Some head and neck squamous cell carcinomas (HNSCC) have a distinct aetiology, which depends on the presence of oncogenic human papilloma virus (HPV). Also, HNSCC contains cancer stem cells (CSCs) that have greater radioresistance and capacity to change replication dynamics in response to irradiation compared to non-clonogenic cells. Since there is limited data on CSCs in HNSCC as a function of HPV status, better understanding of their radiobiology may enable improved treatment outcome.Baseline and post-irradiation changes in CSC proportions were investigated by flow cytometry in a HPV-negative (UM-SCC-1) and a HPV-positive (UM-SCC-47) HNSCC cell line, using fluorescent staining with CD44/ALDH markers. CSC proportions in both irradiated and unirradiated cultures were compared for the two cell lines at various times post-irradiation. To assess repopulation of CSCs, untreated cultures were depleted of CD44+/ALDH+ cells and re-cultured for 3 weeks before flow cytometry analysis.nd generation and at 3 days after irradiation in the 3rd generation. When measured after 10 days, only the 3rd generation of UM-SCC-1 showed elevated CSCs.CSC proportions in untreated cell lines were 0.57% (UM-SCC-1) and 2.87% (UM-SCC-47). Untreated cell lines depleted of CD44+/ALDH+ repopulated this phenotype to a mean of 0.15% (UM-SCC-1) and 6.76% (UM-SCC-47). All UM-SCC-47 generations showed elevated CSC proportions after irradiation, with the most significant increase at 2 days post-irradiation. The highest elevation in UM-SCC-1 CSCs was observed at 1 day post-irradiation in the 2CSC proportions in both cell lines were elevated after exposure and varied with time post irradiation. UM-SCC-47 displayed significant plasticity in repopulating the CSC phenotype in depleted cultures, which was not seen in UM-SCC-1. Head and neck cancers comprise epithelial tumours of the mucosal linings of the oral and nasal cavities, the tongue, paranasal sinuses, salivary glands as well as the pharyngeal and laryngeal areas. Squamous cell carcinoma makes up around 90% of these cancers  which haRisk factors for head and neck cancers include tobacco and alcohol consumption and in countries across South East Asia and the Indian sub-continent, the chewing of betel quid , 7. Of lHead and neck squamous cell carcinoma (HNSCC) are typically aggressive cancers, often involving surrounding normal tissue. Management usually involves a multidisciplinary approach where radiotherapy is a principal intervention. The radiation dose is delivered using conventional or altered fractionation schedules and conformal treatment techniques, e.g. intensity modulated radiotherapy (IMRT), designed to minimise normal tissue complications while aiming for optimal tumour control .HNSCCs contain complex heterogeneous populations where cells demonstrate varied phenotypes and sensitivities to chemotherapy and radiotherapy. A sub-population among these cells has attributes analogous to those of stem cells in normal tissue in that they can self-renew indefinitely and generate other more differentiated cells of the tumour population , 12. TheThe proportion of CSCs in untreated tumours may typically be around 1–10% but this can vary greatly between cancer types, tumours of the same cancer type, and even within the same cancer cell line subjected to different treatments . There iSeveral different markers, both cell surface and functional, have been used to identify CSCs in tumour populations by studies of tumorigenicity using limiting dilution assays –30. ClusIn vitro experiments were conducted in order to identify and measure baseline CSC proportions by the CD44+/ALDH+ phenotype for the two untreated HNSCC cell lines. We also investigated the capacity of CSCs to repopulate in cultures sorted to be negative for the CD44+/ALDH+ phenotype. Finally, time-dependent changes in CSC proportions in surviving cell populations post X-ray irradiation were also investigated in post-irradiated cell generations of each cell line.This study was conducted as a pilot, employing the novel construct of generational cultures of cell lines to test radiobiological responses among the clonogenic population of cell lines as a function of HPV status. Consequently, at this initial stage, representation of HPV status in HNSCC was limited to 1 cell line of each, representing some of the most common presentations of HNSCC and typically, presenting in the same clinic, that would be treated by the same protocol. This experimental work investigated: a) baseline proportions of CSCs in two HNSCC cell lines; b) changes in CSC proportions following depletion by cell sorting; c) changes in CSC proportions post irradiation with 4 Gy X-ray. This dose was selected over the more conventional 2 Gy dose, used as a fractionated dose in HNSCC treatment, to provide a clear cellular response to a smaller number of subsequent exposures. Additionally, the temporal aspect of changes in CSC proportions in HNSCC cell lines after subsequent irradiations was investigated. The Human Research Ethics Committee of the University of South Australia has approved this study. Approval number 0000035359. This study only used commercially purchased laboratory cell lines.st generation. When exposed to 4 Gy X-ray, then re-cultured and passaged, this then becomes the next generation (2nd generation) and so forth for subsequent exposures. This provides for comparison within cell lines as to altered responses after repeated exposures where the unirradiated cells (1st generation) are the control .A. UM-SCC-1 was isolated from a recurrent squamous cell carcinoma in the mouth floor of a 73-year-old male. The cell line originated from the laboratory of Dr. Thomas Carey at the University of Michigan and is negative for the Human Papilloma Virus (HPV).B. UM-SCC-47 is also a squamous cell carcinoma but derived from a primary tumour of the lateral tongue. This cell line also originated from the laboratory of Dr. Thomas Carey at the University of Michigan and is HPV positive for HPV type 16. Oncogenicity is conferred through the expression of viral oncoproteins E6 and E7 .® Darmstadt DE) as a monolayer using RPMI 1640 medium (Sigma-Aldrich® Darmstadt DE) supplemented with 10% foetal calf serum (FCS), 10 mM HEPES, 12.5 μg/ml penicillin and 16 μg/ml gentamycin. Cell flasks were incubated in a humidified atmosphere at 37°C containing 5% CO2 and passaged after reaching exponential growth prior to confluency. Both cell lines were tested for the presence of mycoplasma  and found to be negative.Cell lines were cultured in T75 flasks (Sigma-AldrichCells were stained for both CD44 and ALDH to identify elevated expression of both markers among viable cells. In this study HNSCC CSCs were identified as CD44+/ALDH+ cells.® 450 (affymetrix eBioscience Santa Clara Ca.). Isotype control for this analysis was performed using Rat IgG2b K Isotype Control eFluor® 450.CD44 expression was measured using the fluorescent monoclonal antibody, Anti-Human/Mouse CD44 eFluor™ (STEMCELLTM Technologies Vancouver BC.) was used as per manufacture’s protocol to determine ALDH activity. In the presence of ALDH, the substrate BODIPY-aminoacetaldehyde (BAAA), is converted to BODIPY-aminoacetate (BAA) proportional to ALDH present and is retained in cells. The intensity of the fluorescent expression in cells from this stain is proportional to the cellular ALDH activity. Negative control for this reaction was provided by diethylaminobenzaldehyde (DEAB) to cover background fluorescence at 1 μL in 100 μL cell suspension.ALDEFLUOR7-Aminoactinomycin D (7-AAD)  was used to distinguish between viable and dead cells.™ and DEAB. After incubation at 37°C for 45 minutes, cells were then stained for CD44 and isotype and incubated at 4°C for 30 minutes before staining with 7-AAD, followed by flow cytometry analysis.Cells were initially stained with ALDEFLUORst generation cells of both UM-SCC-1 and UM-SCC-47 were analysed in triplicate by flow cytometry . As mentioned above, cells stained for DEAB and CD44 isotype act as negative controls for ALDH+ and CD44+, respectively, and were used to determine preliminary gating thresholds; additionally non-viable cells were excluded from the analysis.To determine baseline proportions of CSCs in unirradiated cultures, stained 1™ II . Sorted cells from both cell lines, absent of the CD44+/ALDH+ CSC phenotype (CD44-/ALDH-), were re-cultured in T75 flasks for 3 weeks. Flow cytometry was then used to re-measure the same phenotype populations following re-culture and passaging, and the CSC proportions were compared with baseline measurements.In this experiment, repopulation of the CSC phenotype has been investigated in both cell lines following CD44+/ALDH+ depletion by cell sorting. First generation UM-SCC-1 and UM-SCC-47 cells were stained and sorted into phenotypic groups by fluorescence activated cell sorting using a BD FACS Ariast, 2nd and 3rd) from both cell lines were irradiated with 4 Gy. One flask was used for flow cytometric analysis and the other re-cultured to grow the subsequent generation for later re-irradiation and analysis. Additionally, a flask of 1st generation cells was unexposed and used as a sham irradiated control. Irradiation was performed using a 6 MV X-ray beam from a Varian 600C/D linear accelerator  at the Radiation Oncology Department of the Royal Adelaide Hospital. The linear accelerator was calibrated using IAEA TRS398 protocol [™ device  before each radiation treatment. Flasks were positioned on top of 13 mm of solid water  directly above the isocentre of the beam and irradiated with the gantry at 180° to achieve an electronic equilibrium at the cell layer  was used to disassociate adherent cells for flow cytometry analysis with optimal antigen retention [g for 5 minutes  before resuspension and replating then incubated for 24, 48 and 72 hours. At these time points, cells were again disassociated before counting by haemocytometer then aliquoted to 8 flow cytometry tubes, for staining of each generation and control, at 2 × 105 cells per tube. Resulting CSC proportions were compared with baseline values.Following irradiation, TrypLEetention . Cells wIn order to investigate the temporal behaviour of any observed changes in CSC proportions, irradiated generations of both cell lines were also examined for CD44+/ALDH+ fractions by flow cytometry at 10 days post irradiation against matching controls. Controls as well as 2nd and 3rd generations were stained, as described above, to measure CSC phenotype proportions after this latent period for comparison with those of the same culture and treatment after only 3 days.Flow cytometry counting of CD44+/ALDH+ populations were analysed using FlowJo software  to establish final gating and positive phenotype proportions in each sample. Results from FlowJo were analysed using Prism7.01 . Values from triplicate analysis were averaged and reported as mean and standard error of the mean (SEM). Significance of difference for cell sorting and re-culture were tested by one-way ANOVA and Sidak’s test. For flow cytometry post 4 Gy irradiation, significance was calculated using two-way ANOVA and multiple comparisons between generations performed using Tukey’s test. Flow cytometry results post 10 days were tested by one-way ANOVA with Tukey’s tests for multiple comparisons. Significance was considered to be at p < 0.05 .Triplicate analysis by flow cytometry of non-irradiated UM-SCC-47 cell cultures showed a mean population of CD44+/ALDH+ cells to be 2.87 ± 0.219, 5-fold that of the UM-SCC-1 population which was 0.57% ± 0.077 see .After cell sorting to deplete CD44+/ALDH+ cells, the repopulation of CD44+/ALDH+ cells was analysed by flow cytometry following a 21-day culture period . The meaBoth cell lines showed increases in the proportion of CD44+/ALDH+ cells in the surviving populations at each of the 3 time points for both generations of irradiated cells . The extnd generation with an approximately 6-fold increase, which diminished over the subsequent 3 days post-irradiation. A contrary response was observed in the 3rd generation of UM-SCC-1 where the CSC population increased to a level around 6-fold that of the control at 72 hours. UM-SCC-47 showed the greatest elevation in CSC proportions at 48 hours for both the 2nd and 3rd generations where the observed increase was approximately 4-fold. UM-SCC-47 showed a greater number of significant results across the 3 time points for both exposed generations than UM-SCC-1 but less intergenerational difference.The characterisation of CSC increases also varied between the cell lines. UM-SCC-1 showed early significant increases in the 2rd generation showed significance in elevation of CD44+/ALDH+ compared to the control after parallel passage over 10 days. The 2nd generation showed only a slight, non-significant elevation  against ALDH expression (x axis) for both cell lines at each time point. Upper right quadrants show percentages of cells positive for both CD44 and ALDH, which are putative CSCs.(TIF)Click here for additional data file.


