Reversal of cancer gene expression is predictive of therapeutic potential and can be used to find new indications for existing drugs (drug repositioning). Gene expression reversal potential is currently calculated, in almost all studies, by pre-aggregating all tumour samples into a single group signature or a limited number of molecular subtype signatures. Here, we investigate whether drug repositioning based on individual tumour sample gene expression signatures outperforms the use of tumour group and subtype signatures. The tumour signatures were created using 534 tumour samples and 72 matched normal samples from 530 clear cell renal cell carcinoma (ccRCC) patients. More than 20,000 drug signatures were extracted from the CMAP and LINCS databases. We show that negative enrichment of individual tumour samples correlated (Spearman’s rho = 0.15) much better with the amount of differentially expressed genes in drug signatures than with the tumour group signature (Rho = 0.08) and the 4 tumour subtype signatures (Rho 0.036-0.11). Targeted drugs used against ccRCC, such as sirolimus and temsirolimus, which could not be identified with the pre-aggregated tumour signatures could be recovered using individual sample analysis. Thus, drug repositioning can be personalized by taking into account the gene expression profile of the individual’s tumour sample. This is caused by intrinsic and/or acquired drug resistance2. Both are likely enhanced by the existence of inter- and intra-tumour molecular heterogeneity: a recent study demonstrated that different biopsies from the same ccRCC tumour grown in patient-derived xenograft (PDX) mouse models can show different drug sensitivity patterns, and each was associated with markedly different gene expression profiles3.While targeted therapies such as tyrosine kinase inhibitors  and mammalian target of rapamycin (mTOR) inhibitors  have greatly improved the prognosis of metastatic Clear Cell Renal Carcinoma (ccRCC) patients, the average duration of disease control ranges between 8–9 months in the first-line setting and 5–6 months in the second-line setting4. However, combining oncological drugs also tends to prohibitively increase the toxicity, as evidenced by the use of sunitinib and everolimus simultaneously5. Therefore, to design better tolerated and effective combination regimens it might be productive to widen the search to include non-oncological drugs, as they often have a better safety profile. This is not as far-fetched as it may initially seem: aspirin, metformin, itraconazole and many other regular drugs are currently being tested in clinical trials for efficacy in adult malignancies, usually in combination with regular treatments6.To prevent and overcome drug resistance, model systems and clinical experience have shown that combining drugs which target different pathways are superior to single-agent approaches7. This process has been accelerated by the surge of freely available “omics” data which has inspired many researchers to develop computational drug repositioning methods8. One popular method, gene expression signature reversal, is based on the observation that when the difference in gene expression of cells after perturbation by a compound (the drug signature) is negatively correlated to the difference in gene expression between diseased and normal cells (the disease signature), the drug often turns out to be therapeutically indicated for that disease9.The application of already registered drugs and compounds for new indications is called drug repositioning and it has obvious appeal: knowing the safety, toxicity, pharmacokinetic, pharmacodynamic and metabolic properties of a compound significantly reduces the risk and time required to register an indication as compared to a new chemical entity10. If all samples are analyzed collectively, pathways could either be masked or be less prominently expressed in proportion to the incidence of the perturbation. One way to solve this problem is to divide the tumour gene expression signature into subtypes with a statistical technique such as hierarchical clustering, which groups samples with similar expression profiles. If no real biological variability remains within the identified subtypes , the probability of finding a valid result increases due to increased power as bigger groups are compared. However, if subtypes do contain significant biological variability it could interfere with the correct identification of potentially therapeutic drugs. Analyzing individual tumour samples does not suffer from this potentially incorrect binning problem, and could therefore result in more potentially therapeutic drug hits and simultaneously provide information on the proportion of tumour samples which are negatively correlated to each drug signature, at the cost of reduced power when samples are homogeneous. Although development of a method to target drugs based on the gene expression of single tumour samples may not be successful for all tumours due to the existence of inter and intra-tumour heterogeneity, however, such an approach would best approximate the situation of intra-tumour heterogeneity and could be extended to multiple tumour samples in the future.Because of the considerable heterogeneity between and even within ccRCC tumours, it therefore makes sense to reposition drugs based on individual gene expression profiles, as each tumour sample may have a different set of perturbed pathwaysThe goal of this research is to benchmark the results of the gene expression