We previously described – studying transcriptional signatures of hippocampal CA3 explants – that febrile (FS) and afebrile (NFS) forms of refractory mesial temporal lobe epilepsy constitute two distinct genomic phenotypes. That network analysis was based on a limited number (hundreds) of differentially expressed genes (DE networks) among a large set of valid transcripts (close to two tens of thousands). Here we developed a methodology for complex network visualization (3D) and analysis that allows the categorization of network nodes according to distinct hierarchical levels of gene-gene connections (node degree) and of interconnection between node neighbors (concentric node degree). Hubs are highly connected nodes, VIPs have low node degree but connect only with hubs, and high-hubs have VIP status and high overall number of connections. Studying the whole set of CA3 valid transcripts we: i) obtained complete transcriptional networks (CO) for FS and NFS phenotypic groups; ii) examined how CO and DE networks are related; iii) characterized genomic and molecular mechanisms underlying FS and NFS phenotypes, identifying potential novel targets for therapeutic interventions. We found that: i) DE hubs and VIPs are evenly distributed inside the CO networks; ii) most DE hubs and VIPs are related to synaptic transmission and neuronal excitability whereas most CO hubs, VIPs and high hubs are related to neuronal differentiation, homeostasis and neuroprotection, indicating compensatory mechanisms. Complex network visualization and analysis is a useful tool for systems biology approaches to multifactorial diseases. Network centrality observed for hubs, VIPs and high hubs of CO networks, is consistent with the network disease model, where a group of nodes whose perturbation leads to a disease phenotype occupies a central position in the network. Conceivably, the chance for exerting therapeutic effects through the modulation of particular genes will be higher if these genes are highly interconnected in transcriptional networks. Epilepsy prevalence ranges from 0.5–1.0% in developed countries to 1.4–5.7% in developing countries The search for new drug-targets in epilepsy has been impacted by recent advances in functional genomics and systems biology We previously described – studying transcriptional signatures of hippocampal CA3 explants obtained at epilepsy surgery – that febrile (FS) and afebrile (NFS) forms of refractory mesial temporal lobe epilepsy (RMTLE) have different pathomechanisms and constitute two distinct genomic phenotypes as shown by CA3 transcriptional co-expression networks In transcriptional interaction networks, highly connected genes, or hubs, hold the whole network together and are either associated to specific cellular processes or link different biological processes, organizing the interactome Here we developed a methodology for complex network visualization and analysis that allows the categorization of network nodes according to distinct hierarchical levels of gene-gene connections, or node degree, and of interconnection between node neighbors, or concentric node degree (see Methods). We were thus able to study the whole set of CA3 valid transcripts in order to: i) obtain complete transcriptional networks, here called CO networks, for FS and NFS phenotypic groups; ii) examine how CO and DE networks are related; iii) identify genomic and molecular mechanisms underlying FS and NFS phenotypes, contributing for the discovery of potential targets for therapeutic interventions.www.fapesp.br; www.cinapce.org.br). This research has been approved by Hospital das Clínicas da FMUSP and Hospital Albert Einstein, São Paulo, SP, Brazil institutional review boards under numbers 251/05 and CAEE 0122.0.028.174.05. A written informed consent was obtained from all patients. Refractory epilepsy cases were defined as those who have not gained seizure control after treatment with two or more antiepileptic drugs. All patients underwent preoperative clinical, electrophysiological, neuropsychological and neuroimaging evaluations. In this study we compared global gene expression profiles of CA3 explants obtained at surgery room from six FS and twelve NFS patients undergoing corticoamigdalohippocampectomy. None of these patients had mental retardation or first-degree family members with epilepsy or febrile seizure history. Detailed descriptions of the patient's demographic and clinical data and of the procedures for brain tissue collection and preservation for neuropathological (MRI and histopathology) and genomic studies (DNA microarrays) were published elsewhere The RMTLE patients included in this study were selected through the CInAPCe-FAPESP Program . MRI and histological studies were performed in all removed hippocampi for neuropathology analysis and for confirming that the explants for genomic studies were obtained at the proper site Brain tissue explants from CA3 (3–4 mm3) were homogenized with TissueRupter . Total RNA was extracted from the homogenates with RNeasy Lipid Tissue Kit  according to manufacturer's instructions. RNA quality was assessed on the Agilent BioAnalyzer 2100 . All samples were stored at −80°C until used in hybridization experiments.http://www.ncbi.nlm.nih.gov/geo) a MIAME compliant data base, under accession number GSE28674. Differential gene expression data were validated through quantitative real-time polymerase chain reaction In order to determine gene expression profiles, 44 K DNA microarrrays  were used. The procedures for hybridization followed the protocols provided by the manufactureŕs instructions . The images were captured by the reader Agilent Bundle according to the parameters recommended for bioarrays and extracted by Agilent Feature Extraction software version 9.5.3, considering spots with none or only one flag . The selected transcripts were used for analysis using the R software version 2.11.1  and the Lowess test for arrays normalization. We identified 15,615 valid GO annotated genes for the CA3 samples (6 FS and 12 NFS patients). By means of the TMEV software version 4.6.1 http://cyvision.if.sc.usp.br/~bant/hierarchical/), which provides both dynamic and interactive data analysis of complex networks.Transcriptional interaction networks for CA3 differentially expressed GO annotated genes (DE) and CA3 transcriptional interaction complex networks for all valid GO annotated genes (CO) were constructed for FS and NFS groups based on Pearsońs correlation. Pearson correlation identifies sets of genes which co-varies (positively or negatively), thus allowing us to construct networks by considering nodes as genes, with edges inferred if a pair presents high absolute value of correlation. Specifically, we define a correlation threshold that determines if edges are present or absent in the resulting network. This is done in a way that all nodes are connected to the major component and the network is stable in the sense that slight changes in the threshold value do not significantly affect its topological structure. It is an usual approach for constructing transcriptional interaction networks, also applied in other widely used tools ,Traditionally, nodes with particularly high number of connections – the so-called called hubs – are known to play very important roles in most of real networks One way to classify network nodes as VIPs, hubs or high-hubs is by obtaining the node degree, Because most real networks present scale-free distributions, there is no clear definition for setting a degree threshold for which we can classify nodes as being hubs or not Betweenness centrality Insights about the overall structure of complex networks can be obtained by projecting them into 2D or 3D spaces. This allows us not only to visualize but also to get a better understanding of which measurements and techniques may provide relevant information about these structures. In addition, inherent or calculated properties of nodes or edges can be interactively explored, providing more information than those obtained from statistical measurements such as moments and densities.Complex networks can be visualized through projections of nodes represented as circles/balls and edges as lines/tubes connecting the nodes. Force-directed methods can be applied for visualization and are suitable for all kinds of networks, yielding visually pleasing drawings We developed a software to obtain visualizations of large complex networks based on the Fruchterman-Reingold algorithm (FR) By minimizing the energy of the system, one should obtain a set of positions for each vertex in a way that the preferred Euclidian distance between each connected pair is calculated by using Here we chose to use molecular dynamics simulation (MDS) hreshold .Our algorithm starts by assigning random positions – uniformly distributed over the desired metric space – for each node. Then the MDS equation:stem see , while  http://bioinfo.lbhc.hcancer.org.br/cgi-bin/interactomegraph/index.cgi). Data analysis and visualization were accomplished through Cytoscape v 2.8.3.Interactome analyses were performed for selected hubs, VIPs and high hubs in the DE and CO transcriptional networks obtained for FS and NFS groups. These analyses were carried out by using an in house free web tool developed by LA Lima & RD Puga – Centro Internacional de Ensino e Pesquisa – Hospital A.C. Camargo and the proteins annotated in MINT, HPRD and IntAct databases  were constructed as described in the previous section. The frequency distribution of the nodes and their links  and 7A aes of FS  and NFS es of FS  DE netwoBSN, BRUNOL4, BAIAP2, and ADAM23) ABCG4 and ASPHD2) CACNG2, GNG3 and KCNH3MEG3, a maternally imprinted long noncoding RNA gene involved in neuronal differentiation The six selected hubs in the DE network for the FS group (FS-DE network) are genes related to excitatory synaptic function  – are respectively involved in brain-derived neurotrophic factor (BDNF)-induced hippocampal neurite outgrowth CIAPIM1, which is linked to apoptosis inhibition JAK3 that is involved in the differentiation of neural precursor cells DST, acting on neuronal cytoskeleton organization maintenance LIMD2, ALDH2 and PHF20L1) OR4C46, is a G-protein activated olfactory receptor TM6SF2 and C17orf51, code for hitherto uncharacterized proteins.The selected genes of the complex network for the FS group (FS-CO) encompass only high-hubs and VIPs . Three hNAV2, RGS14, SIAE, PCLO, TREML2 and ODZ3  that take place in different hippocampal structures. Complete network analyses bring a potential contribution to understanding neurodevelopmental alterations in epileptogenesis. This issue will be detailed more extensively in the Discussion section.We performed an interactome analysis in order to validate the selected hubs, VIPs