HER-2 represents a relatively new therapeutic target for non small cell lung cancer (NSCLC) patients. The incidence for reported HER-2 overexpression/amplification/mutations ranges from 2 to 20% in NSCLC. Moreover, HER-2 amplification is a potential mechanism of resistance to tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKI) (about 10% of cases). T-DM1, trastuzumab emtansine is an antibody-drug conjugate composed by the monoclonal antibody trastuzumab and the microtubule polymerization inhibitor DM1. The activity of T-DM1 has been studied in breast cancer but the role of T-DM1 in lung cancer remains unexplored.Antiproliferative and proapoptotic effects of T-DM1 have been investigated in different NSCLC cell lines by MTT, crystal violet staining, morphological study and Western blotting. HER-2 expression and cell cycle were evaluated by flow cytometry and Western blotting. Antibody dependent cell cytotoxicity (ADCC) was measured with a CytoTox assay. Xenografted mice model has been generated using a NSCLC cell line to evaluate the effect of T-DM1 on tumor growth. Moreover, a morphometric and immunohistochemical analysis of tumor xenografts was conducted.In this study we investigated the effect of T-DM1 in a panel of NSCLC cell lines with different HER-2 expression levels, in H1781 cell line carrying HER-2 mutation and in gefitinib resistant HER-2 overexpressing PC9/HER2cl1 cell clone. T-DM1 efficiently inhibited proliferation with arrest in G2-M phase and induced cell death by apoptosis in cells with a significant level of surface expression of HER-2. Antibody-dependent cytotoxicity assay documented that T-DM1 maintained the same activity of trastuzumab. Our data also suggest that targeting HER-2 with T-DM1 potentially overcomes gefitinib resistance. In addition a correlation between cell density/tumor size with both HER-2 expression and T-DM1 activity was established in vitro and in an in vivo xenograft model.Our results indicate that targeting HER-2 with T-DM1 may offer a new therapeutic approach in HER-2 over-expressing lung cancers including those resistant to EGFR TKIs. A number of molecular aberrations have been identified in non small cell lung cancer (NSCLC), including EGFR, BRAF, HER2 mutations, EML4-ALK, ROS1 and RET rearrangements in adenocarcinoma; FGFR mutations/amplifications, DDR2 or PIK3CA mutations in squamous cell carcinoma . In partThe acquisition of resistance to tyrosine kinase inhibitors (TKIs) in clinical oncology is a well documented phenomenon that applies to several types of cancers. Almost all NSCLC patients with activating EGFR mutations treated with EGFR-TKI, after an initial response, experience disease progression within 10 to 14 months from the beginning of the therapy . A commoHER-2 represents a relatively new therapeutic target for NSCLC. The potential clinical relevance of HER-2 expression in NSCLC is currently under evaluation , howeverT-DM1, trastuzumab emtansine, is an antibody-drug conjugate composed by the microtubule polymerization inhibitor DM1 (derivative of maytansine) linked with a stable thioether linker to trastuzumab, a monoclonal antibody that targets HER-2 receptor . After bThe activity of T-DM1 has been extensively studied in several human breast cancer cell lines ,17,18 shThe aim of the present study was to test whether T-DM1 activity is affected by HER-2 expression/mutation status and may overcome EGFR-TKI resistance in NSCLC cell lines. To this purpose we evaluated the effect of T-DM1 in a panel of NSCLC cell lines with different HER-2 expression levels, in H1781 cell line carrying HER-2 mutation , and in The total level of HER-2 protein was detected by immunoblotting on cell lysates in a panel of NSCLC cell lines . As shown in Figure 50 = 0.40 ± 0.08 μg/ml, Figure Based on the previous analysis, the effect of T-DM1 and trastuzumab on cell viability was focused on NSCLC cell lines expressing different cell surface levels of HER-2: Calu-3 (very high), H3255 (high) and Calu-6 (low). T-DM1 showed strong anti-proliferative effect in HER-2 over-expressing Calu-3 cells  is one of the main mechanisms of action of specific mAbs directed to ErbB family members in vivo , we examAs previously reported  and indeIt has been reported that cell density can influence the expression of EGFR in breast cancer  and in p6 or 8 × 106 Calu-3 with a mean volume of 161 ± 15 mm3  -32 and 2T-DM1 has been extensively studied in preclinical models of breast cancer ,18 TheseIn a recent review Landi and Cappuzzo  hypothesAll the above observations prompted us to investigate, in a panel of NSCLC cell lines with different levels of HER-2 expression or carrying HER-2 mutation, the effect of T-DM1 on cell proliferation and survival.In agreement with previously reported data in breast cancer models , we docuOur in vitro and in vivo experiments documented that, respectively, low cell density and small xenografted tumors were associated with higher HER-2 expression and thereby greater T-DM1 sensitivity. Thus, the present investigation strongly support the contention that HER-2 expression in NSCLC is regulated by the tumor mass and its structural organization which in turn condition the efficacy of T-DM1. Finally, we suggest that the expression level of HER-2, determined by immunohistochemistry, might represent a predictive factor of response to T-DM1 in tumors carrying wild type or mutant HER-2 receptor.Our results indicate that T-DM1 inhibited cell proliferation and induced apoptosis in NSCLC cells with endogenous or acquired high HER-2 levels but its activity does not seem to be related to HER-2 mutational status. Moreover, a correlation between cell density/tumour size with both HER-2 expression and T-DM1 activity was established in vitro and in an in vivo xenograft model.In conclusion, dual-agent molecular targeting through T-DM1 may be a promising therapy in HER-2 positive lung cancer even in tumors which had developed resistance to EGFR-TKIs.2 and 95% air.The human NSCLC cell lines used in this study were purchased from American Type Culture Collection (ATCC)  and banked at early passage (P2). Furthermore, the cells we culture, are regularly verified on the basis of cell morphology and never cultured for more than 3 months. The PC9, HCC827, HCC827 GR5 and H1781 cell lines were kindly provided in 2013 by Dr P. Jänne . The PC9/HER2c1 was kindly provided in 2013 by Dr.William Pao . All cells were cultured as recommended and maintained at 37°C in a humidified atmosphere of 5% COTrastuzumab and vinorelbine were kindly provided by inpatient pharmacy. T-DM1 was supplied from Genentech Inc.  through a Materials Transfer Agreement. For the in vitro experiments, stock solutions of drugs were prepared in distilled water, stored at 4°C and diluted in fresh medium for use, whereas for the in vivo experiments trastuzumab and T-DM1 were daily dissolved in sterile saline solution (NaCl 0.9%).Tyr1221/1222; p70S6K; p-p70S6K Thr421/Ser424; Akt; p-Akt Ser473; p44/42 MAPK; p-p44/42 MAPK; caspase-7 and 9; cyclin A and B1; Rb; p-Rb were from Cell Signaling Technology . Antibody against cytochrome-c (7H8) was form Santa Cruz Biotechnology Inc. . Antibodies against actin was from Sigma–Aldrich . Antibody against GAPDH was from Ambion . HRP-conjugated secondary antibodies were from Pierce  and chemiluminescence system (ImmobilionTM Western Cemiluminescent HRP Substrate), was from Millipore .Cell protein extraction, solubilization, and analysis by 1-D PAGE were performed as previously described . AntibodOne million of NSCLC cell lines were incubated, for one hour at room temperature, with Isotype control Monoclonal Mouse IgG1/R-PE  or PE mouse anti-Human HER-2  to determine HER-2 protein membrane levels as previously described . After tCell viability was evaluated by tetrazolium dye  (MTT) assay and by crystal violet staining as previously described . Data arc detection were generated using a digitonin-based subcellular fractionation technique as previously described [Apoptosis was assessed by morphological study: stained  or unstained cells were observed using light-, phase-contrast- and fluorescence-microscopy. Western blot analysis was performed to evaluate cleavage products of caspase-7 and caspase-9. Cytosolic and mitochondrial fractions for cytochrome-escribed .Highly purified CD56+ natural killer (NK) cells were obtained by magnetic separation and ADCC was measured with the CytoTox 96 non-radioactive cytotoxicity assay  as previously described .All experiments involving animals and their care were performed with the approval of the Local Ethical Committee of the University of Parma, in accordance with the institutional guidelines that are in compliance with national (DL116/92) and international (86/609/CEE) laws and policies.6 or 8 × 106 Calu-3 cells were subcutaneously injected on the right flank of each mouse. Ten days after cells injection, tumor volume reached an average size of 160 mm3 or 370 mm3 respectively, and animals were randomized into three different groups (n = 6): control, trastuzumab and T-DM1. Once every six days trastuzumab was intraperitoneally administered at a dosage of 15 mg/Kg, whereas T-DM1 was injected into the lateral tail vein at a dosage of 15 mg/Kg. Control mice received intravenous injection of sterile saline solution (NaCl 0.9%) according to the same schedule. Drug dosages were chosen accordingly to previous studies conducted on breast and gastric xenograft models [Balb/c-Nude female mice  6 weeks old, were housed in a protected unit for immunodeficient animals with 12-hour light/dark cycles and provided with sterilized food and water ad libitum ,42. We pt models ,18,22.2)/2. At the end of the experiments, mice were euthanized by cervical dislocation and tumors weighted and collected for immunohistochemical and histological analysis.Tumor xenografts were measured three times per week using a digital caliper and tumor volume was determined using the formula: , Masson’s Trichrome and subjected to immunohistochemistry. A morphometric analysis was performed on the entire section in order to evaluate the volume of neoplastic tissue, connective tissue and vascular interstitium. To better define the fraction occupied by neoplastic cells, sections were stained with pancytokeratin antibody  revealed through biotin-streptavidin-DAB system (Dako). The volume fraction of fibrosis and vascular interstitium was assessed on Masson’s Trichrome stained samples. To this end, the number of points overlying each tissue components was counted and expressed as percentage of the total number of points explored. All these morphometric measurements were obtained with the aid of a grid defining a tissue area of 0.23 mmCombining the entire tumor volume with the above morphometric measurements, the total volume occupied by neoplastic cells, connective tissue and vascular interstitium was computed for each sample.Moreover, immunohistochemical analysis of HER-2 was performed on each tumor of each experimental group. Sections were stained with HER-2 antibody  and revealed through biotin-streptavidin-DAB system. This analysis was performed on the entire tumor by an optical microscope  to evaluate the area occupied by cell expressing HER-2 and their intensity. The latter was expressed as Integrated Optical density (IOD), as detected using a software for image analysis .pos cells was detected by immunofluorescence to document mitotic figures.In addition, the nuclear expression of the phosphorylated form of Histone H3  on PancytokeratinFor all tested epitopes negative controls were represented by immunostaining the sample with an irrelevant antibody or by exposing the sections only to the secondary antibody.t test, and P values are indicated where appropriate in the figures and in their legends. A P value <0.05 was considered as significant. Bliss interaction was calculated as previously described [Statistical analyses were carried out using GraphPad Prism version 5.0 software . Results are expressed as mean values ± standard deviations (SD) for the indicated number of independent measurements. Differences between the mean values recorded for different experimental conditions were evaluated by Student’s escribed . For in ADCC: Antibody-dependent cellular-cytotoxicity; EGFR: Epidermal growth factor receptor; NSCLC: Non small cell lung cancer; TKI: Tyrosine kinase inhibitor.All authors declare that they have no competing interests.DC carried out flow cytometry, cell cycle, experiments on resistant clone and analyzed the results; FS performed the in vivo studies, interpreted the results and performed the statistical analysis; FQ, CF and GG carried out morphometric and immunohistochemical analysis; MB carried out Western blot analysis; CF evaluated cell death; AC isolated and cultured NK cells and carried out ADCC experiments; SLM, MG and CC carried out cell growth experiments; LA was responsible for drug administration in in vivo studies; MT, AA and PGP critically revised the manuscript and assisted with the draft of the manuscript; RRA, designed the project, analyzed the results and wrote the manuscript. All authors read and approved the final manuscript.


Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application.The human pluripotent stem cell registry (hPSCreg), accessible at  The research landscape for human pluripotent stem cells (hPSC) is changing rapidly. The establishment of human embryonic stem cells (hESC) in 1998  has provhttp://hpscreg.eu), established in 2007 with funding from the European Commission, originally aimed to provide transparency and comparability as well as management of ethical compliance in the dynamic yet controversial field of human ESC research , the Human iPSC Initiative (HipSci), WiCell Research Institute, the Korean Stem Cell registry and NIMH stem cell center at Rutgers. An exemplary pipeline established with EBiSC requires registration of an hiPSC line in hPSCreg, which assigns a name together with a BioSample ID , where der.org,  alongsidTo reduce uncertainty and accidental errors during registration, hPSCreg provides extensive help texts for each input.The quality of data available on hPSCreg and its ethical acceptability is of utmost importance. We have established strict criteria to ensure high quality through an open, fair and scientifically rigorous process. This process puts a strong focus on standardization of data input to streamline comparison and validation of data on cell lines from different sources.bonafide provider (i.e. from a recognized research organization) the account will be validated. In the second step, the approved users enter cell line related information. After at least the mandatory nomenclature and minimal information has been provided, the provided information gets manually checked by the hPSCreg data curators. In case of incomplete information or plausibility issues, the provider is contacted for clarification. In the last validation step, the hPSCreg Committee of National Representatives (CNR) is informed and can provide additional feedback on the validity of the data. Only cell lines validated by the hPSCreg data curators and the CNR will be made available to the public.To control data accuracy, hPSCreg uses a three-step registration system Figure . First, Additional validation can be provided by feedback from users. While this is in principle possible already by editing information provided for a cell line in hPSCreg's registration tool followed by manual validation, the feedback process will be expanded and refined in future versions of the Registry.To ease data access, retrieval and understanding, hPSCreg follows a user-centered design approach by implementing an easy-to-use, mobile-friendly and aesthetically pleasing web application. The application's goal is to help users find cell lines and related data as quickly as possible. The interface design is kept at a minimum with focus on content, offering three ways of data discovery: searching, browsing and visual exploration.Text-based searching is designed to be simple but powerful and is omnipresent in hPSCreg, i.e. a search can be started from any page. Search results are ranked according to their relevance by default but can be sorted by name or date as well. The result list shows the hPSCreg name alongside a list of alternative cell line identifiers, the contact institution, the derivation country and date, diseases associated with the specific cell line. Furthermore, the interface implements instantaneous faceted search to limit the search space to cell type, countries, diseases and derivation date.In terms of browsing, hPSCreg provides the possibility to access all available cell lines via facet filter enabled lists just like the search results. Furthermore, cell line provider and hPSC research projects can be browsed through searchable, sortable and filter-enabled lists. For example, providers can be narrowed down by role, i.e. cell line generator, owner or distributor. As projects in which a particular cell line is used can be registered, and all EU-funded projects using hPSC must be registered at hPSCreg , these cTo ease summarizing of available data in hPSCreg and the current state of hESC and hiPSC research around the world, an interactive colored world map has been implemented Figure , visualihttp://hpscreg.eu and cell-line respectively, while the identifier is a standardized hPSCreg name. For example, qualitative meta information of the hESC line WAe009-A  can be accessed at http://hpscreg.eu/cell-line/WAe009-A.To improve hPSC line identification and minimize ambiguities, a simple yet semantically meaningful system for information retrieval is needed. A semantic uniform resource locator (URL) for every registered and validated cell line is provided by hPSCreg. Such a persistent URL will not change over time, allowing their use as uniform resource identifiers (URI) in future application ontologies. Semantic URLs in hPSCreg follow the schema: <ORGANIZATION>/<RESOURCE>/<IDENTIFIER>. Hereby, the organization and resource will be Access to certain information falls under a managed access code, based on the consent provided by the original tissue donor to protect privacy. This includes genetic information and some clinical data, which may be used to identify the donor . ManagedThe hPSCreg web application's architecture can best be described as a four-tier system, consisting of a front-end application, i.e. user interface (UI), a middleware server-side application, a large set of specialized web-services and data stores. We follow the micro-services architecture pattern, for maximum flexibility, helping hPSCreg to scale well in the future. The front-end application, implemented using cutting edge HTML5, CSS3 and JavaScript technologies, communicates with a PHP-based middleware application, running on an Apache web server. The middleware securely handles the communication between UI and a variety of specialized Java-based micro web-services. These micro web-services process, send and retrieve data from the data stores. Data is simultaneously stored in a MySQL database management system and Elasticsearch. MySQL databases, act as the main data storage, while Elasticsearch enables a Google-like search experience. LDAP is used for authorizing users and managing groups.The Registry has implemented a partnership with the European bank of induced pluripotent stem cells (EBiSC) and is used by this bank as a depositors’ portal for the registration of iPSC-lines for deposition in EBiSC. EBiSC is using hPSCreg's registration facility and nomenclature system to achieve standardized data provision and unambiguous naming of all lines. In addition, hPSCreg provides, through an API with the European Bioinformatics Institute (EBI), a BioSamples ID to each line registered for EBiSC. The BS-ID provides a unique identifier for aggregation of any sample related to the cell line, for example the donor, RNA, differentiated cells, lots and batches derived from the line, as well as relationships between samples such as ‘derived from’. Hence the implementation of a standard registration tool, a unique standard name and a BS-ID provides data traceability between different banks and institutions, and comparability down to the sample and lot level.http://grants.nih.gov/stem_cells/registry/current.htm), or restricted to national cell lines, for example in Korean PSC Registry .One increasingly important application of human PSC is their use in drug efficacy studies, based on genetically stratified cohorts of individualized cell lines. This requires access to genetic data from the cells and is currently handled via data access committees. This elaborate process is prohibitive for the design of these kinds of studies aiming to use cells from different sources. Hence, there is a need to develop specific access protocols for potentially sensitive genetic data, which adheres to the standards established by the Global Alliance for Genomics and Health (hPSCreg was established in 2007 as the European human embryonic stem cell registry and is supported by the European Commission. Its initial goal to provide transparency to the field of stem cell research and to establish ethical standards has been extended to cover all fields of human pluripotent stem cell research. The registry now provides comprehensive scientific and ethical information on all hPSC lines and provides these to scientists, regulators, lawmakers and the general public. Future implementations will include integration of standards and validation tools for clinical grade human PSC, the registration of clinical trials using stem cells as well as the provision of APIs for programmatic access to hPSCreg's data and registry. An option for downloading certain parts of hPSCreg is currently tested.The registry will be sustained through further European and international funding, which will allow it to gradually develop additional tools, include more focused information on cell types derived from human PSC, disease-specific lines and implementation of feedback as well as customized data display and documentation tools.