Approximately 18–20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies.TM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2– breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor.A new biosensor-based test  contains supplementary material, which is available to authorized users. Molecularly targeted therapies represent a major advance in cancer treatment. Amongst the most consequential therapies are those targeting human epidermal growth factor receptor 2 (HER2). HER2 overexpression or gene amplification is associated with more aggressive disease progression, metastasis, and a poor clinical prognosis in breast and gastric cancer , 2. CurrHER2 gene copy number and trastuzumab benefit and that a sub-group of HER2- breast cancer patients inadvertently included in a trial intended for HER2+ patients benefited from HER2-targeted therapies / (CF-C)]*100.CELx test data was exported from the RTCA software file for the time versus Cell Index (CI) analysis by TraceDrawer  and Microsoft Excel. The cell index versus time course data essentially fell into one of 3 groups for each cell sample tested: cells with addition of media only (C), cells with addition of growth factor stimulus only (CF), and cells with addition of an antagonist drug followed by a growth factor stimulus (CDF). To permit inter-sample quantitative comparison, the cell index was set to zero for each set of CI versus time course data at the time point of stimulus addition to a cell sample. After the stimulus was added, data were assessed using the CI versus time data by one of the following algorithms:P < 0.05 was considered statistically significant.All dose–response curves were obtained using nonlinear regression curve fitting with GraphPad Prism 6 . Pearson correlation analysis was performed using GraphPad Prism 6 to evaluate the relationships among the variables of interest. +, Claudin4+) and basal  markers as well as estrogen receptor (ER) and progesterone receptor (PR) was performed on the primary samples to confirm epithelial cell identity and that fibroblast content was low. Fluorescence flow cytometry was also used to assess protein expression levels of the cell lines and primary cells used in this study. Antibodies used in this study are described in Additional file Flow cytometric analysis of luminal , reflecting the balance of settling, adhesion, spreading), lag (plateau and stabilization), logarithmic growth (proliferation), and formation of a cell (mono) layer Fig. . Human bCell seeding density is a critical factor in establishing a useful dynamic range for CI values that encompass the spectrum of attachment values observed using different cell lines. The results indicated that 12,500 to 15,000 cells per well in a 96-well format sensor plate is the ideal seeding density, allowing cell-cell contacts that are required for authentic epithelial cell signaling. No significantly proportional increase in CI values was seen when higher densities of cells  were used. Thus, a seeding density of 15,000 cells per well provided a balance between signal magnitude and cell conservation when considering data from numerous breast cancer cell lines and primary cells.50 for EGF is 74.1 pM  , lapatinib (200nM), or vehicle (control buffer) 18 h prior to stimulation with growth factors (NRG1 or EGR). As shown in Fig. al) Fig. . In contal) Fig. . The mea/mL Fig. , with a A panel of pharmacological inhibitors that specifically inhibit different points in the PI3K and MAPK pathways was tested in order to determine which pathway(s) was critically involved in NRG1b- and EGF-directed HER2 signals in breast cancer and thereby specific cellular responses in our CELx HSF tests.Dose–response curves of inhibitory effects of GSK1059615, a selective PI3K inhibitor , on ligaTrametinib, a specific inhibitor of MEK1/2, was also tested for the effect on inhibition of the MEK/ERK pathway on ligand-driven HER2 signals . The resGrowth factor receptor / receptor tyrosine kinase (RTK) signaling networks share many common features, such as interactions among ligands, antagonists (receptor inhibitors), and RTKs, receptor phosphorylation / activation, and activation of downstream pathways. All these factors could contribute to the CELx signals. Verification of the specificity and selectivity of the CELx HSF test was performed by evaluating whether the test response identifies solely HER2-related activity when HER family ligands are applied to the test cells. Additionally, testing was performed to determine whether the activity of antagonists at HER family receptors affects growth factor activity on other receptors and whether antagonists applied to other receptors affected growth factor activity on HER family receptors during the test. For an example of evaluating CELx for receptor cross-talk, the network profile of HER2 signaling was compared with that of insulin-like growth factor 1 receptor (IGF-1R) by utilizing specific agonists and antagonists for IGF-1R in the CELx assays. Using the T47D breast cancer cell line, IGF-1 induced substantial CELx signals through IGF-1R with an average Delta CI of 0.4 Fig. . As an aHER2 gene expression recorded in public databases such as the Cancer Cell Line Encyclopedia (CCLE) [HER2 gene copy number and MDA-MB361 had more than 2.2 copies per cell. Another recent study indicated that MDA-MB361 had amplified gene copy number and would qualify as a clinical HER2+ [After confirming the selectivity and specificity of the CELx HSF test, ligand-driven HER2 signals were surveyed in 10 human breast cancer cell lines overexpressing HER2 (HER2+) and 10 human breast cancer cell lines expressing lower or normal levels of HER2 (HER2-) in order to determine whether CELx HSF test positive (HSF+) and CELx HSF test negative (HSF-) populations exist among HER2+ and HER2- cell types. These cell lines were chosen based on a (CCLE) . Here anal HER2+ . The HERn = 9) and HER2- (n = 10) breast cancer cell lines in the presence and absence of pertuzumab. EGF and NRG1b are both capable of initiating signaling of HER family homodimers and heterodimers without HER2 participation. The antibody pertuzumab’s mechanism of action for disruption of ligand induced signaling is by binding to HER2 and prevention of HER2 dimerization with other HER family members. When pertuzumab was applied to the different cell samples, the results showed different levels of attenuation of EGF and NRG1b signals depending on the cell line. The variable attenuation by pertuzumab is related to the amount of HER2 participation in each growth factor initiated signaling for each of the different cell samples. Thus pertuzumab is an appropriate tool for the determination of HER2 participation in signaling activity measured by the CELx HSF Test and was used for subsequent data analyses. Results for ligand-driven HER2 CELx signals from all HER2+ and HER2– cell lines are presented in Fig. The CELx HSF test was used to determine the amount of HER2 participation in NRG1b- and EGF-driven activity in the HER2+ , which further supports the conclusion that HER2 pathway activity can be independent of HER2 expression status.As further confirmation of the CELx HSF test results for AU565 and BT483, their responses to pertuzumab and lapatinib were evaluated. The evaluation focused on data for NRG1b-driven signaling with these drugs given the results showing the primary importance of this mechanism in HER2 signaling. NRG1b-driven CELx signals and sensitivities to these drugs are presented in Fig. Overall, when comparing the magnitude of HER2 ligand driven signaling activities determined by CELx HSF tests, there existed at least four subtypes of cell lines, including HER2+/HSF+ , HER2+/HSF- , HER2-/HSF+ , and HER2-/HSF- . The CELx curves characteristic of each subtype are shown in Fig. Following initial results with well-established reference cell lines, CELx HSF tests were applied to primary epithelial cells derived from two patients with breast cancer and one healthy control subject as further proof-of-concept. Typical flow cytometry results for primary samples in short-term, zero passage culture confirmed a heterogeneous population of myo and luminal epithelial cells and low stromal cell content  further demonstrated that fresh patient-derived cells could produce sufficient signal upon stimulation, confirming the pathway deconvolution results in a more physiologic setting Fig. . ConsistAccurate determination of HER2 status is critical for optimizing use of HER2-targeted therapies and improving therapeutic outcomes. Existing HER2 tests (either IHC or FISH)  only proBreast cancer cell lines have been widely used as model systems for studies on breast cancer pathobiology and new therapy development –41. NeveFollowing the cell line work, three different samples of primary cells were analyzed to demonstrate the feasibility of applying the CELx HSF test to clinical specimens. For the clinical specimen, FACS data first established that cultured primary cells derived from fresh patient tumor tissue were of the epithelial type with stromal content typically 5% or less. Several biomarkers that define luminal and basal types of epithelial cells were used . The tumReliability, analytical specificity, sensitivity, and accuracy are essential prerequisites for the CELx HSF test to be considered for clinical diagnostic applications. When performing label-free biosensor-based viable cell assays, complexity is inherent and caution was exercised to test whether the signal was limited to a biological response resulting from a single molecule type binding to a single receptor type effecting signaling on a single pathway. In this study, a series of experiments were performed to demonstrate the selectivity and specificity of the assay for cell lines and primary cells.FDA-approved HER2 inhibitors that treat HER2-positive breast cancer in clinical settings were used in this study to serve three purposes. First, the inhibitors helped to identify the specificity of the impedance signal arising from treatment of the cells with growth factors. Second, the anti-HER2 mAb inhibitors isolated the impedance signal arising solely due to HER2 participation in the growth factor activation of HER family pathway signaling. This provides a level of detail regarding the specificity of the selected reagents by using antagonists that work most proximal to signal initiation, receptor dimerization and receptor tyrosine kinase priming, thereby most effectively defining HER2 participation and isolating early signaling events before signal branching takes place. Finally, previous studies suggest differential sensitivities to the HER2 inhibitors among the cells lines used here . Thus, t1 [The data for testing baseline effect of pertuzumab or lapatinib alone on cells Fig. , Panel a1 , 44.Trastuzumab was not selected for evaluation in this study because its primary mechanism of action, as reported by its manufacturer, is not HER2-driven signaling inhibition, but instead