In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition,  Liver cancer is one of the most frequent malignancies and has poor prognosis, ranked as the third leading cause of cancer-related deaths worldwide and thus remains a global health issue . The inc7G-cap structure at the mRNA ‘5’ end, whereas eIF4G acts as a scaffold protein recruiting eIF4E and eIF4A. The latter, the only component of the eIF4F complex with enzymatic activity, is an ATP-dependent DEAD-box RNA helicase that unwinds the mRNA secondary structures in the 5′ untranslated regions (5′-UTRs) to enable ribosome scanning. The helicase eIF4A is required to efficiently translate mRNAs with long and highly structured 5′-UTRs, including those with G-quadruplex structures benzofuran core that suppresses cap-dependent translation by inhibiting eIF4A activity. More than one hundred natural rocaglates have been isolated from lability ,33,34. Tlability ,36, and lability . Silvestlability ,39, and lability ,41,42,43lability . Furtherlability .In this study, we investigated the pathogenetic role of eIF4A1 in human HCC and its potential usefulness as a novel molecular target for the development of novel pharmacological strategies.eIF4A1 mRNA levels in human cancers were investigated using data obtained from The Cancer Genome Atlas (TCGA) and the UALCAN  analysis tool. eIF4A1 mRNA expression was elevated in various cancer types, including colon adenocarcinoma (COAD), glioblastoma multiforme (GBM), esophageal carcinoma (ESCA), and kidney renal clear cell carcinoma (KIRC), among others , eIF4A1 was significantly overexpressed (−10) with comparable incidences for women (P = 2.566 × 10−10) and men  compared to that in normal tissue. Maximal eIF4A1 expression was detected in tumors of young patients between 21 and 40 years of age (−02). Examination of multiple clinicopathological features revealed that HCC patients with high eIF4A1 expression had poorer prognoses. The survival time of patients with high eIF4A1 expression was significantly shorter than that of patients with low or medium eIF4A1 levels . The analysis of HCC cancer stages . Importantly, high eIF4A1 expression was found in N0 and N1 nodal metastasis status , with a clear tendency toward enhanced eIF4A1 expression in N1 specimens.First, g others A. In HCCp = 0.0002; Wilcoxon test; p = 0.0249; Wilcoxon test) and the translation initiator and rocaglate target DEAD-box protein 3 DDX3 [p = 0.0088; Wilcoxon test) in HCC lesions  by immunohistochemistry . In agreOverall, the present data indicated that eIF4A1 is highly expressed in primary HCC lesions and associated with a poor outcome. These findings suggest the usefulness of eIF4A1 as a prognostic biomarker in human HCC.Next, to evaluate the therapeutic potential of targeting eIF4A1 activity in HCC, we challenged human HCC cell lines in vitro with five distinct rocaglate derivatives, namely CR-1-31-B, FL3, Rocaglamide, Silvestrol, and Zotatifin. These drugs promote the binding affinity of eIF4A1 to mRNA species containing polypurine sequence motifs, sequentially impeding the translation initiation during protein synthesis by blocking the formation of the heterotrimeric eIF4F cap-binding complex. Prior to pharmacological treatments, we assessed the endogenous eIF4A1, eIF4A2, and DDX3 protein levels using western blot analysis in various human HCC cell lines. eIF4A1, eIF4A2, and DDX3 proteins were expressed in all HCC cell lines tested .Subsequently, to assess the cytotoxic potential of eIF4A inhibitors, the HLE, HLF, and PLC/PRF/5 tumor cell lines were randomly selected and treated with a range of concentrations between 0 and 200 nM per eIF4A inhibitor, and colorimetric MTT assays were performed 48 h after starting compound exposure. Principally, cellular metabolic activity was measured to indicate cell viability, proliferation, and cytotoxicity. Overall, all five rocaglates efficiently reduced tumor cell viability in the three cell lines in a dose-dependent manner A–C. NotaTo further elucidate the mechanisms of action of eIF4A inhibitors in HCC cell lines, we selected the two most effective drugs, i.e., CR-1-31-B and Zotatifin, for the following experiments. Zotatifin was also selected because, to date, it is the only eIF4A inhibitor in clinical trials. When evaluating cell proliferation, we found that incubation with CR-1-31-B and Zotatifin induced a marked reduction in BrdU incorporation in both HLE and HLF cell lines, with a slightly more pronounced anti-growth effect in the case of CR-1-31-B A,B.Subsequently, apoptosis was determined in the same cell lines treated with the two eIF4A inhibitors. Both CR-1-31-B and Zotatifin induced higher apoptotic cell death in the cell lines than was observed in untreated and DMSO-treated cells A,B.Next, we assessed the effects of CR-1-31-B and Zotatifin on some of the most relevant oncogenic pathways in hepatocarcinogenesis by western blot analysis . Both drInspired by the findings that eIF4A inhibitors did not reduce the activation of mTOR effectors, we tested whether mTOR suppression could synergize with eIF4A inhibitors to hamper HCC cell growth. For this purpose, the HLE and HLF cell lines were treated with the pan mTOR inhibitor, Rapalink-1 , either Subsequently, we assessed proliferation and apoptosis in the same cell lines using Rapalink-1 alone and in combination with CR-1-B-R1 or Zotatifin. Again, the combined treatments resulted in a significant reduction in proliferation A and a rFinally, western blot analysis confirmed that the mTOR inhibitor, Rapalink-1, alone and in combination with the eIF4A inhibitors, CR-1-31-B or Zotatifin, fully inhibited p-RPS6 and p-4EBP1 levels. In addition, Rapalink-1 alone resulted in the downregulation of eIF4A1, eIF4A2, and DDX3, whereas inconsistent results were obtained for these proteins when Rapalink-1 was combined with CR-1-31-B or Zotatifin . These dHCC, the most common liver primary cancer, is characterized by its late diagnosis, increasing incidence worldwide, and limited treatment options . DespiteCumulating evidence indicates that translation dysregulation is critical to tumor development and progression . TherefoFurthermore, we revealed that targeting eIF4A1 using rocaglates induced strong growth suppression of human HCC cells by inhibiting proliferation and triggering apoptosis. Notably, the growth inhibitory activity of these compounds on HCC cells occurred at low nanomolar concentrations, implying their potency, at least in vitro. These data, confirming previous findings obtained in different tumor types, support the targeting of eIF4A1 as a promising therapeutic strategy against HCC .At the molecular level, we discovered that various oncoproteins belonging to the JAK/STAT, ERK/MAPK, AKT, and Hippo/TAZ pathways are markedly downregulated by eIF4A inhibitors. On the other hand, we revealed that treatment with these compounds did not affect the activity of members of the mTOR signaling cascade. Strikingly, exposure of HCC cells to combinations of eIF4A inhibitors and the mTOR inhibitor, Rapalink-1, a third-generation mTOR inhibitor that links rapamycin and MLN0128 , resulteSer235/236 and p-4EBP1Thr37/46, even in the presence of eIF4A inhibitors. Hence, we suggest that a combinatory therapy with eIF4A inhibitors and Rapalink-1, a drug that was originally developed to overcome mTOR resistance mutations, might serve as a refinement strategy for systemic management of HCC by simultaneously and effectively targeting both mTOR signaling and eIF4A-dependent translation. Overall, the present data indicate that eIF4A is a potential prognostic biomarker in HCC patients and a promising therapeutic target in this deadly tumor type. In particular, eIF4A inhibitors displayed formidable cytostatic activity against HCC cells in vitro, further augmented in combination with an mTOR inhibitor. Although the antineoplastic effectiveness of these drugs, alone and in combination, requires validation in vivo, the present data suggest that targeting translation initiation can provide an innovative and valuable strategy in the development of novel treatments against human HCC.Moreover, this is the first study to demonstrate in the setting of HCC that Rapalink-1-induced inhibition of mTOR signaling can be achieved through suppression of two key downstream substrates, p-RPS6n = 25) and longer survival/better outcome , characterized by <3 and >3 years’ survival following partial liver resection, respectively. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of the Medical University of Regensburg .Forty-seven human HCC tumor tissue samples and paired surrounding non-tumorous tissues were collected at the Institute of Pathology at the University of Regensburg . Patient features are summarized in 2 humidified atmosphere. HLE, HLF, and PLC/PRF/5 cell lines were maintained in Dulbecco’s Modified Eagle Medium , and SNU182 and SNU449 cell lines were maintained in RPMI 1640 (Anprotec) supplemented with 10% (v/v) FCS , 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 1% penicillin/streptomycin solution . Mycoplasma-free status for all cell lines was monitored regularly using the PCR Mycoplasma Test Kit I , and cell line authentication was performed by Cell Lines Service . CR-1-31-B , FL3 [The human HCC cell lines HLE , HLF , PLC/PRF/5 , SNU182 , and SNU449  were cultured at 37 °C in a 5% CO-3), FL3 , Rapalin4 cells per well. After an overnight attachment period, cells were exposed to various concentrations of CR-1-31-B, FL3, Rocaglamide, Silvestrol, Zotatifin, and Rapalink-1 alone or CR-1-31-B-Rapalink-1 and Zotatifin-Rapalink-1 combinations for 48 h. Cells treated with DMSO and wells containing only culture medium served as negative and background controls, respectively.Cells were seeded in flat-bottom 96 well plates at a density of 10 x 102 for 2 h. The medium was fully aspirated, and 100 µL of 100% (v/v) DMSO was added per well to dissolve the formazan crystals. After 15 min of shaking at room temperature, the absorbances were measured at 570 and 630 nm using the FLUORstar Omega multiplate reader and MARS data analysis software . All experiments were performed in triplicate or quadruplicate and repeated at least twice. The average absorbance of the DMSO-treated cells was defined as 100% cell viability. IC50 values and standard deviations were calculated using GraphPad Prism software. Average cell viability values of three independent MTT assay experiments were employed to determine dose-effect curves (Fa = fraction affected) and the combination indices of two drugs using CompuSyn PC software version 1 .For the MTT assay, following treatment, 10 µL of 5 mg/mL methyl-thiazolyl-diphenyl-tetrazolium bromide (MTT) solution was added per 96 well and incubated at 37 °C and under 5% COCell proliferation was evaluated in the cell lines at the 48-h time point using the BrdU Cell Proliferation Assay Kit . Briefly, following drug treatment, cells were incubated with 1× bromodeoxyuridine (BrdU) for 2 h and fixed for 30 min at room temperature. The fixing solution was discarded, and cells were incubated with the BrdU mouse detection antibody for 1 h at room temperature. After washing, cells were stained with HRP-conjugated anti-mouse secondary antibody for 30 min at room temperature and washed again. After incubation with TMB substrate solution for a further 30 min at room temperature, stop solution was added, and the absorbance was measured at 450 nm. The results are expressed as fold-change over DMSO control.Apoptosis was determined in the HCC cell lines using