Giardia intestinalis is an important single‐celled human pathogen. Interestingly, this organism has two equal‐sized transcriptionally active nuclei, each considered diploid. By evaluating condensed chromosome numbers and visualizing homologous chromosomes by fluorescent in situ hybridization, we determined that the Giardia cells are constitutively aneuploid. We observed karyotype inter‐and intra‐population heterogeneity in eight cell lines from two clinical isolates, suggesting constant karyotype evolution during in vitro cultivation. High levels of chromosomal instability and frequent mitotic missegregations observed in four cell lines correlated with a proliferative disadvantage and growth retardation. Other cell lines, although derived from the same clinical isolate, revealed a stable yet aneuploid karyotype. We suggest that both chromatid missegregations and structural rearrangements contribute to shaping the Giardia genome, leading to whole‐chromosome aneuploidy, unequal gene distribution, and a genomic divergence of the two nuclei within one cell. Aneuploidy in Giardia is further propagated without p53‐mediated cell cycle arrest and might have been a key mechanism in generating the genetic diversity of this human pathogen. The accurate propagation of genomic information to subsequent generations is crucial for the efficient proliferation of any living organism. Alteration through different mechanisms leads to chromosomal rearrangements or whole‐chromosome aneuploidy, which usually has a detrimental impact on an individual′s health, viability, and reproduction  is an evolutionarily distant binucleated eukaryote and an important human and veterinary pathogen. The coexistence of two same‐sized transcriptionally active nuclei within a Giardia cell provides additional complexity to the karyotype organization of this organism. The transcriptional interplay and coordinated cell cycle progression of the two nuclei is puzzling, as essential data are missing regarding Giardia nuclei genomic organization, including ploidy. Giardia has one of the smallest eukaryotic genomes (11.7 Mb), which is divided into five chromosomes in a haploid set  can be modified to an aneuploid pattern and that the nuclei harbor different chromosome sets , we evaluated the ploidy and karyotype dynamics in different Giardia lines. The analyzed lines showed inter‐ and intraheterogeneous karyotype organization, different cell growth dynamics, and chromosomal missegregations. Thus, Giardia represents a novel noncancer model organism with an aneuploid genome that balances – within a single cell – the effects of different aneuploidy patterns occurring in the two nuclei.In this study, we used a single‐cell approach to characterize karyotypes both within and between Giardia intestinalis lines from assemblage A were examined. Six of these lines originated from the same original WB isolate that was obtained in 1979 from a symptomatic metronidazole‐resistant patient by F.D. Gillin in Bethesda, USA (ATCC 30957), collected for this study from different sources. The line WB‐Meyer was a gift from Prof. E.A. Meyer  in 1989, after which frozen in liquid nitrogen and then maintained in continuous culture since 2013; the line WB‐1W was a gift from Prof. C.C. Wang  in 2001, after which frozen in liquid nitrogen and maintained in continuous culture since 2013; the line WB‐Tach was a gift from Prof. J. Tachezy  in 2013 and has been kept in continuous culture since then. The line WB‐ATCC was retrieved from the ATCC collection (www.lgcstandards-atcc.org) in 2012 and maintained in a continuous culture. Two laboratory lines of a WBc6 clone (ATCC 50803) of the original WB line were used: line WBc6‐Cande was a gift from Prof. Z.W. Cande  in 2012 and kept in continuous culture, and line WBc6‐ATCC was retrieved from ATCC collection in 2013 and kept in continuous culture. The other clinical isolate, Portland‐1 (ATCC 30888), was isolated in 1971 by Prof. E.A. Meyer in Portland, USA, from a human patient; this line was retrieved from ATCC in 2012 and kept in continuous culture. The original Portland‐1 isolate was obtained in our laboratory in 1989 and, since then, has been maintained in continuous culture (labeled HP‐1). Axenic cultures were routinely maintained in TYI‐S‐33 medium (pH 6.8) in screw‐cap borosilicate glass tubes. The cultures were passaged twice per week by inoculating 500 μL of the chilled 4‐day‐old culture into a new tube containing 7.5 mL of prewarmed TYI‐S‐33 medium. For all experiments, only the trophozoite life‐cycle stage of Giardia intestinalis was used unless otherwise stated.Eight Giardia lines, 2 mL of fresh culture with 1 × 104 cells per milliliter was placed in separate wells of a 24‐well Nunclon© plate and maintained in quadruplicate at 37°C under anaerobic conditions  for 24, 48, 72, 96, and 120 h. The cells were counted, and the growth curves were calculated.For cryopreservation in liquid nitrogen, dimethyl sulfoxide was used as a cryoprotectant at a final concentration of 5% (v/v) in culture medium , and the pellet was fixed with a freshly prepared methanol/acetic acid fixative . The suspension was then dropped onto glass slides, air‐dried, mounted to DAPI/Vectashield and observed. The reliability of the counting method was ensured by random blind and independent counting by two researchers. The Giardia lines were examined several times, each from at least four different passages. The differences among the Giardia lines or among individual passages of a line were statistically tested using the Dell Statistica program (Pearson′s chi‐square test at a 0.05 significance level). The null hypothesis was that passages or lines have the same distribution of karyotype variants. To obtain relevant test results, the expected counts for each cross category (karyotype variant in each passage/line) must be greater than five in least 80% of the expected counts and greater than one in the remaining . The events were recorded at a low speed of 200–400 events/s, and at least 10,000 events were recorded for each histogram. The data were analyzed using FACSDiva software.Flow cytometry was performed as previously described  to prevent unspecific probe‐RNA binding was applied for 60 min at 37°C, followed by three changes of 2× SSC for 5 min. The hybridization mixture contained 20 ng of labeled probe, 10 μg of salmon sperm, and 50% deionized formamide  in 2× SSC and was applied at 82°C for 5 min. The single‐color FISH was developed by the TSA‐Plus TMR System according to the manufacturer′s directions  using a dig‐labeled probe and an anti‐dig‐HRP antibody . In the two‐color FISH, a sequential double hybridization signal development was processed according to the manufacturer's directions , as a combination of (1) a dig‐labeled probe, anti‐dig‐HRP antibody, and TSA‐Plus TMR and (2) a biotin‐labeled probe, streptavidin‐HRP, and TSA‐Plus Fluorescein. An Olympus BX51 fluorescence microscope that was equipped with a DP70‐UCB camera was used for observation. For the two‐color FISH imaging, a Leica TCS SP2 AOBS confocal microscope was used. Single‐scan images were processed with Huygens Professional software using the classical maximum likelihood estimation deconvolution algorithm. For chromosome‐specific probes, we selected four single‐copy genes that were annotated in the Giardia database  to a chromosome four as follows: rad50 GL50803_17495; telomerase catalytic subunit (tert) GL50803_16225; isoleucyl‐tRNA synthetase (iso) GL50803_104173; and ubiquitin (ubi) GL50803_13701. The probes rad50 and tert were assigned to one chromosomal end (near 5′end), and the probes ubi and iso were assigned to the opposite chromosomal end (near 3′end). The probes against actin (act), chromosome 2, GL50803_40817, and a probe against ser/thr phosphatase (ser), chromosome 5, GL50803_7439, were used as controls for a specific probe binding to a chromosome of interest. The probe sizes, chromosome localization and primer sequences are listed in Table S5. The presence of a single copy of the respective probe sequence in the genome database was verified through reciprocal BLAST searches at http://giardiadb.org. The PCR products were cloned into the pJET 1.2/blunt cloning vector (Fermentas) and transformed into chemically competent TOP10 E. coli cells (Invitrogen). Purified PCR products that were amplified from plasmids that were isolated from a single bacterial colony  were labeled by random priming with digoxigenin‐11‐dUTP (Roche) or biotin‐11‐dUTP  using the DecaLabel DNA Labelling Kit (Fermentas). The probe binding efficiency was estimated at >300 cells as the percentage of the FISH‐labeled to unlabeled nuclei. This value varied among probes between 94% (rad50) to 98% (ubi) of the positive nuclei. Randomly chosen slides were counted by two persons, and the probe binding patterns were evaluated from at least three independent FISH experiments.The FISH protocol was adapted from Conrad et al. . BrieflyGiardia database (GiardiaDB) was designed as previously described . The term “prevailing karyotype” refers to the most frequent karyotype variant for the given population and time. Moreover, minor karyotype variants were present in all of the examined Giardia lines. The “stable” Giardia lines did not show any change in the prevailing karyotype during long–term in vitro cultivation, in contrast to the “unstable” lines, where the prevailing karyotype changes were observed.We compared six Giardia lines, except for the WBc6‐Cande line. In the unstable lines, the karyotypes deviated more from the 10 + 10 pattern  than in stable lines . The tested lines significantly differed from each other in their karyotypes . Minor variants were newly generated, and some of them became subsequently prevalent. The karyotype of the WB‐Meyer line underwent a change from a prevailing karyotype 8 + 13 to 12 + 14, 8 + 15, and 8 + 14 within 16 months of observation. However, some WB lines with a clonal origin (WBc6‐ATCC and WBc6‐Cande) maintained stable karyotypes, and their prevailing karyotype did not change during long‐term cultivation (in WBc6‐Cande during 4 years of continuous cultivation) . However, the differences in karyotype variant distributions among passages in a stable line WBc6‐Cande were smaller than in the unstable line WB‐Meyer, as apparent from the comparison of Figure Giardia trophozoites of the line HP‐1 did not change its karyotype (9 + 11) in multiple experiments.The Portland‐1 isolate and its line HP‐1 revealed a stable aneuploid karyotype with a prevailing 9 + 11 pattern that has been maintained during 13 years of continuous laboratory cultivation. In contrast, the WB isolate and its laboratory lines, WB‐ATCC, WB‐1W, WB‐Tach, and WB‐Meyer, showed unstable karyotypes during in vitro cultivation  GL50803_16225; isoleucyl‐tRNA synthetase (iso) GL50803_104173; and ubiquitin (ubi) GL50803_13701. The probes rad50 and tert were assigned to one chromosomal end, and the probes ubi and iso were assigned to the opposite chromosomal end. Single‐copy gene probes were previously used for detecting Giardia chromosomes . For design of chromosome‐specific probes, we selected four single‐copy genes that were annotated in the rad50, iso, and ubi detected 2 copies of chromosome 4 in each of the two nuclei in most of the cells ; however, other patterns were also observed  with approximately similar binding frequencies for the three probes (Table S3). Namely, for the rad/iso/ubi probes, respectively, there was a 2 + 1 pattern in 22.5/23.3/19.6% of the cells, a 3 + 2 pattern in 11/9.7/13.7% of the cells, 1 + 1 pattern in 6.5/3.2/1.9% of the cells and a 3 + 1 pattern in 0/6.4/5.8% of the cells. This indicates disomy of chromosome four in a Giardia nucleus in most of the cells; however, other copy numbers and/or chromosomal rearrangements leading to rad50, iso, and ubi gene duplications or losses were present in approx. 40% of the cells. The latter process was also indicated using the tert probe, which labeled the two nuclei with the 2 + 1 binding pattern in 45% of the cells, 2 + 2 (15%), 1 + 1 (14%), 3 + 2 (9%), 2 + 0 (8%), 3 + 1 (5%), and 3 + 0 (4%)  is plausible in some, but not all, cells in the WBc6‐Cande line. The classic diploidy of a nucleus resulting in tetraploidy of a cell, however, must be verified by confirming the 2 + 2 pattern for all five of the Giardia chromosomes in the WBc6‐Cande line. Moreover, the loss of a tert gene copy in one of the nuclei reflects possible chromosomal rearrangements, which may lead to an unequal gene distribution between the two nuclei even in this Giardia line. The specificity of the used probes was also verified by two‐color FISH experiments, which permitted the specific binding of the probes to the opposite chromosome ends  evP‐1 Fig. B and C, P‐1 Fig. D.Giardia cells, we aimed to analyze genes that were previously shown to be involved in the response to aneuploidy. The genome of Giardia does not contain genes for the key tumor suppressor homologs p53 and p73 (p53 family member), which are usually responsible for the slow proliferation and G1 arrest of aneuploid cells. This result is consistent with findings that these regulators evolved first in Choanozoans from the Opisthokonta group  due to the uncorrected merotelic kinetochore‐microtubule attachments are considered the major mechanistic cause of aneuploidy in cancer cells , and pathways to inhibit the proliferation of aneuploids by a G1 cell cycle arrest have not been identified and may be absent from its genome. As a consequence, aneuploidy in Giardia is further propagated and represents a constitutive feature of its karyotype.Regarding the tolerance of aneuploidy, the bioinformatic analysis that was conducted in this study revealed that further aneuploid cell proliferation in Candida sp.  that were derived from one original clinical drug‐resistant isolate WB. This finding raises questions regarding the development of efficient therapeutic strategies. The karyotype heterogeneity and evolution during in vitro cultivation also set up additional requirements for researchers working with this pathogen. The observed genomic instability as well as the tendency to generate new karyotype variants and to outcompete the other karyotypes can all hinder the gene knock‐out and gene‐editing efforts in Giardia, which has so far been unsuccessful in this eukaryotic model.It is difficult to interpret the impact of aneuploidy in The authors declare no potential conflicts of interest.Figure S1. FISH hybridization of probes designed to different Giardia intestinalis chromosomes.Click here for additional data file.Figure S2. FACS analysis of cell cycle progression in Giardia intestinalis lines with stable and unstable aneuploid karyotypes.Click here for additional data file.Figure S3. Proposed model for the karyotype evolution toward aneuploidy in Giardia intestinalis.Click here for additional data file.Table S1. Observed karyotype variants of the WB‐Meyer line during a long‐term in vitro cultivation.Click here for additional data file.Table S2. Observed karyotypes of the WBc6‐Cande line during a long‐term in vitro cultivation.Click here for additional data file.Table S3. The table shows the percentages of probe binding patterns in the two Giardia nuclei observed by single‐color FISH in interphase and mitotic nuclei. N indicates the number of cells, in which the binding pattern was evaluated.Click here for additional data file.Table S4. Outcomes of a bioinformatic search for orthologs of major tumor suppressor factors and checkpoint activators implied in the ploidy control.Click here for additional data file.Table S5. Information regarding the probes used in FISH experiments on Giardia intestinalis chromosomes.Click here for additional data file.


