Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions, as exemplified by DU-145, PC-3, and LNCaP cells, are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments, however, have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions, in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice, co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity, cellular heterogeneity, and histology of original patient tumors. In contrast to PDX models, patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article, we review PCa preclinical model cell lines and their sublines, PDXs, and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine. Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer-related deaths among men in western countries . AndrogeVarious preclinical PCa models have been developed to clarify complex mechanisms underlying treatment resistance of PCa . Three PPatient-derived xenografts (PDXs) are an important preclinical cancer model for overcoming limitations associated with the use of cancer cell lines and allow investigators to obtain preclinical results that more accurately reflect clinical responses in patients. This is because PDXs grown in immunocompromised mice retain the key molecular aberrations present in patient tumors, including mutations, structural genomic events, epigenetic features, and gene expression programs, which drive their three-dimensional growth ,21. The Moreover, transition from monolayer cultures to three-dimensional cultures has increased the successful take rate of patient-derived cancer models . The useIn this review article, we will focus on the in vivo and in vitro PCa models, including original cell lines, original cell line-derived treatment-resistant sublines, PDXs, and patient-derived organoids and spheroids.Since the development of the HeLa cell line, several cancer cell lines have been developed and used for determining mechanisms underlying cancer tumorigenesis and for identifying markers of therapeutic response . Cancer The 1013L cell line was established in 1978 from a primary prostate tumor ,34. UsinE006AA cell line was established in 2004 from a PCa tissue obtained from a 50-year-old African-American (AA) patient who underwent radical retropubic prostatectomy for treating clinically localized PCa . A cultuThe RC-77T/E cell line was derived in 2010 from a radical prostatectomy specimen obtained from a 63-year-old AA patient . This paSeveral studies have reported that some cell lines that were previously believed to be derived from primary tumors are actually derived from cell lines established from metastatic tumors  or other malignant cell lines  ,109,110.The ALVA-31 cell line was initially reported to be established from a biopsy specimen of a primary tumor . HoweverThe JCA-1 cell line was derived from a poorly to moderately differentiated prostatic adenocarcinoma . Howeverpfp−/−/rag2−/− mice have very weak metastatic potential [The DU145 cell line was the first PCa cell line and was derived in 1975 from a brain metastatic prostate tumor of a 69-year-old white man ,43. The otential ,47. MetaThe PC-3 cell line was isolated in 1979 from a lumbar vertebral metastatic prostate tumor of a 62-year-old white man . The PC-6 cells with Matrigel™ is associated with a modest success rate of 50% and tumor doubling time of 86 hours. Tumor formation occurs at a faster rate and is more efficient in male hosts than in female hosts; however, the growth rate of xenograft tumors is independent of the host gender [The LNCaP cell line was established in 1980 from a needle aspiration biopsy of a lymph node metastatic lesion obtained from a 50-year-old white man . The LNCt gender . PCa subARCaP cell line was derived in 1996 from the ascites fluid of an 83-year-old white man with advanced metastatic PCa . The ARCThe MDA PCA 2a and 2b cell lines were derived in 1997 from a bone metastasis tumor of a 63-year-old AA man who had PCa showing androgen-independent growth . Althougpfp−/−/rag2−/− mice [LuCap 23 cell line series was derived in 1996 from xenograft tumors obtained from a 63-year-old white man with stage DI prostate adenocarcinoma  at autopsy . The pat−/− mice . These r−/− mice .The LAPC-4 cell line was established in 1997 from a xenograft model of a femoral PCa metastasis formed in a patient undergoing androgen ablation therapy . These cThe 22Rv1 cell line was derived in 1999 from a xenograft CWR22R isolated from a patient with bone metastasis. The CWR22R xenograft was serially propagated in mice after castration-induced regression and relapse of a parental, androgen-dependent CWR22 xenograft . In nudeThe VCaP cell line was derived in 2001 from a vertebral metastatic lesion through autopsies ,70. ThisThe KUCaP cell line was derived in 2005 from an autopsy specimen of liver metastasis obtained from a 64-year-old Japanese male patient who died because of CRPC . The KUCPC346 cell line was derived from a xenograft model PC346 derived from the transurethral resection of a primary prostate tumor . The PC3The LNCaP-abl cell line was established after 41 passages of the parental LNCaP cells in an androgen-depleted medium . The morThe LNCaP-SF cell line is an androgen-independent LNCaP subline that was established by culturing the LNCaP cells in RPMI-1640 medium supplemented with a steroid-stripped serum for a long period (more than six months) . The LNCSOCS2-AS1 is higher in the LNCaP-LTAD cells than in the parental LNCaP cells. This lncRNA promotes castration-resistant and androgen-dependent growth of the LNCaP-LTAD cells and upregulates androgen signaling in these cells by modulating the epigenetic control of AR target genes [The LNCaP-LTAD cell line was established by culturing the LNCaP cells in a phenol-red free RPMI-1640 medium supplemented with 10% charcoal-stripped FBS for more than 9 months ,78. The et genes . In thisThe C4-2 cell line was isolated in 1994 from a mouse vertebral metastasis of LNCaP xenografts . To geneThe PC346Flu1 and PC346Flu2 cell lines were derived from PC346C cells by culturing in an androgen-depleted medium supplemented with 2% charcoal-stripped FCS and 1 µM hydroxyflutamide . These fThe LNCaP-BicR cell line  was established by culturing the LNCaP cells in RPMI 1640 medium supplemented with 10% FBS and 10 μM bicalutamide for more than 3 months . BicalutThe LNCaP-BicR cell line  was established by culturing the LNCaP cells with increasing concentrations of bicalutamide (1–40 µM) for over 12 months . The LNCThe MR49F is an ENZ-resistant cell line derived by culturing cells obtained from ENZ-resistant LNCaP xenografts in RPMI-1640 medium supplemented with 5% FBS and 10 μM ENZ . The MR4R cell line series, which also shows ENZ resistance, was derived from cells obtained from ENZ-resistant LNCaP xenografts [R) was established by injecting the LNCaP cells in intact male athymic mice to produce subcutaneous tumors, followed by the castration of these mice. After tumor recurrence (CRPC), the mice were treated with vehicle or 10 mg/kg ENZ daily. Although the ENZ treatment decreased tumor growth compared with the vehicle treatment, it did not prevent tumor recurrence. The primary PSA-positive ENZR xenografts also produced nine serially transplanted tumors out of 35 (25.7%) tumors that showed no increase in PSA expression. Different cell lines have been derived from multiple transplanted ENZR tumors . The 42DENZR and 42FENZR cell lines derived from the PSA-expressing tumors show reduced expression of classic AR target genes and increased expression of canonical transcription factors and markers associated with NEPC. These ENZ-resistant cell lines may be used for examining mechanisms underlying the development of both classic AR-driven and AR-undriven PCa phenotypes, especially the transdifferentiation of ENZ-resistant CRPC to NEPC.The ENZnografts . An ENZ-(a) Paclitaxel-Resistant PCa Cell Lines50 of paclitaxel against the DU145-TxR and PC-3-TxR cells is 34.0- and 43.4-fold higher, respectively, than that against both the parental cell lines. Both the cell lines show cross-resistance to estramustine phosphate, docetaxel, vinblastine, and doxorubicin, but not to etoposide and cisplatin. Knockdown of P-glycoprotein, which is upregulated in resistant cells, by using MDR1 siRNA restores paclitaxel sensitivity of the DU145-TxR cells. Microarray analysis has shown that the C-terminal tensin-like protein  gene is downregulated by 10-fold in the PC-3-TxR cells. CTEN knockdown in the PC-3 cells induces paclitaxel resistance, and CTEN overexpression in the PC-3-TxR and DU145-TxR cells restores paclitaxel sensitivity [The DU145-TxR and PC-3-TxR are paclitaxel-resistant PCa cell lines established in 2007 by incubating the parental DU145 and PC-3 cells, respectively, with gradually increasing concentrations of paclitaxel for 2 days, followed by culturing in the absence of paclitaxel until the cells showed appropriate growth . The IC5sitivity . These csitivity ,94,95,9650 of paclitaxel against the PC3PR cells is 2577.3 nM and that against the parental PC-3 cells is 8.6 nM. MSK1 knockdown by using miR-148a decreases the paclitaxel resistance of the PC3PR cells, indicating that miR-148a attenuates paclitaxel resistance in the hormone-refractory, drug-resistant PC3PR cells partly by regulating MSK1 expression. Moreover, reduced miR-34a expression confers paclitaxel resistance to the PC3PR cells by upregulating SIRT1 and BCL2 expression [The PC3PR cell line is a paclitaxel-resistant cell line established from the PC-3 cells . The IC5pression . The PC3pression ,100.50 values. LJ-2618 treatment suppresses the activated PI3K/AKT signaling pathway in the PC-3-Pa cells. Treatment of a PC-3-Pa cell-implanted xenograft mouse model with 3 or 10mg/kg LJ-2618 effectively inhibits tumor growth by enhancing SKP2 degradation and by inducing p27 expression in the tumor tissues.The PC-3-Pa cell line was established in 2018 from the PC-3 cells and shows >100-fold higher resistance to paclitaxel than the paclitaxel-sensitive PC-3 cells . Treatme(b) Docetaxel-Resistant PCa Cell LinesThe PC-3dR cell line was derived by repeatedly exposing the PC-3 cells to docetaxel in vitro . For pro50 values against the respective parental cell lines. The IC50 of docetaxel against the PC-3 D12 cells is 10-fold higher than that