the Cell Death Detection Elisa plus Kit , following the manufacturer’s instructions. All cell line experiments were repeated at least three times in triplicate.TM lysis buffer  containing 1x HaltTM Protease and Phosphatase Inhibitor Cocktail  in the Bullet Blender Storm 24  for 3 min at speed 8, placed in the rotator mixer for 30–60 min at +4 °C and sonicated. Lysis of pellets from the cell lines was performed in the same lysis buffer for 1 h on ice. Protein concentrations were determined using the colorimetric BioRad Protein Assay Dye Reagent Concentrate  with bovine serum albumin (BSA) as the standard.Human HCC tumor and non-tumorous liver tissues were homogenized in 100–250 µL of T-PERTM sample buffer (B0007) and 1× BoltTM reducing agent  by boiling for 5 min at 95 °C. Samples with 10 µL of protein per lane were separated by SDS-PAGE on Bolt™ 4–12% Bis-Tris Mini Protein Gels (NW04125BOX) in 1× Bolt™ MES SDS Running Buffer  and transferred onto nitrocellulose membranes  by electroblotting running program P0 in the iBlot™ 2 Gel Transfer Device . Membranes were blocked in EveryBlot Blocking Buffer  and probed at +4°C overnight with specific antibodies: eIF4A1 ; eIF4A2 ; DDX3 ; p-RPS6Ser235//236 (4856), p-4EBP1Thr3747 (2885), p-STAT3Tyr705 (9145), AKT (4691),), ERK1/2 (4695), p-ERK1/2Thr202/204 (4370), SKP2 (2652), YAP/TAZ (8418), p-YAP/TAZ (13008), PHB1 (2426), PHB2 (14085) ; p-AKTSer473 ; and β-actin . Horseradish peroxidase-conjugated secondary antibody  was incubated for 1 h at room temperature, and blots were imaged with Clarity Max ECL Western Blotting Substrate  using ChemiDoc MP . Band volumes were quantified with Image LabTM software version 6.0.1 (Bio-Rad) and normalized to GAPDH or β-actin.For western blot analysis, protein lysates were denatured in 1× LDS BoltTM Plus 2, Thermo Fisher Scientific) were applied to the gels. After conventional gel electrophoresis, the gel fluorescence of SMA basic L  and SMA label  were imaged. Following blotting, the blot fluorescence of the SMA label  and the blot chemiluminescent signal of the target protein  were detected. Finally, the normalization of gel load , normalization of Smart Label (based on SMA label), and normalization of target protein signal  were conducted using the SPL-normalization template following the manufacturer’s protocol. Results were expressed as SPL normalized target volume. Visualization and quantification were performed using the Chemidoc MP Imaging System and Image LabTM software version 6.0.1 (both from Bio-Rad).High patient-to-patient variation of reference protein expression, such as GAPDH and β-actin, impedes proper quantification of western blot data. Hence, for accurate target protein quantification of human HCC samples, western blotting was performed using the Smart Protein Layers (SPL) Kit Red from NH DyeAgnostics  according to the manufacturer’s instructions. The SPL technology is based on three components: (i) red-fluorescent Smart Label (SMA label) reagent for labeling and visualization of total protein on gels and blots, (ii) bi-fluorescent sample-specific standard Smartalyzer L (SMA basic L) for normalization, standardization, and quantification of total protein per sample, and (iii) a calibrator (CAL) for comparison of fluorescence and target protein signals derived from different gels. Briefly, before gel electrophoresis, every protein sample was labeled with 2 µL of SMA basic L and 1 µL of Smart label working solution. Samples with of 10 µg of protein per sample, 12 µL of calibrator , and 5 µL of Biotinylated Protein Ladder  staining was conducted using a standard protocol on liver sections. Specifically, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) by heating in a microwave on high for 10 min, followed by a 20-min cool down at room temperature. After blocking with 5% goat serum and the Avidin-Biotin blocking kit , the slides were incubated with the anti-eIF4A1 primary antibody  overnight at 4 °C. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and, subsequently, the biotin-conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. Reaction detection was achieved using the Vectastain ABC-Elite Peroxidase Kit  with ImmPACT DAB  as the chromogen. Slides were counterstained with Mayer’s hematoxylin.p values for TCGA data were obtained from the UALCAN analysis tool. The non-parametric Wilcoxon signed-rank test was used for paired sample comparison. Kaplan-Meier curves were evaluated using the log-rank test. For calculation of IC50s, data were transformed to log2, normalized, and non-linear regression (log)inhibitor vs. response–variable slope (four parameters) was performed. For the transformation of SEM to SD, SEM was multiplied by the square root of the sample size n. Tukey’s multiple comparison test was applied for multiple comparisons. Two-tailed values of * p < 0.5, ** p < 0.1, *** p < 0.01, and **** p < 0.001 were considered significant. Lowercase letters denoted statistical significance, as stated in the associated figure legends. All data are expressed as the mean ± SD or SEM.GraphPad Prism version 9.3.1 software  and IBM SPSS version 26 software  were employed to evaluate statistical significance. The Our results, obtained in silico and in vitro, constitute the initial step in an innovative direction to develop pharmacological strategies to treat liver cancer. This is particularly valuable owing to the lack of available tools for efficaciously treating advanced non-surgically resectable HCC. The fact that eIF4A1 was consistently upregulated in HCC in comparison with adjacent non-tumor liver tissue and the magnitude of this change was negatively correlated with the prognosis of HCC patients supports a role of eIF4A1 in HCC malignancy. This led us to evaluate the impact of eIF4A1 inhibitors on HCC cell viability. Interestingly, even at low nanomolar concentrations, these inhibitors triggered a remarkable reduction in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Moreover, the cytostatic effect of eIF4A1 inhibitors was further increased by the pan mTOR inhibitor, Rapalink-1.During the last decade, considerable efforts have been made to characterize the molecular basis for the high refractoriness of HCC to currently available first- and second-line drugs . This, tIn summary, the results of the present study highlight the pathogenic relevance of eIF4A1 in HCC and constitute an encouraging advance in developing new sensitizing strategies. Thus, the usefulness of combining eIF4A1 and mTOR inhibitors with currently used chemotherapeutic agents deserves further investigation.


Angiogenesis is the formation of new blood vessels from pre-existing vessels. Adequate oxygen transport and waste removal are necessary for tissue homeostasis. Restrictions in blood supply can lead to ischaemia which can contribute to disease pathology. Vascular endothelial growth factor (VEGF) is essential in angiogenesis and myogenesis, making it an ideal candidate for angiogenic and myogenic stimulation in muscle. We established C2C12 mouse myoblast cell lines which stably express elevated levels of (i) human VEGF-A and (ii) dual human FGF4-VEGF-A. Both stably transfected cells secreted increased amounts of human VEGF-A compared to non-transfected cells, with the latter greater than the former. In vitro, conditioned media from engineered cells resulted in a significant increase in endothelial cell proliferation, migration, and tube formation. In vivo, this conditioned media produced a 1.5-fold increase in angiogenesis in the chick chorioallantoic membrane (CAM) assay. Delivery of the engineered myoblasts on Matrigel demonstrated continued biological activity by eliciting an almost 2-fold increase in angiogenic response when applied directly to the CAM assay. These studies qualify the use of genetically modified myoblasts in therapeutic angiogenesis for the treatment of muscle diseases associated with vascular defects. Adequate oxygen transport and waste removal are essential for tissue homeostasis. For this to occur, a vast network of well-connected blood vessels to deliver well-oxygenated blood is required, with ischaemia occurring in its absence leading to tissue deprivation and death ,2. AngioTherapeutic angiogenesis is the coordinated delivery of vascular growth factors with the aim of promoting blood vessel growth, leading to revascularisation of previously under-perfused tissues ,8. VascuVEGF-A is a key factor in vascular development and is the single most important regulator of blood vessel formation . VEGF exThe C2C12 immortal muscle cell line has a rapid proliferative and differentiation ability making it an ideal in vitro approach to studying myogenesis . VEGF-A Previous studies have reported the implantation of VEGF-expressing muscle-derived stem cells improves vascularisation and reduces fibrosis in dystrophic muscle ,30 and ihVEGF-A secreting stable cells. The chick CAM is a highly vascularised membrane used for nourishment and gaseous exchange in fertilized chicken eggs [Several angiogenic assays, both in vitro and in vivo have been used in the past to study angiogenesis. These include in vitro assays such as the use of mouse and human endothelial cell lines   and in vken eggs ,38. Withken eggs ,40. Matrken eggs ,42,43.hVEGF, both alone and as part of a dual plasmid co-expressing hFGF4 and hVEGF. We successfully characterised the hVEGF-A expression and the angiogenic potential of stably transfected C2C12 mouse myoblast cells using these models of angiogenesis. The first aim was to establish a stable C2C12 muscle cell line expressing hVEGF-A protein, confirmed through molecular analysis. Secondly, we observed the paracrine angiogenic efficacy of conditioned media taken from these cells using the CAM filter disc and endothelial cell assays. Finally, we investigated the in vivo pro-angiogenic efficacy of these cells when applied to the CAM angiogenic assay. This investigation combines elements of several previous studies, providing an insight into whether hVEGF-A-producing cells can be used as a pro-angiogenic factor with therapeutic potential in the treatment of conditions that experience decreased neovascularisation.We used the CAM and endothelial cell proliferation, migration, and morphogenesis assays to develop an ex-vivo strategy to deliver the angiogenic factor C2C12  mouse myoblast cells and C166  mouse yolk sac endothelial cells were cultured in high glucose Dulbecco’s Modified Eagle medium with sodium pyruvate, supplemented with 1% penicillin-streptomycin antibiotic and 10% fetal calf serum . Cells were maintained in the standard cell culture conditions (5% CO2 and 37 °C) and sub-cultured when necessary, using trypsin/EDTA solution.6) and high (0.2 × 106) densities and subjected to increasing concentrations (0–1000 μg/mL) of G418 to determine the minimum antibiotic concentration needed to kill all cells over the course of 3- 7 days. Cell confluency was assessed by visual inspection, while cell numbers were counted using the Countess™ II FL Automated Cell Counter (ThermoFisher Scientific) following 7 days of exposure. At 1000 µg/mL of G418, there was a visible reduction in confluency and increased cell toxicity in those seeded at both low and high concentrations  resistance gene. The kill curve experiment involved a dose-response where non-transfected C2C12 cells were seeded at low  was received from Prof Jozef Dulak, Department of Biochemistry, Biophysics, and Medical Biotechnology Jagiellonian University, Krakow, Poland. Heat shock was used to transform plasmids into DH5α