Pistacia genus on human tumor cell lines. In particular, the cytotoxic activity of essential oils obtained from P. lentiscus, P. lentiscus var. chia (mastic gum), P. terebinthus, P. vera, and P. integerrima, was screened on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). The results indicate that all the Pistacia phytocomplexes, with the exception of mastic gum oil, induce cytotoxic effects on one or more of the three cell lines. PCA highlighted the presence of different cooperating clusters of bioactive molecules. Cluster variability among species, and even within the same species, could explain some of the differences seen among samples suggesting the presence of both common and species-specific mechanisms. Single molecules from one of the most significant clusters were tested, but only bornyl-acetate presented cytotoxic activity, although at much higher concentrations (IC50 = 138.5 µg/mL) than those present in the essential oils, indicating that understanding of the full biological effect requires a holistic vision of the phytocomplexes with all its constituents.Principal component analysis (PCA) multivariate analysis was applied to study the cytotoxic activity of essential oils from various species of the  Herbal medicine is emerging as a holistic tool in novel personalized medicine approaches, especially in primary and secondary prevention . In addid-Limonene, one of the most widespread natural monoterpenes, exerts in vitro anti-tumor activity against several cancer types: neuroblastoma, leukemia, breast, skin, liver, lung, and stomach cancer [p-cymene, 1,4-terpineol, α-terpinene, α-copaene, α-elemene, α-humulene, and α-caryophyllene, among others, have shown significant cytotoxic activity in different cancer cell lines [Plants are a major source of chemopreventive phytochemicals . Among th cancer ,13,14,15ll lines ,19,20,21Pistacia, a genus of flowering plants from the family Anacardacee, includes several species largely distributed and rich in terpenoids [Pistacia genus comprises more than twenty species and, among them, P. lentiscus, P. terebinthus, P. vera, P. integerrima, and P. lentiscus L. var. chia are more popular and have been extensively used in traditional medicine for their tonic, antiseptic, antihypertensive, and gastro-protective properties [rpenoids ,22, whicoperties ,22,23. Moperties .P. lentiscus L. var. chia (known as mastic gum) which has been reported to exert cytotoxic activity on tumor cells and inhibition of cancer cell proliferation, angiogenesis, and inflammatory response [Most published data are related to the resin of response ,29,30,31Pistacia species have been carried out, mostly using extracts and not terpenes-rich essential oils [P. lentiscus, P. lentiscus L. var. chia, P. terebinthus, P. integerrima, and P. vera) on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). A multivariate analysis approach was then used to investigate the statistical correlations among the different phytocomplex components of the essential oils, allowing the identification of clusters of molecules cooperating in the cytotoxic activity.A number of studies concerning the cytotoxic activity of other ial oils ,36,37,38ial oils . In the P. lentiscus L. , P. terebinthus L., P. vera L., and P. integerrima J.L. Stewart ex Brandis, as identified by the Wiley mass spectra library. A total of 32 samples from different aerial parts of the plants were analysed. Although most terpenes were present in both P. lentiscus and P. terebinthus samples, the oil composition varied considerably, with limonene and cis-ocimene being more meaningfully present in samples from P. terebinthus, while β-phellandrene, 1,4- and α-terpineol, α- and γ-terpinene, and T and α-cadinol were relatively more abundant in samples from P. lentiscus. Collectively, the two species show similar amounts of α- and β-pinene, β-caryophyllene, and butylated hydroxy toluene. Among the samples from other species, those from P. integerrima showed similarities to P. terebinthus in the content of α- and β-pinene and of β-caryophyllene, and to P. lentiscus in the amounts of 1,4- and α-terpineol, endoborneol, and bornyl acetate. Finally, a significant amount of δ-carene appears to be characteristic of all the three species. Oil samples from P. lentiscus var. chia (resin) and from P. vera (nuts) are the ones with the least similarities with the others. The composition of the essential oils indicates consistency with the major terpenes known to be present in these species [Essential oils were obtained by hydrodistillation of aerial parts of the plants, or obtained from producers, and oil composition was determined using GC-MS.  species .Pistacia species was evaluated in vitro on three human adenocarcinoma cell lines: MCF-7 (breast adenocarcinoma); 2008 (ovarian adenocarcinoma); and LoVo (colon adenocarcinoma). Results are shown in The cytotoxic activity of the phytocomplexes from the different P. lentiscus were active, with the exception of the one from P. lentiscus var. chia. Samples from P. terebinthus displayed a high frequency of activity, above 70%, while both samples from P. integerrima were active and