reversal analysis of the tumour subtype and the individual tumour sample signatures against the results of the average ccRCC tumour signature, as this could provide support for the development of an individualized drug repositioning approach based on gene expression.In total, 610 expression profiles from 606 different tissue samples  were included in the analysis . Varying the CPM cutoff or minimum sample requirement around these values does not significantly affect the number of genes remaining in the analysis Figs . FurtherFig. Of the 1,309 drugs tested in CMAP and the 19,812 drugs tested in LINCS, 21 (1.6%) and 388 (2.0%) could not be processed further because the linear model could not be fitted due to the lack of control samples. The genes measured by the CMAP and LINCS arrays shared 6,058 and 502 genes in common with the 11,333 genes included in the tumour gene expression analysis, respectively Fig. . However−16, Wilcoxon rank sum test). Furthermore, the amount of DEG in a drug signature  shows a much stronger correlation with tumour sample negative enrichment rate  than with the amount of negatively enriched drugs calculated with the group and subtype signatures  does reveal significant negative enrichment for 2 out of the 3 mTOR-inhibitors used against ccRCC (sirolimus and temsirolimus). However, the other mTOR inhibitor (everolimus) and the 4 tyrosine kinase inhibitors used against ccRCC present in the LINCS database  did not reveal significant negative enrichment. This could be because these drugs were tested in fewer cell lines (N = 13–20) as compared to sirolimus and temsirolimus (N = 51–57). Furthermore, these tyrosine kinase inhibitors are believed to work because they primarily inhibit the Vascular Endothelial Growth Factor (VEGF) receptor present on non-cancerous endothelial cells12. The nucleotide reverse transcriptase inhibitor tenofovir is associated with nephrotoxicity due to accumulation in the proximal tubules13, which ccRCC is thought to originate from. The similarity in gene expression between ccRCC and proximal tubules cells has been noted before14, and therefore it seems plausible they share the same toxicity as well. Quinine has shown some efficacy as an add-on in breast cancer patients15 and nicotinamide has been shown to substantially reduce the recurrence risk of skin cancer in a RCT16. Lastly, temsirolimus is already in use against ccRCC.Our study also shows that the approach of drug repositioning by gene expression reversal reveals interesting potential drugs for treatment of individuals with ccRCC. Indeed, most of the top 8 of drugs with the highest negative enrichment of tumour sample signatures are already supported by existing evidence. Erlotinib and genistein both inhibit the Endothelial Growth Factor Receptor (EGFR), and the EGFR gene expression profile showed overexpression in 38.2% of tumour samples from an independent cohort of 63 ccRCC patients17. More sophisticated batch effect correction methods than including a factor in the linear model exist, e.g. the use of control genes, could further amplify the signal from the noise18. Different connectivity scoring methods also exist, which could further improve the sensitivity and/or specificity of the pipeline19. Lastly, the selection of tumour sample genes was done by the commonly accepted but arbitrary criterion of a FDR below 1%; ideally this cut-off would be determined from the data, or genes more likely to be actually differentially expressed could be given a higher weight. It will be more difficult to quantify the false negative rate which can be increased by biological factors, e.g. if drugs are tested in cell lines which do not express the drug target(s). However, as the amount of hits that can be validated is likely small, perhaps the focus should mainly be on decreasing the false positive rate.Some potential statistical issues/refinements of the described pipeline remain: the current method of determining the drug signature equally weighs experimental instances equally with different drug concentrations and drug exposure durations, whereas it has been demonstrated that higher drug concentrations and exposure durations induce a stronger effect on the differential gene expression profilesDespite all these potential issues/refinements however, the simulations already make it very clear that it would have been extremely unlikely to have found the same results if all tumour samples came from a single uniform tumour expression profile or a representative combination of the 4 previously identified subtype profiles. Indeed, the power of this approach lies in not having to assume the number of subtypes, whether there are none, 4 or more. Repeated RNA-seq analysis of the same tissue sample, and to a lesser degree a new tissue sample from the same patient, already shows remarkable consistency in connectivity scores calculated with the current method and increases as the P-value of the connectivity score decreases.et al. did publish a similar analysis10, but as this was the only type of analysis they did, it did not demonstrate the superiority to the connectivity mapping of the group or subtype tumour signatures. Furthermore, our analysis contains more than 25 times more, and arguably better characterized, tumour samples and more than 120 times more drug compounds profiled in far more cell lines, resulting in a more comprehensive analysis.To our knowledge, this is the first paper to convincingly demonstrate that using individual tumour sample signatures