against its parental cells. Treatment of the PC-3 D12 cells with BAY 11-7082 inhibitor represses docetaxel-induced increase in NF-κB activity and increases sensitivity to docetaxel. In addition, an analysis of these cells showed that ZEB1 promotes both epithelial-mesenchymal transition (EMT) and docetaxel resistance in these cells by repressing the transcription of E-cadherin [The DU145 R, 22RV1 R, and PC-3 D12 cell lines were developed by culturing their respective parental cells with gradually increasing concentrations of docetaxel over 6 months . The celcadherin .50 of docetaxel against the DU145R and PC-3R cells ranges from 10 to 15 and from 20 to 22 nM, respectively, whereas that against the DU145 and PC-3 cells ranges from 4 to 5 and from 3 to 5 nM, respectively. Results of microarray analysis have shown that 243 overlapping genes are differentially expressed in both the DU145R and PC-3R cells.The DU145R and PC-3R cell lines were developed in 2012 and show 2- to 5-times higher resistance to docetaxel than their respective parental cells . The IC5(c) Cabazitaxel-resistant PCa cell linesThe DU145CR and PC3CR cell lines were established by culturing the DU145 and PC-3 cells, respectively, with increasing concentrations of cabazitaxel (0.3–3 µM) for 2 years . The DU1The DU145-TxR/CxR and PC-3-TxR/CxR cell lines were established by culturing the DU145-TxR and PC-3-TxR cells, respectively, which are paclitaxel resistant (see above), with gradually increasing concentrations of cabazitaxel for 6 months . The DU1(d) Cisplatin-Resistant PCa Cell LinesThe LNCaP/C sublines were established by culturing the LNCaP cells with increasing concentrations of cisplatin . This reTM into nude mice [Exhaustive efforts have been made for years in terms of in vivo tumor formation experiments based on human tumor cell lines, and the usefulness of this model has been shown as exemplified by the evaluation of drug efficacy and the identification of various therapeutic targets for the disease. Nevertheless, the cell line model has a limitation in terms of the in vitro adaptation of cells to culture conditions, which sometimes leads to the discrepancy between the experimental and clinical outcomes. PDXs may more appropriately recapitulate the molecular diversity, cellular heterogeneity, and histology in patient tumors ,25. The ude mice . Recent ude mice . Toivaneude mice . Establiude mice . Althougude mice ,21. In tPfp−/−/rag2−/− mice are completely devoid of functional T- and B-lymphocytes, as well as NK cells. As NK cells are potent defenders against circulating tumor cells, pfp−/−/rag2−/− mouse strain is of particular interest in metastasis research [Flanagan et al. reported the first immunodeficient nude mouse model in 1966 . These mresearch ,169. Altresearch .The Rotterdam PCa models were derived from primary PCa specimens (prostatectomy specimens), transurethral resection specimens, and metastatic lesions  at Erasmus University ,75,121. The LuCaP PDX models were established from primary PCa tumors or PCa metastases derived from operative specimens and metastatic CRPC samples obtained by performing tissue acquisition necropsy at the University of Washington ,139,140.The LAPC PDX models were first reported in 1997 by the UCLA School of Medicine  and wereThe BM18 PDX model was derived in 2005 by subcutaneously xenografting a metastatic PCa tissue, which was curetted from the femoral bone of an 88-year-old man who underwent intramedullary fixation for a pending fracture, into SCID mice . The BM1www.livingtumorlab.com). They report that although survival rate (1st generation xenografts) in PCa is > 95%, the success rate in establishing PCa tissue lines (≥ 5 generations) is 17% (10 of 59). LTL-311 PDX model was first established in 2008 by grafting the tissues under the kidney capsules of NOD/SCID mice. The LTL PDX models include varying types of PCa, hormone-naïve adenocarcinoma, CRPC, and NEPC cells. LTL-352 PDX model was derived from an NEPC patient specimen and expresses an NE marker but not PSA. LTL-331R-G7 PDX model was derived from an adenocarcinoma specimen and shows neuroendocrine features after host castration and passaging. These NEPC models show significant suppression of FOLH1, amplification of SSTR2, and abnormal expression of heterochromatin genes [The Living Tumor Laboratory (LTL) PDX models were established from adenocarcinoma or NEPC tissues obtained from patients at Vancouver Prostate Centre ,149,150.in genes ,150. Genin genes . The LTLin genes , CRPC drin genes , stroma-in genes , and new3 pieces and subcutaneously transplanted into male nude mice along with 50 μL MatrigelTM. The KUCaP-2 xenograft was established ∼10 months after the first inoculation. These xenografts express WT AR and PSA. Tumors derived from the KUCaP-2 PDX model regress immediately after castration but recur after 1–2 months, thus mimicking the clinical behavior of CRPC.The KUCaP-2 PDX model was derived in 2010 from trans-urethrally resected specimens of local recurrent tumors obtained from a Japanese patient by performing radical prostatectomy . The speThe MDA PCa PDX models were derived from primary or metastatic PCa specimens obtained from operative specimens available at The University of Texas MD Anderson Cancer Center ,156,157.The Monash University PDX models were derived using localized primary PCa and CRPC specimens available at the Monash University. The xenograft success rate of localized primary PCa tissues was improved using chimeric grafts with neonatal mouse mesenchyme. The use of the neonatal mesenchyme significantly increased the xenograft survival rate and doubled the proliferation index of xenografted cancer cells . The mouTM and retain their normal genome over time [TM and a cocktail of essential stem cell growth factors supplemented with a transforming growth factor-β receptor inhibitor (A83-01) and p38 MAPK inhibitor (SB202190) can be used as a growth medium for culturing healthy human intestinal or colon cells, and eventually for culturing CRC organoids [Although the use of PDX models helps in overcoming the limitations associated with the use of cell lines, the PDX models are associated with some limitations. Engraftment and drug validation in mice usually requires >6 months. This time delay limits the applicability of PDX models in real-time patient treatment. Moreover, although PDX models are suitable for examining a limited number of drug combinations, they are not amenable to high-throughput screening . Therefover time . Matrigerganoids . Thereafrganoids  and PCa rganoids . Organoirganoids ,176. Linrganoids . Althoug® Human CD45 Depletion Cocktail, followed by the depletion of red and white blood cells by using Ficoll-Paque. Next, the cells were washed, placed in ice-cold MatrigelTM, and plated in the middle of one well of a 24-well plate containing human prostate culture medium. The prostate-specific culture medium contained DMEM/F12 supplemented with GlutaMAX, penicillin, streptomycin, primocin, B27, N-acetylcysteine, mouse recombinant EGF, human recombinant FGF-10, FGF-basic, A-83-01, SB202190, nicotinamide, testosterone, PGE2, Y-27632, noggin-conditioned medium, and R-spondin-conditioned medium [The MSK-PCa model was the first reported patient-derived organoid model of PCa, developed in 2014 by Memorial Sloan Kettering Cancer Center , and wasd medium ,178. TheTM in a 1:2 volume ratio. Fresh tumor tissues from 25 patients with metastatic PCa were used for developing organoid models, with an overall success rate of 16% (4/25). The organoids models were engrafted as patient-derived organoid xenografts (PDOXs) into NOD/SCID mice and were subsequently re-passaged in vitro as organoids from PDOXs. The metastatic tumors obtained from each of the four patients and their matched organoids and PDOXs were classified as NEPC based on their tumor morphology. The organoids and PDOXs were clustered based on the shared expression of CRPC-NE signature genes, including the overexpression of MYCN, PEG10, SRRM4, EZH2, SOX2, BRN2, and FOXA2 genes, and decreased expression of AR signaling genes. These organoid models were used to confirm the effect of EZH2 inhibitor on NEPC.The ORG WCM organoid model was established in 2018 from a biopsy specimen of metastatic PCa obtained from patients with NEPC at Weill Cornell Medicine . For thi® hESC supplement, penicillin/streptomycin, R1881, FGF-basic, and Y-27632 dihydrochloride. In all, 109 spheroid models were established using tissues obtained from 173 patients. These models stayed viable for several months and were amenable to cryopreservation. Almost all the models showed AR, CK8, and AMACR positivity. The viability of these spheroid models markedly reduced upon bicalutamide and ENZ treatment. However, abiraterone treatment had no effect and docetaxel treatment only had a moderate effect on these models.The Saarland University spheroid models were generated in 2018 from radical prostatectomy specimens of organ-confined PCa . For thiIn the last several decades, quantitative analysis of molecular studies has been advanced in the field of prostate cancer research based on the establishment of PCa cell lines and their drug-resistant sublines. A small number of cell lines, however, could be established from cancer tissues with rare possibilities, and their application to preclinical studies was rather limited due to their characteristics usually being apart from the original tumors. Recent advances in culture technology and mouse engineering has improved the success rates for the establishment of patient-derived cancer platforms, including PDC organoid/spheroid and PDX models, which retain the characteristics of the original tumors and tumor heterogeneity. The generation of PDC models is currently still challenging, particularly from primary hormone-naïve PCa, nevertheless, it has preclinical relevance, as PDCs have advantages for large-scale drug screening and its application for PDX reestablishment. Considering current clinical streams towards cancer precision medicine, patient-derived cancer models are powerful and promising “avatars” of the original tumors, and these PCa platforms are favorable for the characterization of the clinical pathophysiology and the determination of the most efficient therapeutic regimen.