competent E. coli cells, and subsequently cultivated. The DNA was then isolated using a Miniprep DNA Elution Kit  according to the standard manufacturer’s instructions. Confirmation of the plasmids was carried out by DNA restriction digests and gel electrophoresis.Transfections with pVEGF-EGFPN1, pEGFPN1, and FGF4/VEGF plasmids were carried out using Lipofectamine 2000 (Invitrogen), in a 1:2.5 ratio of DNA: Lipofectamine, with a DNA concentration of 8 µg for each plasmid. The DNA and Lipofectamine mixture was applied to plates containing C2C12 cells at 90% confluency and incubated at 37 °C overnight. In the case of pVEGF-EGFPN1 and pEGFPN1 plasmids, positive transfection was identified by observing GFP positive cells under FITC light 24 h post-transfection. Plates were trypsinised, serially diluted, and seeded in Geneticin (G418) selective media to screen for positively transfected cell colonies. Plates were screened for antibiotic resistance for four weeks, with media replenished every 3 days until isolated single colonies could be observed. Isolated colonies which showed continued antibiotic resistance were isolated using trypsin and cloning discs and expanded for further study with two clones selected for each pVEGF-EGFPN1 and pEGFPN1 plasmid, and eleven clones selected for FGF4/VEGF.hVEGF-A ELISA kit (Invitrogen) according to the manufacturer’s instructions. Conditioned media from non-transfected C2C12 myoblasts served as a control. Selected cell lines for each plasmid were designated as Myo-GFP, Myo-VEGF-GFP, and Myo-FGF4-VEGF and were each expanded for further investigation into their angiogenic potential.Non-transfected and stable transfected C2C12 cells were seeded and allowed to grow for 72 h. Media taken from each well was then centrifuged at 1000 rpm for 10 min to remove cell debris and frozen in aliquots at −80 °C. Protein levels of VEGF-A were determined using an ® Reagent (Invitrogen). The RNA samples were quantified using a NanoDrop ND 1000 spectrophotometer . A total of 500 ng total RNA was reverse transcribed into first-strand cDNA using Superscript III Reverse Transcriptase with random primers (ThermoFisher Scientific). qPCR was then performed using a StepOne Plus RealTime PCR system . Each 20 μL reaction mixture consisted of 5 μL cDNA, 5 μL SyberGreen, and 10 mmol/L of specific primers  or Myo-GFP, Myo-VEGF-GFP and Myo-FGF4-VEGF cells were applied. Scratches were imaged using EVOS M5000 at 10X magnification and then returned to the incubator at 37 °C for 8 h before being imaged again. Cell migration was quantified by the change in width of the scratch between experimental time points, with the distance measured using Image J software .Endothelial cell migration (Scratch) assay was carried out as described previously ,46. C166Endothelial cell proliferation was measured using a mitochondrial metabolic (MTT) activity assay, similarly to as described previously ,48. C1664 in triplicate in 100 μL of conditioned media taken from Myo (control), Myo-GFP, Myo-VEGF-GFP, or Myo-FGF4-VEGF cells. After 18 h, tube-like structures were observed. Each well was imaged at four non-overlapping locations, and the number of tubes, nodes, and total tube length were measured using Image J software  was greater than 45°, and whether it branched within a ring (⌀ 12 mm) encircling each filter disc. Similarly, in the case of the Matrigel-cell experiments, all blood vessels which approached the on-plant (A = 16 mm2) at an angle greater than 45° were assigned a score of 0, 1, or 2 based on whether they branched within the focal plane of the on-plant. In the case of both experiments, the total score for each on-plant was then determined by the sum of each of these individual scores.Any chicks which had perished during the course of the experiment were excluded from the analysis. With the analyst blinded to the treatment used for each on-plant, angiogenesis as a result was scored by determining the vascular index of blood vessels which grew towards the on-plant, similarly, to as described previously ,62,63. It-test was used to determine statistical significance between the two groups. A one-way analysis of variance (ANOVA) and Tukey’s post hoc test were applied to determine statistical significance between groups, while a two-way analysis of variance (ANOVA) was applied where two factors were present in an experiment. Data are expressed as mean ± S.E.M. or as a scatter plot ± S.E.M., with data considered statistically significant at * p < 0.05; ** p  <  0.01; *** p  <  0.001 and **** p  <  0.0001.All statistical analyses were carried out using Excel and GraphPad Prism software. A Student’s C2C12 muscle cells were transfected with pEGFPN1, pVEGF-EGFPN1, and FGF4/VEGF plasmids. Over the course of four weeks following transfection, G418 antibiotic selection was carried out. Two clones of each pEGFPN1 and pVEGF-EGFPN1 and eleven clones of FGF4/VEGF transfected cell colonies, which showed continued antibiotic resistance were selected and expanded to larger numbers. Based on the brightness of their GFP activity, pEGFPN1 and pVEGF-EGFPN1 clones were referred to as low and high expression pEGFPN1 and p-VEGF-EGFPN1 . Once eshVEGF-A protein expression in stably transfected cells, and to screen cells to identify those with the most potency. To achieve this, the level of hVEGF-A protein released from the stably transfected cell lines into the cell media was measured. Non-transfected C2C12 and pEGFPN1 transfected cells served as controls, where no hVEGF-A expression is expected. High expression pVEGF-EGFN1 transfected cell lines showed a high hVEGF-A secretion compared to pEGFPN1 and non-transfected C2C12 cell lines (p < 0.0001) (hVEGF-A protein expression (p < 0.0001)  and Myo-GFP cell lines, and an almost 50-fold increase compared to Myo-VEGF-GFP cell lines , and Myo-GFP A. A huma 0.0001) . While c 0.0001) . Based oll lines B.hVEGF and hFGF4 expression in the Myo (control), Myo-GFP, Myo-VEGF-GFP, and Myo-FGF4-VEGF cell lines to confirm altered gene expression. qPCR indicated a significant upregulation of hVEGF expression in Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines compared to non-transfected and Myo-GFP cell lines which resulted in undetermined products. A modest increase of hVEGF was seen in the Myo-VEGF-GFP cell line. Meanwhile, a much more apparent increase was seen with the Myo-FGF4-VEGF cell line compared to Myo (control) and Myo-GFP cell lines (p < 0.0001) , Myo-GFP, and Myo-VEGF-GFP (p < 0.0001) cells  C. hFGF4 1) cells D, confirhVEGF-A expression and secretion by Myo-VEGF-GFP and Myo-FGF4-VEGF cells, our next aim was to investigate the angiogenic potential of the conditioned media from these different cell lines. Both hVEGF-A and hFGF play important roles in mediating endothelial cell growth and promoting angiogenesis, as a result, endothelial cell angiogenic assays were employed. This involved monitoring the behaviour of C166 endothelial mouse yolk-sac cells, following treatment with conditioned DMEM taken from each cell line.Following confirmation of p < 0.05 (p < 0.0001) or Myo-GFP (p < 0.0051) cell lines. Myo-VEGF-GFP cell conditioned media elicited a significant increase in cell proliferation compared to non-transfected cell conditioned media (p < 0.05)  C. Interep < 0.05 , Myo-GFP Myo-VEGF-GFP, and Myo-FGF4-VEGF were investigated through an application on developmental day 8 (EDD8) for 72 h.Following confirmation that conditioned media from these stably transfected cells elicited pro-angiogenic effects in both endothelial cell angiogenic assays and the CAM filter disc assay, the focus of this study then moved to the investigation of the angiogenic response of the gene-modified cells directly. To examine this, Matrigel-cell mixtures were applied to the CAM. To begin, a dose-dependent curve using the CAM assay was carried out, with increasing quantities of Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines respectively applied to the CAM for 72 h to establish the optimum cell number required . For botp < 0.05 . Howeverobserved . Based op < 0.001) and Myo-GFP cells (p < 0.0001). Meanwhile, Myo-FGF4-VEGF cells elicited a similar response, resulting in a significant increase in angiogenic observed compared to on-plants containing Myo (control) and Myo-GFP cells, with p < 0.001  and Myo-GFP encapsulated on-plants respectively A. An ang < 0.001 B.VEGF is paramount in vascular development and is the single most important regulator of blood vessel formation ,64. AngihVEGF-A secreting C2C12 mouse myoblast cell lines, and quantify the angiogenic potential of these cells, which may prove beneficial in the amelioration of muscle ischaemia associated with diseases including CLI, PAD, diabetes, cachexia, COPD and muscular dystrophy [We conducted a number of studies in order to establish stable ystrophy ,6.hVEGF. Conditioned media taken from the hVEGF-A secreting cell lines proved efficacious in the induction of angiogenesis in the CAM angiogenic assay, while also resulting in pro-angiogenic behaviours in murine endothelial cell assays of angiogenesis. Finally, following confirmation of the pro-angiogenic benefits as a result of conditioned media, we then demonstrated how these cells provoked a potent angiogenic response in the CAM angiogenic assay.In the present study, we successfully established two stable muscle cell lines including expression of human VEGF-A protein, Myo-VEGF-GFP as a GFP tagged fusion protein, and Myo-FGF4-VEGF as cells dually expressing human FGF4 and hVEGF-A alone and in combination with hFGF4.The efficacy of myoblast-mediated VEGF gene delivery to mouse and rat muscle has been investigated in the past, with varying levels of success. Despite the potent angiogenic potential of VEGF, studies have indicated that the optimum dosage for VEGF is within a narrow limit, with overproduction leading to the formation of disorganized, malformed vessels with irregularly sized lumens and weakened wall permeability ,69,70,71hVEGF-A and hFGF4/VEGFA overexpressing C2C12 mouse myoblast cells, both as a result of stable and transient transfections [hVEGF-A secretions in the dual expression cell line are much higher than in previous reports, confirmed by both ELISA and qPCR [hVEGF-A protein secretion and mRNA from both cell lines, while qPCR alone confirmed the dual expression of FGF4 and VEGF in the Myo-FGF4-VEGF cell line. Myo-FGF4-VEGF cells secreted a significantly higher amount of hVEGF-A protein compared to non-transfected and Myo-GFP cell lines, while in comparison to the Myo-VEGF-GFP cell line, Myo-FGF4-VEGF cells exhibited an almost 50-fold increase in hVEGF-A protein secretion.Previous studies have generated fections ,26. In tand qPCR ,26,74,75hVEGF falls under IRES-dependent second gene expression, as a result, expression of hVEGF would be expected to be lower compared to the single VEGF expression plasmid [hFGF4 gene transcript level is higher than the hVEGF-A expression.Research to date has examined the expression and therapeutic benefits of both VEGF and FGF secretions separately . However plasmid . This cahVEGF-A protein secretion in these studies was observed to be much less potent. In previously published reports, transient transfection with this dual FGF4/VEGF plasmid reported hVEGF-A secretions of approximately 6000 pg/mL in human primary myoblasts [hVEGF secretions of around 5500 pg/mL. Additionally, this was used to ameliorate wound healing and post-ischemic blood flow recovery in the hindlimb ischemic mouse model [The dual FGF4/VEGF plasmid used in this