This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse  is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP) males and territorial, terminal phase (TP) males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.The highly conserved members of the  However, zic2 is one of the five highly conserved zics among vertebrate groups (zic1-5)zic family members play critical roles in upregulating proliferation factors and delaying differentiation during development ic2 is one of the few genes, independent of the hedgehog pathway, linked to the severe and prevalent congenital malformation of the forebrain termed holoprosencephaly in humans (HPE) zic2 patterns binocular vision by specifying the ipsilateral projection of RGCs ic2 mRNA has been detected across a wide range of malignant tissues Zic family members encode zinc-finger transcription factors composed of five tandem Cys2His2 zinc-finger domains zic2 is strongly expressed in cerebellar granule cells in the hindbrain of rodent models and humans zic2 as restricted to the cerebellum and functioning as a marker for granule cell neurons and their precursors zic mRNA signaling in the medial habenula nucleus, olfactory bulb, thalamus, and pontine nucleus of adult mice. Similarly, Brown and Brown zic2 expression in the olfactory bulb, the rostral migratory stream (RMS), and the sub-ventricular zone (SVZ) in addition to hindbrain labeling.In the mature mammalian brain, zic2 mRNA expression across the three sexual phenotypes of the bluehead wrasse, Thalassoma bifasciatum. The bluehead wrasse is a well-studied model for behavioral and neuroendocrine aspects of sex change and alternate sexual phenotypes. This species displays two discrete alternate male phenotypes, initial phase (IP) males and territorial, terminal phase (TP) males, and undergoes socially-induced sex change Our objective in this study is to examine the abundance of neural zic2 was upregulated in terminal phase males relative to both females and IP males . This study focuses on the preoptic area of the hypothalamus and the possibility that zic2 mRNA abundance is elevated specifically in TP males relative to females and IP males in this key integrative area for male-typical courtship and sexual behavior zic2 mRNA expression in the brain of the bluehead wrasse using in situ hybridization.Expression data from bluehead wrasse brain cDNA microarrays generated in our lab indicated that zic2 in the NCBI database . Zebrafish have been shown to possess two zic2 homologs, zic2a (zic2.1) and zic2b (zic2.2), due to genome duplication zic2 cDNA shows the strongest sequence identity to the zebrafish zic2a gene Thalassoma bifasciatum zic2 (HQ423137.1) sequence indicated 84% sequence identity to Danio rerio zic2a (NM131558.1) and 91% sequence identity to Gasterosteus aculeatus zic2 (BT027912.1). Nucleotide sequence alignment of the confirmed zic2 partial nucleotide sequence isolated from Thalassoma bifasciatum  aligned with Gasterosteus aculeatus zic2  is presented in supporting information . Silver grain quantification in the preoptic area indicated the same differences between phenotypes as the film-based optical density comparisons. Grain counts in the preoptic area of terminal phase males were significantly higher than both females and initial phase males . Significant correlations between i) mean absolute density values from autoradiographic film and phosphor imaging quantification  and ii) ranking of values from individuals  demonstrated consistency in relative values between measurement techniques. Optical density measures from film-based autoradiography also correlated well with silver grain quantification data, whether assessed by comparing optical density against silver grain measures directly or the ranking of values from individuals . Comparison across silver grain, autoradiographic film optical densities, and phosphor screen techniques demonstrated a consistent relationship between phenotypes . The model considered values for initial phase males and females together  and demonstrated that only the relationship between females and initial phase males to terminal phase males explained part of the variation observed . Analysis of cerebellar hybridization signal intensity across phenotypes showed no significant difference in zic2 mRNA labeling density . There were also no significant differences in zic2 signal across phenotypes in the habenula .Signaling was assessed for each slide with phosphor imaging screens, autoradiographic film optical density, and silver grain quantification techniques. Clear hybridization signal was apparent in the preoptic area of the hypothalamus in all three sexual phenotypes . Both phZic has been studied primarily in relation to development, but we found widespread expression of zic2 mRNA in the brain of adult bluehead wrasses. Expression was high in the granule cells of the cerebellum, as in other species examined to date, but is also present in a variety of other brain areas. These areas include the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle, and various telencephalic regions. We also found that zic2 mRNA was more abundant in the preoptic area of terminal phase (TP) males than in either females or the female-mimic initial phase (IP) males, but found no significant differences in zic2 mRNA in the habenula or cerebellum across sexual phenotypes.al., 2002 zic2 mRNA labeling throughout the granule cell layer of the cerebellum. Additional signal was noted in the granule layer of the valvula cerebella and crista cerebellaris which, as shown in zebrafish, contain proliferating cells that give rise to cerebellar granule neurons zic2 signaling was present throughout the habenula extending into thalamic and hypothalamic nuclei. With outputs to both dopaminergic and serotonergic targets, the habenula is also an important link between the limbic and striatal forebrain in at least mammals Consistent with the findings of Aruga et zic2 mRNA is also expressed in key integrative areas. Expression in the hypothalamus includes both the magnocellular and anterior and posterior parvocellular divisions of the preoptic area. The preoptic area is a critical region regulating reproductive behavior and function across vertebrate animals and exhibits sexual dimorphisms in a variety of species including bluehead wrasses zic2 mRNA abundances in the preoptic area of TP males than either females or IP males, suggesting zic2 could be important in sex and role differentiation, perhaps through effects on neuroendocrine signaling or neurogenesis. The dopaminergic system has been implicated in the regulation of sexual behavior in a variety of species and two studies have examined the involvement of monoamines in sex change in the saddleback wrasse , a congener of the bluehead wrasse Zic2 functions as a transcriptional repressor of the human dopamine receptor D1  and the promoter region of this receptor is one of the few known direct targets of zic2zic2 expression in the bluehead wrasse preoptic area could potentially influence sexual behavior and function through effects on dopaminergic signaling.In addition to expression in sensory processing regions, zic2 in the bluehead wrasse as in other species. In the developing mammalian brain, the medial septal nucleus, the thalamic nuclei, and preoptic nucleus express zic2zic2 expression in the adult bluehead wrasse brain, potentially reflecting the neural plasticity and high levels of neurogenesis that adult teleosts exhibit. Consistent with this suggestion, regions of cellular proliferation and neurogenesis in the adult zebrafish zic2 mRNA expression in the adult bluehead wrasse. Specifically, these include the telencephalon, preoptic area, habenula, thalamus, ventricular domain of the hypothalamus, torus longitudinalis, corpus cerebella, valvula cerebella, and cerebellum zic2 expression overlaps with markers of neurogenesis in the dentate gyrus of the hippocampus and subventricular zone lining the lateral ventricles zic2 expression in the telencephalon of adult blueheads. The lateral and posterior zones of the telencephalon in teleosts potentially represent a functional homolog of the mammalian hippocampus, and there are descriptions of cellular proliferation and neurogenesis in these regions in fishes Neurogenesis is also likely influenced by zic2 is widely expressed in the adult bluehead wrasse brain, including regions that regulate sexual behavior and function. The function of zic2 remains relatively unstudied in adult animals generally and this work is therefore novel in adult teleosts. We have also provided evidence of sexual phenotype differences in zic2 expression in the preoptic area. Together with evidence of roles in neural differentiation and dopaminergic signaling in other species, these sexual phenotype differences in bluehead wrasses suggest a potential role in their remarkable sexual plasticity. Further research involving mechanistic approaches will be necessary to explore this possibility.In summary, Thalassoma bifascitum) were collected from shallow patch reefs in the Florida Keys National Marine Sanctuary near Key Largo, Florida  under permit during July 2008. Females, IP males, and TP males were captured using a lift net between 0900h–1300h over two consecutive days (see references 21 and 23 for descriptions of lift netting). In order to ensure accurate behavioral phenotyping, fish were observed before capture to verify spawning activity. Captured fish were sacrificed immediately (within three minutes) in a small boat at the capture site using an overdose of MS-222 . Before sacrifice, fish were measured : F = 69.0 mm (62.3–75.8 mm), IP = 68.9 mm (61.4–82.9 mm), TP = 88.9 mm (84.2–94.0 mm) and sexed by visually examining the genital papillae and/or expression of sperm or eggs following manual pressure on the abdomen. Sex was confirmed by direct examination of the gonads on disssection post-sacrifice. Brain dissection and removal was complete within approximately three minutes post sacrifice. Brains were snap frozen and kept on dry ice for transport to the laboratory at NCSU, then stored at −80°C until further processing. Brains were embedded in OCT