of EMT by HGF on both cell lines within three days.The HGF-induced EMT was first evaluated by expression levels of the epithelial marker E‑cadherin and mesenchymal marker N‑cadherin  , while the untreated cells decreased from 30% to almost 0% during the same time period (data not shown).The HGF-induced EMT was also confirmed by the relatively high cell growth rates of both HGF-treated cell lines in FBS-free medium, a condition that the untreated cells could not survive. The number of both treated cells increased from 30% to approximately 90% within three daysN-linked intact glycopeptides (still with N-glycan attached) within 1% false discover rate (FDR), respectively , were identified in these two cell lines within 1% FDR . These intact glycopeptides represented 891 glycosites from 657 glycoproteins, modified with 283 glycan structures (159 compositions) . Up to 80 glycans were identified at the glycosite Asn-69 (NACCST69N#TSQEAHK) of folate receptor alpha (FOLR1). The 283 N-glycans contained 16 high mannose, 121 hybrid, 138 complex, and eight paucimannose subtypes of glycans . Three pairs of representative MS/MS spectra, each containing a spectrum from 20% HCD fragmentation and a spectrum from 37% HCD fragmentation, were provided to demonstrate the principle of glycan type determination in the intact glycopeptide analysis .From SMMC-7721 and HepG2 cell lines, we were able to identify 1474 and 1682 unique  andTable S2). Even though a 1.5-fold change was used as the cutoff, only six increased proteins were identified in the I or M stage compared with the initial E stage in both cell lines . Although more in-depth proteomic analysis may lead to larger numbers of changed proteins identified, the present results clearly indicated that the majority of proteins remained unchanged during the EMT process of both HCC cell lines. Gene ontology (GO) analysis indicated all six proteins were involved in lipids and cholesterol biosynthetic process, in which five of these proteins could interact with each other (-D). These results suggested that increased synthesis of lipids and cholesterol might be involved in the EMT process induced by HGF, which is consistent with the simulative role of lipid and cholesterol to EMT in prostate cancer We first performed quantitative proteomic analysis during the EMT process of both cell lines. The relative abundance of each protein was calculated as the ratio of protein expression at the I or M stages versus that of E stage. Among all protein groups identified from SMMC-7721  and HepG2  cell lines, over 99.7% of them were changed within two folds in the I and M stages (2(I/E) and log2(M/E) ratios revealed significant alterations of glycopeptides with approximately 14% and 9% glycopeptides changed more than two folds in the I and M stages of SMMC-7721 cell line, respectively (Table S3). Compared with SMMC-7721, fewer glycopeptides were altered  in HepG2 cell line . Overall, these results demonstrated a more significant change at glycosylation level than that of protein level during EMT.We then analyzed the quantitative glycoproteomic data to investigate whether glycosylation was changed at the intact glycopeptide level. The distribution of logFigure ). We further analyzed structures of complex glycans using our in-house StrucGP software and found that core-fucosylated glycans accounted for the largest proportion of N-glycans, followed by glycans with terminal sialic acids and di-antennary glycans . Quantification results showed that 20 intact glycopeptides from 9 proteins had more than two-fold increases at I or M stage in both cell lines . Among them, four intact glycopeptides were modified with core-fucosylation, including HLA-C modified by N4H5F1S1 at the glycosite Asn-110, CD63 modified by N4H5F1 at the glycosite Asn-130, and FOLR1 modified by N4H5F1S1 and N4H5F3 at the glycosites Asn-69 and Asn-201, and all of them were elevated more than 2-fold at the I and/or M stages in both cell lines .In order to quantify the alterations of intact glycopeptides, we selected 607 unique glycopeptides detected in both cell lines with PSMs ≥ 5. Among them, glycopeptides modified by high mannose glycans accounted for the largest proportion, followed by complex and hybrid glycans  and 31 glycans . Among these glycopeptides, one was modified with paucimannose glycan, 13 were modified with high-mannose glycans, and the other 37 were modified with 23 hybrid or complex glycans . We interestingly found that core-fucosylation was accounted for 62.7% of all glycans on identified intact glycopeptides, including 7 glycans with both core- and branch-fucosylation. The majority of core-fucosylated intact glycopeptides were up-regulated during EMT in SMMC-7721 cell line, and many were also increased in HepG2 cell line . In addition, many core-fucosylated intact glycopeptides of FOLR1 were also up-regulated in HGF-stimulated Hep3B cells .To examine the relationship between the site-specific glycosylation on FOLR1 and HGF-stimulated EMT process, we mapped Figure S10). After LC-MS/MS analysis, we found that most of the core-fucosylation of FOLR1 were also up-regulated in TGF-β1-treated SMMC-7721 and Hep3B cells, especially the glycosylation of glycosite Asn-201 , which was consistent with the results of HGF treatment. In fact, we also harvested and analyzed TGF-β1-treated HepG2 cells by LC-MS/MS. Unfortunately, the intact glycopeptides from FOLR1 were not identified, which most likely was due to the low expression level of FOLR1 in HepG2 cells .We further measured whether core-fucosylation on FOLR1 was also up-regulated in TGF-β1-treated HCC cells. Three cell lines were also treated with 10 ng/mL TGF-β1, and their whole cellular proteins were harvested at three time points . Western blot analysis showed that the E-cadherin protein level was decreased, while the N-cadherin protein level was significantly increased in all three TGF-β1-treated cell lines, indicating the successfully induction of EMT by TGF-β1 , while the glycan structure was determined based on the B/Y ions of the intact glycopeptide fragmented by the low HCD energy . These results indicated that FOLR1 is a highly core-fucosylated glycoprotein and many core-fucosylated glycans were increased with the EMT in both HCC cell lines, further proved that core-fucosylation of FOLR1 might be involved in the EMT progress of HCC cells.A representative pair of spectra from an up-regulated glycopeptide modified by the glycan HexNAcN-glycans Figure ). Similarly, the expression levels of FUT8 protein were significantly up-regulated in three TGF-β1-treated cell lines . These results indicated that FUT8 was positively correlated with the increase level of core-fucosylated glycans with EMT.It is well-known that FUT8 is the sole enzyme to generate a-1, 6-fucosylated structures (core-fucosylation) on Figure ). As shown in Figure , overexpression of FUT8 significantly increased