as the basis for analysis outperforms analyses based on tumour group or subtype signatures. Zerbini 20. Furthermore, the transcriptomic heterogeneity is unlikely to be well represented in existing cell lines. It could be an option to first grow some of the patient’s own tumour tissue in an and test the drugs using an ex vivo functional assay. When the candidate drugs are marketed drugs which have been tested and approved for clinical use within the dose range an effect can be expected, it might therefore be ethically defensible to directly prescribe the drug off-label in an N = 1 trial. Possibly both scenarios could even be run in parallel, with the ex vivo results providing some external validation and validated alternatives in the event the first prescription failed to provide any benefit. If a database of the results of these N = 1 trials can be created and maintained, it could eventually generate enough evidence to conclude which combinations of gene expression profiles and drugs show a consistent therapeutic benefit.Whether a pathway is causally involved in the survival of the tumour is impossible to determine from the gene expression data alone. Testing the hits on existing ccRCC cell lines is a possibility, but many aspects of ccRCC cannot be replicated using cell linesAll data processing and analyses were performed using R version 3.4.0 using the R/Bioconductor packages mentioned below. FDR was always calculated using the Benjamini-Hochberg method.21. It is automatically annotated with metadata, such as patient information and the molecular subtype as determined by the original TCGA analysis using hierarchical cluster analysis11. The genes were normalized within samples by gene length and between samples to correct for sequencing depth using the EDAseq package (version 2.10.0)22. Only genes which were expressed above 0.5 Counts Per Million (CPM) in at least a sixth of all samples were retained, i.e. selection was irrespective of tissue type. This cutoff was taken from a Bioconductor example workflow, and reduces the high noise inherent in measuring lowly expressed genes23.The mRNA-seq v2 read counts produced by The Cancer Genome Atlas (TCGA) Kidney Renal Clear Cell Carcinoma project were downloaded from the Genomic Data Commons using the TCGAbiolinks package (version 2.5.7)22, and statistical significance was assessed using a likelihood ratio test. Three different types of tumour signature were generated:Tumour group signature: All tumour samples versus all normal samples. This is the conventional comparison, and ideal under the assumption of homogenous tumours.Tumour subtype signatures: All tumour samples belonging to each one of the 4 molecular tumour subtypes versus all normal samples.Tumour sample signatures: Each tumour sample versus all normal samples. This provides a unique signature for each tumour sample.The read counts were modelled using the negative binomial generalized log-linear model available from the edgeR package (version 3.18.1)To estimate the true false positive rate of genes included in the tumour sample signatures, normal sample signatures were generated by comparing each normal sample versus all the other normal samples.24.A preprocessed version of the CMAP database was downloaded using the PharmacoGx package (version 1.6.1) which was corrected for systematic differences caused by the different microarray platforms using the ComBat function in the sva package (version 3.10.0)25.The LINCS database was downloaded directly from the Gene Expression Omnibus (GSE92742) in the Level 3 format. The Entrez gene identifiers were converted to Ensembl gene identifiers using the bioMart package (version 2.32.1), to make them compatible with the tumour expression profiles and the preprocessed CMAP database26The drug signatures were calculated with a linear model using the limma package (version 3.32.5) with drug concentration as a linear parameter (i.e. 0 for controls and >0 for drugged cells) and cell type, perturbation duration (if >2 perturbation durations), batch as categorical variablesGene Set Enrichment Analysis (GSEA), using the log2 fold difference of the tumour genes which are below a 1% FDR in combination with using the landmark genes and tumour cells resulted in the least amount of false positive results out of all tested methods and configurations, when CMAP drug signatures were used to retrieve the corresponding LINCS drug signatures  than the amount which would be expected by chance 95% of the time. FDR correction was applied separately for each decile of drug signature percentage DEG.For the top 8 most frequently negatively enriched drugs, a simulation study was performed to validate which drugs show individual differences in connectivity score different from the group and subtype signatures. 10,000 batches of individual tumour samples of the same size as the original batch and with the same distribution of sequencing depth to the original tumour samples were simulated from the tumour group signature to determine the amount of negatively enriched tumour sample signatures. Another 10,000 similarly constructed batches were sampled from the subtype signatures in the same proportion as found in the original data. Simulation was performed by extracting the mu and size parameters for each gene from the negative binomial generalized log-linear model. Tumour sample signatures were then calculated to determine the connectivity score and associated P-value with each drug.https://gitlab.com/k.k.m.koudijs/personalised_DR_ccRCC).The R code, drug signatures, tumour signatures and the resulting datasets generated during the current study are available in a public GitLab repository (Supplementary figures


Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs  both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis). The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown. In humans, the physiological plasma kallikrein-kinin system (KKS) pathway is composed of high molecular weight kininogen (H-kininogen), plasma pre  and bradykinin (BK). The KKS pathway is very important for inflammatory processes, through BK activities that are mediated by the constitutive G-protein-coupled receptor BK B2 (B2R) present in different cells  and a C-terminal light chain  bound to each other by a single disulfide bond between Cys10-Cys596 , characterized as a six domain multifunctional protein playing antithrombotic, profibrinolytic, proinflammatory, andantimicrobial roles, and is an inhibitor of cysteine proteases and regulator of angiogenesis. After cleavage by plasma kallikrein (KAL) at LysBothrops jararaca venom or trypsin with the globulin fraction of dog plasma. BK corresponds to H-kininogen domain 4 (D4) and it reproduces many of the characteristics of an inflammatory state, such as changes in local blood pressure, edema, and pain, resulting in vasodilation and increased microvessel permeability  was first described by Mauricio Rocha e Silva and co-workers  and a C-terminal light chain (33–36 kD) linked via a single disulfide bond between Cys364-Cys484. The protease domain is in the light chain and contains the catalytic domain   is the zymogen of KAL (EC 3.4.21.34), a serine protease synthesized predominantly in the liver. Its mRNA codes for a single chain protein which, according to the degree of glycosylation in the C-terminal portion, may appear as a doublet (85 and 88 kD) independent of the protein reduction. The classic activation of PK by factor XIIa occurs through cleavage of the ArgKAL plays a central role in a variety of proteolytic systems, such as the intrinsic pathway of coagulation, the KKS, the fibrinolysis pathway, the renin–angiotensin system, and the complement pathways. Therefore, it can be considered an important regulator in the pathogenesis of thrombosis, inflammation, and blood pressure , which inhibits endothelial cell migration and proliferation, both of which play a role in angiogenesis  on domain 6 (D6)  and a His-Gly-Lys-rich region (His475-Lys502); H-kininogen exhibits a higher affinity to heparin (lower KD) than BK-free H-kininogen because the former loses a peptide sequence (Ser372-Arg419) which corresponds to a third cleavage by KAL after BK release from H-kininogen , however, at pH 7.35 cathepsin kininogenase activity is impaired suggesting that H-kininogen is a substrate for cathepsin B under pathophysiological conditions  polysaccharide chains are ubiquitous GAGs in animal cells  and preserve its activity by protecting it from inhibition by α1-antitrypsin and secretory leukocyte peptidase inhibitor and focus the activity of HNE to the pericellular environment  and metalloproteinase-9 (MMP-9) activities. In the pro-MMP-9/TIMP-1 complex, HNE preferentially inactivates TIMP-1 and renders pro-MMP-9 activity through MMP-3  and H-kininogens  GAGs were indicated as putative receptors that accumulate H-kininogen on the cell surface of HUVECs and other cells in a zinc-dependent manner, along with H-kininogen binding proteins, the integrin receptor Mac-1/αPrevious studies concerning KKS purified protein assembly on the ECM of different endothelial cell lines, from rabbit aorta endothelial cells (RAECs) and ECV304 cells, suggest that not only the cell membranes but also the ECM produced by these cells assemble H-kininogen/PK as a complex and form KAL that activates pro-urokinase, which in turn activates plasminogen  could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment  which has very important tradition and contribution in both fields. Both authors work also on cell biology and enzymes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CO and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


E. coli strain MG1655 was investigated as a tumour targeting system in order to produce TNFα specifically within murine tumours. In vivo bioluminescence imaging studies and ex vivo immunofluorescence analysis demonstrated rapid targeting dynamics and prolonged survival, replication and spread of this bacterial platform within tumours. An engineered TNFα producing construct deployed in mouse models via either intra-tumoural (i.t.) or intravenous (i.v.) administration facilitated robust TNFα production, as evidenced by ELISA of tumour extracts. Tumour growth was impeded in three subcutaneous murine tumour models  as evidenced by tumour volume and survival analyses. A pattern of pro-inflammatory cytokine induction was observed in tumours of treated mice vs. controls. Mice remained healthy throughout experiments. This study indicates the therapeutic efficacy and safety of TNFα expressing bacteria in vivo, highlighting the potential of non-pathogenic bacteria as a platform for restricting the activity of highly potent cancer agents to tumours.Systemic administration of the highly potent anticancer therapeutic, tumour necrosis factor alpha (TNFα) induces high levels of toxicity and is responsible for serious side effects. Consequently, tumour targeting is required in order to confine this toxicity within the locality of the tumour. Bacteria have