CHEK1, which encodes for the cell cycle checkpoint kinase CHK1, is significantly overexpressed in SCLC, compared to lung adenocarcinoma. In line with uncontrolled cell cycle progression in SCLC, we find that CDC25A, B and C mRNAs are expressed at significantly higher levels in SCLC, compared to lung adenocarcinoma. We next profiled the efficacy of compounds targeting CHK1 and ATR. Both, ATR- and CHK1 inhibitors induce genotoxic damage and apoptosis in human and murine SCLC cell lines, but not in lung adenocarcinoma cells. We further demonstrate that murine SCLC tumors were highly sensitive to ATR- and CHK1 inhibitors, while KrasG12D-driven murine lung adenocarcinomas were resistant against these compounds and displayed continued growth under therapy. Altogether, our data indicate that SCLC displays an actionable dependence on ATR/CHK1-mediated cell cycle checkpoints.Small cell lung cancer (SCLC) is a difficult to treat subtype of lung cancer. One of the hallmarks of SCLC is its almost uniform chemotherapy sensitivity. However, chemotherapy response is typically transient and patients frequently succumb to SCLC within a year following diagnosis. We performed a transcriptome analysis of the major human lung cancer entities. We show a significant overexpression of genes involved in the DNA damage response, specifically in SCLC. Particularly  Further features of SCLC are its extraordinary aggressiveness and tendency to metastasize early1, the almost universal sensitivity to genotoxic combination chemotherapy, as well as the typically transient and short duration of the resulting response, leading to the early death of affected patients1. Despite recent exome and genome sequencing efforts, no recurrent actionable genomic aberrations have been identified in SCLC, thus far3. However, the study of SCLC genomes has revealed a number of recurrent aberrations, including an obligate bi-allelic inactivation of TP53 and RB1, frequent losses on the short arm of chromosome 3 and the recurrent inactivation of the histone acetyl transferase genes, CREBBP and EP3003. In the absence of effective targeted agents, chemotherapy, typically involving cisplatin and etoposide, as well as whole brain irradiation, remain the main therapeutic principles for extensive disease4. In addition to chemotherapy, early clinical data suggest that targeting DLL3, a ligand involved in Notch signaling, is clinically active6. Furthermore, immunotherapy has similarly elicited pronounced and durable responses in SCLC, likely due to the presence of a high mutational burden, which is known to correlate with immunotherapy response7.Small cell lung cancer (SCLC) is a neuroendocrine lung cancer subtype that is characterized by small cells that express neuroendocrine markers, such as chromogranin or synaptophysinTP53 and RB1 may lead to massively destabilized cell cycle checkpoints in SCLC cells. In response to genotoxic stress, p53, encoded by TP53, undergoes extensive posttranslational modifications, leading to protein stabilization and nuclear translocation, where p53 acts as a transcription factor9. Among the target genes that are trans-activated by p53 are the cell cycle-regulating genes CDKN1A, 14-3-3σ, GADD45α, RPRM and others9. Thus, it is conceivable that loss of TP53 is associated with defective cell cycle control in response to endogenous and exogenous genotoxic damage. Moreover, loss of RB1, which belongs to a family of genes encoding the so-called pocket proteins RB1, p107 and p130, is associated with uncontrolled cell cycle progression, particularly at the G1/S border12. In early G1, RB1 governs the restriction point by sequestering the E2F1 transcription factor12. Upon phosphorylation by CDK4 and 6, RB1 undergoes a conformational change, leading to the release of E2F1, which subsequently drives the expression of cyclin E, which further enhances CDK activity and ultimately drives an irreversible S-phase entry12. Thus, loss of RB1 is thought to lead to a destabilized G1/S border.The combined loss of both 13. The DDR consists of a series of proximal kinases, including ATM, ATR and DNA-PKcs15. Particularly, ATM and ATR relay their signaling activity through the downstream effector kinases CHK2 and CHK1, respectively15. We and others recently identified a third branch of cell cycle checkpoint signaling, involving a kinase pathway in which ATM leads to the activation of TAO1, which in turn activates the p38MAPK/MAPKAP-K2 stress kinase complex20. The three cell cycle checkpoint effector kinases CHK1, CHK2 and MK2 share substrate motif homology, selecting for amino acid sequences with basophilic residues in the Ser/Thr −3 position and hydrophobic residues in the Ser/Thr −5 and +1 position15. One of the most prominent substrates of these checkpoint effector kinases is the CDC25 family of phosphatases, which are inactivated by CHK1/CHK2/MK2-mediated phosphorylation15. CDC25 phosphatases mediate de-phosphorylation and subsequent activation of cyclin dependent kinases (CDKs), which are critical drivers of the mammalian cell cycle22. Thus, DDR-mediated inhibition of CDC25 activity leads to a cell cycle arrest, due to inadequate CDK activity22.In addition to p53- and RB1-controlled transcription-mediated cell cycle control, a kinase based cell cycle checkpoint network exists that, when activated by genotoxic damage, leads to a rapid block in cell cycle progression and the subsequent repair of DNA damage. This signaling network is commonly referred to as the DNA damage response (DDR)CHEK1 mRNA is significantly overexpressed in primary human SCLC, compared to non-small cell lung cancer (NSCLC) samples. We further show that not only CHK1 inhibition, but also ATR inhibition leads to the induction of genotoxic stress and subsequent apoptosis, specifically in SCLC cells, while NSCLC cells display resistance against ATR/CHK1 inhibition. We confirm these results in autochthonous and transplanted murine models of SCLC and NSCLC (both Tp53-deficient), as well as xenograft experiments. Overall, our data further implicate inhibition of the ATR/CHK1 cell cycle kinase axis as a potential drug target for the treatment of human SCLC patients. These data are in accordance with previously published observations reporting an increased expression of numerous components of the DDR network, including ATR and CHK1, both on the mRNA and protein level in SCLC, compared to NSCLC24. Moreover, a recent study demonstrated that the second-generation CHK1 inhibitor prexasertib exerts anti-tumor activity against SCLC in vitro and in vivo24. Furthermore, prexasertib enhanced the effects of cisplatin and the PARP inhibitor olaparib24. Employing proteomic technologies, the group showed that CHK1 and MYC were predictive biomarkers of prexasertib sensitivity24.Here, we show that TP53 and RB13, we sought to further characterize the transcriptional profile of SCLC in comparison to other major lung cancer subtypes. We therefore performed a comparative analysis with the transcriptome sequencing data of 214 patient-derived lung tumors28. In comparison to lung adenocarcinomas (ADC) and squamous cell carcinomas (SqCC), where co-occurring somatic alterations of TP53 and RB1 are less frequent and rather rare26, SCLC tumors exhibited significantly higher expression levels of genes controlling cell cycle regulation and DNA replication, as well as pathways that emphasize the neuroendocrine features of this lung cancer subtype  and 5-fold (4.6-fold), compared to adenocarcinomas and squamous cell carcinomas, respectively , which in the absence of p53 and RB1 may initiate cell cycle arrest and subsequent DNA repair mechanisms15. Intriguingly, and in line with poorly controlled cell cycle progression in SCLC, we find that the mRNAs encoding the phosphatases CDC25A, B and C are expressed at significantly higher levels in SCLC, compared to SqCC and ADC samples , than SCLC cell lines (GI50 = 12.3 µMol). In contrast, NSCLC cells were more resistant to etoposide (GI50 = 29.1 µMol), compared to SCLC clones (GI50 = 14.3 µMol).To further characterize the biological properties of the individual cell lines, we next profiled their sensitivity to the frontline chemotherapeutic agents cisplatin Fig.  and etop33. PF-477736 displays a ~100-fold selectivity for CHK1, compared to CHK233. Similarly, VE-822 is a selective ATR inhibitor with IC50 values of 0.019 µM, 2.6 µM, and 18.1 µM for ATR, ATM and DNA-PK, respectively34. VE-822 displays negligible activity against mTOR and PI3Kγ34. Both PF-477736 and VE-822 are currently being investigated in clinical trials . As shown in Fig. 50 = 1.9 µMol vs. GI50 = 7.2 µMol, respectively). Similarly, SCLC cells were significantly (P < 0.0001) more sensitive to PF-477736, compared to NSCLC cell lines   Fig. . As ATR As cell viability assessments through intracellular ATP measurements do not distinguish between growth retardation and active cell death, we next performed flow cytometry experiments to directly test the hypothesis that ATR- and CHK1 inhibitors induce apoptotic cell death in SCLC cell lines Fig. . We spec35. Corroborating this role of the ATR/CHK1 pathway for genome maintenance in S-phase, Atr and Chek1 constitutive knockout mice were shown to be embryonically lethal37. Furthermore, Chek1 conditional knockouts display signs of genotoxic damage, upon Chek1 deletion38. Lastly, ATR- and CHK1 inhibitors have previously been shown to cause genotoxic damage40. To interrogate the effects of ATR- and CHK1 inhibition on genome integrity, we assessed the induction of genotoxic damage, using indirect immunofluorescence. We specifically employed an antibody to detect phospho-Ser-139 in the histone variant H2AX (γH2AX), an established marker for DNA damage42. These experiments revealed that VE-822 (0.75 µM) and PF-477736 (1.0 µM) inflicted genotoxic damage in both, NSCLC and SCLC cell lines  and 1.5 × 106 NSCLC cells (KP1 and 2) into the lungs of 8–12-week-old syngeneic C57BL/6 J recipient mice. Tumor manifestation was documented through µCT imaging  and 4  , PF-477736  or treated with vehicle control. Tumor volume assessments were performed, using µCT imaging on the day prior to cycle 1, as well as after completion of cycle 2 and 4 Fig. . When trtro Fig. . We notetro Fig. , S4B. In01) Figs , S4C. Th01) Figs . While a01) Figs . PF-477701) Figs . Contrar01) Figs . Median 