compound , coronally cryosectioned at 20 µm, and transferred onto Superfrost slides . In order to allow comparison of antisense and control sense strand treatments across adjacent sections in in situ hybridization, consecutive sections were thaw mounted on a series of six alternating slides. All slides were stored at −80°C until used for in situ hybridization. All experimental methods were in compliance with the guidelines of the Institutional Animal Care and Use Committee of North Carolina State University (NCSU).Adult bluehead wrasses , and sequenced. Comparison of the 727 bp sequence using BLAST  showed greatest sequence similarity to Gasterosteus aculeatus  and Danio rerio zic2a  and supported the identification as zic2. Purified PCR product was transformed into JM109 competent cells . Upon confirmation of the zic2 insert, plasmid was either linearized with ECOR1 for antisense template or HINDIII for sense template. To further assess probe specificity, an alternate 315 bp antisense zic2 template was cut from the same plasmid and used in in situ hybridization.Previous microarray work in our lab generated a bluehead wrasse brain cDNA library  from field-collected fish. in situ hybridization was used to compare the relative abundance of brain zic2 mRNA across sexual phenotypes. The protocol followed was described previously for assessing mRNA abundances in rat brain tissue in situ hybridization was performed using a 727 bp 35S-labeled RNA probe transcribed from template zic2 DNA. The RNA probe was synthesized with a MAXIscript In Vitro Transcription Kit  and labeled with [α-35S]CTP . Antisense probe from both 727 bp and 315 bp zic2 template was transcribed using T7 RNA polymerase with the MAXIscript kit, while sense probe was transcribed using T3 Polymerase . Probe reactions were ethanol precipitated overnight and kept at −20°C for 24 hrs until addition to hybridization buffer. RNAse control slides went through an additional one hour ribonuclease A digestion step   during standard washes that followed overnight pre-hybridization. Probe was added at 190 ng/µl/kb (0.19 µg/kb) for each reaction. A probe concentration level of 170 ng/µl/kb (0.17 µg/kb) was previously shown to be saturating through quantification of zic2 mRNA hybridization signal in the cerebellum (the region of strongest expression) across a range of probe concentrations. Following post-hybridization washes, slides were dried and placed on Kodak BioMax MR film for a 73 hour exposure at room temperature . After film exposure, slides were placed on Kodak phosphor screens  for five days to enable radioisotopic hybridization signal measurement on a Bio-Rad Molecular Imager FX scanner . Two weeks post-processing, slides were dipped in Kodak NTB3 autoradiographic emulsion , dried, and exposed in a light-sealed box at 4°C for three weeks. Exposed slides were developed for four minutes , rinsed briefly with running water, and fixed for 12 minutes  under safelight conditions. After rinsing for approximately 20 minutes under running water, slides were cresyl violet stained and coverslipped with Permount  to observe morphology. All images were taken using a Retiga 2000R, 12 Bit Color Camera  and MCID Image Capture software .Riboprobe zic2 probe  showed identical patterns of labeling to the probe generated from the 727 bp zic2 template, and pre-digestion with RNaseA eliminated labeling. A similar protocol was used to compare relative labeling densities from film exposure of probe dilution test slides. Cerebellar optical density readings across comparable sections from the same animal were submitted to a Wilcoxon signed-rank nonparametric test  in this control. A dilution test intended to ensure use of sufficient probe to saturate endogenous zic2 mRNA indicated comparable hybridization signal levels in the cerebellum with probe concentrations at 0.340 ng/ml/kb, 0.170 ng/ml/kb, and 0.085 ng/ml/kb .Comparison of adjacent sense strand sections to antisense sections from the same animal was utilized to correct for background and identify potential non-specific labeling . Controlzic2 mRNA was mapped in detail for a terminal phase (TP) male bluehead wrasse by comparison of antisense and sense-strand control slides from the same animal. This distribution was compared against other animals of the TP male, IP male, and female phenotypes to assess consistency of zic2 mRNA localization . Neuroanatomical nomenclature is consistent with Wullimann et al.Distribution of Three different quantification procedures were utilized to assess precision and consistency of the results generated. These methods were quantitative regional autoradiography , silver grain quantification, and relative hybridization signal measures from phosphor imaging screens. Quantitative regional autoradiography using MCID software  was employed for a high resolution measurement of signal density  in the cerebellum and preoptic area of the hypothalamus across phenotypes. Labeling density in the cerebellum was quantified and compared across twenty-four animals: eight animals from each of the three phenotypes. Density was quantified in ten sections of the granule cell layer of the cerebellum. A rotatable ellipse (100×75 µm) drawn using MCID software was utilized for the quantification of this region. The elliptically-shaped template was designed to lie within the confines of the granule cell layer in the cerebellum, and template placement was kept consistent across all sections and animals. In the preoptic area, typically, four sections from each animal were used for quantification of optical density, but this ranged from two to six, with sample sizes for the different sexual phenotypes totaling five females, eight initial phase males, and seven terminal phase males. Sample size was lower than cerebellar quantification due to tissue quality or loss of the region of interest in some slide sets. An elliptically-shaped template (120×75 µm) was designed to tightly border visible signal in the preoptic area of the smallest animals. This procedure allowed for conservative measures to be obtained from larger animals, and central placement of the template along the vertically-oriented third ventricle in a position encompassing the entire preoptic area addressed concerns of sampling region uniformity. The area of quantification and placement of the template was kept constant across all sections and animals to ensure accuracy of quantification procedures in both the cerebellum and preoptic area. Background density measures, taken immediately adjacent to the template with the same ellipse, were subtracted from each reading, and density values were recorded for all sections exhibiting hybridization signal.2 circular region of interest was defined over sections showing signal in the preoptic area of the hypothalamus and habenula (following the quantification procedure described), and the hybridization signal of each target area was computed in counts/mm2 by Quantity One 4.6.6. Typically, three to four sections from each animal were used for this lower resolution quantification procedure. Preoptic area hybridization signal was quantified for 5 females, 8 IP males , and 7 TP males. Habenula hybridization signal was quantified for 5 females, 8 IP males, and 7 TP males. Background difference between screens was subtracted from each measurement, and slides from the different sexual phenotypes were randomly distributed across the screens to avoid any potential confounding effect of individual screens on average measured signal intensities. The density values for the phenotypes were independent from the screens  and there was no statistical interaction between the screens and phenotypes .We used Quantity One 4.6.6 analysis software  for measurement of hybridization signal from phosphor screen scans. A 203 µmWe compared hybridization signal across phenotypes at the cellular level by measuring silver grain density in the preoptic area using MCID software . Typically, four or five sections from each animal were used for quantification purposes, but this number ranged from three to six, with sample sizes for the different sexual phenotypes totaling four females, five IP males, and six TP males. A rectangle (245×65 µm) drawn using MCID software was utilized for the quantification procedure. Grain counts were recorded from only one hemisphere of each animal. The template was designed to tightly border visible signal on one side of the preoptic area of the smallest animals and was placed parallel to the third ventricle in a fixed anatomical position. The area of quantification (245×65 µm) and the placement of the template, parallel to the third ventricle and centrally within the proximal preoptic area, was kept constant across all sections. Background silver grain density was measured immediately adjacent and lateral to the sampled area utilizing the identical template and subtracted from each preoptic area measurement taken.Measurements from successive sections in a brain area for individual animals were averaged and mean measurements were analyzed in JMP 7.0.0  by Oneway ANOVA and Tukey-Kramer post hoc tests. To assess body size effects, a multivariate stepwise regression with standard length  and sexual phenotype as factors were utilized. This analysis was performed separately for the film, phosphor screen, and silver grain quantification techniques.Figure S1Nucleotide sequence alignment of the confirmed zic2a partial nucleotide sequence isolated from Thalassoma bifasciatum  aligned with Gasterosteus aculeatus zic2a . Nucleotides indicated with “*” represent identical nucleotides when our clone was aligned with stickleback zic2a sequences from the NCBI database.(TIF)Click here for additional data file.Figure S2Unrooted Bayesian phylogenetic tree of vertebrate zic2 sequences. Numbers at each node indicate 50% or greater clade credibility values. * = only partial sequence available, ** no credibility value available for central node. Scale denotes 0.1 amino acid substitutions per site.(TIF)Click here for additional data file.Text S1Detailed methods for Bayesian phylogenetic analysis of zic2 sequences.(PDF)Click here for additional data file.


BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression (‘SCNA-genes’) in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the  Somatic copy number alterations (SCNA) are a recurrent characteristic of malignant cancers The methods used to detect and characterize SCNA have evolved significantly over the last decades. Initial cytogenetic observations have been supplemented with Southern blots and quantitative PCR. Almost twenty years ago, the availability of BAC clones delineating a tiling path through the entire human genome made it possible to detect SCNA in a genome-wide fashion, but with limited resolution Genomic alterations in many tumors, especially at late stages in their development, are so extensive that the copy-number status of individual genes or chromosomal regions can vary over a very wide range of values. A mixture of chromosomal rearrangements and focal expansions can create genomic landscapes that are very difficult to analyze using standard CGH techniques. Moreover, the exact boundaries of SCNA or the expression status of the genes encoded within them are usually not known, precluding a thorough assessment of their impact on the phenotype of the cancer cells. It has recently been proposed that SNP arrays may be better suited for the determination of copy number states in tumor samples because the analysis of data derived from such arrays can make use of allelic imbalance information in addition to hybridization intensity In the present study, we analyzed the genome-wide copy-number status of seven highly aneuploid metastatic melanoma cell lines and determined the expression of their genes using a sequencing-based approach. We show that a combination of SNP-based and CGH arrays is necessary to obtain a reliable estimate of the true copy-number status of the entire genome in the face of extensive genomic instability, and that the combination of copy-number and expression status provides powerful clues as to the possible role of genes encoded within SCNA in tumorigenesis. Moreover, we show that a protein-based network-guided analysis of SCNA-affected genes with altered expression in our data and two published datasets We analyzed seven low-passage melanoma cell lines that were established from metastases see  togetherIn initial CGH (Agilent 244k) experiments we observed that in LAU-Me275, and other highly hyperploid cell lines, the hybridization ratios between cancer cells and matched controls did not reflect the chromosome-wide aberrations observed in the karyotypes. For example tetraploid regions were measured as triploid or less by the CGH arrays see . We consThe GMM method found only 42 regions in the LAU-Me275 genome that were amplified  and SNP (Illumina 1M) array platforms. Identification of genes within amplifications or deletions was determined as described in A potential problem with SCNA studies performed in isolation is that they cannot assess the expression status of the genes contained within the altered genomic regions. Amplified genes are not necessarily highly expressed, and the exact boundaries of deletions may or may not encompass a gene of interest. We reasoned that the combination of precise copy number determination and gene expression measurement would allow us to highlight with much higher confidence those genes whose expression is affected by SCNA (SCNA-genes) in the melanoma cell lines. We therefore analyzed gene expression in each of the melanoma cell lines by RNA-seq using the Roche/454 pyrosequencing method. Additionally, we performed RNA-seq on a pool of epidermal melanocytes to determine a reference level of expression for each gene in normal melanocytic cells see .KIAA0090, a protein coding gene of unknown function not previously associated with cancer, was affected in three melanomas . By contrast, in LAU-T50B, MDM2 was predicted by SNP array to be diploid (CN = 2), and by CGH to be duplicated (CN = 3). This potential difference between these cell lines is intriguing because they were derived from metastases surgically removed from the same patient at a 12 year interval. We therefore determined the copy number status of the MDM2 gene in these two samples using fluorescent in situ hybridization (FISH). In LAU-Me275, the fluorescence signal indicated that at least 8 MDM2 copies were present at the locus on Chr12 in addition to a homogeneously staining region on Chr5  or cadherin  gene families were affected by SCNA in only one dataset, further reinforcing the idea that different genes can potentially alter the same pathway (WNT or cadherin) in different melanoma samples.An additional benefit of the protein network-guided approach is that it generates a list of genes affected by SCNA that contributed significantly to a given pathway see . Althoughree see . Of the Our goal to identify somatic copy number aberrations in metastatic melanoma cell lines revealed extreme levels of aneuploidy characteristic of this cancer type MDM2BRAF, MDM2, and NRAS, genes known to be important in melanoma PARK2 has been recently described as a tumor suppressor gene in glioblastoma and other malignancies DLL1, HSD17B3 and ULBP have been reported to be associated with cancer, although not as tumor suppressors PTEN, a tumor suppressor gene already known to be deleted in melanoma To enrich for genes that might be involved in the oncogenic process, we focused on two groups: focally amplified genes that were over-expressed relative to melanocytes; and deleted genes with no expression in the melanoma cell lines, but that were expressed in normal melanocytes. In the first group, MC1R (melanocortin receptor), chemokine (e.g. CXCR2), and endothelin receptors. GNAQ and GNA11, in a large proportion of uveal melanomas In an alternative approach to detect recurrent events in these samples, we used a protein network-guided analysis FZD8 and several members of the WNT family). This may reflect interactions between these pathways, an interplay between the WNT and cadherin pathways is known to exist MITF transcription program GRM1 has recently been implicated in the development of spontaneous melanoma in a mouse model, and an autocrine glutamate/GRM1 loop has been described in human melanoma Although annotated as distinct pathways, WNT, cadherin signaling and melanogenesis shared six SCNA-genes in common  can be recognized by cancer-specific cytotoxic T-cells EPHA3 targeting has been reported EPHA3 might be a promising target for therapeutic treatment in melanoma and other cancers.In our analysis e246 see  and amplFRS2, fibroblast growth factor receptor substrate 2, is an adaptor that acts downstream of a limited number of receptor tyrosine kinases, in particular FGF and neurotrophin receptors, RET and ALK, and plays a major role in tumorigenesis FRS2 has been suggested as a therapeutic target in cancer FRS2 was both focally amplified and over expressed in two melanoma samples (LAU-T149D and LAU-Me275) and amplified (CN = 4) in two additional melanomas (LAU-T618A and LAU-Me235). Inspection of its amplification status in larger melanoma collections would be useful to confirm its potential role as a target of interest in melanoma.Using SILAC, we demonstrated a global correlation between mRNA and protein levels across all samples. However, we are aware that the levels of individual proteins may not always reflect mRNA levels, and that the activity of certain proteins, and therefore the activation state of cellular pathways, can be further modulated by post-translational modifications. Further investigations, beyond the scope of the current study, will be required to address these possibilities. Such studies should include a functional assessment of the implicated pathways using various manipulations to activate or inhibit them (e.g. with siRNA or other inhibitors) to determine the role of the pathways in these melanoma cell lines.EPHA3 and FRS2, emerged from our analysis as potential therapeutic targets. These genes were replicated in our analysis of the two published melanoma collections, have been extensively studied in other cancer types, and thus might offer new insights in the treatment of malignant melanoma.In conclusion, we have identified SCNA-genes and pathways potentially altered in our metastatic melanoma samples and two published datasets  which should be investigated by screening larger tumor collections and in functional studies. Two SCNA-genes, Melanoma cell lines were established from metastases from patients with cutaneous melanoma and were used at low passage (<10). Donor matched cells were either peripheral blood lymphocytes (PBL) or Epstein-Barr virus transformed lymphoblastoid (EBV) cell lines. EBV cell lines were karyotyped to ensure genome stability and diploidy. Approval to use these samples for this project was given by the CHUV  ethical committee for clinical research. Melanoma cell lines were cultured conditions in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), and no antibiotics. Human foreskin melanocytes were grown in HAM-F10 medium supplemented with 2% FCS, 5% MelanoMax supplement  and 6 mM HEPES. EBV cell lines were cultured in IMDM/10% FCS medium. All cultures were without mycoplasma. DNA  and RNA (guanidinium/cesium chloride gradient) isolation and karyotype preparations were performed from parallel cultures.MDM2 combined with a chromosome 12 centromeric probe (Kreatech Poseidon FISH probe) to distinguish aneuploidy of chromosome 12 and specific locus loss or gain. Chromosomes with homogeneously staining region (HSR) were identified with the analysis of FISH metaphases in inverted digital images. Copy number estimation of HSR was done using FISH interphases.Cytogenetic (GTG-banding) and fluorescence in situ hybridization (FISH) metaphase analyses of melanoma cell lines were performed using standard protocols. We performed 15 spreads for each melanoma cell line, except for Lau-Me246 CGH arrays were processed according to the manufacturer's protocol  and as described in Martinet et al. The normalization and detection of copy number aberration is detailed in Then probe-level data were segmented using Circular Binary Segmentation Genomic DNA from each of the 7 melanoma and their matched normal cells (either EBV cell line or PBL); as well as two control melanocytes were genotyped on the Illumina Infinium Human1M-Duo arrays. Aliquots of DNA (30 µl at 50 ng/µl) for each sample were processed according to the manufacturer's protocol (Infinium HD Gemini protocol). Subsequently we used the OverUnder algorithm As part of the technical replicate design, we analyzed LAU-Me275 on Affymetrix 6.0 SNP arrays. The experiment was performed in accordance with the manufacturer's instructions. Normalization and copy number prediction were done using the PICNIC algorithm The transcriptome from all seven melanoma as well as a pool of two melanocytes was sequenced using the Roche 454 Titanium technology. mRNA isolation and cDNA preparation were performed following the protocol used by Bainbridge et al. We computed the median copy number at each Refseq gene. To overcome density limitations, we included SNPs that were within 2 kb of the gene boundaries. For CGH arrays, we included probes within 3 kb of the gene boundaries. We defined SCNA-genes as follows. A gene was flagged as within a focal amplification when its CN, as computed from SNP arrays, was ≥4, the difference in CN relative to the chromosomal arm was ≥1, the gene was diploid (CN = 2) in the matched control cell line, and the expression in the melanoma cell line was at least 2-fold greater than that in the control melanocytes. For deletions, a gene needed to have CN<2, as detected by CGH, without expression in the melanoma cell line and CN = 2 with detected expression in the melanocytes.www.genenames.org). Among the 1710 (unique) SCNA-genes, 104 did not have a UniProt ID and 43 could not be mapped onto the network. Using a walk trap community algorithm and permutation approaches (with n = 1000), we were able to extract clusters of proteins from the network  could influence the protein quantification, we performed a self-self experiment with LAU-Me275. Although no significant bias was found see , we excl2 ratios . Next, we computed the Spearman correlation coefficient between the expression and protein logtios see . P valueMicroarray and sequencing data were deposited in NCBI GEO and are available under accession number GSE23056.Figure S1CGH hybridization ratio in a tetraploid region in LAU-Me275. Each plot shows the hybridization log2 ratio at each CGH probe (in gray) obtained using three normalization methods. Ridge refers to the framework from Chen et al. Red segments were obtained using Circular Binary Segmentation. Karyotype analysis of LAU-Me275 revealed 11q amplification (CN≥4), so the expected CGH log ratio would be two. Here the ratios obtained from three different normalizations failed to reflect the amplification .