However immunological insights are restricted due to a lack of ferret-specific reagents and limited genetic information about ferret B and T cell receptors. Here, variable, diversity and joining genes within the ferret kappa, lambda and heavy chain immunoglobulin loci were annotated using available genomic information. A multiplex PCR approach was derived that facilitated the recovery of paired heavy and light chain immunoglobulin sequences from single sorted ferret B cells, allowing validation of predicted germline gene sequences and the identification of putative novel germlines. Eukaryotic expression vectors were developed that enabled the generation of recombinant ferret monoclonal antibodies. This work advances the ferret as an informative immunological model for viral diseases by allowing the in-depth interrogation of antibody-based immunity.The domestic ferret  Effective humoral immunity is contingent upon the phenomenal diversity of antibodies. In mammals, this is derived via genetic recombination of numerous variable (V), diversity (D) and joining (J) gene segments localised to heavy, kappa and lambda immunoglobulin loci. In recent years, the capacity to clone and express antibodies from single B cells has proved a powerful tool to study antibody repertoires in a variety of infectious disease settings in humans –4, and i(Mustela putorius furo) is a critical mammalian model to study pathogenesis and evaluate vaccines against a variety of human respiratory pathogens (reviewed in Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/AReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript “Sequencing B cell receptors from ferrets (Mustela putorius furo)” describes the development of a multiplex PCR approach to single cell-sequence ferret IgG antibodies. This study builds the framework for more in-depth immunological studies in an extremely important animal model for respiratory infectious diseases. The manuscript is scientifically soloid, well planned, written and relatively easy to follow by anyone in the field. The authors efforts to assemble the ferret immunoglobulin germline repertoire has been long needed, particularly in the Influenza research world, and will certainly pioneer similar studies in other respiratory infectious diseases.Overall this manuscript is of extreme value for the scientific community and I strongly recommend its publication in PLOSone. Nonetheless I do have some small concerns:1. The authors report the recovery of IgGH, IgGV� and IgGV� as 121/480, 99/480 and 144/384. Furthermore, it is then stated that 60/480 heavy/light chain pairs were recovered, but it would be important to know if these are equally distributed between � and � light chains. Plate based single cell sorting for BCR amplification is known to be considerably inefficient mostly due to low RNA input or inefficient diversity coverage during amplification. The ratio of successful heavy and light chain pairs to the number of individual heavy or light chain sequences recovered might be a good indicator good diversity coverage.2. In figure 5 the authors report the expression and purification of a Ferret/human chimeric antibody. In spite of the technological ingenuity of such experiment, it would be much more interesting to actually express and purify a full ferret monoclonal antibody. If using influenza infected animals, it would be easy to prove the specificity of this antibody by ELISA. Standing alone this experiment adds very little to the manuscript and actually distracts the reader from its main message.Reviewer #2: The manuscript describes the development of multiplex PCR for obtaining ferret Ig genes and generating recombinant monoclonal antibodies. This is an extremely important development to gain a better understanding of immune responses in ferrets, which have been a valuable model for the study of human respiratory infections. The manuscript is clearly written with well-designed experiments.Some minor comments are:Using bulk RNA-Seq to obtain constant region genes with multiple sub-classes can be problematic. It is mentioned that the sequences from RNA-Seq showed minimal variation compared to the genomic sequences. It will be helpful if this information is made available in some Figure or Table.Sequences of Constant gene segments are not provided in the Supplementary Table 1Line 117 – Should it be supplementary table 2 instead of 1?The sequence data should be deposited in GenbankThis is a very good start but significant more work is required to obtain a complete repertoire of germline Ig genes. Some discussion on how previous studies using template-switching methods such as BASIC, BALDR, BraCeR and VDJPuzle2 may be implemented for ferrets will be useful. In addition, long read sequencing has helped significantly improve our understanding of Ig loci in rhesus macaques . Discussion on using long-read sequencing and germline inference methods such as IgDiscover, partis and Tigger will also be helpful with respect to future directions in this field. Lastly, this work is extremely relevant with respect to the current pandemic. Discussing how ferrets maybe useful for studying viral infections such as COVID-19 will highlight the importance of this work.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  28 Apr 2020Response to ReviewersWe thank the reviewers for their positive comments and suggestions which have significantly strengthened the manuscript. We address each reviewers’ comments below in point-by-point form. Reviewer #1: The manuscript “Sequencing B cell receptors from ferrets (Mustela putorius furo)” describes the development of a multiplex PCR approach to single cell-sequence ferret IgG antibodies. This study builds the framework for more in-depth immunological studies in an extremely important animal model for respiratory infectious diseases. The manuscript is scientifically soloid, well planned, written and relatively easy to follow by anyone in the field. The authors efforts to assemble the ferret immunoglobulin germline repertoire has been long needed, particularly in the Influenza research world, and will certainly pioneer similar studies in other respiratory infectious diseases.Overall this manuscript is of extreme value for the scientific community and I strongly recommend its publication in PLOSone. Nonetheless I do have some small concerns:1. The authors report the recovery of IgGH, IgGV� and IgGV� as 121/480, 99/480 and 144/384. Furthermore, it is then stated that 60/480 heavy/light chain pairs were recovered, but it would be important to know if these are equally distributed between � and � light chains. Plate based single cell sorting for BCR amplification is known to be considerably inefficient mostly due to low RNA input or inefficient diversity coverage during amplification. The ratio of successful heavy and light chain pairs to the number of individual heavy or light chain sequences recovered might be a good indicator good diversity coverage.We have updated the manuscript (Lines 296-297) to reflect the relative proportions of kappa versus lambda light chain pairs recovered. This now reads:“Overall, this initial pilot allowed about 12.5% (60/480) recovery efficiency of functional heavy and light chain pairs from single sorted ferret B cells, with 31/60 pairs utilizing Kappa and 29/60 pairs utilizing Lambda chains.”2. In figure 5 the authors report the expression and purification of a Ferret/human chimeric antibody. In spite of the technological ingenuity of such experiment, it would be much more interesting to actually express and purify a full ferret monoclonal antibody. If using influenza infected animals, it would be easy to prove the specificity of this antibody by ELISA. Standing alone this experiment adds very little to the manuscript and actually distracts the reader from its main message.We agree and have undertaken the recovery of a fully-ferret monoclonal antibody and included both ELISA and viral escape data in the revised manuscript. We have updated the Methods with the following:“For the recovery of antigen-specific ferret B cells, a single ferret was infected with 1000 TCID50 of H1N1 A/California/04/2009 and a single cell suspension of parapharyngeal lymph node cells (pLN) was prepared at 28 days post-infection and cryopreserved in heat-inactivated FCS containing 10% DMSO. Cells were subsequently thawed and stained with Live/Dead Fixable Aqua (Thermo Fisher), surface stains anti-CD11b-BV510 (Biolegend: clone M1/70), anti-ferret IgA/IgM/IgG-FITC , anti-CD8 eFluor450 (eBioscience Clone OKT8), anti-ferret CD4  conjugated to APC-Cy-7. Biotinylated recombinant full length A/California/04/2009 hemagglutinin (HA) probes  conjugated to streptavidin-PE or streptavidin-APC (Invitrogen) were used to sort single HA-specific B cells into 96-well plates and stored at -20oC.”And:“Viral Escape AsssayThe generation of viral escape mutants was based upon previously described protocols . Briefly, 24-well plates were seeded with 2.5 x 105 Madin Darby Canine Kidney (MDCK) cells per well to form confluent monolayers. The next day, serial dilutions of recombinant ferret mAbs were incubated with A/California/04/20090 virus for one hour at 37oC in Flu-media (Dulbecco's Modified Eagle's Medium (DMEM) with 0.8% bovine serum albumin (BSA), 1% penicillin/streptomycin and 0.1% L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin), before adding the virus-antibody mixture to MDCK cells. After 2 to 3 days culture media supernatants from wells with visible cytopathic effect were collected and used to infect a fresh monolayer of MDCK cells in the presence of increasing concentrations of mAb. After serial passaging, culture supernatants were harvested, viral RNA was extracted and the HA gene sequenced. Putative mutant viruses were identified based upon sequence comparison to similarly passaged media-only controls.“We have updated the Results with the following:“Recovery of hemagglutinin-specific monoclonal antibodies from an influenza-infected ferretWe next sought to recover monoclonal antibodies from influenza immune ferrets. From a single ferret infected with H1N1 A/California/04/2009, we cells from parapharyngeal lymph nodes 28 days post-infection, stained with a panel of ferret B cell surface markers and sorted single cells that bound to recombinant HA probes  labelled in two alternate fluorophores (PE and APC) . Due to an inability to identify ferret IgG subclasses, we designed additional primers (Supplementary table 2) targeting IGHJ-gene segments for multiplex PCR amplification of ferret immunoglobulin genes. From 376 sorted HA specific B-cells, we recovered 263 productive, recombined heavy chain immunoglobulin sequences including significant clonal expansions . Representative examples from various antibody lineages were cloned into ferret expression vectors, expressed in mammalian cell culture and screened by ELISA. Two antibodies (3B03 and 4A06), derived from a common lineage, were found to both bind to full length A/California/04/2009 HA by ELISA . Both antibodies  mediated potent hemagglutinin inhibition activity against A/California/04/2009 (data not shown) and similarly when tested using an in vitro neutralization assay, both 3B03 and 4A06 were able to prevent A/California/04/2009 infection of MDCK cells down to an effective concentration of 0.4mg/ml and 0.08mg/ml respectively. In order to better understand the epitope on HA recognised by 3B03 and 4A06, we generated escape viruses by serial passaging of A/California/04/2009 in the presence of ferret mAbs followed by sequencing of the full-length HA gene using standard techniques . Neutralisation resistant virus displayed a single K163E mutation  localised within the canonical Sa epitope  and previously associated in human populations with escape from pandemic H1N1 serum neutralising activity .”And we have included a new Figures 6 and 7:And additional text has been incorporated throughout the discussion.Reviewer #2: The manuscript describes the development of multiplex PCR for obtaining