study has been successfully transfected into myoblast cells in previous reports ,25,74. Hyoblasts  and 2800yoblasts . Howeveryoblasts . An AAV hVEGF-A protein secreted from Myo-VEGF-GFP cells compared to Myo (control) or Myo-GFP transfected cell lines. However, this expression is not as potent as concentrations seen in the Myo-FGF4-VEGF cell lines in the present study. Sun et al., through lentiviral transfection, developed a stable VEGF-165 expressing ADSCs which secreted approximately 920 pg/mL of VEGF [With the aim of therapeutic angiogenesis, VEGF-165 alone has been transfected into several different cell types including fibroblasts , endothe of VEGF . Meanwhi of VEGF . This rehVEGF and hFGF4/VEGF protein expression from Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines, we moved our focus to proving the pro-angiogenic effects these cells could elicit. Previously, various cells have been successfully modified or transfected to overexpress VEGF [hVEGF or hFGF4-VEGF expressing C2C12 cells using the chick CAM assay. In the current investigation, we chose to employ both in vitro murine endothelial cell angiogenic assays, and the in vivo chick CAM assay.Following on from the successful confirmation of ess VEGF ,80,82, Fess VEGF  or both ess VEGF ,74. HowehVEGF protein secretion in affecting the angiogenic response in vitro. The lower hVEGF-A secretion by the Myo-VEGF-GFP cell line is reflected. Consequently, a similar angiogenic response is not as clearly apparent, with Myo-VEGF-GFP conditioned media failing to induce a significant increase in cell proliferation compared to Myo-GFP conditioned media.VEGF-A and FGF have both proven to elicit pro-angiogenic behaviours in endothelial cells in vitro ,84. In tThe potent angiogenic ability of Myo-VEGF-GFP and Myo-FGF4-VEGF cell conditioned media is again seen in the tube formation assay using endothelial cells seeded on a basement media matrix. In vivo, VEGF triggers the release of proteolytic enzymes which degrade the surrounding basement membrane, allowing endothelial sprouts to invade the extracellular matrix leading to tube formation. Following this, endothelial cells migrate, forming tube-like structures which fuse to form an organised and functional blood vessel network . ConditiMeanwhile, only a modest difference in endothelial cell migration, proliferation, and tube formation is seen in the comparison of Myo-VEGF-GFP and Myo-FGF4-VEGF conditioned media, where despite the increased potency of VEGF-A secretion, little difference in these parameters is observed. This reinforces the delicate VEGF dose-responsive effects aforementioned, where overexpression of VEGF can lead to negative effects ,85.While in vitro assays such as the endothelial cell assays can elucidate pro-angiogenic potency, in this study, it was important to examine the efficacy of these cell secretions on the in vivo model of angiogenesis. We chose to use the CAM assay to investigate this pro-angiogenic potential. The CAM assay is a highly versatile, cost-effective model of angiogenesis with a vast vascular network of capillaries, veins, and arteries which can be easily manipulated and observed for experimental study ,39,40.A strong and potent angiogenic effect is seen in this study as a result of conditioned media taken from the stable transfected Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines. This is consistent with other studies where conditioned media from osteoblast-like cells which secreted elevated levels of VEGF-A also induced a positive angiogenic response in the CAM assay .hVEGF and have the potential to induce angiogenesis in vivo. Naturally, the next phase would be to examine the applicability and efficacy both these cell lines could have in a higher in vivo mouse model of disease. Myoblast transplantation is a promising area of therapeutics [The present findings are promising, confirming that Myo-VEGF-GFP and Myo-FGF4-VEGF cell lines secrete elevated levels of apeutics ; howeverapeutics ,88. TherhVEGF protein production, as observed in other published studies [Application of Myo-VEGF-GFP and Myo-FGF4-VEGF cells encapsulated in Matrigel onto the CAM assay resulted in a significant increase in the angiogenic score, similar to the effects seen with cell conditioned media. In this study, the correct dosage of cell number applied was paramount to the most efficient angiogenic response. Higher levels of Myo-FGF4-VEGF cells per on-plant resulted in a reduction in total angiogenic score with a bell-shaped dose-response observed, possibly indicating excessive cell numbers prove to be harmful in the angiogenic potential. This reinforces the potentially deleterious effects of excessive  studies ,69,70. FhVEGF-A and hFGF4 from C2C12 mouse myoblast cells into an in vivo angiogenic system. These reveal several important findings which could potentially provide broad implications in the field of therapeutic angiogenesis in muscle diseases with defects in microcirculation such as diabetes and muscular dystrophies. We have successfully established two pro-angiogenic mouse myoblast cell lines which secrete hVEGF-A alone or in combination with hFGF4, with the latter cell line showing great efficacy. Both of these cell lines have proven to elicit a potent pro-angiogenic effect both in vitro in the murine endothelial cell angiogenic assay and in vivo in the chick CAM model of angiogenesis. Furthermore, the manipulation and application of these cells in higher-level in vivo studies may prove highly beneficial in the amelioration of vascular abnormalities in muscle disease.In summary, this current study demonstrated the ability to deliver growth factors such as


V600E-driven papillary thyroid cancer that closely mimic the heterogeneous pathological progression of the disease seen in patients despite sharing a common driver mutation. We anticipate that these newly developed models will allow for the discovery of novel mechanisms that determine differences in disease progression among BRAFV600E-driven thyroid cancers and facilitate the testing of therapeutic interventions.Laboratory experimental models are essential for advancements in thyroid cancer translational research. In this study, we discuss the development and characterization of six cell-based models of BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a V600EBraf+/−/Pten+/−/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAF V600E is the most common driver mutation of PTC in adults and the second most common driver mutation in children [V600E mutation confers constitutive activation of the kinase, leading to sustained activation of the mitogen-activated protein kinase (MAPK) signaling pathway [V600E mutation with extrathyroidal extension, lymphatic and vascular invasion, local lymph node metastasis, higher clinical stage, persistent disease, reduced avidity for radioactive iodine, and lower survival rates in adults with PTC [Thyroid cancer is the most common endocrine malignancy and the most frequently diagnosed cancer among adolescents and young adults between ages 15–29 . Papillachildren ,9,10. Th pathway ,12. Clinwith PTC ,17,18. Iwith PTC ,20,21. Nwith PTC ,23,24,25Most PTCs are successfully managed with standard multimodal therapy. Patients undergo surgical tumor resection and lymph node dissection for localized disease, often followed by radioactive iodine (RAI) ablation, as adjuvant therapy in patients with high risk of recurrence or metastatic disease. Patients are then placed on thyroid hormone replacement therapy, with or without long-term thyroid hormone suppression, based on risk of recurrence. In the subset of patients with advanced and progressive RAI-refractory disease, oncogene-targeted therapies represent essential advancements in care and continue to be important areas of preclinical and clinical investigation in both adult and pediatric thyroid cancer.G12V-driven models of FTC [V600E-driven PTCs.It remains imperative to develop tumor-specific in vitro and in vivo preclinical models that recapitulate patient disease to prospectively evaluate treatments for efficacy and to decipher molecular mechanisms that confer therapeutic resistance. Although cell lines derived from human thyroid tumors provide a powerful tool to study novel therapies, research using human cells is limited to the tissue culture environment or xenograft models in immunocompromised hosts, which fails to recapitulate the interactions between tumor cells and the immune system in the host. Further, it was discovered that many previously reported thyroid tumor cell lines were either not unique or not actually of thyroid origin, further limiting the utility of some of these models . In conts of FTC  and sougV600EBraf+/−/Pten+/−/TPO-Cre mouse model of PTC. We have previously described a mouse model of PTC whereby thyroid-specific expression of V600EBraf induces development of thyroid tumors with 100% penetrance by five weeks of age [V600EBraf and homozygous Pten inactivation in mice to achieve concomitant MAPK and PI3K/Akt pathway activation. These mice develop PTCs that rapidly progress to PDTC with 100% penetrance and lethality by weaning [V600EBraf and heterozygous Pten. Three cell lines were isolated from well differentiated PTC tumors  and the other three cell lines were isolated from higher grade tumors with more invasive characteristics . This variability reproduces the clinical heterogeneity within the PTC patient population and, thus, allows for evaluation of characteristic features, in a subset of cell lines, that may be critical for disease progression. These cell lines represent novel and physiologically relevant research tools that can be used to evaluate therapeutic strategies and to illuminate factors that impact the progression of disease.In this study, we detail the establishment of six independent cell lines from the s of age . Additio weaning . These m weaning ,31. HereV600EBraf+/−/Pten+/−/TPO-Cre mice of a pure 129/SvImJ genetic background (Jackson Laboratory mouse strain #:002448 here on referred to as 129SvJ). B1865 and B1866 tumor cell lines were established from thyroid tumors of V600EBraf+/−/Pten+/−/TPO-Cre mice of a mixed genetic background. Thyroid tumors or WT thyroids were dissected and minced. The pieces were then resuspended in Ham’s F12 medium  supplemented with 10% fetal bovine serum , 2 mM L-Glutamine (Gibco), Penicillin/Streptomycin/Amphotericin B , 10 µg/mL Piperacillin sodium salt , and 10 µg/mL Ciprofloxacin Hydrochloride . Cells were then seeded into bind tissue culture flasks , and each flask was given an additional 500 μL of FBS. They were maintained at 37 °C in 5% CO2. After 24–48 h, the media were replaced, and the cells were maintained in 10% FBS F12 media supplemented with antibiotic for the next two weeks. WT thyrocytes were only cultured for at most one week before RNA was extracted to limit outgrowth of non-thyrocyte cells in culture. Then, antibiotic-free complete Ham’s 12 media was used. To rule out stromal cell contamination, all tumor cell lines were passaged at least 5 times after seeding and then genotyped using primers specific for BrafV600E to check for complete recombination , Pax8, Slc5a5 (Nis), and B2m were measured using pre-designed probes  and Taqman master mix . A total of four µL of cDNA from tumor samples and independent passages of each cell line and WT thyroids were run in triplicate on the QuantStudio 3 . B2m was used as an internal control to normalize the expression levels of thyroid specific genes. Data analysis was based on the delta-delta