(DOC)Click here for additional data file.Figure S2Correlation between replicates using different normalization schemes. The scatter plots illustrate the correlation at each CGH probe between two replicates. The heatmaps show the correlation for each pairs of replicates. The normalization method is indicated in each plot title. RIDGE refers to the framework from Chen et al.(DOC)Click here for additional data file.Figure S3Gaussian Mixtures identified in four replicates from LAU-Me275. Each histogram shows the distribution of CBS segment log2 ratios, colors highlight the Gaussian components. The number of components identified and the Bayes Information Criterion are indicated in each figure title.(DOC)Click here for additional data file.Figure S4Comparison of CNV detection algorithm on CGH data. Panels are, from left to right: a melanoma with large deletions (LAU-Me280); a melanoma with large amplifications (LAU-Me275); and a control EBV cell line  hybridized using a pool of female references. From top to bottom: CNV classification (following CBS segmentation) using 1) Gaussian Mixture Model (GMM), 2) MergeLevels, 3) the scoring-based approach. Each dot corresponds to a CGH probe with its genomic position on the X axis and its log2 ratio of hybridization on the Y axis. Colors indicate the copy number state: orange< = 1 copy gray = 2 copies, cyan = 3 copies and dark blue more than 3 copies. For the scoring approach distinction is made between 1 copy (orange) and 0 copy (red).(DOC)Click here for additional data file.Figure S5Optimization of Illumina analysis and comparison with Affymetrix prediction in LAU-Me275.A. Pearson correlation between SNP CN, as a function of OverUnder window size. B. Copy number concordance at each SNP for different window sizes of OverUnder. Colors indicate window size parameters, the bar height indicates the total number of SNPs  found in one replicate. The gray bar indicates the intersection between two technical replicates. The percentage of concordance  is shown on top of each bar. C. Copy number prediction on chromosome 1 using OverUnder with a window size of 201 SNPs. D. Copy number prediction on chromosome 1 using an Affymetrix 6.0 array .(DOC)Click here for additional data file.Figure S6Copy number analysis using Illumina SNP arrays. DNA from LAU-Me275 was hybridized to Illumina SNP arrays, and the data were analyzed using the method of Attiyeh et al. The top panel shows genome-wide copy number: dark blue indicates more than three copies; cyan:three copies; gray:copy neutral; orange: deletion. Subsequent panels show chromosome 7 with, from top to bottom: Hybridization log2 ratio; B allele frequency; and copy number prediction.(DOC)Click here for additional data file.Figure S7Intersection between CGH and SNP predictions.A. Intersection between CGH and SNP predictions for genes with more than 4 copies. B. Intersection for genes within deletions. C. Intersection for genes within deletions for which expression was not detected.(DOC)Click here for additional data file.Figure S8Copy number prediction from CGH and SNP arrays, LOH prediction from SNP arrays.(PDF)Click here for additional data file.Figure S9Boxplots of SILAC heavy/light normalized log2 ratios. In all experiments, LAU-Me275 was labeled with the heavy isotope; the unlabeled sample is indicated in the boxplot label on the X axis. ‘NHM’ refers to the pool of normal melanocytes and ‘self-self’ to a control experiment using only LAU-Me275 to check for any bias due to the label/no label culture conditions (no significant bias was detected).(DOC)Click here for additional data file.Figure S10Histogram of unique peptides identified per protein in the SILAC data.(DOC)Click here for additional data file.Figure S11Correlation between mRNA expression and protein levels. Both SILAC and RNA seq log2 ratios are expressed for LAU-Me275 with respect to the sample indicated in each plot title. Spearman Rho correlation coefficient is also indicated in the title. In all experiments, the correlation is significantly positive (p<0.001).(DOC)Click here for additional data file.Table S1Count of genes affected by SCNA.(DOC)Click here for additional data file.Table S2Processed list of SCNA genes in all seven melanoma cell lines.(XLS)Click here for additional data file.Table S3Genomic and transcriptomic data for SCNA-genes in all seven melanoma cell lines.(XLS)Click here for additional data file.Table S4List of pathways significantly enriched in SCNA, and pathway comparison between three melanoma datasets.(XLS)Click here for additional data file.Table S5List of genes contributing to pathway enrichment in three melanoma datasets.(XLS)Click here for additional data file.Methods S1(DOC)Click here for additional data file.


Electroacupuncture (EA) is a novel therapy based on traditional acupuncture combined with modern eletrotherapy that is currently being investigated as a treatment for acute ischemic stroke. Here, we studied whether acute EA stimulation improves tissue and functional outcome following experimentally induced cerebral ischemia in mice. We hypothesized that endothelial nitric oxide synthase (eNOS)-mediated perfusion augmentation was related to the beneficial effects of EA by interventions in acute ischemic injury. EA stimulation at Baihui (GV20) and Dazhui (GV14) increased cerebral perfusion in the cerebral cortex, which was suppressed in eNOS KO, but there was no mean arterial blood pressure (MABP) response. The increased perfusion elicited by EA were completely abolished by a muscarinic acetylcholine receptor (mAChR) blocker (atropine), but not a β-adrenergic receptor blocker (propranolol), an α-adrenergic receptor blocker (phentolamine), or a nicotinic acetylcholine receptor (nAChR) blocker (mecamylamine). In addition, EA increased acetylcholine (ACh) release and mAChR M3 expression in the cerebral cortex. Acute EA stimulation after occlusion significantly reduced infarct volume by 34.5% when compared to a control group of mice at 24 h after 60 min-middle cerebral artery occlusion (MCAO) (moderate ischemic injury), but not 90-min MCAO (severe ischemic injury). Furthermore, the impact of EA on moderate ischemic injury was totally abolished in eNOS KO. Consistent with a smaller infarct size, acute EA stimulation led to prominent improvement of neurological function and vestibule-motor function. Our results suggest that acute EA stimulation after moderate focal cerebral ischemia, but not severe ischemia improves tissue and functional recovery and ACh/eNOS-mediated perfusion augmentation might be related to these beneficial effects of EA by interventions in acute ischemic injury. Despite decades of intense research, current treatments for acute stroke are far from optimal. Electroacupuncture (EA) is a traditional therapy that has been widely applied for treatment of ischemic stroke because it has been shown to improve its outcome in experimental animals Many other factors could have influenced the results of EA studies, including type of stroke, its severity, interval after stroke before treatment began, and amount of time until post treatment assessment After cerebral ischemia, restoring blood supply to the ischemic region as rapidly as possible to save the dying neurons, neuroglial cells and vascular endothelial cells is essential for treatment of ischemic cerebrovascular disease. In the past, studies focused on neurons and paid less attention to vascular endothelial cells. After focal cerebral ischemia, dilating the blood vessels, anti-coagulant therapy and thrombolytic therapy obviously improve perfusion of the surrounding tissues, and can promote recovery of the nervous functions. EA has been shown to increase cerebral blood flow (CBF) and improve microcirculation It is well-known that nitric oxide (NO) synthesized by endothelial NO synthase (eNOS) plays a pivotal role in maintaining CBF in the ischemic cortex. Under most conditions, stimulation of eNOS activity is protective In this study, we investigated whether acute EA stimulation simultaneously targeting Baihui (GV20) and Dazhui (GV14), improved tissue and functional outcome following experimentally induced cerebral ischemia in mice and, if so, what mechanism mediates this protective effect. We hypothesized that eNOS-mediated perfusion augmentation might be related to the beneficial effects of EA via interventions in acute ischemic injury.ad libitum. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Pusan National University Institutional Animal Care and Use Committee (Permit Number: PNU-2011-000420). Computer-generated randomization was conducted by SigmaPlot 11.2  for allocating to EA group or control group. After getting the random number by computer-generated randomization, C57/BL6J male mice were allocated either EA group or control group in a blinded fashion. Anesthesia was achieved by face mask-delivered isoflurane . The femoral artery was catheterized for measurement of the mean arterial blood pressure (MABP) using an MLT844 physiological pressure transducer . The data were continuously recorded using a PowerLab data acquisition and analysis system  and stored in a computer. The depth of anesthesia was checked by the absence of cardiovascular changes in response to a tail pinch. Rectal temperature was kept at 36.5°C–37.5°C using a Panlab thermostatically controlled heating mat . Arterial blood gases and pH were measured before and after EA using i-Stat System . Relative CBF was measured in C57BL/6J and eNOS KO using a PeriFlux Laser Doppler System 5000 . A flexible 0.5-mm fiberoptic probe was affixed to the skull over the brain cortex supplied by the middle cerebral artery (MCA) . CBF measurement was conducted for 5 min before EA stimulation, 20 min during EA and 20 min after EA, giving a total of 45 min. Baseline CBF values measured before EA were defined as 100% flow.Male mice  were housed under diurnal lighting conditions and allowed food and tap water Animals were anesthetized with isoflurane to avoid restraint stress. The transpositional method was used to determine the acupoints in mice. In this method, the veterinary acupoints are located by transforming human acupoints onto the animal anatomy Normal mice were deeply anesthetized with thiopental sodium at 20 min after the end of EA treatment, after which the cortical brain tissues were collected. The amount of acetylcholine (ACh) from brain tissues was then determined using a commercially available ELISA kit according to the manufacturer's instructions .2O2 and non-specific antibody binding was blocked with CAS Block  for 10 min. The preparations were then incubated overnight at 4°C with antibody against muscarinic ACh receptor (mAChR) M3 , after which they were incubated with an avidin-biotin peroxidase complex solution  for 1 h. The immunoreaction products were visualized with a solution of 3,3′-diaminobenzidine tetrahydrochloride , then counterstained with hematoxylin . All samples were visualized using a light microscope .Twenty min after the end of EA treatment, mice were deeply anesthetized with thiopental sodium and subsequently perfused transcardially with cold PBS followed by 4% paraformaldehyde for fixation. The brain of each mouse was then removed and further fixed for 24 h in 4% paraformaldehyde at 4°C followed by cryoprotection in 20% sucrose for 72 h at 4°C. The isolated brains were frozen in an optical cutting temperature medium for frozen tissue specimens  and then stored in the freezer at −80°C until examined. The frozen brains were cut at a thickness of 14 µm using a Leica CM 3050 cryostat . For immunohistochemistry, endogenous peroxidase activity was quenched by incubation with 0.3% H3) - undamaged ipsilateral hemisphere (mm3).Focal cerebral ischemia was induced by occluding the MCA using a previously described intraluminal filament technique Neurological deficit was scored in each mouse at 24 h after the ischemic insult in a blinded fashion according to the following graded scoring system: 0 = no deficit; 1 = forelimb weakness and torso turning to the ipsilateral side when held by tail; 2 = circling to the affected side; 3 = unable to bear weight on the affected side; and 4 = no spontaneous locomotor activity or barrel rolling Vestibulo-motor function was assessed using a wire-grip test 24 h after cerebral ischemia P<0.05 was considered statistically significant. Statistical analysis was performed using SigmaPlot 11.2 (Systat Software Inc).First we checked the data whether normally distributed by conducting normality test using SigmaPlot 11.2 (Systat Software Inc). When data were normally distributed, we planned to use parametric analysis method and if not, we planned to conduct nonparametric statistical analysis. All the data showed normal distribution, therefore we conducted parametric analysis. The data are expressed as mean ± standard error of mean (SEM). Two-way analysis of variance (ANOVA) for repeated measures followed by Student-Newman-Keuls multiple comparisons test were used to compare CBF between the control and EA and between the vehicle and drug-treated groups. Unpaired t-test was used to compare acetylcholine, infarct volume, neurological score and wire grip test between control and EA group. 2, pCO2 and pH) and MABP remained in the normal range throughout the experimental period . Arterial blood gases , and 1 out of 7 mice (14%) in the EA group died at 24 h after 90 min-occlusion, and there were no losses of mice in other surgical groups. Acute EA stimulation significantly reduced infarct volume by 34.5% when compared to the control group mice at 24 h after 60 min-MCA occlusion (moderate ischemic injury), but not after 90-min MCA occlusion (severe ischemic injury), suggesting that EA might be effective for treatment of moderate ischemic injury, but not severe ischemic injury . In addiEA is a potential therapeutic strategy to improve functional outcome in acute ischemic stroke. Here, we provide evidence that acute EA stimulation at Baihui (GV20) and Dazhui (GV14) after moderate focal cerebral ischemia improves tissue and functional recovery, and that ACh/eNOS-mediated perfusion augmentation might be related to these beneficial effects of EA in response to interventions in moderate ischemic injury. Our results showed that EA stimulation increased perfusion in the cerebral cortex, which was almost abolished by a mAChR blocker. In addition, EA increased acetylcholine release and mAChR M3 expression in the cerebral cortex. Acute EA stimulation after occlusion significantly reduced moderate ischemic injury, which was suppressed in eNOS KO, suggesting that the cerebrovascular protective effects of EA are dependent on eNOS. Consistent with a smaller infarct size, acute EA treatment showed prominent improvement of neurological function and vestibule-motor function. These findings support the view that acute EA treatment improves cerebrovascular damage in the acute stage of moderate focal cerebral ischemia, at least partially because of an increase of cerebral perfusion in an ACh/eNOS-dependent fashion.EA is a novel therapy based on traditional acupuncture combined with modern electrotherapy that exets many effects including analgesia, promotion of homeostasis, and changes in the microcirculatory network Although acute EA treatment could induce a protective effect against cerebral ischemic injury, there are several characteristics of EA treatment that differ from other treatment methods, especially the acupoint specificity. The Baihui (GV20) and Dazhui (GV14) acupoints were chosen because the theory of meridians in traditional Korean medicine indicates that they are closely related to the brain and spinal cord and they were commonly used to treat stroke in ancient Korea. Additionally, these acupoints protect against hypoxic-ischemic brain damage in immature rats Our understanding of the mechanisms by which EA stimulation in the acute stage of focal cerebral ischemia improved tissue and functional outcome has been limited; however, several mechanisms for the protective effects of acute EA after ischemia have been suggested. EA administered 15 min after occlusion of MCA enhanced the expression of insulin-like growth factor-1, which might be an important mechanism of neuroprotective effects of EA against cerebral ischemia in monkey Therapies that reinstate CBF to the ischemic territory are effective in acute stroke, suggesting that CBF is a critical determinant of final stroke outcome. Therefore, it is possible that enhancement of CBF might be the underlying mechanism of EA. A large number of animal studies have shown that EA could increase CBF in ischemic injury. The local CBF in the striatum decreased during MCAO, whereas EA improved the CBF significantly in focal cerebral ischemic monkey The mechanism of improved perfusion in the cerebral cortex by EA stimulation is unclear. Several lines of evidence have indicated that the central cholinergic system is sensitive to ischemia, and that even mild hypoxia may impair the synthesis of ACh The NO-dependent dilation described above could be mediated by either one of two types of ACh receptors identified in brain microvasculature, nicotine In summary, our data suggest that acute EA stimulation at the GV20 and GV14 acupoints after moderate focal cerebral ischemia has cerebrovascular protective potential mediated, at least in part, via ACh/eNOS-mediated perfusion augmentation. Although further investigation is needed to elucidate stroke subtypes and the therapeutic window within which EA is efficacious, EA may present a novel therapeutic opportunity during acute stroke. At this time, our results provide a scientific basis to better understand the cerebrovascular mechanisms of EA used in acute treatment of cerebral ischemia in the practice of complementary and alternative medicine.


Effective chemotherapeutics for primary systemic tumors have limited access to brain metastases because of the blood–brain barrier (BBB). The aim of this study was to develop a strategy for specifically permeabilizing the BBB at sites of cerebral metastases.111In-BnDTPA-Tz) was injected intravenously. BBB permeability was assessed in vivo using gadolinium-enhanced T1-weighted magnetic resonance imaging and confirmed histochemically. Brain uptake of 111In-BnDTPA-Tz was determined using in vivo single photon emission computed tomography/computed tomography. Endothelial expression of TNF receptors was determined immunohistochemically in both mouse and human brain tissue containing metastases. Group differences were analyzed with one-way analysis of variance followed by post hoc tests, Wilcoxon signed rank test, and Kruskal–Wallis with Dunn’s multiple comparison test. All statistical tests were two-sided.BALB/c mice were injected intracardially to induce brain metastases. After metastasis induction, either tumor necrosis factor (TNF) or lymphotoxin (LT) was administered intravenously, and 2 to 24 hours later gadolinium- diethylenetriaminepentaacetic acid, horseradish peroxidase, or radiolabeled trastuzumab . Human brain metastases displayed a similar TNF receptor profile compared with the mouse model, with predominantly vascular TNFR1 expression.Localized expression of TNF receptor 1 (TNFR1) was evident on the vascular endothelium associated with brain metastases. Administration of TNF or LT permeabilized the BBB to exogenous tracers selectively at sites of brain metastasis, with peak effect at 6 hours. Metastasis-specific uptake ratio of These findings describe a new approach to selectively permeabilize the BBB at sites of brain metastases to aid in detection of micrometastases and facilitate tumor-specific access of chemotherapeutic agents. We hypothesize that this permeabilization works primarily though TNFR1 activation and has the potential for clinical translation. Brain metastases pose a substantial challenge for chemotherapeutic treatment because of the impermeable nature of the blood–brain barrier (BBB), which limits access of drugs and thus prevents accumulation of clinically effective doses at tumor sites. Substances with good penetration of the BBB have limited activity against breast cancer, one of the most common cancers metastasizing to the brain, whereas the most active therapeutics for breast cancer (including doxorubicin and trastuzumab) appear not to reach the central nervous system (CNS)  because A number of approaches to transiently circumvent the BBB have been investigated for the delivery of chemotherapeutics to brain tumors [for a review see ]. BradykPreclinical studies aimed at increasing drug delivery to systemic tumors have demonstrated the ability of an intravenous bolus dose of recombinant human tumor necrosis factor (TNF), a proinflammatory cytokine, to disrupt endothelial tight junctions in the tumor vasculature through the RhoA/Rho kinase pathway . This apBased on the above findings, we hypothesized that cerebral metastases may provide a unique environment for TNF receptor activation on the associated vasculature and that this might yield a target for specific and local opening of the tumor-associated BBB. The primary aims of this study, therefore, were to determine whether TNFR1 and TNFR2 are expressed on metastasis-associated vasculature, and to determine whether intravenous administration of TNF, or its endogenous analogue lymphotoxin (LT), can permeabilize