the one from P. vera was not. The sample with the most activity was one essential oil from P. lentiscus leaves (sample A: IC50 = 173.9 µg/mL).Most oil samples displayed some cytotoxic activity on one or more cell lines with different potencies. In particular, looking at the results obtained with LoVo cells, all the samples from P. lentiscus on LoVo cells, none of the species was active on all the other cell lines. In particular the activity towards 2008 cells was higher for the samples from P. terebinthus (71%), while P. lentiscus displayed a significant activity in 55% of samples, while the two samples from P. integerrima were active and those from P. vera and P. lentiscus var. chia were not. The two most active samples were both from P. lentiscus leaves (sample A: IC50 = 181.5 µg/mL and sample C: IC50 = 220.4 µg/mL).Apart from P. lentiscus (73%), and it was the only cell line sensitive to the essential oil from P. vera. Essential oils from P. terebinthus were active in 44% of cases, while the two oils from P. integerrima had a weak effect or were inactive. The two most active samples were from P. lentiscus leaves (sample B: IC50 = 239.6 µg/mL), from P. terebinthus galls (sample AB: IC50 = 254.9 µg/mL) and from P. vera nuts (sample A3: IC50 = 290.6 µg/mL).The activity on MCF-7 cells was higher for the samples from Pistacia species phytocomplexes on all the three cell lines tested, in most cases with IC50 values below 500 µg/mL, suggesting that this effect might be a shared feature of the Pistacia genus. There were wide differences in biological activity between samples, regardless of the species and even the anatomical part, likely related to the high variability in oil composition even within the same species, as determined by GC/MS fingerprinting.Taken together the results indicate a dose dependent cytotoxic effect of different The oils did not show a consistent order of potency from one cell line to the other, and in some case the same sample could display high activity on one cell line, while being inactive on the others . Such diIn general, the biological activity of a phytocomplex is related to its chemical composition, with each single compound acting synergistically with the others. Thus, given the high heterogeneous compositions of essential oils, likely due to genetic diversity of the samples , it is dPistacia genus, but most studies have focused on different types of extracts and not on the terpene-rich essential oils, suggesting other types of chemicals and mechanisms were involved [Pistacia species have been shown to exert cytotoxic activity on tumoral cell lines. Pistacia vera and Pistacia khinjuk resin essential oils were found to be active against retinoblastoma cells (Y79) [P. palestina Boiss. Although inactive on MCF-7 cells, displayed cytotoxic activity towards amelanotic melanoma cells (C32) and renal cell adenocarcinoma (ACHN) [P. lentiscus var. chia, although in most cases not with essential oil [P. lentiscus var. chia resin (mastic), we did not observe any cytotoxic activity in our experimental conditions. This discrepancy could be related to the different experimental models used, to the different treatment with the essential oil, or to the relatively stringent threshold we used for the cytotoxic effectiveness (500 µg/mL). To eliminate nonspecific interferences with adhesion of cells to the experimental substrate, a “pre-incubation” strategy was also used. Unlike the other published data, cell cultures were exposed to essential oils only for three hours and then allowed to recover for the following 21 h. The limitation of unwanted effects on cell adhesion, by improving the focus on cytotoxicity, might explain the different results.Terpenes and terpene-rich essential oils have been shown to exert specific cytotoxic activity on several tumoral cell lines ,43,44,45involved ,32,34,38ls Y79) . Essentia (ACHN) . Our res9 . Essential oil ,31. In stial oil ,26. Althn = 7), while LoVo and 2008 cells had six and five, respectively. All the compounds showing a high level of correlation with bioactivity were included at least in one cluster, with the exception of sabinene and α-humulene, the latter a known anticancer agent [P. lentiscus was the species with more clusters of bioactivity-associated molecules in all cell lines. P. lentiscus was also the species with higher principal component scores, with L2 being the cluster with the highest values .PCA analysis was performed with the aim of identifying synergic clusters of compounds present in the phytocomplexes, with the best correlation with biological activity . Four prer agent . P. lentP. integerrima on LoVo cells  is also the core part of I1a and I1b and correlates with activity in all cell lines and, is shared by all L1s P. lentiscus clusters, thus suggesting a common mechanism of P. lentiscus and P. integerrima phytocomplexes throughout the cell lines. Although the aim of the work was not to identify biological activity in single terpenes, given that this particular cluster is shared by all cell lines and at the same time by more than one Pistacia species, we tested the cytotoxic activity of the pure