antibody-dependent cell-mediated cytotoxicity (ADCC). Any results studying the effect of trastuzumab on HER2-driven signaling would thus be confounded by the lack of direct linkage between the activity we are measuring, HER2 signaling, and trastuzumab’s primary mechanism of action (ADCC). Since the CELx HSF Test is designed to assess HER2 participation in HER family signaling, pertuzumab, a known HER2 dimer blocker, was selected instead to confirm the amount of HER2 participation in HER family signaling in this assay.in vitro receptor affinity of ~100pM for EGF and NRG1b [All HER2 CELx signals tested are agonist- and antagonist-concentration dependent within physiological doses in the picomolar to nanomolar range. When a HER2 antagonist (e.g. pertuzumab or lapatinib) is added with agonist, the cells show a significantly attenuated delta CI compared to the signal for addition of agonist only, indicative of a blocked HER2 signaling response. The work employs carefully selected components that have known specificity and well characterized affinity at concentrations that reduce the likelihood of activation of other pathways from high concentrations of agonists. EGF and NRG1b are very specific ligands for HER1 and HER3 receptors. Multiple literature references cite nd NRG1b , 46. ThiFurther dissection of the information from rich CELx data suggests sources of NRG1-driven test signal that is linked to more than just HER2/HER3 heterodimerization. In the SKBr3, HER2+ cells . Although protein or gene based assays provide incremental information, they are still classical endpoint assays that reflect only the relative activity of a limited set of proteins that may be involved in disease propagation, and the results do not describe the dynamics or real-time status of the complete HER2 signaling network in a particular patient. Given that infinite permutations of circumstances are present and each persons’ genomic or proteomic status does not yet describe the  patient , 58, 59. patient . They co patient . Further patient , 58, 59.n = 9) and HER2- (n = 10) breast cancer reference cell lines were chosen. Fluorescence flow cytometry measurement of HER2 protein expression levels demonstrated HER2 expression data largely consistent with published CCLE data on HER2 gene copy number in these cell lines [in vitro. Recent retrospective analyses of previous clinical trials indicated that there is no significant correlation between HER2 gene copy number or total HER2 protein and clinical benefit from trastuzumab [To verify the CELx HSF test concept, HER2+  and CLIA  established guidelines for Laboratory Developed Tests would be required. Finally, the clinical validity of using HER2-driven signaling activity as a diagnostic biomarker must be confirmed in a clinical trial that evaluates whether HER2-breast cancer patients with abnormal HER2-driven signaling benefit from treatment with HER2 signal inhibitors.HER2 receptor levels do not correlate with the functional activity measured by the CELx test. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker with the CELx test to identify HER2-driven cancers not currently diagnosable with IHC or genomic techniques. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy.


Osteosarcoma is the most common and aggressive bone tumor in children. The Endocannabinoid/Endovanilloid system has been proposed as anticancer target in tumor of different origins. This system is composed of two receptors (CB1 and CB2), the Transient Potential Vanilloid 1 (TRPV1) channel and their ligands and enzymes. CB1 is expressed mainly in central nervous system while CB2 predominantly on immune and peripheral cells. We investigated the effects of JWH-133 (CB2 agonist) and RTX (TRPV1 agonist) in six human Osteosarcoma cell lines: MG-63, U-2OS, MNNG/HOS, Saos-2, KHOS/NP, Hs888Lu, by Apoptosis and Migration-Assay. We also compared the effects of these compounds on Caspase-3, AKT, MMP-2 and Notch-1 regulation by Q-PCR and Western Blotting.We observed an anti-proliferative, pro-apoptotic, anti-invasive effect. Our results show that both CB2 stimulation and TRPV1 activation, in different Osteosarcoma cell lines, can act on the same pathways to obtain the same effect, indicating the Endocannabinoid/Endovanilloid system as a new therapeutic target in Osteosarcoma. Osteosarcoma (OS), the most common primary malignant tumor of bone, affects predominantly children and adolescents, exhibiting high invasion and metastasis rate , 2. Due The cell of origin of OS, is still far from clear. During the last 10 years mounting evidence has placed mesenchimal stem cells (MSCs) and/or their immediate lineage progenitors as the most likely cell-of-origin for many types of sarcomas including OS –11.The Endocannabinoid/Endovanilloid (EC/EV) system has been proposed as an anticancer target by several studies –26. ThisHowever, we cannot exclude the effects that these compounds may induce, activating TRPV1 receptor channel. When TRPV1 channel proteins are activated, they mainly cause the calcium ion influx. Intracellular calcium overload mediate cell apoptosis through different mechanism interfering with cell energy production and metabolism, therefore also drugs acting on the TRPV receptors can act as potential target to reduce cell proliferation and survival in cancer –39.Recent studies described the role of EV/EC system in remodelling, differentiation, survival and activity of bone cells. In particular, we have demonstrated a functional cross-talk between CB2 and TRPV1 in human osteoclasts. TRPV1 stimulation, exerts pro-osteoclastogenic effects, whereas CB2 stimulation promotes anti-osteoclastogenic events, suggesting these receptors as new pharmacological target for the treatment of osteoporosis –34. MoreBased on these evidences, we investigated for the first time the expression of CB2 and TRPV1 receptors and the effects of JWH-133 and RTX, (agonists of the EC/EV system) at different concentrations, in six OS cell lines , analyzing the response to these compounds on cell survival, invasion and migration capacity. We observed an anti-proliferative, pro-apoptotic and anti-invasive effect induced by EC/EV compounds in the studied OS cell lineages.We performed a Real Time PCR to evaluate the expression of EC/EV system in untreated MG-63, U-2 OS, MNNG/HOS, Saos-2, KHOS/NP and Hs888Lu prior to the treatments, to be sure the receptors we were going to stimulate, were present. The result demonstrated the presence of mature mRNA for CB2 and TRPV1 receptors , in all cell lines except Saos-2. The reduction in cells migration was statistically significant Figure .To evaluate the possible role of EC/EV drugs on cell invasion and migration we analyzed the expression levels of Notch-1 and MMP-2, after RTX [5 μM] and JWH-133 [100 nM] treatments. The incubation with RTX in the first 24 hours of exposure reduced significantly the expression of Notch-1 in MNNG/HOS and MG-63, while treatment with JWH-133 down regulated this gene in all cell lines except Saos-2. Also MMP-2 showed a significant decrease in all cell lines studied except for Saos-2 after JWH-133 exposure. RTX treatment reduced MMP-2 expression in MNNG/HOS, KHOS/NP and MG-63 in a statistically significant manner Figure .in vitro models as long as they are representative of the original tumor , RTX  and cells were harvested at 24h for mRNA isolation, protein extraction, and Muse® “Annexin V and Dead Cell Assay Kit” (Millipore).OS MG-63, U-2 OS, MNNG/HOS, Saos-2, KHOS/NP and Hs888Lu cell lines were purchased from Sigma Aldrich. The MG-63, KHOS/NP, MNNG/HOS cell lines were cultured in EMEM medium with 1 % Non Essential Amino Acids (NEAA), the Saos-2 and the U-2OS cell lines were cultured in McCoy's medium, the Hs888Lu cell line was cultured in DMEM medium. The complete culture medium containing 10% fetal bovine serum (FBS), supplemented with 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco) and 2mM L-glutamine (Euroclone). Cells were culturedat 37°C in a humidified atmosphere with 5% COResiniferatoxin  and JWH-133 (Potent CB2 selective agonist. Approx. 200-fold selective over CB1 receptors) were purchased from Tocris Bioscience . The powder was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50mM for JWH-133. RTX was dissolved in DMSO to a stock solution of 100mM. DMSO final concentration on cell cultures was 0.01%. Stock solutions were aliquoted and kept at −80°C for long-term storage. OS cell lines were treated with RTX  and JWH-133 . For real time and western blotting studies EC/EV compounds were added alone at concentrations of RTX [5 μM] and JWH-133 [100 nM]. We choose only these treatments considering the results obtained in the apoptosis assay. Non-treated cultured cell lines were maintained in incubation media during the relative treatment time with and without vehicle (DMSO 0.01%).Apoptosis has been evaluated by a fluorometric assay on the Muse cell analyser machine with the “Cell dead and Annexin V Assay Kit”. Test was performed after 24 h of EC/EV compounds exposure. EC/EV active compounds were added alone at the following concentrations: JWH-133 , RTX . The Muse™ Annexin V & Dead Cell Assay utilizes Annexin V to detect phosphatidylserine (PS) on the external membrane of apoptotic cells. A dead cell marker, is also used as an indicator of cell membrane structural integrity, 7-amino-actinomycin D (7-AAD). Briefly, 100μL of a cell suspension (1 × 104 cells/mL) was mixed with100 μL of Muse™ Annexin V & Dead Cell Reagent and incubated for 20 minutes at room temperature in dark. The results, automatically displayed, were analyzed with “Muse 1.4 Analysis” software for data acquisition and analysis.Following treatment with JWH-133 [100 nM] and RTX [5 μM] and incubation for 24 h at 37°C with 5% CO2, OS cells were harvested. Cells without treatment served as the control group. The total RNA was extracted using Quiazol® (Quiagen) following the manufacturer's instructions. EasyScript™ cDNA Synthesis Kit (abm) was used to synthesize from approximately 1000ng mRNA, the first strand cDNA. The transcript levels of Notch-1, matrix metalloproteinase-2 (MMP-2) and Caspase-3 (CASP3) were detected by RT-qPCR using a CFX96 Real-Time PCR system (Bio-Rad) using I-Taq Universal SYBR® Green Master Mix (Bio-Rad). The cycling conditions were 10 min at 95°C  followed by 40 cycles of 15 sec at 94°C (denaturation) and 1 min at 68°C . The β-Actin gene served as the reference gene for the normalization of the real-time PCR products. The PCR primers used to detect each gene were designed using Primer 3 program and synthesized by Sigma Aldrich . The linearity and efficiency of the assays were tested over dilutions of input cDNA spanning five orders of magnitude. Assays were performed in triplicate. The dissociation curve analysis of amplification products was performed at the end of each PCR reaction to confirm the specificity of the amplification. The 2Osteosarcoma MG-63, U-2 OS, MNNG/HOS, Saos-2, KHOS/NP and Hs888Lu cell lines were seeded in a six well plate (500.000 per well). Incubated 24 hours to let them attach in monolayer in the wells. A single scratch was made in each well with a 200 μl sterile pipette tip. The well has been washed with PBS and fresh media containing RTX [5 μM] and JWH [100nM] has been added. Fresh medium without any drug was used as control (Labelled as NT: Non-Treated). Images were taken 24 hours later with an AE2000 microscope (Motic) and analyzed with Motic Images plus 2.0 Software.Proteins were extracted from treated and non-treated OS cell lines using RIPA Lysis Buffer (Millipore) and following the manufacturer's instructions. Akt, pAKT and Caspase-3 proteins were characterized in total lysates from cell line cultures by Western blotting. Membranes were incubated overnight at 4°C with rabbit polyclonal anti pAKT antibody , rabbit policlonal anti Akt antibody  and rabbit anti Caspase-3 antibody . Reactive bands were detected by chemiluminescence (Immobilon western Millipore) on a C-DiGit ® Blot Scanner (LI-COR Biosciences). A mouse polyclonal anti β-Tubulin antibody  was used to check for comparable protein loading and as a housekeeping protein. Images were captured, stored, and analyzed using “Image studio Digits ver. 5.0” software.t test to evaluate differences between quantitative variables. A p value less than 0.05 (*) or 0,01 (**) were considered statistically significant.Results are expressed as means ± S.D. The experiments were run in triplicate. Statistical analyses were performed using Student's