01) Figs .Tp53fl/fl;Rb1fl/fl mice through intratracheal Ad-CMV-Cre instillation, as previously described , or vehicle control , due to compound toxicity.To further substantiate these observations, we next employed an autochthonous mouse model of SCLC. For that purpose, we induced SCLC in rol Fig. . While trol Fig. . This morol Fig. . Collect43, including 61 SCLC, 109 NSCLC and 800 non-lung cancer cell lines. In line with our data derived from primary tumor specimens  database are indicated in Fig. 44. H526, H69 and H82 cells are reported to carry bi-allelic TP53 and RB1 mutations. Intriguingly, a re-analysis of human lung cancer cell lines curated in the Catalogue Of Somatic Mutations in Cancer (COSMIC) database revealed that bi-allelic TP53 and RB1 aberrations can also be detected in a subset NSCLC lines , PF-477736  or vehicle solution was initiated. While vehicle-treated xenograft tumors derived from H-82 -approved second line regimen for SCLC patients47. Further second line options beyond topotecan include taxanes, irinotecan, vinorelbine, and gemcitabine. In addition, temozolomide might be an attractive regimen for the second line situation, as it can be applied orally and displays activity in central nervous system lesions48. There is no internationally accepted consensus on systemic treatment regimens beyond first- and second-line therapy49. Furthermore, recently published exome and genome sequencing efforts have not revealed any recurrent actionable oncogenic genome aberrations3.In the Western world, the first line treatment of SCLC patients presenting with extensive stage disease consists of a platinum-based combination chemotherapy, typically cis- or carboplatin in combination with etoposideCHEK1. This CHEK1 expression in human SCLC samples has recently also been reported in an independent study24. We further demonstrate that ATR- and CHK1 inhibitors display a selective toxicity in SCLC cells, as well as allograft and xenograft SCLC tumors, while NSCLC cell lines isolated from Kras-driven murine lung adenocarcinoma and allograft NSCLC tumors remained largely unaffected, when treated with these compounds , RB1 governs the G1 restriction point through sequestration of E2F112. Thus, combined loss of TP53 and RB1 is likely to promote unrestricted proliferation and a substantially impaired ability to halt the cell cycle in response to DNA damage. In line with a destabilized G1/S border as a common denominator for CHK1 inhibitor sensitivity, other groups have reported that MYC-driven diffuse large B cell lymphoma and CCND1-driven mantle cell lymphoma display a marked sensitivity against the CHK1 inhibitor PF-47773651. Altogether, it is tempting to speculate that SCLC cells display a compensatory upregulation of p53- and RB1-indpendent regulators of cell cycle progression, such as the checkpoint kinase CHK1. We note that a re-analysis of cell lines curated in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database revealed that bi-allelic loss of TP53 and RB1 can also be detected in a subset of human NSCLC cancer cell lines  is currently undergoing evaluation as a single agent in a phase II trial with extensive stage SCLC. Given the data reported in our study, it will be interesting to see whether the results reported in the prexasertib trial will lend further support to the clinical development of CHK1 inhibitors for the treatment of SCLC patients. It has also been reported that ATR- and CHK1 inhibitors display synergistic activity in cancer cells54. It will thus be interesting to study whether combined inhibition of ATR and CHK1 may display selective synergistic activity in SCLC.Intriguingly, numerous checkpoint kinase inhibitors, including CHK1- and combined CHK1/CHK2 inhibitors have been developed and are currently evaluated in phase I and II clinical trials, either as single agents or in combination with chemotherapeutic compounds28, 60 lung squamous cell carcinoma samples26 and 53 SCLC tumor samples3. Gene-specific transcripts were quantified with RSEM55. Differentially expressed genes were determined with the SAM R package at a Q-value < 0.0556. Differentially expressed gene expression profiles were analyzed with DAVID57.Transcriptome sequencing data of human lung tumors was analyzed by referring to the RNA-seq data of 101 lung adenocarcinomas7 PFU Adeno-CMV-Cre intratracheally to Rb1fl/fl;Tp53fl/fl (RP) and KrasLSL.G12D/wt;Tp53fl/fl (KP) mice, respectively30. 10-week-old animals were anesthetized with Ketavet (100 mg/kg) and Rompun (20 mg/kg) by intraperitoneal injection, as previously described58. RP mice were scanned by MRI under isoflurane (2.5%) anesthesia to confirm tumor formation approximately 30 weeks after Adeno-Cre application. KP mice were scanned by µCT imaging  under isoflurane (2.5%) anesthesia to confirm tumor formation 5 weeks after Adeno-Cre application. Mice were kept on a C57Bl6 background. The local authorities and the local animal protection committee approved all animal procedures. All mouse experiments were conducted in accordance with our Institutional Animal Care and Use Committee (IACUC). All animal experiments were approved by the responsible local government authorities, namely the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein Westfalen. All animal experiments were carried out in accordance with the relevant guidelines and regulations.To induce murine SCLC and lung adenocarcinoma, we applied 2.5 × 10C57Bl6mice with 2 × 106 murine SCLC tumor cells  and 1.5 × 106 murine NSCLC tumor cells (KP1 and 2) into the right lung. Tumor formation was verified by µCT imaging 9 days after injection. SCLC- and NSCLC tumor-bearing mice were randomly allocated to receive four cycles of VE-822, PF-477736 or vehicle, as indicated. Tumor volume changes were monitored by µCT imaging under 2.5% isoflurane at the end of two treatment cycles and assessed by OsiriX and DICOM viewer software packages . We normalized the tumor volumes under treatment to baseline tumor volumes, assessed prior to therapy initiation. Survival curves were calculated up to 175 days after the injection of tumor cells. The local authorities and the local animal protection committee approved all animal procedures. All mouse experiments were conducted in accordance with our Institutional Animal Care and Use Committee (IACUC). All animal experiments were approved by the responsible local government authorities, namely the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein Westfalen. All animal experiments were carried out in accordance with the relevant guidelines and regulations.For the allograft experiments, we anesthetized (2.5% isoflurane) and injected 8–12-week-old wildtype 6 tumor cells, re-suspended them in serum-free media and subcutaneously injected them into the flanks of 8-week-old male athymic nude mice . Mice were monitored for the formation of palpable subcutaneous tumors. Upon manifestation of tumors (≥100 mm³), VE-822 or PF-477736 were administered for two cycles following the protocol described above. Diameters of subcutaneous tumors were measured by a digital caliper every second day following initiation of therapy. For tumor volume measurements, mice were anesthetized with 2.5% isoflurane. Tumor volumes were calculated, based on the modified ellipsoid formula V = 1/2 (width × length²). We normalized the tumor volumes under treatment to baseline tumor volumes, assessed prior to therapy initiation. In accordance with our Institutional Animal Care and Use Committee (IACUC), mice with a single tumor volume larger than 1.500 mm³ were sacrificed. The local authorities and the local animal protection committee approved all animal procedures. All mouse experiments were conducted in accordance with our Institutional Animal Care and Use Committee (IACUC). All animal experiments were approved by the responsible local government authorities, namely the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein Westfalen. All animal experiments were carried out in accordance with the relevant guidelines and regulations.We generated 120 xenograft tumors from three different human SCLC cell lines . For each xenograft tumor, we collected 1.5 × 10Mice harboring SCLC or NSCLC tumors were sacrificed at the indicated time points and lungs were fixed in 4% PFA. Formalin-fixed paraffin-embedded (FFPE) murine lung samples were cut into 4 μm thick sections and mounted on slides. After staining with haematoxylin and eosin (H&E) or Ki67 the samples were assessed by two independent board-certified pathologists .58. Human cell lines were obtained from the American Type Culture Collection (ATCC), the German Resource Center for Biological Material (DSMZ), or were a kind gift from Roman K. Thomas . All our cell lines were cultured at 37 °C in a humidified incubator supplemented with 5% CO2. RPMI medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin was the culture medium used for all cell lines. We passaged all adherent cells lines by washing with PBS and subsequent incubation in trypsin/EDTA. By addition of culture medium, we inactivated trypsin and plated or diluted the cells afterwards. Passaging of our suspension cell lines was executed by suitable dilution of the cell suspension and adding fresh culture medium. All cell lines were routinely tested for infection with mycoplasma .Individual murine SCLC and NSCLC tumor nodules derived from individual RP and KP mice, respectively, were isolated and cultured in RPMI medium supplemented with 1% penicillin/streptomycin and 10% fetal calf serum, to derive stable tumor cell lines, as previously described6 cells per dish for SCLC and 750,000 cells per dish for NSCLC. After 48 hours, cells were trypsinized and counted on a Beckman Coulter cell counter. Subsequently, cells were seeded at the above-mentioned density. The experiment was performed over 12 days, resulting in a total of 7 passages. Doubling times were calculated using the following equation: PD = t × Log2/(LogC2-LogC1), where PD = Population doubling; t = time (hours); Log = 10 based log; C1 = first count; C2 second count.To assess population doubling times, triplicates of each of the five murine SCLC cell lines and the five murine NSCLC cell lines were seeded in 10 mm dishes at a density of 1 × 10Cisplatin was purchased from Accord Pharmaceuticals and etoposide was from Hexal Pharma. VE-822 and PF-477736, as well as AZD-7762 were purchased from Abmole and AZD-6738 was from Selleckchem. All small molecule compounds were dissolved in DMSO and stored at −80 °C or −20 °C, according to instructions.Control cohort: daily intraperitoneal injection of PBS/DMSO vehicle solution.VE-822 cohort: weekly for three consecutive days at a dose of 30 mg/kg by oral gavage.PF-477736 cohort: weekly for five consecutive days at a dose of 20 mg/kg by intraperitoneal injection.The ATR inhibitor VE-822  was dissolved in an equilibrated (v/v) mixture of PBS (70%), Polyethylene glycol 300  and Tween 80 (0.5%) at a concentration of 18.0 mg/ml. The Chk1 inhibitor PF-477736  was prepared in a 1:1 (v/v) mixture of PBS and dimethyl sulfoxide at a concentration of 10.0 mg/ml. The following treatment regimens were applied in the allograft and the xenograft mouse model:® (Promega) assays, according to the instructions provided by the manufacturer. In brief, 100 µl of each cell line were plated into 96-well plates at a density of 5.000 cells per well. 24 hours after seeding, triplets of each cell line were treated, as indicated. Each drug concentration was pre-prepared in deep-well plates. 