Ct method.Total RNA from all cell lines was extracted using the Direct-zol RNA Miniprep . Equal amounts of RNA template were reverse transcribed using the Verso cDNA synthesis kit . The differential mRNA expressions of Cells were plated, 300 per well, in quadruplicate into 96-well plates. At designated time points, cell growth was assessed using the Cell Titer-Glo luminescent cell viability assay . Luminescence was determined using the Varioskan Lux . Day 0 represents 18 h after plating. Inhibitor studies were conducted using Dabrafenib methanesulfonate salt  and Trametinib  dissolved in DMSO. To assess the relative viability of cells in the presence of single inhibitors, Dabrafenib, Trametinib, or vehicle (DMSO) were added 18h after plating. Relative viability was assessed using the Cell Titer-Glo luminescent cell viability assay, and concentrations of Dabrafenib 10 nM and Trametinib 1 nM were selected for viability assays of the complete cell line panel.To prepare total cellular protein lysates, 200,000 cells per well were seeded in six-well plates and allowed to attach for 24 h in complete medium. Cells were serum starved overnight before any specific treatment or stimulation. Then, cells were lysate in equal volumes of radioimmunoprecipitation (RIPA) buffer  supplemented with phosphatase and protease inhibitors  and 4× sample buffer  containing 200 mM DTT and boiled for 5 min. A small aliquot was taken after harvesting to measure protein concentration by BCA. To perform the SDS–polyacrylamide gel electrophoresis (PAGE), equal quantities of total cellular proteins from each sample were loaded into 10% acrylamide gels containing SDS-PAGE. Protein sizes were determined by comparison to a set of standards . To perform the immunoblot, proteins were transferred to a PVDF membrane pore size 0.45 μm  after methanol activation. Primary antibodies (1:1000) specific to pERK (9101S), ERK (9102S), pAKT (9271S), AKT (4685S), PTEN (9188T), E-cadherin (14472s), Vimentin (5741S), and β-actin (3700S)  were diluted in 5% bovine serum albumin (BSA) and 0.1% Tween-20 tris buffer (TBS-T) and incubated overnight at 4 °C. Specific binding of antibodies was detected with appropriate HRP-coupled secondary antibodies anti-rabbit (1:3000) or anti-mouse (1:5000) and visualized using enhanced chemiluminescence reagent . Protein expression levels were analyzed and compared by densitometry using iBright Analysis Software . Relative protein phosphorylation levels were obtained by normalizing the amount of phosphorylated protein to that of total protein. Original, uncropped Western blot membrane can be found in Organoids were grown using previously described methods . BrieflyMigration assays were performed in 24-well plates with cell culture inserts with 8.0 μm pores ; 75,000 tumor cells were seeded in the top of each insert in serum free Hams F-12 media. Complete 10% FBS Hams F12 media was added to the bottom chamber of the plate. Cells were then incubated at 37 °C for 18 h. The cells were then fixed using 4% Paraformaldehyde, and the cells were removed from the top of the chamber using cotton tipped applicators. The underside of the chambers was then stained with crystal violet. Images were taken on the EVOS M7000 imaging system (Invitrogen by Thermo Fisher Scientific).Phase contrast imaging was performed using the EVOS M7000 imaging system (Invitrogen by Thermo Fisher Scientific). H&E histology slides were imaged using the EVOS M7000 imaging system (Invitrogen by Thermo Fisher Scientific). Trichrome histology slides were scanned by the CHOP pathology core and images captured using Image Scope. For differential interference contrast (DIC) imaging live cells were imaged with a Zeiss Axiovert 200M platform using a 40× EC Plan Neo Fluar, 1.3 N.A., or a 63× Plan Apochromat, 1.4 N.A. objective  equipped with an Orca ER CCD camera . Images were captured using Slidebook 6 imaging software  using an Orca ER CCD camera (Hamamatsu).5 tumor cells  were pelleted by centrifugation, resuspended in 100 µL of a 1:1 mixture of Hams F-12 and Matrigel , and injected subcutaneously into the right hind flanks of 4–5-week-old male and female wild-type 129SvJ recipient mice. Tumor development was monitored weekly for 15 weeks. Mice were sacrificed due to tumor burden or loss of skin integrity as needed during that period or were collected finally at the end of the 15 weeks. Tumors were collected and fixed in 10% formalin for histological analysis.All animal experiments were performed at the Children’s Hospital of Philadelphia and approved by the IACUC. 5.0 × 10p-values of ≤0.05 were considered statistically significant. All data comparing more than two groups were analyzed via ANOVA analysis with post hoc analysis (indicated in figure legends).All data were analyzed using Prism 8 software . Differences with V600EBraf+/−/Pten+/−/TPO-Cre mice of a pure 129SvJ genetic background  or a mixed genetic background (cell lines B1865 and B1866). Tumors were collected from different sex animals and at a variety of time points from 3–24 weeks of age , Pax8, and Slc5a5 (Sodium Iodide symporter: Nis)  [Tg and Tshr expression levels were similar among the six cell lines but were very low compared to expression in WT thyrocytes. In contrast, Nis expression was variable between the six cell lines but was consistently much lower compared to WT thyrocytes. Like thyroid transcription factors, loss of Tg, Tshr, or Nis expression has been associated with PTC disease progression [Nis expression is particularly relevant clinically as it is associated with disease refractory to RAI ablation therapy [The thyroid epithelial lineage and differentiation status of the cell lines was determined by measuring the mRNA levels of thyroid-specific genes including er: Nis) A. Pax8 asgenesis ,34. The gression ,36,37,38 therapy ,40,41.We next sought to evaluate the proliferation rates of the cell lines in 2D culture using the Cell Titer Glo luminescence-based viability assay B. The CePten. To determine whether levels of PTEN protein expression correlated with basal pAKT levels, Western blot analysis was performed for PTEN. Consistent with their elevated basal pAKT expression, BB57, BB1845, B1865, and B1866 all showed reduced or no expression of PTEN protein  models of tumor growth may be more relevant as they better recapitulate the natural environment and structure of solid tumors. To characterize the growth of these novel cell lines in more physiologic conditions, we used an organoid forming assay D. BB342,Heterogeneity in cell morphology can be observed among the six cell lines A. DespitWe next evaluated the efficacy of MAPK specific inhibitors on the growth of these novel cell lines. Each cell line was treated with a variety of concentrations of either Dabrafenib (BRAF inhibitor) or Trametinib (MEK1/2 inhibitor) for 3 and 6 days . Both ofTo evaluate the growth-inhibiting effects of these drugs in a more physiologic environment, we established 3D organoid cultures of each cell line and assessed the size of the organoids following treatment with Dabrafenib, Trametinib, or DMSO control for 10 days C. AveragV600EBraf+/−/Pten+/−/TPO-Cre de novo tumors in the mouse thyroid gland precludes many in situ experiments. Therefore, the development of a subcutaneous model in immune competent animals would enable temporal models to study disease progression and to potentially serve as a future model for investigating targeted therapeutic interventions. The strengths of the subcutaneous tumor models coupled with the rapidly penetrant phenotype of our de novo V600EBraf+/−/Pten+/−/TPO-Cre murine model [The subcutaneous injection of tumor cells into murine hosts has been proven to be a valuable and convenient method for studying tumorigenesis. The very rapid growth and progression of ne model ,31 led uG12V-driven tumor cells, variable tumor penetrance was observed between each of the cell lines. BB19 and BB57 were the least penetrant, whereas BB342 had an intermediate penetrance and BB184-injected mice developed tumors with 100% penetrance  leads to the development of PTC in mice [Cancer cell lines harboring clinically relevant mutations derived from genetically engineered mouse models are invaluable tools for in vitro and in vivo preclinical research to drive advancements in our understanding of disease mechanisms and treatment approaches in thyroid cancer. Here, we sought to generate cell lines that would recapitulate the heterogeneity of PTC and that would allow us to examine factors that contribute to disease progression. PTC is the most prevalent form of thyroid cancer, and incidence is steadily increasing . BRAFV60tric PTC ,11,12,45tric PTC ,23,24,25 in mice ,31. In aV600EBraf+/−/Pten+/−/TPO-Cre mouse thyroid tumors. These cell lines share the same initiating driver mutations but represent a heterogenous population regarding sex, age, and histologic differentiation status. Additionally, we shown that these cell lines can be used for a variety of in vitro applications including mechanistic studies of targeted inhibitors and formation of 3D organoid cultures, while also demonstrating successful transplantation into syngeneic hosts for in vivo studies. Altogether, our results demonstrate heterogeneity within these cell lines that recapitulate differences observed clinically in the patient population. This novel cell line panel provides an innovative research tool to identify differences among tumor cells with heterogenous responses to treatment and/or invasive behavior despite sharing the same initiating driver mutations. Previous studies have indicated that progression from PTC to PDTC or ATC may be facilitated by mutations leading to the activation of the PI3K/Akt pathway co-occurring with BRAF mutations [Pten (In this study, we described six novel independent murine PTC cell lines that were derived from utations ,23,24,25ns [Pten . These cEpithelial-to-mesenchymal transition is associated with loss of E-cadherin expression resulting in loss of cellular adhesion and polarity that leads to an invasive cell phenotype with increased metastatic potential ,46,47.PrThe current understanding of EMT in thyroid carcinomas suggests that EMT is implicated in the progression from well-differentiated thyroid cancers to PDTC and ATC ,50,51,52In this study, we sought to validate a syngeneic subcutaneous tumor model. This provides a readily accessible method of exploring tumorigenesis that recapitulates the complex interactions between tumor cells, the immune system, and physiologic components of the tumor microenvironment. Previous studies have shown that immune cell recruitment, particularly macrophages, plays a role in thyroid cancer pathogenesis ,54,55. FV600EBRAF mutation is very common in adult PTC and moderately common in pediatric PTC, the mechanisms behind the disparity in clinical outcomes between these two populations is not well-understood. We believe that these cell lines model the heterogeneity seen in the population of patients with BRAF-driven PTC and provide a novel platform to investigate features critical to PTC disease progression and response to therapies. These tools provide a valuable platform for more reductionist and mechanistic approaches to investigate Braf-driven disease progression.Additionally, this model could be used to evaluate the role of age at onset of disease in PTC development and progression by injecting hosts of different age equivalents to compare penetrance, disease progression, and metastasis ,59,60,61V600E-driven papillary thyroid cancer represents an advancement in the development of much-needed validated preclinical models for thyroid cancer research. We anticipate that these cell lines will provide a powerful platform for in vitro and in vivo studies that will lead to the development of precision diagnostic and treatment strategies for patients with newly diagnosed, advanced, or treatment-refractory thyroid cancer. Ongoing work in the laboratory is seeking to identify molecular mechanisms that drive heterogeneity observed in morphology and responses to therapy of these cell lines, despite sharing identical driver mutations.The establishment of six independent cell lines modeling BRAF


