The Ewing sarcoma family of tumors (ESFT) represents an aggressive spectrum of malignant tumour types with common defining histological and cytogenetic features. To evaluate the functional activation of signal transducer and activator of transcription 3 (STAT3) in ESFT, we evaluated its activation in primary tissue sections and observed the functional consequences of its inhibition in ESFT cell lines. STAT3 was activated (tyrosine 705-phosphorylated) in 18 out of 31 primary tumours (58%), either diffusely (35%) or focally (23%). STAT3 was constitutively activated in 3 out of 3 ESFT cell lines tested, and its specific chemical inhibition resulted in complete loss of cell viability. STAT3 inhibition in ESFT cell lines was associated with several consistent changes in chemokine profile suggesting a role of STAT3 in ESFT in both cell survival and modification of the cellular immune environment. Together these data support the investigation of STAT3 inhibitors for the Ewing family of tumors. OverallParaffin-embedded tumor samples were randomly selected from hospital histology archived material. Haematoxylin and eosin stained sections were examined to identify areas of viable tumour. Tissue cores were taken from these areas using a skin biopsy punch (diameter 4 mm) and reembedded to produce 3 tissue arrays, comprising a total of 31 tumour cores.μm-thick sections, as previously described [Immunohistochemical analysis was performed on 4 escribed . As primThe ESFT cell lines A673 and TC32 were kind donations of Professor Sue Burchill, University of Leeds, UK. The ESFT cell line SKNMC, the prostate cancer cell line PC3, and HeLa were obtained from ATCC. Cell lines were maintained in the following media by Gibco/Invitrogen : DMEM , RPMI (cell line TC32), or F12 (cell line PC3) media containing 10% foetal calf serum. For fixing of 20 ng/mL IL-6-treated HeLa cells (E-Bioscience), supernatant was aspirated after 10 minutes activation, and cell pellets were fixed in 10% formaldehyde for 10 minutes before undergoing processing as for routine histopathology specimens. Following processing, each sample was embedded in paraffin.μmol/L. 24 hours later the cells were harvested and lysed using RIPA buffer  containing protease inhibitor cocktail  and sodium orthovanadate . Western blotting was then performed on equal amounts (in weight) of lysate as previously described [For production of lysates the following experimental protocol was followed for ESFT and the PC3 cell lines. 500,000 cells were incubated in 2 mL media for 16 hours. Media was then removed and replaced by 1 mL of fresh media in the presence or absence of the pharmacological STAT3 inhibitor, S3i-201 , at a concentration of 100 μL of media. 16 hours later, treatment was added. Each treatment condition was performed in triplicates. 24 hours later, cell viability was assessed, using 20 μL of the CellTiter 96 Aqueous nonradioactive cell proliferation assay based on the tetrazolium compound [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS . Absorbance was measured using the 96-well plate BIO-RAD Model 680 microplate reader .25,000 cells were plated in 96-well flat-bottom plates suspended in 100 Supernatants for chemokine array analysis were produced by following the experimental protocol for production of lysates, as outlined above. Before harvesting and lysing cells, spent media was removed and stored at −80°C, after centrifugation to remove debris. The supernatants were analysed at a later stage using the Ray Biotech  Human Chemokine Array 1 according to the manufacturer's instructions, using overnight incubations. The spots on the developed films were scanned with a BIO-RAD GS-800 Calibrated Densitometer  and quantified using ImageQuant software . From each spot that represented a chemokine, the background (negative control spots) was subtracted. The chemokine spots were then normalised against positive control spots. The values that were obtained thus were density values expressed as percentage of positive control spots. Further analysis was then carried out using Microsoft Excel 2007 . Chemokines which had changed in control experiments (PC3 cell line) by more than 10% were excluded from further analysis, to exclude potential off-target effects. A change in chemokine level was arbitrarily defined as an increase or decrease by more than 10%, observed in arrays from two independent experiments.705-phosphorylated form of STAT3 (P-STAT3) and first validated its specificity using IL-6-treated or -untreated HeLa cells , or they existed in positively staining clusters , particularly along fibrovascular bundles. The extent of P-STAT3 positive tumor cells amongst diffusely positive tumors was graded as follows: 8 tumors 1+; 2 tumors 2+; 1 tumor 3+.Previous work has described a high incidence of STAT3 activation in ESFT , and we La cells . We nextLa cells . Positivin vitro cell viability growth assays in a panel of ESFT cell lines and in the STAT3-null prostate cancer cell line, PC3. The presence or absence of P-STAT3 in the cell lines was demonstrated by Western blotting as was the ability of the specific STAT3 inhibitor, S3i-201 [μmol/L in ESFT cell lines, but not in the STAT3-negative PC3 cell line. S3i-201 treatment nonspecifically inhibited viability in growth conditions in all cell lines (including the STAT3 negative PC3 cell line) at concentrations of 300 μmol/L and 1000 μmol/L, respectively.We assessed the role of P-STAT3 in contributing to tumour growth by performing  S3i-201 , to abolWe next studied the effects of STAT3 inhibition on chemokine secretion patterns of ESFT cell lines and of the STAT3-negative PC3 cell line. Chemokine arrays were performed on supernatants obtained from cells cultured in the presence or absence of pharmacological P-STAT3 inhibition. For each cell line, two independent experiments were performed. Prior to analysis by chemokine arrays, we confirmed by Western blotting that P-STAT3 had been blocked in the cells from which the supernatants had been harvested (data not shown). Known STAT3-regulated chemokines  were afWe sought to confirm that STAT3 is present in a subset of ESFT tumours and to investigate the role of STAT3 in ESFT. We were particularly interested in the role of STAT3 in contributing to tumour growth and immune regulation, which are classical roles described for STAT3 in adult tumours.in vitro.In our series, we demonstrated P-STAT3 positive tumour cells in 18/31 ESFT cores. In comparison, Lai et al. previously found P-STAT3 positive tumour cells in 25 out of 49 tumour cores [μmol/L (previously reported IC50 of 86 ± 33 μmol/L) S31-201 reduced viability of STAT3 positive ESFT tumour cells, but not of the STAT3 null PC3 cell line. At higher drug concentrations, cell viability was also diminished in the PC3 cell line, indicating STAT3-indepedent effects of S3i-201 at such concentrations. Our findings are consistent with data presented in the original drug discovery paper of S3i-201. Here Siddiquee et al. reported that in breast cancer cell lines S3i-201 inhibited tumour growth, with an IC50 of 86 ± 33 μmol/L and with STAT3-independent cytotoxic effects at higher drug concentrations [We studied whether STAT3 contributes to tumour growth by performing viability assays of cells in growth conditions in a panel of three ESFT cell lines, which harbour constitutively activated STAT3. As a STAT3 negative control cell line, we used in our experiments the prostate cancer line PC3, which is homozygous STAT3 null . As a phtrations . Our obsNext we studied the effects of STAT3 inhibition on chemokine secretion patterns of ESFT cell lines through chemokine arrays performed on supernatants of ESFT cells. We confirmed STAT3 inhibition in the cells that produced the supernatants through Western blotting, and we used a STAT3 null cell line PC3 to exclude nonspecific effects. We found changes in chemokine levels of known STAT3 targets such as IL-8 whose secretion was increased by STAT3 inhibition in all three ESFT cell lines, but not in PC3. In addition, STAT3 inhibition induced a distinct the pattern of changes in chemokine secretion in each ESFT cell line. The significance of these findings is twofold. First, they suggest that STAT3 plays a role in regulating the chemokine environment of ESFT cell lines. Second, our observations highlight that in each ESFT cell lines STAT3 may be integrated into different immunological pathways.in vitro experiments provide evidence that, in ESFT cell lines, STAT3 plays its classical role of contributing to tumour growth and regulating the tumour immune environment. As global efforts are underway to develop clinically usable STAT3 inhibitors, our findings have to be validated in different in vitro and in vivo models of ESFT, to provide a rationale for targeting STAT3 in patients suffering from ESFT.In this study we confirm the finding that a subset of ESFT contains P-STAT3 positive tumour cells. Furthermore, we describe two novel distribution patterns of PSTAT3 positive tumour cells. Our


Her2/neu ECD levels in the supernatants of USC cell lines and in the serum of 38 USC patients and 19 controls were tested using ELISA. The biologic effect of Her2/neu ECD on trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated in 5-h chromium-release assays.Her2/neu expression was evaluated by immunohistochemistry (IHC), real-time PCR and flow cytometry, while c-erbB2 gene. High levels of Her2/neu ECD were found in supernatants of all FISH-positive tumours. In contrast, FISH-negative USC was negative for Her2/neu ECD shedding. Serum Her2/neu ECD levels in patients harbouring 3+Her2/neu tumours were higher than those found in healthy women (P=0.02) or USC patients with 2+ or 1+/negative Her2/neu expression (P=0.02). In cytotoxicity experiments, trastuzumab-mediated ADCC was significantly decreased by the addition of Her2/neu ECD-containing supernatants (P=0.01).Five out of ten USC cell lines overexpressed Her2/neu by IHC and showed amplification of the in vitro and potentially neutralise its therapeutic effect in vivo.FISH-positive c-erbB2 USC cell lines shed high levels of Her2/neu ECD. High levels of Her2/neu ECD in USC patients may reduce trastuzumab-mediated ADCC  Endometrial cancer is the most common female genital tract malignancy in the United States, with an incidence of 43 470 new cases and 7950 deaths in 2010 . Thus, tc-erbB2 is a proto-oncogene that encodes the human epidermal growth factor type II receptor (Her2/neu), a 185-kDa transmembrane protein, composed of three domains: the cytoplasmic tyrosine kinase domain responsible for intracellular signalling, a hydrophobic membrane-spanning region and the extracellular domain (ECD), which includes the binding site for trastuzumab  in 40–60% of patients harbouring USC  is a humanised monoclonal antibody (mAb) directed against the ECD of Her2/neu. This therapeutic agent has been shown to be highly effective in patients with early or advanced/metastatic breast cancer overexpressing Her2/neu (Ten primary USC cell lines (USPC ARK-1 to USPC ARK-10) were established after sterile processing of tumour samples from surgical biopsy specimens, as described previously , as previously described (in situ hybridisation (FISH) analysis was performed in the tumour tissues using the PathVysion Her-2 DNA FISH Kit  according to the manufacturer's instructions, as previously described . The