25 µl of this stock solution was transferred from the deep-well plate to the 96-well plates. Cells were exposed to treatment for 48 hours in a humidified incubator at 37 °C and 5% CO2. After incubation, 125 µl of the CellTiterGlo® reagent were added to each well of the 96-well plate. The contents were mixed for 2 minutes in an orbital shaker to induce cell lysis. After a 10-minute incubation at room temperature, relative cell viability was determined by measuring the ATP content in each well on a luminescence plate reader, equipped with an automated stacking module (Tecan). We normalized luminescence values to luminescence intensities of control wells on the same plate, treated with vehicle solution.To assess cell viability, we employed CellTiterGlo60. All murine SCLC and NSCLC cell lines were seeded into 6-well plates at approximately 50% density and were incubated for 24 hours in a humidified incubator at 37 °C and 5% CO2. Subsequently, triplets of each cell line were either left untreated or were exposed to one of the following inhibitors and dosages: cisplatin , etoposide , VE-822  or PF-477736 . After 48 hours of drug exposure, cells were trypsinized and harvested. After washing them twice in cold PBS, cells were re-suspended in an antibody-binding buffer  and incubated for 15 minutes in Annexin-V (BD 556420) plus propidium iodide (PI) . Annexin-V/PI staining intensity was subsequently assessed by flow cytometry . A minimum of 100,000 events was analyzed for each measurement and apoptotic cells were defined as double-positive.In the flow cytometry experiments reported here, we used Annexin-V/PI staining to quantify the induction of apoptosis, exactly as previously described2, 1 mM EDTA, 0.5% Triton-X100) and incubated with cytoskeleton stripping buffer  under the same conditions. Samples were then blocked in 5% bovine serum albumin (BSA); 2% normal goat serum (NGS), 0.01% Triton X100 in PBS and incubated overnight at 4 °C with a primary antibody detecting γH2AX . After 18 hours of incubation, we executed a 1 hour incubation with a secondary antibody goat-anti-mouse Alexa488  followed by several washes. Finally, we mounted cells with ProLong Gold antifade reagent with DAPI , analyzed them through microscopy, using an AxioImager M1 (Carl Zeiss) and counted the γH2AX-positive cells.Murine SCLC and NSCLC cells were seeded at a confluency of approximately 50% on 6-well plates with a 22-mm glass coverslip on the bottom. The cells were treated with VE-822 (0.75 µM) or PF-477736 (1.0 µM) or were left untreated for 12, 24 or 48 hours. Cells were fixed in 4% PFA (15 minutes on ice) and the coverslips were subsequently washed in PBS. Cells were subsequently permeabilized on ice for 10 minutes in cytoskeleton buffer  and lysates were supplemented with 1% SDS and 100 μM PMSF. Samples were subsequently sonicated for 10 seconds and centrifuged at 12.500 g for 30 minutes. We transferred the supernatants into fresh tubes and examined the protein concentration with a BSA standard curve, using a Bradford assay (Thermo Scientific). Equivalent amounts of protein (up to 90 µg) were boiled at 95 °C for 5 minutes in Laemmli sample buffer, separated using 10% SDS-PAGE and blotted on PVDF membranes . Membranes were blocked in 5% BSA in TBS-T for 1 hour and stained with specific antibodies. The γH2AX antibody  was applied overnight. The β-actin antibody  was applied for 20 minutes. Secondary antibody staining was performed following several washing steps in TBS-T. The secondary antibody was applied for 3 hours. After several washing steps , we imaged the membranes on a digital ChemiDoc XRS+ System (BioRad), using the Amersham ECL Western Blotting Detection Reagent  and analyzed them with the manufacturer’s software . γH2AX expression relative to the vehicle-treated control was analyzed through densitometry, using ImageJ version 1.51 h. Original full-length immunoblot membranes are shown in Figs Statistical analysis was performed using GraphPad Prism Version 5.00  and Excel . We used student’s two-tailed t-test, log-rank (Mantel-Cox) test and one-way-Anova test to determine statistical significance. Significance was considered on p-values below 0.05. Levels of significance were *p < 0.05; **p < 0.01; ***p < 0.001. Errors bars in figures represent standard deviation, if not differently indicated.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information


The use of nutraceuticals is gaining in popularity in human and canine oncology with a relatively limited understanding of the effects in the vastly different tumor types seen in canine oncology. We have previously shown that turmeric root (TE) and rosemary leaf (RE) extracts can work synergistically to reduce neoplastic cell growth, but the mechanisms are poorly understood and require further elucidation.−1 extract individually, or 3.1 μg mL−1 of each extract in combination based on studies showing synergy of these two extracts. Apoptosis, antioxidant effects, cellular accumulation of curcumin, and perturbation of signaling pathways were assessed. The TE + RE combination treatment resulted in Caspase 3/7 activation and apoptosis in all cell lines, beyond the effects of TE alone with the CMT-12 cell line being most susceptible. Both extracts had antioxidant effects with RE reducing reactive oxygen species (ROS) by 40–50% and TE reducing ROS by 80–90%. In addition RE treatment enhanced the c-jun N-terminal kinase (JNK) activity in the C2 cell line and TE + RE exposure increased activated JNK by 4–5 times in the CMT-12 cell line. Upon further examination, it was found that RE treatment caused a significant increase in the cellular accumulation of curcumin by approximately 30% in the C2 and D17 cell lines, and by 4.8-fold in the CMT-12 cell line. This increase in intracellular curcumin levels may play a role in the synergy exhibited when using TE and RE in combination.Three different canine cell lines  were treated with 6.3 μg mLThe use of RE in combination with TE induces a synergistic response to induce apoptosis which is better than either extract alone. This appears to be related to a variable increased TE uptake in cells and activation of pathways involved in the apoptotic response. The use of natural remedies, or nutraceuticals, in the treatment of cancer and a variety of other diseases appears prevalent in human and veterinary medicine. The use of plant extracts has been around for centuries, but investigations into the mechanisms of action across various cancer cell lines are more recent, and appear to be highly variable in cell culture systems –3. The eThe effects of these purified compounds have been examined in vitro in a variety of human cell lines derived from tumors of the colon, skin, and breast tissue , but onlThe objective of this in vitro study was to determine the effects on canine cancer cell death and possible mechanisms by which TE and RE exert anti-proliferative and cytotoxic effects individually and in combination on canine mastocytoma, mammary carcinoma, and osteosarcoma cell lines. Concentrations were chosen based on our prior publication surrounding the effective concentrations for synergy between the two extracts of interest . Markers−1. Fresh extract stock solutions were prepared and used for every experiment.Turmeric extract  and rosemary extract  were solubilized in 100% dimethyl sulfoxide  at 20 mg mL2 for all experiments and passage of cells, unless otherwise noted. Cell lines were cultured in appropriate complete medium as previously described.11 All culture reagents were purchased from Invitrogen, Carlsbad, CA, USA, unless otherwise indicated.Three canine neoplastic established cell lines, representing hematopoietic, epithelial, and mesenchymal tumor types were used for all experiments; mastocytoma C2 , mammary gland carcinoma CMT-12 , and osteosarcoma D17 . These cell lines were chosen for initial screening as representative cell lines of the three major cell lineages of cancer in dogs in hopes of finding a similar global effect across different cell lineages. Cell lines were grown on tissue culture-treated plates  at 37 °C and 5% CO3 cells per well on white walled 96-well tissue culture-treated plates  and incubated overnight in complete medium. Cells were treated the following day with DMSO vehicle control, 6.3 μg mL−1 extract alone, or 3.1 μg mL−1 each extract in combination for 36 h. Chemotherapeutic drugs at a 50% inhibitory concentration (IC50) were used as a positive control; 12.5 nM toceranib phosphate  was used for the C2 cell line, and 0.3 or 0.5 μM doxorubicin hydrochloride  was used for the CMT-12 and D17 cell lines, respectively. Background fluorescence and luminescence was measured in wells containing treatments but no cells. Caspase 3/7 activation was quantified using the ApoLive-Glo™ Multiplex Assay  following manufacturer’s instructions. Briefly, after 36 h of treatment, viability reagent was added to the wells and incubated at 37 °C for 30 m and fluorescence was measured at 400Ex/505Em. Next, Caspase-Glo 3/7 Reagent was added to all wells, incubated for 30 m at room temperature, and luminescence was measured. Fluorescence and luminescence