Exosomes are small endosome-derived vesicles ranging from 30-120 nm in diameter secreted from cells through exocytosis and, in cancer context, are able to prepare metastasis niche and suppress host's immune system. Despite these pro-tumorigenic characteristics, exosomes can be useful for several Biotechnological applications such as biological anticancer vaccines, cancer diagnosis and prognosis. Due to its property of carrying parental cell's protein and active nucleic acids there is an increasing interest to use exosomes to track cancer metabolism alterations in different conditions, for example, during drug administration [One of the most common types of cancer that develops drug resistance during drug treatment is Melanoma. The majority of drugs developed against melanoma targets the oncogenic mutations in BRAF (BRAF V600E) that are present in nearly 60% of cutaneous melanoma tumors. Resistance to BRAFV600E inhibitors are classified in two main groups, Intrinsic Resistance and Acquired Resistance, the second is developed after treatment starts that happens mainly by MAPK reactivation and RTK upregulation .Various methods are being studied to overcome drug resistance in melanoma, but there is a need to detect when the resistant mechanism is arising to orientate which approach to choose. This early detection has demonstrated to be a difficult task especially during advanced stages where there are several metastatic niches.With this aim, our research project was designed to create a method to follow tumor's status through melanoma-derived exosomes. As most types of melanoma overexpress MART-1 we checked its expression, as well as PDGFR-B and others proteins, in PLX4032 (V600E mutated BRAF inhibitor) resistant and sensitive melanoma cell lines to start to understand their potential to be exosomes-associated proteins in the development of acquired resistance detection method[Cells were cultured in exosome depleted media by FBS ultracentrifugation during 70 min at 100.000xg. Sensitive  and resistant  melanoma cell lines to PLX 4032 (vemurafenib) were cultured until 75% of confluence and the supernatant was processed to obtain the exosomes following 1500x g - 10 min, 17000x g - 15min and 160.000x g - 1 hour.To confirm this protocol it was done a Transmission Electron Microscopy (TEM), followed by Flow Cytometry against CD63, an exosome molecular marker. TEM showed an uniform size for exosomes ranging from 60- 90 nm in diameter and Anti-CD63 Flow Cytometry showed a positive population of 99.1%. Western Blots were done with the cells lines cited previously demonstrating that the exosome's protein content of MART-1, PDGFR-B, FGFR3 and others, corresponds to the parental cell.Preliminary results show that exosomes can be useful tools to predict cell's metabolism. Here we present that protein differential expression between sensitive and resistant cell lines can be detected by exosome profile such as PDGFR-B increased expression in resistant lines.


Novel biomarkers for cancer diagnosis and therapy selection are urgently needed to facilitate early detection and improve therapy outcomes. We have previously identified a novel phosphorylation site at serine 506 (PS506) on topoisomerase-I (topo-I) and have shown that it is widely expressed in cell lines derived from several cancers, including lung cancer, but is low in cell lines derived from non-cancerous tissues. Here we have investigated how PS506 expression in lung tissue specimens correlates with their malignant status. We find that PS506 expression is significantly elevated in malignant tumors of non-small cell lung cancer (NSCLC) compared to adjacent, non-cancerous lung tissue and benign lung tumors. PS506 expression was up to 6-fold higher in malignant specimens than in paired non-malignant tissue. Using the well-characterized NIH/NCI 60-cell line panel, we correlate the most elevated expression levels of PS506 in lung, ovarian, and colon cancer cells lines with increased sensitivity to camptothecin, a plant alkaloid that targets topo-I. This is consistent with our earlier studies in a smaller sampling of cell lines and with our finding that PS506 increases topo-I DNA binding. Two widely used chemotherapeutic drugs for ovarian and colon cancer, topotecan and irinotecan, respectively, are derived from camptothecin. Irinotecan has also displayed efficacy in clinical trials of NSCLC. Our results suggest that elevated PS506 expression may correlate with clinical chemosensitivity to these agents in ovarian, colon, and NSCLC. PS506 may therefore serve as a biomarker for diagnosis or therapy selection. New molecular biomarkers for cancer, particularly those relevant to the mechanism of cancer or to cancer therapy, could enhance the power of currently used diagnostic approaches, reduce the risk of over diagnosis, facilitate treatment selection, and greatly improve cancer survival. We have previously identified a novel phosphorylation site on topoisomerase I (topo I) at serine residue 506 (PS506) that is highly expressed in cancer-derived cell lines but is low to undetectable in cell lines derived from normal tissues, making it a possible candidate as a malignancy-associated biomarker for diagnosis .Topo I plays an essential role in DNA metabolism, and is the unique cellular target for irinotecan and topotecan, two widely used chemotherapeutic drugs derived from the plant alkaloid camptothecin (CPT) , 3. By rThe therapeutic relevance of topo I also makes PS506 a potential biomarker for treatment selection. CK2-mediated S506 phosphorylation of basally phosphorylated recombinant topo I generates a topo I with increased DNA binding activity and increased DNA relaxation activity on supercoiled plasmids , 8. We oIn this study we have explored the hypothesis that PS506 expression is a characteristic of malignancy, and that the highest levels of expression correlate with cellular responses to CPT-based drugs. To evaluate the relationship of PS506 to malignancy we have examined tissue specimens of malignant lung tumors, paired non-malignant adjacent lung epithelium, and benign lung tumors. To assess the utility of PS506 as an indicator of responsiveness to CPT-based drugs, we have screened its expression in cell lines from the NCI-60 cell line panel for which CPT sensitivity profiles were available from the National Cancer Institute/Developmental Therapeutics Program (NCI/DTP) data base. The results indicate a highly selective overexpression of PS506 in all malignant lung specimens examined, compared to adjacent non-malignant tissue and benign tumor specimens. In cell lines derived from lung, colon, and ovarian cancers, we find that the highest levels of PS506 expression correlate with increased CPT sensitivity.The pAb506P rabbit IgG was generated to a 21-amino acid phosphopeptide , as previously described [2 in Dulbecco’s Modified Eagles Medium supplemented with 10% newborn calf serum and additives, as previously described [NCI-H358 human bronchioalveolar non-small cell carcinoma cells (H358), catalogue number CRL-5907, were purchased in May 2011 from the American Type Culture Collection and were mycoplasma-free. Cells were grown at 37°C in 10% COFrozen cell pellets from the NCI 60-cell line panel were obtained from the Division of Cancer Treatment and Diagnosis (DCTD) Tumor repository of the National Cancer Institute (NCI) and were stored at -80°C until use. The cell lines for which cell pellets were received are listed in .All tissue specimens used in this study were frozen, anonymous, publicly available archival specimens of non-small cell lung cancer with paired non-malignant control tissue, or specimens from benign lung tumors. All specimens were received from the Western Division of the Cooperative Human Tissue Network (CHTN), Vanderbilt University, Nashville, TN. No identifiable private patient or donor information was supplied with the specimens. Since no data was obtained through intervention or interaction with the individual and since no identifiable private information was supplied to us, this research did not constitute human subjects research as defined by CFR 46.102f and the OHRP Guidance on Research Involving Coded Private Information or Biological Specimens, and was granted exemption from review by the Torrey Pines Institute for Molecular Studies IRB.7 cells/pellet) or two near-confluent 10-cm plates of PBS-washed H358 cells  were lysed by the addition of 350 μl per pellet or 700 μl per plate of cold RIPA buffer  to which complete protease and phosphatase inhibitors  were added just prior to use, and processed as previously described [Cell pellets  were resolved by SDS-PAGE using Criterion 10–20% Tris-HCl precast gels . An equivalent amount of a freshly thawed aliquot of the reference stock of H358 lysate was included in each run as a common control. Following electrophoreses, proteins were transferred to PVDF membranes and immunostained by incubation with primary antibody , followed by appropriate HRP-conjugated secondary antibody (1:1000), and Pierce ECL reagent (Thermo Scientific), followed by exposure to film. Films were scanned using an Alpha Imager and bands were quantified using the accompanying software. PS506 band intensities were normalized to the reference H358 control.Immunoprecipitations were carried out essentially as described . Briefly3, pH 9). Plates were washed with washing buffer  and blocked with TBS containing 1% bovine serum albumin (BSA). Plates were washed once with washing buffer and then incubated for 1 h with serial dilutions (in duplicate) of pAb506P in TBS/1%BSA, washed again, and incubated for 1 h with goat anti-rabbit-HRP. Following another wash step, plates were incubated with HRP substrate 3,3’,5,5’-tetramethyl benzidine  according to the manufacturer’s instructions. Absorbance at 450 nM was read.Serine 506-phosphorylated and non-phosphorylated forms of the 21-amino acid topo I peptide described above see  section 2, 0.001M DTT as suggested by the manufacturer. A parallel control lysate prepared in the presence of phosphatase inhibitors was incubated in buffer lacking alkaline phosphatase.300 μg of cell lysate prepared in RIPA buffer in the absence of phosphatase inhibitors was treated for 1 h at 37° C with 300 units calf intestinal alkaline phosphatase  in buffer adjusted to 0.1M NaCl, 0.01M MgClStatistical analyses were carried out using GraphPad® Prism software .The rabbit polyclonal IgG, pAb506P, raised to a PS506-containing peptide unique to topo I, has been previously shown to recognize the 90 KDa cellular topo I in cancer-derived cell lines as well as the PS506-containing form of recombinant topo treated with protein kinase CK2 . The antpAb506P was used to screen PS506 expression in anonymous specimens of non-small cell lung tumors  and non-malignant lung tissue (as paired specimens from the same patient), as well as benign lung tumors. Specimens were obtained from the Western Division of the Cooperative Human Tissue Network (CHTN) and are listed with their descriptions in (p<0.0001), and the difference between malignant tissue and benign tumor tissue was