comparative threshold cycle method was used to determine gene expression in each sample, relative to the value observed in the lowest non-malignant endometrial epithelial cell sample, using glyceraldehyde-3-phosphate dehydrogenase (Assay ID Hs99999905_m1) RNA as internal control.RNA isolation from all 10 primary USC cell lines and 2 breast cancer cell lines was performed using TRIzol Reagent , according to the manufacturer's instructions. Quantitative PCR was carried out with a 7500 RealTime PCR System using the manufacturer's recommended protocol  to evaluate the expression of 2 immunoglobulin was used as a secondary reagent . Analysis was conducted with a FACScalibur, using Cell Quest software . Using in vitro dose titration experiments with different amounts of trastuzumab (ranging from 0.05 to 1.5 μg ml−1), binding of USC cells was found to plateau at a trastuzumab concentration ranging from 0.5 to 1 μg ml−1  was used for our flow cytometry studies. For staining, a fluorescein isothiocyanate-conjugated goat antihuman F(ab1) μg ml−1 . On the 6 primary tumour cells for each cell line cultured in plasticware  using RPMI-1640 media, supplemented with 10% FBS . After 96 h incubation at 37 °C, supernatants were aspirated, rendered cell free by centrifugation at 1500 r.p.m. for 10 min, and stored at −80 °C before being analysed for Her2/neu ECD by ELISA (see below). The Her2/neu ECD values are expressed in ng ml−1.To evaluate the potential secretion of Her2/neu ECD by primary cancer cell lines, supernatants obtained from all 10 USC cell lines and 2 breast cancer cell lines were evaluated by a commercially available ELISA kit . Briefly, tumour supernatants to be tested for Her2/neu ECD secretion were collected after incubation of 10Her2/neu concentration was measured in the serum samples of 19 healthy female donors and 38 USC patients , using an ELISA for the quantitative detection of human soluble Her2/neu protein, according to the manufacturer's instructions . Serum was collected in all patients at the time of pre-operative visits before their surgery and before any treatment. Patient characteristics are described in Large-scale production of conditioned medium was obtained from supernatants of USPC ARK-2 cell line . Cells were plated into T150 flasks in RPMI medium supplemented with 10% FBS for a total of 6 days. The medium was then collected and centrifuged at 1000 r.p.m. for 7 min to remove insoluble material. Conditioned medium was concentrated 40-fold by ultracentifugation using YM50 membranes  before being used in cytotoxicity assays. Control supernatants collected from FISH-negative USC cell lines were similarly processed before their use.51Cr)-release assay was performed to measure the cytotoxic reactivity of Ficoll-Paque PLUS  separated peripheral blood lymphocytes (PBLs) obtained from several healthy donors against two representative FISH-positive primary USC cell lines , as previously described (μg ml−1) with conditioned medium containing 500 ng ml−1 of soluble Her2/neu ECD or matched control supernatants negative for soluble Her2/neu ECD for 1 h at RT on a rocker. The antibody/supernatant preparations were then added to the target cells (USPC ARK-2 and USPC ARK-10) for 30 min before incubation with the effector PBLs. Controls included the incubation of target cells alone or with PBL or mAb separately. The chimeric anti-CD20 mAb rituximab was used as an additional negative control in the assays.A standard 5-h chromium  were calculated by computing them on the ΔCts and then reverse transforming the results to obtain means (95% CIs) of relative copy numbers. The analyses of differences in Her2/neu ECD shedding in supernatants and in Her2/neu expression levels measured by flow cytometry and IHC were performed using the Wilcoxon–Mann–Whitney test. Kruskal–Wallis test and χ2-analysis were used to evaluate differences in trastuzumab-mediated ADCC levels in primary tumour cell lines. Her2/neu serum concentrations among the different groups of patients  were summarised as means and compared with Student's t-test. Statistical analysis was performed using PASW version 18 . A P-value of <0.05 was considered statistically significant.For qRT–PCR data, the right skewing was removed by taking copy number ratios relative to the lowest expressing normal human endometrial cells sample (‘relative copy number’), logImmunohistochemistry detecting Her2/neu expression was performed on formalin-fixed paraffin-blocks of USC tissues from which the 10 primary cell lines were established. As reported in erbB2 gene amplification was detected in 5 out of 10 primary USC specimens, suggesting that strong receptor expression by IHC and high Her2/neu mRNA level of these tumours (see below) are likely caused by gene amplification. In contrast, the remaining five USC cell lines were found to be negative for c-erbB2 gene amplification  were tested by real-time PCR for evaluating the expression of Her2/neu at mRNA level. High levels of Her2/neu mRNA transcripts were detected in five out of five (100%) of the FISH-positive cell lines tested, with values ranging from 549.8 to 4993.8 . In contP>0.01). USPC ARK-10, a FISH-positive tumour, was particularly remarkable because the gene amplification and high transcript expression levels  did not correlate with a strong surface receptor expression by flow cytometry. Both breast cancer cell lines were found to express high levels of Her2/neu protein by flow cytometry with an MFI of 654 (BT-474) and 265 (SK-BR-3)  using trastuzumab. In addition, as negative controls, several B-cell lines (EBV-transformed lymphoblastoid B-cell lines) established from the same USC patients from which the tumour cell lines had been established were also studied (data not shown). Four out of ten USC cell lines  showed a very high expression of Her2/neu (mean fluorescence intensity (MFI) ranging from 228 to 710), while 6 out of 10 (1 FISH positive and 5 FISH negative) were found to express significantly lower levels of Her2/neu (MFI ranging from 10 to 47) , P>0.01.SK-BR-3) .−6 ng ml−1, range 1.6–9.1 × 10−6 ng ml−1 per 96 h) in five out of five of the FISH-positive primary USC tested. In contrast, Her2/neu ECD was undetectable in the supernatants of the five Her2/neu low-expressing cell lines. SK-BR-3 and BT-474 tumours were found to secrete large amounts of Her2/neu ECD, 6.4 × 10−6 ng ml−1 and 2.9 × 10−6 ng ml−1 per 96 h, respectively.Cell-free supernatants obtained from all 10 USC cell lines and 2 breast cancer cell lines were collected and analysed for quantitative detection of soluble Her2/neu ECD by ELISA. We found high secretion of Her2/neu ECD (mean 4.6 × 10−1) and patients harbouring Her2/neu 2+ USC  or patients harbouring Her2/neu 1+/negative USC . In contrast, the serum Her2/neu ECD levels of the 10 patients harbouring Her2/neu 3+ tumours had a mean±s.d. of 13.18±7.81, ranging from 6.56 to 28.80 ng ml−1 (P=0.02), or the group of Her2/neu 2+ or 1+/negative USC patients (P=0.02).Serum from healthy female donors and USC patients harbouring tumours with different Her2/neu expression was tested for Her2/neu ECD levels by ELISA. No significant differences were found in the serum levels of Her2/neu ECD between healthy donors  varying from 25:1 and 50:1  USC cell lines overexpressing Her2/neu at 3+ levels and harbouring c-erbB2 gene amplification. In contrast, no Her2/neu ECD was detectable in the supernatants of the remaining five USPC cell lines that did not overexpress Her2/neu. Consistent with these in vitro data obtained using primary cell lines, patients harbouring USC overexpressing Her2/neu at 3+ levels were found to have significantly higher levels of Her2/neu ECD in their serum samples when compared with healthy women or patients harbouring tumours with moderate  or low/negative  Her2/neu expression by IHC (P=0.02). In contrast, no difference was found in serum Her2/neu ECD levels between healthy donors and patients harbouring USC with moderate or low/negative Her2/neu expression.In the current study, using multiple techniques we demonstrated high Her2/neu expression in 5 out of 10 primary USC cell lines established in our laboratory. We also confirmed that USC cell lines overexpressing Her2/neu are sensitive to NK-mediated cytotoxicity in the presence of trastuzumab  of Her2/neu has a constitutively activated kinase domain and its presence has been correlated with poor prognosis and decreased responsiveness to treatments (in vivo activity of trastuzumab (in vivo (P=0.01). Our results are, therefore, in agreement with those published by in vivo.Although multiple mechanisms of action have been attributed to trastuzumab including prevention of Her2-receptor dimerisation, inhibition of tumour proliferation, increased endocytotic destruction of the receptor and inhibition of shedding of the ECD, strong experimental evidence suggests that engagement of Fc receptors on effector cells  represents the dominant component of the in vitro. Furthermore, we demonstrated that patients harbouring high Her2/neu-expressing USC have high amounts of Her2/neu ECD in the circulation. On the basis of these results, prospective evaluation of the biological function of Her2/neu ECD in USC patients overexpressing Her2/neu undergoing trastuzumab-based therapy seems warranted.In conclusion, our results show for the first time that primary USC cell lines that overexpress Her2/neu release high amounts of Her2/neu ECD in the culture media and soluble ECD may significantly block one of the most effective mechanisms of trastuzumab activity


Monocarboxylate transporters (MCTs) are transmembrane proteins that facilitate the transport of important monocarboxylates such as lactate across the cell membrane. Thus, these transporters play a central role in tumour metabolism and, as a result, are attractive targets in cancer therapy, which are now starting to be explored in the clinical context.Expression of key metabolic markers was assessed by immunohistochemistry in 480 prostate samples and prostate cell lines. The levels of glycolytic metabolism were assessed using commercial colorimetric assays. The effect of the MCT inhibitor and thioridazine was evaluated on cell viability by using the Sulforhodamine B assay. Finally, ultra-structural studies were performed by classical electron microscopy transmission techniques.Firstly, we observed an overexpression of proteins involved in oxidative phosphorylation and lipidic β-oxidation in localized prostate cancer, which expression was already evident in precursor lesions. Importantly, only proteins involved in glycolytic metabolism were associated with poor prognosis and the same proteins, despite expressed at low levels in localized tumour, were highly expressed in the metastatic samples. Secondly, prostate cancer cell lines showed important differences at metabolic and ultrastructural levels. The less aggressive LNCaP cells exhibited a more oxidative phenotype whereas the highly aggressive and metastatic cell lines PC3 and DU145 were more glycolytic. Finally, the distant metastatic prostate cancer cell lines were more sensitive to CHC (MCT inhibitor) than LNCaP cells.These studies demonstrate differences in the metabolism of prostate cancer cells, which could be relevant on the development of new diagnostic, prognostic and therapeutic strategies involving metabolic targets.Fundação para a Ciência e Tecnologia (FCT).