Cell lines and cell types are extensively studied in biomedical research yielding to a significant amount of publications each year. Identifying cell lines and cell types precisely in publications is crucial for science reproducibility and knowledge integration. There are efforts for standardisation of the cell nomenclature based on ontology development to support FAIR principles of the cell knowledge. However, it is important to analyse the usage of cell nomenclature in publications at a large scale for understanding the level of uptake of cell nomenclature in literature by scientists. In this study, we analyse the usage of cell nomenclature, both in Vivo, and in Vitro in biomedical literature by using text mining methods and present our results.We identified 59% of the cell type classes in the Cell Ontology and 13% of the cell line classes in the Cell Line Ontology in the literature. Our analysis showed that cell line nomenclature is much more ambiguous compared to the cell type nomenclature. However, trends indicate that standardised nomenclature for cell lines and cell types are being increasingly used in publications by the scientists.https://github.com/shenay/CellNomenclatureStudy.Our findings provide an insight to understand how experimental cells are described in publications and may allow for an improved standardisation of cell type and cell line nomenclature as well as can be utilised to develop efficient text mining applications on cell types and cell lines. All data generated in this study is available at  Distinct identification of cell lines and cell types in the literature is important for the reproducibility of studies in order to clearly identify and differentiate information about the cell types or cell lines used in an experiment. The Cell Ontology (CL)  and the Here, we perform a large-scale analysis focusing on cell nomenclature usage in the biomedical literature by using text mining methods. Our aims are to understand how the nomenclature related to cell types and cell lines is used in literature, how both evolved over time, and how this information may allow us to provide better tools and resources for biomedical researchers. To the best of our knowledge, this is the first study with this focus, while several previous studies focused on identifying the cell type and cell line names in text , 6.http://europepmc.org/ftp/archive/v.2017.06/) (~1.5 million) from Europe PubMed Central [Latest archived version of the Open Access (OA) full text articles . We removed a total number of 9 cell type terms and 650 cell type terms from the dictionaries. The final cell type dictionary consists of 3838 term referring to 2180 distinct cell types while the cell line dictionary consists of 76,747 terms belonging to 38,605 distinct cell lines.We used the Whatizit  entity rWe conducted manual analyses on the annotated corpora to evaluate the performance of our text mining approach as well as to understand how the quality of terminological resources on cells like CL and CLO might be improved. To this end, we generated two evaluation sets, each of them comprising randomly selected 50 sentences containing either at least one annotation or a specific keyword, “cell type” and “cell line” for CL and CLO annotation evaluation respectively.Many cell line names consist of only numbers. Many cell line names consist of less than 4 letters Cell line names often look like gene/protein names (e.g. MCF2)Some cell line names look like person names (e.g. Ishikawa)Some cell line names are very generic Annotation errors were due to false positive and false negative cell type/line name callings. Identifying cell lines is a non-trivial problem due to the following challenges:Although we removed several potentially ambiguous terms from the cell line dictionary in the refinement process (see Section 2.1) as they might introduce a high number of false positives into our analyses, we still have false positive annotations of cell lines. For example, ‘ARL6’ is both a cell line name and a gene name leading to a false positive annotation in some of the articles.Missing cell line names  are mainly due to the cell lines which are in CLO’s scope but not yet been incorporated in a CLO release. For example, NCI-60 or LINCS cell lines are currently missing in CLO and cannot be found using our dictionary-based approach. Other missing cell lines are due to their uncovered synonyms by CLO. For example, the cell line name ‘HEK293T’ is not found in literature since the cell line is referred to as ‘293 T’ in CLO and the ontology does not contain the synonym ‘HEK293T’. An additional check revealed that, ‘HEK293T’ exists in the Cellosaurus . Our celCell type nomenclature used for the cell type names is much less ambiguous compared to the cell line nomenclature. Nevertheless, some false positives occur due to overlapping cell type and anatomy terms such as ‘smooth muscle’. Other false positives are due to abbreviations. For example, SMC is used as an abbreviation for ‘Smooth muscle cell’, but in some of the articles, SMC refers to ‘soil microbial community’ . False negatives are mainly due to the cell types which are not yet covered by CL such as cells tagged with cell surface markers, e.g., ‘CD58-positive natural killer cell’, and absent synonyms  cell types and 4907 of 38,605 (13%) cell lines in the open access full text articles.Figures Our analysis can also make artefacts resulting from the evolution of the ontologies visible. The drop in the number of CLO classes in 2011 is due to a restructuring of CLO resulting from the transformation of the original Cell Line Knowledgebase (CLKB)  cell linAs can be seen from Fig. We projected the text mined annotations generated in this study (1277 cell types 59%) and 4907 cell lines (13%)) onto CL and CLO to reveal which branches of the ontologies are well covered and which branches are not referred to in the literature. This analysis is intended to highlight the ontology branches that need to be expanded, either by adding classes or their synonyms, as they are frequently referred to in literature but not available for ontology-based annotations. . Our analysis revealed that, for both CL and CLO, the majority of the classes are referred to in literature by their 10% or less only. Particularly, for CLO which is only 13% of it is referred to in the literature, almost all of the referred classes are represented by their 10% or less. This is potentially a strong signal for the existence of other cell line synonyms or classes preferably used by scientists and need to be added to the ontology. Several poorly represented ‘cell line cell’  classes are shown in Fig. 9% and 49Although, throughout this study, we observed that the terminology underlying CL ontology is better standardised than the terminology underlying CLO, there are still many underrepresented classes which need to be analysed further for their expansion with possible synonyms. For example, as can be seen from Fig. https://github.com/shenay/CellNomenclatureStudy which can be used to prioritise the ontology classes that need to be focused for their further expansion with additional synonyms or sub classes.A complete list of class representation of each individual class is available at The outcomes of our study can provide insights to improve and develop text mining methods so that cell lines and cell types can be captured in literature more accurately, and further lead to guidelines for continuous development of biomedical ontologies as well as recommendations for the use of cell type and cell line nomenclature in literature so that unambiguous understanding, which is a prerequisite for reproducibility, is ensured.. We generated a novel corpus annotated with mentions of cell types and cell lines, which can be used for developing and evaluating text mining methods. For example, our corpus can be used for training of named-entity recognition and normalisation systems that utilise machine learning approaches, as well as for evaluation of existing named entity recognition and normalisation approaches. Furthermore, these datasets can be expanded by using the dictionary-based taggers that we developed, an approach that would be justified based on the high precision our method achieves.Our results will be of most interest to researchers in biomedical text miningOur gold standard corpus may also serve to improve recall by utilizing the positive and negative annotations in the corpus, in a machine learning based annotation tool that learns to distinguish positive and negative occurrences of tokens that may refer to cell types or cell lines based on context. Such an approach would be particularly useful for cell lines as we found the cell line terminology to be highly ambiguous.Our manual analysis further revealed that there are several cell type and cell line names missing in CL and CLO, respectively, which currently might be covered by other resources. Therefore, existing cell line and type resources should be merged to develop a comprehensive dictionary of names for cell biology, which can then be utilised to develop more comprehensive dictionary-based annotation tools.The lack of an authority in cell line naming, or cell line naming conventions, leads to the frequent usage of ambiguous names. This brings limitations to efficient text mining application development.For ontology developers, our most important finding is a set of missing cell type and