MYC is one of the most frequently altered driver genes in triple-negative breast cancer (TNBC). The aim of this study was to evaluate targeting MYC for the treatment of TNBC.The anti-proliferative and apoptosis-inducing effects of the recently discovered MYC inhibitor, MYCi975 were investigated in a panel of 14 breast cancer cell lines representing the main molecular forms of breast cancer.p = 0.047, r = − 0.5385) or ELISA , i.e., response to MYCi975 decreased as endogenous MYC levels increased. MYCi975 also induced variable levels of apoptosis across the panel of cell lines, ranging from no detectable induction to 80% induction. Inhibition of proliferation and induction of apoptosis were greater in TNBC than in non-TNBC cell lines . Finally, combined treatment with MYCi975 and either paclitaxel or doxorubicin resulted in enhanced cell growth inhibition.IC50 values for growth inhibition by MYCi975 varied from 2.49 to 7.73 µM. Response was inversely related to endogenous MYC levels as measured by western blotting (Our findings open the possibility of targeting MYC for the treatment of TNBC. Based on our results, we suggest that trials use a combination of MYCi975 and either docetaxel or doxorubicin and include MYC as a putative therapy predictive biomarker.The online version contains supplementary material available at 10.1007/s10549-022-06673-6. Although the treatment of patients with breast cancer has greatly improved in recent years , the forThe c-MYC oncogene is one of the best studied cancer driver genes, promoting tumorigenesis via diverse mechanisms such as stimulating cell proliferation, blocking apoptosis, altering metabolism and depressing host immunity –7. OveraIn breast cancer, MYC is the most frequently amplified gene , 10, witTargeting MYC for cancer treatment, however, has proved difficult as the protein lacks a suitable site for high-affinity binding of low-molecular-weight inhibitors , 16. BecThe origin and maintenance of the breast cancer cell lines used was as previously described , 20. The5 per well and incubated overnight at 37 °C. Cells were then treated with dimethyl sulfoxide (DMSO) or 10 μM MYCi975. Following 48 h of incubation, they were harvested and stained with annexin-V and propidium iodide using the Annexin-V-FITC Apoptosis Detection Kit (Invitrogen). FACS analysis was performed using a BD FACSCanto ™.Cell growth was assessed using 3--2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as previously described , 20. ForFollowing treatment with MYCi975 or MYCMI6, cells were lysed in RIPA buffer (Sigma-Aldrich) containing 0.1% of Triton X-100 (Sigma-Aldrich) as well as protease and phosphatase inhibitors (Roche). Following 20 min incubation on ice, cell lysates were centrifuged at 21,000 g for 20 min at 4 °C. The resultant supernatants were collected and stored at − 80 °C. Extracted proteins (70 or 100 μg) were then separated on a 10% or 12% handmade SDS-PAGE or precast Bolt Bis–Tris gels (Invitrogen) and transferred onto a PVDF membrane (Cytiva) [For determining proteasome inhibition, cells were incubated with 10 µM MG132 (Sigma-Aldrich) for 22 h, followed by another 2 h incubation with 10 µM MYCi975 or DMSO. The 2 h incubation period with MYCi975 was chosen based on the results shown in Suppl Fig. 1, where degradation of survivin was clearly found after this time. For determining protein half-life, cells were treated with 10 µM MYCi975 or DMSO for 6 h and then incubated with cycloheximide (100 µg/ml). Cells were then harvested at the indicated time points and the relative levels of MYC were determined by western blotting.c-MYC protein was quantified by ELISA using a c-Myc  Human ELISA Kit, according to the manufacturer’s instructions . Absorbance was measured on a plate reader at 450 nm .RNA was extracted from cell lines using Trizol reagent (Sigma-Aldrich). One μg of RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Survivin primers were supplied by Sino Biological Inc. GAPDH (Sigma-Aldrich) was used as a housekeeping-gene control. The amplification process was performed with the Roche Light Cycler 480, according to the manufacturer’s instructions. Changes in gene expression were calculated using the ddCt method.All the raw data were analyzed using Microsoft Excel 2016. Graph Pad Prism 5 was used to graph the calculated data points and calculate statistical values. The significance of data were evaluated using the Student’s unpaired, two-tailed t-test. Spearman’s rank correlation was used to assess the correlation between two groups. Combination Index  was calculated using CalcuSyn software (Biosoft). CI < 1 at 50% or 90% inhibition was used to indicate synergism .p = 0.041).The effect of MYCi975 on cell proliferation was investigated using the MTT assay in a panel of 14 breast cancer cell lines, representative of the major molecular subtypes of this disease. Following 5 days incubation with increasing concentrations of MYCi975, the IC50 values for inhibition of growth varied from 2.49 to 7.73 µM  of apoptosis induced by MYCi975, we first tested its effect on 43 apoptosis-associated proteins using apoptosis antibody arrays . Figures Theoretically, the downregulation of survivin could have occurred at an mRNA or protein level. To establish the mechanism of downregulation, we performed cycloheximide chase experiments . As showAs MYCi975 appears capable of altering survivn levels, we wanted to assess if combined inhibition of MYC and survivin could produce synergistic effects. We treated cell lines with MYCI975 and the survivin inhibitor, YM-155 and evaluated the potential for enhanced interaction between the two drugs using the Chou–Talalay method . As showTo enhance the anti-proliferative effects of MYCi975, we combined it with two drugs widely used to treat breast cancer, i.e., docetaxel or doxorubicin. As shown in Fig. Similar with MYCi975, MYCMI-6 was previously reported to block the interaction between MYC and MAX and inhibit cell proliferation , 25. Of In this study, using a panel of 14 breast cell lines, we showed that the MYC inhibitor, MYCi975 reduced cell proliferation and promoted apoptosis in a cell line-dependent manner. In particular, the effect of MYCi975 on apoptosis was highly variable across the panel of cell lines investigated, ranging from effectively no induction of apoptosis in some cell lines to approximately 80% induction in others. Previously, Han et al.  reportedA potential mechanism by which MYCi975 mediated apoptosis in our work was by reducing expression of survivin. Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins which is believed to decrease apoptosis by inhibition of caspase activity, although the precise mechanism of this inhibition is unclear . By decrFurther evidence for a possible interaction between MYC and survivin was our finding of enhanced inhibition of cell proliferation when MYCi975 was combined with the survivin inhibitor, YM-155. We should state however, that our work does not exclude mechanisms other than a reduction in survivin levels by which MYCi975 might induce tumor cell apoptosis.Furthermore, although YM155 has been clearly shown to reduce survivin expression, it may possess other activities , which mPreviously, the MYC inhibitor, MYCMI-6 was also found to reduce cell proliferation in a diverse range of cancer cell lines , 24. DesAs previously reported with MYCi975 , we alsor = − 0.23, p = 0. 086).From a clinical point of view, a potentially important observation from our work was that the effects of MYCi975 on both proliferation reduction and apoptosis promotion was greater in TNBC cell lines than in non-TNBC cell lines. Furthermore, we found a significant inverse relation between cellular MYC levels and IC50 values for MYCi975, suggesting that MYC protein levels might be used as a predictive biomarker for response, assuming MYCi975 will enter clinical trials. In an earlier study, the MYCi975 analog, MYCi361 was found to exhibit a non-significant trend for an inverse relationship with MYC mRNA in the NCI60 panel of cells Below is the link to the electronic supplementary material.


Gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) form a rare and remarkably heterogeneous group of tumors. Therefore, establishing personalized therapies is eminently challenging. To achieve progress in preclinical drug development, there is an urgent need for relevant tumor models.We successfully established three gastroenteropancreatic neuroendocrine tumor (GEP-NET) cell lines  and two gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) cell lines (NT-32 and NT-38). We performed a comprehensive characterization of morphology, NET differentiation, proliferation and intracellular signaling pathways of these five cell lines and, in addition, of the NT-3 GEP-NET cell line. Additionally, we conducted panel sequencing to identify genomic alterations suitable for mutation-based targeted therapy.Death Domain Associated Protein (DAXX), sensitizing NT-18LM to the Ataxia telangiectasia and Rad3 related (ATR) inhibitor Berzosertib, and a mutation in AT-Rich Interaction Domain 1A (ARID1A), sensitizing NT-38 to the Aurora kinase A inhibitor Alisertib. Small interfering RNA-mediated knock down of DAXX in the DAXX wild type cell line NT-3 sensitized these cells to Berzosertib.We found that the GEP-NEN cell lines exhibit a stable neuroendocrine phenotype. Functional kinome profiling revealed a higher activity of serine/threonine kinases (STK) as well as protein tyrosine kinases (PTK) in the GEP-NET cell lines NT-3 and NT-18LM compared to the GEP-NEC cell lines NT-32 and NT-38. Panel sequencing revealed a mutation in The newly established GEP-NET and GEP-NEC cell lines represent comprehensive preclinical in vitro models suitable to decipher GEP-NEN biology and pathogenesis. Additionally, we present the first results of a GEP-NEN-specific mutation-based targeted therapy. These findings open up new potentialities for personalized therapies in GEP-NEN.The online version contains supplementary material available at 10.1007/s13402-022-00727-z. Neuroendocrine neoplasms (NEN) form a heterogeneous group of malignancies comprising well differentiated neuroendocrine tumors (NET) and poorly differentiated neuroendocrine carcinomas (NEC). NEN originate from neuroendocrine cells ubiquitously dispersed throughout the body, in which the gastroenteropancreatic (GEP) tract represents the major site of primary tumor development , 2. AlthDeath Domain Associated Protein (DAXX), ATP-dependent helicase ATRX, X-linked helicase II (ATRX) and Multiple endocrine neoplasia type 1 (MEN1) genes have been reported. Additionally, germline mutations have been detected in the MEN1, CDKN1B, Von Hippel–Lindau (VHL), Neurofibromin 1 (NF1) and Tuberous Sclerosis Complex 2 (TSC2) genes [Tumor protein P53 (TP53), Retinoblastoma protein 1 (RB1) and Murine sarcoma viral oncogene homolog B (BRAF) genes [Several recent genomic investigations revealed main genes being involved in GEP-NET pathogenesis. Specifically, high occurrences of somatic mutations in the 2) genes , 6. The 2) genes . In contF) genes , 9. DespUnfortunately, clinical options for systemic GEP-NEN therapy are limited. So far, mammalian target of rapamycin (mTOR) inhibitors, multi-targeted receptor tyrosine kinase inhibitors, somatostatin analogues (SSA), and peptide receptor radiotherapy (PRRT) have been approved as targeted anti proliferative therapies in GEP-NET . RegardiAberrant activation of mTOR signaling has been implicated in GEP-NEN tumorigenesis, proliferation and angiogenesis , 13. TheSSA are well-established for the treatment of SSTR-positive GEP-NET patients, achieving prolonged intervals of disease stabilization –23. AddiRegarding GEP-NEC, most patients do initially respond to first line chemotherapy. However, the response duration is limited to a few months and a high rate of treatment failure to second line options is observed . Thus, dA major challenge in developing targeted therapies in GEP-NEN is the paucity of preclinical tumor models resembling the unique genetic and phenotypic tumor features. So far, only few GEP-NEN cell lines are available. Of these BON and QGP-1 are the most widely used cell lines. While BON is commonly regarded as a GEP-NET cell line, it is currently under discussion whether QGP-1 falls under the GEP-NET or GEP-NEC classification. Although NET marker expression is present in both the BON and QGP-1 cell lines, their genomic alterations, including mutations in tumor suppressor and oncogenes , do not normally occur in GEP-NET. Additionally, both cell lines exhibit an extremely high proliferation rate, hardly resembling a GEP-NET phenotype , 28. ThuDAXX and MEN1 genes in the GEP-NET cell lines and in RB1 and TP53 in the GEP-NEC cell lines. Furthermore, we show the feasibility of a mutation-based targeted therapy. Overall, our models provide a valuable research resource for both GEP-NET and GEP-NEC.To overcome this limitation, we have generated three GEP-NET and two GEP-NEC cell lines. Panel sequencing not only identified typical genomic alterations in the Newly established GEP-NET cell lines NT-18P, NT-18LM and NT-36 were generated from a G3 pancreatic NET (PanNET) primary tumor (NT-18P), a liver metastasis (NT-18LM) and a local recurrence 12 months after initial surgery and chemotherapy treatment (NT-36) of a 57-year-old male patient. Besides the confirmed chromogranin A (CgA) and synaptophysin (SYP) expression, histopathological examination revealed Ki-67-based proliferative indexes of up to 70%. The newly established cell line NT-32 was generated from a large cell neuroendocrine carcinoma of the pancreas of a 70-year-old female patient. Histopathology revealed the presence of CgA and SYP and a Ki-67-based proliferative index of 70%. Finally, cell line NT-38 was established from a duodenal small cell neuroendocrine carcinoma of a 78-year-old female patient. Here, histopathological examination revealed expression of SYP, while CgA expression was absent. The Ki-67-based proliferative index was 70%. The use of human tissue for the generation of permanent cell cultures was approved by the local ethical review board and informed consent from the patients was obtained prior to surgery . The present study was conducted in accordance with the Declaration of Helsinki.Primary cell culture was performed as previously described . BrieflyGrowth curve analysis was performed by trypan blue staining and manual cell counting. Briefly, cells were seeded in 24-well-plates in medium supplemented with EGF (20 ng/ml) and FGF2 (10 ng/ml). Cells were counted in a Neubauer counting chamber on Days 1, 3, 7, 14 and 24 and doubling times were determined by exponential growth curve analysis.Transfection and small interfering RNA (siRNA)-mediated knockdown of DAXX expression was performed using Lipofectamine RNAiMAX Transfection Reagent  according to the manufacturer’s instructions. Briefly, 5 pmol of pre-validated, quality controlled and DAXX-specific siRNA (DAXXHSS102654 Sequence (5’ – 3’) (RNA) – GGA GUU GGA UCU CUC AGA AUU GGA U), Stealth, Thermo Fisher Scientific, Massachusetts, USA) was used for transfection. An equal concentration of scrambled RNA (scrRNA)  was used as a control.Immunofluorescence (IFC) was used to detect CgA and SYP in GEP-NET and GEP-NEC cell lines. Additionally, IFC was performed to determine the Ki67-based proliferative index. In general, GEP-NEC and GEP-NET cells were cultured on collagen IV coated glass coverslips and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). Subsequent permeabilization was performed in 0.3% Tween 20 for 5 min at RT followed by blocking in 10% BSA 0.1% Tween 20 in PBS for 1 h. Next, the coverslips were incubated with 1:100 diluted primary antibodies  in a humified chamber overnight at 4 °C. Incubation of 1:400 diluted secondary antibody  was performed for 60 min at RT. After subsequent Hoechst 33,342  incubation for 5 min at RT, the coverslips were mounted on an object slide with a drop of fluorescent mounting medium . Finally, the cells were analyzed by fluorescence microscopy using a BioRevo BZ-9000 apparatus  and the proliferative Ki67 index was determined by counting Ki67 positive cells.MTT analysis was performed to quantify viable tumor cells in culture and to determine IC50 values after treatment with Adavivint, Alisertib, Berzosertib, Olaparib and Trametinib . For dose response analyses, cells were seeded in a 96-well plate under standard culture conditions. After overnight culture, the cells were treated with a dilution series of the various inhibitors. 48 h after the initial treatment, all inhibitors were replenished and after 120 h MTT (5 mg/ml) was added to each well, followed by an incubation for 90 min at 37 °C. Subsequently, supernatants were aspirated, and precipitated formazan dye was solubilized with DMSO. Absorbance was measured at 540 nm using a Tecan infinite F50 spectrophotometer and viable tumor cells were calculated by the mean absorbance relative to DMSO control.CHGA: Hs00900370_m1, DAXX: Hs00985566_g1, ISL1: HS00158126_m1, NEUROD1: HS01922995_s1, NEUROG3: HS01875204_s1, PDX: HS00236830_m1, SSTR1: HS00265617_s1, SSTR2: HS00265624_s1, SSTR3: HS00265633_s1, SSTR5: HS00990407, Thermo Fisher Scientific, Massachusetts, USA) were used to assess gene expression signatures of neuroendocrine differentiation. Samples were analyzed as duplicates in a Step One Plus Real-Time PCR System . Using comparative Ct-analyses, gene expression values were normalized to that of the housekeeping gene GAPDH and were expressed as fold changes.Total RNA was isolated from cell lines using a Nucleospin RNA isolation kit  according to manufacturer’s instructions. The RNA quality was checked by spectrophotometric absorbance measurements (260/280 ratio). Subsequently, pure RNA was converted into cDNA by reversed transcription using a first strand cDNA synthesis kit according to manufacturer’s instructions . Pre-validated and quality controlled TaqMan® probes  using the Burrows Wheeler Aligner (BWA-mem Version 0.7.2-cegat) [Coding and flanking intronic regions were enriched using a SureSelectXT Library Prep Kit (Agilent) and a CeGaT tumor panel (Agilent), and were sequenced using an Illumina NovaSeq6000 system (CeGaT GmbH Tübingen)2-cegat) . Local r2-cegat)  to achie2-cegat) . The rem6 cells per 100 μl M-PER Mammalian Extraction Buffer containing Halt Phosphatase Inhibitor and EDTA-free Halt Protease Inhibitor Cocktail . Protein quantification was performed using a bicinchoninic acid assay according to the manufacturer´s instructions . Per array 1 µg (STK) or 5 µg (PTK) protein and 400 µM ATP were applied. Technical triplicates were analysed, and sequence-specific peptide phosphorylation was detected either in two steps  or in one step (PTK) using fluorescein–labelled antibody PY20 . Signals were recorded using a CCD camera and Evolve software . After quality control the final signal intensities were log2-transformed and used for further data analysis with BioNavigator software version 5.1 . Unless otherwise stated, data are expressed as the average signal intensity (± s. d.) of 144 (STK)/196 (PTK) peptide spots based on end levels of the phosphorylation curve. Significant differences were determined using One-way ANOVA comparisons and Dunnett’s test.To profile kinase activity a PamStation®12  was used as described previously . To analPreparation of whole cells lysates, electrophoresis and immunoblotting were performed as previously described . BrieflyP-values < 0.05 were considered as statistically significant * p-value < 0.05, ** p-value < 0.01. *** p-value = 0.001. **** p-value < 0.0001.) Data are presented as mean ± SD and 95% confidence interval of at least three replicates.Statistical analyses were conducted using GraphPad Prism 8. Shapiro–Wilk test was used to test for normal distribution. Comparisons of parametric data of two or more groups were done using two tailed unpaired t-test or one-way ANOVA, respectively. IC50 values were determined using an inhibitor vs. normalized response (variable slope) algorithm.  PDX and NEUROG3 were exclusively expressed by the well-differentiated NT-3 cell line, whereas NT-18P and NT18LM cells exhibited the highest NEUROD1 expression among all GEP-NEN cell lines tested. ISL1 expression was detected in all GEP-NET cell lines, whereas the GEP-NEC NT-32 and NT-38 cell lines hardly showed any ISL1 expression  updated GEP-NEN classification according to the grade of differentiation and Ki67 positivity. Well-differentiated GEP-NET can be subdivided into GEP-NET grade 1 (G1), grade 2 (G2) and grade 3 (G3) according to a Ki67 index (%) of < 3%, 3%—20% and > 20%, respectively. Poorly differentiated GEP-NEC exhibit a Ki67 index of > 20% and can be further subdivided into a small cell type and a large cell type. To assess the proliferative activity of our GEP-NEN cell lines, we performed immunofluorescent Ki67 staining and growth curve analyses. We found that GEP-NEC cell lines N-32 and NT-38 showed the highest Ki67 indexes with 50.40% ± 4.34% and 44.92% ± 2.65% positivity, respectively. Among the GEP-NET cell lines, NT-36 exhibited the highest Ki67 index with 27.71% ± 2.34% positivity followed by NT-18P and NT-18LM with 25.18 ± 2.35% and 25.57 ± 1.59% positivity, respectively. Consistent with a slowly proliferative G2 tumor, the well-differentiated NT-3 cell line showed the lowest Ki67 labeling index  and protein tyrosine kinase (PTK) activities in NT-3, NT-18LM, NT-32 and NT-38 cells were assessed using microarray-based functional kinomics. We observed a significantly higher number of strongly phosphorylated peptides in the GEP-NET cell lines NT-3 and NT-18LM compared to the GEP-NEC cell lines NT-32 and NT-38, i.e., the STK as well as the PTK array revealed increased overall STK and PTK activity in GEP-NET cells Fig. , B. AnalMEN1, DAXX and ATRX as the main mutated genes being apparently involved in the pathogenesis of GEP-NEN. However, further genetic modifications, especially gain of function and druggable mutations, are very rare and have not yet been elucidated. Thus, a mutation-based targeted therapy could so far not be established. Therefore, and to further characterize our established GEP-NEN cell lines, we performed sequencing of a 700 gene panel. We found that NT-3 cells harbored mutations in LRP1B (c.12010C > G), NSD1 (c.2875G > A) and PARP1 (c.826G > T). The GEP-NET cell lines NT-18P, NT-18LM and NT-36 