In male dogs, uroepithelial cancers include invasive urothelial carcinoma (iUC) and prostate carcinoma (PCA). The inability to distinguish iUC involving the prostate from PCA results in indiscriminate clinical management strategies that could be suboptimal as first-line chemotherapy for iUC (cisplatin) and PCA (docetaxel) differ in people. Prostate specific membrane antigen (PSMA) is a transmembrane protein, and its overexpression has been identified in human prostate carcinoma and neovasculature associated with solid tumor growth. This study investigates whether differential PSMA expression exists between presumptive canine iUC and PCA among cell lines and archived patient samples, which might allow for improved accuracy in disease-based stratification and optimal chemotherapy selection. Additionally, in vitro sensitivities of reported canine iUC and PCA cell lines to uroepithelial directed chemotherapeutic agents were characterized.Normalized PSMA gene and protein expressions were not significantly different between 5 iUC and 4 PCA cell lines. PSMA protein expression was uniformly observed in uroepithelial cancers regardless of anatomic origin from archived patient samples, further confirming that PSMA cannot differentiate iUC from PCA. In vitro sensitivity of cell lines to uroepithelial directed chemotherapeutics revealed that vinblastine exerted the broadest cytotoxic activity.Differential expression of PSMA was not identified between canine iUC and PCA cell lines or archived patient samples, and PSMA alone cannot be used for disease stratification. Nonetheless given its conserved overexpression, PSMA may be a targetable surface marker for both canine iUC and PCA. Lastly, in uroepithelial carcinomas, vinblastine might exert the broadest anticancer activity regardless of cellular origin.The online version contains supplementary material available at 10.1186/s12917-022-03544-6. Pet dogs with uroepithelial carcinomas such as invasive urothelial carcinoma (iUC) or prostate carcinoma (PCA) commonly present with lower urinary signs including hematuria, dysuria, and stranguria. Although uroepithelial carcinomas can metastasize, locoregional disease progression resulting in urinary obstruction is expected to become life-limiting in the majority of pet dogs. Although surgical treatments and radiation therapy have been explored for managing localized uroepithelial cancer in dogs –5, medicProstate-specific membrane antigen (PSMA) is a transmembrane protein that has been shown to be significantly overexpressed in human prostate carcinoma and to a lesser extent in tumor neovasculature of various solid tumor histologies , 14. BasWhile less studied in veterinary medicine, canine PSMA protein characterization and cross-species reactivity of antibodies to canine tissues have been reported –24. In tGiven the divergent first-line chemotherapeutic options in managing human uroepithelial cancers as well as the ability to specifically target PCA using PSMA-directed theragnostics in humans, we sought to determine if PSMA could differentiate between canine iUC and PCA using heavily referenced cell lines and archived patient tissues. Overarching goals of this investigation were to direct the practice of veterinary oncology towards a more customized chemotherapeutic selection process, as well as provide foundational data to justify future exploratory studies evaluating PSMA-targeted approaches for canine uroepithelial cancers and other solid tumors.2 atmosphere).Human PCA cell lines (LNCaP and PC-3) utilized as controls were purchased from ATCC . Canine iUC cell lines  were generously provided by Deborah Knapp (Purdue University). Canine PCA cell lines  were generously provided by Tom Rosol (Ohio University) and CPA was generous provided by Monique Doré . Cell lines were cultured in Dulbecco’s modified eagle medium ) supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Lonza 17-602E) and were grown in standard conditions  and the resultant cDNA was diluted 1:10 in sterile water to a final volume of 200 μL. 5 μL of cDNA was used as a template in a 25 μL PCR also containing 1.25 μL canine specific TaqMan PSMA primer (Cf02738537_g1) or GAPDH primer (Cf04419463_gH) and 1X TaqMan Fast Advanced Master Mix . All quantitative PCR reactions were performed in a 96-well reaction plate , performed in two biologic replicates, and completed using the Applied Biosystems 7500 Real-time PCR Thermal cycler .RNA was isolated using a commercially available kit . The concentration and purity of RNA  was determined using a NanoDrop1000 spectrophotometer . 1 μg/mL and membranes were incubated overnight at 4 °C, followed by secondary labelling using species a specific HRP-linked antibody  for 1 hour at room temperature. Membranes were then developed using SuperSignal West Femto Maximum Sensitivity Substrate  and ChemiDoc XRS+ molecular imager system. All membranes were re-probed for β-actin  for use as loading control.Cell pellets were lysed on ice in Mammalian Protein Extraction Reagent  containing Halt Protease Inhibitor Cocktail . Lysates were quantified using the Pierce BCA Protein Assay kit . 10 μg of LNCaP and 50 μg other PCA cell line samples were then denatured (95 °C for 5 minutes), separated by SDS-PAGE (4–20%), and transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat milk, dissolved in tris-buffered saline-tween 20 (TBST), for 1 hour at room temperature prior to primary antibody incubation. PSMA protein expression was evaluated using a mouse monoclonal, anti-human PSMA antibody  with demonstrated reactivity to canine PSMA [Cell lines were fixed in 10% formalin for 1 hour at room temperature. After 1-hour incubation, cells were washed and pelleted in 2% agarose, then paraffin-embedded. The University of Illinois Veterinary Diagnostic Laboratory database was reviewed for cases of iUC among female dogs (for undisputed iUC confirmation) and carcinomas of the prostate. In total, 12 iUC cases and 10 carcinomas of the prostate were identified to be recut for PSMA immunohistochemistry (IHC). IHC staining was performed using an autostainer . Samples were deparaffinize in xylene and rehydrated in alcohol. Samples were treated with Diva Decloaker and then blocked with Peroxidazed 1 for 5 minutes. A secondary block with Background Punisher was performed for 10 minutes. Samples were incubated with a mouse monoclonal, anti-human PSMA antibody  at 1:1000 dilution for 30 minutes. Samples were washed, incubated with a biotinylated secondary antibody for 30 minutes, washed, then incubated with the chromogen IP FLX DAB. Slides were counterstained with hematoxylin. Well characterized human cell lines served as strong positive (LNCaP) and negative/weak positive (PC-3) PSMA controls.Cell lines were seeded in 96-well plates, at a concentration of 2000–4000 cells per well (optimized to achieve 100% confluency at 72 hours in untreated/control wells), and allowed to adhere overnight (12–16 hours). Medical grade carboplatin, mitoxantrone, and vinblastine were used in these studies. Docetaxel was purchased  and solubilized in DMSO. Treatments ensued for 72 hours with each chemotherapeutic prior to completing SRB assays as previously described . Severaln = 12) were generated with 2 parameter  or 3 parameter (n = 10) fitting. Given that normality was not met using the Shapiro-Wilk test, Wilcoxon rank-sum testing was utilized to determine if IC50 differences existed between iUC and PCA cell lines following 72 hour exposure to each chemotherapeutic agent. RStudio  was used for statistical testing, and significance was defined as p < 0.05.The half maximal inhibitory concentration (IC50) for each cell line histology classification (iUC versus PCA) following exposure to each chemotherapeutic agent  was extracted using AAT Bioquest IC50 calculator  4 parameTo evaluate whether PSMA may be differentially expressed among canine uroepithelial carcinomas, comparative mRNA and protein expression studies were performed across heavily referenced canine iUC  and PCA Next, PSMA expression was characterized in archived iUC samples from female dogs, and carcinomas of the prostate from male dogs. PSMA expression was evident among all patient tissue samples and PSMA staining localized to tumor cells, further confirming PSMA could not solely stratify iUC and PCA in naturally occurring tumor specimens Table . Intrigup-values calculated for each chemotherapeutic agent were carboplatin ; docetaxel ; mitoxantrone ; and vinblastine   as well as docetaxel, the first-line chemotherapeutic for human PCA, were evaluated in cell culture. Figure Although surgery can be utilized in select cases, medical management (NSAIDs and chemotherapy) alone or in combination with radiotherapy will continue to play an important role in managing uroepithelial carcinomas in dogs. Uroepithelial carcinomas in male dogs can be difficult to discern, and first-line chemotherapeutic selection may be suboptimal as iUC and PCA are treated differently in humans. This study sought to evaluate PSMA expression among a panel of canine uroepithelial cell lines and archived patient samples to ascertain whether differential expression exists to help distinguish PCA from iUC involving the prostate. The data generated revealed that PSMA is variably expressed among canine iUC and suspected PCA samples, indicating that PSMA expression cannot be used as a sole distinguishing marker for disease stratification.While there has been longstanding and recognized value of PSMA for the diagnosis and therapeutic management of PCA in people, it has been debatable if PSMA is uniformly and overexpressed in urothelial carcinomas of the bladder in human beings . HoweverThe findings from the current investigation also highlights the potential utility of pet dogs with naturally occurring cancers to serve as a pre-clinical model for PSMA-targeting strategies that can aid disease diagnosis and selective payload delivery of anticancer drugs, small molecule inhibitors, or radionuclides. While limited in number, there have been recent scientific investigations demonstrating the potential value of PSMA expressions in canine hemangiosarcoma (cHSA), a highly vascular solid tumor expected to express PSMA. First, the prognostic value of three immunohistochemical markers, being claudin-5, Ki67, and PSMA were evaluated in 53 splenic cHSA samples with linked clinical outcome data. From this investigation, low PSMA gene and protein expressions correlated with longer survival times . Second,Given the distinction in first-line chemotherapies for treatment of uroepithelial carcinomas in human beings, another aim of the current investigation was to characterize in vitro sensitivity to conventional uroepithelial directed chemotherapeutic agents, hypothesizing that iUC and PCA cell lines would have divergent sensitivities that could help clinicians select top choice chemotherapeutics if PCA could be differentiated from iUC involving the prostate. Interestingly, no obvious contrasting sensitivities of iUC and PCA cell lines to mitoxantrone, carboplatin, vinblastine, or docetaxel were identified. Of particular note, PCA cell lines did not appear to be more sensitive to docetaxel compared to iUC cell lines. These cell culture findings are not incongruent with recently described activity of docetaxel in dogs with epithelial malignancies . In thisAcknowledging the recognized limitations and predictive value of in vitro assays, and guided by the in vitro chemosensitivity data generated in this study, when in doubt clinically, clinicians should consider commonly utilized vinblastine or mitoxantrone as broadly active agents for either iUC or PCA, based upon their capacity to induce cytotoxicity at biologically achievable concentrations across the cell lines tested. While docetaxel also induced cytotoxicity at biologically achievable concentrations in vitro, additional clinical investigations of docetaxel for the treatment of canine urothelial carcinomas are recommended before it may be considered as a first-line therapy in dogs. Carboplatin may also be considered in dogs with newly diagnosed urothelial carcinomas given that it has proven clinical activity as a first-line chemotherapeutic in dogs with urothelial carcinomas . It woulIn summary, PSMA remains a highly promising biomarker for specific tumor types in human beings, and there is gradual exploration of its theragnostic value in veterinary oncology. While PSMA cannot reliably distinguish canine PCA from iUC, its broad expression by uroepithelial carcinomas keeps the door open for future research studies related to PSMA-targeting strategies that include molecular imaging and small molecule inhibitors. Additionally, given no obvious contrasting chemosensitivity profiles between canine PCA and iUC cell lines to common chemotherapeutics targeting uroepithelial cancers, until larger evidence-based clinical trials can be conducted to advise best therapeutic practices for particular uroepithelial cancers in pet dogs, current recommendations for optimal cytotoxin drug selection should be guided primarily by real-time assessment of patient tolerability and objective responses.Additional file 1.Additional file 2.