significant (p = 0.0115). No significant difference was observed between PS506 levels in benign and non-malignant tissues (p = 0.86). These results indicate that PS506 expression is strongly associated with malignancy.The scatter plot in In an earlier study examining PS506 levels in a variety of cancer cell lines, we found that the highest PS506 levels were present in cell lines with the greatest sensitivity to the topo I-targeted plant alkaloid, CPT, with differences of about 2–3-fold between CPT-sensitive and-resistant cell lines . To exteCell lysates from the frozen cell pellets were evaluated for PS506 expression by SDS-PAGE/Western in the same manner as the tissue samples. Bands were quantified digitally and PS506 levels relative to the H358 control value were verified in a second independent experiment and averaged. For the 42 cell lines for which CPT sensitivity data were available, we found that PS506 levels were distributed over a broad range, as was observed for the tumor tissue specimens. The average level of PS506 in all 42 cell lines was 0.37 relative to the H358 common control; a value considerably higher than the average value of 0.24 observed for non-malignant paired tissue or benign tumors . We nextp = 0.028) in the average PS506 level between the most sensitive and the most resistant cell lines, consistent with our earlier studies with a smaller sampling of cell lines [We observed a correlation between PS506 expression and CPT sensitivity in a grouping of 20 non-small cell lung, colon, and ovarian cancer cell lines, representing three cancers for which CPT-based therapies are either FDA-approved  or are being evaluated in ongoing clinical trials  , 31. Tabp = 0.43), possibly due to the contribution of cellular factors independent of topo I, such as CPT uptake, intracellular CPT metabolism and distribution, and the cellular response to DNA damage, all of which can affect the outcome of CPT treatments [We also compared PS506 levels with CPT sensitivity for the larger group of 42 cell lines representing a broader range of cancer types , were not significantly different in PS506 expression. While the absolute levels of PS506 expression in malignant tumor specimens was variable, 71% of malignant tumors displayed PS506 levels above the population average , while only 7% of non-malignant specimens expressed PS506 above the population average.When we evaluated the NCI-60 cell line panel, we also found that the average PS506 expression level across the cell lines was higher than that of the non-malignant tissue specimens . We analyzed the correlation between PS506 expression with cellular sensitivity to the topo I-targeted drug CPT, using CPT sensitivity data available on the NCI/DTP website. In lung, colon, and ovarian cancer cell lines, representing three cancers for which CPT-derived chemotherapeutic drugs are approved or are under clinical testing, we found that the 40% most CPT-sensitive cell lines expressed significantly higher levels of PS506 than did the 40% most resistant cell lines, consistent with our earlier observations in a smaller sampling of available cell lines . IntermeThe development of blood-based assays for PS506 could potentially provide a diagnostic assay for early detection or monitoring of lung cancer or other cancers. Several circulating protein markers are currently in clinical use for certain cancers, primarily for follow-up. These include CA125 for ovarian cancer, CA19-9 for pancreatic cancer, CEA for colon cancer, and PSA for prostate cancer (reviewed in ). CYFRA2The possibility that PS506 may be causally linked to cancer greatly increases its interest as a biomarker. Because topo I possesses DNA nicking/religating activity, its tight regulation is critical to avoid aberrant DNA nicking that could destabilize the genome and promote malignant progression . EctopicA 45 kDa topo I species associated with chemical- or heat-induced necrosis has also been reported in Jurkat leukemia cells . DetectiFurthermore, this study raises a number of interesting questions that warrant further investigation, including the biological relevance of PS506, its role in malignancy, and the basis for its differential appearance in malignant versus normal tissue.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Their resemblance to the primary disease, however, has not been well defined. In the present study, we evaluated five canine UC cell lines via oligonucleotide array comparative genomic hybridization (oaCGH), fluorescence in situ hybridization (FISH), and gene expression analysis.Urothelial carcinoma (UC), also known as transitional cell carcinoma (TCC), of the bladder is the most common neoplasm affecting the canine urogenital system. To facilitate study of the disease Comparison of genome wide DNA copy number profiles of the cell lines with primary biopsy specimens revealed redundancies in genomic aberrations, indicating that the cell lines retain the gross genomic architecture of primary tumors. As in the primary tumors, gain of canine chromosomes 13 and 36 and loss of chromosome 19 were among the most frequent aberrations evident in the cell lines. FISH analysis revealed chromosome structural aberrations, including tandem duplications, bi-armed chromosomes, and chromosome fusions, suggesting genome instability during neoplastic transformation. Gene expression profiling highlighted numerous differentially expressed genes, including many previously shown as dysregulated in primary canine UC and human bladder cancer. Pathway enrichment analysis emphasized pathways suspected to be at the crux of UC pathogenesis, including xenobiotic and lipid compound metabolism.These data support valid use of the canine UC cell lines evaluated by confirming they provide an accurate and practical means to interrogate the UC at a molecular level. Moreover, the cell lines may provide a valuable model for furthering our understanding of aberrant metabolic pathways in UC development.The online version of this article (doi:10.1186/s40575-015-0028-3) contains supplementary material, which is available to authorized users. Urothelial carcinoma (UC), also referred to as transitional cell carcinoma, is the most common bladder neoplasm in the dog . While pPrevious studies have shown that the genomic landscape of canine UC is highly aberrant, with recurrent chromosome copy number aberrations affecting gene dosage on, most notably, canine chromosomes (CFA) 13, 19, and 36 . In addiin vitro model.In addition to genetic risk factors, UC is associated with numerous environmental factors, including flea and tick dips, obesity, pesticides, and cyclophosphamide treatment –9. Whilein vitro model for the study of disease. By facilitating characterization and manipulation of all molecular facets without harming the model animal, cell lines enable researchers to evaluate highly experimental ideas and therapies. Although canine UC cell lines have previously been established for such use [Cell lines provide a useful such use , their rin situ hybridization (FISH). Clinical relevance of findings was validated by comparison of oaCGH profiles of cell lines with those of primary tumor samples, which showed conservation of major genomic aberrations in both sample cohorts. Subsequent gene expression profiling quantified mRNA from copy number neutral and aberrant regions of the genome. KEGG pathway and gene ontology (GO) analysis demonstrated that genes involved in lipid metabolism and cell cycle regulation were enriched among genes differentially expressed between neoplastic cell lines and healthy urothelium, highlighting a potential pathogenesis that is relevant to known risk factors. Our results affirm that the canine UC cell lines are genomically similar to the primary tumor, providing a relevant in vitro model for study of the molecular mechanisms of disease.In the present study, we investigated the molecular profile of five canine UC cell lines with oligonucleotide array comparative genomic hybridization (oaCGH) and fluorescence Five tumor cell lines Table  were estControl samples for expression analysis  were collected during necropsy of dogs with no clinical or histopathological signs of disease at North Carolina State University College of Veterinary Medicine. After collection, bladder tissue was stored in transport media  until sample processing (<1 h). Samples were split in half: one half was fixed in 10 % neutral buffered formalin (NBF) before paraffin-embedding (FFPE) to enable pathologic evaluation, and the second half was used for urothelial cell isolation. FFPE samples were sectioned and stained with hematoxylin and eosin (H&E) for review by a board certified veterinary pathologist. All control samples were found to be histopathologically normal prior to use in subsequent protocols. Urothelial cells were isolated by scraping the bladder mucosa three times with a glass slide and rinsing with 1x phosphate buffered saline (PBS), as described previously . UrothelCells from each of the five lines were cultured in DMEM/F12 without glutamine  and supplemented with 10 % FBS (Mediatech), 1 % Glutamax™ , and 0.6 % Primocin™  to confluence, passaging when confluent. For each line, cells from the same flask were split into three aliquots and used simultaneously for chromosome preparation, DNA isolation, and RNA isolation. Four of the cell lines  were available as low passage (<p7) and so were harvested at an earlier (<p10) and later (>p16) passage to evaluate genomic evolution during culture. One cell line (K9TCC) was available only from p14 and so was harvested at p16 only.DNA from each cell line ("test") was isolated using a DNeasy Kit according to manufacturer’s recommendations . Purified DNA was verified to be of high molecular weight and purity by agarose gel electrophoresis and spectrophotometry . Since all five cell lines were female, a single female "reference" mix was generated by combining equimolar quantities of DNA extracted from ten different, healthy female dogs of various breeds . Isolated DNA was labeled using the Genomic DNA Enzymatic Labeling Kit  as described previously , 13. Flup = 0.05, differential threshold = 1 %).Genomic regions likely to be central to tumorigenesis were pinpointed by the Genomic Identification of Significant Targets in Cancer (GISTIC) algorithm in Nexus Copy Number, which identifies regions unlikely to be aberrant by chance when taking aberration frequency and amplitude into account . A G-scoPreviously published oaCGH data from 31 primary canine UCs was compared to cell line aberrations . None ofCells were harvested using conventional methods of colcemid-induced metaphase arrest (Life Technologies), hypotonic (KCl) treatment (Life Technologies), trypsinization , and fixation, as described previously . Fixed cin situ hybridization (FISH) was used to validate and visualize copy number aberrations indicated by oaCGH. Based on FASST2-called aberrations, 11 genomic regions were selected for FISH analysis: 10 aberrant regions of high frequency (gained/lost in at least three cell lines) and one balanced region on CFA 11, selected as a