Lipolysis is a highly regulated process and is controlled by nervous system, hormones, and paracrine/autocrine factors. Dysregulation of lipolysis is associated with some pathophysiological conditions including diabetes, metabolic syndrome, and obesity. Nowadays, special attention isthereforepaid to study lipolysis using different experimental models. This review summarizes the current experimental methods for studying lipolysis. Culture of preadipocyte cell lines, use of differentiated stroma-vascular cells, primary culture of adipocyte, organ culture of adipose tissue, and microdialysis technique are the most widely used techniques to study lipolysis. The advantages and limitations of using these methods are discussed. It is responsible for the storage of fat as triglyceride (via lipogenesis) during times of energy excess and for the mobilization of triglyceride (via lipolysis) during periods of calories deficit . Hormoneα) , 3–5. Phα) , 4. in vivo estimation of lipolysis, culture of preadipocyte cell lines, primary culture of adipocyte, organ culture of adipose tissue, and microdialysis are of the most used techniques for studying lipolysis in vitro and in situ. This review summarizes the in vitro and in situ techniques for studying lipolysis in animal and humans. Moreover, the advantages and drawbacks of using each method are discussed  and antilipolytic  agents [in vitro studies may not fully reflect in vivo condition due to physiologic responses active in the whole organism. On the other hand, one advantage of this method is that preadipocytes are adherent cell lines and therefore are suitable for studying molecular mechanisms and transcription factors involved in lipolysis process. Also, culture of these cell lines provides an abundant supply of homogeneous cells which make it an appropriate model for screening compounds for their possible lipolytic or antilipolytic effects. Furthermore, newly differentiated fat cells provide a monolayer culture and can be used for studies of long-term regulation of adipocyte functions [There are several murine and human preadipocyte cell lines for studying adipocyte metabolism, for example, 3T3-L1, 3T3-F422A, LS14, LiSa-2, and HPB-AML-I –9. Among) agents , 11. Upo) agents –12. Ther) agents . In addiin vivo conditions of fat tissue complexity more closely than method of preadipocyte cell line culture [Adipocytes are responsible for only 25% of the total cell population of fat tissue. Therefore, nonfat cells comprise a significant part of adipose mass. This part is known as stroma-vascular fraction and includes fibroblasts, macrophages, endothelial cells, blood cells, pericytes, preadipocytes, and mesenchymal stem cells . Culture culture .Primary culture of fat cells was first described by Rodbell . In thisThe chief advantage of this method is the ability to study lipolysis in fully differentiated adipocytes . Also, tin vitro with preservation of hormonal responsiveness and maintenance of gene expression up to 2 weeks [Organ culture system of adipose tissue was first described by Slavin and Elias . In this 2 weeks –39. Lipo 2 weeks –43.in vivo situation and is useful for chronic lipolysis studies. Even, for acute studies, tissues can be culture 24–48 h prior to the treatments, allowing the factors to equilibrate with culture medium and therefore to minimize interindividual variability caused by subject factors such as current health status, hormonal status, and medications [Organ culture system of adipose tissue preserves extracellular matrix and paracrine interactions between different cell types that can influence adipocyte metabolism. Thus, data obtained from this method have a good correlation with those of ications , 34, 40.ications , 33, 34.ications .In organ culture method, like primary culture of adipocyte, suitable fat depot should be selected for studying lipolysis when considering depot-related variations in adipocyte responsiveness –28.Microdialysis technique was introduced over 40 years ago to measure brain neurotransmitters in animal studies. This method was then adapted for adipose tissue to continuous sampling of metabolites  from extracellular space from animals or human. For adipose tissue microdialysis, especial probes are inserted percutaneously after light intradermal anesthesia into the subcutaneous fat. The probes are connected to a microinjection pump and perfused with Ringer solution supplemented with ethanol . For lipolysis evaluation, glycerol concentration in the dialysate is determined and plotted against the perfusion rates , 45. Altin vivo studies. Given the recent increase in attention to study the adipose tissue metabolism, this review was undertaken to compare advantages and limitations of current research methods for studying lipolysis. Several experimental techniques of adipocytes and adipose culture have been described in the litterateur. The techniques are classified into five main categories: culture of preadipocyte cell lines, use of differentiated stroma-vascular cells, primary culture of freshly isolated adipocyte, organ culture of adipose tissue, and microdialysis technique. Use of preadipocyte cell lines, differentiated stroma-vascular cells, or freshly isolated adipocytes provides abundant supply of fat cells which is suitable for screening substances for their lipolytic or antilipolytic activities and for study molecular mechanisms of lipolysis. Therefore, when the possible effect of a substance is tested for the first time, these methods are preferred. Nevertheless, cell lines or isolated cells may not exactly represent the responsiveness and the full spectrum of metabolic characteristics of adipose tissue . On the in vitro and in situ methods for studying lipolysis in the hope that chose of appropriate method for a given study aim will be considered. Taken together, this present review provides an initial comparison of different


Cell lines producing biopharmaceuticals with high yield and high quality in a regulatory compliant environment are a prerequisite for cost effective bioproductions. The development of these production cell lines often includes screening strategies combined with gene amplification and limited dilution experiments, a time consuming process. Especially gene amplification tends to interfere with clonal stability. Limited dilution, especially using a serum-free culture environment is prone to failure and low clone yields.We present here the SEFEX platform technology for the development of non-amplified high yield production cell lines. The strategy is based on a regulatory compliant method for transfection and single cell cloning using a proprietary, fully tested, CHO-K1 host cell line adapted to chemically defined medium. Fast track cell lines were generated by selection of single cell derived clones within 2.5 months after transfection. 1.0 g/L product concentration were achieved after two rounds of process development using these cell lines. Optimized cell lines were developed based on fast track cell lines employing a second transfection. These cell lines were capable for production of 2.6 g/L during an early phase of process development.et al. in this issue [Cell line development with the SEFEX technology comprises steps carried out entirely under serum-free conditions including transfection, selection, and single cell cloning. Fast track cell lines were developed within 2.5 months by transfection and selection of stably transfected cells followed by single cell cloning. During single cell cloning, semi-automated photo documentation at the single cell level is used to assure and document clonality in a regulatory compliant manner. Table is issue . NeverthFigure The SEFEX platform technology for the development of non-amplified high yield production cell lines is based on a regulatory compliant, proprietary CHO-K1 host cell line adapted to chemically defined medium. Fast track cell lines, which were available within 2.5 months produced 1.0 g/L product. Optimized cell lines, which were developed based on fast track cell lines, were capable for production of 2.6 g/L in early phase of process development. Using the SEFEX platform technology, all development steps were carried out under entirely serum-free or chemically defined media conditions including transfection, selection, and single cell cloning. Clonality was assured and documented in a regulatory compliant manner using photo documentation of single cells in cell cloning experiments. The serial transfection cell line development strategy described here provides the possibility to develop production cell lines meeting industrial demands employing simplest process development procedures within minimized time frames.