ferret Ig genes and generating recombinant monoclonal antibodies. This is an extremely important development to gain a better understanding of immune responses in ferrets, which have been a valuable model for the study of human respiratory infections. The manuscript is clearly written with well-designed experiments.Some minor comments are:Using bulk RNA-Seq to obtain constant region genes with multiple sub-classes can be problematic. It is mentioned that the sequences from RNA-Seq showed minimal variation compared to the genomic sequences. It will be helpful if this information is made available in some Figure or Table.Sequences of Constant gene segments are not provided in the Supplementary Table 1Line 117 – Should it be supplementary table 2 instead of 1?Sequences for the ferret constant gene segments have been included within Supplementary Table 1 (Tab – “constant”)The sequence data should be deposited in GenbankRelevant sequence data have been deposited to Genbank (submission ID:2331742)This is a very good start but significant more work is required to obtain a complete repertoire of germline Ig genes. Some discussion on how previous studies using template-switching methods such as BASIC, BALDR, BraCeR and VDJPuzle2 may be implemented for ferrets will be useful. In addition, long read sequencing has helped significantly improve our understanding of Ig loci in rhesus macaques . Discussion on using long-read sequencing and germline inference methods such as IgDiscover, partis and Tigger will also be helpful with respect to future directions in this field. We have expanded the discussion to cover these important sequencing technologies. It now includes the following section:“While we employed a multiplex PCR approach to amplify immunoglobulin genes, the use of template switching based methods such as 5’RACE has been applied to other species such as dogs (39) and may reduce the potential for primer bias driving preferentially recovery of specific immunoglobulin gene segzments. Alternatively, next generation high throughput approaches and tools for the analysis of large RNA-seq data sets such as VDJPuzzle(40), BraCer(41), BALDR(42) and BASIC(43) have been deployed for analysis of antibody repertoires from cats (44) and rhesus macaques (45). While the entire ferret kappa loci is assembled in the current copy of the ferret genome, the lambda and heavy chain loci were distributed across a number of contigs and the gene arrangement of these two loci remains unclear. Recent advances in long-read NGS approaches such as Oxford Nanopore (46) technologies could enable high resolution mapping of these loci in ferrets, as recently demonstrated by the assembly of reference gene loci in rhesus macaques (45) which facilitates the in depth analysis of antigen-specific immunoglobulin gene repertoires and the characterisation of antigenic epitopes (47). Additional germline inference methods such as IgDiscover (48) and TIgER (49) could also be deployed in ferrets to more accurately assess and categorise allelic variation within these outbred animals.”Lastly, this work is extremely relevant with respect to the current pandemic. Discussing how ferrets maybe useful for studying viral infections such as COVID-19 will highlight the importance of this work.We have expanded the discussion to cover the importance of ferrets for coronavirus research. It now includes the following section:“The tools reported in the current study may also be informative for other emerging human respiratory viruses, most notably the current SARS-CoV2 pandemic. Ferrets can be productively infected with SARS-CoV2  and will serve as a critical model for testing therapeutic options and vaccines against the virus. The capacity to characterise immune repertoires and recover ferret mAbs from SARS-CoV2 infected or immunised ferrets might further support global efforts to develop effective countermeasures against COVID-19.”AttachmentPLOS_rebuttal_Final.docxSubmitted filename: Click here for additional data file. 13 May 2020Sequencing B cell receptors from ferrets (Mustela putorius furo)PONE-D-20-05643R1Dear Dr. Wheatley,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Stephen Mark TompkinsAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 18 May 2020PONE-D-20-05643R1 Mustela putorius furo) Sequencing B cell receptors from ferrets (Dear Dr. Wheatley:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Stephen Mark Tompkins Academic EditorPLOS ONE


The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines , induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays , non-cancerous human liver-derived assays  and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs  and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity  with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.The online version of this article (10.1007/s00204-020-02937-6) contains supplementary material, which is available to authorized users. The liver plays a central role in metabolizing exogenous substances. After oral uptake xenobiotics pass through the digestive tract and enter the liver via the portal vein, where metabolism by phase I and II enzymes take place  individually with equal weight, we also analyzed non-differentially expressed genes (non-DEGs). Usually, differentially expressed genes (DEGs) between samples are analyzed to describe the differences between cell types, exposures, time points, or other influences . By RNA-Seq, quantified information of different transcripts of a gene can be obtained  variants for each given gene so that the change in the amount of expression of individual isoforms is not apparent. A previous consensus has been that the majority of genes are regulated through their mRNA levels but NGS has shown that also the selection of individual spliced variants may change, while the sum of all isoforms remains unchanged. Moreover, the ENCODE project revealed through NGS that close to 95% of human multi-exon genes undergo alternative splicing Carninci  to form To our knowledge, previous studies have compared genome-wide expression only of individual cell models, such as e.g. PHH and iPSC derived human hepatocyte-like cells  was analyzed using the Fastqc (version 0.10.1)  were present for all cell models. To eliminate this possible source of bias, we selected three replicates from each cell model which presented the highest similarity with the healthy in vivo liver . Then each in vitro dataset was compared individually to the in vivo dataset for calculating the non-DEGs using the ‘DESeq’ function from DESeq2 R-package  were removed. These isoforms were removed, because their expression level was not sufficient to be considered above the noise at this sequencing depth. This filtration step was performed on isoform counts.Similar expression:Isoforms that differ less than equal to 10% between the average percentage of in vivo and in vitro samples were removed, because we were interested in looking for the isoforms having sufficient differential usage. This filtration step was performed on isoform percentages.No expression in some samples:The isoforms that were not detected in more than 20% of the samples in any one of the datasets (in vivo or in vitro) were discarded, as this would reduce the confidence in the samples that showed expression for those isoforms. If the number of samples for test or control were less than five, we imposed that the transcripts were detected in all samples. This filtration step was performed on isoform percentages.The change in the proportion of the transcripts expressed for a gene represents differential transcript usage. Isoform counts and percentages were calculated using RSEM. The isoform counts were normalized using DESeq2 as explained for gene reads for the selected replicates for each cell model. Considering the number of transcripts assessed, multiple filtering steps were applied to remove the low expressed transcripts (or noise), and transcripts expressed at a similar level from the control (in vivo) and test (in vitro) samples.Isoforms deleted from the count dataset were removed from the percentages dataset and the ones deleted from the percentages were removed from the counts.p value < 0.01 as the calculation at the isoform level has a higher error rate.After the filtration steps, the genes left with only one isoform were removed from both datasets (counts and percentages). The variance was calculated between the test and control samples for all the remaining transcripts using ANOVA in R. Isoform percentages were used to find the variance because percentages are linearly distributed (contrary to the RNA-Seq read count). It was filtered on DTU−. The list of non-DEGsDTU− was mapped onto the KEGG pathways and pathway coverage was recalculated.The highest expressed isoform was identified (highest percentage) in the control samples for each gene and was compared with its expression profile in test samples. The genes with different expression profiles (DTU) were removed from the non-DEGs and were named as non-DEGs8) for community usage. However, to avoid coverage bias in our analysis with the in vitro samples (sequenced at a depth of 33.65*106), only the first 30 million reads of the fastq files obtained from the in vivo samples were used. We compared the full coverage and part of the data and found that the whole sample and sub-selection had similar distribution .RNA from totally of 27 liver tissue specimens from donors without liver diseases, further named “healthy in vivo liver” and 46 samples from cultivated hepatocytes, cell lines, liver slices or iPSC-derived hepatocyte-like cells, so-called in vitro cell models, were sequenced on the Illumina NovaSeq  and the Illumina HiSeq 2000 , respectively. After removing the samples from children or infants for healthy in vivo liver specimens and filtering for sequencing depth 24 in vivo cell models, 38 in vitro samples remained for further analysis (Suppl. Table 1). Since healthy human liver represents a very valuable resource, we generated sequences at very high depth . The samples that had lower protein coding and 3′UTR reads showed an increase in the intergenic and 5′UTR reads. Overall, the samples had similar distributions across different regions. Furthermore, the global similarity of the cell models was evaluated using pairwise Spearman’s correlation for normalized read counts  of the in vitro cell models and in vivo liver were used to identify genes that are not differentially expressed (non-DEGs) Fig. . SimilarTo understand the effect on the biological processes, we then mapped all non-DEGs onto KEGG pathways (Suppl. Table 4a). Pathway mapping data can be used to study the specific processes/pathways of interest for each cell model and provide a metric of the similarity between liver tissue and the individual in vitro systems. Pathway coverage was calculated for the 20 liver pathways  before the incubation period showed the highest coverage for most pathways but after 168 and 336 h of incubation, the coverage systematically dropped. In general, HepaRG 3D and HepG2 demonstrated a much lower coverage with HepG2 having the least. Exceptions were the high DNA repair functions of both tumor cell lines, with a relatively high coverage seen for base excision repair for HepaRG (68%) and nucleotide excision repair for HepG2 (59%). PHH showed a relatively high pathway coverage for all pathways and only small differences before (0 h) and after (24 h) after the cultivation period. For the primary bile acid biosynthesis pathway, PHH showed an increase during the 24 h cultivation period. For cytochrome P450 pathways, microtissues, PHH, and hPCLiS demonstrated a high coverage , hence presenting their metabolizing capacities for exogenous substances. While the number of pathways for which hPCLiS exhibited a low coverage, was six for 0 h and eight for 24 h, it also had the highest coverage for two pathways for 24 h (both cytochrome p450 pathways). IPSC-HLC also showed a high coverage for DNA repair pathways with highest on base excision