copy number neutral control (Log2 = 0) (Table http://bacpac.chori.org/library.php?id=253) containing the regions of interest and labeled with one of five spectrally-resolvable fluorochrome-conjugated dNTPs as described previously [Fluorescence eviously . Multicoeviously . Verifie2 ratios ascertained by oaCGH and also deduced from single locus-BAC probe probe enumeration, correlation coefficients were derived. To calculate the Log2 ratio, the average copy number of the aberrant locus was divided by the average copy number of the CFA11 neutral locus of each cell line. A Log2 of the resultant value was calculated and used as the FISH-derived Log2 for each copy number-aberrant locus. Additionally, a Mann–Whitney U test  was performed using JMP Professional Statistical Software  to compare mean Log2 ratios from both methods.Using Logcell_line ÷ NScontrol_average = relative fold changegene). Significantly over- and underexpressed genes in demonstrated a ± 2-fold change in gene expression in the cell lines relative to the average of control bladders. The resulting gene expression profiles of cell lines and healthy control urothelium were clustered using an agglomerative, unsupervised hierarchical clustering algorithm in Nexus Expression (Biodiscovery).RNA was extracted from cultured cells or control urothelial cells using the RNeasy Plus Mini Kit (Qiagen). RNA was immediately isolated from the healthy, fresh urothelial cells to minimize alterations in post-mortem and post-collection gene expression. RNA purity and integrity was assessed using spectrophotometry (Nanodrop) and a 2100 BioAnalyzer (Agilent), respectively. Samples with 260:230 and 260:280 > 2.0 and RIN > 8.0 were used for microarray analysis and qRT-PCR validation. RNA was labeled using the Quick Amp Labeling kit (Agilent). Purified RNA probes were hybridized to a one-color expression microarray (Agilent SurePrint G3 Canine 4x44k Expression Array). Arrays were scanned at 3 μm . Analysis was performed using the GeneSpring advanced platform  and Nexus Expression . Prior to expression normalization, compromised probes were removed from analysis in GeneSpring, including features which were non-uniform, saturated, or population outliers. Normalized signal values were obtained by log transformation, followed by 75th percentile shift normalization and baseline shift to median of all samples. Normalized signal values (NS) were compared between RNA isolated from the cell lines and from urothelium of two histologically confirmed healthy dog bladders to determine the fold change in expression (NSRPL32) and for a gene within each of the loci assessed by FISH and dysregulated according to the expression array. Ct values for RPL32 were within 0.7 Ct among all cell lines and controls (standard deviation = 0.23), validating its use as a reference gene. Ct Primer template specificity was confirmed by agarose gel electrophoresis of PCR products (single product) and primer melt curve analysis (single peak). An efficiency curve based on five ten-fold dilutions was constructed to evaluate the performance of each primer pair. Primers with efficiency between 90-110 % and linear correlation coefficients over 0.95 were selected for qRT-PCR analysis  were noted on CFA10, 36, and 38, suggesting DNA amplification in these regions. To identify regions differentially aberrant between early and later passage cell lines, a “comparisons” analysis was performed in Nexus Copy Number. The analysis showed only three genomic regions where the aberration frequency among the early and later passage cell lined differed by more than 1 %: CFA1q22, CFA2q24.1, and CFA9q22.3 (p < 0.01). Although copy number neutral in early cell line passages, later passages of the same cell lines demonstrated a copy number gain.Metaphase preparations from each of the five canine UC cell lines revealed a high degree of aneuploidy, with an excess of 100 chromosomes in four of five cell lines Table . Highly Cell line oaCGH profiles were compared to those of 31 previously published primary tumors Fig. . HierarcChromothriptic-like events were noted throughout the genome of all five of the cell lines, most notably on CFA 9 and 10 (>80 % of cell lines) Fig. . CFA 5, 2 ratios , while correlation analysis showed a strong correlation between FISH- and oaCGH-determined Log2 ratios (r = 0.91). FISH enabled appreciation of hyperdiploid status in four of five cell lines , with average total chromosome number among 30 enumerated chromosome spreads ranging from 103 (K9TCC-Pu-In) to 134 (K9TCC-Pu-Mx) total chromosomes (Table 2 > 4. The region probed on CFA 38 demonstrated a translocation to a position adjacent to the CFA 36 locus in two cell lines (K9TCC-PU-An and K9TCC-PU-Sh). In K9TCC-PU-Sh, tandem duplications of CFA 36, juxtaposed with tandem duplications of CFA 38, were present twice on a single aberrant chromosome structure  in our dataset, with 189 genes underexpressed in the cell lines. Among overexpressed genes , “DNA replication, recombination, and repair” was the most highly enriched term (p = 0.01), with 13 overexpressed genes. When evaluating all DEGs, “DNA replication and repair” was the single most commonly dysregulated functional ontology, represented by 10 % of all cell line DEGs.Using the DAVID tool for gene ontology (GO) and pathway analysis, DEG enrichment in functional pathways was evaluated. Among underexpressed genes , the GO term “lipid metabolism” was the only GO term enriched (p = 0.0004), as were 1.8 % (26 of 1444) of all upregulated genes (p = 0.00003). DAVID chromosome association analysis found 4.7 % of overexpressed genes (68 genes) were found on CFA 13 and 4.2 % on CFA 10 (61 genes). Similarly, 3.6 % (66 genes) of underexpressed genes were located on CFA 12 and 1.2 % on CFA 19 (23 genes).KEGG pathway analysis in DAVID highlighted four pathways enriched for underexpressed genes: metabolism of xenobiotics by cytochrome p450, glutathione metabolism, drug metabolism, and fatty acid metabolism, resulting in a KEGG pathway clustering of these pathways with an enrichment score indicative of biological significance . Additionally, 3.7 % of genes with five-fold or greater expression than normal urothelium (10 of 268) were involved in cell cycle regulatory pathways . Furthermore, in all cell lines, abnormally metacentric chromosomes were seen, suggesting prior mitotic dysfunction and/or breakage-fusion-bridge cycles and leading to the formation of new hybrid chromosomes.2 = 0.The hyperdiploid karyotype of the cell lines, excluding K9TCC, further confirmed previous findings that canine UCs are often tetraploid (>50 % of UCs) . Since oIGF2R, CFA1) and a protocadherin cluster . IGF2R and PCDH are both potential adaptations to the culture environment, allowing improved utilization of media sugars and improving culture flask adhesion, respectively. Nevertheless, such few aberrations acquired over 10+ passages represent a very minor effect of repeated passage and cell culture. This characteristic of the cell lines allows experimentation and analysis over time without concern of culture-induced changes, a valuable feature for translational studies.Cells were repeatedly passaged (>10 passages) in order to determine the stability of the UC genomic profile over time. Little change was evident in oaCGH profiles or chromosome structure of cell lines after passaging cells from early to mid-late stage. Only three regions on CFA1, 2, and 9 were significantly different between early and late passages, representing less than 0.006 % of the entire genome in length. Although the region on CFA9 contains no known genes, the regions on CFA1 and 2 contain genes potentially relevant to the culture environment: insulin-like growth factor 2 receptor  is involved in metabolism of organophosphate and pyrethroid insecticides, both associated with UC development [Factors incriminated in the development of UC include exposure to cyclic amines, including carcinogens in cigarette smoke, estrogen, and cyclophosphamide, as well as obesity , 24–27. elopment –30. Simielopment . In addielopment , 33.CYP2C19; CFA28; fold change = −8.1) is a potentially relevant metabolic gene central to the enriched cytochrome p450 KEGG pathway. CYP2C19 is one of the most important cytochrome p450 enzymes and is responsible for the metabolism of numerous cyclic amine xenobiotics, including the pyrethroid-derivatives of hydantoin present in common insecticides [CES1, genetic polymorphisms of CYP2C19 are associated with reduced metabolic capacity in humans, suggesting similar phenomena may occur among genetically distinct dog breeds [Of further interest, Cytochrome P450 2C19 . As a result of its functions as a sister chromatid securin, microtubule nucleation regulator, and AKT activator, among others, PTTG1 overexpression potentiates tumor proliferation and invasion, as well as chromosome instability [FGFR3, an upstream regulator of AKT activity, are implicated in constitutive activation of the AKT pathway commonly seen in human UC [PTTG1 has the potential to be a major driver of AKT activation in canine UC, providing a valuable therapeutic target and highlighting pathway dysregulation similarities in human and dog UC.Among genes overexpressed in the UC cell lines, GO terms were enriched for DNA replication, recombination, and repair. Similarly, KEGG analysis highlighted cell cycle regulator pathways, in which 4 % of genes with expression five-fold above that of normal urothelium were involved . One of the most prominent overexpressed genes in our dataset and a key gene in DNA replication tability –39. Intehuman UC . PTTG1 hr = 0.76). DAVID chromosome analysis showed enrichment of underexpressed genes on CFA19, which is frequently lost in UC cell lines. CFA 19 contains the gene for histamine N-methyltransferase (HNMT), a gene highly under expressed in our cell lines (fold change = −29.6). Although unreported in bladder cancer, reduced HNMT transcription has been reported in other human carcinomas and is posited to be involved in tumorigenesis [HNMT lead to local increases of angiogenesis-promoting PTGS2 . PTGS2 levels are increased in both canine and human UC, and this gene is similarly overexpressed in our cell line data (fold change = +76.9), providing a further mechanistic role for COX-2 inhibitors in UC treatment [COX2 is a downstream effector of an activated AKT pathway, again suggesting a conservation of AKT overactivation in both canine and human UC development.Our expression data also emphasized the potential impact of chromosome copy number on gene dosage and, therefore, gene expression. When analyzing genes located in regions of copy number aberration, it was shown that gene expression often varied directly with copy number change , including the oncogenic transcription factor r cancer  and is oUC Table , providiPABPC1, a gene located in the region of shared high frequency gain on HSA 8/CFA 13 in human and canine UC, was actually underexpressed (average fold change = −2.6) in the cell lines [PABPC1 is highly expressed, at both mRNA and protein levels, in normal urothelium, potentially obscuring subtle differences in expression while reducing their physiologic significance [PABPC1 at the mRNA level, protein studies have shown a relative decrease in protein, espousing the need for protein evaluation alongside mRNA quantification [Despite strong correlations between copy number gain/loss and differential gene expression Fig. , it is nll lines . Howeverificance , 46. Addfication . We cannin vitro, making the cell lines a valuable resource for translational UC research. Future research should involve utilizing the UC cell lines in a functional manner to elucidate mechanisms of UC pathogenesis.The recurrence of specific genetic aberrations in UC emphasizes the importance of continued UC genomic research, particularly in predisposed dog breeds with reduced genetic variation and in association with prominent carcinogenic risk factors. A treatment found to be effective in canine cell lines, which then is effective in dogs, increases the clinical predictive value of the cell lines and, therefore, their value to biomedical research . The pre