the BBB specifically at sites of cerebral metastasis throughout the brain to an extent that allows entry of 1) diagnostic imaging agents and 2) a clinically relevant anticancer drug.Animal procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and with local ethical committee approval. Female BALB/c mice ; Charles River, Margate, UK) were injected in the left ventricle of the heart with 10000 4T1–green fluorescent protein (GFP)  murine mSupplementary Methods (available online) for details of immunohistochemistry and immunofluorescence.Brain tissue sections from mice injected with 4T1-GFP cells were assessed immunohistochemically for TNFR1 and TNFR2 expression, and colocalization with glucose transporter 1 (Glut-1) was determined using immunofluorescence. Six cases of human brain metastasis and one control case of noncancerous neuroinflammatory disease were examined immunohistochemically for TNFR1 and TNFR2 expression. See the Supplementary Methods (available online) for details of full experimental protocol and histology.At 13 days postmetastasis induction in the 4T1-GFP model, mice were intravenously injected with 100 µL saline containing doses from 1 to 5 μg per mouse of either recombinant mouse TNF  or recombinant mouse lymphotoxin  or saline as control. Two to 24 hours later, mice were intravenously injected with of 100 µL of type II horseradish peroxidase (44kDa)  and killed by transcardial perfusion fixation. Brains were excised and prepared for histology. Slides were stained using a modified Hanker–Yates method and counterstained using cresyl violet. Brown staining indicating permeation of HRP was assessed blindly, and a percentage of positive metastases for each group was calculated. See 1-weighted images were acquired using a spin-echo sequence both before and 5 minutes after intravenous injection of 30 µL of gadolinium-diethylenetriaminepentaacetic acid  to assess BBB permeability. Regions of interest (ROIs) containing metastases  and contralateral same-slice nontumor ROIs were segmented using VnmrJ (Varian) on post-Gd images. The mean signal intensity values of the ROIs were quantitated before and after LT or TNF administration . See Supplementary Methods (available online) for details of sequence parameters.BALB/c mice injected with 4T1-GFP cells (n = 3 per group) were anesthetized and positioned in a quadrature birdcage coil. Respiration was monitored throughout, and body temperature was maintained at 37°C. MRI data were acquired using a 7 T horizontal-bore magnet with a Varian Inova spectrometer . Before and 2 hours after the 3-μg intravenous injection of TNF or LT, a set of 10 serial T111In-BnDTPA-Tz, 145kDa) was synthesized as previously described  under anesthesia. SPECT/computed tomography (CT) was performed using a nanoSPECT/CT scanner . Data were reconstructed using InVivoScope (Bioscan) and analyzed using Inveon Research Viewer  and normalized against a muscle ROI to compensate for intersubject variability in radioactivity dose. See Supplementary Methods (available online) for details.Radiolabelled trastuzumab  were intracardially injected (as above) with 10000 MDA231BR-GFP cells  . Mice n. Tissue BBB breakdown frequency in metastases was analyzed with one-way analysis of variance (ANOVA) followed by a Dunnet post hoc test or a Tukey post hoc test. The difference in ratio of signal intensity at sites shown to contain metastases vs equivalent regions in the contralateral hemisphere was assessed with the Wilcoxon signed rank test. Intracerebral radioactivity differences were analyzed with the Kruskal–Wallis test followed by a Dunn multiple comparison test. All statistical tests were two-tailed and considered statistically significant if P was less than or equal to 0.05.2 to 50000 μm2.Cerebral metastases were evident in all mice injected with 4T1-GFP cells. Mice displayed transient piloerection after TNF injection. Cross sectional areas of metastases at day 13 ranged in size from 500 μmExpression of TNFR1 was immunohistochemically localized to the vessels associated with metastases in mice injected intracardially with 4T1-GFP cells , arrows.Supplementary Figure 1A, available online).After intravenous injection of HRP, BBB permeabilization was defined as brown HRP staining within Hanker–Yates-treated tissue sections at metastases larger than 10 cells. One thousand one hundred and two separate metastatic colonies were blindly analyzed to determine their status with respect to BBB integrity. No BBB breakdown was observed in 4T1-GFP–injected mice at nonmetastatic sites before or after cytokine injection , A and D1-weighted image of mouse brain  or TNF  (The Tse brain  before cd images  from micd images . The ratnk test) .111In-BnDTPA-Tz was administered to determine whether cytokine-mediated permeability changes facilitated trastuzumab delivery to metastases. Antibody was excluded from the brain at sites of metastasis when injected systemically with saline   .P < .05, unpaired t test) , and a similar expression profile of TNFR1 and TNFR2 was observed as for the 4T1-GFP model .To further confirm the effects of TNF on metastasis permeabilization, the MDA231BR-GFP model was used. The frequency of BBB breakdown in metastases after cytokine treatment was statistically significantly higher than in noncytokine-treated controls .To determine whether this local cytokine-mediated permeabilization of the BBB in brain metastasis may be clinically translatable, six biopsies from brain metastasis were stained for TNFR1 and TNFR2. Endothelial TNFR1 staining was confirmed in a control human brain sample with nonspecific inflammation , whereasWe have described a new approach to permeabilize the vasculature of brain metastases that is based specifically on the stromal vascular phenotype of these tumors, with a view to enhancing detection and improving the delivery of chemotherapeutic agents. The 4T1-GFP mouse model of brain metastasis was used to generate micrometastases in the brain, which, at the age used here, rarely present with a permeable BBB. To ensure that all metastases within the brain were targeted, the cytokines LT or TNF were injected intravenously and were found to induce permeabilization of the BBB specifically at the sites of brain metastases, as revealed both ex vivo through histology (HRP) and in vivo through contrast-enhanced MRI (Gd-DTPA). We further replicated these findings in a second model of human-derived brain metastasis and again found that systemically administered TNF increased permeability focally at tumor sites. With this method, the commonly used treatment for breast cancer, trastuzumab (145kDa) , which iBrain metastases in mice showed increased expression of both receptors for LT and TNF (TNFR1 and TNFR2) before any cytokine treatment. TNFR1 expression was exclusively found on blood vessels within the metastases and colocalized with the endothelial marker Glut-1. These findings suggest the potential for systemic administration of either TNF or LT to alter BBB function that is likely to be through activation of TNFR1. The downstream effects of endothelial TNFR1 stimulation include alterations to cytoskeletal proteins , cell–ceAt sites of brain metastasis, expression of TNFR2 was also clearly increased but restricted to what appear to be recruited intravascular leukocytes and parenchymal microglia. This receptor expression pattern is in accord with previous studies reporting TNFR2 expression by immune system cells . Given tSupplementary Figure 1, B–D, available online).After treatment with either cytokine, the intravenous tracer HRP was found within metastases in the brain, indicating compromise of the BBB. Increasing cytokine dose was accompanied by an increase in the frequency of metastases exhibiting a permeable BBB. Breakdown of the BBB was specific and local to the sites of brain metastases, and no other permeabilization of the BBB was observed in normal brain tissue. These findings support the concept that upregulation of TNFR1 on tumor-associated vessels alone enables specific permeabilization of the tumor vasculature in response to systemic LT or TNF. This effect was maintained for all of the time points studied here. The greatest number of HRP-positive metastases was evident at 6 hours, whereas the lower number of positive metastases at the 24-hour time point suggests that this is an extended but transient opening of the BBB. The concentration of the administered cytokine rapidly returns to baseline levels , which sLT elicited a less marked response than TNF at each dose and time point, which is in agreement with previous studies that describe a less stable complex formation of lymphotoxin with TNFR1 compared with TNF, thus inducing a lower signaling capability .1-weighted images after treatment with LT or TNF. Although these regions were associated with sites of metastasis shown histologically, other sites of metastasis were not detectable with MRI. The apparent lack of permeability may be because of the limited resolution of our in vivo scans (approximately 160 μm), giving a higher size detection threshold than the histological method (approximately 1 μm). Alternatively, it is possible that the micrometastases must show a particular growth pattern or cell recruitment profile before TNFR1 expression is sufficient to allow permeabilization. Nevertheless, even metastases of approximately 50 μm diameter showed sensitivity to cytokine-induced permeabilization, which is considerably smaller than those currently detectable clinically owing to natural BBB permeability (0.5–1.0cm diameter)  ,35.HER2-overexpressing breast cancer, leading to reduced breast tumor burden and increased patient survival . It is also worth noting the very short half-life of TNF within the circulation  and Strategic Skills Award G1000402).This work was funded by


Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells.We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism.Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH). Human embryonic stem cells (hESCs) are self-renewing and pluripotent cells derived from the inner cell mass (ICM) of a blastocyst prior to implantation. However, although they can be differentiated into any somatic cell lineage, they cannot be genetically matched to putative patients in possible cell replacement therapies. However, it has been recently shown that human somatic cells can be reprogrammed into hESC-like pluripotent stem cells, the so called induced pluripotent stem cells (IPSCs), by the ectopic expression of a combination of pluripotency factors and using a variety of approaches 2 consumption, whereas mitochondria of the TE are elongated and have both higher mitochondrial potential and higher O2 consumption The mammalian embryo resides in a hypoxic environment prior to implantation 2 tensions have been reported to increase the reprogramming efficiency of both mouse and human somatic cells Similarly to ICM cells, embryonic stem cells rely mostly on glycolysis for energy supply. Furthermore, the mitochondria in these cells are rather immature with perinuclear localization. As human pluripotent stem cells differentiate they acquire more mature mitochondria and undergo a metabolic switch from glycolysis to OXPHOS 2 tensions or in the presence of dysfunctional mitochondria pyruvate will be converted to lactate.It is known that glycolysis and OXPHOS function in a coordinated fashion, and that the former generates 18 times more ATP per molecule of oxidized glucose than the former The pyruvate dehydrogenase (PDH) complex, localized in the mitochondrial matrix is the link between glycolysis and the TCA cycle. It is composed of several copies of three catalytic proteins  that catalyze the irreversible decarboxylation of pyruvate to acetyl-coenzyme A and NADH The exact mechanism by which hESCs maintain an anaerobic metabolism even in the presence of oxygen remains largely elusive. However, there is certain putative parallelism with what has been described for cancer cells, which also maintain glycolysis as a key metabolic pathway under normoxia, in detriment to OXPHOS, the so-called Warburg effect Additionally, it would be of interest to determine if and how IPSCs acquire a more glycolytic metabolism upon reprogramming, or if they instead maintain the metabolic features of the original somatic cell type that was reprogrammed. In this study our aim was to answer these questions by further characterizing the energy metabolism of both hESCs and human IPSCs when compared with their differentiated somatic counterparts. Towards that goal we analyzed mitochondrial morphology, glucose-related gene expression, OCR, intracellular ATP levels, lactate production and protein levels of regulatory enzymes relevant in metabolism.In accordance to previous reports our results demonstrate that hESCs have mitochondria consistent with lower activity, at least when compared to differentiated cells. Furthermore we show that mitochondria in IPSCs are morphologically distinct from both hESC and differentiated somatic cell mitochondria. In addition IPSCs are not identical to hESCs in terms of glucose-related gene expression, although they cluster with hESCs rather than with their somatic counterparts. Moreover analysis of OCR, intracellular ATP and lactate levels confirm that human pluripotent stem cells rely mostly on glycolysis to meet their energy demands. Finally our study demonstrates that human pluripotent stem cells express high levels of hexokinase II and have an inactive PDH complex. In all parameters quantified IPSCs seem to be close to hESC, but at slightly lower levels of expression/activity. These results shed light on some of the mechanisms that human pluripotent cells use to maintain high levels of glycolysis under normoxia.In this work we used hESC lines WA07, WA09 and WA01 , human IPSC lines HFF1 iPS, IMR-90 iPS (WiCell Research Institute) and AE iPS (derived in our laboratory). In terms of differentiated fibroblasts, both the IMR-90 human diploid fibroblast and the human foreskin fibroblast (HFF1) strains were obtained from American Type Culture Collection ; these lines were reprogrammed to form the IMR-90 iPS and HFF1 iPS lines noted above, respectively. Finally, differentiated H7TF fibroblasts were isolated from a WA07 ESC-derived teratoma, following injection of WA07 hESCs into immunocompromised mice.2 and 5% CO2. Throughout the work WA07, IMR-90 iPS, H7TF and IMR-90 lines were used in the majority of the experiments in order to compare cell lines that have the same genetic background. The other lines were used in key experiments to consolidate the results trying to avoid any artifact that could be line specific or culture derived.HESC and IPSC lines were cultured in mTeSR™  on matrigel  coated dishes. Both H7TF and HFF1 were cultured in CF1 medium containing: 90% Dulbecco's Modified Eagle's medium; 10% fetal bovine serum (FBS); 1% MEM non-essential amino acids; 1% penicillin/streptomycin (Pen/Strep) and 1% 2 mM L-glutamine . The IMR-90 line was cultured in 90% Eagle's minimal essential medium , 10% FBS and 1% Pen/Strep. All lines were maintained at 37°C, 21% OTCS-SP@ laser scanning confocal microscope .Mitochondria GFP labeling was performed using the organelle lights Mito reagent (CellLight™ Mitochondria-GFP by Molecular Probes/Invitrogen). This system is a baculovirus system that contains a leader sequence for the E1 alpha pyruvate dehydrogenase fused with GFP protein. Cells were plate on coverslips and cultured for three days or 24 hours, for pluripotent and differentiated cells, respectively. Cells were rinsed with PBS, and incubated in PBS containing 82% organelle lights reagent during 30 minutes at room temperature with gentle shaking. Organelle lights mixture was removed and cells were incubated with appropriated media containing 0.1% enhancer solution (provided) for two hours at 37°C. Enhancer solution was removed and replaced by cell culture media. Although the transfection efficiencies were low, 20 hours later, GFP protein could be observed in the mitochondria. Cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) and imaged using a Cells were cultured on matrigel or matrix free conditions for pluripotent cells or differentiated cells, respectively. Cells were rinsed with PBS and fixed with 2.5 % glutaraldehyde in PBS, pH 7.4, for 1 hour at room temperature. The fixative was removed and samples were washed 3 times with PBS at room temperature for 10 minutes each. Samples were post-fixed for 1 hour at 4°C in 1% osmium tetroxide with 1% potassium ferricyanide, followed by three washes with PBS (10 minutes each). Afterwards, samples were dehydrated using a graded ethanol series  with three changes in 100% ethanol (15 minutes each). Samples were then incubated three times with a 100% Epon solution (1 hour each). Finally, samples were incubated for 1 hour with Epon solution, maintained overnight at 37°C and afterwards at 60°C for 2 days. Sections were made using an ultra microtome and imaged in a 1011 CX Transmission electron microscope .ALDO B, FBP1, FBP2, G6PC, GCK, GYS2, HK3, PCK1, PDK4, PGK2, PKLR and PRPS1L1) were not considered in the analysis, as their Ct value was above 30. Gene expression differences were calculated using the -ΔΔCt method. Gene expression was normalized to the housekeeping gene β-actin and fold change was calculated relative to the hESC line WA07. p values were determined using online software provided by SABiosciences.Total RNA isolation using the TRIzol reagent (Invitrogen) and genomic DNA was eliminated with DNA-free kit  as previously described Pluripotent cells were cultured as mentioned above for seven days, whereas differentiated cells were cultured for two days. Cells were enzymatically dissociated with Accutase (Millipore) or TripLe Express  for pluripotent cells and differentiated cells, respectively. Adenosine triphosphate (ATP) was extracted with 0.6 M perchloric acid (Sigma) supplemented with 25 mM EDTA-Na (Sigma) followed by centrifugation. Supernatants were neutralized with 3M potassium hydroxide in 1.5M Tris. ATP separated by HPLC. The detection wavelength was 260 nm and the column was a Licrosphere 100 RP108 5 µm (Merck). The elution buffer for was 100 mM phosphate buffer (pH 6.5) supplemented with 1% methanol. Standard for ATP was purchased from Sigma- Aldrich.Lactate levels were determined using the Lactate assay kit (Biovision). In this assay lactate is enzymatically oxidized and the product of this reaction reacts with a lactate probe (provided) and emits fluorescence at Ex/Em = 535/587 nm. Both pluripotent and differentiated cells were plated and cultured for 48 hours. At this point, fresh media was added to the cells. Twenty-four hours later, 500 µl of medium was collected, and cell numbers were determined. Medium samples were processed following manufacturer's instructions. Fluorescence was determined using a microplate reader . Lactate concentrations were normalized to cell number.293 ; anti-hexokinase II , and anti-Nanog , ECL Advanced Western Blot Detection kit  was used for detection.Cells were maintained on matrigel or matrix free conditions for pluripotent cells or fibroblasts, respectively. Pluripotent cells were dissociated using Accutase (Millipore), whereas for differentiated cells Triple E (Sigma-Aldrich) was used. Total cell extracts were lysed in RIPA buffer (Sigma) supplemented with 1 mM phenylmethylsulphonyl fluoride (Sigma-Aldrich) PMSF and 2x Halt phosphatase inhibitor cocktail . Mitochondrial extracts were isolated using mitochondria isolation kit (Sigma-Aldrich), as previously described by Wieckowski and colleagues Cells were cultured on coverslips for six days or two days for pluripotent and differentiated cells respectively. Cells were fixed in 2% formaldehyde, rinsed in PBS, permeabilized with PBS 0.1 % Triton and incubated with blocking solution  for an hour at room temperature. The primary antibodies used were anti-hexokinase II . Antibodies were incubated overnight at 4°C, followed by three washes of 15 minutes in PBS 0.15 Triton. Respective secondary antibodies were incubated for one hour at 37°C, followed by three washes in PBS 0.1% Triton. Coverslips were assembled in slides using the fluorescence mounting media Vectashield with DAPI . Slides were imaged using a TCS-SP2 laser scanning confocal microscope (Leica).2 consumption was determined using Seahorse XF24 extracellular Flux analyzer as previously described by Qian and Van Houten OMeans and standard error of the mean (SEM) were calculated and statistically significant differences were determined by One-way ANOVA followed by Dunnett's Multiple Comparison test. n refers to sample size. Statistical significance was determined at p<0.05. The p values for the Glucose array were calculated using SABiosciences online software. The test used was a two-tailed Student's t-test, and statistical significance was determine at p<0.05.versus differentiated cells we transduced hESCs (WA07 line) and fibroblasts cells  with a baculovirus system containing a leader sequence for PDH E1 alpha subunit fused with the GFP protein. Mitochondria in the hESC line WA07 showed perinuclear localization  for hESCs (WA07 line), IPSCs (IMR-90 and HFF1 IPSC lines) and fibroblasts cells  . TEM ana2 profiler PCR array. This array contains 84 key genes involved in the regulation of glucose and glycogen metabolism, along with five housekeeping genes. Overall we observed that pluripotent lines tend to cluster together rather than with their somatic counterparts (To further investigate the metabolic pathways used by human pluripotent stem cells and differentiated somatic cells we used two hESC lines (WA07 and WA01), two IPSC lines (IMR-90 IPS and AE IPS), and two somatic lines  and performed the SABiosciences human glucose metabolism RTterparts , heatmapterparts . Statistterparts . For difterparts . We furtterparts , Table 1ENO 1 and ENO 3 were downregulated  in AE IPSC relatively to the WA07 line, whereas the expression of these did not significantly vary for the IMR-90 IPSC line. Instead IMR-90 IPSC displayed up regulation of BPGM , and HK2  when compared to the WA07 line  in comparison with the WA07  and up regulation of ALDO A .Regarding the glycolytic signature, six genes among all lines were either up regulated or down regulated when compared with the WA07 line . Among tA07 line , Table 1the WA07 , Table 1H6PD , PRPS1 , PRPS2 , TKT  in comparison with the WA07 line. Both the H7TF and IMR-90 lines displayed increased expression of G6PD  H6PD , and decreased expression of TKT  in comparison to the WA07 line. In addition the IMR-90 line showed decreased expression of PRPS1 , RPE  and RPIA .In terms of the pentose phosphate pathway seven genes were differentially expressed among all the lines when compared with the WA07 line. No differences were observed