repair (68%) and nucleotide excision repair (66%). For other DNA repair pathways, iPSC-HLC also exhibited a higher coverage than HepaRG and HepG2 cell models.q value (padj) < 0.05 and average counts > 10) to highlight the differences. The volcano plots demonstrated the extent of perturbation in the genes for all cell models . The Network influence score (NIS), defined by the expression of downstream regulated genes, for the transcription factors from all the cell models was calculated using CellNet . These differences were calculated with respect to the cell/tissue profiles of the CellNet. The results show that the transcription factor ATF5 had the highest perturbation for all cell models except the PHH where it was shown to be the least perturbed. In the case of PHH, NR1H4 was the most affected factor. Moreover, in the case of PHH, ATF5 exhibited perturbation in the opposite direction than all other cell models. A similar analysis using the microarray data for freshly extracted hepatocytes, PHH and hiPSC using the microarray data illustrated a different list of TFs being affected. However, different types of data used for the two studies (microarray and transcriptomics) might be the reason for this difference.Furthermore, we also investigated how the cell models behaved over the incubation period. Three cell models, namely, PHH, hPCLiS, and 3D liver microtissues were incubated for different time durations. We computed the DEGs for each cell model over different time points Fig. . PHH andp value < 0.01) at transcript level were then removed from the list of non-DEGs, giving the non-DEGDTU− (Suppl. Table 8). An exhaustive list of DTU for all the cell models can also be found in the supplementary (Suppl. Table 9). The gene for which transcripts had differential usage (DTU) should be removed from the non-DEGs to fine-tune the analyses. This was illustrated for the examples of four genes that were non -differentially expressed but display DTU for the in vitro cell models  was mostly replaced by other protein coding transcripts. For GOLGA8B (Golgin subfamily A member 8B) and ARHGAP21 (Rho GTPase-activating protein 21), it was predominantly replaced by non-coding transcripts. HSPA8 (Heat shock cognate 71 kDa protein) exhibited a different pattern, where the highest expressed protein coding transcript was replaced by other protein coding and non-coding transcripts. The highest expressed protein coding transcript in the case of POLR2F (52%) was reduced to < 2% in all cell models except hPCLiS 0 h (9%) while for HSPA8, it was reduced from 65 to < 3% for all except iPSC-HLC (25%). Similar trends can be seen for GOLGA8B and ARHGAP21  may nevertheless be relevant, because DTU may generate functionally different gene products. Differences in transcript expression (DTU) may be caused by alternative splicing, preference for one transcription start site over the other, spatial availability of transcription factors, and other elements. In standard RNA-Seq analysis, gene expression is assessed by summing the expression of all the transcripts for a given gene, and then it conceals the genes regulated at the splicing level. Genes with significant differential usage .An investigation at this level revealed major changes in transcript usage for all in vitro cell models Fig. . After rTU− Fig. , their cDifferent in vitro liver cell models have been developed for studying the effects of toxic compounds in humans. In the past, these models have been evaluated time and again for specific processes and components, giving a limited overview  reads for building the consensus expression profiles and GRNs  and hence pathway coverage. While globally the results of pathway coverage were similar to non-DEGs, studying the DTUs helped in identifying the genes which were differentially spliced between the in vivo liver and the in vitro system, notably by having a dominant protein coding or non-coding transcript(s). An important point worth mentioning here is that in our methodology, we used a stricter p value cut-off in the case of DTU (0.01), because isoform mapping is known to induce a higher false-positive rate  and in some cases even better than hPCLiS as well. Though these results suggest that they exhibit some similarity to in vivo liver, there are still significant hurdles to overcome before iPSC-derived hepatic progeny reach a high similarity to real hepatocytes. Different strategies to improve HLC differentiation may include chemical engineering of the culture media Supplementary file2 (DOCX 1229 kb)Supplementary file3 (DOCX 89 kb)Supplementary file4 (DOCX 254 kb)Supplementary file5 (DOCX 39 kb)Supplementary file6 (DOCX 324 kb)Supplementary file7 (DOCX 312 kb)Supplementary file8 (DOCX 316 kb)Supplementary file9 (DOCX 330 kb)Supplementary file10 (DOCX 341 kb)Supplementary file11 (DOCX 326 kb)Supplementary file12 (DOCX 90 kb)Supplementary file13 (DOCX 15 kb)Supplementary file14 (DOCX 16 kb)Supplementary file15 (DOCX 6068 kb)Supplementary file16 (DOCX 127015 kb)Supplementary file17 (DOCX 6727 kb)Supplementary file18 (DOCX 31 kb)Supplementary file19 (DOCX 668 kb)Supplementary file20 (DOCX 272 kb)Below is the link to the electronic supplementary material.


Conventional cytotoxic agents and pazopanib are approved for advanced soft tissue sarcomas but have low response rates and modest survival benefits. Recently, immune checkpoint inhibitors have shown clinically meaningful activity. The combination of pazopanib and immunotherapy has shown synergism in various other malignancies but has not been fully explored in advanced soft tissue sarcomas.A 63 year old woman with metastatic undifferentiated pleomorphic sarcoma progressed after two lines of palliative combination chemotherapy—doxorubicin with olaratumab, and gemcitabine with docetaxel. In view of significant symptoms, she was treated with pazopanib in combination with pembrolizumab. She had remarkable radiological and clinical improvement, with a manageable toxicity profile and an ongoing response at ten months of therapy.Undifferentiated pleomorphic sarcoma is an immunologically active subtype of soft tissue sarcoma, which is particularly amenable to immune checkpoint inhibitors. Pazopanib with immune checkpoint inhibitors is a well-tolerated, yet hitherto underexplored combination that may offer significant clinical benefit in advanced sarcomas—this finding warrants further evaluation in clinical trials. The outcomes in metastatic soft tissue sarcoma (mSTS) remain dismal even though various drugs have been added in treatment arsenal during this decade. Conventional cytotoxic agents like doxorubicin, ifosfamide and gemcitabine/docetaxel have modest activity and significant toxicities associated with their use. Pazopanib was the first targeted therapy that broke the dormancy in the landscape of mSTS based upon PALETTE trial and was approved by (US FDA) United States Food and Drug Administration in second line in non-adipocytic STS . SubsequImmune checkpoint inhibitors have shown promising results in many other tumors apart from sarcoma  and are thus being explored in advanced STS. A multicenter phase 2 trial (SARC-028) evaluating pembrolizumab in advanced STS showed an overall response rate of 40% (4/10) in patients with undifferentiated pleomorphic sarcoma (UPS) but was ineffective in leiomyosarcoma (0/10) and moderately effective in liposarcoma (2/10) . The PEMBased upon the available data (which show somewhat conflicting results), liposarcoma and undifferentiated pleomorphic sarcoma are probably the sarcomas in which immunotherapy should be explored. Herein we present the case of a 63 year old patient with metastatic undifferentiated pleomorphic sarcoma who failed two lines of therapy but had a remarkable response with anti-programmed death protein-1 (anti-PD-1) antibody pembrolizumab in combination with the multitargeted small molecule tyrosine kinase inhibitor pazopanib.A 63 year old woman with no known comorbidities, was evaluated in September 2017 for complaints of an insidious onset, gradually progressive painless swelling in the posterior aspect of right thigh. Magnetic resonance imaging scan revealed a well-defined, lobulated soft tissue lesion in posterior subcutaneous compartment of the right knee joint. She underwent excision biopsy of the primary lesion at a local hospital and histopathology was suggestive of undifferentiated pleomorphic sarcoma, with 14–15 mitoses per high power field, no necrosis and FNCLCC grade II  has been implicated not only in tumor angiogenesis and metastases, but also plays a crucial role in maintaining an immunosuppressive tumor microenvironment. VEGF-VEGF receptor interactions have been shown to upregulate intratumoral regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), and tumor-associated macrophages, while suppressing T cell function and interfering with differentiation and activation of dendritic cells . VEGF reAs the patient was in significant distress due to persistent cough, and monotherapy with pazopanib and pembrolizumab is associated with modest objective response rates , we considered combing the two agents in her case. Given the need to achieve a rapid tumor response, and evidence for the combination therapy resulting in meaningful results with acceptable toxicity, we were enticed to offer it to our patient after a discussion of the potential risks and benefits. She has tolerated the dual therapy well and has had no grade 3 or 4 adverse effects. Although each agent’s individual contribution to the good response observed in this patient can only be confirmed or refuted in a randomized trial, the improvement was most likely attributable to the immune checkpoint inhibitor since objective responses with single agent pazopanib are uncommon and typically not well-sustained . InteresIn contrast to immunologically active tumors like melanoma, the tumor microenvironment in soft tissue sarcomas is considered immunologically quiescent. Sarcoma tumor cells have poor antigenicity and easily escape recognition by tumor infiltrating immune effector cells. However, undifferentiated pleomorphic sarcomas have high tumor mutation burden and may provide protein targets for immunotherapy. Extremity and trunk UPS biopsy samples have been shown to have an infiltrate of immune cells at baseline, and this infiltration by CD3, CD4, CD8 and FOXP3 positive cells is further enhanced by radiation therapy . The posIn the SARC028 trial, only 3 out of 70 biopsy samples with advanced sarcoma were positive for PD-L1 expression at 1% threshold—all 3 cases were UPS. Although responses to pembrolizumab were seen even in the absence of PD-L1 expression, 2 evaluable patients with PD-L1 expressing tumors achieved partial response or complete response. The authors concluded that UPS fits the model of an inflamed tumor, with potential benefit from single agent anti-PD-1 antibodies. We may extrapolate these findings to suggest that patients with PD-L1 expressing UPS represent the most likely subset to benefit from anti-PD-1 antibodies. This biomarker analysis may allow better patient selection for consideration of immune checkpoint inhibitors. Outcomes from the expansion cohort of SARC 028 trial including total 40 patients with UPS have now been reported and have shown encouraging results. In these patients who failed one or more prior lines of therapy, pembrolizumab showed an overall response rate of 23% (9/40)—2 complete responses and 7 partial responses . 75% of Our case report adds to the currently scant literature on the results achieved from the combination of an immune checkpoint inhibitor and a VEGF TKI in advanced sarcomas, more specifically undifferentiated pleomorphic sarcoma. Considering that this dual therapy is feasible, well tolerated and can result in sustained response, these findings merit further evaluation in prospectively designed randomized clinical trials. Better understanding of the sarcoma microenvironment and response/resistance pathways is crucial to individualize therapy and improve patient outcomes.