and high-hubs in DE and CO networks. CACNG2, a VIP in the FS-DE network) and quantitative analysis revealed higher expression of this protein in FS cases when compared to NFS group. Stargazin immunoreactivity was observed in the soma of pyramidal cells of cornu ammonis and subiculum, granule cells and hylar neurons of the dentate gyrus (p = 0.0004), CA3 (p = 0.0007) and granule cell layer (p = 0.0012), when compared to NFS group. A detailed analysis of hippocampal neuropathology and immunocytochemistry findings in this series of patients will be published elsewhere.GCL severity was higher (p<0.05) in FS group . Differete gyrus . NeuronaIn this paper we developed a methodology for: i) transcriptional interaction network analysis of differentially expressed non-categorized GO annotated genes; ii) global transcriptional interaction analysis – encompassing all valid GO annotated genes – based on complex network 3D visualization; iii) concentric characterization measuring and node hierarchical categorization. This methodological approach allowed us to: i) construct transcriptional networks for differentially expressed genes without previous gene classification in thematic groups  depends substantially on their gene-gene connectivity and much less on structural gene alterations When a gene-gene interaction network in a disease target tissue suffers a perturbation caused by environmental factors this perturbation triggers a succession of events. In febrile RTMLE, for instance, the release of inflammatory mediators probably drives coordinate changes in the functioning of hundreds of genes in the brain Joint visualization and analysis of CO and DE networks revealed that DE hubs and VIPs are evenly distributed inside the CO networks , as shown in ADAM23 and ABCG4 are common hubs and MEG3 is a common VIP in DE networks). The remaining hubs and VIPs with known biological function in DE networks , which is a hub in the CO-NFS network, exert a redundant role in maintaining synaptic vesicle clustering BSN and NTKR2, a gene that codes for TrkB, the BDNF receptor, and that is also involved in mesial temporal lobe epileptogenesis NTKR2 is a high-hub in the CO-FS network, what points out for its relevance in hippocampal CA3 molecular pathways. On the other hand, valproic acid, a commonly used antiepileptic drug, rebalances TrkB and BDNF brain levels in Bassoon mutant mice and reduces epileptic seizures BSN, BDNF and NTRK2 interconnection in epilepsy, corroborated in this study.The first FS-DE hub to considered here, ADAM23 and BRUNOL4, act on neuronal excitability. ADAM23 controls dendritic arborization in CA1 pyramidal neurons and is a LG1L receptor BRUNOL4  codes for a RNA-binding protein and is primarily expressed in excitatory neurons – including large pyramidal cells in the cerebral cortex and hippocampus – and regulates excitatory but not inhibitory neurotransmission. This gene regulates translation and local abundance of several synaptic function-associated genes. BRUNOL4 deficiency causes complex seizure disorders in mutant mice and humans ADAM23 is an interactor of LGI1, an important antiepileptogenic protein BRUNOL4 codes for a RNA-binding protein highly expressed in the hippocampus that regulates several genes related to synaptic functions BRUNOL4, since alterations in this gene are involved not only in epilepsy but also in other neurological conditions, such as autism Two other hubs, ABCG4, a hub, and MEG-3, a VIP, both associated to brain homeostasis and stress responses. ABCG4 controls neuronal but not astrocytic cholesterol efflux status epilepticus occurs in temporal lobe epilepsy ABCG4 in brain disorders MEG3 , is a maternally imprinted long noncoding RNA gene MEG3 orthologous gene in mice, Gtl2, is strongly expressed in the pyramidal cell layer of the hippocampus and this expression is consistent with a role in neuronal development and differentiation There are also two relevant nodes common to FS-DE and NFS-DE networks: BAIAP2 and ASPHD2. The first is involved in excitatory synaptic transmission and in neurite growth and spine morphogenesis The remaining FS-DE main hubs are CACNG2, KCNH3 and GNG3, with relevant roles in epilepsy. CACNG2, which codes for the protein stargazin, is essential for stabilizing diffuse AMPA receptors in the post synaptic density, an important feature of glutamatergic synaptic transmission KCNH3, which codes for a potassium channel Kv7, is a potent regulator of hippocampal and cortical excitability GNG3 is widely expressed in the brain and required for GABABR neuronal excitability regulation. The role of GNG3 protein, and of other G-protein subtypes, in neuronal excitability regulation is currently being investigated in animal models: GNG3 genetic deficiency causes increased susceptibility to seizures in mice The FS-DE network has three distinctive VIPs, A2BP1 codes for a RNA-binding protein, Rbfox1, that regulates the alternative splicing of neuronal transcripts involved in synaptic transmission and membrane excitation, some of which involved in epilepsy BRUNOL4 in the FS-DE network, its mutation causing epilepsy AMPH1 codes for amphiphysin I, which is an important regulator of synaptic vesicle endocytosis + flow into presynaptic terminals, a phenomenon observed in epilepsy AADACL1 regulates de novo formation of platelet-activation factor (PAF), an inflammation mediator. PAF accumulates in the brain after seizures and causes hippocampal hyperexcitability AADACL1 attenuates hippocampal excitability by reducing de novo PAF formation This network harbors hubs and VIPs with prominent roles in neuronal excitation, synaptic transmission and neuronal differentiation. Two of these hubs, ADAM23 and ABCG4, are also present in the FS-DE network and were commented above. Three out the other four hubs are directly involved in neuronal excitability . It was recently discovered that some LRR-containing proteins, as Elfn1, are involved in controlling hippocampal synaptic functions and, moreover, in similar ways as perfomed by LRR containing-Trk neurotrophin receptors and the antiepileptic protein LG1L C6orf154 is an interesting candidate for further investigations on target-specific synaptic transmission Three of the six NFS-DE VIPs are linked to neural excitability and circuitry control. One of them, GPR68) is associated to astroglial cell functions. MYT1L, it is an important gene for neurogenesis and neural cell maturation NEUROD6 is a gene involved in neuronal stress tolerance in CA1-CA3 pyramidal neurons NEUROD6, a gene responsible for granule cell differentiation in the hippocampus NEUROD6 was found in both groups, being significantly higher in FS cases, as determined by quantitative real-time polymerase chain reaction Among the other three VIPs, two are genes acting on neuronal differentiation and one , regulates histone demethylation BDNF gene expression after seizures CIAPIM1, codes for anamorsin, a cytokine-induced apoptosis inhibitor JAK3 codes for a non-receptor tyrosin kinase and is involved in proliferation and differentiation of neural precursor cells DST, codes for dystonin, a cytoskeletal linker protein Two among the other five high-hubs, LIMD2, PHF20L) and protection of hippocampal neurons against oxidative stress (ALDH2), what may be related to compensatory mechanisms, as discussed above. The other VIP, OR4C46, codes for an olfactory receptor (OR) and is ectopically expressed in CA3. Although functional interpretation for ectopic OR expression based only on transcription information must be taken with precaution 2+ levels Four out of the six FS-CO VIPs  have knoNAV2 is a key player in the processes of neurite outgrowth and axonal elongation ODZ3 codes for a teneurin, which is a neuronal connectivity regulator SIAE codes for a sialic acid acetyl estearase. Sialic acid modifications are involved in hippocampal CA3 neural regulation and network excitability PCLO (Piccolo) acts on the maintenance of synaptic vesicle clustering PCLO acts redundantly with BSN, which is a hub in FS-DE network, on synaptic vesicle clustering and integrity maintenance Some hubs of this network present rather high betweenness centrality values , what caMUS81 codes for a DNA recombination repair endonuclease highly accumulated in nucleoli FARSA codes for an aminoacyl-tRNA synthetase related to axonal development and SAMC1L is highly expressed in hippocampus. This gene regulates Golgi membrane morphology Finally, the three NFS-CO VIPs may be also involved in compensatory mechanisms: Taken together, the results for DE networks reflect a majority of processes linked to synaptic transmission and neuronal excitability, whereas the profile of CO networks show predominance of compensatory mechanisms for the neuronal cell loss/damage associated with epilepsy-induced hippocampal sclerosis Interactome analyses  A–D valiIn conclusion, complex network analysis conducted in this study yielded a broad and more detailed view of genomic and molecular mechanisms involved in RMTLE in comparison to analyses centered on differentially expressed genes. Specifically, hubs and VIPs in DE networks are mostly related to neuronal excitability and, in a broad sense, play pro-epileptic roles. Conversely, hubs, VIPs and high-hubs in CO networks are more frequently related to neuronal differentiation, neuroprotection and synaptic function, in a scenario compatible with compensatory mechanisms that may play a reparatory role, but may also be linked to epilepsy pathogenesis Video S1Complete transcriptional interaction network for FS based on Pearson's correlation of 15,585 GO annotated genes. High-hubs and VIPs are identified by their gene symbols. FS-DE network hubs and VIPs are also shown.(DOC)Click here for additional data file.Video S2Complete transcriptional interaction network for NFS based on Pearson's correlation of 11,233 GO annotated genes. Hubs and VIPs are identified by their gene symbols. NFS-DE network hubs and VIPs are also shown.(DOC)Click here for additional data file.Table S1Functional description of interactome nodes corresponding to hubs and VIPs in FS transcriptional interaction network for differentially expressed genes (DE).(DOC)Click here for additional data file.Table S2Functional description of interactome nodes corresponding to hubs and VIPs in NFS transcriptional interaction network for differentially expressed genes (DE).(DOC)Click here for additional data file.Table S3Functional description of interactome nodes corresponding to high-hubs and VIPs in FS complete transcriptional interaction network (CO).(DOC)Click here for additional data file.Table S4Functional description of interactome nodes corresponding to hubs and VIPs in NFS complete transcriptional interaction network (CO).(DOC)Click here for additional data file.