a natural capacity to grow within tumours and deliver therapeutic molecules in a controlled fashion. The non-pathogenic  The murine recycled prostate cancer cell line TRAMPC1 was kindly provided by Dr. Richard Ciavarra of Eastern Virginia Medical School, Norfolk USA, and propagated as described in .oC (protected from over-exposure to light). Cells were seeded in 500 μl of relevant growth medium at 4 x 104 cells per well in a 24 well flat bottomed plate (Sarstedt) and allowed to grow for 24 h. Wells were treated with varying concentrations of murine TNFα (Sigma-Aldrich) or vehicle (sterile water) in triplicate. Blank wells without cells were also included to account for any background fluorescence. After 48 h, MTT was added to cells at a final concentration of 0.5 mg/ml and incubated for 90 min at 37 oC, 5% CO2. All medium was then aspirated carefully and purple formazan crystals were dissolved upon addition of 150 μl DMSO per well. Each well was mixed with 70 μl each sample and transferred to a 96-well microtest plate (Sarstedt) and absorbance read at 570 nm on an Infinite 2000 spectrophotmetric plate reader (Tecan). An Excel add-in ED50V10-2 was used for calculating half maximal effective concentration of TNFα (ED50), calculated from absorbance readings.The 3--2, 5-diphenyltetrazolium bromide (MTT) assay was optimised for use as an end-point analysis for experiments involving the treatment of CT26, RENCA and TRAMP cell lines with TNFα. Stocks of MTT were prepared by dissolving MTT powder in phosphate buffered saline at a concentration of 5 mg/ml, and stored at -20 Mus musculus tumour necrosis factor alpha (GenBank: BC137720.1) was retrieved from the NCBI database. The gene was then synthesized and ligated into a custom made version of plasmid pSF-OXB20-Br322 by Oxford Genetics Ltd, Oxford, UK, containing a low copy origin of replication, strong promoter, and kanamycin resistance marker (E. coli MG1655-p16lux (herein referred to as MG) [50 + Em300 and incubated at 37 °C.The mRNA sequence for e marker . An emptTo confirm the expression of TNFα by the plasmid constructs in MG and the absence of expression in the equivalent empty constructs, each isolate was grown in 10 mL LB for 18 h at 37 °C with appropriate antibiotics. A Mouse TNFα Single Analyte ELISA Kit (Qiagen) was used to test for the presence of the cytokine in all transformed plasmid constructs in the total bacterial lysate, as per manufacturer’s instructions.All animal procedures were performed according to the national ethical guidelines of the Health Products Regulatory Authority (HPRA). Protocols were approved by the University College Cork Animal Experimentation Ethics Committee (AERR #2010/003 and #2012/015). Health status of all mice was monitored daily for the duration of experiments. There were no deaths outside of humane euthanasia. Mice were humanely euthanized by cervical dislocation upon tumours reaching a size of 1.5 x 1.5 cm in diameter. To minimize suffering or distress during invasive procedures , mice were anaesthetised with isoflourane (2.5% mixture with oxygen).5 CT26, 1 x 105 RENCA, 5 x 105 TRAMPC1) suspended in 200 μl serum-free culture medium was injected subcutaneously (s.c.) into the flank. The viability of cells used for inoculation was greater than 95% as determined by the Nucleocounter system . Following tumour establishment, tumours were allowed to grow and develop and were monitored three times weekly. Tumour volume was calculated according to the formula V = (ab2) Π /6, where a is the longest diameter of the tumour and b is the longest diameter perpendicular to diameter a. When tumours reached approximately 100 mm3 in volume, mice were randomly divided into experimental groups.Mice were kept at a constant room temperature (22 °C) with a natural day/night light cycle in a conventional animal colony. Standard laboratory food and water were provided ad libitum. Before experiments, the mice were afforded an adaptation period of at least 7 days. Male C57Bl (TRAMPC1 model) and female Balb/C (RENCA and CT26 models) mice in good condition, without fungal or other infections, weighing 16–22 g and of 6–8 weeks of age, were included in experiments . At experiment end, animals were euthanized by cervical dislocation. For routine tumour induction, the minimum tumorigenic dose of cells  injection (RENCA and TRAMPC1 model) or intravenous (i.v.) injection via the lateral tail vein (CT26 model). The viable count of each inoculum was determined by retrospective plating on LB agar containing the appropriate selective antibiotic.Inocula were prepared by growing MG-Empty and MG-TNFα with or without the integrated p16Slux aerobically in 100 mL LB broth containing either 50 μg/mL Kan (MG1655) or 50 μg/mL + 300 μg/mL Em (MG1655p16Slux). Overnight cultures were re-inoculated into fresh LB (1/50 dilution) and incubated shaking at 37°C until they reached an OD600 of 0.6–0.8. Cultures were harvested by centrifugation , washed three times with PBS and resuspended in a one tenth volume of PBS. Tumours were administered 10in vivo Bioluminescence Imaging was performed using the IVIS Lumina II  with 2 min integration times at high sensitivity. Following whole-body imaging, the mice were euthanized via cervical dislocation and the subcutaneous tumours were aseptically removed and imaged. For each experiment, images were captured under identical exposure, aperture and pixel binning settings, and bioluminescence is plotted on identical colour scales. Bioluminescent signal was quantified by