Titanium carbide (TiC) reinforced nickel (Ni) matrix composites were processed via mechanical alloying (MA) followed by spark plasma sintering (SPS) process. Mechanical alloying has gained special attention as a powerful non-equilibrium process for fabricating amorphous and nanocrystalline materials, whereas spark plasma sintering (SPS) is a unique technique for processing dense and near net shape bulk alloys with homogenous microstructure. TiC reinforcement varied from 5 to 50 wt.% into nickel matrix to investigate its effect on the microstructure and mechanical behavior of Ni-TiC composites. All Ni-TiC composites powder was mechanically alloyed using planetary high energy ball mill with 400 rpm and ball to powder ratio (BPR) 15:1 for 24 h. Bulk Ni-TiC composites were then sintered via SPS process at 50 MPa pressure and 900–1200 °C temperature. All Ni-TiC composites exhibited higher microhardness and compressive strength than pure nickel due to the presence of homogeneously distributed TiC particles within the nickel matrix, matrix grain refinement, and excellent interfacial bonding between nickel and TiC reinforcement. There is an increase in Ni-TiC composites microhardness with an increase in TiC reinforcement from 5 to 50 wt.%, and it reaches the maximum value of 900 HV for Ni-50TiC composites. Over the past few decades, metal matrix composites (MMC) have attracted considerable interest due to their exceptional physical and mechanical properties, such as high specific modulus, fatigue strength, thermal stability, and wear resistance. These outstanding properties make MMCs suitable for a wide range of applications, including aerospace and automotive industry, and other structural, electrical, and thermal applications . Low coeWhile a lot of work has been done in the area of iron (Fe) and aluminum (Al) based MMCs, there has not been much work in the field of nickel (Ni) based MMCs, although there has been a significant increase in the interest surrounding them. Although Ni is a relatively soft metal that may not appear optimal for applications where high strength is required, it exhibits excellent bonding with harder reinforcement and substrates ,5, makinMechanical alloying is a method of dry alloying powders, which was invented in 1966 and rapidly gained popularity as a novel method of producing alloyed and composite powders . The milThe concept of current assisted sintering is described in several patents issued as early as 1933. Since then, the process has been developed with slight variations going by different names such as Spark Plasma Sintering (SPS), Plasma-Assisted Sintering (PAS) . Part ofThe development and production of nickel-based composites have been studied before. However, the production methods used to create these composites significantly influence the final properties of the components themselves. In the present investigation, Ni-TiC composites have been processed via mechanical alloying followed by SPS processing. There are very limited studies that have been done on understanding the effect of TiC content on microstructure and mechanical properties of Ni-TiC composites. This investigation’s primary objective is to study the effect of TiC content on microstructure, mechanical, and tribological behavior of SPS processed Ni-TiC composites. The present study will form the basis for the systemic selection of TiC content for the processing of Ni-TiC composites with target mechanical and tribological properties.The Ni-TiC composite powder was prepared from an elemental mixture of Ni metal powder  and TiC metal powder  powders alloyed via high energy ball mill . The mechanical alloying has been carried out for 24 h in a tungsten carbide 80 mL grinding bowl and 3 mm size balls with a ball to powder weight ratio (BPR) of 10:1 and 300 rpm. The milling operation carried in two steps involves 20 min of milling and 10mins of the cooling interval to avoid overheating the sample. Steric acid (2 wt.%) was used as a process control agent (PCA) during mechanical alloying to prevent excessive cold welding due to high energy generated during the alloying process. The TiC content inside composites has been systematically increased from 5 to 50 wt.% to investigate its effect on these composites’ microstructure and mechanical properties.The Ni-TiC composite powders were pre-compacted in a graphite mold under a pressure of 5 MPa. The pre-compacted powders were sintered by SPS system  at a temperature of 900–1200 °C for 5 min under a controlled argon atmosphere under a pressure of 65 MPa. The SPS processing parameters for pure nickel and Ni-TiC composites are listed in The microstructures of all samples were analyzed using Scanning Electron Microscopy (SEM) . Grain sizes were measured using ImageJ software, and an average of 50 grains were reported in this paper. The phase analysis of all sintered samples was performed using an X-ray diffraction (1.54 Cu Kα) line of a Rigaku Ultima III x-ray diffractometer. −4 s−1. Dry (unlubricated) wear tests were performed on a ball-on disc tribometer  at room temperature and the standard test method for wear testing pin on disc apparatus has been followed [3N4) ball (diameter 6 mm) was used as a counter body. The wear tests were conducted in the dry atmosphere (relative humidity~50%) using a load of 1 N, sliding velocity of 200 rpm, and track diameter of 5 mm . EDS elemental analysis using SEM was used for a better understanding of wear mechanism and track study.The SPS-processed samples microhardness measurements were carried using a standard Vickers microhardness tester  under a load of 5 N for 10 s. The average of those ten readings is used as the hardness for each sample. The cylindrical specimens with a 3 mm diameter and a height of 6 mm were machined using a wire-EDM machine and then used for the compression tests. The loading surface was polished parallel to the other, and lubrication oil was applied on both surfaces to avoid a barreling effect due to friction. The compression tests were carried out on the Instron 3369 universal testing machine with a strain rate of 1 × 10followed . Silicon3Ti, which cannot hold high sintering temperatures [eratures . The Vickers micro-hardness values for the pure nickel and all Ni-TiC composites have been plotted in The compressive stress versus strain plot for Ni-30TiC composites sintered at 900 °C and 1200 °C is shown in 3N4 ball wear has been observed during the wear testing. A tribolayer formed over the sliding track gives a strong indication of material transfer between the grinding surfaces. EDS map clearly shows the presence of silicon and oxygen along the wear track, indicating two to three-body abrasive wear mechanisms. In three-body wear phenomena, wear debris caught in-between the sliding surfaces, leading to the formation of tribiolayer over the sliding path; this similar mechanism is observed in the tribochemical wear. The chips and cracks and the highest silicon and oxygen presence over the sliding path revealed in EDS maps of pure nickel wear track shown in SEM micrographs and EDS maps of the pure Ni and Ni-TiC composites after the tribotesting are illustrated in 3N4 ball, causing a micro ploughing wear mechanism [On the other hand, Ni-50TiC shows significantly less surface wear and minimum counter-body material detected over the track than all different samples. TiC is a harder material, and Ni-TiC composites showing less wear due to the presence of TiC increases the load-bearing capacity . The flaechanism . The weaPure nickel and Ni-TiC composites have been successfully processed via mechanical alloying followed by spark plasma sintering process. The Ni-TiC composites exhibit 5–50 wt.% TiC reinforcements. The SPS processed pure nickel exhibit homogeneous grain structure, and the TiC reinforcements increase the number of nucleation sites, leading to refinement in pure nickel grain sizes. The refinement in grain size enhances the microhardness of Ni-TiC composites apart from microhardness improvement due to TiC reinforcement. Ni-5 wt.% TiC composites exhibit approximately two times higher microhardness than that of pure nickel. As the TiC wt.% increase in Ni-TiC composites, Ni-TiC composites microhardness increases almost linearly and reaches to the highest value of approximately 900 HV for Ni-50TiC composites. The sintering temperature plays an important role in improving the densification of these composites. The Ni-30TiC composites sintered at 1200 °C exhibits higher microhardness and densification than the sample sintered at 900 °C. Ni-30TiC composites exhibited the highest compressive strength compared to all other Ni-TiC composites primarily due to fine microstructure and stronger interfacial bonding between TiC particles and nickel matrix effectively transfer load from TiC to nickel matrix during deformation. The significant strength improvement in all Ni-TiC composites can be attributed to reinforcement and uniform dispersion of TiC particles, grain size refinement of nickel matrix, stronger interfacial bonding between TiC particles, and nickel matrix. It is clear from the present investigation that TiC reinforcement plays an important role in microstructures as well as microhardness and tribological behavior of Ni-TiC composites. The Ni-TiC metal matrix composites displayed high microhardness and a low coefficient of friction than a pure nickel. Mechanical alloying followed by spark plasma sintering process is a feasible way of processing these composites with improved densification and mechanical behavior without any excess grain growth observed in other conventional methods.