The mitochondrial redox state was determined by the ratio of the intrinsic fluorescence signals from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins , and the glucose uptake was measured using a near-infrared fluorescent glucose-analogue, pyropheophorbide 2-deoxyglucosamide (Pyro-2DG). Significant inter- and intratumor metabolic heterogeneity were observed from our imaging data on various types of breast cancer xenografts. The patterns and degrees of heterogeneity of mitochondrial redox state appeared to relate to tumor size and metastatic potential. The glucose uptake was also heterogeneous and generally higher in tumor peripheries. The oxidized and reduced regions mostly corresponded with the lower and the higher pyro-2DG uptake, respectively. However, there were some regions where the glucose uptake did not correlate with the redox indices. Pronounced glucose uptake and high NADH were observed in certain localized areas within the tumor necrotic regions, indicative of the existence of viable cells which was also supported by the H&E staining. Significant heterogeneity of the redox state indices was also observed in clinical specimens of breast cancer patients. As abnormal metabolism including the Warburg effect (high glycolysis) plays important roles in cancer transformation and progression, our observations that reveal the 3D intratumor metabolic heterogeneity as a characteristic feature of breast tumors are of great importance for understanding cancer biology and developing diagnostic and therapeutic methods.We previously reported that tumor mitochondrial redox state and its heterogeneity distinguished between the aggressive and the indolent breast cancer xenografts, suggesting novel metabolic indices as biomarkers for predicting tumor metastatic potential. Additionally, we reported that the identified redox biomarkers successfully differentiated between the normal breast tissue and the cancerous breast tissue from breast cancer patients. The aim of the present study was to further characterize intratumor heterogeneity by its distribution of mitochondrial redox state and glucose uptake pattern in tumor xenografts and to further investigate the metabolic heterogeneity of the clinical biopsy samples. We employed the Chance redox scanner, a multi-section cryogenic fluorescence imager to simultaneously image the intratumor heterogeneity in the mitochondrial redox state and glucose uptake at a high spatial resolution (down to 50 × 50 × 20 μm Abnormal metabolism and high variability in disease presentations are among the hallmarks of cancer  and two MCF-7 tumor xenografts  were used for simultaneous imaging of Pyro-2DG uptake and the redox state. Pyro-2DG was administered to the anesthetized mice through tail vein injection at a dosage of ~2.5 mg/kg after the tumor-bearing mice were starved for 24 hrs. In approximately two hours, the anesthetized mice were snap-frozen using liquid nitrogen. The snap-freezing protocol maintained the in vivo mitochondrial redox state for the ex vivo redox scanning. The preparation of tissue samples for the redox scanning were previously described  were grown to 6–10 mm in diameter in athymic nude mice for the 3D redox scanning. Another two MDA-MB-231 tumor xenografts  or NADH/(Fp + NADH). To account for the cell or mitochondria density difference at different regions within a tumor, the normalized Pyro-2DG, i.e. Pyro-2DG/(NADH + Fp) was also calculated based on the nominal concentrations of NADH and Fp.i  values were taken as the means of these redox indices in the corresponding more or less oxidized region.In order to obtain the mean values in the more oxidized regions (high Fp redox ratio or Fp/(NADH + Fp)) and less oxidized regions (low Fp redox ratio), the sum of two Gaussian functions Xu et al.. The fitWe have observed significant inter- and intratumor heterogeneity in the mitochondrial redox state in the breast cancer mouse xenografts. As the representative tumors shown in Figure To see how the redox indices change along the depth of each tissue section, they were plotted against the depth of each tumor section of the three tumors in Figure In addition to the section average values, for the aggressive tumors, we also obtained the mean values of the redox indices of the core (defined as high Fp redox ratio regions) and those of the rim (defined as low Fp redox ratio regions) by fitting of two Gaussian functions to their corresponding histograms. The plots in the middle column of Figure Figure Figure Due to complete cell death, we can identify certain central regions where there were no fluorescence signals in all channels. However, within these necrotic centers, there are islands of cells exhibiting active metabolism indicated by strong NADH, Fp, and Pyro-2DG signals, particularly in the large MCF-7 tumor in Figure 2 = 0.68. For the MCF-7 tumor image section in Figure 2 = 0.24. Similar correlations between Pyro-2DG or Pyro-2DG/(Fp + NADH) versus the redox indices (NADH/(Fp + NADH) or NADH) were observed in general for all four tumors. Despite the existing correlations, the distributions of the glucose uptake level versus mitochondrial NADH or NADH redox ratio were relatively scattered. On the correlation plots such as in Figure In general, all four tumors had oxidized core and reduced peripheral regions mostly corresponding with the lower and the higher pyro-2DG uptake, respectively, and showed a positive correlation of the pyro-2DG signals with NADH or NADH redox ratios in peripheral tissue regions. Figure 2 variations within breast tumors measured by needle electrodes did not correlate with the measuring site (tumor center versus periphery)  grossly positively correlated with NADH/(Fp + NADH) in the tumor periphery. Early studies using bioluminescent enzymatic assay demonstrated the heterogeneous spatial distribution of glucose, lactate, and ATP in human breast cancer xenografts in rats  rather than in State 2 where cells were nutrient-starved   and positively correlates with NADH and the NADH redox ratio in general but not in all regions, and that islands of viable cells existed in tumor necrotic/oxidized centers exhibiting both high glucose uptake and NADH. These results indicated that a variety of metabolic states exist in a breast tumor and the metabolic heterogeneity warrants further investigation for developing potential metabolic biomarkers for clinical applications.