creation of regions of interest (ROIs). To standardize the data, light emission was quantified from the same surface area (ROI) for each tumour. Imaging data was analysed and quantified with Living Image Software (Perkin Elmer) and expressed as photons/second/cm2.At defined time-points after bacteria, animals were anesthetized by intraperitoneal administration of 200 mg xylazine and 2 mg ketamine and 2D Following imaging, each tumour was aseptically cut into three sections, one-third of which was immediately homogenized by fine mincing using a scalpel and pushed through a 20 μm pore nylon filter , Oxford, England). The filter was then rinsed with 2 mL of LB broth to create a cell suspension. Serial dilutions were plated in triplicate on LB agar containing selective antibiotics, grown overnight at 37°C and the resulting colonies used to calculate the number of bacterial cells per tissue sample.E. coli antibody  and counterstained using donkey anti-rabbit Alexa Fluor 488-conjugated anti-Ig antibody . Stained sections were mounted in ProLong Gold antifade reagent with DAPI  and visualized using a fluorescence microscope (Olympus BX51).One third of the tumour was snap frozen in optimal cutting temperature compound  using liquid nitrogen and isopentane. Frozen tumour sections (5 μm) were fixed in an ice-cold acetone-alcohol mixture (3:1 ratio), blocked with blocking serum for 45 min at RT in a humidified chamber, stained with purified rabbit polyclonal anti-Snap frozen tumour sections were thawed, weighed and placed into Lysing Matrix A tubes  containing 1 mL of homogenization buffer (50 mL PBS + 1 protease inhibitor cocktail tablets (Roche) + 10% FCS and homogenized using a FastPrep FP120 Cell disrupter . Homogenized samples were centrifuged for 12 min at 14,000 RPM at 4°C and the supernatants collected and stored at -80°C. Cytokine concentrations in the supernatants were measured by a Meso Scale Discovery 7-plex pro-inflammatory cytokine plate  according to the manufacturer’s instructions. The plates were analysed on the MSD Sector 2400 Imager (MSD).Statistical significance was determined with unpaired Student’s T test or Gehan-Breslow-Wilcoxon test for survival curves. All statistical tests were performed using commercially available statistic software . Data are represented by Mean ± (Standard Error of the Mean) SEM, unless otherwise stated. P values of < 0.05 were considered significant .To validate the utility of this platform, a bioluminescent form of MG1655 carrying the lux cassette in its genome (herein referred to as MG) was employed, which has previously been validated in this setting in our laboratory . MG was in vitro by ELISA   and monitored by BLI and immunofluorescence (IF) over time. BLI demonstrated increasing numbers of this strain within tumours for two weeks . IF spec or PBS) .Induction of inflammatory cytokines within CT26 tumours was examined by MSD multiplex cytokine analysis of tumour homogenate. A pattern of pro-inflammatory cytokine induction was observed in tumours of MG-TNFα treated mice vs. controls .Mice bearing subcutaneous flank tumours  were administered PBS, MG-Empty or MG-TNFα i.t  or i.v. (CT26) and tumour volume and survival monitored over time . Therapein vivo study that demonstrates TNFα delivery by bacteria to experimental murine tumours. E. coli MG1655 was employed to deliver and produce the therapeutic biomolecule TNFα inside murine tumours. Initially, both i.t. and i.v. routes of administration were tested to compare tumour targeting, and MG1655 performed well in both. For more clinical potential (for inaccessible and metastatic cancers), the i.v. route was chosen and MG-TNFα was administered to mice bearing CT26 tumours for further qualitative and quantitative analysis. MG-TNFα was capable of targeting tumours and proliferating as evidenced by BLI, IF and cfu counts. TNFα production within CT26 tumours was confirmed by ELISA and therapeutic studies indicated that MG-TNFα can impede tumour growth without inducing significant systemic toxicity.To our knowledge, this is the first in vitro production of TNFα by Clostridium were described [in situ production of therapeutic biomolecules. Bacteria were tolerated well by mice in all experiments without any observable toxicity. The lux imaging system employed provided a robust method to track bacterial viability  making ity Figs  and 3. Wal times , 26–29, nescence .E. coli within tumour tissue of treated mice only. TNFα production was achieved using MG-TNFα as evidenced by analysis of tumour extracts. Immune competent syngeneic mice were utilised for all studies, and downstream local pro-inflammatory effects were apparent following treatment as evidenced by cytokine analysis, suggesting local secretion by immune cells following treatment. In terms of immune responses to the bacterial vehicle, E. coli) was observed for the duration of the experiment  Plasmid Construct (b) ELISA data for TNFα production from MG extracts in vitro. (c)In vitro growth rates of TNFα -producing bacteria, indicating no significant toxic effects on the host bacterium.(EPS)Click here for additional data file.S1 Table7cfu) or low (106 cfu) dose of MG-TNFα via i.v. injection to the tail vein. Mice were monitored at regular intervals post bacteria for the following macroscopic health indicators; Fur texture ; Posture ; and Activity  to create a disease activity index.6–8 week old tumour-free BALB/c mice (n = 3 per group) were administered a high (10(DOCX)Click here for additional data file.


Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in ‘bystander killing’. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation. Ideally, anti-cancer treatments should specifically target and kill tumour cells while leaving normal, healthy tissues relatively unscathed. The therapeutic ratio of the maximum tolerated to the minimally efficacious dose of many of the drugs used in conventional cytotoxic chemotherapy is relatively low, resulting in unwanted side-effects. Nevertheless, these drugs are relatively effective against cancer cells because normal cells have more efficient mechanisms for effluxing cytotoxins, repairing DNA, and clearing dead and damaged cells. Together, these mechanisms limit toxicity even among the rapidly dividing cells of the bone marrow and gastrointestinal epithelium before the next cycle of chemotherapy is given. Immunotherapy has now joined cytotoxic chemotherapy as an effective systemic treatment for cancer, and antibody-based therapy has contributed most significantly to this burgeoning area. Monoclonal antibodies (mAbs) may directly stall or kill tumour cells by binding tumour cell-specific antigens, or indirectly kill tumour cells by inhibiting the immune checkpoint molecules that restrain tumouricidal lymphocytes. Building on these advances, antibody drug conjugate (ADC) therapy has emerged with worldwide marketing approvals for the indications of breast cancer and lymphoma.An ADC is divided into three distinct parts – the tumour-targeting mAb, the linker, and the cyotoxic drug, also known as the payload or warhead . Modern Two ADCs are currently FDA-approved for cancer treatment; ado-trastuzumab emtansine  for the treatment of patients with human epidermal growth receptor 2 (HER2)-expressing, metastatic breast cancer  linker, must be internalised by the target cell for the antibody, rather than the linker, to be degraded before release of the drug. This form of ADC does not produce efficient bystander killing because, typically, it contains the potent tubulin-binding maytansine derivative, DM1, and the cleaved drug product cannot enter surrounding cells because its positive charge prevents it from penetrating the cell membrane  cell in HL and on the non-HL cells of ALCL. In contrast, normal tissue expression of CD30 is limited to activated B and T cells , thymocy+ neoplastic HRS cell of HL represents only a small proportion (0.1–10%) of the total lymphoma cell population, which mainly comprises such reactive cell types as T and B lymphocytes, plasma cells, eosinophils, neutrophils, macrophages, and fibroblasts  had responses with little (⩽15%) or no CD30 expression by central review of immunohistochemistry  achieved 50% partial response rate . Remarkably, one patient with relapsed DLBCL, who had 0% CD30 expression within the tumour biopsy by central review of visual IHC and 1.4% via cIHC, still achieved a complete response after two cycles of brentuximab vedotin  and enzyme-labile (peptide-based) linkers. It is often presumed that these linkers are cleaved only after internalisation by the target cell with subsequent lysosomal degradation releasing free drug. ADCs with these linkers and appropriate drugs can kill not only the antigen-positive target cells but also the surrounding antigen-negative cells. Consequently, these ADCs are useful for treating tumours with heterogeneous antigen expression . Although degradation of the acid-labile linker in the acidic lysosome is the major degradation pathway for drug release, studies have shown that this may also happen extracellularly. For example, targeting the poorly internalising antigen CD20 with an ADC consisting of the acid-labile 4-(4-acetylphenoxy)butanoic acid (AcBut) linker and the DNA minor groove-binding drug calicheamicin was effective in a preclinical lymphoma model, whereas substitution with an acid-stable (amide) linker had no anti-tumour effect when compared with the parental antibody alone (N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) and N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), which contain one or two methyl groups adjacent to the disulphide bond, respectively, resulting in increasing steric hindrance, have been used for ADCs. The more sterically hindered SPDB linker showed superior activity compared with the SPP linker cyclohexane-1-carboxylate (SMCC)  to antibodies targeting poorly internalising antigens  via the cleavable SPP linker were effective in xenograft models and were more effective than ADCs with the same antibody/drug using the non-cleavable thioether linker succinimidyl-4-. The resulting drug conjugates do not require internalisation by the target cell because the drug is released upon reduction or hydrolysis of the disulphide bond within the tumour microenvironment. This technology has used a SIP format of the F8 antibody, which binds the alternatively spliced extracellular domain A of fibronectin, an extracellular tumour angiogenesis marker. In preclinical studies using (SIP)F8 conjugated to the tubulin-binding drug, cemadotin, which is an analogue of dolastatin and chemically related to the aurostatins, some complete tumour regressions were observed at relatively high daily doses of 43 mg kgvs three out of five complete responders) when given at equivalent molar concentrations  because of its increased cell permeability. Consequently, MMAE has the advantage of being able to diffuse out of the target cell and enter surrounding cells to cause bystander killing, something that MMAF cannot do.The vc dipeptide linker, which is cleaved preferentially by the lysosomal protease, cathepsin B, is more stable in both human and mouse plasma than the pH-dependent hydrazone linker, resulting in lower toxicity in mice . Althoug50 of the ADC, the concentration of MMAE released from the targeted cell best determined