Reproducibility and transparency in biomedical sciences have been called into question, and scientists have been found wanting as a result. Putting aside deliberate fraud, there is evidence that a major contributor to lack of reproducibility is insufficient quality assurance of reagents used in preclinical research. Cell lines are widely used in biomedical research to understand fundamental biological processes and disease states, yet most researchers do not perform a simple, affordable test to authenticate these key resources. Here, we provide a synopsis of the problems we face and how standards can contribute to an achievable solution. A major contributor to lack of reproducibility in preclinical research is insufficient quality assurance of reagents used. This article examines the problems surrounding the authentication of cell lines and discusses potential solutions. Statistician George Box famously stated, “all models are wrong.” While referring to mathematical models, there are many parallels from this statement to the biological models used in biomedical research. Whether a biological model relies on cell-free biochemical assays, cell-based assays, or genetically engineered organisms, the validity of the system can, in most cases, be called into question. This is part of the scientific process and should be encouraged.Blind use of a model system without first conducting fundamental quality controls to ensure its validity should never be tolerated. In the absence of such vetting, validity of results cannot be assessed, resulting in work that at best is impossible to interpret and, at worst, generates meaningless data, use of the same misleading model by others, and wasted research resources. Use of invalid models, such as misidentified (MI) or cross-contaminated (CC) cell lines , is an eCell lines require constant quality assurance because, as living models, they can change over time. Many cell lines are decades old—sometimes older than the scientists who work with them. Cell lines can change phenotypic behaviour as they continue to be handled. Cell lines can also be contaminated by microorganisms, including bacteria, fungi, and viruses . Whilst In order to ensure a cell line model is consistent with expectations, three properties should be assessed:Identity (authenticity). Analysis of genotype through comparison of DNA profiles is used to establish the original source of a cell line. If the profile does not correspond to the original source material, the cell line is misidentified. In cases where the DNA profile of the individual who donated the tissue is known, a direct comparison is sufficient. In cases where original profiles are not available, comparison to the earliest possible source of a cell line is needed.Purity (contamination). Contamination is commonplace. Adventitious organisms  and cross-contamination with another cell line can occur but can be detected by a variety of simple tests. Contaminants such as viruses and prions are less common and require more complex testing approaches. The need for further testing is determined by the application; for example, viral testing is needed for safety of the researcher or if a cell line is in vivo or for production of biologicals.Phenotype (characterization). These are the complex traits of a cell line as a result of its genotype and environment. Phenotypic traits of cell lines (such as proliferation rates and drug sensitivity) can change over time in the absence of best practices in cell culture. These traits are often unique to specific cell types and individual patient samples and thus require unique tests.A cell line can be considered as authentic if it corresponds to its original source by comparison of DNA profiles. Authentic cell lines maintained in different laboratories can possess variable phenotypes. Variations in phenotypes do not invalidate the authenticity of the cell line, which is why careful characterization of cells should be performed before experiments are conducted. While standards for identity have begun to be defined, standards to assess contaminants and phenotypic traits have yet to be defined.In this issue, Almeida et al.  discuss Here, we consider the key features of our current research climate that contribute to this problem and how standards are an important factor to help promote positive change.Natural selection favours those who cheat, resulting in an innate tendency to maximize one’s individual fitness to gain advantage. This biological hardwiring—the “selfish gene”—is evident in the behaviour of children and some adults throughout our society. To form an orderly society, we create laws that regulate certain actions to promote safety and ensure individual rights. Even in science, where empirical evidence, objectivity, and integrity are fundamental tenets, there are many examples of individuals engaging in fraudulent behaviour. This and other forms of misconduct are obvious causes of unreliable or irreproducible research, which undermines our discipline .Although fraudulent behaviour is an important concern, there are more pervasive reasons why scientists continue to use invalid models. Generally, when something is not required, it does not get done. When conducting cell-based experiments, why perform an additional test to authenticate a cell line when the results support the hypothesis and the test is not required for funding or publication? If granting organisations, institutions, and journals were to require authentication to receive funding, retain research space, or publish, it is likely more researchers would adopt this practice. Several organisations now encourage or mandate authentication, but this represents only a handful of stakeholders. So why do the majority of stakeholders not follow suit?As Almeida et al. point out, in order to require authentication, there must first be standards to serve as guidelines . StakehoExtensive work has been done to show that STR profiling is suitable for human identification, which the cell authentication community has used as the foundation for its work. STR profiling is based on regions of DNA that contain short repeated sequences. Testing involves PCR amplification of multiple STR loci, followed by analysis of the amplified fragments to determine the number of repeats present at each locus. These numbers can be recorded in database format, allowing scientists to compare results between laboratories. STR loci can vary extensively between individuals across the population. This means that, depending on the number of STR loci examined, the scientist can have a high degree of confidence that the STR profile is unique to that individual. STR profiling of cell lines allows scientists to compare their cell line sample to tissue or DNA from the original donor or to other cell lines to determine whether their sample is authentic or misidentified.Society as a whole equates success with visible achievement, resulting in relentless stress and pressure to produce tangible results. As a result, the academic scientific community spends much of its time searching for scarce resources that often require publications to secure. Pressure to publish and to be seen to generate “novel” findings can encourage behaviour that is inconsistent with fundamental tenets of good science. Hence, it is no surprise that many researchers feel that they do not have sufficient time to assess the quality of their reagents using methods such as cell line authentication .There is a pernicious belief that the quality of scientific work is directly proportional to the quality of the journal in which it is published. This has resulted in a perverse reliance on journal impact factors when assessing career success , a fetisThe peer-review system has evolved to prevent poor or fraudulent science from being widely distributed. It is designed not only to gauge the merits of new scientific discoveries but also to vet the quality of the science conducted and the validity of the data. However, there are significant limitations to what a reviewer can infer from words and images on paper. Much is left to the belief in the integrity of individuals, and there are no written consensus standards for reviewers to follow. Serial protagonists of scientific fraud are often caught not during scientific review but through post-publication analysis.Although the research organisation supports and enables the scientist’s work, the organisation is often missing when it comes to publication. Looking at high-profile cases of deliberate misconduct and retractions because of inadequate scientific rigour, serious questions should be raised regarding the degree of oversight by the fraudster’s mentors, colleagues, and institution. Good institutional practice is needed to improve scientific integrity . There iAs Almeida et al. point out, defining standards and publishing the highly qualified data which drives those standards will be an integral part of this process, particularly for transparent reporting and evaluation of experimental reagents . This prFunding is tight and tied to publications. The typical funding agency calculates funding levels based on personnel costs, consumables, and overheads. Commonly, there is no mention of, or budget allocated for, careful assessment of the quality of reagents. To date, only the Prostate Cancer Foundation (PCF) requires adherence to best practices regarding cell line authentication from its funded investigators. The National Institutes of Health (NIH) have recently adopted a policy on rigor and transparency, requiring applicants to describe methods to ensure the identity of key biological resources such as cell lines. While a welcome step forward, the policy stops short at requiring proof of authentication. Until all funding agencies put in place similar requirements to that of the PCF and support those requirements with specific funding, only small increases will occur in the proportion of labs that perform authentication.Cost is widely seen as a problem for cell authentication, partly because the alternative (doing nothing) is always cheaper. It may surprise some scientists to know that the costs of either STR- or SNP-based authentication are quite low, in comparison to a small aliquot of antibody or chemical reagent. Admittedly, costs can grow dramatically if large panels of cell lines are used. However, there is a general lack of understanding as to how frequently cell lines need testing, making it difficult to predict overall costs. Standards and guidelines help to clarify what is necessary and sufficient for authentication, enabling broader compliance.The current education system teaches scientific facts and theorem from textbooks, with an emphasis on undergraduate training. Postgraduate practical training rests on an ad hoc system of mentor- or peer- based training. Specialised cell culture textbooks and protocols are available, and online written and video protocols are becoming more widely available. However, practical knowledge is mostly learned on the job and is thus dependent on the skill and knowledge of the instructor, which can vary widely from one laboratory to the next. Training is rarely supported by funding or common resources that can be used in many different laboratories. Without a common set of standards to define best practices in cell culture, it is inevitable that individuals will receive inconsistent tutorage and pearls of ignorance will be propagated . To elicThere remain significant barriers to exhaustive adoption of authentication. As is the case for all research, it is difficult to guard against all misconduct; if all that is required from investigators is a table of STR profiles or copies of electropherograms, these can easily be duplicated from publicly available resources by unscrupulous individuals. Requirement of a certificate of authenticity from a certified testing facility would unfairly penalise organisations that have the necessary expertise to perform SNP or other genome-based comparisons and conduct their own internal tests. Journals could require submission of DNA to an independent body for authentication as a prerequisite for publication, but this would increase publication costs, burden the process, and would be open to potential fraud. So we are once again back to reliance on the integrity of the individual scientist.There is hope when we look at the increasing number of stakeholders that require authentication. Early training of scientists will add to that number, as will adoption of new practices to address some fundamental flaws. We can learn from industry to place a greater emphasis on focused, good science rather than publications or big science. The need for investment down the drug development pipeline is of sufficient magnitude that mistakes or misconduct cannot be tolerated. Our goal should be for scientists to view authentication as essential and burdensome as running a molecular weight ladder on their western blots.All the aspirations outlined here are mere fantasy without a set of community-driven standards that can define and equalise the process. Stakeholders in cell line authentication need these standards for a consistent and shared approach to improve quality. In this issue, Almeida et al. describe how this has been accomplished for STR authentication of human cell lines and comprehensively illustrate the benefits of defined standards . They alClearly, our community has a long way to go. It is now up to investigators, funding agencies, journals, and public and private enterprises to insist on the highest standards of transparency and rigor in biomedical research and to provide funding to promote research in these areas.