TYMP S471L (rs11479) and early dose modifications and/or severe adverse events . There was also a significant association between these phenotypes and a signature of DPYD mutations . We did not identify any significant associations between the individual candidate pharmacodynamic markers and toxicity. If a predictive test for early adverse events analysed the TYMP and DPYD variants as a signature, the sensitivity would be 45.5 %, with a positive predictive value of just 33.9 % and thus poor clinical validity. Most studies to date have been under-powered to consider multiple pharmacokinetic and pharmacodynamic variants simultaneously but this and similar individualised data sets could be pooled in meta-analyses to resolve uncertainties about the potential clinical utility of these markers.The potential clinical utility of genetic markers associated with response to fluoropyrimidine treatment in colorectal cancer patients remains controversial despite extensive study. Our aim was to test the clinical validity of both novel and previously identified markers of adverse events in a broad clinical setting. We have conducted an observational pharmacogenetic study of early adverse events in a cohort study of 254 colorectal cancer patients treated with 5-fluorouracil or capecitabine. Sixteen variants of nine key folate (pharmacodynamic) and drug metabolising (pharmacokinetic) enzymes have been analysed as individual markers and/or signatures of markers. We found a significant association between  Folate-dependent one-carbon metabolism is a target for drug groups that are widely used in the treatment of cancer and inflammatory diseases. Two of the drugs, 5-fluorouracil (5-FU) and capecitabine, are central to the medical management of colorectal cancer in both advanced and adjuvant settings; they are used as monotherapies or in combination with other chemotherapeutic and biological agents. Both folate metabolism and the catabolism of 5-FU and capecitabine depend on a number of enzymes that are functionally polymorphic . 5-FU isThe fluoropyrimidine, capecitabine , has been designed for oral administration and to be more specific than 5-FU, thus leading to potential differences in the safety profile -5.The use of 5-FU/leucovorin in randomized controlled trials of adjuvant chemotherapy for colorectal cancer has been shown to improve both disease-free and overall survival as compared to surgery alone ,7. SubseTYMS and methylenetetrahydrofolate reductase (MTHFR) , p=0.023, and OR of 2.15 , p=0.020 in the dominant model (2). An association for severe toxicity and or SHMT1 (rs1979277) heterozygotes; an adjusted OR of 0.40 , p=0.023 compared to CC homozygotes, and OR of 0.47 [0.23-0.97], p=0.041 in the dominant model. No other adjusted results were significant.Adjusted results that we do not emphasise  were (1) an association for dose modification and DPYD and TYMP genotypes) and clinical phenotype data for the 44 participants who had severe adverse events. Nineteen of the 44 participants with severe adverse events carried at least one of the candidate predictive markers. If detection of the combined SNP signature was used as a diagnostic procedure to identify those who would subsequently suffer severe adverse events, the sensitivity would be 45.5 %, with a positive predictive value of 33.9 %. The potential impact of testing for the combined SNP signature and then changing to a different chemotherapy regimen in affected individuals can be estimated in a hypothetical clinical population of 1000 patients. If those patients had similar characteristics to those within our cohort, 233 of them would be combined SNP signature positive. The impact of changing the regimen in these 233 patients would be 79 fewer severe adverse events. Here, 95 patients would still have severe adverse events . However, 156 patients with the combined SNP signature would have had their regimen changed for no specific benefit because they would not have gone on to develop severe adverse events.We present an analysis of functionally important genetic variants in the pharmacokinetic and pharmacodynamic pathways that influence response to fluoropyrimidines. The phenotypes examined were adverse events that were identified by a dose delay or dose modification within 12 weeks due to toxicity, and by CTCAE grade 3, 4 or 5 scores. Each of the pharmacokinetic variants that we consider are compelling candidates as predictive markers because they have a known or putative functional impact on the enzymes needed for drug catabolism or their metabolism to an active form.DPYD variants analysed lead to enzyme deficiency or absence and their functional effects can be observed in heterozygous carriers. However, each DPYD SNP is rare, which reduces their potential clinical utility as predictive markers. In this study therefore we have also considered these DPYD variants as a signature of alternative polymorphisms, and found a strong association with early adverse events  mixed cohort of breast and colorectal cancer patients treated with capecitabine [TYMP SNP, rs470119 [citabine . We pres1479 see  and S2, citabine . In anotrs470119 . UMPS (rs1801019) and CDA (rs2072671). Genetic variants for these loci have previously been associated with severe neutropenia and diarrhoea in patients treated with 5-FU [We found no significant associations or trends for early dose modifications or severe adverse events with the candidate variants of ith 5-FU , and witith 5-FU .MTHFR, DHFR, MTHFD1, and SHMT in addition to TYMS. These are non-synonymous SNPs or variants that affect untranslated control regions; each polymorphism has a demonstrated or putative influence on gene expression or function. Inhibition of thimydylate synthase is an important pharmacodynamic mechanism for fluoropyrimidines but the balance of folate species within the biochemical pathway may depend on the enzyme variants at key branch points . We haveTYMS genotypes, it has been suggested that haplotype rather than genotype analysis may improve the sensitivity and specificity of pharmacogenetic testing. The G>C polymorphism in nucleotide 12 of the TYMS 28 bp VNTR repeat elements has been proposed to affect both expression of TYMS in vitro and levels of 2’-deoxyuridine in vivo. However, the published data are inconclusive, the majority of the possible genotypes have not been examined in relation to 5-FU sensitivity; different methods have been used to test gene expression; and conclusions about the putative functional effects have not been congruent [TYMS variants, rs45445694 and rs16430  for which there is linkage disequilibrium [No significant associations or trends were found for individual polymorphisms that have been classified as low activity thymidylate synthase variants and toxicity within 12 weeks . In the ongruent -45. Anotongruent  compriseilibrium ,32,47. DHFR, SHMT or MTHFD1 genotypes. Associations with an increased risk of dose modification were identified for particular DHFR genotypes and decreased risk of severe adverse events with particular SHMT genotypes in this study. But we do not emphasise these results because they derive from the adjusted dominant model only and there was no concordance between the 2 phenotypes considered . The observational nature of this study means that potential bias cannot be excluded. There are a number of factors that could bias towards the null. This may stem from incomplete recording of adverse events, missing data, or patients having early dose modifications and/or prophylactic interventions before higher grade events occurred. Small effect sizes, coupled with low allele frequency in some instances would have reduced the power of the study to detect any significant association. Conversely, type I error may also occur in erroneously reporting a significant finding when there is actually no true association. This can stem from multiple testing of a diverse range of genetic markers  and is a problem that may be ameliorated through a Bonferroni correction. However, Perneger highlights a number of methodological weaknesses with Bonferroni corrections , and he recommends that Bonferroni correction ‘should not be used when assessing evidence about specific hypotheses’. This poIn conclusion, these data identify and confirm markers that predict toxicity but our analysis of their clinical validity indicates limited utility. This has important implications for helping clinicians and patients arrive at evidence-based decisions on the pros and cons of investing in commercially available genotyping tests for predicting 5-FU toxicity during treatment of colorectal cancer. Only a few pharmacogenetic studies of the genotypes described have considered the role of epistasis but some interactions have been identified that warrant further analysis ,46,49,50Table S1The genotypes at the loci DPYD and TYMP for 44 participants who had grade 3, 4 or 5 adverse events within 12 weeks of starting the chemotherapeutic protocol.Treatment regimes; 1 = 5-FU as monotherapy; 2 = 5FU in combination chemotherapy; 3 = capecitabine as monotherapy; 4 = capecitabine in combination chemotherapy.For the genotype data; 0 = homozygous for the minor allele; 1 = heterozygous; 2 = homozygous for the major (wild type) allele. The genotypes 1236G>A and c1129-5923C>G are in linkage disequilibrium. LFT; liver function tests.(DOC)Click here for additional data file.Table S2Analyses of associations between fluoropyrimidine toxicity and genotype.The results from the test for trend and from dominant and recessive genetic models are shown. (a) Main effect of polymorphisms on fluoropyrimidine dose modification: markers of pharmacodynamics.† Adjusted for age, sex, previous chemotherapy and treatment regime using logistic regression ‡Fisher’s exact estimate used *Test for trend could not be calculated because of 0 observations in one or both phenotype groups for homozygotes in the minor allele.(b) Main effect of polymorphisms on fluoropyrimidine dose modification: markers of pharmacokinetics.† Adjusted for age, sex, previous chemotherapy and treatment regime using logistic regression ‡Fisher’s exact estimate used.(c) Main effect of polymorphisms on grade 3, 4 or 5 toxicity events: markers of pharmacodynamics.† Adjusted for age, sex, previous chemotherapy and treatment regime using logistic regression ‡Fisher’s exact estimate used.(d) Main effect of polymorphisms on grade 3, 4 or 5 toxicity events: markers of pharmacokinetics.† Adjusted for age, sex, previous chemotherapy and treatment regime using logistic regression ‡Fisher’s exact estimate used *Test for trend could not be calculated because of 0 observations in one or both phenotype groups for homozygotes in the minor allele.(DOCX)Click here for additional data file.Table S3Individualised data for each toxicity phenotype.(XLS)Click here for additional data file.


Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permits extensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated with radiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIR to a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liver lobes by regional HIR should be sufficient to correct some inherited metabolic disorders.7) isolated from wildtype F344 rats or Wistar-RHA rats were engrafted into the livers of congeneic dipeptidylpeptidase IV deficient (DPPIV−) rats or uridinediphosphoglucuronateglucuronosyltransferase-1A1-deficient jaundiced Gunn rats respectively by intrasplenic injection 24 hr after HIR (50 Gy) targeted to the median lobe, or median plus left liver lobes. An adenovector expressing hepatocyte growth factor (1011 particles) was injected intravenously 24 hr after transplantation.Hepatocytes (10− rats, 30–60% of the recipient hepatocytes were replaced by donor cells in the irradiated lobe, but not in the nonirradiated lobes. In Gunn rats receiving median lobe HIR, serum bilirubin declined from pretreatment levels of 5.17±0.78 mg/dl to 0.96±0.30 mg/dl in 8 weeks and remained at this level throughout the 16 week observation period. A similar effect was observed in the group, receiving median plus left lobe irradiation.Three months after hepatocyte transplantation in DPPIVAs little as 20% repopulation of 30% of the liver volume was sufficient to correct hyperbilirubinemia in Gunn rats, highlighting the potential of regiospecific HIR in hepatocyte transplantation-based therapy of inherited metabolic liver diseases. Hepatocyte transplantation has been used as an alternative to whole liver transplantation for amelioration of metabolic deficiencies in a number of patients with various inherited liver-based metabolic disorders Despite the encouraging results in the rodent models, whole liver HIR has not been tested as a preparative regimen in clinic trials because the risk of RILD may be greater in primates, including humans. However, recent clinical studies using conformal HIR have shown that partial irradiation of the liver markedly reduces the occurrence of RILD and allows far greater doses of radiation to be delivered safely − recipients by injection into the splenic pulp Initially, for the convenience of identification of the donor cells, we transplanted hepatocytes from F344 rats into congeneic DPPIVMale, 250–300 g wildtype Fisher 344 (F344) rats were purchased from Taconic Farms . Wildtype Wistar-RHA rats, congeneic Gunn rats and DPPIV- deficient F344 rats were bred and maintained on normal rat chow diet and the climate-controlled facility of the Institute for Animal Studies at the Albert Einstein College of Medicine in a 12-hr light/dark cycle. This study was approved by the Institutional Animial Care and Use Committee of the Albert Einstein College of Medicine.An adenoviral vector expressing human HGF was generated by the Cell Culture and Genetic Engineering Core of the Marion Bessin Liver Research Center of the Albert Einstein College of Medicine − rats (n = 3), and the whole median lobe (n = 6) or median and left lobe (n = 8) in Gunn rats, using a Philips orthovoltage unit operating at 320 kVP, 5 mA, and 0.5 mm copper filtration at 200cGy/min. Control Gunn rats (n = 4) underwent laparotomy, but no HIR was delivered. In our study we had to deliver intra-operative HIR to rat livers in order to spatially confine the irradiation dose to specific liver lobes because of the use of orthovoltage animal irradiator and the small volume of liver in rats. However clinical translation of our preparative HIR technique could be easily achieved in humans via external beam RT using modern 3D conformal techniques. Indeed, fractionated external beam RT has been used as a preparative regimen for hepatocyte transplantation in F344 rats using a clinical linear accelerator Regional HIR in rats was performed as reported with some modifications + wildtype F344 rats or ugt1a1+ wildtype Wistar-RHA rats by an in situ two-step collagenase perfusion procedure − Fisher rats and Gunn rats (180–200 g body weight) that had received regional HIR served as recipients for congeneic normal F344 and Wistar-RHA rat hepatocytes, respectively. Twenty-four hours after conformal HIR, 107 hepatocytes were transplanted by intrasplenic injection as described 11 particles) was injected via the tail vein 24 hours after hepatocyte transplantation Donor hepatocytes were isolated from DPPIVThe median lobe subjected to conformal HIR was separated from other liver lobes and snap-frozen in pre-cooled 2-methylbutane at −80°C. Five-micron thick cryostat liver sections were fixed for 5 min in cold 95% ethanol/5% glacial acetic acid (99∶1vol/vol) on ice and followed by a 5 min wash in cold 95% ethanol on ice. The sections were then air-dried and stained for DPPIV histochemistry as previously described Five-micron frozen sections of liver tissues were fixed with ice-cold acetone/methanol for 45 minutes. Then the sections were incubated for 60 minutes at room temperature with the primary antibody  at 1∶50 dilution after blocking with donkey serum. The sections were washed thrice with phosphate-buffered saline (PBS) and incubated for 45 minutes at room temperature with the secondary antibody, Cy3-conjugated donkey anti-rabbit IgG (H+L) at 1∶200 dilution . After extensive washing, the cover slips were mounted onto the slides with Vectashield (Vector Laboratories) and imaged using a Nikon Eclipse TE 2000-S fluorescent microscope.Liver fragments (100–150 mg) from each lobe were homogenized in a glass-Teflon motorized homogenizer and protein concentrations were determined (Bio-Rad protein assay). The proteins (100 µg/lane) were subjected to electrophoresis on 5%–7.5% polyacrylamide gradient SDS/glycine gel. Following electrophoresis, proteins were transferred onto a polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% fat-free milk powder in PBS followed by incubation with the rabbit antiserum directed against the rat ugt1a1 amino-terminal domain. After washing with PBS, the membranes were incubated with a horseradish peroxidase–conjugated goat anti-rabbit IgG (Sigma) and developed using an enhanced chemiluminescence reagent .Approximately 0.4 ml of blood was collected from the rat tail vein before any experimental procedure (pretransplant) and at various time points after transplantation under isoflurane anesthesia, and serum bilirubin levels were determined using an automated clinical microchemistry system .Bile samples were collected from the bile ducts of the Gunn rats after completion of serial blood collection, and were analyzed by reverse-phase high-performance liquid chromatography as described Ugt1a1 enzyme activity in the rat livers was analyzed as described previously t test. P values less than 0.05 were considered statistically significant.Repopulation of donor cells was assessed by counting the number of ugt1a1-stained donor hepatocytes in host liver lobes after ugt1a1 immunohistochemistry. Sampling regions were chosen at random for digital acquisition for data quantitation in a blinded fashion. A total of 20 fields at 200× magnification, each from irradiated and non-irradiated liver lobes of three rats per treatment group were used for each data point. The serum bilirubin values were expressed as the mean ± standard deviation (SD) and the ugt1a1 enzyme activity was expressed as percent of ugt1a1 activity in the wildtype Wistar RHA rat liver in order to estimate the extent of repopulation of host liver by donor hepatocytes. Statistical differences between two groups were determined by a two-sided Student + F344 hepatocytes were transplanted into congeneic DPPIV− rat livers that had been subjected to preparative HIR of one half of the median lobe only. Three months after transplantation, 30–60% of the irradiated region of the median lobe was selectively replaced by DPPIV+ donor hepatocytes, indicating massive proliferation of the donor cells. In contrast, the non-irradiated half of the lobe contained only individual DPPIV+ donor hepatocytes, indicating lack of significant donor cell proliferation  of the irradiated lobe was replaced by ugt1a1atocytes .Three months after hepatocyte transplantation and preparative HIR to the median and left lobe, 52-kd ugt1a1-immunoreactive bands were observed in all four lobes of Gunn rat livers. However, the ugt1a1 content in the irradiated median and left lobe was much higher than that of unirradiated right and caudate lobe .Following transplantation, ugt1a1 activity in the liver homogenates was assayed with bilirubin as the acceptor substrate. In Gunn rat livers that received regiospecific HIR prior to hepatocyte transplantation, ugt1a1 activity in irradiated median and left lobes was about 25–40% of the activity found in control Wistar-RHA rat livers (2.6±0.14 µmol/g liver per hour). These values were significantly higher than in the unirradiated lobes of Gunn rat livers, which were less than 5% of the activity in livers of Wistar-RHA rats (P<0.01). Similarly, ugt1a1 activity in the median lobe of Gunn rats that had received preparative HIR to that lobe only was about 21–24% of the activity in wildtype RHA rats, and was significantly higher than the activity in the unirradiated left, right, and caudate lobes. The ugt1a1 activity was undetectable in untreated Gunn rats .Crigler-Najjar syndrome type 1 (CN1) is a potentially lethal autosomal recessive disorder characterized by life long non-hemolytic unconjugated hyperbilirubinemia. The disorder results from a deficiency of ugt1a1 activity, which is critical for glucuronidation and excretion of bilirubin We then performed further preclinical studies to achieve proliferation of the engrafted hepatocytes within the liver. Host hepatocytes in CN1 patients, as in most other liver-based inherited metabolic disorders, have normal proliferative capacity. As the liver to body weight ratio is maintained within a tight limit by strong physiological mechanisms, the engrafted wildtype cells can proliferate only by replacing the host hepatocytes. Massive hepatic repopulation has been achieved by genetic, chemical and physical means to limit the mitotic capacity of the host As the chemical methods used to inhibit host hepatocyte proliferation are not readily translatable to clinical use, we investigated preparative HIR for achieving liver repopulation by transplanted hepatocytes. These studies showed that whole liver HIR in combination with a mitotic stimulus, such as partial hepatectomy or HGF expression, results in massive liver repopulation and complete correction of the metabolic defect in Gunn rats − recipient rats demonstrate that the area of the liver to be repopulated can be precisely mapped by targeting preparative HIR to that area. Percent repopulation of the liver was somewhat lower in the Gunn rat recipients than in the DPPIV- rats, which could have resulted from the strain difference. Nevertheless, we show that as little as 20% repopulation of 30% of the liver volume in the Gunn rat recipients was sufficient to correct hyperbilirubinemia. Repopulation of twice that volume reduced serum bilirubin levels more rapidly, but ultimately the long-term effect was similar with both HIR groups. Excretion of bilirubin glucuronides in the bile confirmed the function of the repopulating donor cells in vivo. In addition, there was a significant unconjugated bilirubin peak in the bile of the transplant recipients. A similar pattern is also found in human subjects with Gilbert syndrome, who have a partial deficiency of bilirubin glucuronidation In contrast to the treatment of the liver by drugs or chemicals, modern conformal irradiation technology permits HIR delivery to precisely targeted regions. This should permit repopulation of specific regions of the liver, keeping the remaining liver unperturbed. To take advantage of this unique characteristic of irradiation, it would be important to estimate the volume of the liver that must be repopulated to achieve a cure of a metabolic liver disease. Our results in the DPPIVAlthough we used an adenoviral vector to express HGF, for clinical application the mitotic stimulation will need to be achieved by alternative methods. Humanized activating antibodies to c-met, which have a much longer half-life in the circulation are being evaluated toward this goal in preclinical studies.In conclusion, we have shown for the first time that targeted irradiation of liver lobes can be used to precisely delineate a fraction of the liver volume to be repopulated by transplanted hepatocytes. Partial repopulation of one third of the liver volume using this method cured hyperbilirubinemia in the Gunn rat model of CN1. Our finding is highly relevant to potential clinical application of preparative HIR for hepatocyte transplantation because irradiation of a limited liver volume is tolerated much better than whole liver irradiation in the clinic. Modern conformal irradiation techniques can precisely delineate regions of the liver to be irradiated, thereby avoiding the development of RILD. In combination with newer approaches to providing mitotic stimulation to the engrafted hepatocytes, regiospecific HIR could pave the way for a safe and highly effective hepatocyte transplantation-mediated treatment regimen for a large number of liver-based inherited metabolic diseases.


EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.Two of the signature genetic events that occur in human gliomas,  EGFR amplification, for example, is a signature genetic event for human glioblastoma, and yet few cell populations derived from human glioblastoma biopsies ever maintain EGFR amplification in culture EGFR amplification appears to be selected for in certain xenograft systems when generated directly from primary tumors EGFR amplification may represent a small percentage of cells in the primary tumor, engraftment into rodents intracranially appears to select specifically for these cells for reasons that are currently not well understood EGFR amplified cells is in great excess of the cells that do not harbor amplification units Human glioblastoma is a disease that exhibits underlying genetic heterogeneity even within individual tumors. Amplification of specific receptor tyrosine kinases is one visible measure of the genetic diversity of cells within a tumor, but the biological properties of these cells remain ill defined, in part, because few IDH mutations, in particular oligodendrogliomas. Attempts to passage them in culture have led to only a few successes, and some additional cell cultures appear to lose the cells harboring IDH mutation A second tumor genotype that has been difficult to establish in experimental systems are gliomas with There are many possibilities for the apparent disappearance or absence of these genotypes in culture. In addition, to understand why we might lose these genotypes in culture could broaden our understanding of essential parameters for the growth of these tumors in the human brain.EGFR amplified tumor cells in serum free conditions and that under adherent serum conditions, a nestin/SMA positive cell population consistently emerges from human EGFR amplified xenografts and IDH mutated primary human biopsies. Our results indicate that these culture conditions favor the growth of normal cell types at the expense of certain tumor cells in vitro.In this work, we discovered that a progenitor-like cell population outgrows specifically Patient material was obtained from surgeries performed at the Haukeland University Hospital . Written consent was obtained from patients with procedures that were approved for the project (project number 013.09) by the Regional Ethics Committee . All animal protocols were approved by authorities in an AAALAC accredited facility at the Haukeland University Hospital and in accordance with the national regulations of Norway.Tumor tissue (<1 g) was gently cut with scalpel blades, rinsed in Hank's buffered saline (HBSS), and enzymatically dissociated at 37°C for one hour, or until single cells could be observed, in 5 ml of HBSS containing Liberase DH  and 100 2. For osteogenic and adipogenic differentiation, monolayer cultures were incubated in appropriate medium for 21 days , fixed in 4% paraformaldehyde, and stained with alizarin red or oil red to detect for bone and fat, respectively.Biopsy spheroids from GBM xenografts were prepared and cultured as described previously in vitro human umbilical cord vein endothelial cell-pulmonary artery smooth muscle cell (HUVEC-PaSMC) co-culture angiogenesis assay has been described previously 4, HUVEC 10×103, 200The 4OAc and 2.5 volumes of 100% ethanol. DNA was resuspended in Nuclease-Free water (Qiagen).DNA was isolated from patient material that had been immediately frozen in liquid nitrogen subsequent to surgery. Frozen sections were prepared, and every fifteenth section was stained with H&E to ensure >60% tumor cell content. Samples were treated with proteinase K overnight at 50°C in ATL buffer (Qiagen) and then DNAse free RNAseA  for 5 min at room temperature. Reactions were extracted with phenol:chloroform:isoamyl alcohol 25∶24∶1 saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma) and precipitated in 2.5 M NHSpheroids/cell pellets were washed with PBS and fixed in 4% formalin overnight at room temperature. Cells were centrifuged for 10 min. at 300×g, and formalin was discarded. The cell pellet was then stained with 20 DNA was digested using the restriction enzymes RSA1 and Alu1 and labeled using the BioPrime aCGH Genomic Labeling Kit (life technologies) and Cy3 and Cy5 dyes ), following standard protocols for Agilent CGH+SNP. A female HapMap sample with a known genotype  provided by the Coriell Repository  was used as a reference for each of the CGH+SNP experiments. Labeled DNA was competitively hybridized to SurePrint G3 Human 2×400 k CGH+SNP microarrays  following standard Agilent protocols. The slides were scanned at 3 http://www.dsmz.de. Profiles were analyzed to determine that they matched the original biopsy, and that they did not match any of the cell lines listed in the DSMZ STR database.Oligodendroglioma cell populations used for implantation or culture experiments were fingerprinted to confirm that all cell populations were derived from the matched patient biopsy. The AmpFlSTR Profiler Plus PCR Amplification Kit (life technologies) was used according to the manufacturer's protocol. This kit amplifies nine tetranucleotide short tandem repeat (STR) loci and the amelogenin (gender determination) locus in a single reaction. The samples were run and allele sizes interpreted on an ABI3100 Genetic Analyzer (life technologies). The fingerprinting profiles were run through the online international reference STR profile database for human cell lines at the German collection of microorganisms and cell cultures DSMZ Immunohistochemistry of paraffin sections was performed as described previously FISH analyses of paraffin sections were performed with the Vysis LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe  using the DAKO Histology FISH Accessory Kit (DAKO).2, perfused intracardially with 0.9% NaCl, and sacrificed when symptoms developedNude immunodeficient rats (rnu/rnu Rowett) were fed a standard pellet diet and were provided with water ad libitum. Biopsy spheroids or cell supensions were stereotactically implanted into the right brain hemisphere as described previously Axial (T2w) RARE sequences were acquired as described previously EGFR amplified patient biopsies  were used, was that cells without EGFR amplification also appeared to have “lost” human chromosome 7, as the chromosome 7 FISH probe (green) also did not hybridize to the cells in serum monolayer cultures. The selection process to the human chromosome 7 deficient cell population occurred rapidly in cultures from P8 and P6, where most of the EGFR amplified cells had already disappeared after one month . In P22,hemistry  and S2.EGFR amplification no longer remained, or were dramatically reduced in serum monolayer. In contrast, serum spheroid cultures preserved EGFR amplified cells, as described previously In effect, tumor cells with in vitro. To verify that rat stromal cells outgrew EGFR amplified tumor cells from the three xenografts under different in vitro conditions, we used human- and rat-specific anti-nestin antibodies to detect and differentiate between cells from the different species. Human nestin was used as a marker for the presence of human tumor cells, as it is highly expressed in the tumor cells of our xenograft model One possibility for this result was that the cells taking over the serum monolayer cultures were no longer human, but instead from the rat, and could represent a stromal cell population that has a growth advantage EGFR amplification in derived cultures are contradictory EGFR amplified cells are not preserved in standard NBM culture, others described the opposite EGFR amplified xenografts in NBM, three different scenarios emerged. From P8 tumors, similar to serum monolayer cultures, a cell population was generated that appeared to be derived from the rat, as the chromosome 7 probe had also disappeared along with amplification and expression of EGFR  without EGFR amplification . Under aEGFR amplified glioblastoma xenograft P8 when implanted into the brain of nude rats resulted in highly invasive tumors as described previously Serum spheroid cultures derived from the EGFR amplified xenograft cultures, we performed immunostaining on cells from serum monolayer cultures derived from P8, P6, and P22 as well as NBM cultures derived from P8 with SOX2, GFAP, beta-tubulin-III, and SMA. Stromal cells in P8 NBM cultures were positive for SOX2 and negative for GFAP and beta-tubulin III, indicating that these cells are most likely nestin+ SOX2+ neural progenitors . Serum free cultures yielded viable spheres that did not proliferate in vitro but in one case, engrafted IDH1/2 mutation of the primary tumor. Three out of four oligodendrogliomas and one oligoastrocytoma were all mutated at codon 132 of IDH1 whereas one oligodendroglioma did not harbor mutations at the canonical codons 132 or 172 of IDH1 or IDH2IDH mutation. In none of these cases did we observe the cells in monolayer to harbor IDH mutations . As this cell line harbors additional mutations that are typical for GBM, the cultured cells seem to be tumor cells; however, it is still unclear why they are non-tumorigenic EGFR amplified glioma cells in adherent serum conditions have failed and led to the assumption that EGFR amplification is “lost” during the culturing process EGFR amplified tumor cells can be easily outgrown by another cell population, which in case of our xenograft model was a stromal cell population. Thus, the proposed “loss” of EGFR amplification in monolayer cultures by Pandita et al. EGFR amplified tumor cells at the expense of other cells, which in the case of human biopsies might also be other tumor cells that do not harbor EGFR amplification. A more recent study by Fael Al-Mayhani et al. showed that EGFR amplification is also lost in serum-free monolayer cultures on a defined extracellular matrix, while other chromosomal aberrations are preserved, indicating that EGFR non-amplified tumor cells had a selective growth advantage even in these cultures EGFR amplification as also observed previously Adherent serum cultures have been the standard growth conditions for glioma cells since the 1970s. Although it has been shown that this culturing procedure, in contrast to serum-free cultures, does not faithfully preserve the patient genotype in vivo precursor/counterpart of the stromal cells that we find in monolayer in vitro, but they resemble to some degree mesenchymal stromal cell populations derived from glioma model systems and other solid tumor types We cannot accurately identify the in vitro as opposed to in vivo. In serum monolayer, for example, the cells may lose SOX2 and in so doing, become mesenchymal stem cell-like. Interestingly, when assessing the differentiation potential of cortical stem cells in vitro, investigators found that plating density affected cellular fate. Lower plating density of the stem cells generated a cell population that was SMA positive rather than neurons, astrocytes, or oligodendrocytes One additional hypothesis is that the adherent cells are derived from a neural stem/progenitor cell type that becomes differentiated and grows efficiently in vitro and raises the question of the origin of stroma in human gliomas.In conclusion, stromal cell populations with stem cell-like features can be isolated from human primary tumors and xenografts. While the true cellular origin of these cells is currently unknown, they have a proliferative advantage under certain conditions over tumor cells. This result indicates that care must be taken when preparing cells from glioma tissue Figure S1Rat stromal cells have a selective growth advantage over EGFR amplified cells in serum monolayer culture. (a) FISH with an EGFR/Chromosome 7 probe in red and green, respectively, and immunohistochemical staining with antibodies against EGFR. In monolayer culture, both EGFR amplification and the chromosome 7 probe are strongly reduced after three months in cultures from P22 xenografts. (b) Immunohistochemical staining with antibodies against human-specific and rat-specific nestin. The time course shows that after three months, nestin-expressing rat cells take over at expense of human tumor cells in serum monolayer culture from P22 xenografts. Nestin-expressing human cells are predominant in serum spheroid cultures. Scale bars 50 µm.(TIF)Click here for additional data file.Figure S2Rat stromal cells have a selective growth advantage over EGFR amplified cells in serum monolayer culture. Quantification of EGFR, human and rat nestin expressing cells from P22 cultures (3 months) in three random high power (400×) microscopic view fields (HPF) in each group. Values represent mean ± s.d.(TIF)Click here for additional data file.Figure S3Tumor cells with strongly reduced EGFR expression derived from P22 xenografts have a growth advantage in serum free cultures. (a) FISH with an EGFR/Chromosome 7 probe in red and green, respectively, and immunohistochemical staining with antibodies against EGFR. In P22 cultures both EGFR amplification and the chromosome 7 probe are preserved after two months in culture, but EGFR expression is strongly reduced compared to 1-month-old cultures. Quantification of EGFR expressing cells in three random high power (400×) microscopic view fields (HPF) in each group. Values represent mean ± s.d. (b) Immunohistochemical staining with antibodies against human-specific and rat-specific nestin. In P22 cultures human-nestin positive tumor cells have a growth advantage over rat cells. Scale bars 50 µm. Quantification of human and rat nestin expressing cells in three random high power (400×) microscopic view fields (HPF) in each group. Asterix indicates 0%. Values represent mean ± s.d.(TIF)Click here for additional data file.Figure S4Cultures of an EGFR non-amplified GBM. Cultures derived from P3 xenografts show polysomy/gain of chromosome 7, but no EGFR amplification. Immunohistochemistry for EGFR is negative. Scale bars 50 µm.(TIF)Click here for additional data file.Figure S5EGFR non-amplified tumor cells have a growth advantage over rat cells under all growth conditions. Immunohistochemical staining with antibodies against human-specific and rat-specific nestin. Cultures derived from P3 xenografts show many strongly human-nestin positive and only few rat-nestin positive cells under all conditions. Scale bars 50 (TIF)Click here for additional data file.Figure S6EGFR non-amplified tumor cells have a growth advantage over rat cells under all growth conditions. Quantification of human and rat nestin expressing cells from P3 cultures (3 months) in three random high power (400×) microscopic view fields (HPF) in each group. Asterix indicates 0%. Values represent mean ± s.d.(TIF)Click here for additional data file.Figure S7Stromal cells derived from GBM cultures are non-tumorigenic. Cells from the different culture conditions derived from P8 xenografts were implanted into the brain of nude rats. T2- weighted MRIs and H&E sections show invasive tumors derived from serum spheroid cultures, while cells derived from monolayer and NBM cultures are non-tumorigenic. Scale bars 50 µm.(TIF)Click here for additional data file.Figure S8Characterization of rat stromal cells in vitro. (a) Cells from P22 monolayer cultures were stained with antibodies against GFAP, beta-tubulin III, SOX2 and SMA. The cells are strongly positive for SMA indicating rat stromal cells. The GFAP, beta-tubulin III, and SOX2 positive cells are most likely human tumor cells as there was not a complete selection towards rat stromal cells in P22 monolayer cultures. Scale bars 50 (b) Quantification of GFAP, beta-tubulin, SOX2 and SMA expressing cells from P22 monolayer cultures in three random high power (400×) microscopic view fields (HPF) in each group. Values represent mean ± s.d.(TIF)Click here for additional data file.Figure S9Stromal cells derived from oligodendroglioma cultures are non-tumorigenic. Cells derived from oligodendroglioma monolayer cultures were implanted into the brain of NOD/SCID mice. H&E section from a NOD/SCID mouse brain shows the injection site devoid of tumor cells. Scale bar 50 (TIF)Click here for additional data file.Figure S10Characterization of stromal cells derived from oligodendroglioma. (a) Control PaSMC promoted capillary network formation of co-cultured endothelial cells transduced with GFP for visualization, whereas derived monolayer cultures from oligodendrogliomas did not. (b) Monolayer cells from oligodendroglioma were cultured in control medium (DMEM with 10% serum), and osteogenic and adipogenic media for 21 days. Cultures were fixed and treated with alizarin red and oil red to identify bone and fat, respectively. Scale bars 50 (TIF)Click here for additional data file.Table S1Clinical and genomic information from primary GBM patient biopsies.(DOCX)Click here for additional data file.Table S2Derived serum monolayer cell populations from the oligodendroglioma and oligoastrocytoma cases analyzed in the study.(DOCX)Click here for additional data file.


Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in  DNA methylation occurs predominantly as covalent modification of cytosines within a CpG sequence context. It represents the most stable epigenetic mark and has an impact on different biological processes in both healthy and diseased cells, including e.g. neural cell differentiation Pou5f1 , encoding a homeobox protein essential in the maintenance of pluripotency Ddah2 gene to be an epigenetic biomarker for neural stem cell differentiation in vitro induced neural stem cells (NSCs), as well as 4 embryonic and 4 adult murine tissues using a custom mouse DMH array.Much effort has been made to understand the dynamics of DNA methylation during neural cell differentiation and the identification of epigenetic biomarkers that capture different aspects of cellular differentiation processes. ESCs are of particular interest in this context as they have previously been shown to acquire characteristic epigenetic marks during their differentiation from ESCs to NSCs and, subsequently, to tissues Numerous methods have been developed for genome-wide DNA methylation profiling in vitro induced NSCs derived from this cell line We studied the DNA methylation profiles of E.14 ESCs and To explore the DNA methylation distribution across the murine genome, we studied the relationship between the feature location and methylation content in all samples . After aWe studied the variation of DNA methylation profiles among ESCs, NSCs and embryonic brain and analyzed the data to identify candidate markers which potentially play a role during neural cell differentiation. First, we separated the features on the DMH array according to their association to the TSS. Hence, we obtained two independent datasets that can be interpreted independently. In this study, we focused on TSS-associated features. Similar analyses were also performed on non TSS-associated features (data not shown).Sox10) and cell proliferation , as well as genes related to the development of the brain and the neural system . The complete list of tDMRs in each group and their associated genes are provided in supplementary Next, we studied if the identified tDMRs were related to the degree of neural differentiation. We selected 382 candidate tDMRs according to the following criteria: p-value lower than 0.05, effect size higher than 0.5 and minimum DMH score higher than 0.5 in at least one group. Unsupervised clustering of these candidates revealed distinct groups among NSCs, ESCs and embryonic brain samples . VariancTaken together, our data strongly suggest that DNA methylation may play a major role during murine neural stem cell differentiation. To explore whether the genes associated with tDMRs were functionally related, we used the MAPPFinder software To validate the tDMRs identified by murine-specific DMH with a different technology, we used direct bisulfite sequencing (DBS) DBS validation of these 51 tDMRs was done on amplicons from five different biological samples: ESCs, NSCs, embryonic neural tissue , adult neural tissue  and adult non-neural tissues  . To evalIn a previous study, we used an affinity capture method (MeDIP-chip) to screen mouse chromosome 17 for neural differentiation biomarkers in ESCs and NSCs Pou5f1, Ddah2, Nr4a2, Itpka, Btbd17 and Emx2) were hypermethylated in NSCs, while 2 genes (Fam179a and Fbxl17) were hypermethylated in ESCs. We focused our analysis on two genes that have been reported as epigenetic biomarkers for neural stem cell differentiation: Pou5f1Ddah2Pou5f1 (upper panel) and Ddah2 (lower panel), respectively. For the Pou5f1 gene, we studied two regions covering the exon 1-intron 1 junction (Region 1) and intron 1 (Region 2), respectively. We studied 3 regions for the Ddah2 gene, Region 3 covering the exon 1-intron 1 junction and Regions 4 and 5 located within the gene body. In general, the results obtained by DMH and MeDIP-chip were comparable for these regions. For example, we detected differential methylation in Region 2 by both methods, DMH and MeDIP , the concordance highlights the strength of the findings. However intrinsic technical limitations as discussed by Irizarry and colleagues The comparison of DMH and DBS data for differentially methylated regions resulted in a correlation of 68%. This correlation, however, decreased to 44% when the DMH features contained only a single DNA methylation-sensitive restriction site. In DBS amplicons corresponding to DMH fragments with a single restriction site, only the methylation value for the CpG within the site is considered. When DBS amplicons corresponded to DMH fragments containing multiple restriction sites, methylation values were averaged for the CpGs included in those sites.. Therefore, we expect that fragments with single restriction sites may be more sensitive to variability, due to intrinsic sample factors, technical performance or other experimental parameters. As the proportion of fragments containing single and multiple restriction sites fragments in our DBS analysis . There are three possible major sources for such measurements. 1) The variance around 100% is much larger than in the lower methylation range. As DMH utilizes a genome-wide PCR, it might lead to broader peaks at the fully methylated side of the mainly bimodal methylation distribution and thereby, extending the extrapolated rates beyond 100%. 2) The enzymatically methylated reference DNA might partially be incompletely methylated, which would lead to an over-estimation at the affected sites. 3) Since DHM assesses the total amount of methylated DNA, an increase in the copy number of a methylated region will also lead to an increased amount of detected DNA and therefore to a significantly higher methylation percentage. Thus, tissue- specific copy number increases with respect to the 100% calibrator DNA  will result in scores higher around than 1.5 for copies in triplicate and beyond for sites with multiple copies. Thereby, it is very likely that linear interpolation to estimate the exact copy number is not possible with data from the DMH array used in this study. Interestingly scores beyond 1.5 seen in cancer tissue and cancer cell line DMH experiments have been used successfully to obtain consistent information about regions with copy number effects, especially if such measurements were observed at multiple neighboring sites (unpublished data). Therefore it might also be of interest to investigate DMH scores larger than 1.5 as found in the mouse cell lines used in this study.Jag1, Tcf4) or have a function related to the neural system , which likely supports a major role for DNA methylation in neural stem cell differentiation that was unknown in most cases.Many of the identified tDMRs may represent novel epigenetic markers for neural cell differentiation. Among the genes associated with tDMRs between ESCs and NSCs, many have a known function in neural differentiation . Husbandry, housing and experiments of animals comply with the French legislation and the European regulations for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The institution is approved to carry out animal work and the personnel handling the animals have been trained and licensed accordingly. All the work with animals has been done at the Specified Pathogen Free central mouse facility at the Institut Curie. This facility has been accredited by the French National Authority (Accreditation number #B75-05-18). Further ethical approval was not necessary as the study did not require in-vivo experimentation, but the molecular study of tissues obtained from sacrificed animals and established murine cell lines.Adult mice were euthanized and organs  were dissected. Embryonic mice were 13.5 days old and 4 tissues were dissected . All adult and embryonic tissues were obtained from F1 (C57BL/6×DBA/2J) mice. Adult mice DNA samples were taken from one mouse, while embryonic DNA corresponds to pools of 5 to 8 embryos. DNA was isolated from embryonic and adult mouse tissue using the MinElute kit .For the analysis of ESCs, we used an already established murine cell line (E14) derived from C57BL/6 mice DNA from each biological sample was split into two aliquots and both aliquots were processed in parallel. These two technical replicates were then hybridized to two arrays. Each pair of replicates showed a high correlation . DMH scores used for further analysis were calculated using the averaged signals of the two technical replicates. DNA methylation profiles in NSC and ESC were assessed in two independent cultures for each cell type. The embryonic tissue group consisted of limbs (n = 1), spinal cord (n = 1), hindbrain (n = 2) and forebrain (n = 1). The adult tissues group consisted of 4 different tissues: liver (n = 1), spleen (n = 1), kidney (n = 1) and heart (n = 1). Pairs of biological replicates, when available, also showed high correlation in their respective DMH scores .Csp6I, MseI and BfaI) and adaptors were ligated to the generated ends. The adaptors were prepared by annealing the two oligos (5′-AGGCAACTGTGCTATCCGAGGGAT-3′ and 5′-TAATCCCTCGGA-3′). Next, fragments were digested using a cocktail of DNA methylation-sensitive restriction enzymes , which cleave unmethylated sites while leaving methylated sites intact such that subsequent PCR with primers complementary to the adaptors amplifies only the intact fragments. PCR products are then hybridized to microarrays.Hypermethylated regions were enriched using DMH as originally described by Huang et al. In this work, we used a newly developed Affymetrix mouse array. This array was custom-designed (Affymetrix CustomSeq®) and contained 51,243 probesets, with each probeset consisting of 8–10 individual probes. The mouse DMH array was designed containing orthologous regions to the human DMH array in order to enable also human-mouse comparative studies. The probesets cover CpG-rich loci in 5′-untranslated regions, exons and introns of known genes, as well as in intergenic regions, across all murine chromosomes. Approximately 50% of the probes are located in know promoter regions. Furthermore, the array contains probesets for loci that do not contain any relevant methylation-sensitive restriction sites serving as internal calibration controls in addition to Affymetrix-designed control oligos. Data were analyzed according to the methods previously described Genomic DNA was bisulfite converted and PCR amplified as previously described For each selected candidate tDMR, 2 amplicons were designed and each covered at least one restriction site contained in the corresponding DMH feature. The quality of the amplicons was evaluated by assessing the quality of the bisulfite sequencing reads first on technical DNA and the completeness of amplicon coverage. The better performing amplicon for each tDMR was then selected for the panel of amplicons for the validation analysis.Methylated DNA Immunoprecipitation was performed of DNA isolated from ESCs and NSCs according to standard protocols MAPPFinder was used to correlate microarray data of differentially expressed genes to pathways annotated in GenMAPP and to Gene Ontology (GO) annotations Table S1tDMRs and their associated genes.(XLS)Click here for additional data file.Table S2tDMR-associated genes in ESCs, NSCs, and embryonic brain.(XLS)Click here for additional data file.Table S3MAPPFinder results for over-represented GO groups in ESC and NSC tDMRs.(XLS)Click here for additional data file.Table S4Primer sequences for direct bisulfite sequencing (DBS).(XLS)Click here for additional data file.