Breast cancer is a genetically and phenotypically complex disease. To understand the role of miRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer cell lines to identify miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations.Using a microarray carrying LNA™ modified oligonucleotide capture probes), expression levels of 725 human miRNAs were measured in 51 breast cancer cell lines. Differential miRNA expression was explored by unsupervised cluster analysis and was then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer.ERBB2 overexpression and 24 with E-cadherin gene mutations, which are frequent in this subtype of breast cancer cell lines. In contrast, 31 miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in breast cancer cell lines that are not of luminal origin. Thirty miRNAs were associated with INK4 p16status while only a few miRNAs were associated with BRCA1, PIK3CA/PTEN and TP53 mutation status. Twelve miRNAs were associated with DNA copy number variation of the respective locus.Unsupervised cluster analysis using the most variably expressed miRNAs divided the 51 breast cancer cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of breast cancer cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group, 39 miRNAs were associated with ERBB2 overexpression, INK4a p16or E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of these genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.Luminal-basal and epithelial-mesenchymal associated miRNAs determine the subdivision of miRNA transcriptome of breast cancer cell lines. Specific sets of miRNAs were associated with  Numerous lines of evidence indicate that breast cancer is genetically and epigenetically not just one disease but a diverse group of diseases with diverse clinically relevant biological and phenotypical features. Recent technological advances in molecular profiling have led to the identification of an increasing number of molecular subtypes in breast cancer, each with distinct co-regulated and anti-regulated genes. However, the biology of these molecular subtypes and their underlying genetic drivers may be affected by numerous biological factors, including miRNAs.miRNAs are a class of small nonprotein-coding genes that regulate the expression of genes post-transcriptionally via sequence-specific interaction with the 3' UTR of target mRNAs, resulting in inhibition of translation and/or mRNA degradation ,2. A larin vitro cultured conditions and in vivo when raised as xenografts .Total RNA (200 ng) was reverse transcribed, copied into double-strand cDNA and labeled to yield biotin-labeled cRNA using the 3' IVT express Kit  according to the manufacturer's instructions. Biotin-labeled cRNA was subsequently fragmented and loaded onto an Affymetrix GeneTitan instrument. The hybridization cocktail was applied to Human Genome HT_HG-U133_Plus_PM GeneChip 96-well arrays. All steps including hybridization, washing and scanning were carried out automatically inside the instrument. The raw data (.CEL files) were normalized by the RMA method using the default settings of the Affymetrix Expression ConsoleESR1, GATA3, TFF3, and FOXA1; ERBB2-positive cell lines that showed higher expression of ERBB2, GRB7, and STARD3; basal-like cell lines, which were characterized by higher expression of KRT5, KRT17, BST2, and FABP7; and normal-like/claudin-low cell lines that did not show KRT5 and KRT17 expression and have low expression of claudin 3, claudin 4, and claudin 7 genes. The ERBB2-positive cell lines were further designated as luminal-ERBB2-positive and estrogen receptor (ER)-negative/ERBB2-positive cell lines, because the former in addition to ERBB2 overexpression also show ESR1 gene expression on microarray while the latter do not.For subtype classification of the cell lines, Perou and colleagues' intrinsic gene set of 496 genes  were matmiRNA expression profiling was performed using miRNA microarrays according to a previously published method . In brieGenomic DNA from all cell lines was extracted using the QIAamp DNA Mini Kit (Qiagen). Genomic DNA (500 ng) was used as the starting material to capture genome-wide chromosomal aberrations with the aid of the Affymetrix Genome-Wide Human SNP 6.0 array technology. The steps were performed according to Affymetrix's recommended protocols. In summary, after digestion of the genomic DNA using either restriction enzymes NspI or StyI, adaptors were ligated to the obtained DNA fragments. These fragments were subsequently amplified using PCR, fragmented, end-labeled with biotin and hybridized onto GeneChip SNP 6.0 arrays. After hybridization the arrays were washed and scanned to generate the raw data (.CEL files) using the Affymetrix Genotyping Console™ software. The chromosomal gains and losses were calculated using SNP copy number variation (CNV) on the same chromosome. The chromosomal regions containing gains or losses were correlated with the expression level of the miRNAs located on the same genomic regions. The SNP data were deposited in the GEO data repository [GEO:GSE41313].INK4a, BRCA1, E-cadherin, PIK3CAp16, and PTEN and promoter hypermethylation analysis of E-cadherin were previously reported for all cell lines except for HCC cell lines. These were separately analyzed according to the previously published methods [Protein expression data of ER, progesterone receptor, ERBB2, epidermal growth factor receptor, and cytokeratin (CK) 5, CK8-18, CK19, and CK14 were used from previously published work , except  methods  (data prt test in BRB-array tools 3.7 developed by Dr. Richard Simon and the BRB-ArrayTools Development Team). A permutation value of P < 0.05 was considered statistically significant and used to select differentially expressed miRNAs for supervised hierarchical cluster analysis. The associations between continuous variables were tested using Spearman Rank correlation . The Kruskal-Wallis test was used to evaluate differences among groups and P < 0.05 was considered statistically significant.Hierarchical clustering analyses of significant miRNAs were performed using Cluster 3 software  and the The 51 human breast cancer cell lines that were used in this study are listed in Table A Pearson correlation based on the top 10% of variably expressed genes classified the cell line cohort into two obvious groups . The basal-like cell lines within the ER-negative/basal-group expressed basal and luminal cytokeratins , whereas all normal-like/claudin-low cell lines lacked the expression of these cytokeratins except for SK-BR-7 that exhibited CK8-18 expression (Table miRNA expression profiling was carried out using microarrays containing LNA™ capture probe sets against 725 human miRNAs. After filtering out miRNAs that were undetectable in most of the cell lines (57%), 413 miRNAs remained and were selected for further analysis. Unsupervised hierarchical clustering based on the most variable 87 miRNAs (with standard deviation ≥ 0.75) revealed two prominent cell line clusters: a minor cluster that included 18 cell lines and a major cluster that included 33 cell lines  was significantly higher expressed in the minor cluster as opposed to other miRNAs  showed significantly higher expression in the major cluster compared with the other miRNAs (fold change ≥ 1.5)    showed significantly higher expression in both ER-positive cell lines and primary tumors, and one miRNA (hsa-miR-532-3p) showed significantly lower expression in both ER-positive cell lines and ER-positive tumors  and ER-negative  cell lines, respectively, we determined whether differential expression of miRNAs between the two clusters is driven by ER status of the cell lines. For this analysis, we grouped cell lines into ER-positive and ER-negative based on mRNA expression of ER and performed supervised cluster analysis on these groups. We identified 79 differentially expressed miRNAs between ER-positive and ER-negative cell lines (8 of 33 aP < 0.05)   were highly expressed (fold change ≥ 2)  . Notably, the INK4a p16miRNA signature in the ER-negative/basal-group of cell lines appeared to be pronounced, and when used for clustering could almost completely separate the cell lines mutant for the INK4ap16gene from those that were wild-type. One should, however, emphasize that the tumor suppressor genes INK4a p16and ARF p14are co-localized and are often simultaneously lost amongst cell lines. We therefore cannot determine which of these genes might drive the differential expression of these miRNAs.In our cohort, 13 cell lines had lost the PIK3CA mutant cell lines  and eight PTEN mutant cell lines . Both PIK3CA and PTEN are components of the PI3K signaling pathway and mutated mutually exclusively in clinical specimens [PIK3CA or PTEN mutant versus wild-type cell lines for these genes did not yield a considerable number of differentially expressed miRNAs. We therefore combined all cell lines harboring mutations either in PIK3CA or PTEN and compared them with wild-type cell lines. As PIK3CA/PTEN mutations are present in both the luminal-group and the ER-negative/basal-group of cell lines, we therefore performed multiple analyses. First, we analyzed all cell lines and found 49 miRNAs, which were differentially expressed between mutant and wild-type cell lines . Finally, we analyzed differentially expressed miRNAs between TP53 mutant cell lines (n = 39) and wild-type cell lines (n = 9) and found 18 to be associated with the TP53 mutation status  , hsa-miR-22 (17p13.1), hsa-miR-93 (7q22.1) and hsa-miR-383 (8p22) - with DNA CNVs in breast cancer cell lines are shown in Figure Previous studies show that DNA CNVs in breast tumor tissues can lead to differential expression of genes and miRNAs -35. To iHuman breast cancer cell lines are renewable resources that are extensively utilized as reliable workhorses to explore biological functions of clinically relevant molecules in breast cancer. Extensive molecular characterization and gene mutation analysis by us and other researchers have suggested that breast cancer cell lines have retained significant molecular features that are commonly observed in clinical breast tumors ,37. ThisIn this study, we demonstrated that global miRNA expression profiling can assign our cell line collection into two clusters. These two clusters predominantly mirror the ER-based dichotomy present in human breast cancer cell lines, which may point to the fact that, like mRNA, miRNA expression profiling allows the discrimination between ER-positive and ER-negative cell lines. This suggests that a significant number of miRNAs may be under the control of ER regulation. In line with this, we observed a trend of finding ER binding sites closer to the significantly differentially expressed miRNAs between ER-positive and ER-negative cell lines than nonsignificant miRNAs  appear to show consistently higher expression across all the ER-positive cell lines (fold change ≥ 1.5), and as yet only hsa-miR-26a has been implicated in breast carcinogenesis whereas hsa-miR-26a and hsa-miR-92b have also been implicated in brain tumors [hsa-miR-26a miRNA downregulates EZH2 and is therefore related to a favorable outcome on tamoxifen in metastatic breast cancer [CDK4 and CENTG1 oncogenes and forms an integrated oncomir/oncogene DNA cluster, which promotes glioblastoma tumor growth via RB1, PI3K/AKT, and JNK pathways [hsa-miR-92b has been found to be exclusively overexpressed in primary brain tumors but serves as a biomarker to discriminate brain primary cancer from metastasis [hsa-miR-191, hsa-miR-492) have been linked to hepatic cancer. The increased expression of hsa-miR-191 stimulates proliferation in hepatocellular carcinoma cell lines and its therapeutic targeting suppresses tumor masses in vivo [hsa-miR-492 miRNA has been shown to be processed from the keratin 19 gene and upregulated in metastatic hepatoblastoma [Among these potentially novel ER-related miRNAs, four miRNAs , hsa-miR-155, and hsa-miR-622 miRNAs. Several studies have implicated these miRNAs to be involved in epithelial-mesenchymal transition (EMT), to be related to the stem-cell-like