Nature: Cell Death and Discovery, Mahbub et al.1 have demonstrated that polyphenols can synergistically enhance the action of the topoisomerase II inhibitors: doxorubicin and etoposide in leukaemia cells. A reduction of glutathione (GSH) was strongly associated with sensitising cells to the pro-apoptotic effects of polyphenols when used in combination with doxorubicin or etoposide. Importantly, when polyphenols and topoisomerase II inhibitors were combined, it was possible to induce a synergistic decrease in cell proliferation (measured as ATP levels), cell-cycle arrest and induction of apoptosis in leukaemia cell lines.1Recently published in cis-stilbene)2 were combined with doxorubicin or etoposide in two lymphoid (CCRF-CEM and Jurkat) and two myeloid (THP-1 and KG1a) cell lines. These cell lines were selected as they had been identified as the most sensitive and most resistant to polyphenol-induced apoptosis;2 in addition, two non-tumour control haemopoietic stem cells (HSCs) (CD133+ and CD34+) were investigated.Five polyphenols that had been previously shown to induce apoptosis in leukaemia cells  in breast cancer cell lines, and raised the potential negative effect of a polyphenol-rich diet with certain antitumour regimes. The results of Mahbub et al.1 raise the possibility of a similar effect in myeloid malignancies treated with topoisomerase II inhibitors, in that dietary polyphenols may prevent etoposide/doxorubicin-induced antitumour activity. The mechanism of action of polyphenol-mediated antagonism of topoisomerase II inhibitors is unclear. However, it is known that GSH is contra-indicated for other chemotherapy agents, such as for cisplatin, where GSH supplementation inhibits the action of cisplatin. However, GSH-mediated depletion appears unrelated to cisplatin insensitivity in myeloid leukaemia cell lines.11 This is, however, in contrast to most other tumour models, suggesting that alternate multi-drug resistance mechanisms may be a feature of myeloid leukaemia cell lines. Similarly, recent work has shown that antioxidants can increase the metastasis of melanoma in mice,12 which raises the possibility that in some cancer types, polyphenols and other antioxidants could be detrimental. Thus, it is fundamental to tailor any treatment, be it with novel antitumour agents such as polyphenols or standard chemotherapy, to the specific cancer types and investigate any possible treatment interactions.The observed polyphenol-mediated antagonism of topoisomerase II inhibitors in myeloid leukaemia cells is especially important as polyphenols are abundant in a balanced diet. This antagonistic effect of polyphenols has been previously observed by Lui


Besides brain tumors, most animals also developed aggressive fibrosarcomas, likely triggered by Cre activation of mutant PDGFRα in fibroblastic cell lineages. Importantly, in the brain tumors and cell lines derived from brain tumor tissues, we identified a high prevalence of DM Pdgfra gene amplification, suggesting its occurrence as an early mutational event contributing to the malignant transformation of OPCs. Amplicons extended beyond the Pdgfra locus and included in some cases neighboring genes Kit and Kdr. Our genetically defined mouse brain tumor model therefore supports OPC as a cell of origin for malignant glioma and offers an example of a defined temporal sequence of mutational events, thus providing an entry point for a mechanistic understanding of DM gene amplification and its functionality in gliomagenesis.In primary brain tumors, oncogenes are frequently amplified and maintained on extrachromosomal DNA as double minutes (DM), but the underlying mechanisms remain poorly understood. We have generated a mouse model of malignant glioma based on knock-in of a mutant PDGF receptor α (PDGFRα) that is expressed in oligodendrocyte precursor cells (OPCs) after activation by a Cre recombinase. In the tumor suppressor INK4/Arf Focal amplification of genomic DNA is a pathological hallmark in many solid tumors, including glioblastoma, the most frequent and deadly form of primary brain cancer3457PDGFRA gene910111213PDGFRA amplification is often associated with point mutations or structural variants that are thought to increase the intensity of PDGFRα signaling . A number of mouse glioma models have been generated by overexpressing secreted PDGF ligands in the brain  PDGF receptor α (PDGFRα) is frequently encountered in glioblastoma and commonly associated with amplification of the Pdgfra promoter, which, in the CNS, is active in OPCs−/− background, a common tumor suppressor deletion in glioblastoma, mice developed spontaneous primary brain tumors between 15–30 weeks of age. Brain tumors displayed pathological characteristics of human high grade gliomas (WHO grade III), thus providing direct evidence for OPC as a cell of origin for malignant glioma. Importantly, we identified a high prevalence of amplification of the Pdgfra mutant allele as DM in not only advanced, but also early stage grade III gliomas, thus supporting RTK amplification as an important early event in the malignant transformation of OPCs. In summary, our study describes a novel glioma model that provides an example of a defined temporal sequence of mutational events in the malignant transformation of OPCs, starting from an activating RTK mutation and loss of a tumor suppressor, followed by amplification of the mutant RTK in the form of DM.We have developed a new glioma mouse model based on cell-autonomous activation of PDGFRα in oligodendrocyte precursor cells (OPCs). Specifically, we utilized a conditional knock-in of a PDGFRα point mutation that reduces auto-inhibition of the kinase domainPdgfra promoter after Cre recombinase-mediated excision of a STOP cassette. We crossed the conditional mutant mice to Nestin-Cre or GFAP-Cre transgenic lines, leading to Cre activation of mutant PDGFRα in all forebrain areas  or D842V (termed K for “kinase domain”)18in areas . The kno areas 19. Further areas 19 and muta−/−, a frequent tumor suppressor mutation in glioblastoma−/− mutations developed normally until early adulthood and displayed normal numbers of OPC , animals developed brain tumors only rarely, and cohort mice with heterozygous INK4A/Arf mutation alone (INK4A/Arf+/−) never developed brain tumors : brisk mitotic activity, hypercellularity, cellular pleomorphism, and marked nuclear atypia, i.e. hyperchromatic nuclei with irregular contours . Tumor cFurther analysis by immunofluorescence revealed several consistent molecular features, such as increased PDGFRα expression, high proliferative index by Ki67 staining , and expFor further analysis on a cellular level, we derived cell lines from forebrain tissue of mutant and control mice and cultured them in neural stem cell media under spherogenic conditions, which allows for selection of the so-called glioma stem cells . Cell lines derived from advanced brain tumors are expected to consist predominantly of GSCs, whereas cell lines from wild-type brain will only consist of normal neural progenitors. Cell lines derived from tissue of early stage gliomas with lower numbers of tumor cells may consist of either GSCs, normal neural progenitors, or both cell types.Pdgfra gene copy numbers by qPCR. We observed Pdgfra amplification in 9 of the 15 lines derived from tumor-bearing mice, including all 5 lines from mice bearing advanced gliomas, and 4 lines from mice bearing early stage gliomas , indicating a preferential amplification of the PDGFRα J/K mutant allele , the copy numbers of the mutant allele approached the total number of all t allele . In the t allele .Pdgfra gene, or encompasses a larger chromosomal area, we first probed for amplicons 50 kb upstream and downstream of the Pdgfra locus. Of the 6 cell lines tested, 5 lines showed amplification extending beyond the Pdgfra locus by at least 50 kb in both directions, and one line showed amplification only downstream of the Pdgfra locus, indicating that amplicons are in general larger than 100 kb  on metaphase spreads obtained from glioma cell lines using probes for the Pdgfra locus (located on chromosome 5) and for the centromere of chromosome 5 . In all Pdgfra-amplified glioma cell lines, we found that in addition to the two Pdgfra loci on the chromosome 5 pair, FISH signals were also abundant on small extrachromosomal pieces of DNA 26SorPDGFRα J/K: Antibodies and their dilutions used for immunostaining of brain sections: goat anti-PDGFRα ; rabbit anti-PDGFRα ; rabbit anti-Ki67 ; rabbit anti-Olig2 ; rabbit anti-Sox2 ; goat anti-Sox10 ; mouse anti-Nestin ; rabbit anti-Ng2 ; rabbit anti-NeuN ; rabbit anti-GFAP ; mouse anti-APC, clone CC1 .Secondary antibodies were coupled with Alexa fluorophores (Jackson Immunoresearch) or biotinylated for ABC signal amplification and peroxidase DAB staining (Vector Labs).For mRNA in situ hybridization, cryosections were hybridized with Digoxigenin-labeled riboprobes, which were synthesised from the plasmids pBS-Pdgfra-ISH-1.6 kb and pCR4-Plp1-ISH-2.0 kb, and probe hybridization was detected with AP-conjugated anti-Dig Fab fragments (Roche) and NBT/BCIP staining.Hematoxylin & Eosin staining was performed with Shandon Gill #2 Hematoxylin and Eosin-Y (Thermo Scientific).The anterior third of the forebrain of mice was dissected and tissue was minced into small pieces with a scalpel blade and then incubated in Accutase (Invitrogen) for 15 min at 37°C. Cells were gently dissociated with a 5 ml pipette and the cell suspension was filtered through a 40 μm cell strainer  and then cultured in Neural Stemcell media (Stemcell Technologies) for several days until spheres of >200 μm had formedOligodendrocyte precursor cells (OPC) were isolated from cortices of P5 mice by Papain digest and selective immunopanning with anti-PDGFRα antibodies, and then cultured in poly-D-lysine coated tissue culture dishes with defined OPC media containing PDGF-APdgfra gene locus, respectively . Cells and tissue sections were hybridized with FISH probes according to manufacturer's instructions (Empire Genomics) and counterstained with DAPI.Cell lines were cultivated over night in presence of 0.1 μg/ml colcemid (Invitrogen) before cytogenetic preparation by KCl treatment and fixation. The mouse BACs RPCI-23-222P8 and RPCI-23-413L22 were used as templates for synthesis of FISH probes for the chromosome 5 centromere and Foxp2 locus. Primer sequences (5′-3′): Pdgfra: GGGGAGAGTGAAGTGAGCTG and CATCCGTCTGAGTGTGGTTG; PdgfraJ/K mutant allele only: CAAACTCTTCGCGGTCTTTC and CGAAGTTATATTAAGGGTTCCG; Pdgfra 50 kb upstream: GATGCTGAGACACTCCTCTTG and GACTTGCTTAGTGCATCTGTATTG; Pdgfra 50 kb downstream: GATAGAGGCCCATGTCTTCTTAC and GCAGACTAACATGACAGGAGAA; Egfr: CCTGGAAGAGACCTGCATTATC and GTTAAACCCACTACTGAGACAGG; Kit: CAGCACATAGCCCAGGTAAA and CAACTCTTGCCGAGCTGATA; Kdr: CTCCATGACATAAGGCCTACAC and CTGCCACAAGCCTACTGATAA; Mdm2: GGAAGTCGATGGTTGGGAATAG and AGCTGACAGAGAATGATGCTAAA; Foxp2: AGCTCCTTTGCCTTCTCCACTCTT and AGGGCAATGAAGCCAGTCTGTACT.Gene copy numbers were determined by qPCR of genomic DNA from cell lines with a SYBR green reaction mix (Quanta Biosciences) on an ABI7900 device and normalized to a PCR for the Graphpad Prism 5 software was used to calculate Kaplan-Meier survival curves and bar graph analyses. Data are presented as mean ± SEM.H.Z., L.E.O., P.S. and R.H.F. designed research; R.F., Y.H., J.T., M.J., L.E.O. and R.H.F. conducted experiments; H.Z., V.N., N.T., L.E.O., P.S. and R.H.F. analyzed data; H.Z. and R.H.F. wrote the paper. All authors read and approved the final manuscript.Supplementary Info