the protein and mRNA expression level of N-cadherin, which is a key marker of EMT, while significantly decreased the protein and mRNA expression of E-cadherin, indicating that up-regulation of FUT8 could trigger the EMT of HCC cell lines, a similar effect as the HGF or TGF-β1 treatment. We next investigated whether aberrant FUT8 expression promoted the migration and invasion of three cell lines using trans-well invasion assay and crystal violet stain. As expected, overexpression of FUT8 consistently increased the invasive ability of three cell lines .To demonstrate the relationship between FUT8 and EMT, we further over-expressed FUT8 in three cell lines in the absence of HGF and TGF-β1 treatment . Western blot and qRT-PCR results showed that knockdown of FUT8 could cause a significant increase of E-cadherin and a significant decrease of the N-cadherin in stable cell lines in the absence or presence of HGF treatment , revealing that the EMT of three cell lines induced by HGF could be relieved by knockdown of FUT8. To further assess the role of core-fucosylation in migration and invasion of HCC cells, we established stable cell lines of FUT8 knockdown in three cell lines. Then, the effect of FUT8 knockdown on migration and invasion of HCC cells were determined in three cell lines through matrigel trans-well invasion assay. Consistent with our observations, the invasive ability of the stable cell line of FUT8 knockdown was significantly reduced . In addition, the mass spectrometry data showed that overall core-fucosylation were significantly increased in FUT8 overexpressed SMMC-7721 and HepG2 cells , and decreased in FUT8 knockdown cell lines . And the core-fucosylation rate of FOLR1 were also significantly increased with FUT8 over-expression, and decreased with FUT8 knockdown in SMMC-7721 cells . These results provided strongly evidence that increased core-fucosylation is mainly regulated by FUT8 during EMT of HCC cell lines.To further consolidate the above observations, we silenced endogenous FUT8 in three cell lines by short hairpin RNAs targeting FUT8 (shFUT8) . In addition, both overexpression  and knockdown  of FUT8 didn't affect the expression of FOLR1 at mRNA and protein levels, but positively affected the overall core-fucosylation of proteins  as well as core-fucosylation on FOLR1 .To further examine the effects of FUT8 overexpression and knockdown on FOLR1 expression and core-fucosylation of the attached Figure ). Conversely, the amount of green fluorescence of folate was significantly reduced by Cy3-labeled siFUT8 compared with that of control cells . These results indicated that FUT8 can positively regulate uptake capacity of folate in HCC cells, possibly via the increased core-fucosylation on FOLR1.Previous studies have shown that out of three functional folate receptors in human , only FOLR1 exists on the apical surface of polarized epithelial cells, mediating mainly cellular uptake of folate by binding and releasing folate from outside to inside of cells -B) and protein  expression of FUT8 in SMMC-7721 and HepG2 cell lines. Folate increased the expression of N-cadherin and decreased the expression of E-cadherin both in a dose-dependent manner , indicating that “super-physiological” dose of folate solely can directly promote the EMT of HCC cells.Furthermore, we measured the effect of different concentrations of folate on the status of HCC cell lines. Different concentrations (100~1200 μM) of folate did not affect the mRNA  and protein levels  of FOLR1 were significantly increased in four stable cell lines including FOLR1 WT, N69A, N161A and N201A compared with the RFP, respectively. After treated with HGF for 24 h, the EMT progressions were partially blocked in all three glycosite-mutated cell lines in SMMC-7721 and HepG2 cells, according to the mRNA (-D) and protein  alterations of E-cadherin and N-cadherin. Among three glycosites, the blocking effect of the mutation at the glycosite Asn-201 was the most significant.To further determine which glycosite of FOLR1 is critical for cellular uptake ability of folate, we mutated asparagine residues to alanine one by one at three Figure -G). These results suggested that the folate uptake capacity of FOLR1 was maximally affected by site-specific glycosylation of FOLR1, in particular the glycosylation of glycosite Asn-201. In addition, these results further confirmed that the folate uptake capacity of HCC cells could be enhanced by the core-fucosylation of FOLR1.To further evaluate effects of site-specific glycosite mutations of FOLR1 on uptake capability of folate, four stable cell lines were incubated with 300 μM FITC-FA. Compared with FOLR1 WT cells, the amount of green fluorescence from folate was significantly decreased in FOLR1 N69A, N161A and N201A cells . Within this pathway, our glycoproteomic data provided substantial evidence to reveal that the folate uptake capacity of FOLR1 can be positively regulated by site-specific core-fucosylation attached at FOLR1, which established a direct connection between the overexpression of FUT8 (and increased core-fucosylation) induced by HGF or TGF-β1 and the EMT of cancer cells stimulated by “super-physiological” dose of folate, which were only reported disjointly in previous studies Based on above results, we concluded that HGF treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans in three HGF or TGF-β1 treated HCC cell lines, complemented by molecular biology approaches, we provided solid evidences to show that the folate uptake capacity of FOLR1 is enhanced by its increased site-specific core-fucosylation, and therefore links these two stimulation factors into one complete pathway EMT has been recognized as an important step toward the invasion and metastasis enhancement of HCC for decades. In previous studies, many alterations including the up-regulation of FUT8 Core-fucosylation, which was synthesized by the glycosyltransferase FUT8, has long been recognized as an important modification on N-linked glycans. It plays essential roles in modulating the affinity activity of many cell surface receptors with their ligands  et alAmong three known functional folate receptors, FOLR1 and FOLR2 are located at the plasma membrane of the human cells, while FOLR3 is a secreted protein In conclusion, with site-specific glycoproteomics and molecular biology approaches, we demonstrated that HGF/TGF-β1 significantly up-regulated the expression of FUT8, which led to the increasing of core-fucosylation on FOLR1. This further increased the cellular uptake of folate and promoted the EMT of HCC cells. With glycosite mutations, we further identified Asn-201 as the most critical core-fucosylated glycosite for folate uptake and EMT progression. Based on these results, the core-fucosylation of FOLR1 especially at the glycosite Asn-201 may represent another promising marker and therapeutic target for HCC monitoring and treatment.


Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines. Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases that, together with their associated regulatory cyclins, control central cellular processes. CDKs can generally be divided into kinases that are involved in the molecular control of cell cycle progression  or transcription  [In addition, CDK7 plays diverse roles in the regulation of transcription. As a component of the general transcription factor II human (TFIIH), CDK7 phosphorylates Ser-5 of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) to facilitate promotor escape and transcription initiation ,5,6. CDKDue to its dual role in proliferation and transcription, CDK7 represents an especially intriguing target amongst the CDKs for potential therapeutic strategies in oncology. An aberrant increase of CDK7 levels has been detected in many different cancer types, e.g., gastric, pancreatic, colorectal and breast cancer, and often correlates with aggressiveness and poor prognosis ,21,22,23The fact that THZ1 also targets CDK12 and CDK13 at concentrations used to affect CDK7 activity has, however, complicated a clear attribution of cellular phenotypes to CDK7 inhibition and fueled the search for more selective CDK7 inhibitors . SeveralKRAS gene, resulting in constitutively active KRAS and consequent proliferative signaling [CDKN2A , TP53 (50% of cases) or SMAD4 (50–60% of cases) loci [CDKN2A gene encodes for the tumor suppressors INK4 and ARF (ADP-ribosylation factor). INK4 acts as an inhibitor for CDK4 and CDK6 in the cell cycle, while ARF stabilizes p53 through inhibition of MDM2-dependent proteolysis [The progression of healthy cells to pancreatic ductal adenocarcinoma (PDAC) requires the accumulation of several genetic mutations . More thignaling . Additioes) loci . The CDKteolysis ,39,40. Tteolysis . SMAD4 iteolysis ,43,44. Bteolysis .Here, we studied the mechanisms underlying cell-specific responses to limited CDK7 inhibition using a panel of PDAC lines and LDC4297 as a highly potent and specific kinase inhibitor. Specific focus was on two representative cell lines, Mia-Paca2 and Panc89, originating from different stages and tissues of human pancreatic tumors and reacting to CDK7 inhibition with distinct sensitivity. Under the conditions of limited inhibition, we observed a marked impact on transcription rates, while T-loop phosphorylation of CDKs was moderately influenced, none of which, however, accounted for differential sensitivity of PDAC lines. RNAseq and protein analyses—following long-term inhibition with limiting concentrations of the CDK7 inhibitor—eventually revealed broad regulation of expression of cell cycle genes. The latter correlated well to both downregulation of cell cycle activity and differential sensitivity of PDAC lines in viability assays. Further linked to sensitivity, expression of the critical cell cycle regulators E2F1 and NFY declined over days. Our findings provide a mechanistic explanation for cooperative downregulation of cell cycle genes, illuminating the basis for novel programmatic roles of CDK7 in cancer cells.A representative panel of nine human pancreatic cancer cell lines was chosen to investigate the effects of non-covalent CDK7 inhibition by LDC4297 . The panDifferences in sensitivity between cell lines did not obviously relate to the main genetic modifications involved in the transformation of PDAC cells . As one We next studied the effect of LDC4297 treatment on cell cycle progression. Being primarily interested in the mechanisms causing strong CDK7 dependency, we chose to focus on the comparison of one intermediate (Mia-Paca2) and one good responder (Panc89/T3M-4) cell line. A specific reason for the choice of these cell lines was that they differed in sensitivity at low LDC4297 concentrations  is available. Not unexpected, Palbociclib induced a marked increase of the G1 population in both Mia-Paca2 and Panc89 lines a,b. Co-tIn the proliferation analysis, co-treatment with Palbociclib further reduced growth in the low LDC4297 concentration range (0.025–0.1 µM) c,d. HoweGAPDH and Rpl31) revealed a marked (50%) drop in transcription rates after 2 h exposure at 0.1 to 0.5 µM LDC4297. Importantly, transcription rates for both genes were reduced similarly in Mia-Paca2 and Panc89 cells, reasoning against a general difference in transcriptional impact of CDK7 inhibition between the two cell lines  or exon-intron (ei) junction primers . Validatll lines a. This wll lines b, but whNext, RNAseq analysis was conducted with Mia-Paca2 and Panc89 cells. These were treated for three days with 0.1 µM LDC4297, a concentration that elicited both robust effects and showed differences between the two cell lines compare a. CDK7 iExamination of the Myc protooncogene, a key inducer and effector gene in cancer and established player in PDAC , showed We subsequently asked whether Myc is also differentially regulated in other PDAC lines. This relates to the question whether Myc control critically determines the viability properties within the initially defined “good and poor” responder groups. In brief, Myc responded to the inhibitor in all lines tested (AsPc1 and Panc1 as members of the low responder group and PT45 and BxPc3 as good responders) and this response was at least as good as the one seen in Mia-Paca2 cells f. FurtheOur next focus was on genes related to the cell cycle machinery. A closer manual inspection of RNAseq data eventually revealed broad regulation of cell cycle control genes. These included the upregulation of cell cycle inhibitor genes (CDKN1A (p21), 1C (p57), 2A (p16), 2B (p15)). Furthermore, the expression of critical cell cycle kinases CDK1, CDK2, CDK4 and WEE1 (G2 checkpoint kinase), CDK-tyrosine phosphatases CDC25A and CDC25C (cell cycle division 25A und C), CyclinB1 and many other genes related also to S-phase control/DNA replication dropped significantly . RepreseImmunoblots conducted with Mia-Paca2 and Panc89 cell lysates lent further support to this hypothesis. CDK1, CDK4 and WEE1 as well as CyclinB1 were increasingly and selectively downregulated over time in Panc89 cells at limiting LDC4297 concentrations . Importantly, the two good responders BxPc3 and PT45 showed marked downregulation of CDK1, CDK2, CyclinB1, WEE1 and less strict also of CDK4. In contrast, the poor responders AsPc1 and Panc1 responded moderately, thereby more closely resembling Mia-Paca2 cells d. In theThe E2F family and the CCAAT box binding complex NFY are critical regulators of cell cycle genes, among them CDK1, CDK2 and CDC25C ,52,53,54Collectively, our study discloses a striking correlation of sensitivity towards CDK7 inhibition with the broad regulation of gene expression of cell cycle-associated genes between the cell lines Mia-Paca2 and Panc89 that could well explain the sensitivity differences seen for these cell lines at low inhibitor concentrations. This includes downregulation of critical drivers of the cell cycle and of relevant regulator families, as well as upregulation of CDK inhibitors, with the latter possibly being related to the parallel downregulation of Myc. Several critical cell cycle genes and the Myc protein were also regulated in a larger panel of PDAC lines, reasoning for a general importance. The concomitant transcription regulation of both—cell cycle genes and relevant