phenotype, and to be associated with a switch in paclitaxel responsiveness [Another major contribution to the overall miRNA profiling of this cell line cohort is the identification of a signature of 42 differentially expressed miRNAs, which discriminates between basal-like and normal-like/claudin-low breast cancer cell lines. These miRNAs together with the ER-associated miRNAs are the major determinants of the overall clustering of the cell lines. Importantly, this signature includes all four members of the siveness -47. In bsiveness ,49. Impohsa-miR-200 family and the mRNA expression level of the ZEB1 transcription factor, which is a well-known target of this miRNA family  [hsa-miR-155 and hsa-miR-622, which were also linked to enhanced tumorigenesis in various cancer types besides breast cancer [in vivo when raised as xenografts in nude mice . Importantly, this signature includes three previously unknown miRNAs  found to be associated with cell lines frequently showing EMT-like characteristics.Interestingly, we observed a significant inverse correlation between the expression levels of all miRNAs of the CD24low) -53. Othet cancer ,55. In cE-cadherin mutation and promoter hypermethylation include distinct miRNAs. This may point to an existence of unique biology at the miRNA level in tumors that show E-cadherin inactivation due to gene mutation rather than those that lose E-cadherin expression due to promoter hypermethylation. Important to note is that E-cadherin promoter hypermethylated cell lines include all normal-like/claudin-low cell lines, and most EMT-related miRNAs were also associated with E-cadherin promoter hypermethylation.We observed that the miRNA signatures associated with INK4a p16mutant cell lines can strongly discriminate between mutant and wild-type ER-negative cell lines. INK4a P16is a tumor suppressor gene located on chromosomal band 9p21 that has been frequently altered in many human cancers [INK4a P16regulates cell cycle progression by targeting CDK4/6 through the pRb signaling pathway. Interestingly, this signature includes some miRNAs that are involved in cell cycle regulation. For instance, hsa-miR-100, which is highly expressed in the INK4a p16wild-type cell lines, targets the RBSP3 gene that in acute myeloid leukemia regulates the cell cycle through partial modulation of pRB/E2F1 [hsa-miR-34a has been reported as a suppressor of cell proliferation and migration in colon cancer [BRCA1, TP53 and PIK3CA/PTEN; these observed associations require further independent validation in breast cancer specimens.With respect to common genetic aberrations present in breast cancer, our study reveals differentially expressed miRNA signatures associated with commonly mutated tumor suppressors and oncogenes. Most significantly, a signature of 30 miRNAs associated with  cancers . P16INK4pRB/E2F1 . hsa-miRn cancer  and its n cancer . BesidesOur finding that the differential expression of miRNAs is associated with ERBB2-overexpressing luminal-type cell lines is also intriguing since the miRNAs showing elevated expression in ERBB2-positive cell lines (fold change ≥ 1.5) are not located on the ERBB2 amplicon (17q12) and thus may be regulated indirectly by genes in the ERBB2 amplicon. It is also important to mention that a majority of these miRNAs have already been implicated in various types of epithelial carcinoma including breast cancer -64. One hsa-miR-130a, hsa-miR-93, hsa-miR-383, hsa-miR29c, hsa-miR-382, hsa-miR-31) were already found to be located in regions that exhibited DNA copy number abnormalities in breast cancer tumors [hsa-miR-22 previously shown to be regulated by ER [Finally, we also identified 12 miRNAs to be associated with CNVs in breast cancer cell lines  in breast cancer cell lines and thus these miRNAs may contribute to the function of these oncogenes/tumor suppressors. Finally, certain miRNAs themselves are located in genomic regions, which frequently show genetic aberrations, implying that these miRNAs themselves may have a potential driver role or contribute to known drivers in these genomic regions. Our current findings call for further validation of these signatures in clinical specimens. Importantly, our study provides a unique molecular miRNA portrait of human breast cancer cell lines, which can be exploited for functional studies of clinically important miRNAs.In summary, our analyses show that the dichotomy in breast cancer cell lines is, in line with the general consensus in the field, related to ER status. However, part of this dichotomy may be related to ER-independent luminal versus basal differentiation status of cell lines. Secondly, another prominent dichotomy observed among breast cancer cell lines discriminating basal-like cell lines from normal-like/claudin-low cell lines involves miRNAs related to EMT and/or stemness. Furthermore, we reveal sets of miRNAs associated with genes frequently amplified (ERBB2) or mutated  miRNA differential expression associated with PIK3CA/PTEN mutations in all, the luminal-group, and the ER-negative/basal-group of breast cancer cell lines. (B) miRNA differential expression associated with BRCA1 mutation. Yellow and blue, high and low overall similarity of samples in miRNA expression, respectively. Figure S3 showing differential expression miRNAs between cell lines mutant and wild-type for the TP53 gene. Cell lines with TP53 heterozygous mutation (OCUB-F) and with undetermined TP53 mutation status  were excluded from the analysis. Yellow and blue, high and low overall similarity of samples in miRNA expression, respectively. Figure S4 showing comparison of median genomic distances of ER binding sites and significantly and nonsignificantly differentially associated miRNAs with ER status of the cell lines. The Mann-Whitney test was used to compare the median genomic distances of ER binding sites and miRNAs.Click here for file


There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance. At the same time, the reduction in sequencing costs leading to the democratization of molecular testing is already resulting in many patients having their tumors typed at a molecular level. The tumor characterization efforts are no longer rate limiting; rather how to interpret and “act” on the data is now the major limiting factor. These challenges must be overcome before emerging technological advances in tumor characterization can deliver maximum clinical impact. A key step in the process is the identification of biomarkers that would predict response to treatment and the parsing of actionable driving molecular aberrations from noise. These challenges can be solved by implementing algorithms that help analyze the data, in parallel to establishing large scale humanized model systems for high throughput target discovery and validation that will also inform an accelerated drug development and clinical trial process. Robust predictive biomarkers for combinatorial molecular medicine are urgently needed to change the clinical trial landscape from the current state of low therapeutic efficacy in large clinical trials and unselected populations, to high efficacy small clinical trials enriched for target populations. This approach has the potential to make clinical trials smaller, faster, and cheaper, while increasing the benefits for individual patients.A major emerging challenge in the wake of the tsunami of data generated by efforts to characterize tumors at the molecular level , and 14 compounds tested on 500 cell lines (McDermott set) were obtained from public databases  [11]– being used as a second selection criterion. These approaches were applied to the training and test sets respectively.Observed co-mutation rates were compared to predicted co-mutation rates; Co-mutations that occur http://rana.lbl.gov/EisenSoftware.htm) Cluster software was used to adjust GI data prior to hierarchical clustering. For each of the 37 compounds, GI values were first median centered then normalized; this produces a scaled growth inhibition score that is a potency-independent means of comparing response profiles across compounds. The data was then hierarchically clustered using Pearson’s correlation as a metric based on the average distance between nodes. Treeview was used for visualization of the resulting clusters. The Cluster and Treeview software are available from the Eisen laboratory (Pearson’s Chi-square test (for co-events) and Fisher’s exact test (for single events) were used to assign two-sided P values at 95% confidence interval to describe the correlations between gene mutations/copy number aberrations and drug sensitivity. For the determination of statistically significant single genomic events, with only a few tests run, this obviated the need to correct for multiple comparisons. When the space of two co-events was determined in the 310 cell lines , S/R, was computed for any gene aberration present in more than 12% of the sensitive or resistant lines. Any gene frequency that was altered at more than 1.5 fold in sensitive vs resistant lines was considered to be associated with sensitivity to the drug (S/R>1.5) or resistance to the drug (S/R<0.67), respectively. This identified genomic events associated with sensitivity or resistance to specific drug intervention; the statistically significant events are found in Table S6 in As an example, lines that were sensitive to the MEK inhibitor GSK1120212 were 2.93 times, 2.35, 1.92, and 1.67 times more likely to harbor a BRAF, KRAS, APC mutation or CDKN2A(p14) mutation, respectively , whereas resistant lines were 2.57 more likely to harbor an event in RB1 (P = 0.0024) . BRAF anLines sensitive to the AKT inhibitor GSK690693 expectedly harbored mutations in the PI3K pathway, including PIK3CA, PTEN, ERBB2, and also FBXW7, TET2, and BRCA2 alterations  again suggesting unexpected genomic biomarkers of response to AKT inhibitors that could increase the number of patients likely to benefit; there were no single events significantly associated with resistance  (PIK3CA aberrations conferred resistance to the BRAF inhibitor AZ628 (P = 0.042) an observation that was confirmed in the test set (P = 0.05), probably through bypass activation of the parallel PI3K pathway, recapitulating results from experimental intervention = 0.001) .Although there was a strong association of single genetic aberrations with response to therapeutic agents, this association was not absolute with a number of cell lines containing any specific event being scored as either sensitive or resistant. Thus there must be additional events that cooperate with the mutational status to determine sensitivity and resistance to targeted therapeutics. Other than the associations noted above and aberrations in PTEN and ERBB2 potentially contributing to the clustering of EGFR family inhibitors and aberrations in PTEN and CDKN2A being associated with drugs targeting the PI3K and IGF1R pathways, there were no clear aberrations driving the majority of the drug response clusters . Again tTo identify potential cooperating events, we first identified genomic events that co-occurred beyond what is expected if they were independent and the association due to chance factors alone. This defines co-events that were likely under selective pressure during tumor initiation or progression (or during adaptation to culture) and thus have a high probability of being drivers of the cancer phenotype. Molecular lesions whose co-occurrence leads to a proliferative or survival advantage beyond what is observed for separate single occurrences will be co-selected and the frequency of the co-event will increase in the population . Based oTo determine whether co-events predicted response to therapeutic agents, we compared predicted to observed frequencies for each drug in sensitive lines and resistant lines respectively, and also determined which co-events occurred at different frequencies in sensitive vs resistant lines , while BRAF::TP53 and BRAF::CDKN2A were associated with sensitivity in both sets . These results are consistent with recent demonstration that RAF inhibitors inhibit ERK