cell line names and synonyms in CL and CLO. The ontologies can be improved by adding these synonyms and labels, for example by comparing the ontologies’ current content against other available cell type and cell line resources and adding the ones which are covered by the other resources but not by CL or CLO. Furthermore, our analysis shows that scientists sometimes create new names for entities used in their studies without explicitly reusing names already covered by standard resources. Using a machine learning based system to identify cell line and cell type names in text could reveal additional synonyms and new names that can be used for expanding the ontologies.Further manual analyses either on the dictionary-based annotated or machine learning based annotated text would reveal preferred names by the scientist which should be used for refining the existing labels and synonyms in the ontologies. Additionally, our analysis on the distribution of the text mined cell line and cell type annotations based on the ontology classes uncovers the well or poorly represented classes in the literature. Outcomes of such this analysis can be used to refine the terminology used in the ontologies.In the interest of reproducibility of research results, it would be beneficial if authority for naming convention for cell lines would be established. Alternatively, scientists should be encouraged to consider the usage of a given name in their publications if it already exists in standard resources such as the CLO.For a new cell type or cell line which is not covered by standard resources, scientists should consider clear and effective communication while naming their entity. Currently, there is an overlap in names between cell types or cell lines and gene and protein names as well as with names used in other domains, which is a bottleneck in efficient scientific communication and information dissemination in the domain. Further analysis that can be conducted on our results could support new discoveries. For example, text mining can be applied on cell types and cell phenotypes to reveal their relations with diseases such as cancer.Last but not least, scientists, ontology developers and text miners should follow the FAIR principles   to eitheWe performed a large-scale analysis of cell nomenclature usage in the biomedical literature. To this end, we used dictionary-based text mining methods to analyse the terms from CL and CLO in publications. Our approach identifies 59% of the cell type classes in CL and 13% of the cell line classes in CLO in the literature. We find that cell line nomenclature is much more ambiguous compared to the cell type nomenclature. Nevertheless, there is an increasing adoption of standardised nomenclature for cell lines by the scientists.Our study presents insights into the past and current trends for the cell nomenclature usage in the biomedical literature and helps better standardisation of the nomenclature in knowledge bases as well as supports development of text mining applications relevant to cell types and cell lines.In future, we plan to expand our analysis by covering other cell lines and cell types available in resources such as Cellosaurus and CellFinder to gain deeper insights into how CL and CLO might be improved.


Combined γH2AX and SCE analysis was used to evaluate endogenous DNA DSB levels (and their subsequent repair) in 9 primary sarcoma cell lines and compared with well-established commercial lines. All the sarcoma cell lines had elevated γH2AX and SCE levels, but there was no correlation between the DNA DSB frequency and subsequent SCE. Typically, radioresistant osteosarcoma cells had relatively low γH2AX frequency but high SCE counts suggestive of efficient DNA repair. Conversely, liposarcoma cells derived from a radiosensitive tumour had high H2AX but relatively lower SCE levels that may imply inefficient DNA DSB repair. To our knowledge, this is the first report that correlates H2AX and SCE levels in primary sarcoma cell lines and may provide insight into potential response to DNA-damaging treatments.Sarcomas are rare heterogeneous malignancies of mesenchymal origin characterised by complex karyotypes but no specific abnormalities. Recurrence is common, and metastatic disease carries poor survival despite standard DNA-damaging radiotherapy or chemotherapy. DNA double-strand breaks (DSBs) are either repaired by mechanisms such as homologous recombination (HR) or result in cell death by apoptosis. Endogenous  Soft tissue sarcomas (STSs) are a group of rare, heterogeneous malignancies of mesenchymal origin that affect around 2300 people a year in the UK . They coSTSs fall into one of two large genomic classes: The first is characterised by known, specific abnormalities such as chromosomal translocations or gene mutations and comprises around 20% of STSs. The majority of sarcomas, on the other hand, are characterised by complex seemingly random DNA copy number aberrations across the entire genome. They have no known specific abnormalities (a notable exception is well-differentiated liposarcomas that frequently carry chromosome 12q amplification), and their complex karyotypes are believed to be the result of genomic instability, a hallmark of the cancer phenotype .in vitro mitosis. Considered an endpoint of DNA repair by homologous recombination, measurement of SCE is a well-established and sensitive method for detecting DNA damage in the form of double-strand breaks (DSBs) induced by genotoxic agents [Sister chromatid exchange (SCE) analysis is a method that allows the physical quantification of exchanged genetic material between sister chromatids during c agents –9. The cc agents . Once stγH2AX, which accumulates at the sites of damage and recruits other DNA repair proteins. Phosphorylation of the histone protein H2AX is well recognised as an early step in the cellular recognition of DNA DSB for subsequent repair [γH2AX foci at sites of DNA damage can be visualised and enumerated as another measure of DNA repair in eukaryotic cells [γH2AX formation has been observed in human cells where they are believed to be the result of endogenous DNA damage that causes collapsed replication forks and DNA double-stand breaks (DSBs) during mitosis. The frequency of endogenous DNA DSB repair has, therefore, been widely used as a surrogate measure of genomic instability in these cells [An even earlier event in the DNA DSB repair process is the phosphorylation of the histone molecule H2AX at serine 139 to t repair . Using pic cells . Spontanse cells .γH2AX analysis which is done on interphase chromosomes, the specific timing requirement of metaphase spread preparation for SCE analysis makes this method technically challenging, and the majority of published studies utilising SCE analysis have focused on peripheral blood lymphocytes, which have relatively predictable doubling time. The observed frequency of endogenous SCE in normal human tissue and peripheral blood lymphocytes (PBLs) is around 6–8 per cell [Unlike per cell , 16. Eleper cell . Individper cell , with anper cell . Subsequper cell –22.in vitro disease model are increasingly being recognised. Cellular adaptation with prolonged culture in artificial conditions and a lack of cellular heterogeneity that is characteristic of in vivo tumours are important factors that are believed to account for poor correlation of tumour cell line response with clinical outcomes. To ameliorate these important limitations, a widely accepted alternative to commercial cell lines is those directly derived from tumours (primary tumour cells) in early culture with less adaptation to artificial conditions. Ethical and logistic constraints associated with obtaining fresh tumour tissue , variable rates of successful establishment in culture and unpredictable subsequent in vitro behaviour make it rather challenging to utilise them for research in general and SCE analysis in particular.SCE data on cancer cells are derived largely from haematologic malignancies, such as leukaemia, where tumour cells are prominent in the circulation  while onγH2AX and SCE, respectively, as surrogates for genomic instability and compared to those observed in long-established, commercially available sarcoma cell lines, low SCE tumour cells, and nontumour cells.In this study, we assessed endogenous genomic DNA damage/repair in a cohort of primary STS cell lines representing a range of subtypes. The frequency of early and late endogenous DNA DSB repair was measured using Primary STS cell lines were developed from patient samples collected at the Royal Hallamshire Hospital, Sheffield, as previously described . InformeµM 5′-bromo-2′-deoxyuridine (BrdU) for approximately two cell cycles. This duration varied between 3 and 7 days depending on the proliferation rate of the cells, and multiple attempts were required to identify the required duration, particularly for the primary cell lines. Cell cycling was arrested in metaphase by addition of 10 µg/ml colcemid and incubating for 2–4 hours . Chromosomes were then harvested by trypsinization and centrifuged and resuspended in 0.075 M potassium chloride (KCl) at 37°C for 40 minutes before fixing in a 3 : 1 methanol to acetic acid solution. Metaphase chromosome spreads were prepared on cold wet slides before staining with an adapted Perry and Wolff method with incubation in Hoechst 33258 dye for 15 minutes and exposure to UV-light for 12 minutes. Stained metaphases were visualised with a BH-2 light microscope, and images were captured with a Cohu® high-performance CCD camera  and Powergene® software . The number of chromosomes and exchanges in each metaphase spread was recorded. Chromosomes that were clearly overlapping or twisted were excluded from the exchange count, as is consistent with previous reports.SCE analysis was performed as previously described . Cell cuSCE counts were performed on up to 30 harlequin-stained metaphase chromosome spreads  where avγH2AX foci formation was again performed as previously described [γH2AX antibody  diluted 1 : 500 in 10% goat serum in PBS. Cells were subsequently washed on a shaker with PBS, and the coverslips were inverted onto microscope slides and mounted with Vectashield containing DAPI . Slides were stored in the dark at 4°C, and the number of foci per nucleus in 100 cells was counted on a UV spectrum-red fluorescent Nikon image analysis microscope at 100x magnification.Analysis of escribed . Viable Nine primary cell lines, representing four soft tissue sarcoma subtypes, were analysed for SCE . Two of n) karyotype by multiplying observed SCE by 46 and then dividing by the observed chromosome number.Tumour cells were frequently hyperdiploid with significant inter- and intratumour heterogeneity in terms of chromosome number . To accon) metaphase spread, respectively, as expected  that were nearly double those seen among the other sarcoma cell lines  foci by immunofluorescence was used for the detection of endogenous DNA