The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin  and platelet-derived growth factor BB  were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used. Coronary artery disease is the major cause of mortality and morbidity in industrialized nations. Beside coronary angioplasty/stenting, bypass surgery is frequently performed ,2,3. ForBefore novel devices are considered for in vivo implantation, they usually undergo a series of in vitro studies to investigate relevant material properties with respect to their functionality and safety in contact with blood  . Since aHUVEC cultured under conditions that prevent adherence and spreading stop growing and lose viability ,19,20. TConcerning these cellular components, endothelial cell based in vitro models for biomaterial evaluation provide only limited capability for clearance of apoptotic cells or cell fragments, comprising the possibility for the accumulation of cell remnants and mediators, which may can stimulate degenerative or even regenerative processes in nearby viable cells. Recently, it was shown, that apoptotic HUVEC release two types of extracellular vesicles upon caspase-3 activation: apoptotic bodies and exosome-like nanovesicles loaded with immunostimulatory RNAs causing inflammation after injection into mice . FurtherIt is clear from previous studies, that nonadherent HUVEC can have a major impact on the endothelialization of biomaterial surfaces. However, a possible release of signaling molecules from nonadherent dying HUVEC was often neglected in previous biomaterial endothelialization studies, which may have led to misleading conclusions for the in vitro assessment of cardiovascular implant material candidates. Therefore, HUVEC survival rate, aggregate formation and mediator release in anoikis are highly relevant aspects for initial in vitro biomaterial evaluation. The decision for further use of a candidate material may depend on these experiments and an inaccurate assessment of the endothelialization potential could exclude a promising biomaterial from further investigation. Therefore, all influencing factors present in the test system should always be taken into account for a secure evaluation. We addressed this topic in our study by investigating HUVEC behaviour under nonadherent conditions within the initial period of an in vitro biomaterial endothelialization study, concerning time dependent progress of HUVEC proliferation, anoikis, inflammatory activation and secretion of mediators, which may influence adherent HUVEC in the same cell culture.n = 6 for each condition in two independent experimental series with three HUVEC seedings each according to the scheme displayed in In this study, the cellular aspect regarding delayed or incomplete endothelialization was considered in the context of the in vitro evaluation of biomaterial surfaces. The focus was placed on the behaviour of nonadherent endothelial cells in the time frame of a rotational medium change in order to gain insights into possible influences of these cells on their surrounding environment under standardized conditions. In the study, polystyrene-based cell culture materials were used, which are established in in vitro cell culture systems. In tissue culture plates (TCP) the surface is optimised for most adherent cells due to its high hydrophilicity and surface charge. Low attachment plates (LAP) are hydrogel coated polystyrene used for suspension cell models and effectively prevent cell adhesion due to their hydrophilic, nonionic and neutrally charged surface properties. Both material surfaces thus represent the extremes in terms of endothelialization and were therefore deliberately used in this study to determine the behaviour of nonadherent HUVEC, without subjecting the cells to serum deprivation, as has been done in other studies to induce anoikis . HUVEC sIn step A, HUVEC from standard culture were labelled with CellTrace-CFSE proliferation marker see  and seedIn step C, HUVEC controls (W-48h and T-48h) were reseeded on TCP for 48 h until analysis. W-48h served as growth control for adherent HUVEC under standard cell culture conditions, whereby T-48h samples were additionally treated with recombinant human TNF-α (rhTNF-α) as positive control to induce inflammatory activation and artificial apoptosis induction. Nonadherent HUVEC samples S-2h till S-48h were reseeded into six well low attachment plates (LAP), where they remained in suspension without adhering for up to 48 h. Periods in suspension are indicated by the time code of the sample description . S-0h samples were used for HUVEC analysis directly before transfer in nonadherent conditions.In step D, supernatant and HUVEC were harvested for analysis of different parameters, such as cell morphology, proliferation, viability status, cell integrity, and mediator release.5 cells/mL; supernatant: 4.9 ± 2.5 · 103 cells/mL). About 86% of HUVEC in the supernatant showed a positive trypan blue staining indicating a high fraction of dead cells . In contrast, only four percent of the adherent HUVEC were dead , which would remain in culture after medium change.In prior to the comparative study performed experiments with HUVEC grown on TCP, automated cell counting showed that HUVEC were 99% adherent after 48 h and only a small percentage of cells remained nonadherent in the supernatant  and 56,842 ± 27,368 cells/cm2 for the rhTNF-α treated HUVEC (T-48h)). During nonadherent conditions CellTrace-CFSE staining intensity remained constant during the first eight hours of sedimentation, but then progressively decreased over time ; W-48h: 2.7 ± 0.2 · 10−8 Abs450nm/cell). Solely rhTNF-α treated adherent HUVEC showed always a higher LDH signal compared to all other samples (T-48h: 9.0 ± 0.7 · 10−8 Abs450nm/cell).Lactate dehydrogenase (LDH) activity was determined in the HUVEC supernatant  in orderVasoactive mediators—prostacyclin (PGI2) and thromboxane A2 (TXA2)—as well as relevant pro- and anti-inflammatory mediators, cytokines, chemokines and growth factors in the supernatant were quantified and when reasonable calculated as average secretion per cell, allowing a comparison between adherent and nonadherent HUVEC . There wBlood-material interactions are critical to the clinical success of cardiovascular devices like vascular grafts. Among other complications, thrombosis and clot formation remain to be major challenges in clinical application of vascular grafts ,27. One The study revealed that HUVEC undergo under nonadherent conditions phenotypic changes. Without the ability to adhere to the biomaterial matrix surface HUVEC progressively formed aggregates within a few hours and showed early evidence for apoptosis. HUVEC viability was significantly decreased already after two hours. Compared to TCP grown HUVEC, nonadherent endothelial cells showed higher numbers of Annexin-V and DAPI positive cells after eight hours, accompanied by distinct blebbing and increased occurrence of cell debris see . Within The proliferation studies showed a time-dependent decrease of the proliferation marker intensity, which indicated in the first hours of cultivation a proliferation of the nonadherent HUVEC comparable to HUVEC grown on TCP. However, stagnant HUVEC number under nonadhering conditions, progressive loss of membrane integrity and increasing rates of apoptosis and cell death demonstrated the fatal HUVEC development. In our study, about 40% of the HUVEC showed evidence for apoptosis/cell death already after eight hours and nearly 90% after 24 h. In HUVEC cultures grown at nonadherent conditions, apoptosis/cell death events were associated with significant releases of physiologically relevant amounts of the vasodilator PGI2 and the growth factor PDGF-BB. The accumulation of these mediators, but no secretion of, e.g., IL-6 or IL-8 in the supernatant suggests that the stress exerted by the condition of nonadherence did not imply an inflammatory activation. Such an inflammatory response could be demonstrated by treating the HUVEC with rhTNF-α, leading to an upregulation of the secretion of inflammatory mediators such as IL-6, IL-8 or the chemokine CCL2 (MCP-1) and subsequent induction of apoptosis. The baseline values for PGI2 were well in line with earlier in vitro studies with adherent HUVEC ,39,40. TWith respect to in vitro biomaterial evaluation studies, the release of PGI2 by nonadherent HUVEC is expected to influence surrounding viable adherent endothelial cells. PGI2 can induce the release of VEGF  followedThe current study clearly revealed that PGI2 and PDGF-BB were mainly released by the nonadherent HUVEC (PGI2: 7-fold higher and PDGF-BB only detected for nonadherent HUVEC) and not by the adherent HUVEC. In contrast, IL-6, IL-8 and MCP-1 were only released by the adherent HUVEC after inflammatory activation by rhTNF-α  indicating that TCP did not induce an inflammatory response of the HUVEC ,48. StudIn context with earlier studies, HUVEC in anoikis associated with the release of mediators such as TGF-β and cell remnants , have already been shown to elicit different responses in adherent HUVEC in the immediate environment ,25,26. TTherefore, both static as well as dynamic in vitro endothelialization studies should be considered more critically and in more detail with regard to all cellular factors presented in the experimental setup. see  A possib2) in tissue culture polystyrene flasks  with endothelial basal medium EBM-2 supplemented with EGM-2 Single Quots® kit and 2 vol.-% FBS to EGM-2 full medium . HUVEC were used for no longer than four passages [Commercially available primary human umbilical vein endothelial cells  were cultivated under static cell culture conditions  and incubated under standard cell culture conditions for 48 h. Subsequently, nonadherent HUVEC were harvested by isolation of the supernatant and additionally rinsing of the culture with PBS, whereby the supernatant was pooled with the PBS wash fraction. Remaining adherent HUVEC were harvested quantitatively from the wells by trypsin/EDTA treatment . After centrifugation of the supernatant and the adherent fraction at 220× g for 5 min, the cell pellets were resuspended in PBS and the HUVEC were quantified and characterized by trypan blue staining and automated cell counting using a Countess™ II FL Automated Cell Counter .Prior to the comparative study of HUVEC at nonadherent or adherent conditions, a preliminary study was performed to count adherent and nonadherent as well as viable and dead HUVEC grown on TCP, a material that is often used as control . HUVEC w2 from step A in a first incubation period (step B) in six well TCP culture plates ), as prove for typical HUVEC adherence and proliferation (see g for 5 min) to eliminate remaining cells or debris and stored immediately at −20 °C until secretion profile analysis. Remaining cells/supernatant from nonadherent HUVEC were harvested, quantified and analyzed for proliferation as well as apoptosis/cell death by flow cytometry, according to the protocols described below.Studies with nonadherent HUVEC were realized by transferring freshly CellTrace-CSFE labelled HUVEC see  with a dtion see . After c6 cells/mL were stained for 30 min under slightly shaking in the dark at 37 °C. Subsequently, HUVEC were washed in a four-fold excess of EGM-2 and further used for cell culture as described in the study design  was added in a final concentration of 1 µM prior to flow cytometric measurements. Flow cytometric analysis of HUVEC was performed with a MACSQuant® Analyzer 10 and MACS Quantify 2.6 software . FlowJo software  was used for further data analysis. Data acquisition was performed without exclusion of small particles  to gather information about HUVEC microparticle release, such as apoptotic bodies, during progress of the experiment as well as to detect the appearance of cell aggregates. Calibration beads in the scale between 6 to 15 µm from the Flow Cytometry Size Calibration Kit  were used as basis for further analysis of HUVEC by flow cytometry concerning apoptosis/cell death as well as proliferation determination by CellTrace-CFSE labelling, to identify the lower size limit of single HUVEC compared to cell debris. Single cell size of viable nonadherent HUVEC was concluded to range between 14 and 19 µm from light microscopy pictures (n = 109) and confirmed by published data [HUVEC apoptosis was detected using an Annexin-V/Alexa647 apoptosis detection assay . After cultivation period of step C, HUVEC were harvested, washed with cold PBS and 3 · 10hed data . FluoresStructural integrity of HUVEC membranes during incubation under nonadherent conditions was examined by lactate dehydrogenase (LDH) activity in the extracellular fluid by using the Cytotoxicity Detection Kit LDH  according to the manufacturer’s instructions. Secretion of the antagonistically acting vasoactive mediators prostacyclin (PGI2) and thromboxane A2 (TXA2) was quantified from the supernatant of nonadherent and adherent HUVEC by use of a competitive inhibition enzyme immunoassay for the stable hydrolysis product of PGI2  and TXA2 . Similarly, the concentration of prominent pro- and anti-inflammatory mediators , cytokines , chemokines  and growth factors  were quantified from the supernatant by magnetic bead-based Bioplex™ Cytokine Assay , whereby EGM-2 served always as background control.n = 6 in sum). Statistical significance between HUVEC samples in nonadherent conditions was calculated with a one-way ANOVA for repeated measures subsequently followed by Tukey multiple comparison test using Prism 8 . Further, statistical significance was calculated between S-48h and W-48h as well as W-48h and T-48h respectively with a two-tailed paired t-test. Statistical significance was assumed for p < 0.05 for all statistical analyses.For all samples, arithmetic means and standard deviations are given. Data were collected in two experimental series each with three independent HUVEC seedings in separated wells  and no further proliferation occurred. Combined with progressive blebbing and loss of cell membrane integrity, signaling mediators like PGI2 and PDGF-BB were increasingly released from intracellular storage. However, there was no evidence for an inflammatory activation of HUVEC solely by anoikis induced by cultivation under nonadherent conditions.These results show that studies for assessing the endothelialization of implant materials in vitro under static conditions, and maybe dynamic conditions as well, should always take into account the fraction of nonadherent HUVEC still suspended in the cell culture medium. HUVEC remaining in suspension can significantly influence the morphology and function of already adherent HUVEC and thus influence the assessment of the endothelialization capacity of implant materials, possibly distorting the assessment of the results.