was measured using SpectraMax M3 Microplate Reader .Cells were plated at a density of 4 × 10Cells were plated on 60 mm tissue culture-treated plates  and incubated in complete medium until 60% confluent. Cells were then treated with medium, DMSO vehicle control, extract alone, or extracts in combination. Cells were treated for 12 h (reactive oxygen species generation), 24 h (curcumin accumulation), or 48 h . All flow cytometric analysis was performed on BD FACSCalibur .Cell cycle effects were analyzed after 24 h (data not shown) and 48 h treatment using propidium iodide staining to label DNA content. Briefly, cells were detached with Accumax cell dissociation solution , collected in tubes with 1% fetal bovine serum (FBS) in Phosphate Buffered Saline (PBS) and centrifuged for 5 m at 300 rcf at 4 °C. The cell pellet was washed twice with 1% FBS in PBS, filtered, and resuspended in 70% cold ethanol for overnight fixation. The following day, samples were centrifuged for 10 m at 500 rcf at 4 °C, resuspended in cold PBS. Samples were centrifuged again for 5 m at 300 rcf at 4 °C and resuspended in DNA staining solution . Samples were then incubated for 30 m at room temperature and analyzed with an excitation of wavelength of 488 nm and emission of 617 nm. Only C2 and D17 cell lines were analyzed due to the presence of frequent doublets with CMT-12 cells resulting in an artificial accumulation in the G2/M phase.2, pH 7.4) at a density of 1 × 106 cell mL−1. Annexin-V 488 conjugate and 7-Aminoactinomycin D (7-AAD) were added to the cell suspensions and incubated for 15 m at room temperature. After the incubation, ABB was added to the cell suspension and kept on ice until fluorescence analysis. Events labeled only Annexin-V positive were considered to represent apoptotic cells; events labeled Annexin-V positive and 7-AAD positive were considered to represent necrotic cells.Apoptosis and necrosis was measured after 48 h treatment using Annexin-V and 7-AAD staining. Briefly, cells were detached with Accumax dissociation solutions , collected and centrifuged for 10 m at 500 rcf at 4 °C. The cell pellet was washed once with PBS before resuspension in Annexin Binding Buffer  have been implicated as antioxidants, Dihydrorhodamine123  assay was used to determine the amount of reactive oxygen species (ROS) present after 12 h treatment with each extract according to literature . BrieflyThe cellular accumulation of curcumin was measured by exploiting the auto-fluorescent properties of this compound . After 2−1 extract alone, or 3.1 μg mL−1 each extract in combination. Cells were harvested and lysed at 12 h and 24 h after treatment using Mammalian Lysis Buffer  and sonication, and then centrifuged for 5 m at 14,000 rcf at 4 °C. The supernatant was collected and the protein concentration was determined using the Bradford assay . Samples were equilibrated to a common volume (μg μL−1) in MLB and 5× laemmili loading buffer . For each protein of interest, 30 μg total proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on gels ranging from 6 to 15% based on the molecular weight of the protein of interest. The proteins were then transferred to 0.45 μm pore size polyvinylidene fluoride membrane  for 1 h at 333 mA and then blocked in 5% milk in Tris-buffered saline/0.05% Tween-20 solution (TBST). Membranes were incubated overnight in primary antibody solutions at a dilution of 1:1000 in TBST on a rocking platform at 4 °C. Primary antibodies included mouse anti- phosphorylated-gamma H2A.X and extracellular regulated kinase (ERK) ; mouse anti- Thr202/Tyr204 phosphorylated p44/42 MAPK (ERK1/2) and STAT3 ; rabbit anti- protein kinase B (AKT), Ser473 phosphorylated-AKT, stress-activated protein kinase/jun-N-terminal kinase (SAPK/JNK), Thr183/Tyr185 phosphorylated-SAPK/JNK, focal adhesion kinase (FAK), Tyr397 phosphorylated-FAK, Tyr576/Tyr577 phosphorylated-FAK, Tyr925 phosphorylated-FAK, Src, Tyr416 phosphorylated-Src, Tyr527 phosphorylated-Src, mammalian target of rapamycin (mTOR), Ser2448 phosphorylated-mTOR, Janus kinase 2 (JAK2), Tyr1007/Tyr1008 phosphorylated-JAK2, Ser727 phosphorylated-signal transducer and activator of transcription 3 (STAT3), Tyr705 phosphorylated-STAT3, B-Cell CLL/Lymphoma 2 (BCL2), and BCL2-Associated X Protein (BAX) . Membranes were washed three times with TBST and incubated at room temperature for 1 h in the corresponding secondary anti-mouse IgG or anti-rabbit IgG horseradish peroxidase-conjugated antibody at a dilution of 1:2000 . Membranes were washed three times with TBST and visualized with a chemi-luminescent reagent . Digital images were captured using an imaging system . After images were collected, membranes were washed three times in TBST and incubated with a 1:10,000 dilution in TBST of the house-keeping antibody β-Actin (Sigma-Aldrich) for 1 h at room temperature. Membranes were washed, incubated with mouse secondary antibody at a dilution of 1:2000, and imaged as described.Cells were plated on 100 mm tissue culture-treated plates (LPS) and incubated overnight in complete medium until 60% confluency was reached. Cells were treated the following day with DMSO vehicle control, 6.3 μg mLFor all flow cytometry experiments, 10,000 events were collected per sample and then gated based on a forward-scatter/side-scatter plot. Minimally three independent experiments were examined for each treatment through the different assays  and analyzed with Cell Quest software (BD Biosciences). DMSO was compared against cells in media alone showing no significant differences, therefore DMSO treated cells were used as the control sample for all comparisons. The geometric mean fluorescence (GMF) from each treatment was compared to the DMSO treated samples and represented as fold change for all experiments using GMF due to the differences in fluorescence intensity across cell lines. In addition, for measurements of ROS, an unstained control was used to determine the baseline GMF of each extract. This value was subtracted from the GMF of stained samples to correct for any shift due to auto-fluorescence of the extract with cells alone. Caspase 3/7 activation was determined as caspase activation per total viable cells for each treatment. Raw data from the viability portion of the assay  were normalized to the vehicle alone treatment for each cell line, considered to represent 100% proliferating cells. The ratio of caspase activation to viable cells is represented as fold increase over DMSO treatment alone. Each of the treatment conditions were completed in duplicate and averaged in four independent experiments. Western blots were run in three independent time course experiments and densitometry was completed using ImageJ . Values p < 0.05.All statistical analyses were performed using JMP Pro . The residuals of all statistical models were found to be normally distributed therefore parametric statistics were utilized. The fold-change data from caspase 3/7 activation, percent of apoptotic cells, intracellular ROS level and curcumin accumulation assays and the ratio data from western blot assay were processed using analysis of variance with Tukey’s method for multiple comparisons between all treatment conditions . In the case of cell cycle dynamics, Dunnett’s method was used to control for multiple comparisons when studying the percent of gated events difference between single treatment or dual combination and DMSO control only at each time point. Differences were considered statistically significant at −1 TE resulted in a significant decrease in S phase (DNA replication) in the D17 cell line compared to DMSO control. Treatment with 6.3 μg mL−1 RE induced a significant decrease in G1/G0 phase in the D17 cell line, a reduction in S phase in both cell lines, and an increase in G2/M phase (cell division) in the D17 cell line. The combination treatment using 3.1 μg mL−1 both extracts induced a small decrease in S phase in only the C2 cell line, and a modest increase in G2/M phase in only the D17 cell line. While these differences were significant, the mild alterations in cell cycle in the D17 and C2 of 5–10% decrease in the G1/G0 and increase in G2/M phases with RE and less consistent yet similar changes with both TE and RE combined were not considered large enough to continue examining pathways related to cell cycle arrest as observed in prior studies using these cell lines [The effects of TE and/or RE on cell cycle progression were measured on C2 and D17 tumor cell lines using propidium iodide staining. Cell cycle dynamics were analyzed after 24 h and 48 h of incubation with the different treatments; no significant difference was seen between these two time-points therefore only data from the 48 h time point is shown Fig. . Represell lines .Fig. 1Ef−1) resulted in a significant increase in apoptotic cells in the C2 and CMT-12 cell lines as determined by Caspase 3/7 activation, 2- and 2.5-fold, respectively  was used, a significant increase in Annexin-V positive cells compared to vehicle control was seen in the 3 cell lines, but this was not significant compared to 6.3 μg mL−1 TE alone in C2 cell line. However, in the CMT-12 and D17 cell lines, the combination treatment induced a significantly greater percentage of apoptotic cells, 40% and 13%, respectively, compared to 6.3 μg mL−1 TE alone (13% and 7%) and 6.3 μg mL−1 RE alone (5%)  in all the three cell lines  with TE reducing ROS by about 75–90-80%, respectively and RE reducing ROS by about 50–40-40%, respectively. The dual combination treatment using half the concentration (3.1 μg mL−1 each extract) was as effective as 6.3 μg mL−1 TE alone in all three cancer cell lines  significantly increased the GMF in the C2 and D17 cell lines, 1.7- and 1.8-fold, respectively; while when using RE with TE the increase was 2.2 and 2.3 fold, respectively . The addition of RE at the same concentration to TE resulted in a significant increase in GMF of 4.8-fold in the CMT-12 cell line beyond that of TE alone .Observation from previous flow cytometry experiments showed an unexpected increase in the GMF when cells were treated with TE alone when excited at a wavelength of 488 nm, whereas no change was observed when RE was used alone Fig. . A simil−1 TE resulted in an increase from a densitometry value of 1.1 at 12 h to 1.5 at 24 h in p-SAPK/JNK in the C2 cell line, stable