double-strand breaks (DSBs). We performed γH2AX analysis on eight primary STS cell lines. The osteosarcoma (U-2 OS) and retinal epithelial (hTERT-RPE1) cell lines were used as tumour and nontumour cell line controls, respectively. The results were correlated with the corresponding endogenous SCE levels and are summarised in Detection of phosphorylated H2AX . U-2 OS, the osteosarcoma cell line, that had almost double the number of SCEs seen among the other sarcoma cell lines surprisingly had relatively few γH2AX foci (γH2AX foci (>65% of cells showing >10 foci) was observed in dedifferentiated liposarcoma cell lines Shef-DDLPS 01 and Shef-DDLPS 02, whose SCE levels were only moderately elevated with medians of 13 and 14 per 2n metaphase spread, respectively  of the leiomyosarcoma cell line Shef-LMS 01 (γH2AX foci are believed to represent DNA DSBs resulting from replication fork collapse/stress during the cell cycle [ws variant may have been repaired via other cellular mechanisms such as nonhomologous end-joining (NHEJ), which is also preceded by γH2AX formation [In support of the genomic instability suggested by their elevated SCE levels, ol cells . These ring foci , 34. In f-LMS 01  even thof-LMS 01 . Endogenll cycle , while Sormation , 36, or DDLPS 02 that had high endogenous γH2AX foci but relatively low SCE counts  to neoadjuvant radiotherapy that was seen on histologic examination of the parent tumour for this cell line (data not shown). Conversely, the U-2 OS cell line which had approximately double the SCE frequency observed in the other sarcoma cell lines had relatively few endogenous γH2AX foci  would be useful to further elucidate the impact of endogenous genomic instability on treatment response in these sarcomas.Similarly, there was no correlation demonstrated between the frequency of SCE and endogenous H2AX foci among all the cell lines in this study when evaluated by Spearman's test. An illustrative example was the dedifferentiated liposarcoma cell line Shef-E counts . A possi2AX foci , an overin vitro culture were assessed when SCE analysis of four primary cell lines was repeated after 4 months (Temporal changes in genomic instability with 4 months . The resγH2AX and SCE in sarcomas. Given that these tumours are so heterogeneous, it is likely that a number of explanations could exist including highly efficient homologous recombination, increased apoptosis, or alternative DNA repair pathways. Importantly, the stage is set for further studies that will improve our understanding of the mechanisms for genomic instability among sarcomas and how to utilise this for choosing appropriate treatments.This study is the first of its kind in sarcomas and confirms that genomic instability is indeed characteristic of these tumours. It also highlights potential inter- and intratumour differences in DNA damage and/or responses as well as potential markers for the prediction of response to radiotherapy among these rare and heterogeneous tumours. Overall, the data, while not sufficient to make firm conclusions about their utility as biomarkers, suggest that there is still much to understand about the relationship between


Apoptotic induction was significantly increased using platinum together with BPTES compared to either treatment alone. Our findings suggest that targeting glutamine metabolism together with platinum based chemotherapy offers a potential treatment strategy particularly in drug resistant ovarian cancer.Ovarian cancer is characterized by an increase in cellular energy metabolism, which is predominantly satisfied by glucose and glutamine. Targeting metabolic pathways is an attractive approach to enhance the therapeutic effectiveness and to potentially overcome drug resistance in ovarian cancer. In platinum-sensitive ovarian cancer cell lines the metabolism of both, glucose and glutamine was initially up-regulated in response to platinum treatment. In contrast, platinum-resistant cells revealed a significant dependency on the presence of glutamine, with an upregulated expression of glutamine transporter ASCT2 and glutaminase. This resulted in a higher oxygen consumption rate compared to platinum-sensitive cell lines reflecting the increased dependency of glutamine utilization through the tricarboxylic acid cycle. The important role of glutamine metabolism was confirmed by stable overexpression of glutaminase, which conferred platinum resistance. Conversely, shRNA knockdown of glutaminase in platinum resistant cells resulted in re-sensitization to platinum treatment. Importantly, combining the glutaminase inhibitor BPTES with platinum synergistically inhibited platinum sensitive and resistant ovarian cancers  Epithelial ovarian cancer is the most common type of ovarian malignancy and often diagnosed in advanced stages of disease. The standard treatment includes cyto-reductive surgery followed by systemic platinum and taxane-based chemotherapy . The majLike most cancers, ovarian cancer is dependent on the increased utilization of energy sources to maintain ATP levels and intermediates required for enhanced cellular metabolism, cell growth and proliferation . GlucoseThe MYC oncogene encodes the transcription factor c-Myc which controls the expression of multiple key genes involved in the regulation of metabolic pathways including glycolysis and glutaminolysis . Given tin vitro deoxyglucose ([C-14]DG) and [H-3]glutamine ([H-3]GLN) during cisplatin treatment. We evaluated two paired cell lines: the cisplatin sensitive A2780 cell line and its cisplatin resistant derivative CP70, together with the cisplatin sensitive OV81.2 cell line, which is a primary cell line derived from a high grade serous ovarian cancer patient. The cisplatin resistant derivative OV81.2-CP10 (referred to as CP10 henceforth) was derived by propagating OV81.2 cells in the presence of cisplatin for 10 passages thus selecting for resistant clones .The baseline uptake of [C-14]deoxyglucose showed little difference between the paired cisplatin sensitive and resistant cell lines Figure , whereasTo better understand the mechanism regulating the dependence on glutamine utilization in the cisplatin resistant cell lines, we analyzed the expression of the high affinity glutamine transporter (ASCT2) and glutaminase (GLS), which converts glutamine to glutamate. Western blot analysis showed increased expression of the glutamine transporter ASCT2 and glutaminase (GLS) in cisplatin resistant cell lines compared to the sensitive cell lines . We observed a 2-fold higher OCR in cisplatin resistant CP70 cells compared to A2780 and a 3-fold higher OCR in cisplatin resistant CP10 cells compared to OV81.2  and glutaminase (GLS) , 25, 26.To evaluate the effect of different levels of glutaminase (GLS) expression on cisplatin resistance we over-expressed GLS in the cisplatin sensitive A2780 and OV81.2 cell lines. Western blot analysis confirmed a significantly higher glutaminase expression in the GLS transfected cells compared to empty vector control Figure , and thiThe role of glutaminase in cisplatin resistance was further investigated by suppressing the expression of GLS using shRNA. CP10 cells were transduced with a lentivirus containing shRNA against GLS (shGLS) or a lentivirus containing a control shRNA. Immunoblot analysis confirmed that the GLS shRNA effectively reduced glutaminase protein expression in resistant cells, which was comparable to the level in the cisplatin sensitive cell lines Figure . This leThe increased glutamine dependency of platinum resistant cells correlates with the upregulated glutamine utilization compared to cisplatin sensitive cell lines. Coupled to the observation that cisplatin treatment also induces glutamine uptake in sensitive cell lines, we therefore assessed if inhibiting glutaminase (GLS) with BPTES improves the therapeutic effectiveness of cisplatin treatment and is re-sensitizing drug resistant cells to the toxic effects of cisplatin. BPTES is a potent and selective allosteric inhibitor against both splice variants of the GLS gene, GAG and KGA, but not the hepatic form of glutaminase encoded by the GLS2 gene , 27. In To validate the clinical relevance of our findings, we analyzed the relationship between glutaminase and glutamine transporter ASCT2 expression levels and outcome of ovarian cancer patients using the PrognoScan database . In threCurrent platinum-based chemotherapy in ovarian cancer falls short due to the common development of drug resistance. This is often related to reduced platinum accumulation in ovarian cancer cells, linked to increased expression of multidrug resistance associated transporters . In ovarWithin cell signaling pathways, the c-Myc transcription factor is a main regulator of energy metabolism and is often amplified or overexpressed in many types of cancers including ovarian cancer , 41. StuA key finding of our study is the glutamine dependency of platinum resistant ovarian cancer cell lines. Platinum resistant cells were approximately 4-times more sensitive to glutamine deprivation compared to sensitive cells. In addition, platinum resistant cells were characterized by upregulated expression of important proteins in the glutamine metabolic pathway including glutamine transporter ASCT2 and glutaminase (GLS) Figure . We alsoin vitro migratory and invasive capabilities in a panel of eight ovarian cancer cell lines DG and [H-3]GLN at 1 μCi/ml to each well and incubated for 20 mins. Cells were then washed twice in ice cold PBS, harvested with trypsin, and viable cell numbers determined using the Vi-CELL counter. The cell lysates were collected and added to tubes containing 2 ml of scintillation fluid and the cell associated [C-14] and [H-3] radioactivity was measured using the β scintillation counter . Radioactivity uptake is expressed as nanomoles uptake of tracer per 10 000 live cells and expressed relative to untreated controls. All experiments were performed in triplicate and repeated 3 times.Cells were plated at 1 × 102  was determined using the XF24 Analyzer . Drug-treated cells were harvested and 100,000 cells were plated out on the cellTak  treated microplates. The rate of change of dissolved O2 in the medium immediately surrounding the cells was measured using the XF24 Analyzer. The contribution of glutamine in oxidative phosphorylation was assessed using specific inhibitors to glutamine metabolism or stable over expression or knockdown of GLS.The rate of change of dissolved Ohttp://www.prognoscan.org/) and publicly available Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) datasets with the accession numbers GSE 9891, GSE 26712, and GSE 17260, the relationship between GLS expression levels and overall or progression free survival rates was evaluated in ovarian cancer patients [Using the PrognoScan database . Differences between treatment groups or experimental factors were assessed by analysis of variance (ANOVA) using GraphPad Prism Software Version 5  at a two-sided 5% significance level.