Obesity in aging demonstrated similar expression of GWAT p53, p16, p21, Timp1 and Tgfβ1 in both sexes. Our studies suggest that even with aging, female GWAT shows an attenuated inflammatory response compared to males due to an efficient oxidative metabolism combined with an active tissue remodeling state.Aging, like obesity, is associated with metabolic and inflammatory alterations within adipose tissue in older individuals. Younger females are protected from adipose inflammation, but older post-menopausal females exhibit exaggerated visceral adiposity correlated with increased disease risk. Obesity accelerates the onset and progression of age-associated diseases, but it is unclear if aging and obesity drive adipose tissue dysfunction in a sexually dimorphic fashion. We investigated adipose tissue metabolism and inflammation in a diet-induced obesity model in young and old mice. We identified age related sex differences in adipose tissue macrophages (ATMs), fibrosis and lipid metabolism in male and female visceral fat depot (GWAT). Although aging normalized body weights between the sexes, females remained protected from proinflammatory ATMs and stimulated lipolysis failed to adversely affect the inflammatory state even with obesity. Older obese males had augmented CD11c Aging is associated with an increased risk for cardiovascular diseases, especially in older men and women , 2. StudAdipose tissue, while being a primary energy storage site, is now recognized as an endocrine organ that plays a key role in obesity-induced systemic inflammation. Adipocyte hypertrophy is an adaptive response to nutrient excess that maintains adipose tissue nutrient buffering capacity and protects other tissues from lipotoxicity. In obesity, inflammation is initiated by the inability of adipose tissue to buffer dietary lipids resulting in lipotoxicity mediated by the ectopic deposition of lipids in other organs , 6. LipoObesity-induced inflammation is specifically characterized by the infiltration and retention of immune cells within the adipose tissue known as adipose tissue macrophages (ATMs). Obesity leads to a shift in the activation state of ATMs from an M2-polarized state in lean animals that may protect adipocytes from inflammation to an M1 proinflammatory state . InfiltrAnother impact of aging related to sex differences is an overall decline in levels of estrogens and progesterone in females and testosterone in males , 17. Pos3-adrenergic receptor (ADRB3) agonist led to an additional varied inflammatory response in the older females. Our studies show that older obese females were still protected from proinflammatory macrophages and stimulated lipolysis did not adversely affect the inflammatory state. However, our studies suggest that efficient oxidative metabolism in older obese female adipose tissue compared to males combined with an active tissue remodeling state lead to this attenuated inflammatory response even with aging.Understanding the effects of aging on immune responses within the adipose tissue is important. The majority of experimental studies on inflammation have been performed using genetic- or diet-induced obesity in young male animals. This study includes young and aged mice of both sexes to investigate the effects of sex and aging on adipose tissue mass and pro-inflammatory responses. We identified age related sex differences in inflammation, fibrosis and lipid metabolism in aging male and female visceral fat depots. We also investigated whether excess free fatty acid (FFA) accumulation with stimulated adipose tissue lipolysis by a βTo determine differences in age induced metabolic changes, we used young (6-week) and old (10 months) mice that were placed on normal diet (ND) or high fat diet (HFD) for 24-week . Our study included both male and female mice in order to assess any sex differences in aging and HFD responses. We then examined body adiposity, adipose tissue, and liver mass. Diet and age had significant effect on all parameters . Concurrently with increase in liver weights (top right) than females (bottom right).Both young and old female mice on HFD showed significant visceral adiposity as demonstrated by larger GWAT fat pads in grams and as a percentage compared to males . Amongst weights  and 1F, These results suggest that aging led to a shift in fat accumulation in the liver in older males with obesity compared to the adipose depots, while older obese females continued to expand their adipose fat depots.top). CLS were also depicted by H&E staining (bottom) to be numerous and clustered closer together in males compared to females.In rodent models of obesity, macrophage accumulation depicted by macrophage clustering around adipocytes, crown-like structures (CLS), are observed in large numbers in the male GWAT with very few in the male IWAT after HFD exposure. In contrast, female GWAT shows very few pro-inflammatory macrophages with HFD –25. To astaining ; bottom +ATMs in the young obese males than females. Aging led to an increase in CD11c+ATMs in obese males but females showed no significant changes in the GWAT . CLS were considerably fewer in the female old GWAT. ADRB3 stimulation with CL did not show any drastic increase in CLS in older female GWAT (bottom).Previous studies from our lab and others show that FFAs are potential ligands for inflammatory pathways suggesting a link between lipolysis induced FFAs and inflammation , 28. We  females . To deteale GWAT , bottom.+ ATMs in both older male and female GWAT, but lipolytic stimulation did not affect the overall ATM numbers  were both significantly higher in old females on HFD but in older obese males only Mgl1 showed an increased expression , as well as alter soluble and ECM-bound factors, thereby impairing stem cell homeostasis and promoting cell senescence . Senesce obesity , 33. The and age –5F. p53 ale GWAT  and 5E. ung mice . p53, p1ale GWAT –5F. Obesale GWAT –5F. ThesMmp2 and Mmp9 expression in the GWAT were significantly affected by diet and age (5H). Mmp2 and Mmp9 expression were higher in older obese female GWAT  with women having an increased turnover than males [In order to further understand differences in flexibility of adipocytes in aging obese females we turned to evaluations of senescence in both sexes. Unexpectedly we found that both obese males and females showed similar upregulation of senescent markers- an males . A limitIn summary, our studies demonstrate that aging and obesity in aging induce sexually dimorphic inflammatory responses owing to altered lipid metabolism and adipose tissue remodeling even with the same HFD exposure. Since older female adipose depots have enhanced lipid storage, it cannot be ruled out that over time females may eventually develop greater lipotoxicity resulting in increased susceptibility to secondary health issues that increase in incidence with aging, such as cardiovascular disease and ischemic stroke. Overall, the results of our study highlight the importance of using aged animals of both sexes in obesity-related investigations and emphasize the need for sex-based intervention strategies in older individuals.ad libitum either a control ND consisting of 13% fat  or a HFD of 60% of calories from fat  starting at 6-week of age (young) or 10 months of age (old) for 16 to 24-week duration. Animals were housed in a specific pathogen-free facility with a 12h light/12h dark cycle and given free access to food and water. All animal use was in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and approved by the University Committee on Use and Care of Animals at the University of Michigan , an ADRB3 selective agonist or saline twice (4 h between each injection). Mice were sacrificed 14 h after the second injection [Young mice used in the experiments were male and female C57Bl/6J purchased from Jackson Laboratories. Old mice were either aged from mice purchased from Jackson or obtained from the National Institute of Aging (NIA). Mice were fed njection , 59. LivAntibodies used for immunofluorescence included polyclonal anti-caveolin  and anti-Mac2 . For histology, tissues were formalin fixed, paraffin embedded, sectioned at 5μm and stained with hematoxylin and eosin. Hematoxylin-eosin (H-E) staining was performed by the University of Michigan’s Comprehensive Cancer Center Histology Core.Adipose tissue fractionation and flow cytometry analysis were performed as described previously. SVF cells were stained with CD64 PE, CD45.2 e450, and CD11c-APC-Cy7 (eBioscience), and gating was performed for macrophage populations and by CD45 gates to determine ATMs , 61.Gapdh expression was used as an internal control for data normalization. Samples were assayed in duplicate, and relative expression was determined using the 2-ΔΔ CT method. All primers used are listed in RNA was extracted from adipose tissue and liver using Trizol LS (Life Technologies) and cDNA was generated using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). SYBR Green PCR Master Mix (Applied Biosystems) and the StepOnePlus System (Applied Biosystems) were used for real-time quantitative PCR. Estradiol and testosterone levels were assessed in fed serum samples in young and old, male and female mice on 16 weeks of ND or HFD. Testing was performed at the Ligand Assay and Analysis Core Laboratory (University of Virginia).in vivo lipolysis measurement, serum FFA was measured using the HR NEFA series (Wako Diagnostics). Serum free glycerol was measured using free glycerol determination reagent (Sigma-Aldrich). Serum TG was measured with Infinity triglyceride determination kit (Thermo Scientific) following manufacturer’s instructions. Liver triglycerides were homogenized, extracted by a modified Folch method and assayed.Serum insulin levels were determined with an ultrasensitive insulin ELISA kit . For Briefly, deparaffinized and rehydrated GWAT sections were stained by dissolving Sirius Red F3B (Sigma-Aldrich) in a saturated aqueous solution of picric acid (Sigma-Aldrich) at 0.1% w/v for 60 minutes at room temperature. Thereafter, these slides were washed in acidified water containing 0.5% glacial acetic acid (Sigma-Aldrich) twice for 5 minutes. After which the excess water was wicked and sections dehydrated in 3 changes of 100% ethanol, cleared with xylene, and mounted with Permount (Fisher Scientific). Bright-field images of stained GWAT sections were quantified using the ImageJ software (NIH) as per the protocol described on its website .100 mg of adipose tissue was homogenized in 0.5 M acetic acid. The homogenate was transferred to an eppendorf tube and incubated at 4°C overnight on a shaker to allow acid solubilization. Following which the homogenate was centrifuged at 10,000 rpm and the supernatant collected with a syringe to get rid of excess fat. Rat tail collagen was used a standard for the assay. For the assay, 1ml of sircoll dye (0.1% Direct Red 80 in saturated picric acid) was added to the samples and standards and incubated on mechanical shaker for 1h. thereafter tubes were centrifuged at 10,000 rpm for 15 mins. The unbound dye solution was aspirated leaving the pellet behind to which 1 ml of 0.5M NaOH was added, vortexed and incubated on a shaker for 30 mins to release the bound dye in solution. 