In the original article, there was an error. The name of parental cell line and antibiotic selection used should read “(U2OS)” and “neomycin selection with G418” respectively.Materials and Methods, Cell Culture, paragraph one:A correction has been made to the Mycoplasma contamination. Hypoxia was achieved by incubating cells in 1% O2, 5% CO2, and 94% N2 in a Ruskinn SCI-tive workstation, without agitation.”“Human U2OS osteosarcoma (U2OS-HRE-luc) cells have been described by us previously (17) and stably express a luciferase reporter construct under the control of a hypoxia response element, allowing us to monitor HIF/HRE activity alongside measuring mitochondrial endpoints. U2OS osteosarcoma (U2OS) cells were used to generate stable independent clonal cell lines (WT.cl1 and WT.cl3)-expressing CHCHD4.1 cDNA (CHCHD4 (WT)-expressing cells), CHCHD4-C66A/C668A cDNA (CHCHD4 (C66A/C68A)-expressing cells) or pcDNA3.1 (control) by neomycin selection with G418, using constructs we have described previously (9). HeLa cells were purchased from ATCC. All cell lines were maintained in Dulbecco's modified eagle medium containing glucose (4.5 g/L) (Life Technologies) and supplemented with 10% fetal calf serum , penicillin (100 IU/mL), streptomycin (100 μg/mL), and glutamine (6 mM), all purchased from Life Technologies. Cell lines used were authenticated and routinely confirmed to be negative for any The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines  in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation.50) assay, respectively.The virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID5 to 106 FFU / 106 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 104 to 105 FFU / 106 cells. DF-1 produced minimal infectious titre.The isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 10This study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture. Orthobornavirus and family Bornaviridae. There are five recognized species of avian bornavirus: Passeriform 1 orthobornavirus, Passeriform 2 orthobornavirus, Psittaciform 1 orthobornavirus, Psittaciform 2 orthobornavirus and Waterbird 1 orthobornavirus [Waterbird 1 orthobornavirus species contains two viruses, aquatic bird bornavirus 1 and 2 (ABBV-1 and ABBV-2). ABBV-1 was first identified in a retrospective post-mortem evaluation of Canada geese and trumpeter swans with neurological disease from Southern Ontario, Canada, using RT-PCR and immunohistochemistry (IHC) on archived tissues [The term avian bornavirus encompasses a diverse group of viruses within the genus  tissues . Since t tissues , 5, as w tissues , 7.ABBV-1 is an enveloped negative-sense single stranded RNA virus with a genome of approximately 9 kb, which contains six genes encoding for five structural proteins and one non-structural protein, arranged in order from the 3′ to 5′: nucleocapsid (N), non-structural X protein, phosphoprotein (P), matrix (M), glycoprotein (G), and RNA-dependent RNA polymerase (L) . ThroughThe isolation of viruses in cell culture is considered the “gold standard” for virus identification , and theThe current avian bornaviruses invitrome, which is defined as the collection of cell cultures known to support avian bornaviruses replication , is listThere were two goals of this study. The first was to describe the isolation in DEF and phylogenetic characterization of infectious ABBV-1 derived from the brain of naturally infected Canada goose from Ontario, Canada. The second was to characterize ABBV-1 replication in immortalized avian cell lines in order to identify a suitable cell line to routinely propagate the virus.Anas platyrhynchos domesticus). The three immortalized cell lines were: duck embryonic fibroblasts (CCL-141) [Four cell culture systems were used, one primary and three immortalized cell lines. The primary culture was duck embryonic fibroblasts (DEF) from Pekin duck (CCL-141)  and chicCCL-141) , both obCCL-141) .2. The method of cell propagation was the same also for persistently infected cells with ABBV-1 (see below).For routine propagation, DEF, CCL-141, QT-35 and DF-1 were grown in maintenance media . For passaging, cells were washed with phosphate buffer saline  and dissociated with either trypsin-EDTA (Hyclone) diluted to 0.125% in PBS or TryPLE (Gibco). DEF were passaged every two to 3 days and the immortalized cell lines every five to 8 days, at a split ratio of 1:2 or 1:3. All cultures were incubated at 37 °C in an atmosphere of 10% COBranta canadensis), a kind gift from Dr. Dale Smith of the Ontario Veterinary College. Virus isolation was carried out as previously described [6 cells), according to published protocols for detection of the ABBV-1 M gene [ABBV-1 was isolated from the brain of an infected Canada goose , virus titration , and protein extraction and western blotting .To better characterize ABBV-1 replication in DEF, cells in a 15 cm dish were incubated with an approximate 10The comprehensive materials and methods for sequencing and phylogenetic analysis of ABBV-1 is provided in Additional file 1: Materials and Methods section. A brief description is provided as follows. ABBV-1 was sequenced using Sanger sequencing  and assembled using Geneious, version 8.0. Phylogenetic analysis was done using 29 aligned nucleotide sequences of the N gene from representative avian bornavirus strains, using the Neighbor-Joining method  in the MTwo independent infection experiments in immortalized cell lines were performed to characterize the growth and spread of ABBV-1 through cell culture, and establishment of a persistently infected cell populations upon infection. A graphical summary of the experimental outline is reported in Additional file 3.49 FFU in T25 culture flasks (experiment 1), or 103.26 FFU in 15 cm dishes (experiment 2). Cultures were then passaged every 5 to 8 days, for a total of 13 passages. At defined passage intervals, some of the cells were either added to 12-well or 24-well tissue culture plates (Nunc) to be examined by immunofluorescence staining (passage 1–3), harvested for RNA extraction and RT-qPCR (passage 2–7), virus titration , and protein extraction and western blotting .Briefly, uninfected confluent CCL-141, QT-35 and DF-1 were infected with ABBV-1 in 10% FBS/DMEM at 10Cell-free virus from persistently infected cell lines  was harvested using a freeze and thaw method. Briefly, cells were detached from flasks with trypsin and centrifuged at 300 x g for 5 min. After centrifugation, cells were resuspended in 5 mL of 2% FBS/DMEM and subjected to three cycles of freezing (− 80 °C) and thawing. The mixture was centrifuged again at 2000 x g for 5 min to pellet cellular debris. The supernatant was collected and stored at − 80 °C.50) assay in 96-well plates, using one of the four cell cultures  depending on the experiment (see below). Ten-fold serial dilutions of virus suspension in 2% FBS/DMEM were added to each well (200 μL per well). Five to 7 days post-infection, immunofluorescence  cell line from which it was collected, or the same batch of primary DEF (heterologous), using IFA in a 96-well plate format. Homologous titration was used to determine the capacity of each cell line to produce virus and report titre, and heterologous titration with DEF was used to normalize reporting of viral titre produced in different cell lines. Titres of cell-free virus were determined using the Karber method of 50% tissue culture infectious dose  [50-RT-qPCR titration was used to enhance the sensitivity of detection relative to conventional TCID50-IFA.For one of the collected passages of ABBV-1 from immortalized cell lines, a RT-qPCR based variation of the TCIDRT-qPCR)  was used50. A monospecific antibody (Pacific Immunology) raised in rabbit against a peptide spanning residues 332 to 354 of the ABBV-1 N protein (Cys-KEAQLARYRRREVTRGEDGAHLS) was used at 1:1000 concentration. The secondary antibody was a goat antibody against rabbit IgG conjugated with AlexaFluor488 (Invitrogen) used at 1:1000 dilution. Briefly, supernatant was removed from the plates / wells, cells were washed twice with PBS and fixed with 50% alcohol:acetone mixture for 20 min at -20°C. Cells were then washed twice with PBS and blocked for 1 to 3 h with 5% goat serum diluted in PBS-T (PBS with 0.1% Tween-20). After blocking, cells were incubated with the primary antibody diluted in blocking buffer for 1 to 3 h at room temperature or overnight at 4°C. Cells were then washed three times with PBS-T and finally incubated with the fluorophore-conjugated secondary antibodies at room temperature in a dark chamber for 1 to 3 h. After incubation, cells were washed three times, incubated with DAPI (1.43 mM diluted in PBS) for approximately 5 min and observed under a fluorescent microscope. Uninfected cells were used as a negative control.Immunofluorescence assay (IFA) was conducted to evaluate presence of ABBV-1 in infected cells and single wells in 96-well plate format for assessing TCIDWestern blot was conducted to determine the relative amount of viral N protein. Briefly, infected cells were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer . After 30 