regulatory factors—may well establish a novel programmatic basis for proliferation control of human tumor cells by CDK7.The importance of CDK7 for proliferation of cancer cells is intimately linked to activation of CDKs (CAK function), which is setting the pace in cell cycle progression, as well as to an essential role during pausing and early elongation of RNA polymerase II ,55,56. BCancer studies on CDK7 gained momentum upon discovery of the covalently binding inhibitor THZ1 that blocks growth of various cancer entities, among them triple negative breast cancer, Myc-associated tumors, non-small cell lung carcinoma and also pancreatic cancer ,58. In sA second PDAC investigation, conducted with THZ1 while this investigation was ongoing, emphasized the influence of transcription repression especially of NF-κB target genes as a possibly pioneering event in CDK7 inhibition . SurprisAnother potentially compensatory mechanism indicated by the RNAseq data relates to the TGFβ pathway. The TGFβ ligands TGFβ1 and TGFβ2 are upregulated as are the corresponding receptor genes upon LDC4297 treatment . DiffereMyc levels in turn are broadly correlated with the repression by CDK7 in PDAC lines. This is an important effector pathway of our inhibitor class given that Myc has been described as a critical factor for PDAC formation in vivo ,61. Myc The perhaps most striking observation of this study, the broad regulation of cell cycle genes, has been developed from the Mia-Paca2–Panc89 comparison. Here, expression regulation remained generally correlative to LDC4297 sensitivity, at least for certain key genes like CDK1, CDK2 and cyclin B1 within the broader PDAC panel. Of note, individual cell cycle genes have been identified as CDK7-dependent in recent studies based on genetic knockdown and non-covalent inhibitor studies. Impact on E2F-controlled genes after ablation of CDK7 or inhibition with the inhibitor YKL-5-124 has been reported previously although without further follow-up studies ,65. AddiWe have not yet extended the full RNA and protein analysis to the broader panel of PDAC lines. Ongoing preliminary RT-qPCR studies investigating the second strongest responder cell line PT45, however, initially confirm the observation of a broad control of cell cycle genes including E2F1 and also NF-Y subunits (data not shown). Already the selected protein analysis lends support to the hypothesis of a more general role of cell cycle gene expression control by CDK7 in pancreatic cancer. Potential therapeutic applications will certainly rely on the use of limiting CDK7 inhibitor concentrations as applied here in this study. Thereby, our non-covalently binding inhibitor class also offers options for combinatorial treatments, as exemplified here for the CDK4 inhibitor Palbociclib. It will now be important to further validate both the relevance of our findings and the applicability of selected inhibitors  within t2 incubator. Cells were passaged every 2–3 days using 0.25% trypsin. MRC5 cells were cultured in MEM supplemented with 10% FBS and antibiotics, and HEK293FT cells in DMEM containing 10% FBS, antibiotics and 1% L-glutamine. The following inhibitors were used: LDC4297 , Ro-3306  and Palbociclib , TGFbi , Galunisertib .PDAC cell lines  were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS  and antibiotics (penicillin/streptomycin) at 37 °C in a 5% CO® Reagent  was added directly to the cells and luminescence was measured according to manufacturer’s instructions using a Berthold plate reader.300–1000 cells per well were seeded in white, tissue culture-treated 96-well-plates and treated with inhibitors 24 h later. After the indicated time periods, CellTiter-GloCells were cultured as described above and treated with inhibitors for the indicated times. Medium plus inhibitor was refreshed at day 3 of treatment. To perform FACS analyses, cells were harvested by trypsinization. For cell cycle analyses, 100 µL of cell suspension were diluted with 900µL DAPI solution  and cells stained for 15 min at room temperature. Cells were then analyzed by FACS. For analysis of apoptosis rates, cells were stained with an Apoptose Staining Kit AnnexinV-FITC  according to manufacturer’s instructions. The results were evaluated using the FlowJo software.®800CW (α-mouse), IRDye®680RD (α-rabbit) or Jackson ImmunoResearch, West Grove PA, USA: anti-mouse-HRP (115-035-044) and anti-rabbit-HRP (111-035-045).Whole cell extracts were generated using RIPA lysis buffer , 0.5% sodium deoxycholate, 0.1% SDS, 1% IGEPAL) supplemented with phosphatase- and protease inhibitors and 1 mM DTT/15 mM β-Mercapoethanol. Protein concentrations were determined using Bradford-Reagent  and adjusted accordingly. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes using a semidry blotting system. After blocking with Odyssey blocking buffer  diluted 1:1 with PBS or 5% milk/TBST, membranes were probed with primary antibodies at a dilution of 1:1000 overnight. Protein bands were visualized on a Licor Odyssey CLx system (Image Studio Software version 5.2 LI-COR) or using ECL . The following antibodies were used: α-Tubulin , Myc , pCDK1 Thr161 , CDK1 , pCDK4 Thr172 , CDK4 , CDK2 , GAPDH , Actin , Wee1 , E2F1 , pCDC25C S216 , NFY-B  and CyclinB1 . All primary antibodies were diluted 1:1000. Secondary antibodies were purchased from LI-COR: IRDyeg at 4 °C. The aqueous phase was transferred to a fresh tube and mixed with 0.5 ml isopropanol, followed by 10 min incubation and centrifugation for 10 min at 12,000× g at 4 °C. The pellet was washed twice with 75% ethanol, centrifuging at 7500× g for 5 min at 4 °C. Pellets were finally air-dried and dissolved in RNAse-free water. RNA integrity was assessed using the Agilent Bioanalyzer RNA Nano . Libraries were prepared via NEBNext mRNA enrichment and the NEBNext Ultra RNA Prep Kit. Quality of the libraries was controlled via Agilent Bioanalyzer HighSens DNA Chip followed by NEBNext qPCR library quantification and equimolar pooling. Samples were sequenced using an Illumina NextSeq500 system (v2 chemistry). For analysis the R package DESeq2 was used as previously described [p-value < 5% were categorized as significant.Total RNA was prepared from TRIzol  lysates. Briefly, 1 ml Trizol samples were mixed with 200 µL chloroform, incubated for 3 min and then centrifuged for 15 min at 12,000× escribed . As a reFor additional gene expression analyses, cells were lysed in TRIzol reagent and RNA isolated as described above for RNAseq analysis. 500–1000 ng RNA was used for reverse transcription . For analyses addressing transcription rates, RNA was extracted using the Direct-zol RNA Mini Prep kit  including a DNA digest step and cDNA was generated with the PrimeScript RT Kit with gDNA eraser  according to manufacturer’s instructions. qPCRs were performed using SYBR Green  on a StepOnePlus Real-Time PCR System (Thermo Fisher). For data collection and analysis, a 7300 System SDS Software version 2.3 was used. RT-qPCR Primers were as follows . PrimersStatistical analysis was conducted using Prism Software (GraphPad Prism version 8). Unless stated otherwise, all n numbers represent independently performed experiments, and statistical tests are two-sided, unpaired TTESTs. Where representative experiments are shown, experiments have been performed at least two times independently.