between the WA07 line and both the WA01 and AE IPSC lines. The IMR-90 IPSC line showed up regulation of DLD  , and SUCLG2  when compared with the WA07 line  and IDH1  for H7TF and IMR-90, respectively) when compared with the WA07 line , DLD , DLST , IDH3B , MDH1 , MDH1B , PDHA1 , PDHB , SDHC  and SUCLA2 , and up regulation of ACO 1 .Regarding the TCA cycle signature we observed that 17 genes were differentially expressed when compared to the WA07 line , Table 1 p<0.05) . The IMRA07 line . Both thA07 line , Table 1PDP2 . In addition the IMR-90 displayed decreased expression of PDK3 .With respect to genes involved in the regulation of TCA cycle activity two genes were differentially expressed among all lines and the WA07 line . No signTo further understand mitochondrial contribution to the energy metabolism of pluripotent cells we analyzed protein expression of mitochondrial complexes II, III and V in pluripotent vs. differentiated somatic cells. We observed no statistical significant differences in mitochondrial complexes II, III and V between the various pluripotent lines. Intriguingly, we observed that pluripotent cells have higher protein levels of mitochondrial complexes, II, III and V than differentiated cells . Indeed,2 consumption rate (OCR) using the Seahorse XF24 extracellular flux analyzer. We have used a pharmacological profiling approach, by combining the use of three mitochondrial inhibitors  and the Seahorse instrument as previously described by Qian and Van Houten In order to determine if the differences observed in mitochondrial complex expression could be translated in terms of higher mitochondrial activity in pluripotent cells we determine O0 subunit of the ATP synthase. This binding blocks the proton conductance resulting in loss of electron transfer and O2 consumption. Addition of oligomycin resulted in decreased levels of OCR in all cell types  is the amount oxygen consumption that is linked to ATP synthesis in the mitochondria, and represents the mean basal levels of oxygen consumption minus the mean of the two values following oligomycin treatment (measurements 3 and 4). Oligomycin inhibits ATP synthase by binding to the oligomycin sensitivity-conferring protein (OSCP) at the Fll types . Nonethell types .2 consumption rates when compared to the other cell types . In contrast both hESCs and IPSCs showed a considerably reduced response to FCCP than differentiated cells (H7TF and NDHF). These results suggest that mitochondria in pluripotent cells, although functional, present a low activity rate, and that the differences in mitochondrial content noted previously do not directly account for differences in activity. Indeed we observed that mitochondria membrane potential is lower in hESCs when compared to their differentiated counterparts (data not shown).Rotenone is a mitochondrial inhibitor that prevents electron transfer from complex I to ubiquinone by blocking the ubiquinone binding site Total ATP levels are originated both from anaerobic and aerobic respiration. In order to determine intracellular ATP levels in human pluripotent stem cells and differentiated cells we performed HPLC . Our resMammalian tissues harbor four hexokinases isoforms (I–IV) with particular kinetics and tissue specificity. Both hexokinase I and II have an N-terminal hydrophobic domain that allows binding to the outer mitochondrial membrane. Pedersen and colleagues have shown that mitochondrial-bound hexokinase II is responsible for the high glycolytic profile of rat hepatoma cells PDK1 gene expression in pluripotent vs. differentiated cells , and somatic cells. In agreement with previous reports our results demonstrate that mitochondria in hESCs appear immature with fewer cristae, and low electron density matrix, when compared to differentiated somatic cells Glucose-related gene expression analysis revealed that both hESC lines analyzed in this study have identical glucose-related metabolism signatures. In contrast IPSCs are not identical to hESCs, although they cluster together, rather than with their somatic counterparts. Interestingly both IPSC lines analyzed in this study showed different metabolic gene expression signatures, with IMR-90 IPSCs having higher transcripts levels. It should be noted that that the cocktail of transcription factors used for the reprogramming of both lines differ Regarding the glycolytic signature of human pluripotent stem cells and somatic cells we observed that H7TF fibroblasts displayed similar glycolytic signature to their pluripotent counterpart the WA07 hESC line, with only four genes significantly downregulated. Interestingly the IMR-90 fibroblasts displayed 10 genes differentially expressed (six genes significantly downregulated and four genes up regulated when compared to the WA07).de novo nucleotide synthesis. The pentose phosphate pathway can be divided in two major branches, an oxidative branch and a non-oxidative branch G6PD and H6PD are significantly up regulated in differentiated cells. The enzymes encoded by these genes catalyze the first step in the oxidative branch of the pentose phosphate pathway that involves the conversion of glucose 6-phosphate to 6-phosphogluconate with concomitant production of NADPH. Besides its role as a cofactor in many biosynthetic reactions, NADPH is essential to regenerate reduced glutathione from glutathione disulfide G6PD and H6PD transcripts levels may reflect high requirement for NADPH, either as reducing agent for biosynthetic pathways or instead as a cofactor in the cell antioxidant defense. The latter hypothesis is supported by the fact that differentiated cells have a higher mitochondrial activity and therefore a higher potential for ROS production. Furthermore, we observed that human pluripotent cells have higher expression levels of genes related to the non-oxidative branch, such as TKT, and RPIA. These results suggest that human pluripotent cells preferentially use the non-oxidative branch of the pentose phosphate pathway in order to obtain ribose-5 phosphate which is important for nucleotide synthesis. A similar pattern has been previously described for cancer cells Cell division requires not only ATP but biosynthetic precursors derived from glycolysis, the pentose phosphate pathway and the TCA cycle. For instance, glucose 6-phosphate can be diverted from the glycolytic pathway to the pentose phosphate pathway in order to generate ribose 5-phophate for 2 than the differentiated line H7TF. However, IMR-90 fibroblasts displayed similar basal OCR levels than those found in pluripotent lines. These results could be related to the fetal origin of the IMR-90 fibroblasts, as the fetal lung is exposed to a rather hypoxic environment Interestingly, the TCA cycle signature revealed that most of these genes are downregulated in differentiated cells, when compared to pluripotent lines. Besides its role in oxidative catabolism of carbohydrates and fatty acids, the TCA cycle provides precursors for many biosynthetic pathways, including precursors for amino acid and nucleotide synthesis. It is possible that up regulation of TCA cycle-related genes in human pluripotent stem cells is related to the high proliferative rates and concomitant need for nucleotides rather than need for reducing agents for mitochondrial activity. This issue needs to be explored further. Regardless, higher levels of genes encoding TCA cycle proteins might be indicative of increased electron transport capacity and cellular respiration. Consistent with increased level of genes encoding proteins of the TCA cycle we observed that pluripotent lines have higher levels of mitochondrial electron transport complexes than differentiated cells. The transcription factor c-Myc is crucial for the maintenance of embryonic stem cell self-renewal and its ectopic expression allows the reprogramming of somatic cells to an embryonic stem cell like state Hexokinase II catalyzes the first reaction of glycolysis and it has been previously demonstrated that its expression is restricted to the inner cell mass of the blastocyst, and that HK2 knock down results in mouse embryonic lethality around E.D. 7.5 On the other hand, the PDH complex is a crucial step in regulation of metabolism since it constitutes the link between anaerobic metabolism and the TCA cycle. Phosphorylation of the PDH E1α subunit leads to inactivation of the PDH complex and consequently results in lower levels of acetyl CoA to enter the TCA cycle. Tellingly, pluripotent lines have higher levels of phosphorylated PDH E1α. Phosphorylation of PDH complex can be carried out by four PDHKs and in this study we analyzed PDHK1 expression levels. Although we did not observe an increase in PDHK1 gene expression in pluripotent lines when compared to differentiated cells we did observe an increase in PDHK1 protein levels in pluripotent cells. These results suggest that PDHK1 protein stability differs between pluripotent and differentiated cells. Papandreou and colleagues have previously demonstrated that under hypoxic conditions hypoxia inducible factor (HIF-1) up regulation resulted in increased expression of PDHK1 leading to inactivation of the PDH complex. This in turn resulted in reduced substrate availability to enter the TCA cycle and led to decreased oxidative phosphorylation 2 consumption. This suggests that IPSC somatic cell reprogramming to IPSC may result in differences at the metabolic level, when compared to the pluripotent standard of hESC. While epigenetic and transcriptomic differences have been mentioned above Overall our results demonstrate that human pluripotent cells have a greater reliance on glycolysis than differentiated cells. In addition our study suggests that this can be mediated by increasing hexokinase II levels and inactivation of the PDH complex. Interestingly these metabolic strategies involving features of anaerobic metabolism under normoxia are also found in many types of tumor cells In addition we also observe that not all the differentiated lines display the same metabolic profile and this might have an impact in the reprogramming efficiency of various somatic cell types, and on the characteristics of differentiated cells obtained from different IPSC lines. These results stress the importance of the complete characterization of these cell lines, and suggest that there may be other links to tumor cells besides those already described.Figure S1Comparison of OCR in two differentiated cell lines. A) Oxygen consumption rate (OCR) was determined by Seahorse XF24 analyzer for IMR-90 and NHDF lines, the former also listed in B) Basal OCR . E) Reserve capacity . Measurements show that NHDF cells show higher values than IMR-90 cells, similarly to what is described for the other differentiated cell line (H7TF) listed in (TIF)Click here for additional data file.Figure S2p-values for the glucose metabolism gene expression array in human pluripotent stem cells vs. differentiated cells. Statistical analysis was performed using SABiosciences online software and Student's t test was applied. Significance was determined at p<0.05.(PDF)Click here for additional data file.Table S1Average Ct values for gene expression. Differences were calculated using the -ΔΔCt method and gene expression was normalized to the housekeeping gene β-actin. Average Ct values were determined using online software provided by SABiosciences.(PDF)Click here for additional data file.