min incubation, the cell lysates were centrifuged at 10,000×g for 15 min at 4 °C. The supernatants were collected, and protein concentrations were determined using the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions.For denaturing SDS-page and western blots, cell lysates were incubated for 10 min at 95 °C on a hot plate to denature proteins. Proteins (1 μg) were separated by 12% SDS-PAGE at 120 V for 1.5 h, transferred to a PVDF membrane with 1x Towbin buffer at 25 V for 1 h . Membranes were then blocked with 5% skim milk in PBS-T at 4 °C overnight. The rest of the procedures were performed as previously described  and all RT-qPCR was performed on cell cultures infected with ABBV-1 to quantify viral RNA levels over multiple passages after infection. RNA from samples was extracted with RNeasy Mini Kit (Qiagen) according to manufacture’s protocol. Purified RNA was reverse transcribed and amplified using a Luna Universal Probe one-step RT-qPCR kit (NEB) with primers and probes targeting the ABBV-1 N gene. The primer sequences were: forward (5′-ATG CAC TTG CAC TCT TAG AC-3′), reverse (5′-TCC CCA TAA AAC CTC CCA AC-3′), and probe (5′-6-FAM-CCC TGC CCG CAG AGA GAA ATT CCA T-BHQ-3′). The cycling conditions were as follows: 55 °C for 10 min reverse transcription; 95 °C for 1 min initial denaturation, and 40 cycles of 95 °C for 10 s denaturation and 60 °C combined annealing and extension. Cycle threshold (ct) less than 35 was considered to be positive.Statistical analysis was performed to compare the mean yield titre of multiple passages between persistently infected immortalized cell lines within each independent infection experiment. Since cell-free virus stocks can be harvested from persistently infected cell lines at any and even multiple combined passages after persistent infection, comparing the mean yield titre of multiple passages (as opposed to only one specific passage) between different cell lines accounts for the variability of titre between passages and more accurately determine the cell line that supports better virus production. This was done to compare persistently infected CCL-141 and QT-35 but not DF-1. DF-1 did not produce measurable titre in all but one passage. The titres from all measurable passages were averaged and means compared using Student’s t-test. Results of homologous and heterologous titration were tested separately. Only measurable titres were included in the statistical analysis and measurable titres from all passages of each cell line is provided in Table 3.85 to 104.72 FFU / 106 cells. Infection was further confirmed by detecting an approximately 40 kDa protein band in cell lysate   search of the 9006 bp assembled genome sequence showed 97 and 99% identity to European ABBV-1 isolate AF-168  and Nort50 to compare infectious titres. Additional confirmation of ABBV-1 replication was done by western blotting for the N protein. Throughout all the passages, no morphological differences between the infected and uninfected cells were observed under phase contrast light microscopy for any of the cell lines. The other results are described in the sections below.The replication of ABBV-1 in DF-1, CCL-141, and QT-35 was monitored and compared qualitatively and quantitatively in two independent infection experiments  and 104.48 to 104.86 (heterologous titration) FFU / 106 cells (Table 2.43 to 103.18 (homologous titration) and 101.93 to 103.09 (heterologous titration) FFU / 106 cells  was implemented to increase the sensitivity of the titration assay. This is to confirm the lack of titre in DF-1 is not due to possible low sensitivity of TCID50-IFA. The method was completed on passage 8 post-infection for DF-1 and for CCL-141 and QT-35 as positive controls. For DF-1, the titre was once again not detectable, confirming the titration results of the regular TCID50. The positive controls, CCL-141 and QT-35, had titres of 104.91 and 102.07 FFU / 106 cells, respectively.A PCR-based variation of the TCID2.94 to 104.57 (homologous titration) and 103.11 to 104.66 (heterologous titration) FFU / 106 cells (Table 1.82 to 102.90 (homologous titration) and 102.40 to 102.66 (heterologous titration) FFU per 106 cells   from Ontario, Canada was isolated on DEF and characterized in DEF and immortalized avian cell lines: DF-1, CCL-141, and QT-35. DEF were initially used to isolate the virus because it was previously shown to support ABBV-1 replication . The ful50 assays. No CPE were observed at any point during passaging of infected cell lines; however, immunofluorescence showed ABBV-1 signal in both the cytoplasm and nucleus, the latter being often more intense. Nuclear-localized replication is a distinctive feature of both avian [ABBV-1 replicated in primary DEF and immortalized cell lines, CCL-141 and QT-35, producing detectable infectious titre. In DF-1, the virus maintained low-level genomic material and protein that was detectable by RT-qPCR and western blot, respectively, but generally did not yield infectious virus particles as determined by TCIDth avian , 49, 50 th avian –53.Anas platyrhynchos domesticus), as in our case. The titre of ABBV-1 from DEF was in a range from 104.83 to 106.07 FFU / 106 cells in our study; how this titre compares to those in previous publications is unknown, as the ABBV-1 titres from DEF have not been specifically reported [5 FFU/mL (or 105.81 FFU/mL) [4 FFU/mL (or 104.90 FFU/mL) [.Primary DEF were able to become infected, support replication and yield ABBV-1 infectious particles. This is consistent with what is reported in the literature (Table reported . For oth FFU/mL)  and 8.0  FFU/mL) , 32. HowOf the three immortalized cell lines examined, CCL-141 , also deThe QT-35 quail cell line supported ABBV-1 replication, however, the infectious titre was lower than the one produced from either CCL-141 or DEF. One potential reason for the lower capacity of ABBV-1 to replicate in QT-35 may include the differentiation stages of the cell lines. CCL-141 and DEF were derived from duck embryos , while QOne surprising finding was the observed intensity of the ABBV-1 N protein band in QT-35 at later passages relative to the low titre produced by this cell line in those passages. In CCL-141, visually intense N protein bands were also detected, but the titre was higher than in QT-35. In contrast, in DF-1, the faint N protein detection correlated with the lack of virus titre, which was lower than titre in QT-35. This finding was consistent in both independent infection experiments. For QT-35, it is possible that high levels of N protein, relative to infectious virus production, might have affected viral assembly or genome replication by altering the optimal stoichiometric interactions of other viral proteins or causing N protein aggregation. While these speculations need to be experimentally verified, altered N protein expression can impair RNP formation and decrease virus titre, as seen with mammalian 1 bornavirus and other negative-sense single-stranded RNA viruses –63.50 assay reliably. The general difficulty of avian bornavirus to produce abundant infectious virions in chicken cells may reflect host restriction and explain the lack of reports documenting natural avian bornavirus infection in chickens.DF-1 consistently supported low level ABBV-1 gene and protein expression, but no detectable infectious virions up to the 13 passages examined, with one exception. Data in the literature suggest that chicken cells do not support avian bornavirus replication as well as duck or quail cells. For example, PaBV-1 and PaBV-4 did not replicate in chicken embryo fibroblasts but replicated in DEF . ReplicaIn summary, a Canadian isolate of ABBV-1 (named ABBV-1 UoG-CG) was isolated in cell culture for the first time and was shown to replicated best in primary DEF compared to other three immortalized avian cell lines . Of the immortalized cell lines, CCL-141 produced the highest titre and therefore can be used as an alternative to primary embryonic fibroblast cultures for producing ABBV-1, albeit with a lower virus yield. DF-1 could be infected but yielded the lowest infectious titre. By characterizing the infection and replication of ABBV-1 in these cell lines, this research expands the repertoire of known cell culture systems available for the study and propagation of ABBV-1.Additional file 1: Figure S1. Schematic diagram of cell cultures infected with ABBV-1 over multiple passages (P). Infected CCL-141, QT-35 and DF-1 were monitored over 13 passages using four different techniques, immunofluorescence, RT-qPCR, titration, and western blot, at the passages specified in the diagram. Figure S2. Phylogenetic analysis of partial N gene sequence of ABBV-1 UoG-CG. Neighbor-Joining phylogenetic analysis of ABBV-1 UoG-CG partial N gene sequence (1036 positions segment) with 28 other ABV partial N gene sequences. Bootstrap test with 1000 replicates are shown next to the branches. The optimal tree with the sum of branch length = 1.04980780 is shown. ABBV-1 isolate UoG-CG is indicated by arrow. Supplementary Materials and Methods. Table S1. Primers used for sequencing ABBV-1 UoG-CG.


Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS. For those with metastatic OS at diagnosis, outcome is worse, with only 20–30% surviving for more than five years3 and is even more dismal for those with recurrent disease4. There is a paucity of early phase clinical trials available for OS patients and therefore, new or repositioned agents are needed to improve the outcome of patients. With this in mind, recent work has suggested that a significant proportion of OS, perhaps as high as 80%, exhibit genomic features reminiscent of a ‘BRCAness’ phenotype5. BRCAness describes tumours that possess histopathological and molecular characteristics similar to those found in patients with germ-line BRCA1 or BRCA2 gene defects, including a distinctive DNA repair defect arising from loss of Homologous Recombination (HR) and drug sensitivity effects associated with this HR defect, including exquisite PARP inhibitor sensitivity6. Using an analysis of whole-exome sequencing from 31 treatment naive osteosarcoma tumour samples, Kovac et al. identified large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours and recurrent mutations in tumour suppressor genes associated with homologous recombination and related DNA repair processes, including FANCA BRCA2 and ATM5. A subsequent analysis of OS tumour cell lines by Engert, Kovac et al.7 suggested that two OS tumour cell line models (MG63 and ZK58) with genomic loss of heterozygosity characteristics somewhat reminiscent of BRCAness, exhibited sub-micromolar IC50 sensitivity to the clinical PARP inhibitor talazoparib . However, whether the scale of PARP inhibitor sensitivity seen in OS models resembles that in BRCA gene mutant tumour cell lines was unclear.Whilst there have been a number of successes in developing targeted approaches to carcinomas, for many patients diagnosed with hard to treat sarcomas, radical surgery and chemotherapy regimens still represent the standard of care, and survival rates have not significantly changed for some time. This is particularly true for patients diagnosed with osteosarcoma (OS), where there has been little improvement in overall survival in the past 20 years, with five year survival remaining approximately 70% for young patients with localised diseaseBRCA1 mutant tumour cell lines and to a tumour cell line model with a CRISPR-Cas9 mutagenesis-engineered PARP inhibitor resistance-causing revertant mutation in BRCA1. We find that that the majority of OS tumour cell lines, with the exception of LM7, do not exhibit profound sensitivity to PARPi that might be associated with a BRCAness phenotype.Despite the need to identify novel targeted approaches in OS and the availability of a number of relatively well characterised tumour cell lines derived from these tumours, there is a limited amount of publically available information describing the small molecule sensitivity of these models. Such information could provide the starting point for discovering novel treatment approaches for this particular sarcoma subtype. With this in mind, we describe here as a resource, a dataset detailing the chemosensitivity of 18 OS tumour cell lines, and an additional 70 tumour cell lines derived from non-OS cancer histologies, to 79 small molecule inhibitors used in the treatment of cancer or in late stage development. As part of this resource, we describe the sensitivity of OS tumour cell lines to three chemically distinct PARP inhibitors, comparing their sensitivity to BRCA1 mutation (BRCA1 p.P724fs*12) as well as a PARP inhibitor resistant SUM149-subclone, SUM149.B1*.S, that has partially restored BRCA1 function because of a CRISPR-Cas9 mutagenesis-engineered BRCA1 intragenic reversion mutation8. This BRCA1 reversion mutation restores the native open reading frame of the BRCA1 gene, recapitulates RAD51 nuclear localisation (a key molecular event in BRCA1-mediated DNA repair) and causes profound PARP inhibitor resistance8.In order to generate a resource that could be used as a starting point to investigate sensitivity to targeted agents in OS, we compiled a panel of 18 well-characterised OS tumour cell lines (TCLs) and subjected these high-throughput drug sensitivity screening, alongside 70 tumour cell lines derived from the following carcinomas: breast (34) head and neck (11), synovial sarcoma (5), cervical (4), lung (4), large intestine (3), prostate (3), soft tissue  (3), haematopoietic (1), central nervous system (CNS) (1), and hepatic (1)  gene in OS tumour cell lines has previously been shown to predict enhanced sensitivity to FGFR inhibition by the pan-FGFR inhibitor NVP-BGJ39810. To assess the ability of our screen data to identify this known chemosensitivity effect, we interrogated the screen data from OS tumour cell lines for two different FGFR inhibitors, AZ4547 and PD173074. We noted that, in general, OS TCLs exhibited greater sensitivity to AZD4547  or FGFR1 gain (HU09 and NY) were significantly more sensitive to both AZ547 and PD173074 than those OS tumour cell lines without FGFR1 copy number alterations  tumour suppressor defects in OS TCLs  effects, that are somewhat reminiscent of mutational patterns seen in tumours from cancer patients with germ-line 13, but also enhanced sensitivity to the DNA damaging agents doxorubicin, gemcitabine and camptothecin  and a BRCA1 promoter hypermethylated tumour cell line HCC3814 to display PARPi sensitivity, as did a number of synovial sarcoma tumour cell lines , as described earlier  TCLs, we extracted from the dataset the AUC Z scores for three clinical PARP inhibitors; rucaparib, olaparib and talazoparib. When we compared the sensitivity profiles of TCLs to the three different PARP inhibitors, we found these to be highly correlated Fig. . When asnce Fig. . We alsoier Fig. .Figure 2BRCA1 mutant SUM149 cells  and to display a similar level of PARPi resistance as the BRCA1 revertant SUM149 subclone  CAPAN1 pancreatic ductal carcinoma cells16 (ANOVA p = 0.3757) but significantly greater sensitivity to olaparib than BRCA1 mutant SUM149 cells (ANOVA p = 0.0119), the BRCA1 revertant SUM149 subclone, and a previously validated PARPi resistant BRCA2 revertant CAPAN1 subclone, CAPAN1.B2*.S8  and RECQL418 .The nuclear localisation of the DNA recombinase RAD51 to the site of DNA damage is a key step in the DNA repair processes mediated by BRCA1 and BRCA2 and is critical in repairing the DNA lesions caused by PARP inhibitors. This nuclear localisation of RAD51 can be estimated by microscopy-based imaging of discrete nuclear RAD51 fociWe describe here chemosensitivity profiles for a panel of OS tumour cell lines. Our intention in carrying out this work was to generate a reference dataset that could be used for a variety of applications, ranging from the selection of tumour cell models with defined drug sensitivity/resistance phenotypes for subsequent experiments, through to the identification of drug sensitivity effects associated with molecular features found in these diseases. Given the “resource” nature of the work, we have included within this report, both raw and processed data so that subsequent analyses can be facilitated. For example, as the molecular profiling of these tumour cell line models is enhanced, for example by the addition of proteomic profiles of OS tumour cell lines, the utility of the chemosensitivity profiles we describe here will no doubt be enhanced.BRCA1 mutant breast tumour cell lines, the LM7 model does display evidence of BRCAness and a PARP inhibitor sensitivity phenotype. Although this is a singular model, the LM7 cell line therefore does provide a reagent for investigating mechanisms that cause PARP inhibitor sensitivity in OS.As part of this work, we highlight how the dataset can be used to assess PARP inhibitor sensitivity in OS tumour cell lines. By comparing the PARP inhibitor sensitivity of OS tumour cell lines to the scale of sensitivity in breast tumour cell lines with BRCA1 defects and also isogenic tumour cell models with a well-established and clinically relevant mechanism of PARP inhibitor resistance, we have been able to make a comparative analysis that suggests that whilst most OS tumour cell lines do not exhibit the exquisite level of PARP inhibitor sensitivity seen in BRCA1 mutant and BRCA1 revertant clones; the synthetic lethal effects of PARP inhibitors in in vitro cell culture often require drug exposure over several cell cycles , but the five-day exposure appears sufficient here.It is important to describe the limitations of the resource we provide. Using three-dimensional tissue culture approaches would assess whether the drug sensitivity and resistance phenotypes we see here are robust in the face of the loss of an adherent surface or changes in cell morphology and organisation often more apparent in three dimensional, as opposed to two-dimensional culture systems. We also note that more prolonged drug exposures, beyond the five-day drug exposure used here, might also elicit distinct effects, especially for agents whose cell inhibitory properties are amplified by drug exposure over multiple cell cycles. Nevertheless, we did find that the five-day drug exposure used was sufficient to document differences in PARP inhibitor sensitivity between et al., 2016; Jones et al., 2017). For OS tumour cell lines, G292 (clone A141B1), MG63, CAL72, HU03N1, OSA/SJSA-1, NOS1, SAOS2, U2OS, HU09, NY and HOS were gifts from Ultan McDermott at the Wellcome Trust Sanger Institute (WTSI); LM7, OHSN, OSH25HAL (HAL) MHM, and KPD were gifts from Ola Myklebost, Univeristy of Bergen, Norway. Head and neck cancer cell line models were obtained from Susanne Gollin and Theresa Whiteside (University of Pittsburgh), Tom Carey (University of Michigan) and Hans Joenje . All cell lines were maintained as per the suppliers’ instructions. STR typing of 10 loci was performed on each cell line using the GenePrint 10 system (Promega) and used to confirm the identity of cell lines prior to storage.The origin of non-OS tumour cell lines is described in  Cell-Titre Glo (Promega) to media. After 10 minutes incubation at room temperature, Cell-Titre Glo generated luminescence was captured using a Victor X-Light plate reader. Luminescence values from each well were normalised to the median of signals from wells exposed to DMSO only  to generate surviving fractions (SF). In total, each tumour cell line was screened three times, generating triplicate SF data sets  values were calculated from dose-response survival curves  (as9) where Alow is the activity at the lowest concentration of drug used, up to the maximum tested concentration  values were used for all analyses. Proteins were identified as differentially expressed if they were associated with a p < 0.05 effect (two sided heteroscedastic t-test) and an absolute difference in log2(LFQ) greater than 3.Proteomic abundance for the osteosarcoma tumour cell lines was performed as follows: Following lysis, protein purification, and tryptic digest, peptides were separated by liquid chromatography and measured mass spectrometer. Label-free proteome quantification was performed using the MaxQuant software environmentAll processed data is present in Supplementary Tables Supplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8


In this study, a novel scorpion venom‐derived peptide named Gonearrestide was identified in an in‐house constructed scorpion venom library through a combination of high‐throughput NGS transcriptome and MS/MS proteome platform. In total, 238 novel peptides were discovered from two scorpion species; and 22 peptides were selected for further study after a battery of functional prediction analysis. Following a series of bioinformatics analysis alongside with in vitro biological functional screenings, Gonearrestide was found to be a highly potent anticancer peptide which acts on a broad spectrum of human cancer cells while causing few if any observed cytotoxic effects on epithelial cells and erythrocytes. We further investigated the precise anticancer mechanism of Gonearrestide by focusing on its effects on the colorectal cancer cell line, HCT116. NGS RNA sequencing was employed to obtain full gene expression profiles in HCT116 cells, cultured in the presence and absence of Gonearrestide, to dissect signalling pathway differences. Taken together the in vitro, in vivo and ex vivo validation studies, it was proven that Gonearrestide could inhibit the growth of primary colon cancer cells and solid tumours by triggering cell cycle arrest in G1 phase through inhibition of cyclin‐dependent kinases 4 (CDK4) and up‐regulate the expression of cell cycle regulators/inhibitors—cyclin D3, p27, and p21. Furthermore, prediction of signalling pathways and potential binding sites used by Gonearrestide are also presented in this study. It has been proven that peptides/proteins are generally the major components of venoms, and many of these have either shown high potential or indeed had such realised, to defeat diseases such as bacterial/fungal infections, cardiovascular disorders, diabetes and cancers., in vivo and ex vivo experiments to lend further substance to the validation of this approach.Based on this platform, we have identified a panel of novel potential anticancer peptides in scorpion venoms. Anticancer peptide research has been performed at a low level for around 50 years now with limited success and understanding of their mechanisms of action is still in the initial stage. To reveal the potential anticancer mechanisms of candidate peptide, a recently invented transcriptome‐centric strategy was employed to predict their putative functions and targeted signalling pathways. Compared to traditional mechanistic study approaches, this was able to monitor the collective responses of all relevant genes without specific mechanistic or targeting hypotheses.In this study, we initially isolated a panel of potential anticancer peptides from venoms using this state‐of‐the‐art high‐throughput platform. Subsequently, a transcriptome‐centric method was applied to address and to reveal the putative anticancer mechanism of the lead peptide candidate, followed by in vitro, in vivo and ex vivo experimental validation. In addition, we hypothesized as to the involvement of specific signalling pathways and potential binding sites through bioinformatic analyses and use of 3D modelling construction software. Finally, one peptide candidate (Gonearrestide) was identified to affect cancer cell proliferation in vitro and reduce tumour growth in vivo, while negligible cytotoxicity was observed on normal human epithelial cells and erythrocytes. These current data suggest that Gonearrestide has a high potential for development as an anticancer drug. In addition, the findings presented here have further validated the ever‐increasing potential of this high‐throughput platform and transcriptome‐centric mechanistic study strategy to reveal additional novel anticancer peptide drug candidates.22.1Androctonus mauritanicus (AMa) and Androctonus australis (Egypt) (AAu), were purchased from Latoxan, Valence, France. The lyophilized venoms were stored at −20°C prior to use.Venoms from the scorpions, 2.2® mRNA DIRECT™ Kit (Ambion by Life Tech), then subjected to a cDNA library construction procedure using a NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc.). According to the instructions, the cDNA library construction consisted of several main steps. First of all the mRNA fragmentation was achieved by incubating at 94°C with random primers. Then the RNA fragments were subjected to first strand cDNA and second strand cDNA syntheses. After purification with 1.8X Agencourt AMPure XP beads, end repair of the cDNA library was performed by incubating with the end repair reaction buffer and the nucleotide A was added to the 3′ end of the DNA fragments to avoid self‐ ligation. Therefore, the adapters with the nucleotide T at the 3′ end were ligated to the DNA fragments. At last, the DNA fragments with adapters were enriched by PCR reactions and purified by AMPure XP beads. The quality of the cDNA library was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies) with an Agilent DNA 1000 kit (Agilent Technologies). The quantity of the cDNA library was validated by qPCR with a KAPA SYBR® FAST qPCR kit (KAPA Biosystems). The cDNA library was then loaded into a flow cell with oligos complementary to the adapters to generate clusters through bridge amplification. Finally, the transcriptome was obtained by performing RNA Sequencing on the Illumina MiSeq platform. The raw data obtained from the Miseq platform were analysed as follows. Firstly, the index primers used to identify different samples were removed. Secondly, the data were transferred to the program FastQC 0.1.0.1 to filter out the reads with low quality and less than 25 nucleotides. The filtered data were saved as fastq files. At last, the clean reads were de novo assembled using Trinity 2.0.6 software to obtain the transcriptome with default parameters.Five mg sample from each lyophilized scorpion venom was dissolved in 1 mL of cell lysis buffer (Thermo Fisher Scientific). Polyadenylated mRNA was extracted by use of a Dynabeads2.3® 12 10/300 GL column (Sigma Aldrich) to filter out large proteins with molecular masses higher than 10 KD. After this, Tris (2‐chloroethyl) phosphate (TCEP) was used to reduce disulphide bonds, and iodoacetamide (IAA) was used to alkylate free cysteine residues. At last, desalting was performed using PierceTM C18 Spin Tips (Thermo Scientific). Proteomics was performed using the Q Exactive™ Hybrid Quadrupole‐Orbitrap Mass Spectrometer (Thermo Scientific) with a nano‐LC system. Samples were loaded onto the Acclaim pepMap100, 75 μm × 2 cm C18 trap column (Thermo Scientific) using LC‐MS water . Samples were trapped onto the column at 6 μL/min for 5 minutes for pre‐concentration, then the trap column was connected to an EASY‐Spray column, 15 cm × 75 μm ID, 3 μm‐C18 particle sizes (Thermo Scientific) by a 10 port valve automatically. Eluting was performed at a flow rate of 300 nL/min with the gradient mobile phase buffer . Eluting peptide cations were converted to gas‐phase ions by electrospray ionization with a spray voltage of 1.8 kV and capillary temperature of 300°C. Full MS scans of peptide precursors from 300 to 1800 m/z were performed at 70 K resolution, while the AGC target was set to 3 × 106 ion counts with a max injection time of 200 ms. MS2 was performed by an isolation window at 2 m/z with the quadrupole, high energy collision (HCD) fragmentation with normalized collision energy of 27. The resolution was 17.5 K, while ion count target was set to 106 and the max injection time was 110 ms. The top 20 precursor ions were selected in each cycle and only those precursors with a charge state +2 to +5 were isolated for MS2 fragmentation. The fixed first mass of MS2 spectrum range started at 100 m/z. The C‐trap under fill ratio was 2%, which converted to