signaling in cells with mutant BRAF but unexpectedly enhance signaling in cells with wild-type BRAF, and that KRAS activation appears to promote RAF dimerization and resulting MEK activation, compatible with resistance seen here in the context of KRAS::NOTCH1, KRAS::STK11 and KRAS::SMARCA4  For the BRAF inhibitor AZ628  double g−6, test set), while ERBB2::TP53, KDR::STK11  were associated with sensitivity to the intervention !)], and 6871 observed two co-events. Negative differences furthest from zero are mutually exclusive events where the observed frequency is zero; the rest are partially exclusive events  in cell lines were associated with a specific drug response phenotype. For the 294 cell lines (GSK set) for which genomic information was available, we computed the occurrence of each mutation-lineage pair; there were 2132 such pairs (e.g. BRAF::Melanoma). We tabulated all the lineages that co-occurred with each mutation , and for each drug how many of each pair occurred in sensitive vs resistant lines , and compared that, respectively, with the chance random distribution between sensitive and resistant, at P< = 0.05.As an example, we examined the response of tumors harboring the same mutation across lineages to the MEK inhibitor GSK1120212. The single aberrations that were significantly associated with response above  were tesThese observations were reproduced when tested in other drugs. For the AKT inhibitor GSK690693, PIK3CA aberrations predicted resistance only in colon cancer cell lines, and PTEN mutations predicted resistance in CNS and melanoma cell lines but there were no associations when they occurred in other lineages   showed a trend to resistance regardless of lineage  Translating the cancer genome into highly efficacious targets to improve patient outcomes is a major emerging challenge. The last ten years in cancer research have delivered many breakthroughs, among them, first, the demonstration that oncogene-addicted tumors can be specifically targeted with single agents to successfully halt and revert cellular proliferation, at least transiently Single targeted interventions have had limited success in cancer http://www.cancergenome.nih.gov). Our data suggests that combined targeting of specific co-aberrations in the mountain and in the tail will be needed to maximize response and minimize the emergence of resistance.One of the major emerging challenges post data production from massively parallel sequencing is the need to differentiate “driver” from “passenger” mutations. Genetic aberrations that are conserved horizontally across individuals (i.e. hot spot mutations) demonstrate selection for a change in function and identify genomic aberrations that play an important role in tumorigenesis providing one mechanism for identifying driver mutations. Cancer is a microevolutionary process . We usedAs opposed to the Knudson double hit theory where two consecutive hits are required to initiate cancer It is expected that the emerging understanding of the molecular mechanisms of cancer will transform the clinical trial landscape from a trial and error system to a system based on a rational targeting of the molecular lesions that drive cancer. Our results suggest that early in the drug development process, a drug should be tested on a large panel of highly characterized human cell lines to screen for genomic biomarkers that are associated with sensitivity and resistance to the drug . The first inflection point is determined mathematically as described in the text; cell lines to the left of the inflection point are defined as sensitive, those to the right as resistant. Figure S2, Unsupervised hierarchical clustering of the sensitivity  of 310 cell lines to 37 targeted drugs. Magnification of Figure S3, Unsupervised hierarchical clustering of the sensitivity  of 310 cell lines to 23 targeted drugs from the GSK set. To decrease noise, the same data as in Figure S4, Unsupervised hierarchical clustering of the sensitivity  of 141 cell lines to 14 targeted drugs from the McDermott set. To decrease noise, the same data as in Figure S5, Flow diagram for the analysis of interaction of mutation and lineage in predicting response to a drug. Figure S6, Hierarchical clustering of the sensitivity of 18 distinct cancer lineages to 37 targeted drugs. 302 cancer cell lines corresponding to 18 lineages with more than 3 cell lines tested per lineage were included. For each of the 37 compounds, GI values were first median centered then normalized to account for differences in potency. Hierarchical clustering was performed on the median for each lineage and drug. Increasing sensitivity of a lineage is indicated by the increasing intensity of the green signal, and increasing resistance is indicated by the increasing intensity of the red signal. Lineages not screened with a particular compound on a minimum of 2 cell lines are indicated in gray.(PDF)Click here for additional data file.File S2Figure S7, Genomic event prevalence by lineage of origin for the GSK set (n = 294). The frequencies of the individual genomic events (mutation and/or copy number aberration) for the 262 affected genes were tabulated as a heatmap against the lineage of origin for 294 cell lines with genomic information available comprising 20 lineages with more than one cell line per lineage . The occurrence of a mutation and/or copy number aberration for one gene in one cell line was counted as one occurrence. The frequency of each genomic event is indicated by the increasing intensity of the green signal.(XLSX)Click here for additional data file.File S3Tables S1–S26.(XLSX)Click here for additional data file.


Tubifex. We investigated the 4d lineage in the gastropod Crepidula fornicata, an established model system for spiralian biology, and in a closely related direct-developing species, C. convexa.Animals with a spiral cleavage program, such as mollusks and annelids, make up the majority of the superphylum Lophotrochozoa. The great diversity of larval and adult body plans in this group emerges from this highly conserved developmental program. The 4d micromere is one of the most conserved aspects of spiralian development. Unlike the preceding pattern of spiral divisions, cleavages within the 4d teloblastic sublineages are bilateral, representing a critical transition towards constructing the bilaterian body plan. These cells give rise to the visceral mesoderm in virtually all spiralians examined and in many species they also contribute to the endodermal intestine. Hence, the 4d lineage is an ideal one for studying the evolution and diversification of the bipotential endomesodermal germ layer in protostomes at the level of individual cells. Little is known of how division patterns are controlled or how mesodermal and endodermal sublineages diverge in spiralians. Detailed modern fate maps for 4d exist in only a few species of clitellate annelids, specifically in glossiphoniid leeches and the sludge worm C. fornicata and C. convexa. We present a new nomenclature to name the progeny of 4d, and report the fate map for the sublineages up through the birth of the first five pairs of teloblast daughter cells (when 28 cells are present in the 4d sublineage), and describe each clone’s behavior during gastrulation and later stages as these undergo differentiation. We identify the precise origin of the intestine, two cells of the larval kidney complex, the larval retractor muscles and the presumptive germ cells, among others. Other tissues that arise later in the 4d lineage include the adult heart, internal foot tissues, and additional muscle and mesenchymal cells derived from later-born progeny of the left and right teloblasts. To test whether other cells can compensate for the loss of these tissues , specific cells were ablated in C. fornicata.High-resolution cell lineage tracing techniques were used to study the 4d lineage of Crepidula.Our results present the first fate map of the 4d micromere sublineages in a mollusk. The fate map reveals that endodermal and mesodermal fates segregate much later than previously thought. We observed little evidence of regulation between sublineages, consistent with a lineage-driven cell specification process. Our results provide a framework for comparisons with other spiralians and lay the groundwork for investigation of the molecular mechanisms of endomesoderm formation, germ line segregation and bilateral differentiation in  Endomesoderm is an ancient cell type that gives rise to a wide array of tissues in bilaterians and its origin and diversification are the subjects of great interest and debate -3. One o, but 4d gives rise to mesoderm in all embryos that have been studied 54]). NotCrepidula[et al. [et al. [The origin of heart tissue in mollusks is controversial, and has been ascribed to the 2c and/or 4d micromeres in different studies ,77,86-88Crepidula. Hejnol a[et al.  found th [et al.  was carrIlyanassa, Render [Ilyanassa[et al. [Ilyanassa. 4d ablations in Ilyanassa are particularly informative because, mostly, only 4d-derived tissue is affected, as most  of the organizing activity of the D quadrant resides in the 3d macromere [Crepidula serves as the principle embryonic organizer and when it is ablated the embryo becomes radialized and does not form a heart [In , Render  reported, Render . Yet it Ilyanassa,76,90. Ma[et al.  also obsacromere ,91. By c a heart ,92. Cons2.2/R2.2 cells are the progenitors of the germline in Crepidula. This claim is based on several lines of evidence, including comparisons with other model systems, their behavior during gastrulation and their position in the larva. There are claims that the germ cells arise from the pericardial mesoderm such as in the gastropod Viviparus[Crepidula we now know that the heart tissue must arise from a different set of precursor cells than those that give rise to the germline (see above).It is generally accepted that the germline arises from the 4d lineage in spiralians ,73,93-96Viviparus. Their c2.2/R2.2 cells are mitotically quiescent; we never observed either of these two cells dividing up through the veliger stage. The 2mL2.2/R2.2 cells break away from the mesodermal bands during gastrulation and appear to make amoeboid-like movements while forming dynamic filopodia , after which staining disappears. Rabinowitz et al. [in situ as well as with antibody staining and found that both the mRNA and the protein were initially expressed ubiquitously, then became restricted to the 4d cell. IoNanos protein is mostly associated with the teloblasts, but interestingly it persists longest in the 4dL112 and 4dR112 cells, equivalent to 2mL2/R2 cells in our nomenclature . After these teloblasts each form three rounds of mesodermal progenitors, the two remaining teloblasts  each divide equally giving rise to two cells, and these in turn enter a long period of quiescence. Woods [Sphaerium, the germline comes from the equivalent of the 3 M teloblasts.The origin of the germline within the 4d clone has been examined in a few other spiralians. In z et al.  reportede Figure . This ise et al.  suggestee et al. ,53. In tm, Woods ,53 did ae. Woods ,53 indicHelobdella and Tubifex, a combination of lineage tracing, ablation and in situs for nanos and vasa have shown that the germline arises from M teloblast-derived segmental mesoderm (sm) blast cell clones contributing to midbody segments [Crepidula, Ilyanassa or Sphaerium. The germline is known to arise from the 4d lineage in the polychaetes Capitella[Platynereis[et al.