Introduction. Zerumbone is a phytochemical compound of the ginger plant Zingiber zerumbet with cytotoxic effects in various cancer cell lines. To date, zerumbone has shown an antiproliferative effect in oral squamous cell carcinoma cells lines. However, the effect of combination with radiation or cisplatin in head and neck squamous cell carcinoma (HNSCC) is unclear. The aim of this study was to investigate the effect of zerumbone alone, and in combination with irradiation and cisplatin on HNSCC cell lines. Methods. The three HNSCC cell lines SCC25, Cal27 and FaDu were treated with zerumbone, radiation and/or cisplatin. Cell viability and clonogenic assays were performed. The interaction between zerumbone and radiation or cisplatin was evaluated using the combination index. Apoptosis was measured by flow cytometry and cell migration was assessed using a wound healing assay. Results. Treatment with zerumbone resulted in a dose dependent induction of cytotoxicity and apoptosis in all three cell lines. The combination with cisplatin revealed a synergistic to additive effect in Cal27. The clonogenic assay showed a significant radiosensitizing effect in all three cell lines. The wound healing assay showed a reduction of cell migration in Cal27. Conclusion. The natural compound zerumbone shows a cytotoxic and proapoptotic effect on HNSCC cell lines. Furthermore, zerumbone enhances the radiation effect in all three cell lines and thus may be a suitable candidate for combination therapy in HNSCC. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with an incidence of approximately 900.000 new cases per year . The aveNatural sources play a great role in the discovery of many anticancer drugs. For instance, taxanes and vinca alkaloids were initially isolated from the madagaskar periwinkle and the Pacific yew tree bark, respectively . The advZingiber zerumbet Smith. In Asian traditional medicine the plant is used to treat a wide variety of diseases and symptoms. The anticancer properties of zerumbone have been reported in several studies in vitro and in vivo in various cancers [Zerumbone is a natural compound of the ginger plant  cancers –9. In co cancers . Other p cancers . Reporte cancers –9.The aim of this study was to investigate the effect of zerumbone in the HNSCC cell lines SCC25, Cal27 and FaDu especially in combination with (the standard therapeutic options in HNSCC treatment) cisplatin and irradiation.Zerumbone was obtained from Sigma Aldrich . It was dissolved in dimethyl sulfoxide  as a 100 mmol/l stock solution and stored at -20 °C. Further dilution was done with RPMI medium immediately before treatment. Cisplatin (CIS) was taken from ready-to-use infusions.2.The HNSCC cell line FaDu was obtained from the American Type Culture Collection . Cal27 and SCC25 were obtained from the German Collection of Microorganisms and Cell Cultures . The cell line FaDu derived from a human pharyngeal squamous cell carcinoma whereas SCC25 and Cal27 are from human squamous cell carcinomas of the tongue. The cell lines were kept in RPMI medium  supplemented with 1% penicillin–streptomycin  and 5% fetal bovine serum  in a humidified atmosphere with 5% CO3 cells were seeded on 96-well plates in triplicates and incubated for 24 h. Subsequently, they were treated with 0—96 µM zerumbone. For the combination experiments, cells were treated with cisplatin 0—32 µM or a combination of zerumbone and cisplatin (ratio 3:1) as well as with irradiation 0—8 Gy alone or in combination with zerumbone. 0,1% DMSO was used as vehicle control. After 72 h of incubation the percentage of living cells was assessed using the Cell Counting Kit-8 assay  according to the manufacturer’s protocol.A cytotoxicity assay was performed to assess the cytotoxic effect of zerumbone. 3 × 105 cells were seeded on 6-well plates and incubated for 24 h. Then, cells were treated with 0, 4, 8 and 16 µM zerumbone. After 24 h and 48 h, apoptosis and necrosis were measured using the Annexin-V Apoptosis Detection Kit . Apoptosis was defined as Annexin + /propidium iodide − (Ann + /PI-) and necrosis/late apoptosis was defined as Ann − /PI + and Ann + /PI + .To assess the induction of apoptosis, flow cytometry analysis was performed. Briefly, 10Cells were seeded on 6-well plates and after reaching a confluence of approximately 80%, a scratch was made using a 1 ml pipette tip. After washing twice with warm PBS, cells were treated with 0, 5, 10 and 20 µM zerumbone. Two regions per scratch were marked and photographed subsequently after the scratch and again after 24 h of incubation using CellSens Software . The wound healing area was measured using ImageJ macro “MRI Wound Healing Tool” .Irradiation was carried out using a 150 kV x-ray machine  at a dose rate of 2 Gy/min at room temperature. The focus-object distance measured 52 cm and the field size was 20 × 20 cm. Thermoluminescence dosimeters were used for dosimetry assessment.2 cells and incubated for 24 h. Next, cells were treated with 0, 2.5 or 5 µM zerumbone and/or irradiated with 0, 2, 4, 6 or 8 Gy and incubated for 72 h. Afterwards, the medium was replaced with drug-free medium. 10 days later, cells were washed with phosphate-buffered saline, fixed with ethanol 96% and stained with crystal violet. Colonies containing more than 50 cells were viewed as survivors and counted.The clonogenic assay was performed according to the protocol by Franken et al. . Six-welAll experiments were carried out at least three independent times. The statistical analysis for cytotoxicity assays and flow cytometry were performed using GraphPad 5.0 software by Prism® . The interaction of zerumbone and cisplatin and short term combination with irradiation were analyzed using the Combination Index according to the Chou-Talalay method using CompuSyn software (ComboSyn Inc.) . The clo50 at 4.42 µM, followed by FaDu cells with an IC50 at 8.60 µM. SCC25 was the least sensitive cell line with an IC50 at 9.22 µM.The three HNSCC cell lines SCC25, Cal27 and FaDu were treated with zerumbone (0–96 µM) for 72 h and cell viability was measured using CCK-8. Treatment with zerumbone showed a dose dependent inhibition of cell viability in all three cell lines Fig. . Cal27 cFlow cytometry was performed to assess apoptosis and necrosis. Cells were seeded into 6-well plates, treated with 0, 4, 8 or 16 µM zerumbone and incubated for 48 or 72 h. All three cell lines showed a dose-dependent increase of apoptosis and necrosis . CI < 1 indicates synergy, CI > 1 an antagonistic effect and CI = 1 represents an additive effect. Treatment combination in SCC25 and FaDu showed an antagonistic effect for all doses. The CI for Cal27 revealed a synergistic to additive effect up to the combination of 4.88 µM zerumbone and 1.63 µM cisplatin (CI 0.65—1.08) and an antagonism for higher doses  and irradiation (0—8 Gy) for 72 h. Treatment with zerumbone enhanced the radiation effect in all three cell lines at different concentrations Fig. . When anThe long-term effect of combination treatment was assessed with the clonogenic assay. Treatment with zerumbone and/or radiation led to a decrease of clonogenic survival in all cell lines. Moreover, all three cell lines showed a significantly increased inhibition of colony formation at 2.5 and 5 µM (all p < 0.001, Fig. Zingiber zerumbet is commonly used in traditional medicine in Asia to treat a wide variety of symptoms and diseases. Zerumbone is the major component of its essential oil and has been researched in many studies in vivo and in vitro. It shows a cytotoxic effect in cancer cell lines such as lung cancer [HNSCC is one of the sixth most common carcinoma worldwide and survival rate only marginally improved over the last decades. Thus, new treatment options are needed to improve overall survival. The investigation of natural products has led to the discovery of several well-known chemotherapeutic agents. The ginger plant g cancer , colorecg cancer , leukemig cancer  and oralg cancer . Howeverg cancer , 16, 17.50 values between 4.4—9.2 µM. Additional flow cytometry analysis showed upregulation of apoptosis in all three cell lines. Migration was reduced in Cal27 at 10 µM. In concordance, Zainal et al. showed an antiproliferative effect in oral cancer cells between 0.8—4.9 µM and a proapoptotic and antimigratory effect [In this study, the effect of zerumbone on the HNSCC cell lines SCC25, Cal27 and FaDu was investigated. Zerumbone showed antiproliferative effects in all three cell lines with ICNext, the combinatory effect of zerumbone and cisplatin at a ratio of 3:1 was investigated, since cisplatin is the standard chemotherapeutic agent in HNSCC treatment . CombineAnother pillar of HNSCC treatment is radiotherapy. In this study, combination experiments of zerumbone and radiation showed an additive or synergistic effect at different concentrations in all three cell lines. Furthermore, the clonogenic assay showed a significantly reduced colony formation by combination of both treatments. Likewise, a radiosensitizing effect was found in colorectal carcinoma , prostatIn conclusion, zerumbone shows a cytotoxic and proapoptotic effect, and inhibits cell migration in HNSCC cell lines. Moreover, this is the first study to describe a radiosensitizing effect of zerumbone in HNSCC cell lines. These results implicate that zerumbone might be an attractive treatment option in HNSCC especially in combination with radiation. However, further preclinical and clinical studies are required to assess the full potential of zerumbone.