the intensity threshold of 1.7 × 104 for the precursor selection. The dynamic exclusion duration was set to 20 seconds and isotope exclusion was turned on. The LC‐MS/MS raw data were then transferred to PEAKS (version7.5) software for de novo sequencing with default parameters.A second set of 5 mg lyophilized scorpion venom sample was dissolved in phosphate buffer , and subjected to AKTA Avant 150  fractionation using a Superose2.4The software, PEAKS (version8.0) , was used to interrogate the transcriptome database with parameters set as: precursor ion mass tolerance, 3 ppm; fragment ion mass tolerance, 0.01 Da; no enzymes employed; a variety of post‐translational modifications (PTMs) including cysteine carbamidomethylation and oxidation, were used and false discovery rate (FDR) was at ≤1%. The peptides identified high confidence and accuracy, constituted the prospective venom‐derived peptide libraries.2.5® TA Cloning® Kit (Invitrogen) and sequenced using an ABI 3100 automated sequencer. The nucleic acid sequences were isolated by employing the primers below, which were designed to highly‐conserved domains of the 5′‐untranslated regions of previously‐characterized venom peptide cDNAs from scorpions: (i) Primer AMa_Mauri1 (5′‐CATTACTATGTTGATTGTCGATGAAGTC‐3′); (ii) Primer AAm _H2 (5′‐AGTTTGGCACTTCTCTTCGTGAC‐3′); (iii) Primer And_Uni1 (5′‐TAGWCCTGSTRGTYARTCCGA‐3′).A third set of 5 mg lyophilized scorpion venom sample was dissolved in 1 mL of cell lysis/mRNA protection buffer, and polyadenylated mRNA was isolated from each by magnetic oligo‐dT beads as described by the manufacturer (Thermo Fisher Scientific). The isolated mRNA was subjected to 5′‐ and 3′‐rapid amplification of cDNA ends (RACE) procedures using a SMART‐RACE kit . The 3′‐RACE reactions were purified and cloned using a TOPO2.6The prospective novel venom‐derived peptide libraries were subjected to BLAST analyses against the NCBI non‐redundant database and the peptide sequences which produced hits with the sequences on this were then subjected to Pfamscan online searching to identify whether the sequences aligned to any reported toxins. The peptide sequences reported before were removed manually during this step. The novel peptides were manually filtered by removing very short sequences and finally, the venom‐derived peptide library was constructed with all the peptide sequences selected as above. The selected peptide regions were chemically‐synthesized by solid phase peptide synthesis (SPPS) technology.2.72 culture flasks , and cultured in an incubator under 5% CO2/95% air at 37°C. The anti‐proliferative effects of these novel peptides on human cancer cells were measured by employing MTT cell viability assays. Peptides were screened at a concentration of 10 μmol/L with various cell lines: Pancreatic cancer cell lines MIA Paca‐2, PANC‐1, AsPC‐1; Colon cancer cell lines HCT116, DLD‐1, Hke3, Dks8; Breast cancer cell lines MCF‐7, MDA‐MB‐231; Bladder cancer cell line HT‐1376; Human retinal epithelial cancer cell line ARPE‐19; Brain glioma cell line U‐251. Two different normal epithelial human cell lines (colon cell line FHC and breast cell line MCF 10A) were also employed in the screening assays. Peptides with anti‐proliferative activities were then selected and further studied in a dose‐dependent manner in the concentration range from 250 to 1 μmol/L.Cancer cell lines were maintained in their culture medium with 10% (v/v) foetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. All cells were seeded into 75 cm2.8Human erythrocytes were washed with PBS until the supernatant was clear, and then samples of a 4% (v/v) suspension were treated with peptide concentrations ranging from 1 to 250 μmol/L at 37°C for 1 hour. Lysis of cells was assessed by haemoglobin release, measured by optical density changes at λ550 nm, and calculated compared to positive controls. Positive control groups and negative control groups were treated with equal volumes of Triton X‐100 and PBS instead of peptide, respectively.2.9Cells were treated with different peptide concentrations from 250 to 10 μmol/L for 24 hours. LDH Cytotoxicity Assay Kit  was then employed to detect the cytotoxicity of peptides according to the manufacturer's instructions. The supernatants were transferred, mixed with the reaction buffer and LDH release was detected using a microplate reader set at 492 nm and calculated compared to positive controls. Positive control groups and negative control groups were treated with equal volumes of Triton X‐100 and PBS instead of peptide, respectively.2.10™ C6. The blank control group was treated with an equal volume of PBS.Cells were treated with peptides at the concentration of 250 μmol/L for 24 hours, after which the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen) was employed to detect apoptosis according to the manufacturer's instructions along with use of a flow cytometer BD Accuri2.11™ Continuous Live‐cell Imaging & Analysis System was employed to record cell growth for three days. The instrument can automatically generate cell growth curves, which are calculated by the cell area attached to the bottom of each well according to the images taken by the instrument every 2 hours. Blank control group was treated with equal volumes of PBS.Cells were seeded into 6‐well plates and treated with peptides at 250 μmol/L for 24 hours, after which the IncuCyte ZOOM2.12http://ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna//Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz) using the splice aligner tophat2 (v2.1.1) with default parameters.q_value cut‐off of <0.05, were considered as significantly differentially expressed. Further GO enrichment and pathway visualization of differentially expressed genes was performed by the DAVID online tool (https://david.ncifcrf.gov) and KEGG mapper (http://www.genome.jp/kegg/tool/map_pathway2.html), respectively.Samples were prepared by treating cells with Gonearrestide at 250 μmol/L for 24 hours. Blank control groups and negative control groups were treated with equal volumes of PBS and negative control peptide, respectively. For each group, two replicates were employed. Samples were collected and total RNA was extracted, DNA library preparation was performed as previously described under “Transcriptomics procedures”. Transcriptome sequencing and analysis procedures were performed as follows: TruSeq RNA‐seq libraries were sequenced on the Illumina HiSeq2000 platform. The raw reads were mapped to the human genome  for 5 minutes at room temperature. Density of cells in each cell cycle phase was recorded by flow cytometry using a BD Accuri2.14Cells were treated with peptides at 250 μmol/L for 24 hours. The blank control group was treated with an equal volume of PBS, and regarded as time‐point 0 hour. Cells treated with peptides were then collected at different time‐points  and washed with cold PBS. RIPA buffer  was used to lyse cells for 30 minutes on ice, after which cells were centrifuged at 20 000× g for another 30 minutes in a 4°C pre‐cooled centrifuge. The supernatants were stored at −80°C prior to use. Equal amounts of protein samples at each time‐point, including those from the blank control group, were subjected to SDS‐PAGE and then transferred to a membrane. Membranes were then blocked with 5% non‐fat dry milk in TBST (0.1% Tween) for 2 hour at room temperature, and then incubated with primary antibodies  at 4°C overnight. The next day, TBST (0.1% Tween) was used to wash membranes three times, after which secondary HRP conjugated antimouse/anti‐rabbit antibody was incubated with membranes for 1 hour at room temperature. After washing with TBST (0.1% Tween) another three times, Immobilon Western HRP Substrate was employed for chemiluminescent detection in Western blots.2.15Cells were cultured in an 8‐chamber slide and treated with fluorescently labelled peptides for 1 hour. After this, cells were stained with CellMask Plasma Membrane Stains (Invitrogen) and Hoechst dye (Thermo Fisher) according to the manufacturer's instructions, followed by washing with PBS and fixation with 3.75% formaldehyde. The locations of peptides affecting cells were recorded by confocal laser scanning microscopy.2.16Nude Mice used in this study were obtained from the Animal Facility in the Faculty of Health Sciences at the University of Macau (UM). All the experimental protocols were conducted according to the UM Ethics Committee for Animal Experiments with the approved application UMARE‐035‐2016.6 HCT116 colon cancer cells were xenotransplanted by subcutaneous injection (s. c.) into the flanks of 4 week‐old female nude mice. After 3 days of implantation, mice were then divided into four groups: Group 1 (n = 5) was treated with Gonearrestide at a high concentration (100 μg/tumour) by peritumoral (p. t.) injection for another 2 weeks; Group 2 (n = 5) was treated with Gonearrestide at low concentration (50 μg/tumour) while Groups 3 and 4 (n = 5) were treated with an equal volume of PBS and negative control peptide, respectively. Tumour volume measurement was performed during 2 weeks of treatment. To determine tumour volume by use of an external caliper, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined. Tumour volumes, based on caliper measurements, were calculated by the modified ellipsoidal formula2). At the end of the experiment, tumours were removed under anaesthesia and mice were then euthanized. Tumours were snap‐frozen for further histological analysis. On the one hand, tumours were fixed and cut into slides. Immunohistochemical staining of tumour slides were performed by de‐paraffinizing in xylene and rehydrating in graded ethanol, followed by incubation in sodium citrate buffer (pH 6.0) for high‐pressure antigen retrieval. Afterwards, slides were incubated in 3% hydrogen peroxide to block endogenous peroxidase activity, and incubated with cell cycle checkpoint antibody CDK4 at 4°C overnight. On the second day, the slides were incubated with secondary antibody and subjected to diaminobenzidine staining. After counterstaining with 20% haematoxylin, slides were dehydrated and mounted on cover slips. The IHC‐stained tissue sections were reviewed under a microscope. In addition, mRNA and proteins were isolated from frozen tumours for qPCR and Western blot analysis. The procedures were for both techniques were described before.In vivo anticancer activity of Gonearrestide was evaluated in an HCT116 colon cancer xenograft model. 