[Platynereis, the germline comes from the first two daughters born from the teloblasts , which come to reside in the posterior growth zone of the larva.In segments ,95. ThusCapitella,65,66 anatynereis, but foris[et al. showed tCrepidula, these embryos exhibit early unequal cleavage divisions and the D quadrant is specified at the four-cell stage. In Crepidula, however, the early cleavage divisions appear equal and the D quadrant is specified later in development by virtue of inductive interactions from the animal micromeres. There is, however, a bias in terms of which quadrant normally becomes the D quadrant, as only one blastomere (the presumptive D cell) inherits the small polar lobe during the second cleavage division. Whether or not these small polar lobes play a role in germline specification remains to be determined.By all accounts, it appears that the germline arises from 4d in spiralians Table , but theIlyanassa in terms of the progeny of the ML (which she referred to as ME1) and MR (which she referred to as ME2) cells. ME1 (ML) contributed to the formation of the larval retractor muscle and the intestine. ME2 (MR) contributed to the formation of the retractor muscle, intestine, the kidney and the heart. Here we have not been able to detect any obvious differences in the fates between the two teloblasts. In fact, the earlier lineages exhibit a great deal of symmetry. It is unclear, however, if there are any later asymmetries that may arise from progeny of the 5ML and 5MR teloblasts after the birth of 5mL/R.Render  reportedD-F; note that we did not manage to remove 5mL/R). The retractor muscles are formed mainly by 2m cells and some later-born progeny of the 5M teloblasts. Removal of 2mL and 2mR cells leads to disorganized larval retractor muscles, but they are still visible  is specified autonomously at or very near their birth [We have observed that, in general, when 4d daughter cells are removed, the structures derived from them are missing and little effect is seen in other lineages Figure . However8ir birth ,107,108.Ilyanassa, the 4d cell gives rise to intestine, heart and various other structures [Ilyanassa. In this species, the 3d macromere carries out a majority of the organizing activity, but the 4d cell has some organizing activity as well, which might explain some of the ectodermal defects in 4d ablations [Crepidula, one might expect to see analogous ectodermal defects when it is ablated. On the contrary, we have not observed dramatic ectodermal defects in our ablations of 4d sublineages, including no obvious reduction of the shell.In ructures . When 4dructures . The sheructures  suggestsblations . Since tC. convexa is a direct-developing species [C. fornicata, there is accelerated development of the head, foot and the adult shell. Furthermore, we and others have observed that the foot does not form an operculum [C. fornicata, do form an operculum, as do direct-developers produced from small eggs that increase in size by consumption of nurse eggs , but these are subsequently lost at metamorphosis. No adult calyptraeids have an operculum [C. convexa does form some transient larval structures, such as components of the larval kidney complex, that include the external absorptive cells and the autofluorescent crystal cells, which are eventually lost at or shortly after hatching. The cleavage pattern of the teloblasts is identical to that in C. fornicata, as has been observed through the formation of the 4mL/R and 4ML/R teloblasts , segmentation  and the formation of a coelomic cavity . A crucial intermediate step between the spiral cleavage program and the clade-specific generation of the bilaterally symmetric adult  body plan is the bilateral division of the 4d cell. Although formation and bilateral division of the 4d cell is often considered one of the most conserved features of spiralian development  divides making exceptionally large M teloblasts that undergo highly asymmetric divisions and the daughter cells are always born at the same spot off of the teloblast, generating a coherent chain of cells ,62,81,82he clone ,21. For eloblast ,112, whieloblast ,113,114.Studying spiralian development in a phylogenetic framework has the potential to reveal how novel characters arise within a stereotyped cleavage program. The 4d lineage gives rise to numerous tissues, some of which are almost certainly homologous across spiralian phyla such as the heart, germline and intestine. Other 4d-derived tissues are specific to particular groups such as the retractor muscles in mollusks, the larval kidney complexes in caenogastropods, or the proboscis of glossiphoniid leeches. Clearly, these evolutionarily novel tissues can be readily incorporated into the early division of the 4d lineage, since the founder cells of each arise within the first few divisions of the teloblasts (this work and ). HavingCrepidula, multiple teloblast sublineages give rise to endoderm, while other sublineages give rise to mesodermal fates. Likewise, in Ilyanassa, 4d gives rise to both mesoderm and endoderm [Sphaerium, separation of endoderm and mesoderm happens at the birth of M (4d) and D (4D); 4d gives rise exclusively to mesoderm (and germline) and 4D gives rise exclusively to endoderm . If Together, these studies uncover significant diversity in terms of the contributions of 4d in these different spiralian embryos. In reviewing the information summarized in Table The authors declare that they have no competing interests.All authors contributed to the design and execution of the experiments. DCL and JQH prepared the first draft of this manuscript. All authors contributed to revisions of subsequent drafts. JQH prepared the figures. All authors read and approved the final manuscript.Minus 2mL and 2mR Heart. This movie depicts a typical veliger larva that forms after removal of both the 2mL and 2mR derivatives of 4d. The larva is depicted as a right lateral view with the shell that surrounds the opaque visceral mass located to the left and the more transparent head and velum to the right. Note the presence of a beating heart towards the top of the larva, which is located between the visceral mass and the head.Click here for file


It is widely accepted that the (now reversed) Bush administration’s decision to restrict federal funding for human embryonic stem cell (hESC) research to a few “eligible” hESC lines is responsible for the sustained preferential use of a small subset of hESC lines  in basic and preclinical research. Yet, international hESC usage patterns, in both permissive and restrictive political environments, do not correlate with a specific type of stem cell policy. Here we conducted a descriptive analysis of hESC line usage and compared the ability of policy-driven processes and collaborative processes inherent to biomedical research to recapitulate global hESC usage patterns. We find that current global hESC usage can be modelled as a cumulative advantage process, independent of restrictive or permissive policy influence, suggesting a primarily innovation-driven (rather than policy-driven) mechanism underlying human pluripotent stem cell usage in preclinical research. Human embryonic stem cells (hESCs) have attracted much research attention over the last 15 year. Despite of the existence of a steadily increasing number of hESC lines, several studies have noted a preferential use of only a few lines . This policy decision has stirred much controversy In 2001 the Bush administration decided to restrict federal funding through the National Institutes of Health (NIH) to research using hESC lines derived prior to August 9However, the scientific basis for these claims remains obscure We analysed more than 2,300 peer-reviewed studies that documented the experimental use of identifiable hESC lines. Thus, we chose to study the actual use of hESC lines in successfully completed basic and preclinical research projects. This is in contrast to other studies, which have considered a variety of proxies for actual hESC usage including: material transfer agreements on shipping of cell lines from selected providers Database searches for hESC research papers were performed as previously reported Database searches for papers reporting derivation and experimental use of human induced pluripotent stem cells (hiPSCs) were performed according to reference Assignment of papers to specific countries was performed according to the academic affiliation of the corresponding author. A detailed paper list is available on request.We compared our empirical findings with simulations of a Yule-Simon process In our simulations we assumed that all studies use the same number of hESC lines (N = 2). We selected this parameter since it is close to the observed mean number of cell lines used per study in the main connected component of the empirical hESC usage network . To compensate for these minor differences we increased the number of studies in our simulation to 2,927 to arrive at the same overall number of citations.N independent draws with an associated probability p. The newly generated stem cell study is then added to the stem cell research network. If a study does not use a novel line, then the lines employed in the study are chosen from the preexisting cell lines already in use. We took the probability of selecting an individual line from the pool of preexisting hESC lines to be proportional to the number of times each cell line has been previously used in other studies. To estimate the probability with which a novel line is chosen, we ran our model with probabilities of 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3 for introducing a new hESC line into the simulated network. We selected a probability of 0.15 based on comparison with the empirical dataset. The resulting networks are directed bipartite graphs, which are not necessarily connected. In order to obtain statistics of usage we generated 1,000 random simulated instances of our model.In our simulations see Fig. 2B,We used the power.law.fit method from the igraph package with xmin = 3 to estimate the exponent for the in-degree distribution of the network We first analyzed the connected components of the networks. The biggest component contains the largest number of publications. We found the remaining components were dominated by cell lines that were used only in a single study and often appeared in manuscripts that reported the derivation of a large number of new hESC lines. We conclude that the biggest component reflects most hESC research activity. Empirical network analysis and visualization was performed in R as in reference cluster global usage we selected only those countries with at least 30 publications to avoid spurious effects due to small sample sizes. The distances between the 14 identified countries were calculated as one minus the Spearman correlation of the hESC usage patterns. Hierarchical clustering was then performed in Matlab  using average linkage. The assignment of a nationality to study was based on the affiliation of the corresponding author.. The z-score was calculated based on the mean and standard deviation of all cell lines that were first published in the same year.For each cell line, the number of studies it was used in and the year of first publication were determined. A linear regression model using the year of first publication as the independent variable and the logarithm of usage frequency as the dependent variable was calculated using robust linear regression in R using the MASS package We first analysed the usage of hESC lines in 2,338 peer-reviewed studies that reported original experimental research using identifiable hESC lines published worldwide through the end of 2011. This data confirmed the continuing preferential use of a few prominent hESC lines derived early in the history of the field Fig. 1A.. Rather, we found that hESC use correlates strongly with year of derivation  and the use of specific hESC lines  Fig. S3C). It should be pointed out that our model does not contain a discernible “policy term”, which could be interpreted as governmental “power” driving stem cell research through laws or regulations towards the usage of only few “eligible” lines.To do this we constructed a simple mathematical model . We find that our cumulative advantage model  used prominent formerly NIH approved hESC lines introduced early in the field (Table S1). While 28 (15.1%) used other hESC lines in addition to formerly NIH approved lines, 143 CIRM-funded studies (77.3%) were exclusively based on formerly NIH approved hESC lines. Only 14 studies (7.6%) were performed without use of any formerly NIH approved hESC lines. Since 110 of the 185 CIRM-funded studies were also supported by NIH grants, we investigated more closely those 75 CIRM funded studies that were performed without additional NIH funding. In 63 (84%) of these studies, at least one formerly NIH approved hESC line was used, and in 50 studies (66.7%) formerly NIH approved hESC lines were used exclusively. Only 12 studies (16%) were performed without the use of any formerly NIH approved hESC line. This is in accordance with recent analyses of state-funded stem cell research grants, including those from CIRM, which have suggested a similar preference for formerly NIH approved lines at the grant application stage of the research process Table S2).To further analyse at least anecdotal evidence for a policy-driven model, we examined the interesting case of the California Institute for Regenerative Medicine (CIRM): a multi-billion dollar research organisation established, in part, to counteract the possibly negative effects of political funding restrictions, and enable California-based researchers to derive and use cell lines other than those eligible for NIH funding during the Bush presidency Table S3). This was also true for CIRM funded studies, despite independent funding. These results indicate a firm attachment to early hESC lines, particularly those derived in the US by Thomson and colleagues in 1998 To investigate this further we performed a hypergeometric test to determine if there are any statistically significant differences in use of formerly NIH-approved hESC lines in CIRM-funded, NIH-funded and worldwide studies. We found that in both the US and worldwide there was a significant preference for the use of early derived formerly NIH approved lines  research. Thomson et al.  