UCP2) in combination with other chemotherapeutic agents might be more potent in HCC patients.Our results suggest a critical role for mitochondrial uncoupling in gemcitabine (GEM) resistance in hepatocellular carcinoma (HCC) cell lines. Hence, synergistic targeting of uncoupling protein 2  in hepatocellular carcinoma (HCC) patients. However, GEM alone or in combination with oxiplatin hardly renders any survival benefits to HCC patients. Mitochondrial uncoupling protein 2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) generation, thus mitigating oxidative stress-induced apoptosis. We demonstrate in the present study, using a panel of HCC cell lines that sensitivity to GEM in HCC well correlate with the endogenous level of UCP2 protein expression. Moreover, ectopic overexpression of  The uncoupling protein (UCP) family is a sub-category of mitochondrial anion-carrier proteins super-family and can be found in both animal and plant species . Genome Whereas UCP1 is a key regulator of adaptive thermogenesis, UCP2 and UCP3 also process important function to decrease the reactive oxygen species (ROS) that is produced by electron transport ,7. ROS iUCP2 overexpressing colon cancer cells are more resistant to CPT-11, ((4S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo-1H-pyranoindolizinoquinolin-9-yl -1′-carboxylic acid ester hydrochloride), a topoisomerase I inhibitor and (b) genipin, a specific UCP2 inhibitor, enhanced sensitization to anthracyclin to drug-resistant leukaemia cells [UCP2 expression in rendering cellular resistance to GEM and the anti-tumoral effect of GEM treatment associated to UCP2 inhibition.Gemcitabine  is used in combination with oxiplatin as chemotherapeutic agents in HCC. However, using GEM rarely results in survival benefits to a patient with HCC, highlighting the need for identification of novel targets that dictate response to chemotherapy, the regulation of which in turn may improve sensitivity to GEM. Two evidences suggest that UCP2 targeting might be a potential therapeutic strategy for HCC, (a) ia cells ,17. In tHuH6, Hep3B, HepG2 cell and HLE HCC cell lines were purchased from A.T.C.C. and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Gibco). Transfection of cell with plasmid DNA or siRNA was performed by using Lipofectamine 2000 (LifeTechnologies), according to the instructions of the manufacturer.UCP2 was obtained from LifeTechnologies. A scramble siRNA sequence was included as a control. UCP2 ORF was cloned from cDNA of HepG2 by PCR and ligated to pCDNA3.1 expression vector (LifeTechnologies).The siRNA targeting GEM and FCCP (4-trifluoro-methoxy-phenyl-hydrazone) were obtained from Sigma and solubilized in sterile water and 95% ethanol respectively and stored at −20°C until use.4 unit/l heparin (SETH) medium and then homogenized. The resulting homogenate was cleared by centrifugation for 10 min at 600 g. The supernatant containing mitochondria was centrifuged again for another 10 min at 14000 g to pellet the mitochondria. After centrifugation, the mitochondrial pellet was re-suspended in SETH-medium for further analysis.Isolation of mitochondria from different cell lines were as previously described . BrieflyCells or mitochondrial extract were lysed by the Laemmli sample buffer as previously described ,20. The A), were obtained for three different experiments and expressed as mean ± S.D.Cell proliferation was quantified using a mitochondrial colorimetric assay  as per the manufacturer's recommendations. The absorbance was measured at 570 nm and post-measurement corrected were performed by subtracting absorbance at the reference wavelength of 690 nm. The results, expressed as relative absorbance . Fluorescence was measured at excitation of 430 nm and emission of 590 nm. The probe is live-cell permeant and is rapidly and selectively targeted to the mitochondria where it becomes fluorescent after oxidation by O2•−. The values were normalized on cell proliferation by MTT.The non-fluorescent MitoSox Red probe  was used to evaluate mitochondrial superoxide production as per manufacturer recommendations. Briefly, 5×103) were seeded in 96-well plates and were treated with the various compounds at the indicated concentrations for 48 h. Cells were then labelled in culture with the fluorescent inhibitor/substrate of fluorescent labeled inhibitor of caspases (FLICA) -Glu(OMe)-Val-Asp(OMe)-fluoromethylketone)  for 60 min at 37°C. Cells were washed twice with wash buffer and fluorescence measured at excitation of 485 nm and emission of 535 nm. The values were normalized to cell proliferation assessed by MTT.Cells/well (5×10To examine the roles UCP2 played in HCC cells, we first checked the endogenous UCP2 expression in a panel of four different HCC cell lines, HuH6, Hep3B, HepG2 and HLE. Whereas, HuH6, Hep3B and HepG2 showed robust endogenous levels of UCP2 protein expression, HLE showed marked suppression of UCP2 expression compared with the other three cell lines A. IntereUCP2 in HuH6 cells were confirmed by immunoblotting (UCP2 reversed sensitivity to GEM-induced cell growth inhibition (UCP2 with two different siRNAs targeting UCP2, but nor a scrambled control siRNA, significantly increased sensitivity to GEM-induced cell growth inhibition and mitochondrial superoxide generation (UCP2 is a feasible option only in those instances when it is in abundance or inhibition of even smaller amounts as found in HLE cells has an impact on cell growth. As shown in Since HLE cells showed low endogenous UCP2 expression and HuH6 cells exhibited robust endogenous UCP2 protein expression, these two cell lines were chosen for the subsequent gain- and loss-of-function studies respectively. Successful overexpression in HLE cells and siRNA-mediated silencing using two different siRNAs targeting blotting A. Ectopihibition B and sighibition C in HLE neration B and 2C.neration A, we nexUCP2 could sensitize certain cancer cell for chemotherapy in leukaemia cells [UCP2 also inhibits cell apoptosis induced by ROS. This led us to examine whether disruption of UCP2 in HuH6, Hep3B and HepG2 cell could sensitize the cells to apoptosis induction. Apoptosis induction was evaluated by determining caspase-3 and caspase-7 activity. Caspase-3 and caspase-7 activities were higher in each of the three cell lines post transfection of either of two siRNAs targeting UCP2, but not the scrambled control (UCP2 expression sensitized the cells to apoptosis.It has been reported that modification of ia cells . Moreove control . Taken tUCP2 expression sensitized HCC cell lines with robust UCP2 protein expression to apoptosis induction, which implied that UCP2 may be a therapeutic target for chemotherapy in hepatic carcinoma (UCP2 is known to suppress ROS level which is overexpressed by various types of cancer cells including HCC cell lines. However, the role it plays in HCC cell lines as well as its relation with drug-resistance is not well defined yet. In the present study, relative expression level of endogenous UCP2 dictated sensitivity to GEM-induced inhibition of cell growth. In addition, inhibition of the arcinoma .Gardenia jasminoides known as Genipin [In the current study, we have used siRNAs to silence UCP2 expression. An alternative to this would be to use chemical inhibitors. There are two known classes of chemical inhibitors of UCP2, one the more commonly tested extract from  Genipin  and the  Genipin . Even th Genipin ,25. To tUCP2 represents a major adaptive response in cancer cell [UCP2 in cancer cell sensitized drug-resistant cancer cells to cytotoxic agents [It had been demonstrated that cancer cells employed numerous strategies to nullify toxicity generated by chemotherapeutic drugs . Among tcer cell . Severalc agents ,22. ThosUCP2’s role in the cellular ROS level, previous studies also demonstrated UCP2 could regulate the function of p53 [UCP2 in HepG2 cell could restore the p53 function therefore to sensitize the cell for apoptosis induction. It however remains to be determined what causes sensitization in the Hep3B and HuH6 cell lines. Whether UCP2 could act as a p53 antagonist in HepG2 cell and how UCP2 inhibit p53 function merits further investigation. Moreover, although our study indicated that targeting UCP2 alone may be an innovative strategy for cancer therapy, earlier studies have shown that the chemosensitivity was varying among different HCC cell lines [UCP2 could act synergistically with other chemotherapeutic agents for HCC therapy. In conclusion, our study provides new insight for the role of UCP2 in HCC.On the other hand, in addition to n of p53 ,27, the n of p53 ,28. Unlin of p53 , studiesn of p53 ,31. Sincn of p53 , it is pll lines . Hence,


The Brazilian National Cancer Institute (INCA) estimated last year almost forty thousand new cancer cases of laryngeal carcinoma, melanoma, tracheal, bronchial and lung cancer. It is known that multidrug resistance and unspecific toxicity are the major challenges for commercial anticancer drugs . In thistrans-chalcone; Licochalcone A; 4-Methoxychalcone and 3'-(trifluoromethyl)chalcone were tested against four different cell lines: Hep-2, B-16, A549 and 3T3. The cell culture was carried out by using Dulbecco's Modified Eagle Medium for A549 and 3T3, RPMI 1640 for Hep-2, and F-10 for B-16. All medium were supplemented with fetal calf serum. Each cell line was plated in a 96-well plate (3x104 per well) for 24 hours before adding the medium with chalcones in a 5 different concentration treatment . After 48 hours, compounds were withdrawn and a solution of fresh medium containing 3--2,5-diphenyltetrazolium bromide (MTT) was added. After 4 hours of incubation, the product (formazan) was solved by DMSO. Hereafter, the plates were measured through 550nm wavelength analysis, using an ELISA reader. Treatments were compared to negative control (medium with 0.25% DMSO) and positive control (2.5 µg/mL doxorubicin). Cytotoxicity was calculated by the formula: percent cytotoxicity = ×100% [50 values were also determined. Data were analyzed by the software Sisvar.The cytotoxicity of chalcones was evaluated by the colorimetric MTT assay, which determines the reduction of tetrazolium into insoluble formazan by mitochondria of viable cells . For thi50 values for A549 were 81.29 µM, 81.34 µM, 85.40 µM and 46.13 µM for trans-chalcone, 3'-(trifluoromethyl)chalcone, 4-Methoxychalcone and Licochalcone A, respectively. Hep-2 showed up the most susceptible cell line with IC50 values below 10 µg/mL for trans-chalcone, Licochalcone A and 3'-(trifluoromethyl)chalcone. IC50 values for Licochalcone A, trans-chalcone, 4-Methoxychalcone and 3'-(trifluoromethyl)chalcone for tumor mouse cell line B-16 were, respectively, 25.89 µM, 45.42 µM, 50.15 µM and 61.54 µM; for non-tumor mouse cell line 3T3 were 33.42 µM, 48.40 µM, 64.34 µM and 43.44 µM. All IC50 values for 4-Methoxychalcone were higher than 50 µM. Thus, among the chalcones tested 4-Methoxychalcone was the least cytotoxic and Licochalcone A the most effective for all cell lines, followed by trans-chalcone and 3'-(trifluoromethyl)chalcone.The IC