Sarcomas (SARC) have been found as rare and heterogeneous malignancies with poor prognosis. PTBP1, belonging to the hnRNPs family, plays an essential role in some biological functions . However, the role of PTBP1 in SARC remains unclear. In this study, the aim was to investigate the potential role of PTBP1 with a focus on SARC.  The expression, prognostic value, possible biological pathways of PTBP1, and its relationship with immune infiltration and drug sensitivity were comprehensively analyzed based on multiple databases. PTBP1 was further validated in osteosarcoma as the most prominent bone SARC. The expression of PTBP1 was investigated through IHC. The prognostic value of PTBP1 was verified in TARGET-OS databases. CRISPR/Cas9-mediated PTBP1 knockout HOS human osteosarcoma cell lines were used to assess the effect of PTBP1 on cell proliferation, migration, metastasis and cell cycle by CCK-8, Transwell migration, invasion, and FACS experiment.  PTBP1 was highly expressed and significantly correlated with poor prognosis in several cancers, especially in SARC, which was validated in the clinical cohort and osteosarcoma cell lines. The genetic alteration of PTBP1 was found most frequently in SARC. Besides, PTBP1 played a role in oncogenesis and immunity through cell cycle, TGFB, autophagy, and WNT pathways at a pan-cancer level. Knockout of PTBP1 was observed to negatively affect proliferation, migration, metastasis, and cell cycle of osteosarcoma in vitro. Furthermore, PTBP1 was significantly correlated with tumor immune infiltration, DNA methylation, TMB, and MSI in a wide variety of cancers. Moreover, the potential of the expression level of PTBP1 in predicting drug sensitivity was assessed.  PTBP1 is highly expressed and correlated with prognosis and plays a vital pathogenic role in oncogenesis and immune infiltration of various cancers, especially for SARC, which suggests that it may be a promising prognostic marker and therapeutic target in the future. Sarcomas (SARC) are rare heterogeneous malignancies with over 50 histologic subtypes. They account for approximately 1% of all malignancies and are generally classified as soft tissue sarcoma (STS) and bone sarcoma (BS) . The trePolypyrimidine Tract-Binding Protein 1 , a member of the hnRNPs family, can mediate RNA maturation, translocation, localization, and translation. Its aberrant expression has been detected in numerous diseases. The most extensive role of PTBP1 is a major inhibitory splicing factor involved in regulating alternative splicing (AS) of precursor mRNA (pre-mRNA), leading to exon skipping . PTBP1 hIn this study, the correlation between the expression of PTBP1 and prognosis, genetic alterations, potential biological pathways, immune infiltration, and drug sensitivity in human cancers was comprehensively analyzed using multiple databases, with a focus on SARC. PTBP1 was found to be upregulated, positively correlated with malignant biological behavior, and significantly correlated with poor prognosis for patients with SARC, which was validated in osteosarcoma. We also found that PTBP1 might be potentially involved in signaling pathways that regulated oncogenesis and tumor immunity, and its expression was correlated with DNA damage repair systems, DNA methylation, TMB, and MSI at a pan-cancer level. Our findings reveal that PTBP1 might be a prognostic and immune infiltration marker in numerous cancers, especially in SARC.https://xena.ucsc.edu/) and Genotype-tissue expression . Oncomine (https://www.oncomine.org) was adopted to validate the expression difference of PTBP1 in pan-cancer settings, which was a web-based data mining platform assembling 86,733 samples and 715 gene expression datasets , (ab133734), Abcam) originated from Abcam Trading Co., LTD.Human bone marrow stromal  stem cell line hBMSC and human osteosarcoma cell lines  originated from American Type Culture Collection (ATCC). Cell lines were cultured following the instructions. Cas9/sgRNA of PTBP1 was synthesized by OBiO Technology (Shanghai) Corp., Ltd. Human osteosarcoma cell line HOS was transduced with Cas9 lentiviral particles of sgRNAs. Cells were selected with Puromycin. The specific procedures followed the operation manual of OBiO Technology (Shanghai) Corp., Ltd. RT-PCR and Western blot assay were performed to confirm the knockout efficiency of Cas9 virus. Specific primer sequences included PTBP1 , synthesized by Tsingke Biotechnology Co., Ltd., Beijing, China. Cell proliferation, migration, and invasion between HOS and HOS-PTBP1-knockout cell lines were detected by CCK-8 (Cell Counting Kit-8), Transwell migration, and invasion assay. Cell cycle distribution was analyzed by fluorescence-activated cell sorting (FACS) experiment. All experiments were performed as triplicates and repeated at least three times.P < 0.05). Further analyses of the expression level of PTBP1 among different clinical stages of cancers showed that PTBP1 was correlated with advanced clinical stage in ACC, ESCA, and KICH, opposite trend in KIRC, MESO, and SKCM   . The aboThe cBioPortal website was used to explore the genetic alteration frequency of PTBP1 among different cancers , and PTBConsidering the prevalence of genetic alteration of PTPBP1 in SARC, SARC patients were assigned to high- and low-expression groups according to the level of the expression of PTBP1. The top 20 most frequently altered genes in each group were presented by waterfall plots Figures . TP53 alAccordingly, we hypothesize that PTBP1 mutational events occur in multiple tumors, particularly in SARC and may play critical roles in modulating cell cycle, tumorigenesis, and immunity.KEGG and HALLMARK enrichment analysis was conducted to explore the biological function of PTBP1. We found that cell cycle-related pathways , MYC targets, IL-17 signaling, and spliceosome pathway were most enriched in PTBP1 high-expression group , while sWe further explored the correlation between the expression of PTBP1 and well-recognized tumor-related pathways . Since TGFB is an evolutionarily conserved signaling pathway regulating various cellular processes  , the corAutophagy plays an opposing environment-dependent role in cancer, and interventions to regulate autophagy have been proposed as an option for cancer treatment . As depiThe similar results reemerged when we turned to WNT signaling pathway-related genes, which are known to control myriad biological phenomena throughout the development and adult life of all animals  Supplem.To sum up, PTBP1 upregulates cell cycle-related signaling pathways in SARC and pan-cancer and is closely and positively correlated with TGFB, autophagy, and WNT signaling pathways at the pan-cancer level.Tumor immune cell infiltration is an independent predictor of the tumor immune status and survival . To furtImmune checkpoint plays an essential role in cancer immunotherapy  using checkpoint inhibitors . As depiP < 0.05)  .Roozendaal and Mebius  reportedSubsequently, we utilized commonly recognized immune signatures to quantify the level of immune cell infiltration and analyzed its correlation with the expression of PTBP1 and found that the expression of PTBP1 has different correlations with immune cells in pan-cancer. It is consistent that PTBP1 is positively correlated with resting CD4 memory T cells, resting NK cells, and M0 macrophages, but negatively correlated with CD8 T cells, plasma, activated NK cells, and memory B cells .In addition, immunotherapy has significantly improved patient outcomes, but the occurrence of T-cell exhaustion seriously affects the therapeutic efficacy of immunotherapy . AccordiDespite the promising results of Chimeric Antigen Receptor (CAR) T Cell therapy in treating B-cell lymphoma and acute lymphoblastic leukemia , the cliP < 0.05).In SARC, deep deletion of PTBP1 reduced infiltration of CD8+ T cell and neutrophil and arm-level deletion of PTBP1 significantly reduced the infiltration of CD4+ T cell and macrophage . The anaP < 0.05).As a new pillar of modern cancer treatment, immunotherapy has revolutionized cancer management. TMB and MSI were taken as established immunotherapeutic response prediction in multiple tumor types . TMB repMSI is a consequence of DNA MMR deficiency . MMR genDNA methylation is a form of chemical modification of DNA capable of changing gene expression without altering the DNA sequence, which can be detected early, frequently, consistently, and in specific cell types in cancer . Our cirAn overview of the above findings demonstrated that PTBP1 could upregulate TMB, MSI, and MMR in various cancers and mediate DNA methylation in pan-cancer and SARC.Drug resistance remains a major factor limiting outcomes for cancer patients. Early chemo-therapeutics can achieve initial successes, whereas drug resistance may quickly develop, leading to disease relapse . To findOsteosarcoma was the most common bone SARC, the most common primary bone malignancy in pediatric population. We divided osteosarcoma patients from TARGET database into PTBP1 high- and low-expression group . Kaplan-Meier analysis showed that PTBP1 correlated with poor prognosis of osteosarcoma . ConsistTo further investigate the effect of the expression level of PTBP1 on tumor cell function in osteosarcoma, we constructed a PTBP1 knockout HOS (HOS-PTBP1-KO) cell line by CRISPR/Cas9 system. The mRNA expression  and protBesides, given the significant correlation between the expression of PTBP1 and cell cycle pathway, FACS experiment was used to assess the cell cycle of HOS cell line with or without PTBP1 knockout , LUAD, BLCA, and KIRC –45, whicGenetic alteration accumulation is considered to drive the progression of normal cells to invasive cancer and metastatic disease, through hyperplastic and dysplastic stages . The genα pathway [In-depth mechanism research of PTBP1 revealed that PTBP1 was significantly correlated with the regulation of cell cycle, E2F targets, G2M checkpoint, MYC targets, DNA replication, ribosome, and spliceosome pathways in pan-cancer, suggesting that PTBP1 may play an important role in maintaining tumor cell viability to promote tumor proliferation. Similar results were found in SARC alone. Suckale et al.  demonstr pathway . The aboβ-catenin signaling by downregulating the expression of PTBP1. PTBP1 was also reported to regulate the phosphatase and tensin homolog-phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PTEN-PI3K/Akt) pathway and autophagy, thus inducing the proliferation, invasion, and metastasis of breast cancer cells [We conducted correlation analyses between the expression level of PTBP1 and genes relating to the most popular pathways  and found that PTBP1 was positively correlated with almost all the genes of TGFB, autophagy, and WNT pathways in pan-cancer. Sheng et al.  reporteder cells . HoweverDespite the remarkable success of immunotherapy in several types of solid cancers  , the cliMSI and TMB have been considered established biomarkers to assess the response to ICPIs in multiple tumor types . We founDrug resistance refers to an impairment to cancer therapy, and developing novel drugs against drug resistance is time-consuming and challenging. Drug repurposing is more economical based on existing research data. The process of repurposing can provide novel insights into disease pathogenesis and discover new opportunities for pharmaceutical interventions . Since PThe further validation in osteosarcoma datasets of TARGET database suggested that PTBP1 is correlated with poor prognosis of osteosarcoma. Osteosarcoma tissue IHC staining and osteosarcoma cell line western blot analyses proved the overexpressed PTBP1 in tumor compared with normal tissues and cell line. PTBP1 knockout was also performed in HOS cell line by CRISPR/Cas9 system. CCK8, Transwell migration, invasion, and FACS experiments revealed that knockout PTBP1 could inhibit the cell proliferation, migration, and invasion and facilitate G0/G1 phrase accumulation of osteosarcoma.However, there are some deficiencies of this study. TCGA-SARC dataset is mainly composed of soft tissue sarcoma, but there are differences between osteosarcoma and soft tissue sarcoma in pathophysiology and clinical features. In addition, the TARGET-OS dataset lacks multiomics data, so the dataset cannot be analyzed in depth.This study revealed the prognostic value of PTBP1 in SARC and PTBP1 can promote cell proliferation, migration, invasion, and cell cycle in vitro experiments. Besides, PTBP1 was closely involved in cell cycle, TGFB, autophagy, and WNT pathways at a pan-cancer level. The high expression of PTBP1 is correlated with worse prognosis by mediating immune infiltration of cancer cells. The above findings may bring novel perspectives to the treatment of tumor patients.