HDACi and knocking down of E2F5 as tumor suppressors inhibited cell proliferation, migration invasion, and blocked the cell cycle in gastric cancer cells by suppressing BCL2. The results conclude that the anticancer mechanism of HDACi was determined by regulating E2F5 via targeting BCL2. (4) Conclusions: Our results suggest that the HDAC–E2F5–BCL2 signaling axis might be a novel potential biomarker in gastric cancer.(1) Background: Gastric cancer (GC) is the most common high death-rate cancer type worldwide, with an enhanced prevalence and increased rate of mortality. Although significant evidence on surgery strategy has been generated for the treatment of GC, conclusions are still uncertain regarding profound metastatic or persevering gastric cancer. Therefore, it is essential to develop novel and effective biomarkers or therapeutic targets for the diagnosis of GC. Histone deacetylations (HDACs) are important epigenetic regulators that control the aberrant transcription of critical genes that are mainly involved in cell proliferation, cell migration, regulation of the cell cycle, and different signal pathways. (2) Methods: Expression analysis of HDACs family members and E2F5 in gastric cancer cell lines was determined by RT-PCR and Western blotting. The cell proliferation was determined through an MTT assay. Cell migration was determined using a wound-healing assay. Flow cytometry experiments were used to determine cell-cycle analysis. The statistical software OriginPro 2015  was used to analyze data. A  Gastric cancer is the most common destructive tumor in the digestive tract, with increased morbidity and mortality, mostly in developing countries . TherefoFor gastric cancer patients, HDAC inhibitors are being developed as drugs and are showing increased efficacy. Any protein that bonds with HDACs, therefore, has the potential to coactivate the function of enzymes. Individual HDACs interfere with the expression or function of cells to obtain biological reactions . Since HAlthough there have been some in-depth studies, the specific molecular mechanisms of gastric cancer have not been fully evaluated. The inactivation of HDACs in gastric cancer acts as a core transcriptional mechanism that manages the development of the cell cycle and cyclins’ regulation. Interestingly, E2F5 can enhance and inhibit cell proliferation, transcription, activation, or repress the cell transcriptions that depend on the cell environment. In essence, E2F5 connects the cell cycle to the post-transcriptional mechanism and plays a crucial part in controlling cell growth and the transformation of biological processes that are incriminated in cancer development ,9. The B®, Thermo Fisher, Waltham, MA, USA) and F-12K medium supplemented with 10% fetal calf serum  in a humidified 37 °C chamber with 5% CO2. The cell culture medium was replaced every 4 h during subculture and the culture plate was washed with Dulbecco’s phosphate-buffered saline . KATO-III and N87 cells were treated with histone deacetylase inhibitor (HDACi) and transfected with E2F5 and NC siRNA purchased from Ribobio . The compositions of HDACi are detailed in Cell lines, including MGC-803, MKN28, MKN45, KATO III, AGS, N87, and GES-1, were obtained from Bioresource Collection and Research Center . All cell lines were maintained and subcultured in RPMI 1640  followed by qRT-PCR using the SYBR Green PCR Master Mix (Bio-Rad). We employed a CFX96TM Quantitative Real-Time system with a total reaction volume of 20 µL. The reactions were incubated using either a Bio-Rad qRT-PCR 96-well or 8-well tube at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. All PCRs were performed in triplicate, and the cycle number at which the reaction crossed the threshold cycle (Ct) was determined for each gene. The relative amount of each gene was measured relative to GAPDH RNA using the 2ΔCt, equation. Enlisted primer sequences are provided in Total cellular RNA from the abovementioned cell lines was extracted and purified using a Total RNA extraction kit 1  following the manufacturer’s instructions Thereafter, cellular RNA was quantified using NanoDrop  (260/280 = 1.8), and the cDNA was synthesized using an iScript5 cells were transferred into 6-well plates 48 h post-transfection, washed with warm PBS and digested using trypsin. The reaction of trypsin was neutralized with DMEM followed by centrifugation at 1000 rpm for 5 min. Next, the cells were treated with 70% ice-chilled ethanol and placed at −20 °C overnight. The next day, after fixation and washing with cold PBS twice, cell pellets were resuspended in 500 µL PBS containing 50 mg/mL propidium iodide, 0.1 mg/mL RNase A, and 0.05% Triton X-100, and incubated at 37 °C for 30 min in the dark. Cell cycle distributions were detected using FACSCalibur . For cell-cycle analysis, n = 10,000 cells.To analyze the various phases of the cell cycle, KATO III and N87 cells were treated with HDACi and transfected with E2F5 siRNA and negative control siRNA. A total of 5 × 10Western blot analysis was used to investigate the expression of the target protein. In brief, extracted total lysate from the cells was washed thrice with PBS and then lysed in lysis buffer (50 mM Tris-base (pH 7.5), 0.5 M NaCl, 1 mM EDTA (pH 8)), 1 mM β-mercaptoethanol, 1% NP40, 1% glycerol, and protease inhibitor . The concentration of total cellular proteins was determined by the Bradford method . Thereafter, the protein samples were resolved on 10–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane . The membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated with specific primary antibodies .g for 10 min. Then, the supernatant was discarded, and the pellet was dissolved in fresh medium. Cells were counted down with a