100ul of the dissolved dye solution was transferred to a 96-well plate and the absorbance was measured at 540nm.All values are reported as the mean +SEM unless otherwise stated. Statistical analyses were performed in Prism 8 (GraphPad). Before performing the statistical test, we assumed equal variability of differences among the groups and also checked for outliers by ROUT’s test with Q= 1%. Depending on the dataset, ordinary one-way analysis of variance (ANOVA) with Sidak’s post-test or Student’s t-test was performed. Ordinary two-way analysis of variance (ANOVA) was performed with factors of diet and age/treatment with Bonferroni-Dunn’s post-test to determine significance. The test performed is indicated in the figure legend. Statistical significance is indicated by *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Supplementary FiguresSupplementary Table 1


BRAF have made it an oncogene of interest in pediatric cancer. Previous studies found that BRAF mutations as well as KIAA1549–BRAF fusions are common in intracranial low-grade gliomas (LGGs). Fewer studies have tested for the presence of these genetic changes in spinal LGGs. The aim of this study was to better understand the prevalence of BRAF and other genetic aberrations in spinal LGG.The mitogen-activated protein kinases/extracelluar signal-regulated kinases pathway is involved in cell growth and proliferation, and mutations in BRAF and additionally screened for other possible genetic abnormalities of interest. At SickKids, BRAFV600E was assessed by droplet digital polymerase chain reaction and immunohistochemistry. BRAF fusions were detected by fluorescence in situ hybridization, reverse transcription polymerase chain reaction, or NanoString platform. Data were correlated with clinical information.We retrospectively analyzed 46 spinal gliomas from patients aged 1–25 years from Children’s Hospital Colorado (CHCO) and The Hospital for Sick Children (SickKids). CHCO utilized a 67-gene panel that assessed KIAA1549–BRAF. All 13 (100%) patients with confirmed KIAA1549–BRAF survived the entirety of the study period (median [interquartile range] follow-up time: 47 months [27–85 months]) and 15 (83.3%) fusion-negative patients survived (follow-up time: 37.5 months [19.8–69.5 months]). Other mutations of interest were also identified in this patient cohort including BRAFV600E, PTPN11, H3F3A, TP53, FGFR1, and CDKN2A deletion.Of 31 samples with complete fusion analysis, 13 (42%) harbored KIAA1549–BRAF was seen in higher frequency than BRAFV600E or other genetic aberrations in pediatric spinal LGGs and experienced lower death rates compared to KIAA1549–BRAF negative patients, although this was not statistically significant. KIAA1549–BRAF fusion was the most common aberration identified in the low-grade glioma cohort.KIAA1549–BRAF fusion had excellent long-term survival.Patients with Spinal cord low-grade tumors may be good candidates for MEK inhibition.BRAF mutations as well as KIAA1549–BRAF fusions, which is helpful in predicting outcome and treatment responses in this group. Fewer studies have evaluated spinal cord LGG for similar genetic aberrations. This study retrospectively investigated the genetic landscape of a cohort of pediatric spinal LGGs for the presence of BRAF aberrations as well as other genetic mutations of interest. BRAF fusions were the most prevalent change identified. All patients with this fusion had excellent survival. This pediatric population suffers from severe complications as a result of treatment needed to maintain tumor control, and better understanding the genetic landscape of this tumor could potentially inform treatment and predict long-term outcomes in this population.Intracranial low-grade glioma (LGG) in the pediatric population has been well researched for the genetic aberrations that define this population. They are known to have 1 Patients diagnosed with spinal cord LGG have a high overall survival (OS) with progression-free survival (PFS) ranging from 34% to 88%.2 Despite low mortality, there is high morbidity associated with spinal LGG treatment. Specifically, patients can suffer from significant neurologic and orthopedic complications from surgery, chemotherapy, and radiation. Defining the mutational characteristics of spinal cord LGG could provide predictive information regarding clinical outcomes and potential therapy options.Low-grade gliomas (LGGs) of the spinal cord are rare in pediatric patients. Tumors arising in the central nervous system (CNS) make up 20% of all pediatric cancer, but intramedullary spinal cord tumors are only 2%–10% of CNS lesions.3–6 which drives a number of processes including cellular proliferation, differentiation, mortality, stress response, apoptosis, and survival.7 There are a number of mutations and fusions in this pathway that result in constitutive over-activation of the MAP/ERK pathway.8The most common genetic changes in intracranial LGGs or glioneuronal tumors involve the mitogen-activated protein kinases/extracelluar signal-regulated kinases (MAPK/ERK) pathway,KIAA1549–BRAF fusion.4 LGGs occurring elsewhere in the brain have a higher percentage of tumors with a point mutation in BRAF at codon 600 (BRAFV600E and variants such as BRAFV600D). This mutation is also seen in many CNS tumors including desmoplastic infantile gangliogliomas, diffuse astrocytomas, gangliogliomas, pleomorphic xanthoastrocytomas, and epitheloid glioblastomas.9Cerebellar pilocytic astrocytomas often exhibit MAPK/ERK hyperactivation as a consequence of a 10  BRAFV600E inhibitors like vemurafenib (NCT01748149) and dabrafenib (NCT01677741) and MEK inhibitors like trametinib (NCT03434262) are being explored to treat these tumors in phase 1 and 2 trials. Tumors with the KIAA1549–BRAF fusion are considered to be RAS-independent and are resistant to first-generation RAF inhibitors (vemurafenib and dabrafenib).12 This makes MEK inhibition a better option for these patients. In addition to the potential for the development of resistance to targeted therapies,14 progression following single- and multi-agent chemotherapy remains a concern in these patients with 5-year PFS estimated between 43% and 53.2%.15–17At present, gross total surgical resection (GTR) is the first line of treatment for LGG, but adjuvant therapy could include chemotherapy  and potentially radiation therapy. These are particularly considered if GTR is not feasible.KIAA1549–BRAF fusion and BRAFV600E mutations. Sixty-seven secondary genes were also screened for mutations for the tumors from CHCO. We evaluated clinical data and performed genetic testing on 46 spinal LGG (WHO grade 1 or 2) with available tumor samples and mapped PFS and OS of these patients to see if an association between aberrations in BRAF, treatment, and OS could be identified.To identify potential new therapy options in pediatric low-grade spinal cord tumors, we evaluated whether these tumors identified at 2 institutions  harbor targetable lesions such as the 18Fifty-five patients were identified as having intramedullary spinal cord tumors between 1995 and 2016. Of these patients, 46 patients ranging in age from 1 to 25 years (median age at diagnosis: 9.5) had a confirmed WHO grade 1 or 2 spinal cord tumor at CHCO  and SickKids . Nine patients from CHCO were excluded from analysis after it was determined that they had high-grade spinal cord malignancies. Of note, samples included from SickKids were recently also analyzed in a wider study of LGG in multiple CNS locations.Patient charts were retrospectively analyzed for age, diagnosis, location of tumor, past genetic tests completed on a patient’s tumor, treatment, subsequent relapses, treatment at relapse, and date of last follow-up or death. Pathology reports and imaging for each patient were subsequently analyzed to verify an accurate diagnosis, location of tumor, and genetics run at the time of biopsy/resection at CHCO. SickKids considered initial diagnosis and subsequent treatments for each patient.Primary patient samples were obtained and collected from CHCO and SickKids in accordance with local and federal human research protection guidelines and institutional review board (IRB) regulations. Ethical standards according to the Helsinki Declaration were followed and the work was approved by local IRB committees . Written informed consent was obtained