compounds on LoVo cells, where the activity of the phytocomplexes seemed to be stronger. Bornyl acetate was the only tested molecule showing a cytotoxic activity when used alone (IC50 = 138.5 (130.8 to 146.7 µg/mL), although its potency was not as strong as that corresponding to the actual concentration range in the oils. The other compounds displayed no activity in the pre-treatment paradigm used (data not shown), although p-cymene and spathulenol had been previously shown to have anti-tumoral activity [P. lentiscus): α-copaene, α-cadinol, τ-cadinol, cadina-1,4-diene, α-elemene, α-amorphene, germacrene A, α-muurolene, β-elemene, and alloaromandrene. In this cluster α-copaene and β-elemene are known cytotoxic compounds for tumoral cells [P. lentiscus and P. terebinthus . Ten more compounds are differentially cooperating in clusters of cytotoxic activity towards all cell lines, some in the same group, some in totally different contexts , the last one being the only clustered molecule shared by P. vera with other species. Other compounds’ bioactivity are not shared by all three cell lines, thus suggesting a different cell sensitivity and cell-specific mechanisms. Two such molecules, β-caryophyllene and d-limonene, previously shown to have cytotoxic activity and considered promising potential anti-tumorals [The active cluster identified for activity ,42. Thisal cells . α-Terpial cells ,17. Otheal cells ,17. α-Hual cells , is prestumorals ,15,42,45In summary, PCA allowed the identification of 46 compounds in the phytocomplexes correlated with potential biological activity, most of which had not been previously reported for their in vitro cytotoxic activity on tumoral cells (80%). The compounds are distributed within different clusters of molecules potentially cooperating to achieve the cytotoxic activity on the cell lines.P. lentiscus is the species with the most clusters, which is in line with its higher potency and frequency of activity with respect to the other species tested. Only two of such clusters are common for all the cell lines, they are characteristic of P. lentiscus, and one is also shared with P. integerrima. In other cases clusters are shared only for one or two cell lines. In the case of P. vera, only one cluster could be identified. These similarities and differences suggest that the cytotoxic effect is complex. Some are common for all three cell lines which suggests a shared mechanism for all three adenocarcynomas, but some are not and P. lentiscus seems to display the most variability, while samples from the other species seem more consistent throughout the different cell lines. This behaviour points to different mechanisms exerted in a cell-specific fashion.Taken together, the PCA results indicate that the cytotoxic activity of the phytocomplexes can be highlighted in several clusters of likely-synergizing compounds. Keeping in mind that the results with PCA do not necessarily explain the full activity of the whole phytocomplex, the analysis allowed the identification of substances, mostly terpenes, sesquiterpenes, and fatty acids, present in the essential oils likely playing a central role in the in vitro anti-tumoral effect. Of these, some had been previously shown to have such activity when used as single compounds. Almost all the substances identified cooperate with others, thus forming clusters of activity, often different for one or more components in terms of cell sensitivity.Finally, although all the compounds identified had a positive correlation with the activity, none of the single clusters or the single components are, by themselves, sufficient to justify the whole activity, and at least all the molecules identified contribute to the biological effects.Methanol, ethyl acetate, formic acid, acetonitrile, and HPLC-grade solvents were purchase from Carlo Erba Reagents Srl . The solvents were of analytical grade. Double distilled water was used in the HPLC mobile phase. All other reagents, including single purified terpenes, were obtained from Sigma Aldrich srl. .P. terebinthus flowers  which were collected in Provence , aerial parts  of the plants were collected in three different Italian regions: Pistacia terebinthus L.: Veneto, Colli Euganei (45°21′50.01″ N 11°40′13.51″ E) ; Pistacia lentiscus L.: Tuscany, San Giuliano Terme (43°45′45″ N 10°26′29″ E), Pisa , Tuscany, Montepescali, Maremma (42°52′58.04″ N/11°05′11.97″ E/42.882788° N) ; Pistacia vera L.: Sicily, Mount Etna (37°45′04.45″ N 14°59′38.49″ E) (sample M). Other essential oils were purchased: P. lentiscus essential oil from Portugal (sample A) was obtained from Lifetree Aromatix, John Steel, 3949 Longridge Ave. Sherman Oaks, CA, USA; P. integerrima J.L. Stewart ex Brandis essential oil was obtained from Bluebell Herbal Products, Bhaktapur, Nepal ; P. lentiscus var. chia resin (mastic) essential oil, was obtained from “Anemos” Benetos John Galatoulas George Co, Chios, Karfas RD, Kontari, Chios, Greece (sample L).With the exception of P. lentiscus, P. terebinthus, and P. vera) were collected, rinsed, dried, and frozen at −21 °C within three hours from collection to preserve their