harbored mutations in DAXX (c.1884dupC), MEN1 (c.85 > T), MSH6 (c.1095G > A), MECOM (c.1839delA), PIK3C2A (c.4694 T > A) and TCF3 (c.302A > G). Additionally, NT-36 cells exhibited a frameshift mutation in the RAD50 (c.2165dupA) gene. Among all GEP-NET cell lines tested, NT-18P carried a TP53 (c.472C > A) mutation exclusively. The GEP-NEC cell line NT-32 showed a BRAF gain of function mutation (c.1457_1471del), whereas a frameshift mutation was detected in the RB1 gene (c.1696-8_1699del) and stop codon mutation was detected in the TP53 gene (c.586C > T). NT-38 cells harbored loss of function mutations in the APC (c.4037C > A) and ARID1A (c.1681C > T) genes, and a gain of function mutation was detected in the RAF1 (c.776C > T) gene  /lymphoid enhancer-binding factor (LEF) [nM) Fig. .ARID1A wild type cell line NT-32 showed an IC50 of 9.6 µM   Fig. . AdditioµM) Fig. .DAXX mutated GEP-NET cell lines NT-18P, NT-18LM and NT-36 were exposed to the ATR inhibitor Berzosertib , the IC50 of the DAXX deficient cell line NT-18LM was 338.2 nM   to overcome replicative mortality . Interestib Fig. . We founnM) Fig. .RAF1 and BRAF was detected in NT-38 and NT-32 cells, respectively. Concordantly, administration of the MEK1/2 inhibitor Trametinib led to a significant reduction in viable cells with IC50 values of 76.2 nM  for NT-32 cells and 16.6 nM  for NT-38 cells, respectively  and 74.8% (p = 0.0001), respectively   and 68.90% (p = 0.0077), respectively   Fig. , B. Like5%) Fig. , F. In a5%) Fig. , G.Fig. Additionally, PARP cleavage was investigated to assess the effect of Berzosertib and Alisertib on apoptosis. We found that administration of 250 nM and 500 nM Berzosertib induced PARP cleavage after 24 h and 48 h, being particularly strong after 48 h. Specifically, we observed an 11.1-fold higher PARP cleavage in 500 nM Berzosertib treated NT-18LM cells compared to that in DMSO control cells Fig. . For AliDAXX wild type NET cell line NT-3. Quantitative RT-PCR revealed a siRNA-mediated reduction in DAXX expression of 30.89%  in proliferating cells   Fig. , K, L. H77) Fig. .Fig. 6ATGEP-NEN represents a minority among cancers of the digestive system and their rarity impedes the development of novel treatment regimens through large clinical studies. In addition, there is still a scarcity of reliable preclinical in vitro tumor models. Especially, to unravel molecular mechanisms underlying the unique tumor biology of GEP-NEN, well-characterized GEP-NET and GEP-NEC cell lines are urgently needed. To address this problem, we generated five novel GEP-NEN cell lines and showed that personalized molecular targeted therapies based on genetic profiling are feasible.ISL1 and NEUROD1. Additionally, these cell lines express SSTR2 and SSTR5, further indicating their neuroendocrine origin. Regarding the GEP-NEC cell lines, NT-38 cells lack expression of CgA, whereas both NT-32 and NT-38 cells express SYP. Importantly, all GEP-NEN cell lines maintained their neuroendocrine differentiation and morphology over 30 passages, rendering them into stable and continuous cell lines. Although NT-32 cells exhibit a heterogenous morphology, this morphology was consistent over the passages. Based on intra-tumoral heterogeneity, it is conceivable that some cells in culture respond differently to growth factors present in the culture medium. With a Ki67 proliferation index of 20%—30%, the newly established GEP-NET cell lines exhibit an unambiguously lower proliferation rate than that of the most commonly used PanNET cell lines BON and QGP-1. Despite the expression of SYP as a marker of neuroendocrine differentiation in both cell lines, they exhibit atypical GEP-NET mutations . Additionally, BON and QGP-1 exhibit a Ki-67-based proliferation index of > 90%, which hardly resembles the clinical nature of GEP-NET. Consequently, our cell lines represent a better model to investigate the role of GEP-NET specific mutations, thereby achieving a better understanding of GEP-NET biology. While there are additional GEP-NET cell lines  available, recent studies have shown that only GOT1 and P-STS exhibit a true neuroendocrine phenotype. A lymphoblastoid origin was postulated for the other three cell lines, further underpinning the challenging intricacy of GEP-NET cell line generation [Establishing reliable GEP-NEN cell lines has remained exceptionally difficult, mainly due to their low proliferative behavior and unresolved issues regarding optimal culture conditions. We successfully generated three GEP-NET and two GEP-NEC cell lines. All GEP-NET cell lines exhibited a neuroendocrine phenotype as confirmed by the presence of CgA and SYP, and expression of the NET-specific transcription factors neration . More reneration . DUE1 an. observed a diminished adhesion and migration of PanNET cells after pharmacological inhibition of Src kinase family members [Using kinomics analyses, we found a significantly higher frequency of tyrosine-phosphorylated peptides in the GEP-NET cells NT-3 and NT-18LM compared to that in the GEP-NEC cells NT-32 and NT-38. In particular, we found that members of the SrcB subfamily  and the ABL kinase were strongly activated in the GEP-NET cells NT-3 and NT-18LM. Recently, upregulation of LCK was reported in primary and metastatic PanNET samples, while LCK expression was absent in normal islet cells . Intrigu members . Additio members . Regardi members . However members . CollectDAXX mutations. DAXX was originally identified as a Fas death receptor binding protein mediating JNK-induced apoptosis [DAXX mutated cell line NT-18LM with the ATR inhibitor Berzosertib showed a reduction in viable cells, a reduction in proliferating cells and a significant induction of apoptosis. SiRNA-mediated knockdown of DAXX sensitized the DAXX wild type cell line NT-3 to ATR inhibition. However, apoptosis was not induced by Berzosertib in siDAXX transfected NT-3 cells. It is conceivable that transfected NT-3 cells develop only a partial ALT phenotype accompanied by a lower responsiveness to Berzozertib compared to DAXX mutated tumor cells, since siRNA-mediated DAXX knockdown resulted in only a partial reduction in DAXX expression. Even though a recently published study showed that ALT positive cells do not exhibit a general sensitivity to ATR inhibitors, we were able to demonstrate a response of DAXX mutated/knockdown cells to ATR inhibition [DAXX mutation this could be clinically developed into the first personalized therapy for GEP-NET patients.Genomic alterations resulting in an aberrantly activated growth of tumor cells, have been widely used as therapeutic targets in many cancers. Whereas, GEP-NET lack a classical oncogene addiction, GEP-NEC oncogene addiction has so far not been well studied. Thus, establishing targeted treatments remains challenging. Using our newly established GEP-NEN cell lines, we identified potentially druggable genomic modifications paving the way for the design of novel treatment strategies for GEP-NET and GEP-NEC patients. Specifically, we found that NT-18P, NT-18LM and NT-36 cells harbor deactivating poptosis , 47. Howpoptosis , 49. A rpoptosis . Althougpoptosis –52. Intepoptosis , 53. Berpoptosis –56. TreaARID1A. ARID1A represents a subunit of the SWI/SNF chromatin remodeling complex which is involved in regulating the accessibility of transcription factors, DNA polymerases and DNA damage repair proteins to specific regions within the genome. As a tumor suppressor, ARID1A was already identified in various cancers as being able to modulate cellular proliferation, invasion and metastasis [. revealed synthetic lethality in ARID1A deficient colorectal cancer cells to Aurora kinase A inhibitors. Alisertib, a specific Aurora kinase A antagonist has already been found to induce a potent reduction in tumor growth in solid as well as hematologic neoplasms [. in breast and colon cancer cells [A potential druggable genomic alteration was also found in the GEP-NEC cell line NT-38. Here, we detected a loss of function mutation in tastasis . In a prtastasis . Intrigueoplasms , 60. Coner cells . UnfortuBRAF and RAF1 mutations in NT-32 and NT-38 cells, respectively. Both cell lines showed a response to the MEK inhibitor Trametinib, but not to the BRAFV600E-specific inhibitor RAF709. In GEP-NEC, the incidence of BRAF mutations is estimated to be 5%—25%. Two recent case reports of high-grade colorectal NEC showed an initial response to combined BRAF/MEK inhibition, but both experienced disease progression after a short treatment period [BRAF mutations in GEP-NEC proved to be more complex than expected. Our GEP-NEC cell lines represent an ideal tool to investigate MEK/RAF inhibitor treatment response.We also identified t period –64. HencAPC mutation accompanied by increased activation β-catenin. Treatment of NT-38 cells with the Wnt inhibitor Adavivint led to a strong reduction in cellular viability in our study. Comprehensive molecular profiling of GEP-NEC identified APC mutations, particularly in colorectal NEC, with similar frequencies to those in colorectal adenocarcinomas. Consequently, it can be assumed that the primary site of GEP-NEC development is a determinant factor in the occurrence of this specific mutation. Whether APC mutations represent potential therapeutic targets is questionable as, so far, effective Wnt inhibitors have been shown to impair healthy tissue homeostasis and regeneration [Deregulated Wnt/β-catenin signaling has been implicated in neuroendocrine tumorigenesis, and prior studies showed antitumor properties of Wnt inhibitors in GEP-NEN cells –68. Indeneration . TherefoObviously, our study has limitations. First, we presented only a small number of cell lines. Thus, given the well-known heterogeneity of GEP-NEN, these cell lines do hardly capture the full spectrum of GEP-NEN biology. Secondly, we did not study how the identified mutations affect tumor biology, therapeutic response to established therapies and tumor progression. Particularly, in vivo studies of xenografted cells will enhance our knowledge in this regard. Nevertheless, establishing continuous cell lines will maintain a crucial role in cancer research, especially for mechanistic and therapeutic studies .DAXX- and ARID1A-mutated in vivo xenograft models and investigate how ATR and Aurora kinase A inhibition reduces growth in DAXX- and ARID1A-mutated tumors on a molecular basis.Future studies will include molecular and functional genomics to identify genes and expression profiles associated with malignant progression and therapy resistance. Another great opportunity of the new cell lines is to perform unbiased drug screening aiming to identify novel treatment concepts for GEP-NEN. Additionally, we will verify the treatment efficacy of Berzosertib and Alisertib in We successfully generated three GEP-NET and two GEP-NEC cell lines proving them as a valuable resource to further investigate GEP-NET and GEP-NEC. These models allowed us to assess novel therapeutic strategies. In particular, pharmacological inhibition of ATR and Aurora kinase A in DAXX and ARID1A deficient cells may represent a promising therapeutic strategy in GEP-NEN patients.A) PTK array and (B) STK array. Upstream STK kinase analysis of pooled NT-32 + NT-38 vs NT-3 (C) or NT-18LM (D)  > 0: higher kinase activity in NT-3 or NT-18LM; specificity score (log2) > 1.3; white to red bars: statistically significant changes. (DOCX 395 kb)Supplementary Figure 1 Box plots summarizing the log2-transformed signal intensities of (Below is the link to the electronic supplementary material.