hemocytometer in each group, and then inoculated into four 96-well plates at a density of 2 × 102 cell per 200 µL DMEM with 8 repeated walls. After 24 h, the MTT solution was added to each well and incubated at 37 °C for 4 h. The reading was taken at intervals of 24 h. The incubation was stopped, and the culture supernatant was discarded. Dimethyl sulfoxide (DMSO) was added to each well, and the plates were shaken for 10 min in an enzyme-linked immunosorbent detector. The absorbance (OD) value of each well was measured at wavelength of 490 nm at each point. Cell viability curves were determined with time as the x-axis and OD values as the y-axis.Cell viability curves are mostly observed using MTT -2, 5-diphenyltetrazolium bromide) assay to determine the proliferation of treated cells. After 48 h transfection, cells were washed with warm PBS and trypsin was added to the cells. Then they were placed in incubator for 2–3 min to detach the cells from surface of culture dish. Then, fresh culture medium was added to neutralize the trypsin. The cells were transfected to a tube and centrifuged at 1000× p values < 0.05 were considered statistically significant for all comparisons. The difference was found to be statistically significant .Each of the experimental results was revised separately three times with each performed in triplicate. Statistical analysis of data was carried out using the GraphPad Prism 7 software . ANOVA and Student’s t test were used. All the numerical data were determined as the mean ± standard deviation, and p < 0.05) (p < 0.05)  and a normal cell line (GES-1). The qRT-PCR results concluded that the relative mRNA expressions of HDACs in gastric cell lines were remarkably increased when compared with the normal cell line (*  < 0.05) A. Simila < 0.05) B. For fu < 0.05) C. Such cp < 0.005) and N87 (** p < 0.005) cell lines compared with the blank as a control (p < 0.005) and N87 (** p < 0.005) cell lines compared with the blank (Histone deacetylase inhibitor (HDACi) plays an important role in inhibiting the expression of HDACs in different human cancers. The gastric cell lines (KATO III and N87) were treated with HDACi to investigate the expression of HDACs and E2F5 at both mRNA and protein levels. The qRT-PCR results showed that HDACi downregulated the mRNA expression of HDACs in KATO III (**  control A–D. Simihe blank A–D. For he blank E. Moreovp < 0.005) and N87 (** p < 0.005) cell lines when compared with the blank as a control (p < 0.001) and N87 (*** p < 0.001) cell lines, by downregulating the expression of E2F5 in comparison to the blank (p < 0.05)  A,B.p < 0.001, *** p < 0.001)  ((E2F5 plays a significant role in the progression and development of human cancer. Alteration in the expression of E2F5 results in the activation of the expression of BCL2 in gastric cancer cell lines. The KATO III and N87 cell lines were transfected with E2F5 target siRNA and negative control (NC) siRNA to determine the expression of E2F5 and BCL2 at both RNA and protein levels. The qRT-PCR results revealed that knocking down of E2F5 decreased the mRNA expression of E2F5 and BCL2 in KATO III cell line (*** < 0.001) A,C and N 0.001)   and N87 (** p < 0.005) cell lines compared with the negative control (p < 0.05) (p < 0.05)  C,D. In a < 0.05) A,B.In the current study, we demonstrated the expression of HDAC through E2F5 targeting BCL2 downregulation, contributing a novel validation of the mechanism and treatment of HDAC inhibition. HDAC utilizes their targeted measures through post-translation acetylation of core nucleosome histones, accordingly, to modulate the expression of the target gene . This anWe recognize E2F5 as a straight target of BCL2 in gastric cells. A notable increase in E2F5 at the protein level guides the ectopic expression of BCL2. More essentially, current research demonstrates that the HDAC inhibitor could reverse PELP-mediated suppression of MIR-200 family members, suggesting an uninterrupted association between the HDAC inhibitor and family members of miR-200 in breast tumors ,17. ThisMoreover, HDAC decreases cell proliferation, invasion, and migration by suppressing the expression of E2F5 and BCL2. We showed that the HDAC inhibitor was inadequate to completely neutralize the inhibitory effect of proliferation, migration, and invasion of gastric cancer cell lines. One viable cause is that HDACs inhibition fails to completely block the increased level of E2F5 induced via BCL2. Regarding earlier studies on ductal carcinoma, it was described that the upregulation of CRKL protein takes part in integrating signs for invasion, proliferation, and migration of widely virulent breast carcinoma cell lines, and disclosed a remarkable connection between increased proliferative breast cancers and low outputs . Our finEven though the previous observation of E2F5 was altered in various cancer cells, the mechanisms have not been completely explained . For theIn conclusion, the knowledge gained from our study is that HDAC inhibitors impede gastric tumor cell migration, proliferation, and suppression of other cellular functions of tumors by regulating E2F5 through direct BCL2 targeting. This aspect of HDAC inhibitors’ regulation is anticipated not only to widen our understanding of basic cellular occurrence but also to configure advanced mechanisms of therapeutic agents required for the therapy of human diseases such as cancers. Our data somewhat show the conception of gastric cancer clinical therapy via HDAC inhibitors, and suggest that the HDAC–E2F5–BCL2 signaling axis might be a new diagnostic indicator in gastric cancer. However, further studies are required to better elucidate the molecular intricacies and the clinical interest in gastrointestinal practice.