However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals.The key role of cell cultures in different scientific fields is worldwide recognized, both as Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated.In order to evaluate the suitability of 13 different cell lines for Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin  Enterovirus genus, belonging to the Picornaviridae family, known as Poliovirus and composed by a 7,500 nucleotides (+) single stranded RNA molecule [Poliovirus were identified and classified as type 1, type 2 and type 3 [Poliovirus study greatly improved, taking advantage of cell cultures to isolate the virus from infected people [Poliomyelitis is a highly contagious disease caused by a virus of the molecule ,2. Threed type 3 ,5, Poliod people ,7, microd people ,9 and sid people –14.in vitro cultivation, resulting in the appearance of novel biochemical, biological and genetic characteristics that differ from primary or diploid cell ones. This represents an important issue in order to establish the biosafety of the cell lines used as substrates and to monitor the possible transmission of animal pathogens to human recipients [Immortalization of animal and human cells, derived from primary cell cultures, is a phenomenon mainly due to genetic mutations or infections by oncogenic viruses, which can result in the appearance of transformed features and tumorigenic properties. Furthermore, cells can undergo several modifications during cipients .Papillomavirus, revolutionized the study of Poliovirus biology. On the other hand, Vero cells, widely used in Poliovirus vaccine manufacturing, became immortalized through a spontaneous, unknown process and they acquired tumorigenic properties with increasing in vitro passage levels [in vitro establishment of two African green monkey kidney derived cell lines, named BS-C-1 and CV-1, gave rise to transformed colonies and tumor formation in the rat model [Among continuous cell lines, the human HeLa cell line, naturally contaminated by human e levels –18. Moreat model .Poliovirus vaccine production. In this respect, thirteen simian cell lines have been screened in vitro and in vivo for transformation and tumorigenicity features and their permissiveness to Poliovirus infection investigated, in comparison with other well-established substrates.The aim of this research was to identify cell lines free of any transformation ability and tumorigenicity, suitable for www.ibvr.org) and are available upon request. All these are continuous, spontaneously immortalized cell lines, exception made for CYNOM-K1, CV-1 (finite cell lines) and 4MBr-5 (EFG-dependent line). The investigations were performed at the passages indicated.All the investigated simian cell lines reported in Moreover, seven cell lines used as controls or as substrates are reported in a separate section of Poliovirus (see Poliovirus propagation section).Furthermore, MRC-5 and LLC-MK2 cell lines were selected to prepare “master” batches of three types of ® Automated Cell Counter . Finally, 1x105 viable cells of each cell line were seeded in a 75 cm2-flask and incubated at 37°C in 5% CO2 in the below reported culture media, enriched with 10% (v/v) of Fetal Bovine Serum .Cells were cryopreserved in vapor phase nitrogen until use. After thawing at 37°C, they were diluted in MEM culture medium , free of antibiotics, supplemented with 4mM L-glutamine (Sigma-Aldrich) and centrifuged at 125 g for 5 minutes at 20°C, in order to remove the dimethyl sulfoxide cryoprotectant agent. Cells were stained with Trypan Blue (Sigma-Aldrich), counted and checked for viability by a CellometerBGMK, BS-C-1, CYNOM-K1, HeLa, HEp2, LCP, LLC-MK2, MA-104, MARC-145, RK13.6 and Vero cell lines were amplified in MEM, while FRhK-4, FrP3, RC 37 and 4647 cell lines in D-MEM (Sigma-Aldrich). NCTC cl 3526 cell line was maintained in NCTC 135 medium (Thermo Fisher Scientific) and CV-1 in Eagle’s basal medium in Hanks’ BSS with amino acids and vitamins (Sigma-Aldrich). 4MBr-5 were propagated in Ham’s F K12 medium(Thermo Fisher Scientific) with 2 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 30 ng/ml epidermal growth factor, while 3T3BALB/c and MRC-5 cells were grown in MEM supplemented with 1 mM sodium pyruvate (Sigma-Aldrich).Mycoplasma infection was evaluated using the commercial MycoSensor PCR Assay Kit  according to the manufacturer's instructions.All cell lines were tested for bacteria, fungi and yeast contamination. Each cell suspension was inoculated in Agar Sabouraud, Triptic Soy Agar and Brain Heart Infusion microbiological media (Sigma-Aldrich). The mixtures were incubated for 5 days at 30°C and for 3 days at 37°C, respectively, and observed daily in order to detect any contamination. Human Cytomegalovirus), HIV (Human Immunodeficiency Virus), HSV-1 (Herpes Simplex Virus1), HSV-2 (Herpes Simplex Virus2), EBV (Epstein-Barr Virus), HBV (Hepatitis B Virus) and HCV (Hepatitis C Virus) were examined by artus® RG PCR Kit , according to the manufacturer’s instructions. HHV-6 (Human Herpesvirus 6), HHV-7 (Human Herpesvirus 7), HHV-8 (Human Herpesvirus 8) and HPV (Human Papilloma Virus) were evaluated by a Real Quality PCR kit , following the manufacturer’s instructions. Finally, Influenza virus type A was examined using the primers M-for (5’-AGATGAGTCTTCTAACCGAGGTCG-3’), M-rev (5’-TGCAAAAACATCTTCAAGTCTCTG-3’) proposed by van de Brand and colleagues [5’-TET- TCAGGCCCCCTCAAAGCCGA-BHQ1-3’, [Virus contamination of each cell line was investigated by Real-Time PCR assay. HCMV  and theIn vitro testing for adventitious agents was performed in compliance with the European Pharmacopoeia recommendations (Cell substrates for the production of vaccines for human use) [2. After 7 days of growth, medium was renewed, while monolayers were observed daily for CPE for 14 days. On day 14, treated LCC-MK2 cells were removed from the incubator and tested for haemadsorption of guinea pig and chicken erythrocytes. Briefly, cell monolayers were washed and duplicate wells were overlaid with 0.5 ml of 0.5% (v/v) of guinea pig and chicken erythrocytes and, after 30 minutes of incubation at room temperature, examined for adsorption. As positive control H/A/WSN/33 (VIR RE RSCIC 50) influenza virus was used.man use) . Cell cuCell cultures were also examined for the presence of retroviruses, using the Reverse Transcriptase Assay, colorimetric kit  for the quantitative determination of the viral Reverse Transcriptase (RT) activity. According to manufacturer’s instructions, a calibration curve was prepared from HIV-1 RT included in the kit. For the lysis of the retroviruses, 40 μl of supernatant were mixed with 40 μl of Lysis Buffer. After 30 minutes of incubation at room temperature, 20 μl of the reaction mixture were added to each reaction and HIV-1 RT standard tube. Finally, samples were incubated at 37°C for 15 h. Samples and HIV-1 RT dilutions were transferred into the wells of the MP modules and incubated for 1 h at 37°C. The solution was completely removed and the strip was rinsed 5 times with 250 μl of Washing Buffer per well for 30 s. 200 μl of anti-DIG-POD working solution were added and incubated for 1 h at 37°C. The solution was completely removed and the washing steps were repeated. Finally, 200 μl of ABTS Substrate Solution were added and the plate was incubated at room temperature for 30 minutes. The absorbance was measured at 405 nm by using a Gen5 microplate reader  and the effective RT activity was extrapolated from the standard curve.Species of origin for the considered cell lines was evaluated by isoenzyme analysis, using the Authentikit System , according to the manufacturers protocol. Briefly, cells were submitted to the extraction process by dilution of cell pellet in a specific buffer included in the kit. The enzyme activity was quantified by addition, after serial steps, of the Quench-A-Zyme Reagent by spectrophotometer (BioTek). The profiles of glucose-6-phosphatedehydrogenase, lactate dehydrogenase, nucleoside phosphorylase, malate dehydrogenase, mannose phosphate isomerase peptidase B, and aspartate aminotransferase isoenzymes were evaluated by electrophoresis. Species of origin was determined by comparing the migration distance of the analyzed isoenzyme systems with the reference distances provided by the kit .Pharmacopoeia [5cells/well suspension diluted in a mixture composed by 50% MEM supplemented by 20% FBS, and 50% of 0.6% agar noble. Plates were incubated at 20°C to allow solidification and then maintained at 37°C in 5% CO2 for 4 weeks and inspected daily by optical microscopy. Samples were considered negative if no cell growth was observed in soft agar, while the presence of one or more multicellular aggregates was accounted as transformation evidence. HEp2 and 3T3BALB/c cells were used, respectively, as positive and negative control.All the selected cell lines were tested for tumorigenicity according to the European acopoeia . Soft agacopoeia . Brieflyin vivo (Nu/Nu mice) assay, according to the European Pharmacopoeia [in vivo assay, according to 3Rs principles. For the same reason, the in vivo test was not performed on BS-C-1and CV-1 cell lines because literature data has already reported their capacity to induce tumors in laboratory animals [in vivo assay.Potential tumorigenic evolution of the cell line that resulted to be negative in soft agar assays was evaluated by acopoeia . The exp animals , 20. Finad libitum. Each group was injected subcutaneously with 0.2 ml of 107 cell suspension of each cell line. Five mice from each group were sacrificed 20 days after the injection, while the others were observed daily for 12 weeks. At the end of the observation period, the animals were humanely euthanized . A necropsy was carried out on each mouse with the aim of detecting tumors at the injection site and in other organs . The local area of