To prospectively investigate the relationship between the apparent diffusion coefficient (ADC) and cellularity in lung cancer.b values of 50 and 1000 s/mm2). The median mean ADC (ADCmean) value and median minimum ADC (ADCmin) value within each primary tumour were calculated and compared with the median nucleo-cytoplasmic ratio (NCR), which was selected to represent the cellularity. The correlation between the NCR and ADCmean/ADCmin was calculated with SPSS 18.0 software.Sixty patients histopathologically confirmed with lung cancer  underwent diffusion-weighted magnetic resonance imaging of the chest ×10−3 mm2/s, and (14.9±2.6) %, respectively, in adenocarcinoma; (0.88±0.10)×10−3 mm2/s, (0.73±0.12)×10−3 mm2/s, and (20.6±4.4) %, respectively, in squamous cell carcinoma; and (0.89±0.13)×10−3 mm2/s, (0.67±0.13)×10−3 mm2/s, and (18.3±3.5) %, respectively in small cell lung cancer. The NCR of squamous cell carcinoma and small cell lung cancer is greater than that of adenocarcinoma . There was an inverse relationship between ADCmean/NCR and ADCmin/NCR .The mean ADCmean values, ADCmin values and median NCR were (1.07±0.12)×10There is a significant inverse relationship between tumour cellularity and ADC in lung cancer. However, tumour cellularity most likely is not the sole determinant of the ADC. Lung cancer comprises almost 25% of all cancer deaths worldwide, and its incidence rates have risen dramatically over the last few years in vitroDiffusion-weighted imaging (DWI), which tracks the microscopic rate of water diffusion within tissues, is a new means of monitoring tumour progression and response to treatment. Because it provides information about tissue cellularity and the integrity of cell membranes, DWI has benefits over traditional anatomical MRI techniques Thus, the purpose of our study was to prospectively assess whether there is a relationship between the ADC and the histopathological tumour cellularity in lung cancer.The Medical Research Ethics Committee of the Third Military Medical University  reviewed and approved the present study. Written informed consent was obtained from each participant prior to the study. Between February 2012 and April 2013, 79 consecutive patients suspected of having lung cancer were assessed for eligibility if the lesions in size were >20 mm. The size of the lesions was measured on CT. Sixty patients  histopathologically confirmed with lung cancer were included . The pri2 during free breathing. To minimise the influence of respiratory movement on data quality, two approaches were taken: (1) respiratory training, and (2) guided free breathing (instructions from the imaging radiographer). Fat was suppressed by placing a frequency-selective radiofrequency pulse before the pulse sequence. All of the ADCs  were measured by two chest radiologists with 8 and 11 years of experience. In outlining the ROI for measurement of the DWI metrics, we attempted to draw on the solid part of the tumours by imaging of the CT-guided biopsy (Siemens Plus 4), see All MR imaging was performed on a 3.0 Tesla scanner  in combination with an 8 channel phase array coil. DWI was obtained during free breathing Histopathologic specimens were coated with standard marking ink  and fixed in 10% buffered formaldehyde for 24 hours. The histopathology results were the standard of reference for the analysis of cellularity. All histopathologic specimens were reviewed by a histopathologist with 20 years of experience in histopathology.Tumour cellularity was calculated from 5 arbitrarily selected high-power fields in each specimen using a computer program  and the following algorithm: first, digitised high-power (×40 objective) fields were taken from the original microscopic images with a 512×512 display matrix and 8-bit grey scale, which were obtained using a digital microscope camera . Binary image data were then derived from the sample images using a threshold value estimated from histogram analysis of the sample images. Finally, the software calculated the cellularity based on the number of separate dots produced by the binary procedure. The automated counting system performed to within 5% tolerance in all specimens. The automated settings were identical for all specimens and cell counts. The results for the 5 high-power-field cell counts were used to derive the median cell count for the specimen. This method of measuring cellularity is similar to the methods used by other investigators who compared diffusion-weighted imaging features with tumour cellularity Statistical analyses were performed using statistical software . The Kolmogorov-Smirnov test was used to assess the deviation from normal distribution for all data. Intraclass correlation coefficients (ICC) were calculated to evaluate the inter-observer variance in the DWI metrics measurements. To compare the ADCmean values and ADCmin values among histologic types of adenocarcinoma, squamous cell carcinoma and small cell lung cancer, analysis of variance was used. The post hoc hypothesis testing was performed according to the Fisher protected least significant difference method. In addition, Pearson correlation coefficients were calculated to evaluate the correlation between ADCmean values/ADCmin values and the tumour cellularity of lung carcinomas. A P value of less than 0.05 was considered to indicate a statistically significant difference.−3 mm2/s in total, (0.89±0.13) × 10−3 mm2/s in small cell lung cancer, (1.07±0.12) × 10−3 mm2/s in adenocarcinoma, and (0.88±0.10) × 10−3 mm2/s in squamous cell carcinoma. Each set of data was in line with or approximated a normal distribution . The analysis of variance showed significant differences among three groups (P<0.05). The ADCmin value of the primary tumours was (0.80±0.15) × 10−3 mm2/s in total, (0.67±0.13) × 10−3 mm2/s in small cell lung cancer, (0.86±0.13) × 10−3 mm2/s in adenocarcinoma, and (0.73±0.13) × 10−3 mm2/s in squamous cell carcinoma. Each set of data was in line with or approximated a normal distribution . The analysis of variance showed significant differences among three groups (P<0.05).All DWI images clearly showed that there were no significant susceptibility artefacts and that they were suitable for further evaluation. All ADCmean, ADCmin and tumour cellularity values were successfully obtained –3. The IThe NCR of the primary tumours was (16.7±4.0) % in total, (18.3±3.5) % in small cell lung cancer, (14.9±2.6) % in adenocarcinoma, and (20.6±4.4) % in squamous cell carcinoma. Each set of data was in line with or approximated a normal distribution . The analysis of variance showed significant differences among three groups (P<0.05). Among these, the NCRs of squamous cell carcinoma ((20.6±4.4) %) and small cell lung cancer ((14.9±2.6) %) were higher than that of adenocarcinoma ((14.9±2.6) %), and the difference was statistically significant . The NCR of squamous cell carcinoma cells was greater than that of small cell lung cancer cells, but the difference was not statistically significant (P = 0.198).Correlation analysis showed that the ADCmean value and lung tumour cellularity was significantly negatively correlated , see et alWe have performed correlation analyses for both ADCmean and ADCmin with tumour cellularity and have shown that both ADCmean and ADCmin had a negative relationship with tumour cellularity in lung cancer. Matoba Our results experimentally verifies the negative relationship between ADC and cellularity in a more quantitative manner and may be of value for more accurately modeling the diffusion process. Higher nuclear/cytoplasm ratio will reduce extracellular space which may lead to lower apparent diffusion coefficient et alBoth ADCmin and ADCmean values have been reported to correlate inversely with glial and nonglial tumour cellularity as a result of the restricted diffusion of water et alet alOur results show that the tumour cellularity of different histological types of lung cancer is not the same. The results from the studies of Matoba The indicators to evaluate tumour cellularity included cell density, nuclear-cytoplasmic ratio and nuclear cell ratio. Cell density was calculated by dividing the total area of tumour cell nuclei by the area of the histology section. The nuclear-cytoplasmic ratio was calculated by dividing the percentage of the nuclear area by the percentage of the cytoplasmic area. The nuclear cell ratio was calculated by dividing the percentage of the nuclear area by the percentage of the cell area. Compared to cell density, the nuclear-cytoplasmic ratio and the nuclear cell ratio can not only reflect cell density but can also reflect the nuclear/cytoplasm ratio and large nuclei, as all of these factors affect the ADC values There are some limitations to this study. First, the study only included adenocarcinoma, squamous cell carcinoma and small cell lung cancer, and other rare histological types of lung cancer, such as large cell lung cancer, could not be included. Second, the total cell density was considered for comparison, so tumour cells were not separated from non-tumour cells. Physiologically, however, non-tumour cells also affect the water diffusivity within the tissue, and most previous reports did not distinguish tumour cells. Third, this study only focussed on the correlation of tumour cellularity and ADC value in the biopsy site, which cannot represent the whole tumour.In conclusion, our study showed that the ADCmean value and the ADCmin value were significantly negatively correlated with tumour cellularity in patients with lung cancer. However, tumour cellularity most likely is not the sole determinant of the ADC.Figure S1Flowchart illustrating the selection of studies.(TIF)Click here for additional data file.


EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the  EGFR gene is either mutated or shows altered expression in a variety of human cancers. Lung is the most frequent cause of cancer-related mortality worldwide leading to over a million deaths each year . In brief, the cells were treated with 8–12 concentrations of erlotinib each in combination with 8–12 concentrations of MET or AXL inhibitors. In particular the ratio of erlotinib with SU11274 was 1:4, while the ratio of erlotinib with PHA-665752 or PF-04217903 was 1:1. The ratio of erlotinib with the AXL inhibitor was 10:1 as previously reported , in which Dx1 is the dose of Drug 1 that inhibits cell survival at x% and Dx2 is the dose of Drug 2 that inhibits cell survival at x% and D1 is the portion of Drug 1 that also inhibits cell survival at x% in combination with Drug 2 and vice versa. The doses which produced a particular effect (Dx) can be calculated from the Median effect equation: Dx = Dm [fa/1-fa)]1/m, where, Dm, fa and fu (1-fa) represent: the median dose, fraction affected and fraction unaffected, respectively. Dm was estimated from the antilog of the X-intercept of the median effect plot, where X = log (D) versus Y = log (fa/fu); which means Dm = 10−(Y-intercept)/m, m being the slope of the median effect plot.The synergy of erlotinib and MET or erlotinib and AXL inhibitors was evaluated by the Chou-Talalay method [50 values were determined using a sigmoidal dose response inhibition variable slope method.GraphPad Prism software 6.0c was used for MTT data analysis. The regression trendline were fitted using a non-linear regression method and ICIn the drug combination studies, dose–effect curve parameters, CI values, Fa-CI plot  were calculated by CompuSyn program .In the xenograft experiments the statistical significance of the results was evaluated by two way analysis of variance and Bonferroni multiple testing to compare differences in tumor fold growth between ERL-resistant and HCC827 parental cell line. All statistical tests were performed using GraphPad Prism and the threshold for statistical significance was set at P-values lower than 0.05.in vitro models of acquired resistance to the EGFR TKI, erlotinib. Both cell lines harbor EGFR activating mutations in the tyrosine kinase domain, the HCC827 cell line a deletion in exon 19 (ΔE746-A750) and HCC4006 a deletion (ΔL747-E749) and a point mutation (A750P) in exon 19. HCC827 and HCC4006 cell lines are both highly sensitive to TKIs targeting the EGFR. TKI dose-response curves and IC50 values are shown respectively in 50 in line with previous reports [In order to investigate mechanisms leading to resistance to EGFR-targeted therapy, two NSCLC cell lines HCC827 and HCC4006 were used to derive  reports , 45. Pacon 19 ΔE7-A750 andetion ΔL7-E749 and reports –53 had n50−25/50x IC50), while in “protocol 2” cells were continuously exposed to a high dose of erlotinib (1 μM). The selection outcomes were monitored periodically by cell growth inhibition assays by using cells in “drug holiday” for at least 1 week. Five erlotinib (ERL)-resistant cell lines were isolated from the parental HCC827 cell line, three with “protocol 1”  and two with “protocol 2” (RA2 and RB2) and one cell line was obtained from HCC4006 cell line by using “protocol 2” (RC2.2). Several attempts to derived additional HCC4006 ERL-resistant cell lines with both protocols failed.It has been suggested that the features of drug-resistant cells may vary depending on the protocol used for the selection process . Therefo50 > 10 μM)  , Table 1ll lines , Table 1To investigate the tumorigenicity of the ERL-resistant cell lines and verify whether erlotinib could inhibit their anchorage-independent growth we performed soft agar colony formation assays. All HCC827 cell lines formed colonies similar in number and size (diameter > 50 μm), but only the ERL-resistant cell lines formed colonies in presence of erlotinib . The HCC6 cells. In those experimental conditions, only small nodes and not in all mice were obtained (data not shown). Overall the data demonstrate that HCC827 ERL-resistant cell lines and, to a lesser extent, RC2.2 cell line are tumorigenic in anchorage-independent systems. Importantly, even if the selection protocols used monolayer tissue culture conditions, the derived NSCLC cell lines grow in presence of erlotinib in 3D model systems, more closely resembling in vivo tissues conditions.To further investigate the tumorigenicity of the NSCLC cell lines, we performed xenograft assays in athymic nude mice . SimilarEGFR and KRAS oncogene mutations, nucleotide sequence analysis of exons 19–20 of the EGFR gene and codons 12/13 and 61 of the KRAS gene was performed with specific primers listed in EGFR exon 19 in both parental and ERL-resistant cell lines and we excluded the presence of an EGFR T790M gene mutation, the most common cause of acquired resistance to erlotinib in NSCLC patients  were wild type in all our NSCLC cell lines /protein kinase B (AKT) . HER2 amAmong RTK signaling pathways, previously reported to be involved in mechanisms of ERL-resistance, we focused our studies on AXL and MET receptors.AXL is considered a potential relevant target in NSCLC therapy and targeting strategies with TKIs, aptamers or molecules modulating its turnover are under very active investigation –68. More50 values indicate that AXL inhibitor alone or in combination with erlotinib did not impair the cell viability of ERL-sensitive and -resistant cell lines  [50 similar to the one recorded in the ERL-sensitive parental cell line  –72, PHA-274 (SU) –74, and 274 (SU) –77, and ell line . Furtherell line . CI analell line . To valiell line .MET gene amplification and receptor activation are not sufficient to predict efficacy of erlotinib plus MET inhibitor treatment in NSCLC with acquired resistance to erlotinib and that other biomarkers are required for optimal treatment choice.Overall the data suggest that To further investigate the biochemical mechanisms of the combined treatments, the expression of MET, EGFR, HER3 and AKT and their phosphorylation status were investigated by western blotting. In all cell lines, as expected, a strong decrease of MET phosphorylation (Y1234/1235) was observed upon treatment with SU inhibitor or SU in combination with erlotinib . FurtherIn summary these data suggest that this TKIs combination treatment can impair both MET and EGFR signaling pathways but that a strong impact on cell survival can be obtained only in RB1.1 ERL-resistant NSCLC cell line. Ongoing comparative genome hybridization and RNA expression array analyses could be instrumental to identify possible mechanisms leading to differential response to drug treatment combination and/or biomarkers to predict treatment efficacy.MET gene amplification and receptor activation are not sufficient to predict a positive effect of drug TKI cocktails on ERL-resistant NSCLCs highlighting the necessity to search for novel biochemical and molecular markers guiding treatment choice in ERL-resistant patients and in ongoing clinical trials.We have isolated and characterized six ERL-resistant EGFR mutant NSCLC cell lines. We reported alteration in EGFR amplification in one cell line, AXL overexpression and/or MET overexpression and activation in ERL-resistant cell lines, consistently with previous studies indicating that multiple mechanisms may contribute to EGFR TKI treatment resistance. Moreover, our pilot study with EGFR and MET inhibitors demonstrate that S1 Table(DOCX)Click here for additional data file.


AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including  Neuroblastoma (NB) is a solid tumor of the peripheral nervous system occurring mostly in infants and younger children. Accounting for 8-10% of all childhood tumors, NB is responsible for 10-12% of cancer-related deaths in children . PatientDevelopment of targeted treatment approaches has been challenging due to the heterogeneous nature of this cancer and an insufficient understanding of the biology of high-risk neuroblastoma. The anaplastic lymphoma receptor tyrosine kinase, ALK, is mutated in approximately 10% of all spontaneous cases and currently the only therapeutically-targetable receptor tyrosine kinase in neuroblastoma .MYCN and/or structural aberrations of the genome, which are difficult to inhibit directly. Recent research has thus been focused on finding surrogate targets that assist MYCN in driving neuroblastoma. Inhibition of either the PI3K signaling pathway or the serine/threonine kinase AURKA was shown to decrease the stability of the MYCN protein and reduce the proliferation of neuroblastoma cell lines and xenografts [In aggressive neuroblastoma, tumorigenesis is largely driven by amplification of the oncogene nografts -8. Both nografts , 10 and MYCN-amplified (MNA) cell lines with wild-type TP53 (ampMYCN/wtTP53) were highly sensitive to barasertib. Additionally, barasertib effectively decreased tumor volumes and prolonged survival in an MNA mouse xenograft model of human neuroblastoma. Therefore, our studies have uncovered a novel targetable susceptibility in high-risk neuroblastoma with potential clinical application.In this study, we identified actionable targets in neuroblastoma using a combination of high-throughput RNAi and small molecule drug screens. We found knockdown of the mitotic kinase AURKB and its pharmacological inhibition with barasertib (AZD1152-HQPA) to be highly effective in suppressing neuroblastoma cell growth. Most importantly, MYCN-amplification (MNA) status: two MNA (IMR32 and IMR5) and two non-amplified (SK-N-AS and NB-EB) cell lines. Due to the known off-target binding capabilities of siRNAs, we combined Common Seed Analysis (CSA) [GABRD, TINAGL1, and KIF2B) were excluded from further consideration due to their low expression. Thus, we identified 58 vulnerability genes whose knockdown resulted in significant reduction of neuroblastoma cell survival.In order to identify genetic vulnerabilities in neuroblastoma, we first performed a high-throughput synthetic lethality screen using a druggable genome siRNA panel that contained 13904 siRNAs to target 6877 genes Figure . We seleis (CSA)  with Redis (CSA)  to minimwww.ingenuity.com). MYCN/MYC and the interacting transcription factor E2F1 were among the strongest expression activators of the vulnerability genes whereas the tumor suppressor TP53 was predicted to be negatively regulated within this network  to drive mitosis . In addiewed in ). FurtheBIRC5/survivin, CHEK1, KIF11 and PLK1 demonstrated non-selective activity in either subtype , compared to an average of 89% AUC in MYCN-non-amplified lines (median IC50 of 38.3 μM). Therefore, our drug screen demonstrated that vulnerability to aurora kinase inhibition closely linked to with MYCN amplification.In parallel to the siRNA screen, we treated the same four cell lines with 465 small molecules included in the Mechanism Interrogation PlatE (MIPE) compound library  to disco subtype , which w subtype . On the n Figure  and 2B. n Figure . MNA celMYCN status was recently shown for the AURKA inhibitor MLN8237 in neuroblastoma [AURKB promoter by chromatin immuno-precipitation sequencing (ChIP-seq) in an NB cell line with an inducible MYCN-expression construct [AURKB gene indicating that MYCN regulates AURKB expression directly  and a homozygous deletion of exons 10 and 11, respectively. In contrast, TP53-mutants SK-N-DZ and SK-N-BE (2c) exhibited the highest protein levels of TP53, which is likely due to an altered stability of the mutant protein. Barasertib elicited a similarly strong response in the ampMYCN/wtTP53 cell line LAN-5 as in IMR5 and IMR32  were substantially less sensitive. Interestingly, MYCN-non-amplified SK-N-AS which lacks TP53 expression was highly resistant to the maximum dose of barasertib and thus featured the highest normalized AUC of all cell lines. These data indicated that AURKB inhibition by barasertib was far more effective in MNA NB cells with wild-type TP53.In our drug screen, barasertib was highly selective and effective in IMR5 and IMR32 cells inhibiting growth in low nanomolar concentrations with an almost complete maximum response rate Figure . A recenAS Table . WesternAS Table . TP53 wa2 Figure , Table 1TP53 wild-type IMR5 cells, whereas TP53 mutant SK-N-BE (2c) showed neither an induction of TP53 nor CDKN1A. Inhibition of AURKB did not change protein levels of MYCN. Therefore, these data indicate that upregulation of TP53 and its downstream target CDKN1A resulting from inhibition of AURKB likely induces cell death in MYCNamp/TP53wt neuroblastoma cells.In order to examine the downstream effect of AURKB inhibition in NB cells, we performed immunoblotting on protein lysates of cells treated with different concentration of barasertib. We found that cell lines responded to AURKB inhibition with time- and concentration-dependent loss of phosphorylation of histone H3, an AURKB downstream target Figure . FurtherMYCNamp/TP53mut SK-N-BE (2c). Barasertib induced a profound G2/M-arrest as well as polyploidization, albeit at different concentrations, in both cell lines as determined by flow cytometry  and effective (EC50) concentrations of the response curves, respectively   Figure . DespiteAURKB and 57 other essential genes for neuroblastoma cell survival by interrogating an siRNA library focused on target druggability. The importance of AURKB was demonstrated by the decrease of cell viability after AURKB knockdown in 4 NB cell lines and supported by our discovery of numerous vulnerability genes which interact with the mitotic kinase and the CPC. By screening a collection of oncology-relevant small molecules in parallel, we identified the AURKB-selective inhibitor barasertib and seven other classes of agents that specifically inhibit other vulnerability genes discovered in the RNAi experiments. Whereas AURKA, BIRC5, CHEK1, KIF11, PLK1 and the proteasome are currently evaluated in clinical trials or were preclinically examined by the PPTP and other groups [in vivo testing in neuroblastoma.In this study, we identified r groups , 24-28, r groups  but has MYCN-amplified and TP53 wild-type subtype. Previously, synthetic lethality between barasertib and members of the Myc family of transcription factors was shown in MYC-amplified small cell lung cancer cell lines and MYC-overexpressing RPE cells [AURKB knockdown nor overexpression were shown to affect MYCN protein levels [In this study, we demonstrate that barasertib is efficacious in a panel of neuroblastoma cell lines with a preference for the PE cells , 31. In PE cells . NeverthPE cells , our ChIPE cells , neithern levels  which isMYCN induces the expression of the tumor suppressor TP53  which isTP53 mutational status [TP53 status showed no correlation with sensitivity to barasertib by itself but in combination with MYCN amplification, TP53wt cell lines were significantly more sensitive.The effect of TP53 on AURKB function is less well-understood but it was suggested that activity of AURKB inhibitors depends on the l status , 39. TP5l status . Others l status . In our TP53wt IMR5 which, despite CDKN1A induction, arrested in G2/M phase contrary to an expected G1/S phase arrest. However, MYCN-mediated overwriting of the TP53 post-mitotic checkpoint has been commonly observed in MYCN-amplified/wt-TP53 neuroblastoma cell lines [We found that barasertib induced upregulation of TP53 and CDKN1A protein levels in a dose-dependent manner in ll lines , 41. MutMYCN-amplified IMR32 xenografts. Despite the potential treatment-related death of two mice, the drug-treated group showed complete tumor suppression and remained tumor-free throughout the trial, which warrants future studies to use this compound for neuroblastoma with wild-type TP53 and MYCN amplification.In a variety of solid tumors and leukemias, barasertib has shown significant tumor growth inhibition when used preclinically as monotherapy and when combined with standard chemotherapeutic agents or ionizing radiation , 42-49. AURKB expression with poor prognosis for neuroblastoma patients. We further showed an AURKB inhibitor barasertib is more efficacious in neuroblastoma with MYCN amplification and wild-type TP53. Finally, we demonstrated barasertib significantly suppressed tumor growth in a human neuroblastoma xenograft mouse model. Therefore, our study established AURKB as a rational target for patients with highly aggressive MYCN-amplified, TP53 wild-type neuroblastoma.In conclusion, our study demonstrated the importance of AURKB for neuroblastoma cell survival and the association of high MYCN-amplified (MNA) cell lines, IMR32, IMR5, LAN-1, LAN-5, SK-N-BE (2c), SK-N-DZ, as well as the MYCN-non-amplified cell lines SK-N-AS, NB-EB, SK-N-SH and SH-SY5Y were verified by short tandem repeat (STR) analysis. SK-N-DZ and SK-N-BE (2c) were cultured in DMEM+ GLUTAMAX  and all other cell lines in RPMI medium 1640 (1x) + GLUTAMAX  medium supplemented with 10% fetal bovine serum or fetal calf serum and the antibiotics penicillin and streptomycin.AURKB, were obtained from Ambion . Non-silencing negative (Silencer Select Negative Control No. 2) and lethal positive siRNA controls were obtained, respectively, from Ambion and Qiagen.The druggable genome siRNA library targeting 6877 genes with two or more siRNAs/gene was obtained from Qiagen . For the verification screen, siRNAs targeting candidate genes, including 2 for 96 hours. Dimethyl sulfoxide (DMSO) at a final in-plate concentration of 0.1% was added after 24h.The high-throughput RNAi discovery screen was performed using reverse transfection as described previously . BrieflyFor verification screening, daughter plates with 0.8 μmol of siRNA in 2 μl DMSO were printed from the master plates obtained from Ambion. Control siRNAs were added manually to each assay plate. RNAimax was diluted in RPMI. Cells were added in RPMI with 20%FBS . In both screens, CellTiter Glo  was used to measure total cell viability in relative luminescence units (RLU) with an EnVision plate reader .For both screens, cell viability was measured by CellTiter Glo  96h after siRNA transfection.For the confirmation of aurora B kinase-specific knockdown, cells were reverse transfected under the same conditions as the verification screen with three aurora B Silencer Select siRNAs , independently.10p-values assigned to the corresponding genes based on an iterative hypergeometric distribution formula. Genes targeted by siRNAs significantly distributed at the top of the ranking (log10p < −1.301) as determined by RSA and with at least one siRNA active below the estimated significance cutoff were selected as potential hits for each cell line.Raw data obtained from the discovery screen were first corrected