Traditional treatment options are not always effective. During thepromising minimally invasive electroporation-based therapies, biological cellmembranes are exposed to an external, sufficiently high, pulsed electric fieldwhich creates so-called nanopores into the lipid bilayer of the cell membrane.These pores can either be permanent (irreversible electroporation (IRE)),leading to apoptosis, or repairable (reversible electroporation (RE)), withcontinued cell function. In tumor therapy, RE is used to increase the diffusionof a chemotherapeutic drug during electrochemotherapy. For both IRE and RE, thesuccess of the treatment is dependent on application of the appropriate electricfield. Therefore, this study aims to define the pulse parameters and thresholdsfor IRE and RE on hepatocellular carcinoma (HepG2) cellsin-vitro setup, HepG2 cell viability , and the peak temperature were measured after electroporation withthe different IRE and RE pulsing protocols, to determine the most successfulsettings for IRE and RE. A CAM/PI flow cytometric assay was performed to confirmcell permeabilization for the RE pulsing protocols with the highest cellviability.In a custom-made µs pulse length, and 100 ms interval) with an electricfield strength of 4000 V/cm was needed as threshold for almost complete celldeath of HepG2 cells. A RE pulsing protocol  with an electric field strength of 1000 V/cmwas needed as threshold for viable and permeabilized HepG2 cells. The low peaktemperatures  within this study indicatedthat the reduction in HepG2 cell viability was caused by the applied electricfield and was not a result of Joule heating.The results indicated that an IRE pulsing protocol (70 pulses, 100 Although preferredas method, resection of the primary tumor and its metastasis turned out to be onlysuitable for 25% of the patients in our department.5 This emphasizes the necessityof new treatment methods.Hepatocellular carcinoma (>80% of primary liver cancers) is a leading cause ofcancer-related death in many parts of the world.6 The ablationmethod can be either thermal (such as radiofrequency ablation (RFA) and microwaveablation (MWA), most commonly used for liver metastases) or nonthermal (such aselectroporation (EP)-based therapies). Nonthermal EP-based therapies seem the mostpromising, mainly due to their possibility to induce apoptosis instead of necrosis,which can lead to a superior immune effect, and their avoidance of thermal sideeffects.7The latter makes it possible to operate in proximity to vulnerable structures suchas blood vessels, nerves, and ducts.9 In EP-based therapies,biological cell membranes are exposed to an external, sufficiently high, pulsedelectric field (PEF), which can lead to a rapid and large increase in electricconductivity and permeability, creating so-called nanopores into the lipid bilayerof the cell membrane.10 When these PEF are applied to cells, two differentphenomena are observed: irreversible electroporation (IRE) or reversibleelectroporation (RE).10In this era, minimally invasive interventional cancer therapies are a popular area ofresearch. These techniques limit the size of incisions needed resulting in lesspain, decreased risk of infection, and fastened recovery of the patient.811 At present, there are notmany IRE devices on the market, so the classic NanoKnife® System, which used to be the only approved system for IREtreatment in Europe for years, remains widely used.12 It can be used to treat avariety of soft tissue tumors. Several applicator designs have been developed, butthe system combined with straight monopolar needles remains the mostpopular.13 Hence, the need for further research and development, toimprove this promising treatment.During the clinical treatment modality IRE, the cell membrane cannot repair theinduced nanopores because of their size and amount, which causes the cell to undergoapoptosis, the natural cell death.15 Most antitumor drugs are (nearly) nonpermeant, due to theirphysico-chemical properties and lack of membrane transport mechanisms, which limitstheir antitumor effectiveness.16 ECT is an established therapyoption in the clinic for the treatment of (sub)cutaneous tumors.18 The treatmentprotocol was standardized in the framework of the European Standard OperatingProcedure on ECT (ESOPE), which also introduced the widely used Cliniporator™  to perform ECT in clinical practice.22 More research needs to bedone, to extend the application of ECT to deep-seated tumors like livercancer.10During RE, the cells can repair their phospholipid bilayer and continue with theirnormal cell functions. In tumor therapy, those hydrophilic pores are used toincrease the diffusion of a chemotherapeutic drug which is known aselectrochemotherapy (ECT).523 In both IRE and ECT, thesuccess of the treatment is dependent on application of the appropriate electricfield. The electric field can be affected by the following parameters:tissue-specific electrical properties, geometry and positions of electrodes, andelectric pulse parameters.25Working closely with radiologists, our department focusses on developing an improvedelectroporation probe, which enables the combination of IRE with ECT treatment forliver cancer.in-vitro.Therefore, this study aims to define the pulse parameters and thresholds for IRE andRE on hepatocellular carcinoma (HepG2) cells in-vitro experiments was performed to define the pulseparameters and thresholds for IRE and RE.A translational study with several HepG2 in-vitro experiments. The cells were subcultured bytrypsinization twice a week (1:5 ratio) in Eagle's minimum essential medium(EMEM)  supplemented with 10% fetal bovine serum (FBS) (100µL/mL) and 1% penicillin/streptomycin (100 U/mL and 100µg/mL). The cells were grown in a humidified incubator at37°C with 5% CO2.The human Caucasian hepatocellular carcinoma cell line HepG2 was used for allin-vitro, using acustom-made setup designed in our department. The setup consisted of aphotopolymer spacer (RGD720) with a thickness of 0.2 cm (serving as 0.7 mL cellsuspension reservoir), placed between two block electrodes (1.4571 stainlesssteel) and PVC isolators, powered by the ECM 830 Square Wave ElectroporationSystem   . This seFirst, the cell viability and heat production were measured after electroporationon HepG2 cells with different pulsing protocols, to determine the mostsuccessful pulse parameters and threshold for IRE and RE. In addition, cellpermeabilization was examined to confirm pore formation for the selected REpulsing protocols with the highest cell viability.2 1 mM). 500 µL cell suspension wasadded to the spacer in the in-vitro electroporationsetup.HepG2 cells were harvested, and a suspension of 1 million cells/mL wasprepared in HEPES electroporation buffer , 1000, 2000, 3000, and4000 V/cm.For the IRE experiments, the following fixed parameters were used: 70 pulses,100 µs pulse length, 1000 ms interval. The electricfield strength was varied between 0 (control), 500, 1000, 2000, and 4000V/cm.For the RE experiments, the following fixed parameters were used: 8 pulses,100 The number of dead and living cells, as well as the viability of the HepG2was measured at 0, 5, 10, and 15 min after application of the differentpulsing protocols. This was done using a 0.4% Trypan Blue  dye exclusion assay (1:1 ratio) on a LUNA-FL Dual Fluorescence CellCounter .p-value of<0.05 was set for statistical significance (*). For both IRE and RE, theexperimental test series consisted of nine independent experiments, measuredin duplicates (n = 9). The sample size was based onpreliminary data.The collected data were exported to a spreadsheet for further analysis.Control groups were set to 100% viability. Accordingly, the cell viabilityat 0, 5, 10, and 15 min after application of the different pulsing protocolswas normalized to the corresponding control group and visualized usingGraphPad Prism 5 . Results were displayedin graphs as mean ± standard deviation (SD). A two-way ANOVA with Bonferronicorrection was performed and a two-tailed µL cellsuspension (1 million HepG2 cells/mL in HEPES buffer) was added to thespacer in the in-vitro electroporation setup. For both theIRE and RE experiments, the same corresponding fixed and variableelectroporation parameters were applied during the viability measurementsdescribed above.In accordance with the viability experiments, 500 p-value of <.05 was set for statistical significance(*). For both IRE and RE, the experimental test series consisted of sixindependent experiments (n = 6).While applying these different pulsing protocols to the cells, thetemperature in °C was closely monitored using the Testo 882 Thermal ImagingCamera . To ensure reproducibility, the thermal camerawas used in combination with the Velbon Vel-flo 9 PH-368 panhead and VelbonDV-7000 tripod , at a 30-degree angle with aε of 0.96. For each pulsing protocol, the peak temperature was determined,the data was exported to a spreadsheet for further analysis and visualizedusing GraphPad Prism 5. Results were displayed in graphs as mean ± SD. A1-way ANOVA with Bonferroni correction was performed and a two-tailedµL cell suspension (1million HepG2 cells/mL in HEPES buffer), with 40 µM PI was added to the spacer in thein-vitro electroporation setup. To confirm cellpermeabilization for two selected reversible electroporation pulsingprotocols, the same fixed electroporation parameters were applied as duringthe viability measurements described above. The electric field strength wasvaried between 0 (control), 500 and 1000 V/cm.Generally, a Calcein acetoxymethyl ester (CAM)/Propidium Iodine (PI) stainingis used as a life/death staining, but the combined staining of cells withthese two