phytochemical composition until the moment of the distillation. The material was then hydrodistilled within a few months. All the samples, before distillation, were minced to obtain the maximum extraction yield and make the process of diffusion of the essence easier [Plant parts were recognized by one of the authors (G.I.), University of Padua, Italy. Voucher specimens were deposited at the Department of Pharmaceutical and Pharmacological Sciences of the University of Padova . Plant parts , branches, galls or nuts (250 g), berries (100 g), and flowers (270 g). The yield for the various parts of the plant, express as percent of initial wet weight was: leaves (0.05%), branches (0.06%), nuts (0.02%), berries (0.11%), flowers (0.08%), and galls (0.40%).P. lentiscus, P. terebinthus, P. vera, P. integerrima, and P. lentiscus var. chia was determined by gas chromatography analysis coupled with a mass spectrometer detector (GC/MS), with a Hewlett-Packard 6890-5973 system .The chemical composition of the essential oils obtained from The identification of compounds by GC-MS was performed considering not only the matching of mass spectra fragmentation but also the retention index. These two parameters allow the identification of phytoconstituents of the essential oil. In particular, the separation was achieved using a HP-5 MS capillary column (30 m × 0.25 mm i.d. × 0.25 film thickness), with a stationary phase of 95%polydimethylsiloxane. GC-MS analysis carried out under the following conditions: carried gas, He; flow rate, 0.8 mL/min; split ratio, 1:10; injection volume, 1 µL; injector temperature, 200 °C. The temperature programme was as follows: 60 °C with 3 min initial hold, and then to 280 °C at a rate of 3 °C/min, and finally held isothermally for 5 min. The ionization mode was electronic impact at 70 Ev. Identification of the components was achieved by comparison of their mass spectral fragmentation patterns with those stored in the data bank (Wiley Library) ,49,50,51Pistacia oils were dissolved in DMSO and then diluted in culture medium to obtain stock solutions which were stored at −20 °C. Each final stock solution contained 1% DMSO, 9% essential oil, and 90% culture medium. All the procedures were carried out under sterile conditions. Before each experiment the stock solutions were diluted with growth medium and used immediately. The final DMSO concentration was 1%.2) 5%, at 37 °C).Cytotoxicity was evaluated on three human adenocarcinoma cell lines: ovarian (2008) breast (MCF-7), and colon (LoVo). The cell line 2008 was kindly supplied by Prof. G. Marverti  and was maintained in RPMI 1640 medium . MCF-7 cells were supplied by the Experimental Zooprophylactic Institute of Lombardy and Emilia  and were grown in Eagle’s Minimum Essential Medium . LoVo cells were supplied by Dr. G. Toffoli , and were grown in HAM’s F12 (Lonza). L929 fibroblasts  and HFFF2 cells, a human Caucasian foetal foreskin fibroblast derived from a 14–18 old human foetus , were used as healthy controls to verify the specificity of the cytotoxic effects for cancer cells (data not shown). L929 and HFFF2 cells were grown in RPMI 1640 medium  and in DMEM  respectively, and 1% penicillin/streptomycin  (Seromed-Biochrom KG). All cultured media were supplemented with 10% heat-inactivated FCS  (20% for HFFF2 cells), 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) (Seromed-Biochrom KG), and 1% 200 mM glutamine . Cells were maintained under humidified conditions  were seeded in 96-well tissue plates  and treated for 3 h with different concentrations of Pistacia essential oils, with single compounds, or with vehicle . The cells were then rinsed with sterile PBS and incubated in culture medium for 21 h. The cytotoxic effect was evaluated by MTT reduction assay after 21 h of incubation. An amount of 20 µL of MTT solution (5 mg/mL in PBS) was added to each well, and plates were incubated for 4 h at 37 °C. DMSO (150 µL) was added to all wells and mixed thoroughly to dissolve the dark-blue crystals. The absorbance was measured on a micro-culture plate reader  using a test wavelength of 570 nm and a reference wavelength of 630 nm. The 3 h pre-incubation followed by recovery model was chosen after some preliminary tests aimed at identifying the best conditions to assay for specific cytotoxic effects. Cisplatin was used as the positive control. IC50 values below 500 µg/mL were taken into consideration for the cytotoxic activity of each phytocomplex tested. This value was chosen as threshold in line with recent literature on the subject and other works on cytotoxicity of Pistacia essential oils, although some authors have used different thresholds [Cells . IC50, dose-response curve best-fit, Pearson correlation, and linear regression analysis were performed using GraphPad Prism Version 3.0. Principal component analysis (PCA) was performed using XLSTAT® software . PCA was performed on a correlation matrix (Pearson (n)), computed with the contribution (%) of each compound to the IC50 of each oil sample, calculated with the following formula: The results were statistically evaluated by Student’s 50 value takes into consideration