for all specimens collected.Baseline characteristics were reported as count and proportion (%) or median (interquartile range [IQR]), separately by grade 1 and 2 tumors. Time to death was defined as the time from diagnosis to death from any cause or time from diagnosis to the last follow-up if a patient survived. Time to progression was defined as the time from diagnosis to tumor progression or death, with censoring also at the last follow-up date. OS and PFS were estimated at time points of interest using Kaplan–Meier curves. Cox proportional hazard models were fit for time to death and time to progression, and log-rank statistics were reported for differences between tumor grade and KIAA1549–BRAF positivity. All statistical analysis was performed in R version 3.6.1.n = 15) or from frozen material (n = 7) using the Agencourt FormaPure Kit (Beckman Coulter). For mutational analysis, TNA was then processed via the Archer VariantPlex Solid Tumor library preparation kit that is designed to amplify selected regions in 67 genes (ArcherDx). All manufacturer-recommended cutoffs for quality control were used and only samples that met appropriate quality levels were sequenced. Libraries were sequenced via the Illumina MiSeq or Illumina NextSeq (Illumina). Raw sequence data were processed for mutational calling by using the Archer Analysis software package . For fusion analysis, 23 FFPE and 7 frozen samples had TNA processed via the Archer FusionPlex Solid Tumor library preparation kit that is designed to detect gene fusions and novel isoforms involving selected exons of 53 genes (ArcherDx). Libraries were sequenced via the Illumina MiSeq (Illumina). Raw sequence data were processed for fusion calling by using the Archer Analysis software package . SickKids analyzed FFPE samples (n = 18) as previously described.18 In short, KIAA1549–BRAF fusion was tested by NanoString, reverse transcription polymerase chain reaction, and fluorescence in situ hybridization. BRAFV600E was tested by droplet digital polymerase chain reaction (ddPCR) and immunohistochemistry. Other genetic alterations were tested by Sanger and ddPCR.Samples at CHCO were analyzed as follows. Total nucleic acid (TNA) was extracted in a CLIA-certified laboratory from formalin-fixed, paraffin-embedded (FFPE) processed material .Among all participants, 85% survived the entirety of the study period . Among the 7 (15%) who died, the median time to death was 26 months (IQR 17–40). The time-to-death and time-to-progression curves are shown in Patients treated with resection-only  had the n = 7; n = 12) was suspected to be due to samples being very old coupled with suboptimal tissue fixation resulting in TNA that did not pass the manufacturers quality control measures. Of those analyzed, there were 9 different genetic mutations and/or fusions identified in 21 patients including mutations in BRAF, PTPN11, H3F3A, TP53, FGFR1, and CDKN2A  and SickKids (N = 18). In total, 13 (42%) patients tested positive, 6 from CHCO and 7 from SickKids. Twelve of the patients diagnosed with the fusion had the diagnosis of pilocytic astrocytoma and one had an astrocytoma not otherwise specified  who were confirmed to be negative for the KIAA1549–BRAF fusion, although this difference was not statistically significant.Time to death was not significantly different P = .12; . InteresKIAA1549–BRAF fusions are a hallmark mutation in pediatric pilocytic, pilomyxoid, and diffuse astrocytomas of the brain.20 Other studies have demonstrated that BRAFV600E mutations are common in grade 2 pleomorphic xanthoastrocytomas, pleomorphic xanthoastrocytomas with anaplasia, grade 1 gangliogliomas, grade 3 anaplastic gangliogliomas, and pilocytic astrocytomas in the brain. More specifically, BRAF rearrangement has been found in 75%–80% of cerebellar pilocytic astrocytomas, and BRAFV600E mutations have been identified in non-cerebellar regions of the brain.21 Most recently, a large analysis of LGG  demonstrated how molecular studies can be used to stratify these tumors into risk categories.18 To date, there are few studies specifically addressing the presence of KIAA1549–BRAF fusions and BRAFV600E point mutations in low-grade spinal cord tumors. One study by Gessi et al.22 found that spinal gangliogliomas do not harbor the BRAFV600E mutation in high frequency. Another study by Wang et al.23 acknowledged that very little work has been done in regards to understanding the role of BRAF mutations in spinal cord tumors. Ryall et al.18 reported the presence of both BRAFV660E, FGFR1 single nucleotide variants, as well as KIAA1549–BRAF and FGFR1–TACC1 fusions in spinal cord LGG. Our data support these previous studies and add to the overall understanding of the genetic landscape of these tumors.Previous studies have revealed that BRAF fusions are often correlated with a favorable prognosis in cerebellar LGG. One group, in particular, found that pediatric patients who underwent STR but harbored KIAA1549–BRAF fusion had a positive clinical outcome in comparison to patients who had STR and were negative for KIAA1549–BRAF fusion.20 Our results support these findings. Of the 13 patients with confirmed KIAA1549–BRAF fusion, none died during follow-up, regardless of treatment group. Of the 18 patients who confirmed negative for the KIAA1549–BRAF fusion, 3 of the patients died putting the 5-year estimated survival rate at 76% with 7 patients experiencing a relapse. This further supports that identification of genetic mutations and fusions in pediatric spinal LGG may be useful in prognostication18 and provide information for therapy choices as more targeted therapies are being studied.BRAF alterations. A number of clinical trials are evaluating the efficacy of inhibitors of the RAS/RAF/MEK/ERK signaling pathways as well as small molecule inhibitors of BRAFV600E in these tumor types (NCT01748149).19 The specific BRAF alteration is correlated to response to treatment; KIAA1549–BRAF fusions are RAF-independent and BRAFV600E mutations are responsive to autophagy and small molecule inhibitors.14 Two of the tumors evaluated in this study were found to harbor BRAFV600E and would have the potential for BRAF-targeted inhibition. Identification of KIAA1549–BRAF fusion mutations could help to inform treatment decisions to include MEK inhibition. Recent data have demonstrated significant responses to MEK inhibition in this patient population.24There is increasing use of targeted therapy against BRAF mutations and fusions in low-grade spinal cord tumors. Additional mutations were found in FGFR1, CDKN2A, H3F3A, TP53, and PTPN11. This is consistent with previous reports of alternate mutations found in LGG, but in far less frequency.25 Research has found that some low-grade intracranial tumors harbor alterations in FGFRs which involve fusions with TACC genes and FGFR1 tyrosine kinase domain duplication (FGFR1-TKDD) resulting in upregulation of MAPK/ERK and the PI3K pathway.26 Similarly, our data found a FGFR1 mutation in a patient diagnosed with grade 2 astrocytoma NOS who experienced a relapse of this primary tumor 3 months from diagnosis but has survived to 60 months since diagnosis. Another patient with a pilocytic astrocytoma had a confirmed FGFR1 mutation. Genetic screening studies have found that FGFR1-TKDDs are found predominantly in diffuse gliomas,27 although it can also be seen in other tumors such as pilocytic astrocytomas26 and dysembryoplastic neuroepithelial tumors.29 Recently, the Consortium to Inform Molecular and Practical Approaches to CNS tumor Taxonomy—Not Official WHO (cIMPACT-NOW) reported that diffuse gliomas characterized by FGFR1 alterations occur primarily in children and that these should be classified as diffuse glioma, FGFR1-mutant tumors.30 A comprehensive evaluation of LGG of the CNS found FGFR1 mutations in 1.5% of tumors analyzed and that these often co-occurred with other genetic alterations.18 FGFR1-activating mutations and fusions have also been reported in pediatric spinal tumors.31 At present, there are FGFR inhibitors in a clinical trial including erdafitinib which is being studied in the current pediatric MATCH Trial (NCT03210714).The primary aim of this study was to investigate the prevalence of H3F3A.6  H3F3A K27M has proven to be a hallmark of many HG midline gliomas and occurs in about 20% of pediatric glioblastomas.32 It was detected in one of our patients diagnosed with a WHO grade 1 ganglioglioma. This patient experienced a relapse and was subsequently diagnosed with an anaplastic ganglioglioma WHO grade 3 and ultimately passed away 19 months from the original diagnosis. This would suggest a concern for a poor clinical outcome with this mutation regardless of primary pathologic diagnosis. At present, there are no treatments to specifically target this mutation but could have potential importance in the prognosis of these tumors.33In a previous study, 1.9% of patients with low-grade spinal cord tumors demonstrated mutations in 34 A last potential limitation to this study is that diagnoses were made strictly based off of histology rather than based on other molecular profile data.Limitations of this study include the small patient cohort. Another limitation was that as a retrospective study, some samples were old and that was coupled with suboptimal fixation methods resulting in poor quality DNA and RNA for mutational and fusion assays.The genetic aberrations reported here add to the available information to better understand the molecular underpinnings of low-grade spinal cord tumors. Knowledge about the distinct genetic landscape of these tumors could potentially help to inform treatment choices and predict overall prognosis in patients with pediatric spinal tumors.vdaa103_suppl_Supplementary_Figure_1Click here for additional data file.