dyes enables quantification of viability and permeability. Forthis flow cytometric assay, 500  µM (CAM) and incubated for 10 min at RT. A BDLRSFortessa™ Cell Analyzer  flow cytometry devicewas used to process the samples. CAM signal was measured in the FITCchannel, and PI signal was measured in the Texas RED channel. For eachsample, 15,000 events were acquired, and the raw fluorescence data were thenquantitatively analyzed and visualized with the FlowJo™ software . The cells were characterized as viable and nonpermeabilized, viable and permeabilized , dead, and unstained debris . The test seriesconsisted of three independent experiments, measured in duplicates(n = 3).After electroporation, the cells were incubated for 5 min at room temperatureand washed with FACS-buffer . The cells weresuspended in FACS-buffer containing 0.2The HepG2 cell viability was measured at 0, 5, 10, and 15 min afterelectroporation with the different IRE and RE pulsing protocols, as previouslydescribed.P < .001). Application of 4000 V/cm resultedin less than 1% cell viability, indicating almost complete cell death . Application of 4000 V/cm caused, withapproximately 78%, the lowest percentage of cell viability . The total temperature increase was 6.9°C  to 1000 V/cm (23.5°C),did not significantly increase the temperature. The increase from 1000 to 2000(24.7°C), 2000 to 3000 (26.4°C), and 3000 to 4000 V/cm (30.1°C) didsignificantly increase the temperature (as 6.9°C .P < .01). The total temperatureincrease was 1.3°C  to 500 (22.6°C), 500 to1000 (22.7°C), and 1000 to 2000 V/cm (23.1°C) did not significantly increase thetemperature. The increase from 2000 to 4000 V/cm (23.7°C) did significantlyincrease the temperature (as 1.3°C .A CAM/PI flow cytometric assay was performed to confirm cell permeabilization forthe RE pulsing protocols with the highest cell viability .After exposure to an electric field strength of 0 V/cm (control) and 500 V/cm,respectively, 82.2% and 82.8% of the cells were characterized as viable andnonpermeabilized , whereas 17.8% and 17.2% were characterized asviable and permeabilized . After exposure to an electric fieldstrength of 1000 V/cm the majority of cells, 94.3%, were characterized as viableand permeabilized, whereas only 5.7% remained viable and nonpermeabilized. Thenumber of dead cells , and unstained debris  wasneglectable in all experimental groups.2729 Severalin vitro model systems for IRE have been used in literature,including artificial membrane systems, isolated single cells, and cellsuspensions.30 The latter is the most widely used, as it allowsobservation of a group of cells under external electrical stimulation, reflectingthe average behavior within a cell population.30 In contrast to classic EPcuvettes, a custom-made cell suspension setup, designed in our department, was used.This setup had been successfully used in previous studies, and, in order to comparethe results, the conditions were kept similar.26 Moreover, classic plasticcuvettes do not have a heat sink effect due to their low heat conductivity, whereasour setup with stainless-steel electrodes does. This is closer to reality, as theliver also has heat sink characteristics due to its vascular structure and highblood flow rate.31 To maximize the area of a homogeneous field and to minimizeeffects on the borders and corners as well as boundary effects of the electricfield, it was made sure that the surface of the electroporated zone was largecompared to the marginal zone. Also, the suspension reservoir was not completelyfilled, so the suspension was not placed on the edge of the electrodes.The human Caucasian hepatocellular carcinoma cell line HepG2 was chosen because it isa good representation of the patient population that is treated in our departmentand literature.32 Therefore, pH changes due to pulsing were minimized byusing a buffered, low-conductivity solution and stainless-steelelectrodes.33 Although used widely in EP studies, an increase in metalions present in stainless steel electrodes  has recently shownto decrease cell viability in RE studies.34 So, ion metal release fromthe electrodes during pulsing should be kept minimal.It is demonstrated that electroporation buffer composition can influence cellviability.26Standard IRE and RE pulsing protocols were used to compare the results with theliterature and increase the translatability to the clinic. Previous studies from ourdepartment also indicated that these protocols were the most effective.35 This accounts for allsamples, as pore closure time is practically independent of the field by which thepore was induced.35The Trypan Blue dye exclusion assay was used to measure the cell viability at 0, 5,10, and 15 min after electroporation with the different IRE and RE pulsingprotocols. Incubation times were kept short, to avoid any toxicity caused by TrypanBlue. Based on the biological principle, conducting viability measurements withtrypan blue after permeabilization of cells may not be optimal. However, poreclosure time is completed within tens to hundreds of nanoseconds, so should notinterfere with the viability measurements afterwards.Both the control groups for the IRE and RE electroporation experiments had a high andsteady starting viability of 91% and 90%, respectively, which indicates a healthycell culture needed for reliable results.For the IRE experiments, no significant difference was found in cell viabilitybetween the different measuring time points. Within the RE experiments a significantdifference was found, but given the close values, small standard deviation, anderror of the technique, the difference can be disregarded. This proved that thereduction in cell viability was caused by the applied electric field and could notbe attributed to external factors.18 Although the pore formationof electric stimulated membranes is not yet completely understood, it is assumedthat the electric field causes lipid molecule rearrangement on the bilayer thatsupports pore formation.36Regardless of the measuring timepoint, for both the IRE and RE experiments, thestepwise increase of electric field strength from 0 to 4000 V/cm resulted in asignificant reduction of HepG2 cell viability. These results were expected, asincreasing the electric field strength leads to the creation of more and biggernanopores, which eventually lead to cell death.26 The variationin threshold for different cell lines, is in line with literature, and can beexplained by differences in their size and membrane composition, as lipiddifferences in the outer leaflet of the cell membrane can potentially influence thelevel of permeabilisation.38 The variation in threshold among different types of cancercells might be an important factor when considering the treatment planning for IREin the clinic. Nevertheless, 637 ± 43 V/cm, a value determined by Davalos et alsolely on rat liver tissue, is widely accepted as threshold for IRE in most celltypes.39 In addition, it would be ideal if electroporation settings invitro were directly translatable to human. But based on literature (1000–2000 V/cm)and our results (2000–4000 V/cm), the IRE thresholds for cells in suspension arehigher than those measured in tissue.38 This could be explained bythe compactness and spherical shape of cells in suspension, compared to their nativemorphology in tissue. Currently, a similar IRE pulsing protocol with a maximum of1400 V/cm electric field strength (between the two needle electrodes) is used withthe NanoKnife device for the treatment of liver cancer in the clinic.For the IRE experiment, application of 4000 V/cm resulted in less than 0.5% cellviability, indicating almost complete cell death. When comparing this threshold tothe data from previous studies performed in our lab with the same setup anddifferent human pancreatic and cholangiocellular cancer cell lines, the HepG2 cellshave a similar threshold to BxPc3 cells, Panc1 cells, CCLP1 cells, and SNU1079cells, but a higher threshold than MiaPaCa2 cells (2000 V/cm).40 Therefore,the peak temperature was measured during the IRE and RE experiments, with a totaltemperature increase of 6.9°C and 1.3°C, respectively, starting at ∼23°C basetemperature. Temperature induces cell death differently; apoptosis occurs around43°C to 50°C, whereas necrosis occurs at 50°C or higher.41 This means that thelow-temperature changes within this study will not induce cell-death; thus, thisdata indicates that the reduction in HepG2 cell viability was caused by the appliedelectric field and was not a result of Joule heating.Although IRE is classified as a nonthermal ablation method, reports have shown thatJoule heating can lead to thermal damage, especially near the needles.2022For RE, induction of cell death due to the applied electric field is unwanted. Thegoal is to use a field that creates a majority of viable and permeabilized cells.Therefore, the RE pulsing protocols with the highest cell viability were chosen forfurther analysis on their pore formation. Application of a RE pulsing protocol withan electrical field strength of 0 V/cm (control) and 500 V/cm resulted in,respectively, 82.2% and 82.8% viable and nonpermeabilized cells, whereas an electricfield strength of 1000 V/cm resulted in 94.3%, viable and permeabilized cells. Thismeans that an electric field strength of 500 V/cm was not high enough to induce poreformation in the HepG2 cells, but 1000 V/cm was. This finding is also in line withthe standardized treatment protocol for (sub)cutaneous tumors in the framework ofESOPE , currently used in theclinic.µs pulselength, 100 ms interval) with an electric field strength of 4000 V/cm was needed asthreshold for almost complete cell death of HepG2 cells. A RE pulsing protocol  with an electricfield strength of 1000 V/cm was needed as threshold for viable and permeabilizedHepG2 cells. The low peak temperatures within this study indicated that thereduction in HepG2 cell viability was caused by the applied electric field and wasnot a result of Joule heating.To conclude, an IRE pulsing protocol (70 pulses, 100