the intrinsic contribution of the compound to the cytotoxic effect of the phytocomplex and allows the evaluation of its relative potency in the context of the phytocomplex. The resulting values were used as variables in the matrix, and are the vectors in the biplot graphs of principal components, while the samples of the different Pistacia species represent the observations in the matrix and the points in the graph. The clusters of molecules in a given species were identified in the graphs according to four vector characteristics: correlation with the Pistacia species, positive distribution with respect to one or more given principal component, distance from the origin, and angle widths between vectors.Correlating the amount of each compound with the relative ICPistacia. The phytocomplexes in essential oils from four different species of Pistacia  consistently, though differently, induced cytotoxicity on the three cell lines of human adenocarcinoma, with variable potency and tropism depending on the species, and no significant difference among samples from different aerial parts. In general, this suggests that the in vitro anti-tumoral effect is a shared feature of the Pistacia genus, although there are differences that also indicate patterns of activity characteristic for each species and for each adenocarcinoma.This is the largest study conducted to investigate the in vitro anti-tumoral effect of different species from the genus 50 of the considered essential oils. Merging the chemical composition data and the results of the biological assay by a multivariate approach offers a new approach in the evaluation of the bioactivity of these complex mixture. This approach allows the identification of clusters of compounds that, when present together in the mixture, are correlated with increased bioactivity.PCA was used to investigate the correlations between the molecular components of the phytocomplexes, most of them terpenes, sesquiterpenes, and fatty acids, and the observed biological activity. This approach appears to be favourable because it takes into consideration the relative contribution of each compound of the phytocomplex to the final effect on the tumoral cells creating a correlation with the measured ICMolecular cooperation is the most important aspect of the biological activity found in natural products, where a holistic approach is needed to understand their therapeutic potential. This was confirmed in our PCA study and we found several clusters of bioactivity-associated molecules in each species, likely to specifically synergize on each cell line. Two such clusters were shared for the three adenocarcinomas, indicating the presence of at least two common pathways of cytotoxic activity, while all the others were different, thus suggesting specific cell line sensitivity and different species-specific mechanisms observed in vitro. Among the molecules identified only about 20% were compounds already known for their anti-tumoral effect as single drugs, while the others were novel findings. Among the new molecules identified, mostly terpenes, bornyl acetate showed significant cytotoxic activity even when used alone, but to a much lesser extent if compared to the dose range present in the essential oil, thus highlighting the importance of cooperation between different molecules in the phytocomplex to achieve the final effect.


The ability of an organism to respond to physical stresses and return to homeostasis (i.e. resilience) has been suggested to correlate with longevity. Here, we investigated whether this extends to resilience at a cellular level using primary fibroblasts isolated from tail skin of genetically heterogeneous young adult UM-HET3 mice. Cells isolated from each individual mouse (cell line) were tested in their response to concentrations of agents or conditions predicted to induce a cellular challenge, including paraquat, hydrogen peroxide, antimycin a, cadmium chloride, mdivi-1, thapsigargin, and nutrient starvation. Cell viability was monitored in real-time using an incucyte S3 live cell analysis system and we addressed the response following challenge as a marker of resilience. Cellular uptake of ethidium homodimer-1 was used to determine the loss of viability. Cellular bioenergetics were assessed using a seahorse XF24. We found that cell lines that were resistant to paraquat were also resistant to antimycin a, and hydrogen peroxide. Cell lines that were resistant to nutrient starvation were also resistant to mdivi-1. Indices of cellular bioenergetics status including ATP production rate and cell respiratory control ratio, revealed potential relationships with resiliency. Taken together, our data indicate that skin fibroblasts retain individual physiological programs that may in part explain the patterns of resiliency or sensitivity to a stressor at the organismal level. Since the cell lines tested in this study were obtained from living mice, future work will investigate whether these patterns of resiliency change with age and elucidate their utility in predicting future health outcome.


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