injection, together with the organs, were collected for histological examination and stained by haematoxylin-eosin, as described by Ferrari et al. [For the test, 70 athymic, 30-day-old male mice (Nu/Nu genotype), received from Harlan Laboratories, were used. They were subdivided in seven groups of 10 mice each. Five groups were inoculated with FRhK-4, MA-104, CYNOM-K1, 4647 and 4MBr-5 cell lines; one group was injected with the HEp2 cell line (positive control) and, finally, a further group was inoculated with the 3T3BALB/c cell line (negative control). All animal experiments were conducted at IZSLER, Brescia. The animals were housed on sterile bedding and with water and feed i et al. . In the Poliovirus attenuated strains LsC 2ab Sabin type 1 (VIR RE RSCIC 48), P712 Ch 2ab Sabin type 2 (VIR RE RSCIC 182) and Leon 12alb Sabin type 3 (VIR RE RSCIC 183) were gently provided by Dr. Medici (University of Parma). LsC 2ab Sabin type 1 was cultivated in MRC-5 cells, while P712 Ch 2ab Sabin type 2 and Leon 12alb Sabin type 3 in LLC-MK2. The viruses were inoculated in the selected cell line seeded in a 75 cm2 flask at 1 MOI with 0.5 ml of culture medium. Infected cells were incubated at 37°C in 5% CO2 for 60 minutes and then added to 20 ml of culture medium supplemented by 3% (v/v) FBS; finally, samples were incubated at 37°C in 5% CO2 for five days. Cultures were observed daily in order to detect CPE and frozen at -80°C when CPE reached 80%. Subsequently they were thawed at room temperature, centrifuged at 1,000 g for 30 minutes at 4°C, distributed in aliquots, and stored at -80°C. The infectious titers were calculated by the Reed and Müench method [Poliovirus batches were the following: Poliovirus LsC 2ab Sabin type 1: 106.24 TCID50/ml; Poliovirus P712 Ch 2ab Sabin type 2: 107.24 TCID50/ml; Poliovirus Leon 12alb Sabin type 3: 107.74 TCID50/ml.The h method . The infPoliovirus. To do this, 4x104 viable cells/cm2 were seeded in 25 cm2 flasks, incubated in MEM added to 10% (v/v) of FBS at 37°C in 5% CO2. At 80% confluence, cells were infected at 0.1 MOI. At 80% CPE, flasks were frozen at -80°C, thawed, centrifuged at 1,540 g for 20 minutes at 4°C and supernatant was distributed into aliquots and used to evaluate the infectious titre, according to Reed and Müench formula [FRhK-4, CYNOM-K1, 4MBr-5 and 4647 cell lines resulted to be devoid of transformation/tumorigenic evolution and were investigated for permissiveness to  formula .2 for 24 hours. The virus suspensions, collected from the different cell cultures, were diluted from 10−1 to 10−8 in culture medium and each dilution was inoculated into the corresponding cell line. Five wells were inoculated with each dilution at a volume of 100 μl/ well. Control cells were added with culture medium. The plates were incubated at 37°C, 5% CO2 for 60 minutes and then each plate was added to 100 μl/well of culture medium supplemented by 3% (v/v) FBS and incubated at 37°C, 5% CO2 for 7 days. Cell cultures were checked daily for CPE and the infectious titers were evaluated at the end of the observation period. In parallel sessions, Poliovirus infection has been performed on Vero and HeLa cells, as reference lines for the production of Poliovirus, and the obtained infectious titers were compared. These tests were performed in triplicate and the mean infectious titers were calculated.For virus titration, each cell line was seeded in 96-well plastic plates and incubated at 37°C in 5% COPoliovirus infectious titers were checked by one-way ANOVA, followed by a Dunn's post-hoc test for multiple comparisons. The significance threshold was set at P<0.05 .Differences between datasets of The cell lines tested proved to be free of microbial and mycoplasma contamination. No virus, including adventitious agents and retroviruses, has been detected in the selected cell lines. Results are reported in The isoenzyme test confirmed the monkey origin of all cell lines selected for the study, whereas the MRC-5 proved to be of human derivation as expected. No cross-contamination was detected.in vitro transformation ability. The results of the investigation showed that BGMK, FrP3, LLC-MK2, MARC-145, NCTC cl 3526 and RC 37cell lines induced transformed colonies in soft agar medium. These colonies began to appear at about 7 days after seeding  no tumors were detected in skin of the inoculum site. The tissues and organs of the animals injected with either CYNOM-K1, FRhK-4, 4MBr-5, 4647 cell lines, or those treated with the negative control 3T3BALB/c cells, did not show any inflammatory process ; moreover, neoplastic cells were observed in the vessels. These alterations were restricted to the  process . In factinoculum site (data not shown). The results of the in vitro and in vivo tests are summarized in Concerning MA-104 cells, the presence of a foreign body granuloma was detected at the injection Poliovirus types replicated in all the investigated cell lines, as well as in Vero and HeLa cells, tested simultaneously as reference substrates for Poliovirus propagation and vaccine manufacturing. The infectious titers obtained in different assayed cells relative to attenuated Sabin strains type 1, 2 and 3, are reported in 50/ml data are reported as log10 mean ± standard error of the mean and analysed as indicated in material and methods section. Concerning LsC 2ab Sabin type 1 and P712 Ch 2ab Sabin type 2, TCID50/ml infectious titers ranged between 107.24 and 108.16, while Leon 12alb Sabin type 3 between 103.83 and 108.50. They replicated into cells without showing any dissimilarity between the different substrates, exception made for the observed tendency of all investigated cell lines in being more permissive to Poliovirus Leon 12alb Sabin type 3 propagation than HeLa and Vero. In particular, FRhK-4 cells showed to be significantly more sensitive to Leon 12alb Sabin type 3, compared with the simian reference cell line Vero (TCID50/ml mean: 108.50 versus 103.83).All Pharmacopeia, U.S. Food and drug Administration, World Health Organisation) [in vitro spontaneous transformation). For these reasons, cell cultivation history has to be clearly reported and transformation and tumorigenicity properties properly evaluated. Currently, the use of several tumorigenic cell lines is accepted for the manufacturing of viral vaccines, upon risk-benefit evaluation. For example, Vero cells are still used in Poliovirus vaccine production, although recent data showed growing evidence of tumorigenicity acquisition during serial subcultivation [Cell cultures are widely used as substrates for the production of many biotechnological and biological products for human use, such as viral vaccines. Since the use of biological material may lead to potential contamination with known and unknown extraneous agents, cell substrates must be properly tested in order to avoid adverse features in patients. In this respect, the guidelines provided by the International Regulatory Agencies  , 28–30 rtivation –18.in vitro and in vivo tumorigenic evaluation and Poliovirus amplification.The aim of this study was to characterize different simian cell lines and assess their suitability for large-scale production. In particular, different aspects were investigated: microbiological evaluation, species of origin, potential Microbiological results demonstrated the absence of viral and mycoplasma contamination and the isoenzyme test confirmed the monkey origin for all the analyzed cell lines.in vitro and in vivo tumorigenic evaluation permitted to express some interesting considerations.The most crucial point regarding in vitro transformed colonies. Inconclusive findings were found for MA-104 cell line, since its soft-agar colonies appeared to be morphologically different from those observed in positive control.Among the thrirteen cell lines investigated, only six  did not induce in vitro transformation results do not completely overlap with in vivo tumorigenicity ones, these cell lines were also injected into athymic mice, resulting free of any tumorigenic characteristics. In particular, CYNOM-K1, FRhK-4, 4MBr-5, 4647 cell lines did not show any evidence of macroscopic pathological tumors; necropsy was performed in order to collect sample for histological analysis. The obtained outcomes demonstrated the absence of pathological lesions either of neoplastic or inflammatory origin for all the selected cell lines.Since the in vivo tumorigenicity test confirmed the data obtained from the transformation assay performed through the in vitro test and they both indicate no transformation features. The lack of previous studies reporting in vivo investigation of the tested lines allows no comparison of the results.BS-C-1 and CV-1 cell line were not assayed because already published data reported their tumorigenic features –20. AmonPoliovirus as well as Vero and HeLa cells, with even superior performance in the case of Leon 12alb Sabin type 3. Since they resulted not transformed, devoid of tumorigenicity and characterized by a good replication capacity, these cells may represent alternative substrates for Poliovirus production to primary cell cultures from monkey kidneys, or other potentially tumorigenic cell lines, as Vero [in vitro growth of these cell lines. It was noticed a different trend in the doubling time of each biological substrate. As described in literature [FRhK-4, CYNOM-K1, 4647, 4MBr-5 cell lines allowed the growth of the three viral types of attenuated Sabin  as Vero . However as Vero , 33, makterature , Vero cein vitro passages and properly tested according to the good laboratory practice and international guidelines.These results outlined the importance of a deep and constant monitoring of biological substrates, in order to highlight all potential risks related to the uncontrolled serial amplification. Cell batches for biological manufacturing should be maintained at established