1 x 102.17P values were calculated by student's t‐test from the mean values of the indicated data. Significant differences were marked with asterisks .Statistical analysis was performed using GraphPad Prism 6 software. 33.13.1.1Androctonus mauritanicus (AMa) and Androctonus australis (Egypt) (AAu), respectively. As these two scorpion species do not have their genomes assembled so far, the transcriptome data were de novo assembled by use of Trinity software. Consequently, there were 112 235 and 46 360 nucleic acid sequences, respectively, obtained for each species . In total, there were 130 peptides matched with UniProt database, which indicated the robustness of our data.3.1.4There were eight peptide sequences isolated through the molecular cloning approach, including three novel peptide sequences. All these eight peptide sequences were found in the overlapping region of the transcriptome and the proteome results, which validated the new high‐throughput approach. The nucleotide and translated open‐reading frames of the three novel sequences were saved Figure .3.1.5The overlapping regions described previously were subjected to a variety of bioinformatic analyses and the filtered results were saved Figure . As a re3.23.2.1In total, 22 peptides were screened with 19 cell lines to identify their potential anti‐proliferative activities, after which four peptides  were found to inhibit the proliferation of human colon cancer cell lines HCT116, DLD‐1, Hke3, Dks8 and the human glioma cell line U‐251. Two types of normal human epithelial cell lines, including the colon cell line FHC and the breast cell line MCF 10A, were also employed here and it was found that the peptides had negligible effects on their proliferation even at highest concentrations. Peptide 13 , indicating that Gonearrestide was a worthy candidate for further study with the view to its clinic application  is released when cell membrane integrity is compromised. In this assay, Gonearrestide did not induce LDH release from HCT116 cells after 24 hours of incubation. This result indicated that Gonearrestide did not disrupt the cell membrane and did not induce cell death. At the same time, the LDH results were consistent with the RNA sequencing data along with the combination of MTT results. Taken together, these data confirmed that Gonearrestide affected cancer cell proliferation through inhibition of growth  with HCT116. Localization of Gonearrestide within the cell should result in green fluorescence signals from the FITC‐labelled peptide. As shown in Figure 3.3.3Annexin V and PI staining along with flow cytometry can differentiate individual cells into different stages. The results of these experiments were shown in Figure 3.3.4As shown in Figure 3.4As Gonearrestide had the best activity on the cancer cell line HCT116 with no obvious cytotoxicity on normal human cell lines and erythrocytes, it was chosen for further experiments to identify its molecular mechanism of action. There were three groups employed in this assay: HCT116 treated with Gonearrestide (HCT116_Gonearrestide), HCT116 treated with a negative control peptide (HCT116_NC) and HCT116 treated with PBS (HCT116_BC). The negative control peptide was one of the previously identified peptides which showed no anticancer cell activity.P value < .05, tool: DAVID) enriched under common genes  have been listed  and phosphatidylethanolamine (PE) can enhance membrane poration by a peptide with anticancer properties.4Naja naja venom exerted a cytotoxic activity on MCF‐7 and MDA‐MB‐231 cells, and thereby induced cancer cell death directly.2, isolated from snake venom from Walterinnesia aegyptia and Vipera ammodytes meriodionalis, respectively, induced apoptosis on a broad spectrum of cancer cell lines.Venom from animals has been proven to be a useful source of drug candidates in natural drug discovery to fight against diseases.Androctonus mauritanicus (AMa) and Androctonus australis (Egypt) (AAu), have been constructed. To confirm the validity of this platform, a traditional cloning approach was also applied in parallel. The traditionally‐derived cloning data provided evidence of the robust nature of the described high‐throughput screening platform. The coupling of transcriptomic and proteomic/peptidomic approaches using bioinformatics produced venom‐derived peptide panels, which could be used to explore the potential therapeutically useful peptides present in respective venoms.To increase the identification of potential venom‐sourced anticancer peptides, a high efficiency and low‐cost screening platform is urgently required. In this study, a high‐throughput screening platform consisting of transcriptome and proteome sequencing is described and has been of proven efficacy. Based on the use of this platform, complete peptide libraries of venoms from the scorpions, Within the scorpion peptide panels, bioinformatics filtration and MTT screening were initially applied to detect novel candidate anticancer peptides. As a result, peptide 13(Gonearrestide) was identified and found to have a potent anticancer activity against human colon cancer cell line HCT116 but with negligible observed cytotoxicity against normal human epithelial cells and erythrocytes. To study the anticancer mechanisms of Gonearrestide, RNA sequencing was used to detect the transcriptome responses in cells following Gonearrestide treatment. Transcriptome sequencing provided a number of advantages over conventional chemical screening strategies. This approach did not require prior identification of specific drug targets. This meant that, based on a multi‐target, pathway‐centric approach, any potential therapeutic targets could be assessed in the transcriptome landscape. Compared with phenotypic screening, transcriptome screening offers more specific gene expression responses providing crucial clues to potential molecular mechanisms.The RNA sequencing results indicated that Gonearrestide could combine with cancer cell membranes followed by inhibition of cell proliferation through a cell cycle arrest effect. Although well‐designed in vitro, in vivo and ex vivo assays, the transcriptome‐centric prediction that Gonearrestide could arrest HCT116 cancer cells in G1 phase and inhibit tumour growth, was proven. The signalling pathway used involved effects on PIP3 and PTEN, resulting in the inhibition of the Akt pathway. PIP3 is a phospholipid that is bound within the layers of the cell membrane and after combining with the cell membrane, Gonearrestide can affect PIP3 by electrostatic attraction to its anionic trisphosphate group. PTEN catalyses the dephosphorylation of PIP3 to produce PIP2, which is a major negative regulator of Akt signalling. It is a tumour suppressor and the phosphatase activity of this protein may also be related to cell cycle regulation preventing cells growing and dividing too rapidly. From the previous anti‐proliferative screening results, it could also be seen that Gonearrestide showed a better anticancer effect on the PTEN wild‐type cell line, HCT116, when compared with the PTEN‐mutated cell lines, PC‐3 and SW872, with cell viabilities of 55%, 84% and 80%, respectively. The inhibition of Akt induced downstream dysregulation of CDK4/6 and CDK2, thereby promoting cell entry from G1 to S phase. In addition, the peptide‐lipid interaction generated by docking also proved the hypothesis that Gonearrestide could bind to PIP3 through electrostatic attraction and resulted in alerting the downstream cell cycle regulation.Current results showed that Gonearrestide could inhibit cell proliferation in vitro through a cell cycle arrest effect and could reduce tumour growth in vivo, while possessing little observable cytotoxicity on human epithelial cells and erythrocytes. Hence, this peptide was deemed to have a great potential to be proceed to clinical trials as an anticancer drug candidate. To enhance the efficiency of anticancer peptide discovery, we emphasize the broad utility of our high‐throughput platform for venom‐derived peptide library construction and selection and also the interfacing of this with a transcriptome‐centric mechanistic study approach. The validity of this approach has been proven by well‐designed experiments described and employed in this study. Moreover, within the scorpion peptide panel, several additional anticancer peptides are awaiting further investigation.The authors confirm that there are no conflict of interest. Click here for additional data file.