activation from 12 h to 24 h in the CMT-12 cell line . In the D17 cell line only a minor non-significant increase was observed . Activated SAPK/JNK increased from 12 h to 24 h in the C2 cell line  and the CMT-12 cell line (1.2 at 12 h to 1.5 at 24 h) which was not significant over time. Minimal change was seen in the D17 cell lines after treatment with 6.3 μg mL−1 RE, 1.1 at both time points. The greatest increase in p-SAPK/JNK was seen with the combination of 3.1 μg mL−1 each of TE and RE in the CMT-12 cell line where densitometry values increased from 1.0 with DMSO treatment to 4.3 after 12 h incubation and 4.8 after 24 h incubation (p < 0.05 from DMSO treatment). Although there were similar increases in SAPK/JNK activation with TE and dual treatment of TE/RE  in C2 and D17 cell lines, these were not significantly different from DMSO control at 12 h or 24 h incubation time points. These results demonstrate possible mechanisms behind the observed susceptibility differences across the three cell lines, particularly in light of the heightened response of lesser doses of RE and TE in combination when compared to higher concentrations each extract independently in the CMT12 cell line.After examination of several cell signaling pathways, no consistent trend was seen in the phosphorylation status of the variety of signaling proteins, alterations in the mitochondrial proteins involved in apoptosis or markers of DNA damage (data not shown). However, changes in Thr183/Tyr185 phosphorylated-SAPK/JNK  [Bioactive molecules derived directly from plants, or modeled after plant compounds, continue to be an active area of cancer research. The majority of these studies have been focused on human and rodent cancer models and the effects of these plant extracts and select compounds vary depending on species and cell origin , 24. Fewndently) . In agrePrior studies have shown the autofluorescence of curcumin can be examined with flow cytometry . An incr2/M phase. This was attributed to cell clumping in this cell line due to the fixation method used and use of propidium iodide causing cell-doublets to be inappropriately represented as G2M phase. There were some mild alterations in cell cycle in the D17 and C2 cell lines showing decreases in the G1/G0 and increases in G2/M phases with RE and the combination treatment. There were minimal to no cell cycle changes with TE extract alone, therefore further examination of cell cycle pathway analysis was not pursued. Prior literature has shown that curcumin can have significant effects on cell cycle dynamics through the upregulation of cyclins or cyclin dependent kinase activity [The global effects of both TE, RE, and the two in combination showed no appreciable alteration in cell cycle kinetics. Of the three cell lines used in our experiments, only the C2 and D17 cells could be examined as a single cell suspension for cell cycle dynamics, while the CMT-12 cells displayed an artificial accumulation of cells in the Gactivity –28. In tIn line with pathway disruption and cell cycle dynamics, TE and RE are often thought to be antioxidants, however curcumin has been shown to cause oxidative damage to DNA and induction of the DNA damage response pathways that are intimately involved in cell cycle alteration or apoptosis , 30. OurFurther examination of the cellular effects into cell signaling pathways previously implicated in TE and RE treatment were performed –35. ConcDifferences in treatment responses were observed in the three cell lines. Our results showed an increase in phosphorylated SAPK/JNK after 12 h and 24 h of treatment with TE alone. RE induced a significant increase in phosphorylation after 12 h and 24 h of treatment in the CMT-12 cell line, while in the C2 cell line this increase was only seen at 12 h and returned to baseline by 24 h. The dual combination treatment had the greatest effect in the CMT-12 cell line, resulting in phosphorylated SAPK/JNK at levels greater than either extract alone (even using twice the concentration). Only the CMT-12 cell line showed sustained activation of SAPK/JNK with the combination treatment which may be the underlying reason behind the increased susceptibility of this cell line. SAPK/JNK has been implicated as a therapeutic target in certain contexts and patterns of activation whereby constitutive activation appears to be beneficial towards a pro-apoptotic response in a variety of cell lines and animal models –49. The Although the results of these experiments are promising, the clinical utility is complex due to the absorption, transformation and elimination kinetics of these compounds in general. The use of highly bioavailable curcumin is currently being examined and has 10–15% bioavailability . CarnosiThe results of this study provides some insights into possible mechanisms by which TE and RE induce apoptosis across three canine neoplastic cell lines. Our results indicate that different tumor types are likely to have a differential response to such interventions. The enhanced susceptibility found in the CMT-12 mammary cancer cell line may be due to the increased accumulation of curcumin when the combination treatment was used. In addition, sustained activation and signaling through the SAPK/JNK pathway may play a role in this cell line’s increased sensitivity to apoptosis. The results of this study warrant further investigations into the pharmacodynamics and pharmacokinetics of these extracts in dogs when incorporated into feed to determine if clinical trials are feasible.


Chromosomal translocations are a hallmark of cancer cells and give rise to fusion oncogenes. To gain insight into the mechanisms governing tumorigenesis, adequate model cell lines are required.de novo (CD74-ROS1) or existing translocations are reverted back to the original configuration (BCR-ABL1). To this end, we co-apply two guide RNAs to artificially generate two breakpoints and screen for spontaneous fusion events by PCR.We employ the versatile CRISPR/Cas system to engineer cell lines in which chromosomal translocations are either generated The approach we use is efficient and delivers clones bearing translocationsin a predictable fashion. Detailed analysis suggests that the clones display no additional undesired alterations, implying that the approach is robust and precise.The online version of this article (doi:10.1186/s12864-016-3084-5) contains supplementary material, which is available to authorized users. Chromosome abnormalities are a characteristic feature of cancer cells. Recurrent chromosome rearrangements, like translocations, are often found in various tumour types, and there is overwhelming evidence that they are key drivers of tumorigenesis . BalanceThe first ever identified chromosome translocation in cancer was the Philadelphia chromosome originating from a balanced translocation between chromosomes 9 and 22 . The PhiChromosome rearrangements involving ROS1 gene have been found in 1–2 % of patients with non-small cell lung cancers. Several fusion partners are known for ROS1, among which CD74 is the most common . The CD7Streptococcus pyogenes that can be reprogrammed by short RNAs  to induce a DNA double-strand break at any position that is followed by a protospacer adjacent motif (PAM) (NGG for S. pyogenes Cas9). Repair of the double-stand break can either occur by non-homologous end joining or by homology-directed repair, depending on whether a homology template is present or not. The CRISPR/Cas system has been used for genome engineering in a large number of species and for a wide variety of purposes  and 4 L20 (BCR-ABL1 reversion) were selected for further analysis at the genomic DNA level. PCR analysis indicated that the mutant clones contained the desired translocation and that this translocation was absent in parental eHAP cells  and FKBP rapamycin binding (FRB) domain system . AlternaWhile the high efficiency we observed is desired when engineering chromosomal translocations, it may be undesired in other instances, e.g. when engineering double knockouts. We suspect that at least in some instances, the simultaneous delivery of two guide RNAs will cause translocations and other genomic rearrangements as unwanted side effects. While these can easily be assessed in clonal cell lines that proliferate almost indefinitely, they may be more difficult to detect in scenarios where cell populations are more heterogeneous.We used the haploid human cell line eHAP for the experiments presented here. In our view, this choice offers three major advantages, all of which are based on the fact that the cell carries a single genome copy: (i) Genotyping is a lot more straightforward than in diploid cells, (ii) Reversion back to wild-type configuration is hampered because a suitable homology template is missing and (iii) Cell lines derived contain 100 % allele burden with regard to the chromosomal translocation. However, we believe that this approach would be equally feasible in diploid cells. To exemplify this, we transduced diploid 293 T cells with two gRNAs for CD74 and ROS1 and detected both the CD74-ROS1 fusion and the reciprocal ROS1-CD74 fusion by PCR  to guaranty cell line clonality. Monoclonal cell lines were expanded using Microlab STAR Line robotic system (Hamilton Company).For the primary PCR screen, genomic DNA was prepared using DirectPCR Lysis reagent (PeqLab) containing Proteinase K (Sigma). Genomic DNA was isolated from selected clones using the Gentra Puregene Cell Kit (Qiagen) according to manufacturer’s instructions. PCR was performed using GoTaq Polymerase (Promega) according to manufacturer’s instructions. PCR products were analysed using 1 % TAE agarose.Total RNA was purified using RNAeasy Plus Mini Kit (Qiagen) and cDNA was synthesized using SuperScript® III Reverse Transcriptase (Thermo Fisher Scientific) according to manufacturer’s instructions.Spectral karyotyping was performed and analyzed by WiCell Cytogenetics. In brief, metaphase chromosome spreads were prepared on slides according to standard cytogenetic procedures. These slide preparations were then hybridized according to the DNA spectral karyotyping hybridization and detection protocol provided by Applied Spectral Imaging (ASI) using the supplied SKY probes and detection system. Images were captured and analysed using the HiSKYV spectrum imaging system from ASI.


Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response.Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an  In addition to their extensive proliferation and genetic stability in culture, these human cells can give rise to primary cell types relevant for drug response, including cardiomyocytes, hepatocytes and neuronsTo date, most lines of hPSCs available are of European or Eastern Asian ancestryThe Brazilian population results from 500 years of admixture among the original Native Americans, Europeans (mostly Portuguese), and sub-Saharan Africans, most of which were brought to the country as slaves78910We have previously established five lines of hPSCs from surplus human embryos generated for reproduction purpose and stored in private human reproduction clinics in Brazil121315in vitro into tissues from the three embryonic germ layers , homogeneously express the pluripotency markers OCT4, NANOG and SSEA-4, and to differentiate m layers . In addim layers .Genomic ancestries of five lines of hESCs derived from embryos generated in Brazil for reproductive purpose and of the eighteen hiPSCs were determined by the analysis of 192 ancestry informative markers and comparison with reference populations . The datCYP1A2, CYP2C8, CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP2B6, CYP2A6), known for its role in drug metabolism. We found a high level of heterogeneity of ancestry among the hPSC lines, with all CYP genes investigated having cell lines with representatives from African and European ancestries, and all loci except CYP2C8/CYP2C9/CYP2C19 having at least one instance in which it lies in a region of Native American ancestry  are mosaics comprised of segments of the three ancestries . We inveancestry .Human PSCs are a valuable model system in which to study Mendelian conditions, where a single gene mutation triggers the clinical phenotype45Analysis of the genomic ancestry of all lines of hESCs derived in Brazil confirmed our initial hypothesis that the embryos available for research do not represent the ethnic diversity of the Brazilian populationMoreover, we also analyzed the ancestry of 2 hESC and 4 hiPSC in the region containing eight genes from the CYP family, responsible for the metabolism of the majority of drugs and xenobiotics. We looked specifically at CYP family members that have polymorphisms described in different frequency between the ethnic groups, and different levels of metabolic response reportedin vitro clinical trials and other studies of drug response, the availability of extensive clinical data of each participant permits the generation of different collections of cells based on phenotypes of interest. For example, a group of 4,116 participants being treated for hypertension was identified, 11% of those being resistant to pharmacological interventionThe ELSA-Brasil cohort study represents a unique opportunity for the generation of collections of hiPSCs for different research purposes. The study collects data from 15,000 35–74 year old participants from three regions of Brazil  every 3–4 years in the form of interviews, electrocardiogram, blood sample, blood pressure, as well as other clinical, biochemical and genetic testsWe derived lines of hiPSCs with novel combinations of genomic ancestry, including Native American and African. In addition, we have established a collection of primary cells from a well clinically characterized population ready to be reprogrammed into hiPSCs for different research purposes. The new hiPSC lines described here significantly increase the ethnic diversity and genetic admixture of the currently available hPSCs.Participants of the ELSA-Brasil in São Paulo were invited to participate in this research. Exclusion criteria were current or recent (<4 months prior to the first interview) pregnancy, intention to quit working at the institution in the near future, severe cognitive or communication impairment, and, if retired, residence outside of a study center’s corresponding metropolitan area. The first examination was carried out from 2008 through 2010. Annual telephone surveillance for outcomes is now in its seventh year, and the first follow-up examination was conducted form 2012 through 2014. From the 5061 participants, 1872 were randomly chosen to be included in the present analysis. Participants signed informed consents. The project was carried out in accordance with the guidelines for research on human subjects, and approved by the Ethics Committee of the University Hospital, University of São Paulo.Oct4, Sox2, Klf4, cMyc, Lin28 and SV40-T, using the Human CD34+ nucleofector kit and the Nucleofector II device, both by Lonza  following manufacturer’s instructions. Reprogrammed erythroblasts were incubated in MEF-coated plates in MEF medium and FBS ES-Cell Qualified  with basic fibroblast growth factor  overnight. Then, they were transferred into embryonic stem cell (ESC) medium containing Knockout DMEM (Life Technologies), Knockout Serum replacement , Antibiotic-antimycotic (Life Technologies), Glutamax 200 mM (Life Technologies), MEM non‐essential amino acid solution (Life Technologies), 2‐mercaptoethanol (Life Technologies) supplemented with bFGF (20 ng/ml) and Sodium butyrate (0.25 mM). hiPSC colonies were passaged from a 6 well MEF-coated plate into Matrigel (BD)-coated plates with E8 medium (Invitrogen), using Gentle Cell Dissociation Reagent (Stemcell Technologies) and 10 μM ROCK inhibitor Y-27632 (Stemgent).Human embryonic stem cell lines BR-1, BR-2, BR-4, BR-5 and BR-6 were cultured as described121323Human Pluripotent Stem Cell Transcription Factor Analysis Kit from BD according to the manufacturer’s instructions. Cells were incubated with human monoclonal antibodies specific to Nanog-PE, Oct3/4-PerCP-Cy5.5 and Alexa 647-Sox2 and corresponding isotype controls for 30 minutes and washed twice with PBS. Fifty thousand events were acquired in BD Accuri C6 flow cytometer using the kit template. Cells were gated on light scatter properties and analyzed for expression of key pluripotency transcription factors using BD Accuri C6 software.Cells were fixed and labeled using Cells from passages 6 and 29 were fixed and immunostained following standard protocolsin vitro differentiation, hiPSC and hESC were grown to confluency and harvested by dispase. Cells were washed and re-suspended twice in E8 medium and transferred to non-tissue culture treated 6 well plates coated with 1% agarose. Day 5 embryoid bodies (EBs) were transferred to a 6 well plate coated with 0.1% gelatin, and cultured for a further 15 days in differentiation medium . Differentiated cells were harvested with Trizol and RNA extraction was performed usingRNeasy kit (Qiagen). One mg of RNA was used for subsequent reverse transcriptase reactions using High Capacity cDNA kit (Life Technologies). Pluripotency and trilineage differentiation potential was assessed using the TaqMan® hPSC Scorecard™ kit 384w following manufacturer’s instructions and run on a StepOne Plus System (Life Technologies), as described® hPSC Scorecard™ analysis software (Life Technologies).To demonstrate spontaneous Absence of vector DNA integration into hiPSC lines was confirmed by PCR analysis as described® Real-Time PCR (Applied Biosystems). We used as reference ancestral populations those from the Human Genome Diversity Project (HGDP) (Center SHG. Human Genome Diversity Project): Pima, Maya as Native Americans; and from HapMap project : Africans - YRI , LWK , ASW ; European - CEU (Utah Residents (CEPH) with Northern and Western European ancestry) and TSI .Analysis of genomic ancestry was conducted using the Admixture programet al.Local ancestry analysis was performed by genotyping samples with the Axiom Human Origins Array  according to manufacturer’s instructions (Affymetrix). As parental populations we used a sample of 30 unrelated African and 30 unrelated Europeans from 1000 Genomes Project (1000 g) and the 30 unrelated Native Americans from the Centre d’Étude du Polymorphisme Humain and the Human Genetic Diversity Panel (CEPH-HGDP). The SNPs from these three datasets (1000 g+HGDP and present study) were filtered and paired according to Nunes How to cite this article: Tofoli, F. A. et al. Increasing The Genetic Admixture of Available Lines of Human Pluripotent Stem Cells. Sci. Rep.6, 34699; doi: 10.1038/srep34699 (2016).