for plate variability by normalizing against the non-transfected reference wells. Robust Z-scores for each siRNA were calculated . A modifTo employ the knowledge on seed-based off-target binding, Haystack analysis was implemented to identify 3′-UTR regions of genes containing complementary sequences to the most active seeds . Only ge10p-value < −1.301 were considered verified.Analysis of the verification screen data was adapted to the expectation that a high number of siRNAs would be active in each cell line. The 639 siRNAs were normalized by subtracting the positive control and dividing by the negative control. The relative viabilities were then ranked by RSA similar as described for the discovery screen. Genes with a logCells were plated in 6-well plates and treated with DMSO after overnight incubation. RNA was isolated from harvested cells using the Trizol method and the QIAGEN All Prep DNA/RNA mini kit  according to the manufacturer's protocol. In short, Trizol was added to the plates and the cell lysates transferred into tubes. Chloroform was added for phase separation. The aqueous phase was transferred to a fresh tube and 70% ethanol was used to precipitate the RNA before applying the mixture to Mini RNeasy columns for filtration. RNA was quantified with a Nanodrop spectrophotometer and RNA integrity was measured with an Agilent 2100 Bioanalyzer.Library preparation for PolyA-selected mRNA sequencing was performed according to Illumina's TruSeq RNA sample preparation v2 guide . Libraries were sequenced on an Illumina HiSeq2500 using the 100-bp paired-end sequencing protocol. After fastq files were generated using CASAVA , RSEM was applied to estimate the expression levels of each gene using the hg19 reference transcriptome . ExpressChIP sequencing experiments were performed using the Tet-inducible MYCN3 cell line using doxycycline (1 mg/ml) for MYCN induction as previously described . Actin set.al. [2, viability was measured with CellTiter Glo  using a ViewLux imager . Relative luminescence units (RLU) for each well were normalized to the median RLUs of the DMSO control wells set as 100% viability and median RLUs of the 2 mM-bortezomib control wells as 0% viability. Normalized dose responses were fitted to a 4-parameter nonlinear model applying a custom gird-based algorithm that can handle outliers [The high-throughput screen of the Mechanism Interrogation PlatE (MIPE) compound library to identify small compound inhibitors was conducted according to Mathews et.al. , 60. A foutliers . RetestiFor immunoblotting, the samples were harvested by trypsinization and lysed in 2x SDS-loading buffer and PBS. DNA was sheared by sonication. The antibodies used were as follows: phospho-Ser10-Histone H3 (Cat.# 9715), AURKB (Cat.# 3049) and Cleaved Caspase-3 (Cat.# 9661) from Cell Signaling, TP53 (Cat.# sc-126) and GAPDH (Cat.# sc-32233) from Santa Cruz Biotechnology, MYCN (Cat.# OP13L) and CDKN1A (p21/WAF1) (Cat.# OP64) from Calbiochem , vinculin (Cat.# V9131) from Sigma-Aldrich, and PARP (Cat.# 556494) from BD Biosciences. Secondary antibodies were obtained from LI-COR  and Thermo Scientific Pierce . The Odyssey Imaging System from LI-COR was used for signal detection.IMR5 and SK-N-BE (2c) were plated into T25 flasks for cell cycle analysis and allowed to attach overnight at subconfluent concentrations after 48h. IMR5 cells were treated with 8 nM, 12 nM, 16 nM and 20 nM of barasertib whereas SK-N-BE (2c) cells were treated with 20 nM, 50 nM, 100 nM, 500 nM and 1 μM of the compound. The Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit  was used for cell cycle assessment according to the manufacturer's protocol. In short, cells were treated with 10 μM EdU (1:1000) for 2h before collection after 24h and 48h. Cells were fixed, permeabilized, and washed before incubating the samples with the Click-iT reaction cocktail for 30min at RT. FxCycle Violet  was used to counterstain DNA. Flow cytometric analysis was performed on a BD LSRFortessa Cell Analyzer . Data were acquired with the BD FACSDiva software and analyzed with FlowJo version 10 .MYCN-amplified tumors. Animal studies were performed with 4- to 6-week-old female NOD scid gamma (NSG) mice . Five million IMR32 neuroblastoma cells stably expressing luciferase in 100 μl 1:1 HBSS:Matrigel solution  were subcutaneously engrafted into the right flank of each mouse to generate an in vivo neuroblastoma xenograft model. Barasertib was obtained through the Developmental Therapeutics Program, the Division of Cancer Treatment Diagnosis and Centers, National Cancer Institute. Barasertib was dissolved in a solution vehicle made of 30% PEG400, 0.5% Tween 80, 5% Propylene glycol , and administrated to the mice via intraperitoneal injection at 50mg/kg four times a week for two consecutive weeks. Mice were monitored weekly by palpation twice and luciferase imaging using a Xenogen VivoVision IVIS 100 system . Tumor size was measured by caliper twice a week and tumor volume was calculated using this formula: (long axis x short axis2)/2. The endpoint of the experiment was any tumor axis reaching 20mm. To investigate target-specific effects of barasertib in vivo, mice of the control arm were treated with either a cycle of four daily doses of 50mg/kg barasertib or vehicle control immediately after the endpoint of the tumor growth study was reached. Protein isolation from these tumors for immunoblotting was done in RIPA-buffer containing phosphatase inhibitors (sodium orthovanadate and NaF) and protease inhibitors  using a gentleMACS Dissociator  for tissue homogenization.A human neuroblastoma xenograft mouse model was used to examine the efficacy of barasertib for treating


Prnp−/−) mice. So far, six types of Prnp−/− mice have been generated, demonstrating the promiscuous functions of PrP. Recently, other PrP family members, such as Doppel and Shadoo, have been found. However, information obtained from comparative studies of structural and functional analyses of these PrP family proteins do not fully reveal PrP functions. Recently, varieties of Prnp−/− cell lines established from Prnp−/− mice have contributed to the analysis of PrP functions. In this mini-review, we focus on Prnp−/− cell lines and summarize currently available Prnp−/− cell lines and their characterizations. In addition, we introduce the recent advances in the methodology of cell line generation with knockout or knockdown of the PrP gene. We also discuss how these cell lines have provided valuable insights into PrP functions and show future perspectives.Elucidation of prion protein (PrP) functions is crucial to fully understand prion diseases. A major approach to studying PrP functions is the use of PrP gene-knockout ( Prion diseases, also called transmissible spongiform encephalopathies (TSE), are a group of neurodegenerative disorders that affect both humans and animals -knockout (Prnp−/−) mice and cells derived from these mice   into abnormal PrP (PrPSc) constitutes a fundamental feature of prion diseases  is the most important factor for prion infection. This finding has been verified by experiments using PrP gene   is due to ectopic expression of Dpl (derived from Prnp/Prnd chimeric mRNAs through intergenic splicing) as a result of the disruption of the splicing acceptor of Prnp exon 3  as type 2, while the latter non-ataxic Prnp−/− mice  as type-1  and susceptibility to ischemic brain injury , encoded by Sprn. These PrP family proteins are all subject to endoproteolytic processing  mice. In addition, Prnp/Sprn knockout mice survived to over 600 days of age without any severe abnormality, suggesting the existence of a discrete signaling pathway of Prnp and Sprn to maintain neuronal survival. Sho was also found to be expressed in the trophoblast cells of the placenta  embryo and their wild-type counterparts suggested that Sho has functions complementary, not necessary overlapping, with those of PrP, associated with cellular movement and hematological system development and differentiation , a NF-E2 related factor 2 (Nrf2), amyloid precursor protein 1 (Aplp1), F-box protein-6, neural F-box protein 42 kDa (NFB42), postsynaptic density 95 kDa (PSD-95)/ SAP-90 associated protein, protein tyrosine phosphatase non-receptor type-21, and predicted protein KIAA0443 was reported , which is generated by the abnormal processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1) and involved in the pathogenesis of Alzheimer's disease  T-antigen gene . The retrovirus particles produced from the transfected ψ2 packaging cells were used for immortalization by introduction of the SV40 large T antigen gene. The hippocampal primary cultures derived from Prnp−/− and Prnp+/+ mice were infected with the above retrovirus, then incubated, selected, expanded and finally subjected to limiting dilution to obtain cloned cell lines. Three Prnp−/− cell lines, HpL2-1, HpL-3-2 and HpL3-4, and 3 Prnp+/+ cell lines HW8, HW9, HW19, were thereby obtained  were established using the gene transfer of oncogenes by our group  at 0.4–6 mM, retinoic acid (RA) at 0.1–10 μM, and phorbol 12-myristate 13-acetate (PMA) at 160–1600 nM, for up to 10 days of culture. These compounds all induced morphological changes, i.e., elongation of neurite-like processes, in both HpL and HW cells. Among the tested compounds, the morphological change was best observed by stimulating the HpL and HW cells in medium with dcAMP or PMA without fetal calf serum (FCS). To further characterize the cell types of the cell lines, reverse transcription polymerase chain reaction (RT-PCR) was applied to monitor the expression of neurofilament genes. Treatment with PMA resulted in the amplification of the predicted size products corresponding to neurofilament (NF)-68K and NF-200K genes. The NF-68K gene was transcribed in all 3 HpL and 3 HW cell lines with or without PMA treatment. After treatment with PMA, the NF-200K gene was transcribed in all three HW cell lines and in only one of the HpL cell lines (HpL2-1). The transcription of GFAP, a glial cell marker, was not detected in HpL or HW cells. Taken together, the results suggest that all six of these cell lines belong to the neuronal precursor cell lineage.Prnp. Moreover, levels of superoxide anion generated from HpL3-4 cells after serum deprivation were suppressed by reintroduction of Prnp  enhanced autophagic flux, such as the formation of autophagic vacuoles, was observed in HpL3-4 cells in which Prnp was reintroduced. However, autophagy was impaired in HpL3-4 cells transfected with empty vector   in HpL3-4 cells, there was no comparison with the HpL3-4 counterpart in which Prnp was reintroduced . The results obtained from the study of HpL cell lines are consistent with those derived from other studies using primary cultures and other cell lines, which also showed the involvement of PrP in neuroprotection and differentiation  analysis to search for PrP-interacting proteins and identified 28 new hits  with ectopic Dpl expression. To investigate whether PrP inhibits apoptotic neuronal cell death in the absence of Dpl, neuronal cell lines were established from the brain of type-1 Prnp−/− mice (Zrch I), which did not show ectopic Dpl expression . Moreover, PrP expression was found to upregulate cellular SOD activity in NpL2 cells. Therefore, we concluded that ectopic Dpl production did not affect the anti-apoptotic and anti-oxidative functions of PrP. Thus, it was confirmed that the presence of PrP is directly correlated with protection against oxidative stress.As described earlier, PrP exhibits a neuroprotective effect against apoptosis induced by serum deprivation in HpL3-4 cells, which are derived from type-2 Prnp−/− mice by lipofection of SV40 large T antigen-expressing vector and found higher proliferation of neuronal cell lines from Prnp−/− mice (Zpl) than those derived from Prnp+/+ mice (ZW) . Moreover, Zpl cells showed higher levels of apoptosis and autophagy compared to ZW cells after serum deprivation , octapeptide repeat (OR)-deficient PrP, did not display a protective activity in Zpl cells, indicating the importance of the OR region in the neuroprotective activity of PrP. These results are consistent with the HpL3-4 studies using deletion PrP mutants as described in the following section.Kim et al. established neuronal cell lines from hippocampal neurons of Zrch I Prnp−/− derived cell line, obtained by fusion of Prnp−/− cerebellar cells and mouse neuroblastoma cells -mediated knockdown of Prnp, to investigate the gene silencing effect of Prnp  showed increased SOD activity compared to control GpL1 cells transfected with empty vector (GpL1-EM). Thus, this study extended the notion that PrPC prevents apoptosis by its anti-oxidative function in not only a Prnp−/− neuronal cell line but also a Prnp−/− astroglial cell line after serum withdrawal.PrPC function in independent cells, we have investigated whether PrP expression on the cell surface of astroglia influences neuronal survival because neurons and astroglia are closely coupled in their metabolic activities  compared to GpL1 cells transfected with empty vector (GpL1-EM). Similar results were observed when we used both NpL2 cells transfected with Prnp (NpL2-PrP) and NpL2 cells transfected with empty vector (NpL2-EM). These findings suggest that the neuronal cell viability depended on the PrP expression in feeder astroglia in the co-culture system. Taken together, astroglial cell lines are useful for revealing the mechanism of PrPC not only in independent astroglial cells but also in the function of the neuron-glia relationship.In addition to PrPSc accumulation  or lymphocytes, can act as a replication site or as a reservoir for prions  from FVB/N Prnp+/+ and Zrch I Prnp−/− mice were transformed with a replication-defective retrovirus encoding SV40 large T antigen by a similar method previously used for the establishment of HpL, NpL, and GpL cell lines  and MplZ4-3 (from Zrch I Prnp−/− mice) for comparison, because these cells expressed macrophage specific proteins (F4/80 and MOMA-2) and displayed phagocytotic properties. Because it was previously reported that Prnp−/− neuronal HpL and NpL cells and astroglial GpL cells are sensitive to oxidative stress in serum-free conditions, MWF3-3 and MplZ4-3 cells were subjected to serum deprivation. The MplZ4-3 cells died after withdrawal of serum, whereas most MWF3-3 cells survived under the same culture conditions. These findings suggest that PrPC increases the survival rate of macrophages.Macrophages express PrPPrnp−/− macrophage cell line, we have analyzed their cell morphology and phagocytotic activity. The Prnp−/− macrophage cell line (MplZ4-3) showed shorter pseudopodium extension and less phagocytotic activity of latex beads than a Prnp+/+ macrophage cell line (MWF3-3). These findings are consistent with the results using peritoneal macrophages from Prnp−/− and Prnp+/+ mice using latex beads  , and are highly similar to peripheral macrophages. These cells play important roles as the major inflammatory cell type in the brain, and respond to pathogens and injury and contribute to destroying pathogens as well as removing damaged cells. Several studies have indicated that microglia are implicated in the pathogenic events of prion diseases  derived from continuous cultures of abdominal skin explants of Ngsk Prnp−/− mice. SFK cells showed decreased expression of Ras- and Rac-related proteins compared to Prnp+/+ skin fibroblast cell lines (SFH)  from Ngsk Prnp−/− mice by immortalization with the chemical mutagen 3-methylcholanthrene  and the hydrophobic region (HR) (amino acid residue 112–145 in mouse PrP) domains.PrP contains disulfide links (S–S), Asn-linked glycosylation sites (CHO), a signal peptide sequence (SP) and a GPI anchor attached to its C-terminus Brown, . In addivia histidine residues located in the OR domain. The binding of copper to PrP is thought to be indispensable for the SOD activity of PrP GGG(G/-)WGQ, is highly conserved among mammals and can selectively bind copper , PrP (Δ#1: HpL3-4-Δ#1), PrP(Δ95-132) (Δ#2: HpL3-4-Δ#2), PrP(Δ124-146) (Δ#3: HpL3-4-Δ#3), PrP(1-95)-Dpl (ORDpl: HpL3-4-ORDpl), PrP(1-124)-Dpl (ORHRDpl: HpL3-4-ORHRDpl) or empty vector alone (EM: HpL3-4-EM)]. Western blotting confirmed that the cell clones expressed the deletion mutants and fusions at similar levels to those of clones expressing the full-length protein. In the absence of serum, HpL3-4 cells undergo cell death with features of apoptosis, whereas Prnp−/− cells transfected with Prnp are resistant to serum deprivation , miRNA (micro-RNA), bi-shRNA , or shRNA  that is overexpressed, mutated or deleted can proliferate and be maintained in culture medium, resulting in the establishment of a cell line (Obinata, Prnp, resulting in the production of Prnp+/− or Prnp−/− cell lines.If knockout of d Figure . Recent Obinata, . AlternaObinata, , ZFN (ZiObinata,  or TALENObinata,  can be uPrnp−/− cell lines are mainly used for two experimental purposes; analysis of PrPC function and the study of prions. For example, there are several reports of the successful production of monoclonal and polyclonal antibodies against PrP by immunizing Prnp−/− mice with recombinant murine PrP (Zanusso et al., C and PrPSc (Prusiner et al., Prnp (Krasemann et al., Prnp−/− mice with the derived Prnp−/− cell lines transfected with Prnp or mutated Prnp, specific antibodies to wild-type or mutated PrP could be readily obtained. Furthermore, to obtain specific antibodies to PrPSc, immunization of Prnp−/− mice with prion-infected Prnp−/− cells transfected with Prnp could be a promising approach.Prnp−/− cell lines are useful for producing susceptible cells to target species of prion agent after Prnp transfection of the target species. Importantly, endogenous PrP is not present in the Prnp−/− cell line. Thus, interference by endogenous PrP does not occur in a Prnp−/− cell line. The Prnp−/− cell line transfected with deletion mutants of PrP can be used for investigating important regions of PrP for prion infection. Previous results using the HpL3-4 cell line and chandler scrapie strain showed that OR is an essential region for the production of PrPSc during the early stage of infection (Sakudo et al., Sc formation (Maas et al., Sc. Taken together, these results suggest OR, α-helices and the surrounding region contributes to the susceptibility of prion infection. Newly generated PrPC and PrPSc antibodies together with cell lines that are susceptible or non-susceptible to target species of prion agents will be extremely useful for exploring prion biology.There is a species barrier in prion susceptibility, which is determined by the difference between the species of the host expressing PrP and species of prion agent (Beringue et al., C. By exploiting the combination of Prnp−/− cell lines, comparison of PrP-expressing and PrP-non-expressing cells by OMICS will provide invaluable information on the effect of PrP-deficiency and fundamental differences caused by PrP-deficiency. The accumulated knowledge using these systems will be of great help in understanding PrPC functions, which is also important in terms of clarifying the etiology of prion diseases.Finally, we would like to emphasize that development of “OMICS” such as genomics, epigenomics, proteomics, metabolomics and transcriptomics will enable us to obtain a global view of the network of processes that are regulated by PrPAll authors contributed to the conception of this review and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation.To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines . In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected.A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes. Novel therapeutic options have been made possible with production of animal cell culture-derived biopharmaceuticals. An enormous amount of insight has been published on production of recombinant proteins (including antibodies) since the first products have been licensed for application in humans 1986 and 1987. CHO cells are the main substrate for recombinant proteins and therefore most metabolic studies describe these cells . RegardiSeveral virus strains and viral vectors, e.g. Modified Vaccinia Virus Ankara (MVA), only replicate efficiently in avian cells . AccordiMetabolic models have been developed for a variety of cell lines to study physiological states or changes in metabolism as a response to different cultivation strategies, medium composition or stimuli like virus infection or accumulation of toxins. For example, the human cell lines HEK293 ,8 and AG13C labelling experiments. The higher experimental effort of such studies comes with the advantage of an increased number of constraints and uniquely resolvable flux distributions )CellNetAnalyzer, a MATLAB toolbox with graphical user interface facilitating metabolic network analysis [http://www.mpi-magdeburg.mpg.de/projects/cna/cna.html, and the network project files will be made available on this site within CellNetAnalyzer’s model repository.All computations presented in this study were performed with our software analysis . It can Patent applications covering avian cell lines (including AGE1.CR.pIX) have been filed by I. Jordan.VL and OH conceived the experiments or calculations, interpreted the data and drafted the manuscript. IJ and YG edited the manuscript and participated in planning the experiments. HB helped in data analyses and manuscript drafting. SK contributed to the conception of the study and edited the manuscript. UR helped in drafting the manuscript and generally supervised the project. All authors read and approved the final manuscript.Table S1. Amino acid composition of cell protein from CR.pIX cells and other cell lines. Table S2. Calculated rate ranges  or rates  for CR.pIX cells cultured in stirred tank reactors. Table S3. Measured concentrations, standard deviations of validated assays, and measurement devices.Click here for file