RB cell lines (Y79 and WERI-Rb1) were cultivated under three different in vitro conditions and apoptosis, proliferation and cell growth, as well as expression profiles of two epithelial-mesenchymal transition (EMT) markers, were analyzed. EMT gene expression profiles were not generally changed, whereas apoptosis levels, tumor cell proliferation, and in vitro growth were significantly influenced by different cell culture conditions. In order to optimize the time-limited chick chorioallantoic membrane (CAM) assay, we investigated two different time points of tumor cell inoculation (embryonic development day EDD8 and EDD10) as well as three different cell concentrations. We showed that inoculation at EDD8 led to decreased tumor formation and chicken viability, whereas different cell concentrations did not change size and weight of developing tumors. Our findings demonstrate that medium conditions in vitro as well as the starting point for CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines. Retinoblastoma (RB) is a primary intraocular malignancy in childhood. Relapses may develop and cause secondary cancers during later development. This study was set up to identify optimal cell culture conditions for RB cell growth  However, different cultivation protocols for the same RB cell lines have been published so far [2 concentrations, have a huge impact on the tumor cell growth, viability and gene expression, e.g., of epithelial-mesenchymal transition (EMT) markers. Therefore, it is comprehensible that experiments using the same RB cell lines are still not verifiable across different labs as they are carried out using different cultivation protocols.Retinoblastoma (RB) is the most common malignant pediatric intraocular tumor , represed so far ,5,6,7,8.in vivo studies of tumor cell growth and dissemination [The chick chorioallantoic membrane (CAM) assay is an established model system for mination ,10 as wemination ,12. The mination . The higmination . The extmination ,13. Prevmination ,15. Our group successfully established the CAM assay for the analysis of RB tumor growth and metastatic spread ,16,17. Hin vitro culture conditions. Additionally, we re-investigated our established in ovo CAM protocol with three RB cell lines  to compare different conditions for in ovo tumor growth of RB cells and to identify the most suitable experimental setup.Thus, in the study presented, we systematically investigated differences in growth behavior and gene expression of two RB cell lines  grown under three commonly used RB1 gene was performed for all retinoblastoma cells lines.The human RB cell lines Y79  and WERI2 conditions:(1)2.RB medium composed of Dulbecco’s modified Eagle’s medium  with 15% fetal bovine serum , 100 U penicillin/ml and 100 µg streptomycin/ml , 4 mM L-glutamine , 50 µM ß-mercaptoethanol  and 10 µg insulin/ml . Cultivation at 10% CO(2)2.DMEM medium composed of Dulbecco’s modified Eagle’s medium  with 15% fetal bovine serum , 100 U penicillin/ml and 100 µg streptomycin/ml  and 4 mM L-glutamine . Cultivation at 5% CO(3)12 and PABA  with 15% fetal bovine serum , 100 U penicillin/ml and 100 µg streptomycin/ml  and 4 mM L-glutamine . Cultivation at 5% CO2.RPMI medium containing biotin, vitamin BTwo cell lines (Y79 and WERI-Rb1) were cultivated at 37 °C and 95% humidity using the following three different media and COin ovo CAM assays  were cultivated in RB medium as described above. No approval from an ethics committee was required for work with the human cell lines.The RB cells used for the ® 488 . In order to detect changes in apoptosis levels, cells were stained with 4′,6-Diamidino-2-phenylindole  and pycnotic nuclei were counted as described previously by our group [Cell proliferation was investigated by 5-Bromo-2′-deoxyuridine  incorporation. Therefore, 10 µM BrdU was added to the cells 4 h prior to paraformaldehyde  fixation procedure. Afterwards, cells were incubated with a rat anti-BrdU antibody  and proliferating cells were finally detected using a goat anti-rat antibody labeled with Alexa Flourur group .5 RB cells were seeded in 500 µl of each medium  with supplements in a 24-well plate and vital cells were counted manually using the trypan blue exclusion method. Cells were seeded in triplicates and counted at four time points .To determine growth kinetics, 3 × 10vimentin and EpCAM expression, a SYBR™ Green PCR assay  was used with specific primers 5′-TCTGGATTCACTCCCTCTGGT-3′ (forward) and 5′-TCAAGGTCA TCGTGATGCTGA-3′ (reverse) for whole vimentin, 5′-TCAGAATGAT GTGGACATAGCTGA-3′ (forward) and 5′-CCCCATTTACTGTCAGGTCCA-3′ (reverse) for whole EpCAM and 5′-ACCCACTCCTCCACCTTTGA-3′ (forward) and 5′-CTGTTGCTGTAGCCAAATTCGT-3′ (reverse) for human GAPDH (hGAPDH) as an endogenous control. RT-PCRs were conducted in triplicates in 20 μL of SYBRTM Green PCR Master Mix  using the following program: 95 °C for 15 min; 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 34 s and 40 cycles.RNA isolation from RB cell lines was performed using the NucleoSpin RNA II kit . For quantitative real-time PCR analyses, cDNA was synthesized with the QuantiTect Reverse Transcription Kit  following the manufacturer’s protocol. For analysis of 6, 2 × 106 and 3 × 106 cells) were inoculated onto the chick chorioallantoic membrane (CAM) at embryonic development day (EDD) 8 and EDD10 eggs as described below and previously published by our group [In order to test for changes in the tumor formation capacity and mortality rate of the chicken embryos, different RB cell concentrations  was placed onto the traumatized CAM area. The window in the eggshell was sealed tightly with tape and the egg was returned to the incubator until EDD17. in ovo experiments if the chicken is not intended to live beyond hatching. In our study CAM assays were only performed until day EDD17 and thus, prior to hatching. Thus, the duration of the CAM assay was limited to a 7–9 day time window. Seven to nine days after grafting (EDD10-17 or EDD8-17), chick embryos were anesthetized by cooling on ice and sacrificed by decapitation. Mortality rate of the chicken embryos were determined and CAM tumors were excised, measured, and photographed. According to the German Animal Experiment and Welfare Guidelines, no ethics approval is required for t-test or one-way ANOVA and Newman-Keuls Post-test. * p < 0.05, ** p < 0.01 or *** p < 0.001 were considered significantly different. For the statistics on the growth curves a free web interface http://bioinf.wehi.edu.au/software/compareCurves/ was used (Accessed on 1 December 2021), which uses the compareGrowthCurves-function from a statistical modeling package called ‘statmod’, available from the R Project for Statistical Computing: http://www.r-project.org (accessed on 1 December 2021), previously described elsewhere [All assays were performed at least in triplicate and data represent means ± SEM from independent RB cell cultures. Statistical analyses were conducted using GraphPad Prism 6. Data were analyzed by Student’s lsewhere .The established human RB cell lines Y79 and WERI-Rb1 grew in suspension under all conditions analyzed and appeared as small epithelioid round cells , mainly 2 compared to cells grown in RB medium and 10% CO2  8 and EDD10, a highly vascularized CAM area was identified by candling the eggs, marked and the eggshell was opened. After inoculating different RB cell concentrations  onto the dropped CAM, eggs were re-incubated until EDD17  did not change the tumor size  and EpCAM  to detect changes in cellular fate upon different medium conditions. We could show that with regard to potential EMT changes the identity of the RB tumor cells did not significantly alter or differ between the medium conditions except for vimentin expression being upregulated in WERI-Rb1 cells cultivated in RPMI medium and downregulated in Y79 cultured in DMEM medium compared to RB medium conditions. Taken together the results of the EMT marker expression analyses support our observations that the morphology of the RB cells did not change as no shift towards a mesenchymal or epithelial cell morphology was discernible.We therefore compared three different cultivation settings commonly used for RB cell cultures ,5,6,7,8 in vitro cell cultures, in vivo experiments are inevitable to investigate tumor formation in a more natural environment. One in vivo model system, which offers a plethora of advantages over the standard murine model, is the in ovo chicken chorioallantoic membrane assay. The CAM is at least partially immune-deficient and thus, allows for growth analyses of different tumor entities and species without specific or nonspecific immune response. Additionally, the rich blood vessel network of the CAM promotes survival, growth, and rapid vascularization of CAM tumors formed by inoculated cancer cell, xenografted normal or tumor tissues (for review see: [Beside iew see: ,12,17,21in vitro as well as time point of CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines. RB medium combined with a 10% CO2 atmosphere turned out to be the most suitable in vitro culture condition for RB cell lines. EDD10 is the preferential starting point for CAM inoculation assays due to better overall embryo survival and tumor development. It seems that the cell concentration used for inoculation experiments does not significantly influence in ovo tumor development of RB cells. Taken together, it would be desirable to optimize and standardize experimental in vitro and in ovo conditions in order to gain reproducible research results. In summary, we could show that medium conditions