MYCN amplification and 11q-deletion are important, although incomplete, markers of high-risk disease. It is therefore relevant to characterize additional alterations that can function as prognostic and/or predictive markers. Using SNP-microarrays, a group of neuroblastoma patients showing amplification of one or multiple 12q loci was identified. Two loci containing CDK4 and MDM2 were commonly co-amplified, although amplification of either locus in the absence of the other was observed. Pharmacological inhibition of CDK4/6 with ribociclib or abemaciclib decreased proliferation in a broad set of neuroblastoma cell lines, including CDK4/MDM2-amplified, whereas MDM2 inhibition by Nutlin-3a was only effective in p53wild-type cells. Combined CDK4/MDM2 targeting had an additive effect in p53wild-type cell lines, while no or negative additive effect was observed in p53mutated cells. Most 12q-amplified primary tumors were of abdominal origin, including those of intrarenal origin initially suspected of being Wilms’ tumor. An atypical metastatic pattern was also observed with low degree of bone marrow involvement, favoring other sites such as the lungs. Here we present detailed biological data of an aggressive neuroblastoma subgroup hallmarked by 12q amplification and atypical clinical presentation for which our in vitro studies indicate that CDK4 and/or MDM2 inhibition also could be beneficial.In neuroblastoma,  The clinical behavior varies greatly, ranging from spontaneous regression or maturation to aggressive tumor growth with fatal outcome despite intensive multimodal therapy. Poor prognosis is especially apparent among patients with high-risk neuroblastoma (HR-NB), with less than 50% 5-year survival3.Neuroblastoma (NB) is a pediatric cancer that affects 1 in 8000 children and, despite its relative rarity, is a leading cause of cancer-related death in childhood. It derives from immature cells of the sympathetic nervous system originating from the neural crest. The primary tumors commonly manifest in the abdomen along sympathetic ganglia or in the medullary region of the adrenal gland. Over half of these patients already have metastasis at diagnosis, the most common sites being bone marrow (70%), cortical bone (56%), regional and distant lymph nodes (31%), liver (30%), and intracranial and orbital sites (18%), while rarer locations are lung (3%) or central nervous system (0.6%)6, persistent copy number alterations (CNAs) are common, so analysis of somatic chromosomal aberrations is crucial7. Tumors with good prognosis are generally near triploid and display aneuploidy with whole chromosome gains and losses . High-risk tumors with poor prognosis are predominantly near di- or tetraploid and are associated with recurrent segmental chromosomal aberrations (SCAs), such as deletion of chromosome arm 11q or amplification of the MYCN oncogene (MNA)7. Other genomic features linked to adverse outcome include 1p-deletion, 1q-gain, distal 6q-deletion, 19q loss, and 17q-gain11. Further genomic alterations related to poor prognosis are those associated with telomere maintenance, such as aberrations in ATRX or TERT genes, as well as genetic alterations in the RAS-MAPK pathway, oncogenic mutations of ALK, or low-frequency mutations in TP5313.The clinical heterogeneity of this disease is also reflected at the molecular level, at which distinct genomic features of the tumor are critical for stratification and treatment decision. Whereas there is a relative paucity of recurrent single-nucleotide variants (SNVs)15. Apart from the MYCN locus amplification, a focal amplification of the ALK gene has been detected in 1–2% of the tumors, almost exclusively in association with MYCN amplification16. In addition to MYCN and ALK, other amplification regions have been described, although they are infrequent, including various chromosome segments such as 7q21, 11q13, and 16q2217. The oncogenic contribution of these amplicons is still unclear, emphasizing the need for further characterization of these regions. We and others have previously also reported high-grade amplification of chromosomal regions 12q13.3–14.1 and 12q15 in a small subset of tumors, sometimes in combination with gain of the chromosomal region 11q13 that includes the CCND1 gene encoding Cyclin D22. Amplification of chromosome 12 regions and over-expression of both MDM2 and CDK4 are frequently detected in lipo- and osteosarcoma, in which this amplification is associated with poor prognosis27. This has also been reported in other cancers including bladder28, breast29, brain30, and lung31 cancers and melanomas31. Among the different malignancies displaying 12q amplification, two regions mentioned before located at 12q13.3–14.1 and 12q15 are commonly co-amplified. These loci are centered on two genes with a seminal role in cancer development: cyclin-dependent kinase 4 (CDK4), a key regulator of cell cycle progression, and mouse double minute 2 (MDM2), a proto-oncogene involved in p53 regulation. However, cases in which these amplicons appear independent of each other have also been reported32.MNA is a well-defined biomarker present in 25–30% of all NB, and MNA analysis has been mandatory in clinical practice since the 1980s due to its prognostic and diagnostic valueTo further elucidate the extent, frequency, and role of the amplification of chromosomal regions in 12q in NBs, we present a detailed analysis of the genetic and clinical features of this subset of NB cases. Furthermore, through cell-based assays, we demonstrate that the pharmacological inhibition of MDM2 and CDK4 is feasible and that these inhibitors decrease proliferation. Therefore, MDM2 and/or CDK4 inhibition could provide an alternative targeted therapy in this patient group.Among genomic profiles derived from SNP microarrays of NB tumors from 436 Swedish patients, we identified nine patients with tumors displaying structural rearrangements and high-grade amplification involving chromosomal regions in the 12q chromosome arm. Eight additional patients with 12q-amplified NB tumors from Poland, Austria, Norway, and Spain as well as two 12q-amplified cell lines were included in the study. Although the genomic profiles of the individual cases differed in the number of amplified regions in chromosome 12, and in the size and junction sites of these amplicons, two loci in 12q13–14 and 12q15 were consistently co-amplified in 16 out of the 19 NB cases  of the amplified loci at 12q14.1 was located at Chr12: 58.135–58.270 Mb (defined by 73R6 and AuGJ), harboring the genes TSPAN31, CDK4, MARCH9, CYP27B1, METTL1, METTLE21B, TSFM, AVIL, and CTDSP2  was located at Chr12: 69,166–69,270 Mb (defined by AuGJ and SpHM) and contained the genes NR_073494, MDM2, and CPM   gene is located distal to MDM2 at 69.86–69.97 Mb within the 12q15 chromosomal region. The complete FRS2 gene was included among the 12q15 amplicons in the tumors of 14 cases. To summarize, co-amplification of CDK4 and MDM2 was seen in 15 cases (including the two cell lines), amplification of CDK4 in the absence of MDM2 was seen in three cases, and MDM2 amplification in the absence of CDK4 was seen in one case  was amplified in 18 out of the 19 cases and centered on the 2q14.1 waMYCN (seven cases including the two cell lines), multiple loci in 1p (one case), ALK (one case), and CCND1 (cell line LS)  indicates that high expression levels of CDK4, CYP27B1, and TSFM are associated with worse outcome, although not at a statistically significant level. Analysis of two datasets consisting of 88 and 498 NB tumors, publicly available in the R2 database, indicates that high expression of these three genes is associated with low overall survival, with the strongest association between adverse outcome and high expression levels for CDK4    did not include As shown in Table 33 . Instead, lung metastases were favored (6/10 = 60%), that is an uncommon feature reported by the international neuroblastoma risk group project to be present in 3.6% of all NB (100/2808)34 . Kaplan–Meier analysis of 270 NB cases show that patients with 12q-amplified NB has extraordinary poor outcome in comparison to patients with tumors of other genomic subgroups   in our cohort as compared to the HR-NBL1/SIOPEN study reporting bone marrow involvement in 76.7% of included cases (375/489)l NB 100/80834 (p n = 3) and whole exome sequencing  were performed in tumors with 12q amplification from eight patients. Somatic calling for the four tumors for which constitutional DNA was available for sequencing  detected an average of 18 nonsynonymous mutations , we focused on rare nonsynonymous variants in 574 genes with established connections to cancer  have recently been shown to be recurrent in non-MYCN-amplified NBs, which are also connected to adverse outcome12. The ATRX SNVs included a novel nonsense variant in exon 13  in case SpHM and a novel missense variant in exon 30  in case AuHH, which is predicted to be damaging by altering the protein function. Moreover, an uncommon hemizygous ATRX missense variant (rs200709847) was detected in 34R0 , while segmental alterations in the proximity of the TERT locus were detected in three cases: 16R0, SpAI, and in the NGP cell line. The breakpoints for the segmental alterations in the two non-MYCN-amplified tumors were located approximately 100 kb proximal to the TERT transcriptional starting point, while the corresponding breakpoint in NGP was located 1 Mb from TERT.In addition to sequence analysis, re-examination of array data showed intergenic CNAs identified by WGS analysis of three primary tumors were in complete concordance with SNP microarray profiles Fig. , includiTP53 mutations39 by Sanger sequencing or by RT-PCR. The four cell lines , SK-N-DZ, and SK-N-FI) displayed pathogenic homozygous missense mutations, rendering p53 inactive, whereas NGP has a TP53 heterozygous mutation which allows a wild-type function of p53  was used, while two highly selective inhibitors, ribociclib and abemaciclib, were used for CDK4 inhibition. MDM2 inhibition showed that five out of ten cell lines were sensitive to Nutlin-3a treatment with 50% cell viability reduction already present after 24 h, while the remaining cell lines were resistant to Nutlin-3a even after 72 h of treatment  cell lines after 24 h of treatment  cell lines, the synergy score of three cell lines , and SK-N-DZ) is close to 0, meaning that there is no difference between the single and combination treatments. These cell lines experienced a slight additive effect at specific concentrations of the combined drugs. The remaining two p53mut cell lines (SK-N-AS and SK-N-FI) presented an antagonistic additive overview synergy score having a negative score in the most synergetic area. SK-N-AS displayed a later response to CDK4/6i with abemaciclib than did the rest of the cell lines, while SK-N-FI showed no significant response to CDK4/6i or MDM2i after 72 h of treatment , which have a higher expression than that of the experimental control. Regarding the phosphorylation of RB at Ser807/811, a similar pattern was found, except for KELLY, a cell line that had no changes in the expression at all. In the case of NGP, a decrease in pRB Ser807/811 has been observed , and SK-N-AS).Downstream proteins of the CDK4/6 and MDM2 pathways were analyzed to verify the effect of the inhibitors used for each cell line at its respective IC50 value. CDK4/6 inhibition by both abemaciclib and ribociclib led to a marked decrease in RB phosphorylation levels at Ser780 in most used cell lines, except for the wt cell lines. MDM2i also produced an overexpression of PUMA in all p53wt cell lines as well as in KELLY and SK-N-DZ. We also detected that, after Nutlin-3a treatment, all cell lines (with the exception of SK-N-BE(2)) experienced an increase in p53 , also implicated as an oncogenic driver in the 12q15 locus, was amplified in 14 out of the 19 cases with 12q amplification  is a proliferative disadvantage for the cell. This could be due the risk of including less advantageous genes present in the larger amplicon or the replicative burden of carrying large amplification. Among the investigated cases with 12q-amplification, no one displayed one single, large continuous amplified segment including CDK4, MDM2 and FRS2. Instead, the tumors most often presented two distinct amplification peaks; one centered at CDK4 and another centered at MDM2 (and