Lung cancer remains the leading cause of cancer-related death worldwide, with one-third diagnosed with locally advanced (stage III) disease. Preoperative induction chemo-radiotherapy is key for the treatment of these patients, however conventional cisplatin based approaches has apparently reached a plateau of effectiveness. In the search for new therapies, the targeting of tumor metabolism is revealed as an interesting option to improve the patient’s responses. Here we describe the importance of PGC-1alpha and GAPDH/MT-CO1 ratio levels as surrogates of the Warburg effect from a series of 28 stage III NSCLC patients, on PFS, OS and PET uptake. Moreover, our results show a great variability between tumors of different individuals, ranging from very glycolytic to more OXPHOS-dependent tumors, which compromises the success of therapies directed to metabolism. In this sense, using 3 different cell lines, we describe the relevance of Warburg effect on the response to metabolism-targeted therapies. Specifically, we show that the inhibitory effect of metformin on cell viability depends on cell’s dependence on the OXPHOS system. The results on cell lines, together with the results of PGC-1alpha and GAPDH/MT-CO1 as biomarkers on patient’s biopsies, would point out what type of patients would benefit more from the use of these drugs. Histologically, we can differentiate two types: small-cell lung cancer and non-small cell lung cancer (NSCLC), being the latest the most common, counting for the 80–85% of the cases4. Thus, preoperative induction chemotherapy is key, because it can early eradicate distant micro-metastases and the down-staging increases the number of patients whose tumors are considered resectable, which contributes to improve survival5. After chemotherapy, surgery is potentially curative for these patients, being offered to the vast majority of patients with resectable N2 and to some patients with N3 node involvement (TNM staging system).Approximately one-third of patients with NSCLC are diagnosed with locally advanced (stage III) disease. Currently, the therapeutic strategy for these patients is the neo-adjuvant multimodal treatment, since local therapy alone (surgery or radiation) leads to poor overall survival because most of the patients die of distant metastases6. Over the last few years, based on genetic alterations present in the tumor, new targeted therapies focused on the inhibition of key pathways for tumor proliferation have started to appear. However, these therapies do not cover most patients and have not yet reached the clinical routine for stage III patients. Thus, a need for new effective chemotherapy regimens that can be combined with local therapies (surgery and/or radiation) is still open7. In this sense, there is a considerable interest in identifying novel targeted agents that interfere with other hallmarks of NSCLC, such as tumor metabolism, which has recently aroused great interest.Sadly, conventional treatment of NSCLC has apparently reached a plateau of effectiveness in improving survival of patients, and treatment outcomes must still be considered disappointing8. This predilection of glycolysis over the complete oxidation of nutrients via oxidative phosphorylation (OXPHOS) in the presence of oxygen (such as that performed by differentiated tissues), has to do with the facilitation to incorporate nutrients into biosynthetic pathways that support a rapid cell proliferation9.The Warburg effect describes the preference of tumor tissues to obtain energy through glycolysis regardless of oxygen availability12. However, despite its importance, the PGC-1alpha role in glucose metabolism reprograming in NSCLC has not been studied14.PGC-1alpha is a transcriptional coactivator responsible for mitochondrial biogenesis. Thus in general terms, high levels of PGC-1alpha are associated with more OXPHOS-dependent metabolism of the cells, whereas low levels are associated with a more glycolytic phenotypein vitro and in vivo of its possible clinical utility16. In fact, several clinical trials based on this have been done or are currently ongoing, although their clinical benefits have not been as successful as one might expect17.Since the Warburg effect was described, many drugs targeting cancer metabolism have been developed. Unfortunately, there is a current unavailability on cancer drugs in the clinic targeting the tumor metabolism for lung cancer, despite the existence of scientific evidence Remarkably, at the moment there are no good markers of response, most probably because mechanisms at the molecular level are still not clear. Thus, the characterization of these biomarkers is fundamental in order to identify patients who can benefit more from certain drugs targeting metabolism. This could explain why the clinical trials carried out have not been successful.Therefore, knowing that the fundamental basis of treatment and possible cure of these patients is the neoadjuvant chemotherapy, we set out to identify the role of glucose metabolism in the stratification of patients at this stage, and in a second phase, to link these molecular findings with the sensitivity to different metabolic treatments.To evaluate the importance of Warburg effect on NSCLC patients, we first analyzed the prognostic value of PGC-1alpha and GAPDH/MT-CO1 ratio mRNA levels on Overall-Survival (OS) and Progression Free Survival (PFS). In order to select a group of patients with similar characteristics, we focused on a cohort of 28 stage IIIA NSCLC patients. In this cohort the median PFS and OS were 18 [95% CI: 10.1–26.1] and 22.2 [95% CI: 8.9–35.4] months respectively.PGC-1alpha is responsible for mitochondrial biogenesis. On the other hand, the GAPDH/MT-CO1 ratio is indicative of the balance between glycolysis and OXPHOS metabolism. GAPDH  is a key enzyme in glycolysis and MT-CO1 is one of the three mitochondrially encoded subunits that form the core of the Complex IV of the OXPHOS system.Our results reveal a decreased OS for patients with PGC-1alpha low levels, median 15,4 months [95% CI: 7–23 months], compared to high levels patients with an OS superior to 24 months Fig. .Figure 1With respect to the GAPDH/MT-CO1 ratio, the patients were grouped into tertiles, grouping the medium and high levels in the same category since they behaved similarly compared to the low tertile. The median OS for low levels was higher than 24 months while for medium or high levels were 22.2 and 17.9 respectively (data not shown). Opposed to PGC-1alpha, low levels of the GAPDH/MT-CO1 ratio are indicative of a higher OS compared to the medium-high expression group: low ratio OS >24 months versus medium-high ratio 19.4 months [95% CI: 11.3–27.4 months] Fig. .Although studies on PFS were not statistically significant, a clear difference was observed in the same sense as for OS, being high PGC-1alpha levels and low GAPDH/MT-CO1 ratio good prognostic factors. Comparing the PFS medians for both markers these differences are clearer: low PGC-1alpha 8,5 months [95% CI: 6.7–10.2] vs high PGC-1alpha 22 months [95% CI: 15.6–28.4], and medium-high GAPDH/MT-CO1 ratio 11,9 months [95% CI: 7.4–16.3] vs low GAPDH/MT-CO1 ratio 22 months. These obvious differences in median PFS are lost with the disease course, as the disease progresses in all patients Fig. .These results clearly indicate that a more glycolytic metabolism  confers a worse prognosis in both PFS and OS. Furthermore, the expression levels of both variables correlate inversely with a negative correlation index of 61% Fig. .On the other hand, from the diagnostic positron-emission tomography (PET) images of the patients, the maximum standardized uptake value (SUVmax) levels of the tumor lesions were calculated. Due to the low number of PET images, the cohort was expanded to include other stages, thus obtaining a total of 22 patients with SUVmax information. The results show an increase in SUVmax levels in patients with medium-high levels of GAPDH/MT-CO1 Fig. .Finally, to directly assess the role of the Warburg effect on the proliferative capacity of tumor cells, we analyzed the number of mitosis in hematoxylin-eosin tumor sections and correlated them with the expression levels of PGC-1alpha and GAPDH/MT-CO1 ratio as metabolism biomarkers. These data demonstrate an inverse correlation of the number of mitosis with PGC-1alpha levels (correlation of 61%) and a direct correlation with GAPDH/MT-CO1 ratio levels (correlation of 45%) Fig. .0 cell line, derived from the A549 cell line, lacks the mitochondrial DNA (mtDNA) and serves as negative control of the OXPHOS function.To understand the molecular role of the Warburg effect in NSCLC, we decided to move on to an in vitro model using established cell lines. Three cell lines  from different origins and with different mutational profiles were chosen Fig. . The A54First, we set out to determine whether these cell lines have different aggressiveness as a consequence of their different origins: A549 primary tumor, H1299 lymph node metastasis and H460 pleural effusion. For that we characterized their migration capacity through wound healing assays and their clonogenicity. Our results showed an increased clonogenicity for H1299 and H460 cell lines compared to A549 cell line Fig. . Moreove0 line (without OXPHOS function) is unable to grow with galactose as sugar.Next, as a first approach to define their glucose metabolism, namely more glycolytic- or OXPHOS-orientated, we compared the cells abilities to grow in the presence of either glucose (Glc) or galactose  as carbon source Fig. . Our resThen, to study further in-depth their metabolism and to verify that they behave in a similar way to the patients , we analyzed different parameters such as oxygen consumption, reactive oxygen species (ROS) levels, glucose consumption and PGC-1alpha and GAPDH/MT-CO1 ratio levels.Consistent with previous findings, the oxygen consumption rate showed a similar behavior to the Gal/Glc ratio, with significant differences between the H460 and A549 or H1299 cell lines. Despite the decrease in oxygen consumption of H1299 cells compared to the A549 cells, this difference does not become statistically significant Fig. .Figure 3ROS levels are associated with defects in the OXPHOS function and with a higher degree of tumor progression. Our results indicate that H1299 has the highest ROS levels among the cell lines, followed by H460 cells which have higher ROS levels than A549 cells Fig. .Glucose consumption was assessed with the 2-NBDG fluorescent probe, similar to the fluorodeoxyglucose (FDG) used in patient’s PET images. We observed a significant increase in glucose consumption for the H1299 and H460 cell lines with a more glycolytic profile, although no differences were observed between them Fig. .Finally, PGC-1alpha and GAPDH/MT-CO1 levels were evaluated Fig. . A549 ceOnce the cell lines were metabolically characterized and verified that the relationships between metabolism and aggressiveness parameters were similar to those found in patients, we aimed, as a proof of concept, to test their sensitivities to two drugs (2-deoxy-glucose and metformin) targeting different types of metabolism.It has been reported that metformin acts as an inhibitor of Complex I of the OXPHOS system. To check the effect of metformin on the OXPHOS function, we treated all cell lines with 1 mM or 10 mM metformin for 24 hours Fig. .Figure 40, whose MIMP remained constant with the treatments.Our results showed a significant decrease in mitochondrial inner membrane potential (MIMP) for all cell lines dependent on the concentration of metformin present. The only cell line that did not present this effect was the A549ρ0 are not sensitive to the effect of metformin at low doses since there are no significant differences compared to untreated cells. Consistent with this result, the clonogenicity assays showed greater metformin sensitivity for A549 than for H1299 and H460 cell lines, which exhibited reduced metformin sensitivity , showing a decrease in the number of viable cells for A549 cell line for both