C and D, Table S4). Five of these lines  were indeed derived and distributed significantly before their first peer reviewed publication date (see Table S4). The remaining two lines (HUES9 and WIBR3) illustrate two longstanding innovation trends in hESC research: 1) the HUES9 line originates from the Melton lab We finally asked if our cumulative advantage model could be useful for identifying emerging hESC usage trends from empirical data. To do this we fitted a regression model to the observed hESC usage distribution in order to compare actual hESC first publication dates with predicted hESC distribution dates based solely on observed usage frequencies per year. We combined this method with a z-score procedure previously proposed for the identification of outliers  In this study, we analysed human pluripotent stem cell usage patterns by investigating a comprehensive dataset based on published hESC research and proposed a simple mathematical model that qualitatively explains hESC usage patterns. Our analysis shows that a simple first-mover generative mechanism, that does not rely on funding policy as a causative factor, can naturally explain the observed dominance in use of a small number of hESC lines. Although our analysis does not exclude the influence of policy decisions on hESC usage, the results question accepted wisdom connecting stem cell usage patterns exclusively to US funding policies In agreement with a purely policy-driven interpretation of hESC usage, our model favours a set of early “founder” hESC lines. However, in our model preferential use of these lines is not due to political restrictions but rather arises naturally due to collaborative processes within the scientific community. We argue that if hESC use is a purely policy-driven process then qualitatively different patterns of usage of “approved” and “restricted” cell lines should be apparent in differing political environments. However, when analysing worldwide usage patterns we observed no such distinctions  and the cumulative advantage process used here is only one of several possible models that could have been applied While much effort has led to improvements in hESC lines and cultures since they were first derived by Thomson and colleagues Our outlier analysis Fig. 1 aTaken together, our analyses question a substantial impact of the Bush policy on use of specific hESC lines in basic and preclinical research both globally and in the US. We conclude that policy decisions concerning present and future funding for both hESC and hiPSC research should account for alternative generative mechanisms underlying human pluripotent stem cell usage.Figure S1Hierarchical clustering of global stem cell usage patterns. Nations with at least 30 experimental hESC publications were included in the analysis . Spearman rank correlation was used to assess similarity in hESC usage patterns . When assigning a study to a country we used the institutional affiliation of the corresponding author. Countries with diametrically opposing policies on hESC use cluster together despite regulatory differences. The policy classification for 2005 is according to reference http://mbbnet.umn.edu. Note that the displayed policy classification is only a relatively coarse-grained measure for complex national hESC usage regulations.(TIF)Click here for additional data file.Figure S2Global hESC co-citation network. Full hESC co-citation, including all peer-reviewed studies involving experiments with identifiable hESC lines published from 1998 to 2011. The network is a bipartite graph in which both hESC lines and studies are drawn as circles. There is an edge between a cell line and a study if the study uses that cell line. Most studies belong to the largest connected component (in blue). Several considerably smaller components (in orange) contain work with hESC lines that rarely appear in other publications. The main component network is displayed in more detail in (TIF)Click here for additional data file.Figure S3Observed and simulated network parameters. We simulated a cumulative advantage process for the usage of stem cell lines (as shown in Online Methods) 1,000 times and compared the parameter distributions with the actual observed data points (marked red). (A) Histogram of the distance (area enclosed between the curves) between each simulation of our model and the empirically observed stem cell usage data. The simulated result with the smallest distance to the observed usage data is given in black in (B) Histogram of estimated power law exponents for each simulation of our model. The estimated empirical exponent (1.94) is shown as a red line. (C) The frequency with which “founder” lines (the first 5 simulated lines in each simulation) and subsequent lines were used in simulated networks. (D) Histogram of the maximum number of citations received by a cell line in our simulations . The value observed for the empirical hESC co-citation network (the H9 line which received 996 citations) is shown in red line. (E) Histogram of the number of hESC lines in the largest connected component of the simulated co-citation networks. The value observed for the empirical hESC co-citation network (794) is shown in red.(TIF)Click here for additional data file.Table S1Use of hESC lines in studies (co-)funded by the California Institute for Regenerative Medicine. Statistics of hESC use for all published hESC studies with CIRM-funding  are shown. CIRM funding started in 2006, so studies published from 2008 were investigated. Funding information was taken from the appropriate section of the papers. The percentage share in the total number of papers published is in brackets. Note that more than one hESC line is used in most studies; thus the percentages add up to more than 100.(DOCX)Click here for additional data file.Table S2Use of hESC lines in research papers from Germany. Only cell lines that were used in at least two studies are listed. Numbers in brackets indicate the percentage share of the 56 hESC research papers from Germany published through the end of 2011. Of all the hESC lines that became accessible to German scientists after the amendment of the German Stem Cell Act in 2008, only one (HUES2) was used in more than one published study . In most studies more than one hESC line is used; thus the percentages add up to more than 100.(DOCX)Click here for additional data file.Table S3Enrichment analysis for formerly NIH approved hESC lines. Statistically significant differences in use of formerly NIH-approved hESC lines by comparison with other hESC lines were identified using hypergeometric enrichment analysis. Significant p-values indicate enrichment of use of the cell lines indicated in the column headers. We observe a highly enriched usage of the formerly NIH approved hESC lines in the US both before and after policy changes in 2009. This pattern is repeated in CIRM funded studies. We note a trend in the CIRM subset towards an increased number of studies using exclusively non eligible lines (i. e. other lines than the formerly approved NIH hESC lines). (DOCX)Click here for additional data file.Table S4Characteristics of the seven hESC lines highlighted in .(DOCX)Click here for additional data file.


The study is aimed at i) optimization of techniques for preparing microcapsules; ii) generation of multicellular tumor spheroids (MTS) by culturing tumor cells in the microcapsules; iii) study of anticancer treatment effects for both photodynamic therapy (PDT) and anti-cancer drug screening. The model allows to estimate drug doses or parameters for PDT To form MTS, tumor cell lines  were encapsulated in polyelectrolyte microcapsules (200-600 μm), and cultivated for 3-4 weeks . MicrocaMTS based MCF-7 cells were prepared and used to study effects of PDT. To study the effect of irradiation parameters on cell viability, 2 photosensitizers (PS), namely photosense and chlorine e6 were used. Phototoxicity of PS depended on PS concentration and light energy density in both monolayer culture (MLC) and MTS. Study of cell morphology in MLC and MTS before and after PDT revealed that light energy density increase within the range of 30-70 J/cm2 resulted in cell apoptosis. However, cell survival in MTS was much higher than this in the MLC. MTS were also used to test some antitumor therapeutics . An enhanced cell resistance in MTS compared to MLC both for normal and Dox-resistant cells  were observed. MTS were also proposed to evaluate cytotoxicity not only of novel therapeutics but also nanosized drug delivery systems .


Resistance to cancer chemotherapy is a major problem. Herb-drug combinations can offer a new potential to overcome such resistance. To quantitatively assess chemosensitizing effects of herb-drug combinations (HDC), a new approach that takes into account both the chemosensitizing effect (CE) and “safety” considerations of the HDC is proposed and used in describing the cytotoxic activity of Tripterygium wilfordii-docetaxel (TW-Dtx) combination on prostate cancer cell lines.50 of herb (H), drug (D) alone and in combination  in resistant cells were obtained. CE and chemosensitizing utility index (CUI) were calculated as: CE = IC50D IC50CD; CUI=CE (IC50H/ConH) whereas ConH is the H concentration in the combination.The effect of two extracts of TW with Dtx on Dtx resistant PC3 and DU145 cell lines were compared. Cell viability (cytotoxicity) was determined using sulforhodamine B assay after incubation of the cell line. The IC50CD (nM) from TW extract A-Dtx treatment in resistant PC3 cells were 3.8 , 5.8, and 5.8, respectively versus 17.1, 22.0, and 1.5, respectively from TW extract B-Dtx treatment when low ConH relative to IC50H was used. The corresponding values from extract A versus B-Dtx treatment in resistant DU145 cells were >1.3, >4.5 and 75.3 versus >27.6, >55.2 and 3.63, respectively. However, CE values can dramatically increase with higher ConH. The values for CE (fold change), CUI (fold change), and IC50CD are preferred and may prove to be a useful practical tool for assessing chemosensitizing effect of other HDCs.Based on the above CE, CUI or ICCD values, TW extract B-Dtx appeared to be consistently superior to extract A-Dtx combination. However, assessment based on CE value alone may be misleading since it can change dramatically with ConH used. CUI together with IC