FRS2). The specificity of this complex, yet highly specific amplification of these genes, suggests a very strong combined tumorigenic driver effect based on cooperative function.In this study we investigated the amplification of two common regions at 12q13.3–14.1 and 12q15 detected in a subgroup of NB tumors. These two regions at chromosome 12 presented co-amplification in 15 out of 19 analyzed NB samples, and gene expression analysis showed that most of the genes in the amplicons were over-expressed compared with other NB tumors  and palbociclib (PD0332991) , while a trial of ribociclib (LEE011) has recently been completed47. MDM2 targeting agents such as HDM201 (Novartis) are being evaluated in NB , while RG7112 (Hoffman-La Roche) is in trial in adult solid cancer . It has been suggested that combinatorial therapies may increase the efficacy in patients with dysregulation of both MDM2 and CDK4. Synergy between CDK4/6 and MDM2 inhibitors has been observed in preclinical studies in liposarcoma48, melanoma,49 and breast cancer50, although contradictory results have been reported regarding the synergistic effects of a combined MDM2 and CDK4 targeting strategy in sarcoma51.The adverse outcome seen for most patients with 12q-amplified tumors , intra renal primary site and, presence of an inferior cava tumor thrombus and lung metastasis, suggesting an advanced Wilms’ tumor (WT). The deceptive morphology initially also favored the idea of this tumor. The amplifications in the mentioned regions might promote clinical features related to metastatic homing and tumor invasion that mimic WT. The co-amplification of CDK4, which is further potentiated by MDM2 as well as FRS2 amplification in some cases. This study provides new information about the possibility of using MDM2i and CDK4/6i drug combination in p53wt NBs, including tumors with MDM2 and CDK4 amplification, although the effect of MDM2 inhibition is highly dependent on the presence of functional p53 activity.In summary, we present a subgroup of NB characterized by amplification at chromosome 12q with subsequent overexpression of most genes within the amplified regions. Atypical features were observed associated with this subgroup that might correlate to a cell cycle deregulation phenotype given the persistent amplification of All experiments were performed in accordance with the approved guidelines and regulations. Tumor tissue was collected after written or verbal informed consent was obtained from parents/guardians according to ethical permits approved by the local ethics committees of Karolinska Institute and Karolinska University Hospital, the Experimental Research Ethics Committee of the University of Valencia/INCLIVA and the Spanish Society of Pediatric Hematology and Oncology, the University of Vienna, Wroclaw Medical University, and Oslo University Hospital. Genomic DNA was extracted from fresh frozen tumor or blood using the DNeasy Blood & Tissue kit  according to the manufacturer’s protocol. The patients’ clinical and genetic backgrounds are described in Table 64.The handling of specimen followed recommendations in standard protocol. Fresh material was immediately taken for biologic research and the fixed tumors were treated according to conventional pathology practice to diagnose neuroblastic tumors. Classification followed the International neuroblastoma Pathology Classification (INPC)All tumor samples in the present paper had unfavorable histology; undifferentiated, poorly differentiated or anaplastic/large cell.n = 436) were screened using Affymetrix SNP microarrays  as described earlier44. In addition, 12 NB cell lines were also screened using SNP microarray analysis: IMR32, KELLY, NB69, SH-SY5Y, SK-N-AS, SK-N-BE(2), SK-N-DZ, SK-N-FI, SK-N-SH , SH-EP , as well as the two 12q-amplified cell lines LS and NGP . There were nine patients with tumors displaying high-grade 12q-amplification in the Swedish cohort. In addition to these samples, a selection of patients from Norway (n = 1), Spain (n = 3), Austria (n = 2), and Poland (n = 2) with tumors sharing a similar 12q-amplification pattern were also included .The Swedish samples  at Clinical Genomics, SciLife Laboratories, Stockholm, Sweden for average read depths of 62, 32, and 74 for tumor samples 45R1, 59R9, and 73R6, respectively, and of 30, 36, and 36 for constitutional DNA, respectively. Mapping, realignment around indels, and variant calling were carried out using the Sentieon suite of bioinformatics tools  (Sentieon version v201808.03). The Canvas tool (version 1.38.0.1554)53 was used to call CNAs while structural variants were called using the Manta tool (version 1.1.1)54, with filtering based on germline variation, artifacts caused by problematic regions or, presence in the SweGen Variant Frequency dataset (https://swefreq.nbis.se/) or in our in-house set of normal controls. Patient specific origin of tumor-normal pairs was verified using a previously developed Python script that calculate the shared fraction from 400,000 SNPs65. In addition to WGS, whole exome sequencing (WES) was performed on NB tumors from five additional patients together with constitutional DNA from one of these (17E2) using the SureSelect All Exon V5 50 Mb kit  according to the supplier’s protocol. Thereafter, pair-end sequencing was performed on a HiSeq2500 sequencing system (Illumina) targeting an average of 50–60× raw read depth . Read trimming and mapping to the human reference genome were done using the CLC Genomics workbench .WGS was performed on from three patients with tumors displaying 12q amplification, as evaluated from SNP microarrays. DNA from tumor material and matched constitutional DNA were subjected to sequencing and bioinformatical handling as described previously66. All genomic positions are given according to the human reference genome hg19/GRCh37, which was also used for mapping both WGS and WES.Only high-quality called SNVs with a minimum of 10% variant allele frequency and a total read coverage of ten were considered for further analysis and evaluation. All synonymous variants or variants in non-coding regions were excluded except those affecting canonical splice sites. For further filtration, only variants present in 574 genes with established connections to cancer  method67 with the geometrical mean of the five endogenous controls. Fold change was calculated comparing 12q-amplified tumors (n = 5) and 12q non-amplified tumors (n = 31). Mean expression levels were calculated and compared gene-by-gene by two-tailed independent-sample t-test.Total RNA was isolated from 36 tumors and two cell lines (LS and NGP) using ToTALLY RNA (Ambion/Thermo Fisher Scientific) prior cDNA synthesizing using the High Capacity cDNA Reverse Transcription Kit . cDNA corresponding to 250–500 ng of total RNA was loaded in triplicate onto TLDA cards (Applied Biosystems) to determine the expression levels of 31 genes : the Versteeg (n = 88) data derived from GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix) and the SEQC dataset (n = 498) derived from transcriptome sequencing.Kaplan–Meier plots were generated for each assay with median expression value as cutoff. Overall survival (OS) was defined as the time between diagnosis and death of any cause at follow-up. The OS of the patients was compared with gene expression levels using a log rank test, with a TP53 mutational status was confirmed by Sanger sequencing and RT-PCR.All NB cell lines were cultured under standard practice without antibiotics and routinely tested for mycoplasma. The cell line identity was verified by SNP microarray analysis , SK-N-DZ, SK-N-AS, SK-N-FI, SK-N-SH, SH-SY5Y, NB69, LS, and NGP. The cell lines were plated, 24 h before treatment, in 96-well plates at a density of 5000–10,000 cells/well depending on the doubling time of each cell line. The cells were treated with the selective MDM2 antagonist drug Nutlin-3a  and/or the selective CDK4/6 inhibitors abemaciclib  or ribociclib . Concentrations of the tested inhibitors ranged from 20 to 10,000 nM (two-fold dilutions); DMSO was used as a control in an amount corresponding to the levels present in the highest used drug concentrations. The cell viability was measured by means of fluorescence using Presto Blue SH cell viability reagent (Thermo Fisher Scientific). Cell proliferation was monitored for 24, 48, and 72 h. The experiments were carried out in six replicates at each concentration and repeated at least three times. The concentrations that caused 50% inhibition of cell growth (IC50) were calculated using GraphPad Prism, version 8.4.3 .Ten NB cell lines with different genetic backgrounds70. The derived synergy score data can be directly interpreted as the proportion of the cellular response associated with the drug interaction. As synergy scores near 0 have limited confidence, synergy scores between − 10 and 10 are considered to indicate additive interaction, with positive and negative score values denoting synergy and antagonism, respectively: synergy scores larger than 10 are considered to indicate synergetic interaction, whereas synergy scores below − 10 are considered antagonistic interaction70.To study a possible synergy effect on cell proliferation after dual MDM2 and CDK4/6 inhibition, cells were treated for 72 h with either (a) Nutlin-3a combined with ribociclib or (b) Nutlin-3a combined with abemaciclib. The concentration ranges were from 0 to 6000 nM for ribociclib and from 0 to 2000 nM for Nutlin-3a and abemaciclib. The viability was normalized as the percentage of inhibition prior to calculating the synergy score for drug combination responses, which were calculated based on the HSA reference model using SynergyFinderSer807/811 (#702097) from Thermo Fisher Scientific; and p-RBSer780 (#9307), p21 (#2947), and KU80 (#2573) from Cell Signaling Technology . Protein levels were quantified using ImageJ software60 with normalization against KU80.Proteins from cells treated for 72 h with corresponding IC50 concentration for respective cell lines and control cells were extracted using RIPA lysis buffer supplemented with proteinase and phosphatase inhibitors. Protein amount was quantified using the Pierce BCA Protein assay kit (Thermo Fisher Scientific). The same amount of total protein sample (20 µg) was separated from each sample using SDS-PAGE and transferred onto 0.2-µm PVDF membrane. In order to optimize the number of proteins analyzed from a single membrane the blots were cut in multiple pieces according to size. Uncropped blot images are presented in Supplementary Fig. mut vs. p53wt) was analyzed using Student’s t-test. P values < 0.05 were considered statistically significant.The Kaplan–Meier estimates are based on data generated from 270 neuroblastoma cases with available clinical outcome and the analysis was performed using the IBM SPSS statistics for Windows, version 25 while the other statistical analyses were performed using GraphPad Prism, version 8.4.3 (GraphPad Software). The IC50 values and corresponding 95% confidence intervals (95% CIs) were calculated using dose–response sigmoidal curve with variable slope analysis . The difference in the IC50 values of two groups (p53Supplementary Figures.Supplementary Tables.


Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies. Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs with 80% to 100% mortality in infected piglets and results in enormous economic losses in the swine industry worldwide. PEDV was first described in the 1970s in England , which aPEDV is an enveloped, single-stranded, positive-sense RNA virus, which primarily infects and replicates in the villous enterocytes of the swine small intestine . Its infPEDV entry into host cells is mediated by the spike glycoprotein, which is considered as the main determinant of viral host and tissue tropism. It has been reported that PEDV infects multiple cell lines from different species including bat, rat, human, duck, and monkey in vitro. In addition to Huh7 cells, human embryonic kidney 293 (HEK293) cells are susceptible to infection with the PEDV vaccine strain CV777 and field isolate LNCT2 . Chen etContinuous cell lines that originate from mammalian tissues are for the production of various types of biological pharmaceuticals. Identification and characterization of different cell lines that are susceptible to PEDV are critical to understanding the entry of PEDV into cells and to facilitate the discovery of the functional receptors for PEDV, which are not determined yet . In thishttps://depmap.org/portal/download/all/, accessed on 9 August 2021). The gene expression level was presented as log2 (TMP + 1)  for each gene. Pearson’s correlation coefficient of each cell line with Huh7 transcriptome was calculated using the cor function with default parameters in R (v4.2.1) (https://www.r-project.org/).The up-to-date processed CCLE (Cancer Cell Line Encyclopedia) expression data (CCLE_expression.csv) and cell lines annotation data (sample_info.csv) were downloaded from the CCLE database , Huh7 , HepG2 (Procell), Hep3B217 , and BHK21 (Procell) were cultured in DMEM  containing 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin (Gibco). Li-7 (Procell), SNU182 , SNU387 (MeiSenCTCC), and SNU761 (MeiSenCTCC) were cultured in RPMI 1640  supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). The PEDV-SD strain was isolated and stored in our laboratory. Recombinant rPEDV-EGFP was produced by transfecting infectious cDNA clones into BHK21 cells as previously described . The primer sequences for each gene are provided in Total RNA was purified from different cell lines infected with PEDV-SD or mock using TRIzol . The reverse transcription reaction was carried out with 1 μg of total RNA using a PrimeScript first-strand cDNA synthesis kit . Quantitative real-time PCR was performed using SYBR Premix Ex Taq  and a LightCycler 96 instrument . The relative quantities of the genes were calculated using GAPDH as a reference, using the formula: 2Huh7, HepG2, Hep3B217, SNU387, SNU761, SNU182, and Li-7 cells were precultured in 12-well plates to 80% confluence and infected with rPEDV-EGFP at an MOI of 0.1. After 2 h of incubation, the supernatant containing virus was discarded. Cells were washed three times with PBS and cultured in fresh DMEM supplemented with 2% FBS. EGFP expression was observed at 48 h after rPEDV-EGFP infection by fluorescence microscope .50. The viral growth curves were generated with GraphPad Prism 9.The different type cells were cultured in 6-well plates and infected with PEDV-SD at a multiplicity of infection (MOI) of 0.1. Cells were incubated with 10-fold diluted virus supernatant with DMEM for 2 h, then washed twice with PBS, and supplemented with 1 mL DMEM with or without 5 μg/mL of trypsin. Virus supernatants of two wells for each virus were collected at 12, 24, 36, 48, 60, and 72 hpi for virus titration by TCIDDifferent cell lines including Vero, Huh7, HepG2, Hep3B217, and SNU387 were precultured in 6-well plates to 100% confluence and incubated with 10-fold dilutions of PEDV for 2 h. The cells were washed three times with PBS and cultured in DMEM supplied with 1% of ultrapure low melting point agarose , and then inverted after the medium was completely solidified. Three days post-infection, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet to visualize plaques.Cells were seeded in 6-well plates. When they reached 80% confluency, cells were infected with PEDV-SD at an MOI of 0.01. After 24 h post-infection, cells were washed with cold PBS three times before being fixed with cold methanol for 10 min, and then the cells were incubated with 5% BSA at 37 °C for 1 h prior to primary antibody  incubation for 1.5 h, followed by incubating with donkey anti-rabbit IgG (H+L) Alexa Fluor 594  for 1 h. Cell nuclei were stained with DAPI  for 5 min at room temperature. The images were captured with a fluorescence microscope .p < 0.01 unless otherwise stated. All of the downstream statistical analyses and generating plots were performed in R (v4.2.1). Pearson’s coefficient was calculated using the cor function with default parameters in R. Heatmaps were generated using ComplexHeatmap [p-values < 0.05 were considered to be significantly enriched. All enrichment plots were generated using enrichplot package. Venn diagram was generated using VennDiagram package.The three cell lines, HepG2, Hep3B217, and SNU387, as well as Huh7 were infected with PEDV at an MOI of 0.01. PEDV- and mock-infected samples were collected with two biological replicates for all four cell lines. Total RNA was extracted from porcine intestinal organoids using the miRNeasy Minikit  at 6 h post-infection. Total RNAs were sent to the OEbiotech  for sequencing including library construction. We obtained 40 million at least of 150 bp paired-end reads for each RNA sample. Clean reads were obtained from raw reads by filtering out poor quality reads and removing adapters contamination using Cutadap (v1.16), and reads with <10% quality threshold and <20 Phred score were also removed. Then, clean reads were mapped to the human reference genome (GRCh38/hg38) using HISAT2 . DifferexHeatmap  based ont-test. The significance level  was set at <0.05 (*), <0.01 (**), and <0.001 (***).All data were analyzed with R and GraphPad Prism 9  and provided as mean ± SD unless otherwise indicated. Statistical analyses were performed using an unpaired Student’s The physiological state of a cell is an approximate reflection of its transcriptome, it is a reasonable assumption that similar global gene expression can be highly correlated with cell physiological state. The Cancer Cell Line Encyclopedia (CCLE) provides large-scale mRNA-seq expression data for over 1300 cell lines. Since Huh7 is an established cell culture infection model for PEDV ,23, we fTo confirm that this above result was not unique to the GFP-expressing PEDV, HepG2, Hep3B217, and SNU387 cells were infected with the field strain PEDV-SD isolated by our group , which i6 TCID50/mL to 107 TCID50/mL | > = 1, the numbers of differentially expressed genes (DEGs) in PEDV-infected cell lines were identified compared to mock-infected cell lines. The volcano plots of the results showed the significantly differentially expressed genes in different cell lines after PEDV infection. There were 1,573 DEGs (751 upregulated and 822 downregulated), 824 DEGs (502 upregulated and 322 downregulated), 1,896 DEGs (893 upregulated and 1003 downregulated), and 1,752 DEGs (799 upregulated and 953 downregulated) in PEDV-infected Huh7, HepG2, Hep3B217, and SNU387 compared to their corresponding mock-infected cells, respectively  experiments using HepG2, Hep3B217, and SNU387 as well as the Huh7 control infected with 0.01 MOI of PEDV and evaluated the transcript landscape of these cell lines at 6 h after viral challenges. PEDV- and mock-infected samples were collected with two biological replicates for all four cell lines. The correlation of PEDV- and mock-infected cell lines were calculated based on all genes expression (FPKM). As shown in ectively . We thenectively D. KEGG pectively E.PEDV infection induces inflammatory cytokines and chemokine in Vero cells . We furtp < 0.01) in PEDV-infected-Huh7, -HepG2, -Hep3B217, and -SNU387 compared to their corresponding control, respectively. The top 15 significant enrichment pathways were shown  to evaluate the global change of transcriptome for all three cell lines as well as for the positive control Huh7 in response to PEDV infection. GSEA analysis based on the KEGG database showed that there were 51, 14, 36, and 44 pathways with significant enrichment  which provides a total of 1,040 curated human genes related to the innate immune response. Among the DEGs in PEDV-infected cell lines, there were 171, 89, 153, and 192 immune-related genes in PEDV-infected Huh7, HepG2, Hep3B217, and SNU387 compared to their mock controls, respectively. Among all innate-immune-related DEGs, most of them were upregulated  is a well-recognized proinflammatory cytokine that is critical in the innate host defense against infectious agents. There were increased pro-inflammatory cytokine  ,37 respoIn conclusion, our studies identified HepG2, Hep3B217, and SNU387 cells were highly susceptible to PEDV. PEDV could efficiently replicate in these cell lines as it does in Vero cells. Moreover, PEDV infection activated potent inflammatory cytokines and interferons in HepG2, Hep3B217, and SNU387 cells, which highly simulated its infection in the small intestine and in Vero cells. All these data above suggest that HepG2, Hep3B217, and SNU387 cell lines can be used as suitable models for PEDV studies.


Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intra-species cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling; however, the relatively low STR mutation rate in Drosophila melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TEs) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and ovarian somatic sheath Drosophila cell lines. Two independent downstream analyses of the next-generation sequencing data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research. Therefore, we used six modENCODE lines derived from various Drosophilamelanogaster developmental stages: S2R+, S2-DRSC, Kc167 (embryonic origin); ML-DmBG3-c2 ; mbn2 ; and CME W1 Cl.8+ (wing disc origin) in our analysis. Two other non-modENCODE cell lines—S2-DGRC and ovarian somatic sheath (OSS)—that are ordered frequently from the DGRC were also included.Based upon these findings, we investigated if the genome-wide distribution of these six TE families could form the basis for a reliable protocol to authenticate D. melanogaster cell lines. The developed gTED protocol was able to generate distinct TE genomic distribution signatures for all the cell lines tested. Moreover, using the gTED protocol, we were able to authenticate blinded samples from the Drosophila research community, thus validating the protocol. Moreover, the gTED signatures of up to 50 passages of S2R+ cells do not cluster in a passage-dependent manner, indicating that this protocol could be used to authenticate cell lines with up to 50 passages. Moving forward, we aim to expand the repertoire of cell lines assessed for their TE genomic distribution. We now have a protocol that can be adopted by the Drosophila research community to authenticate their cell lines and provide the necessary standards as per NIH guidelines.Here, we present data supporting the utility of a genomic TE distribution (gTED) protocol to authenticate Drosophila developmental stages: embryonic—S2R+ , S2-DRSC , Kc167 ; L3 larval CNS origin—ML-DmBG3-c2 ; larval circulatory system origin—mbn2 ; and wing disc origin—CME W1 Cl.8+  (Our protocol development included six modENCODE lines derived from various CL_Z790) . Two othDrosophila RNAi Screening Center, Harvard University  data were used as initial candidates for primer design. These six TE families are all LTR retrotransposons, which insert as full-length elements containing an identical LTR that provides a reliably known junction for PCR at each terminus of the TE .Full length consensus sequences for all six TE families from VCF file was generated using bcftools consensus (v1.9) with variable sites encoded as ambiguities.Both the 5′ and 3′ LTRs from the full-length TE consensus sequence for each family were extracted.https://github.com/primer3-org/primer3; last accessed: 11/28/2021) using the following parameters: PRIMER_LIBERAL_BASE=1; PRIMER_MAX_NS_ACCEPTED=1; PRIMER_NUM_RETURN=10; PRIMER_GC_CLAMP=1; PRIMER_DNA_CONC=25; PRIMER_SALT_MONOVALENT=50; PRIMER_MIN_TM=60; PRIMER_OPT_TM=62; PRIMER_MAX_TM=65; PRIMER_SALT_DIVALENT=2; PRIMER_DNTP_CONC=0; PRIMER_TM_FORMULA=1Primers for nested PCR were detected with primer3 (v2.5.0) . Briefly, equal volumes of the products of Reactions A and B Nest PCRs containing either the TE 5′ and 3′ flanking regions were combined and used as the template. The Index PCR was performed by using the Illumina i5 primer and the NEBNext® Multiplex Oligos to add i7 adaptor and index  . Then, tnd index . FinallyPaired end sequencing was performed on an Illumina NextSeq 500 with a 150-cycle midi-cycle kits. The first read in a pair  corresponds to flanking gDNA; the second read in a pair  corresponds to TE sequence. Raw sequencing data was submitted to SRA (SRP323476).D. melanogaster genome (version 6.30) in which the TEs were N-masked (D. melanogaster genome (version 6.30) using NCBI blastn (version 2.2.26) with the following parameters: -a 10 -e 1e-100 -F “m L” -U T -K 20000 -b 20000 -m 8. R1 reads that did not map with a uniquely best match to the genome were subsequently excluded. Simultaneously, the R1 reads were mapped to the TE consensus sequences. The initial goal was to identify any valid junction where we could explicitly identify the transition from a unique genomic context into a TE, aka a TE junction . By design, R2 reads occur inside the TE and can be used to demultiplex individual fragments by TE of origin from a multiplex PCR. To do this, R2 reads were aligned to a database of the consensus sequences used for primer design of the relevant TEs using Bowtie2 (v2.3.5.1); the corresponding R1 reads from the same fragment were then demultiplexed into TE-specific bins based on the best alignment of R2. R1 reads were then mapped with Bowtie2  to the complement and reverse-complement junction .Read datasets were analyzed in their entirety or by random sub-sampling using vsearch (v2.14.2) (https://github.com/mondegreen/DrosCellID.git (last accessed: 11/28/2021).Code and notes on running the TE detection and clustering pipeline are available at: 297, copia, mdg3, mdg1, roo, and 1731) was sufficient to replicate the clustering observed when data from all TE families was used  test the validity of this protocol by assessing the identities of blinded samples, both internal and those provided by the Drosophila community, and (3) assess if cell lines subjected to extensive passaging retain the unique cell-specific gTED signatures.Previous analysis of available WGS data revealed that gTED can reliably cluster cell lines based on their genotype and laboratory of origin  enrichment protocol described in the dmethods . Brieflydmethods . The promdg3-specific primers was very low, mdg3 was excluded from further analyses. Normalized counts of reads mapping near TE junctions for the remaining five families were then used to hierarchically cluster all the cell lines. Reads mapping close to the identified TE junctions, whether at 5′ or 3′ end or both, were included in further analyses  or generated internally at DGRC. All blinded samples, as well as triplicates of an internal control for S2R+ (DGRC_Blinded_control_1-3), were processed as outlined in the Materials andmethods section.To assess the value of the developed gTED pipeline and validate it, we next queried if the cell line-specific gTED signatures could be employed to determine the identities of blinded samples . The bliDrosophila samples submitted to DGRC by the community.Of the eight external cell lines processed from two different donating labs, six robust gTED signatures were obtained . HoweverDrosophila vs non-Drosophila cell lines, and (3) D. melanogaster cell lines previously uncharacterized by the gTED protocol can be identified as such, without providing a false identification.All three internal blinded cell lines had unique gTED signatures that clustered distinctly relative to all previously-characterized gTED signatures . NeverthExtensive passaging of cell lines can potentially alter cellular genomes . Upon geDrosophila cell lines to help researchers validate their reagents as per the NIH mandate. Our novel protocol allowed us to define unique gTED signatures that could identify each of the Drosophila cell lines that were tested here. In addition, the resolution obtained from the gTED signatures allows for distinguishing between S2 sublines. Data presented here demonstrate that the gTED signatures of the replicates of most cell lines cluster together, outlining the reproducibility of the gTED protocol while also underscoring the value of having replicate samples for reliable cell line identification. Crucially, accurate identification of blinded samples donated by the research community validated the gTED protocol in a real-world setting.The aim of this study was to develop and test a cell authentication protocol that could reliably identify the most commonly used D. melanogaster cell line using the gTED protocol, an established gTED signature is a prerequisite. Toward this end, we have now established gTED signatures for the widely distributed lines, S2R+, S2 DGRC, S2 DRSC, Kc167, and ML-DmBG3-c2 lines  cells (D. melanogaster cell lines using the gTED protocol. However, detecting low levels of inter- or intra-species contamination might be a more challenging pursuit. A D. melanogaster cell line contaminated with low levels of a mosquito cell line is unlikely to be detected with gTED, necessitating using other methods for such specific instances. A future avenue is to explore the sensitivity of the gTED protocol to intra- or inter-species contamination. In addition, it will be imperative to determine if we can determine low levels of contamination of Drosophila cell lines containing unique gTED signatures.Mosquito cell lines included as blinded samples helped clarify that the gTED protocol can discriminate non-nsposons . PresencD. melanogaster cell lines (Our analysis also demonstrated that the genomic distribution of TEs is largely unchanged over 50 passages in S2R+ cells. The narrow window into the passaging-associated genomic structure provided by the gTED protocol is most likely not representative of more complex genomic and/or transcriptomic changes that the extensively passaged cells might have undergone. Nevertheless, S2R+ cells passaged continuously for up to 50 times can still be identified with the gTED protocol. Among the S2 lines assessed in this study, it has been proposed that the S2R+ line is possibly the closest to the original Schneider line (The gTED protocol relies on TE-specific amplification from gDNA and therefore provides a cost-effective strategy as compared to performing WGS for the same purpose. In addition, post-sequencing downstream analyses can be performed more quickly because of the smaller amount of sequencing data produced by the gTED protocol. The reduced cost and computing time requirements of the gTED protocol allowed multiple triplicate sample processing, thus aiding the resolution of sample mislabeling that was detected with one of the replicates of S2 DGRC and S2 DRSC .Drosophila cell lines. We demonstrate that the developed gTED protocol can assign distinct signatures to the various Drosophila cell lines tested. Blinded and extensively passaged samples can now be authenticated employing the gTED protocol. Researchers working with Drosophila cell lines can independently authenticate cell lines being used in their laboratories using the protocol and code described in this study. Alternatively, DGRC will implement a cost-based service for the research community to access and authenticate their cell lines for both publications and research funding. Ultimately, our goal is to include more cell lines from the DGRC repository into the gTED pipeline and generate gTED signatures for all cell lines deposited with the DGRC.In summary, utilizing the genomic distribution of five TE families, we have developed the gTED pipeline to facilitate the authentication of All data necessary for confirming the conclusions in this paper are included in this article and in G3 online.jkab403_Supplementary_DataClick here for additional data file.