Pediatric Non-Hodgkin Lymphomas (NHL) are a diverse group of malignancies and as such treatment can vary based on the different biological characteristics of each malignancy. Significant advancements are being made in the treatment and outcomes of this group of malignancies. This is in large part due to novel targeted drug therapies that are being used in combination with traditional chemotherapy. Here, we discuss several new lines of therapy that are being developed or are in current use for pediatric patients with NHL. Pediatric Non-Hodgkin lymphomas (p-NHL) are a diverse group of diseases in their morphologic characteristics, clinical behavior, and biological features. Improvement in the outcomes of p-NHL has been seen over the past few decades. The use of multidrug chemotherapy and radiation therapy, intensification of treatment, improvement in supportive care, and better imaging and staging systems have resulted in the cure of more than 75% of patients, representing one of the most significant success stories in Pediatric Oncology. More recently, tremendous progress in the understanding of cancer cell biology and its microenvironment have resulted in the development of biologic agents, also called “target” therapies, that are more specific in targeting cancer cells either directly or via enhancement of the immune system. Many clinical studies have focused on those biologic agents in combination with traditional chemotherapy with the goal of improved outcome, or reduced acute or long term complications associated with non-Hodgkin lymphoma (NHL) therapy. In this review, we will discuss different biologic agents that are currently available for patients with NHL. Many of these drugs are already under investigation in p-NHL; however, some of these are currently limited to adult trials but given promising results, may be incorporated into future pediatric trials.et al. [B-lymphocyte antigen CD20 is an activated glycosylated phosphoprotein expressed on the surface of all B cells, and on greater than 90% of B-NHL cells. Rituximab is a chimeric murine/human anti-CD20 monoclonal antibody. It has a human glycosylated IgG1 with a κ constant region and light and heavy chain variable regions isolated from a murine anti-CD20 monoclonal antibody. This chimeric antibody binds to CD20 positive cells and induces rapid depletion of CD20 normal B cells and lymphoma cells [et al.  conducteet al. . After tet al. ,6,7,8,9.et al. [Pediatric NHLs also express CD20 in the majority of cases. CD20 is positively present in 100% of the pediatric Burkitt and Burkitt-like lymphomas, and 98% of cases with diffuse large B-cell lymphomas (DLBCL) ,12. Lesset al.  reviewedet al. ,15,16. Set al. . A phaseet al. ,19, impret al. . No signet al. . Current90Yttrium-Ibrutumomab tiuxetan is a monoclonal antibody for radioimmunotherapy. It is made up of an immunoglobulin IgG1 mouse monoclonal antibody that is attached to a β radioactive isotope, 90yttrium (90Y). 90Yttrium is a high energy isotope that gets delivered intracellularly via the anti-CD20 antibody in CD20+ cells, leading to cell death [versus ibrutumumab tiuxetan [90yttrium-ibritumomab tiuxetan has been tested in children and adolescents with (r/r) CD20+ lymphomas in a phase I setting [ll death . A phasetiuxetan . Responstiuxetan . Within  setting . Ibritumin vitro and in vivo CD30 positive models [Activated T- and B-cells express CD30, a cell membrane protein of the tumor necrosis factor (TNF) receptor family, which has a role in apoptosis regulation. CD30 has been found to be expressed in a variety of malignancies, including embryonal carcinoma , Hodgkine models . Subseque models , leadinge models .The initial experience with BV is limited to case reports of pediatric patients with (r/r) HL or ALCL . Safety Currently, Children’s Oncology Group is conducting a randomized phase II trial of BV or crizotinib (discussed herein) in combination with standard chemotherapy (best arm of ALCL99) for newly diagnosed pediatric patients with ALCL.Nelarabine is a nucleoside analog and an effective pro-drug of ara-G . It is d2) administered on Days 1, 4, 8, and 11 added to four-drug induction chemotherapy with vincristine, dexamethasone, pegylated l-asparaginase, and doxorubicin in children with relapsed ALL showed acceptable toxicity, and 67% of the patients (six out of nine) achieved CR [Bortezomib has been described in the treatment of multiple myeloma and is known to reversibly block proteolytic activity in proteasomes. It blocks NF-κB activation by stabilizing the lkB and thus allowing lkB to continue its inhibitory effect over NF-κB. NF-κB activation occurs frequently in childhood ALL. In vitro studies have shown promising results with bortezomib with T-cell ALL cell lines , likely ieved CR . A phaseieved CR . Bortezoin vivo [Histone deacetylase (HDAC) inhibitors are a class of drugs that have been shown to induce tumor cell apoptosis and/or cell cycle arrest, and to induce differentiation in a variate of tumor-derived cell lines and to block angiogenesis in vivo . SAHA siin vivo  as well in vivo . Clinicain vivo ,57, inclin vivo . SAHA hain vivo , and to in vivo  and pediin vivo . Recentlin vitro and in vivo cytotoxicity related to increased surface expression of MIC A/B and IL-2 activated natural killer (NK) in various ALL and NHL cell lines [Romidepsin is a structurally unique, bicyclic class I HDAC inhibitor that has been approved for the treatment of (r/r) primary T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) since 2009 . Althougll lines . Interesll lines .Clinical activity was demonstrated in a phase I trial that included three patients with CTCL (one patient achieved PR) and one patient with PTCL (CR), with reasonable tolerability and demonstrated increase in Sésary cell histone acetylation ,68. Clinet al. studied the use of belinostat in 22 adult patients with (r/r) DLBCL and found an ORR of 10.5% and good tolerability. This trial included correlative studies and will serve as the base for a SWOG trial combining HDACI with R-CHOP [Belinostat is a pan-HDACI available for the treatment of (r/r) PTCL since 2014  after a h R-CHOP . The safNPM1-ALK fusion protein) translocation [et al. published the results of a phase I trial of pediatric patients with (r/r) solid tumors or anaplastic large cell lymphoma and found good tolerance in children at a dose of 280 mg/m2 twice a day. Of note, single-agent crizotinib use resulted in objective antitumor activity in patients with activation ALK aberrations, including seven out of nine patients with ALCL reaching CR. Grade 3–4 adverse reactions included neutropenia, abnormal liver enzymes, and lymphopenia [Crizotinib is a tyrosine kinase inhibitor that targets the anaplastic lymphoma kinase (ALK) receptor. The ALK gene fusion was originally identified in anaplastic large cell lymphoma and encodes a cell surface tyrosine kinase that serves as a potential therapeutic target. It is constitutively activated in the majority of pediatric anaplastic large cell lymphoma (ALCL) cases due to the t  inhibitor. Activation of the B-cell receptor (BCR) signaling pathway leads to initiation of B-cell malignancies. BTK is required for BCR signaling and it is selectively and irreversibly inhibited by ibrutinib. Initial studies using ibrutinib showed objective response in B-cell NHL animal models . BurkittIbrutinib also has shown promising results in a primary mediastinal B cell lymphoma (PMBL) cell line. When treated with ibrutinib the total BTK protein expression was decreased. Cell proliferation was significantly decreased in the PMBL cell line when treated with ibrutinib alone, and in combination with dexamethasone, rituximab and carfilzomib .In a phase II adult study for relapsed/refractory follicular lymphoma, ibrutinib alone had an overall response rate of 30% and therapy was reported as well tolerated . In trean = 25), overall survival of 19.8 months (95% C.I. not reached), and PFS of 2.3 months (95% C.I. 1.8–3.7) .Idelalisib is a selective phosphatidylinositol 3 kinase δ inhibitor. The phosphatidylinositol 3 kinase δ (PI3δ) pathway is the most frequently activated pathogenic signaling in human cancer. PI3δ plays an important role in B-cell proliferation and survival. The effects of idelalisib were initially demonstrated in a variety of immature and mature B-cell malignancy cell lines with resulting decreased AKT phosphorylation, increase in poly (ADP-ribose) polymerase and caspase cleavage and induction of apoptosis . In 2014Tumor cells can evade the immune system by expressing Program Death (PD-1) ligands on the cell surface . Anti-PDThe treatment of NHL has been significantly influenced by the recent development of biological therapies. Considerable long-term complications are observed among pediatric survivors exposed at an early age to chemotherapy and/or radiation therapy. New biological agents may become an essential component of therapy to improve quality of life and reduce the burden of long-term complications in survivors. Up to now, the use of most of these new agents has been limited to adult patients or is being tested in the setting of refractory or relapsed disease only; continued effort to develop clinical trials in pediatrics is needed with the hopes that these may be successfully incorporated into upfront therapy for these patients. With targeted therapies such as those discussed here, it is possible that we will witness significant changes in the standard therapy of pediatric patients with NHL in the next several years.


No abstractThe online version of this article (doi:10.1186/s13058-014-0490-y) contains supplementary material, which is available to authorized users. Clustering the transcriptomic profile of 587 triple-negative breast cancer (TNBC) cases extracted from 21 breast cancer microarray datasets relying on the lack of transcript-level expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2), Lehmann and colleagues have categorized the TNBCs into seven subgroups [+, ER+PR+, Her2+, ER+PR+Her2−, ER−PR−Her2+, luminal A, luminal B and non-TNBC (if not specified) or matched adjacent normal, unmatched normal breast or parenchyma tissues as analyzed. The ethnicity/origin as provided by the authors/deduced, the frequency of observation of the molecular alterations in terms of the number of patient samples analyzed per study, fold values of expression, experiment platform used and the reference to corresponding research articles are provided for the curated records.The TNBC tissues are categorized into lymph node-positive, lymph node-negative and lymph node metastatic tissues, or otherwise as TNBC-not specified. TNBCDb hosts experimentally reported alterations in these tissues compared with ERTNBCDb also hosts the comparative molecular profile of 39 TNBC cell lines compared among themselves as well as with 55 non-TNBC cell lines as a tool for selection of appropriate cell lines for specific studies on the basis of their molecular background. Effective visualization of genes differentially regulated in TNBC tissues and cell lines and, further, the signaling pathways , biologiConsidering the heterogeneity of breast cancers, we believe the TNBCDb will serve as a platform for selection of therapeutically relevant molecular entities from the tissue and cell line information and also for the selection of appropriate cell lines for evaluation of therapeutic targets in the direction of personalized therapy. We request suggestions from the scientific community to improve and keep this resource up to date with more information/clinical parameters through an online portal . We beli