doses in a concentration dependent way. On the contrary, it appears that cell lines H1299, H460 and A549ρity Fig. .0 cell line is extremely sensitive (there are no viable cells after treatment), followed by H1299 and H460 cell lines which reduce their viability to approximately 50%. Finally, the A549 cell line is the least sensitive, reducing its viability only 20% compared to untreated cells. In the same way, the number of colonies after 2-deoxy-glucose exposure is lower for H1299 and H460 compared to A549 cells  and tumors with an energetic profile more based on their OXPHOS system . In addition, our results confirm that the Warburg effect has a clinical relevance in NSCLC, both in PFS and OS. The majority of the studies in stage IIIA, even with the heterogeneity of these patients, show a PFS and OS in the order of months, which vary in function of diverse prognostic factors. In our cohort of patients the median OS was 22 months similar to other stage IIIA patient cohorts19. The inverse relationship of PGC-1alpha and GAPDH/MT-CO1 ratio with PFS and OS of patients coincides with the function of these genes related to glucose metabolism and with the Warburg effect. Indeed, both variables correlate inversely, reinforcing this affirmation.Thereby, our results demonstrate that the glycolytic phenotype correlates with a worse prognosis in both PFS and OS in stage IIIA NSCLC patients. Similar conclusions for stage I NSCLC patients were obtained from the proteomic-based bioenergetic cellular indexin vitro studies with cellular models to find markers that define sensitivity to drugs directed to glucose metabolism.Next, due to the impossibility of directly asses drug response in patients, we performed 20.Prior to determine their drug sensitivity, we characterized their aggressiveness and biochemical function. Similar to the results in patients, our cell lines with different aggressiveness showed different metabolisms. A549 cell line showed the weakest malignant phenotype, with low clonogenic potential, migration capacity and a growth rate. On the other hand, H1299 and H460 showed higher aggressiveness than A549 cell line for most of the analyzed parameters. Regarding to metabolism characterization, A549 cell line showed a more OXPHOS metabolism, with higher Gal/Glc index, greater oxygen consumption, lower glucose consumption, elevated levels of PGC-1alpha and lower levels of GAPDH/MT-CO1, whereas the tumor cell lines H1299 and H460 turned out to be more glycolytic, with lower Gal/Glc index, lower oxygen consumption, greater consumption of glucose, reduced levels of PGC-1alpha and elevated GAPDH/MT-CO1 ratio. Nevertheless, there are differences between H1299 and H460 cell lines for some of these parameters although not enough clear to observe a pattern, which make it impossible to conclude differences between these two cell lines. However, the relationship between higher glycolysis with increased malignancy described in our cell lines is similar to other tumor types21. In our study we demonstrate that this compound reduces the MIMP of cells, most probably as a consequence of Complex I inhibition, since Complex I is mainly responsible for MIMP maintenance. Thus, treatment with metformin does not reduce MIMP in A549ρ0 cells, since it is maintained by another mechanism and not through Complex I activity22. Furthermore, when A549 cell line (the one with higher OXPHOS function and highest metformin sensitivity) lacks the mtDNA and therefore is forced to lose its OXPHOS dependence, is no longer sensitive to metformin. On the other hand, H1299 and H460 cell viability is not severely affected, even though metformin decreases their MIMP. This is probably because their MIMP and the associated OXPHOS function are not so important for their growth, as demonstrated by their biochemical parameters. In contrast, 2-deoxy-glucose, which inhibits hexokinase, and blocks the glycolytic pathway, affects the cell lines that are more glycolysis-dependent, such as H1299, H460 and A549ρ0 but does not disturb the more OXPHOS-dependent A549 cell line.Finally, we measured cell lines’ sensitivity to metformin and 2-deoxy-glucose. We propose that metformin affects the cells more when they are more OXPHOS-dependent. Recently, it has been described that metformin is an inhibitor of the complex IOur study point out, through PGC-1alpha levels, an easy manner of assessing the metabolic phenotype in patients and also suggest a correlation with sensitivity to metabolism targeted drugs. The approach proposed, focused more on the phenotype than on the tumor genotype, avoids the difficult interpretation of complex genomic results, even more for a phenotype that can be regulated by multiple genes, as well as, by epigenetic and microenvironmental factors. Further studies will clarify the relationship of these factors in lung cancer, defining the tumor metabolism in a more comprehensive fashion. This knowledge could have implications for the patients’ diagnosis, follow-up and treatment.in vitro drug-sensitivity in cell lines would suggest that PGC-1alpha and GAPDH/MT-CO1 levels could be used to predict patient response to treatments based on targeting glucose metabolism on lung cancer.To summarize, our results indicate that exist variability in glucose metabolism pathways in lung cancer patients’ tumors and established cell lines. In this respect, PGC-1alpha mRNA levels inversely correlate with GAPDH/MT-CO1 ratio. Interestingly, both markers have a clinical relevance in prognosis (PFS and OS), supporting the relationship between a more glycolytic metabolism with a more aggressive phenotype. Furthermore, the All the experiments carried out in this study complied with current Spanish and European Union laws and the principles outlined in the Declaration of Helsinki. The study and experimental protocols were approved by the Hospital Universitario Puerta de Hierro Ethics Committee and written informed consent was obtained for all the patients recruited.A cohort of 28 stage IIIA NSCLC patients was studied. Clinical data from these patients: Overall-survival (OS), Progression-Free survival (PFS), SUVmax levels, etc. as well as molecular data from diagnostic biopsies, were obtained for the analysis.Unfortunately we were only able to obtain PET images for 8 of these patients. Thus, the cohort was increased for SUVmax correlations, retrieving the SUVmax levels from another 17 patients to a total number of 25 patients with SUVmax data .23. Per sample, 10 HPF were analyzed by two independent anatomical pathology technicians and the mean was calculated.The number of mitotic figures was evaluated in the hematoxylin-eosin diagnostic slides using high-power field (HPF) at × 400 magnification0 cells were kindly provided by Dr. Rafael Garesse, from UAM (Madrid). All cells were cultured routinely in DMEM, with 10% Fetal Bovine Serum (FBS), penicillin streptomycin (Gibco) and uridine (Sigma). The mutational landscape of TP53, KRAS, NRAS, PIK3CA, LKB1 was retrieved from COSMIC database.A549, H1299 and H460 were obtained from the ATCC. A549 ρCells were cultured in a 24-well plate until they reached 90% confluence and then scratched with a 200 µL pipette tip. The wounded area was photographed at the start (t = 0 h) and at time point (t = 16 h). Pictures were analyzed using Image J software and the migration rate was calculated as the distance recovered by the migration front per time unit (µm/h). Assays were performed in at least 3 independent experiments.The RNA from cells was extracted using the “RNeasy mini Kit with DNAse” (Qiagen). The RNA extraction from FFPET biopsies was performed using the “High Pure FFPET RNA isolation Kit” (Roche). cDNA was synthetized using the “NZY First-Strand cDNA Synthesis Kit” (NZYtech).24.mRNA expression was evaluated by qRT-PCR using the following Taqman® gene expression assays: PGC-1alpha(Hs01016719_m1), GAPDH (Hs02758991_g1), MT-CO1 (Hs02596864_g1). TBP (Hs00427621_m1) was used as endogenous controlDoubling Time Software v1.0.10 (http://www.doubling-time.com). Assays were performed by duplicate in at least 3 independent experiments.Cell growth was evaluated after seeding 6 well plates at a density of 25,000 cells/well and growing the cells for 4 days in DMEM containing either 4.5 g/L glucose (Glc) or 0.9 g/L galactose . The cells were harvested and counted every 24 hours. Population doubling times were calculated using 3 cells per well of a 96-well plate were cultured in DMEM overnight. The next morning the medium was replaced for DMEM containing the different metformin treatments according to figure legends and incubated for 48 hours. Cell viability was assayed using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s protocol. Metformin (Sigma) was diluted to 1 M stock, aliquoted and frozen until its use. Assays were performed by duplicate in at least 3 independent experiments.3 × 103 cells per well of a 96-well plate were cultured in DMEM overnight. The next morning the medium was replaced for a solution  containing 5 mM 2-deoxy-D-glucose or 10 mM glucose25. After 6 hours of incubation the percentage of viable cells in 2-d-G relative to glucose was evaluated using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s protocol. Assays were performed by duplicate in at least 3 independent experiments.6 × 1050.000 cells per well were seeded on 6-well plates. The next day medium was replaced for DMEM containing the different metformin or 2-d-G treatments. After 72 h incubation cells were trypsinized and 1,000 single viable cells were plated in 100-mm Petri dishes. The cells were then grown for 14 days and the number of colonies counted using 2% crystal violet. Assays were performed in at least 3 independent experiments.6 intact cells was measured at 37 °C using a Clark-type oxygen electrode (Hansatech Instruments) as described26. The mean of at least three independent experiments is shown.The basal respiration of 4 × 102DCFDA, Invitrogen). The mitochondrial inner membrane potential (MIMP) was evaluated using tetramethylrhodamine ester . Glucose consumption was evaluated using 2-NBDG (Molecular Probes). 2-NBDG is a derivative of glucose modified with a fluorescent amino group at the C-2 position. The C-2 position is the same position that the 18F atom is bound to in FDG. The 2-NBDG is uptaken into the cells and then decomposed into non-fluorescent forms27.Cytoplasmic ROS were assessed using 2′,7′-dichlorodihydrofluorescein diacetate  and incubation at 37 °C for 30 min in the dark, cells were collected in DMEM and analyzed immediately with a MACSQuant® (Miltenyi-Biotec) flow cytometer. Forward and side scatter were used to gate the viable population of cells, and the mean fluorescence intensity was determined with FLOWJO software (TreeStar). Assays were performed by duplicate in at least 3 independent experiments.For these assays, 0.75 × 10All the statistics were carried out using the SPSS software version 15.0. The relationship between the cumulative probability of OS, PFS and analyzed predictors, was calculated with the Kaplan-Meier method. The population was divided based on the median or tercile. The medium and high terciles for GAPDH/MTCO1 ratio were grouped since they behaved in a similar way.Bivariate correlations studies between PGC-1alpha, GAPDH/MT-CO1, and mitosis number in patient studies were analyzed using the Pearson correlation test. Differences in SUVmax levels between GAPDH/MT-CO1 ratio groups were evaluated using ANOVA.ANOVA followed by Bonferroni post-test for multiple comparisons were used to analyze the differences in clonogenicity, migration, galactose to glucose growth ratios, oxygen consumption, ROS levels, glucose consumption and PGC-1alpha or GAPDH/MT-CO1 ratio mRNA levels among the cell lines. 2-tailed Student’s t test was used to analyze differences in MIMP, cell viability and clonogenicity after metformin treatment. In all cases, values of P < 0.05 were considered statistically significant.The datasets generated during the current study are available from the corresponding author on reasonable request.