The last two lines of


Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. Recent genome-wide sequencing projects have shown that EAC is one of the most highly mutated solid cancers with a high degree of heterogeneity , including cancers of the gastro-esophageal junction, represent a substantial health concern in Western countries due to its increasing incidence and poor prognosis. To date, there are no widely accepted animal models for EAC and a limited number of cell lines are all that are available forCell lines were obtained through commercially available repositories except JH-EsoAd1, which was a kind gift from Hector Alvarez .All cell lines were from a certified source  and verihttp://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to assess the quality of the sequence data. Additional alignment, duplication and insert size metrics quality metrics are reported inhttp://broadinstitute.github.io/picard). Original BAM files are available in the European Bioinformatics Institute (EBI) repository .Genomic DNA was prepared from cultured cells with AllPrepDNA/RNA Mini Kit (Qiagen) according to manufacturer’s instructions. A single library was created for each sample, and 90-bp paired-end sequencing was performed at Beijing Genomic Institute  according to Illumina  instructions to a typical depth of 30×, with 94% of the known genome being sequenced to at least 10× coverage and achieving a Phred quality of 30 for at least 80% of mapping bases. FastQC 0.11.2 (https://www.broadinstitute.org/gatk) to generate a high-confidence set of SNV and indel calls. These were analyzed with Ensembl Variant Effect Predictor  to annotate with genomic features and consequences of protein coding regions  was used to call and filter single nucleotide and indel variants compared to the reference genome. In brief, the steps run were as follows: 1) local realignment of reads to correct misalignments around indels using GATK RealignerTargetCreator and IndelRealigner tools; 2) recalibration of base quality scores using GATK BaseRecalibrator tool; 3) SNV and indel calling using GATK HaplotypeCaller which determines haplotype by re-assembly within regions determined to be active, i.e. where there is evidence for a variation, and uses a Bayesian approach to assign genotypes. Hard filters were applied to the resulting call set using recommendations available from the GATK documentation (regions . For theCopy number (CN) analysis was carried out using Control-FREEC . This approach reduced the number of variants by an order of magnitude from the original GATK pipeline (from a median of 4.1×106 to 1.3×105). Yet, the abundance of called variants compared to a range of 4,8×103-6×104 reported in human EAC , EGFR . Among DNA repair genes all cell lines carry benign missense variants of ATM and missense variants of uncertain significance in BRCA2. MSH2 is affected by a missense variant in SKGT4, splice site variants in CP-D, JH-EsoAd1, and UTR variants in ESO51 and OACP4 C . Copy nuin vitro models that have a germline reference genome to identify clearly the somatic changes . Accession numbers: CP-D ERS1158083; SK-GT-4 ERS1158082; OE33 ERS1158081; OACP4 C ERS1158080; OACM5.1 ERS1158079; JH-EsoAd1 ERS1158078; FLO-1 ERS1158077; ES051 ERS1158076; ES026 ERS1158075.BAM files are available at the European Nucleotide Archive  oesophageal adenocarcinoma cell lines. This is an adequate platform to present these data and the fact that the authors make all raw BAM files easily accessible to the community means that this study is particularly valuable to colleagues looking to contrast cell lines with particular genomic aberrations or different neo-antigenic burdens. Such studies always come with the known caveats ofI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have performed whole genome sequencing of eight esophageal adenocarcinoma cell lines and one esophageal high grade dysplastia cell line to an average depth of 30x.  The authors have made the BAM and VCF files available through the EBI repository and this will be an excellent resource for researchers working on this cancer.  We feel the methods used are appropriate and most of the analyses described are informative.  We do however have a few suggestions for the authors to address, these are listed below:Clarify the % of variants that fall in each sequence context, coding, intronic, regulatory, intergenic.  We assume this should sum to 100%.In the next sentence there is a “(“instead of a ” ,” “in front of the 21% and 11% respectively”Table 1: ploidy state of CP-D, should this be hypotetrapoid?3 to 4.8x103Paragraph 2 of this section: Change 4,8x10MuTect was used as variant caller in the Dulak paper and SomaticSniper was used in the Weaver paper.  The authors should explain that they can’t use a somatic variant caller as these require a "normal" sample and also that application of a different caller for this cell line project may also make comparisons with the Dulak and Weaver papers less powerful. Dataset validation WGS section:There isn’t an ARID1A UTR variant shown for any of the cell lines in Figure 2b yet the authors mention 1 of the 9 cell lines has such a variant in the text.  On a related note we think the authors should consider the relevance of including UTR and synonymous changes in figure2b.  We don’t think that these are considered in the Dulak and Weaver papers and are, as far as we understand, unlikely to be functional.Second sentence of the second paragraph needs clarifying.  Presumably missense mutations were found in MET and EGFR? IH-EsoAd1 should be JH-EsoAd1 in the same sentence.Authors should make more of the fact that they have sequenced whole genomes whereas the COSMIC cell line project has only sequenced cell line exomes.  The authors could perhaps highlight the useful extra data that is available from this sequencing effort, such as identification of mutations in putative regulatory regions and germline variants.  Both classes of variants will be of interest to researchers working on understanding the genetics of oesophageal adenocarcinoma and wishing to identify appropriate cell models to work with. Analysis of putative EAC driver genes:We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. in vivo.The authors have examined the DNA sequences of 8 oesophageal adenocarcinoma cells lines and one high-grade dysplasia cell line, The authors should be congratulated for tackling this important unmet need in oesophageal cancer research and publishing these important findings in such an accessible manner. As the authors state, oesophageal adenocarcinoma seems to be one of the cancers carrying the most mutations, and although several cell lines, including those utilized in this study are commonly used for laboratory studies, there has never been a systemic study of the genetic abnormalities in these cells lines. The data in this study does fill that important gap, allowing comparisons between them and the cancer The methods are appropriate for the study and well-described and the abstract accurately represents the contents of the study. The results are appropriately and clearly presented. The conclusions appear to be sound based on the data presented and most importantly the paper provides the data to enable other researchers to build on these data and hopefully further refine laboratory models for oesophageal adenocarcinoma.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The PI3K-AKT pathway has been shown to complement activation of the MAPK pathway in melanocyte transformation in preclinical models. We have utilized clinical specimens and cell lines to examine the regulation, functional role, and clinical significance of the PI3K-AKT pathway in melanoma.All analyses of clinical specimens were performed under protocols approved by the MD Anderson Institutional Review Board. Identities of cell lines were confirmed by STR-fingerprinting, and cell lines were genotyped by sequenom analysis for hotspot mutations. Reverse phase protein array (RPPA) analysis was performed by the MD Anderson RPPA Core Facility.Proteomic analysis of frozen melanoma clinical specimens and cell lines demonstrated that phosphorylation (activation) of AKT (P-AKT) correlated inversely with PTEN protein expression. Confirmatory analysis of PTEN expression by an immunohistochemical (IHC) assay on archival tumor samples identified a linear correlation between PTEN measurement by IHC and RPPA. Significantly increased expression of P-AKT was observed in melanomas with complete loss of PTEN protein expression compared to tumors with any other pattern of PTEN expression. Integrated analysis of BRAF/NRAS mutation status, PTEN expression, and clinical outcomes was performed using lymphadenectomy specimens from patients who underwent surgery for stage IIIB/C melanoma. Complete loss of PTEN expression correlated with shorter overall survival and time to brain metastasis on univariate and multivariate analyses. Testing of human melanoma cell lines demonstrated that cells with baseline or compensatory activation of the PI3K-AKT pathway were generally resistant to apoptosis induction following BRAF or MEK inhibition. Extended molecular profiling found that a subset of melanoma cell lines with de novo and acquired resistance to MEK inhibitors are characterized by increased expression of PGC1alpha and a metabolic phenotype of high oxidative phosphorylation (OxPhos). The resistant high OxPhos cell lines all demonstrated synergistic growth inhibition and apoptosis induction with combined targeting of MEK and mTORC1/2. mTORC1/2 inhibition decreased PGC1alpha expression in vitro and in vivo, and caused cytosolic localization of MITF. Treatment with a MEK inhibitor and an mTORC1/2 inhibitor demonstrated marked synergy in a BRAF-mutant, MEK inhibitor resistant human melanoma cell line in vivo.Activation of the PI3K-AKT pathway correlates with clinical outcomes and resistance to MAPK pathway inhibitors in melanoma. These findings support the rationale to determine the clinical benefit of inhibiting the PI3K-AKT pathway in this disease.