the extent of in vitro tumour cell killing . This anti-tumour effect was mediated by the Fc-mediated uptake and processing of the ADC by the TAMs, which then resulted in extracellular release of MMAE and bystander tumour cell killing. It is therefore clear that immune cells, in particular macrophages, are required for immunotherapeutic efficacy, and these effects may be dependent on Fc-mediated phagocytosis. However, ADCs with site-directed mutation to reduce Fc-binding still show efficacy, meaning that in some cases Fc-dependent effector functions are not solely required for tumour responses to ADCs (The internalisation of ADCs after target antigen binding by tumour-associated myeloid or immune cells may also play a role in ADC catabolism. For example, a specific antibody bound to CD30ADCs are revolutionising the field of cancer treatment by harnessing the specificity of tumour-targeting mAbs with the stability and high potency of advanced linker/drug combinations. Although it has been established that internalisation and intracellular processing of ADCs provide one mechanism of action, it is becoming clear that this is not the only mechanism of ADC action and other processing mechanisms independent of direct cell-surface antigen internalisation can produce effective ADCs.Depending on the linker type and drug combination, non-internalising ADCs may have additional advantages by exploiting pathological features inherent to the microenvironment of many tumours such as hypoxia, necrosis, excess reducing equivalents, acidity, an abundance of both active extracellular proteases and protease-rich tumour-infiltrating myeloid cells. By providing the setting for conditional activation of ADCs, tumour properties other than antigen specificity can enhance the tumour selectivity of ADCs. Finally, a strong rationale exists for combining ADCs with other cancer therapies that either act upon or react to elements of the tumour microenvironment. For example, cytotoxic chemotherapy drugs can increase tumour expression of ADC-catabolising cathepsin B (


Varicella or chickenpox was not a notifiable disease until 2005 in Sri Lanka and only a few studies have been conducted on the epidemiology of VZV infection in the country. The antiVZV IgG sero-prevalence among antenatal women is extremely limited and thus a selected group of antenatal clinic attendees were chosen to determine the exposure rate to VZV infection.Women attending the antenatal clinic at Teaching Hospital, Peradeniya, Sri Lanka were selected for the study and 3 mL of venous blood was collected from 181 participants and the demographic data was obtained through a pretested questionnaire. Sera of the women were then tested for the presence of antiVZV IgG using ELISA . Data was analysed using the SPSS statistical software for Windows, Version 12.0.n = 61) were from urban, 41.8% (n = 59) were from rural and 14.9% (n = 21) were from estate populations . Out of the 88 antenatal women with a positive history for varicella, 85 (96.6%) were positive for anti-VZV IgG. The highest number of antiVZV IgG positivity was seen in the 31–35 age group, which was 85.0% of the total number of antenatal women included in that category. An increase in the antiVZV IgG sero-prevalence with increasing age was also noted in the study sample.Of the 181 antenatal women who took part in the study, 141 were positive for antiVZV IgG giving a sero-prevalence of 77.9% for the past exposure to VZV. Of the 141 antiVZV IgG positive women, 43.3% (Exposure rate of VZV infection as confirmed by antiVZV IgG in the present study sample of antenatal women was 77.9%. Age specific, population based future sero-prevalence studies should be conducted in Sri Lanka to understand the anti-VZV IgG status in the country. Varicella zoster virus (VZV) is a ubiquitous virus that infects humans. VZV causes a highly contagious disease called varicella or chickenpox, which has an attack rate of 65 to 85% in susceptible individuals followed by household exposure , 2. AlthVZV infections occur worldwide but the epidemiology differs greatly between tropical and temperate climates. In temperate countries, children are predominantly affected by VZV infections and the sero-conversion to anti-VZV IgG occurs usually during late childhood and thus adults show up to 90 to 95.0% serological evidence for the past exposure to the virus . AvaccinIn tropical countries, the VZV infections are common in adolescents and adults . The cliVaricella was not a notifiable disease until 2005 and only a few studies have been conducted on the epidemiology of VZV in Sri Lanka. Based on a study carried out in a selected urban and rural population in Colombo, none of the children below 5 years in the rural area had detectable anti-VZV IgG. Only 10.0% of the children in the urban population had sero-positivity for anti-VZV IgG. In the same study, a sero-positivity of 17.0% and 24.0% for anti-VZV IgG was observed among children below 15 years in urban and rural populations, respectively. In those aged 60 years, only 50.0% in the rural population were immune to VZV whereas in the urban population, 78.9% were immune to VZV .There is limited data on the antiVZV IgG sero-prevalence among antenatal women in the country. The economic drain posed by varicella and its complications are currently not studied and the incidence data on varicella are sparse in Sri Lanka. Varicella vaccine is not included in the National Immunization Programme in Sri Lanka . HoweverThe objective of the present study was to determine the exposure rate to VZV infection in a selected group of antenatal clinic attendees at a Teaching Hospital in Sri Lanka. The study also aimed to determine theexposure rate to VZV infectionin urban, rural and estate communities; to determine the exposure rate to VZV infection in antenatal women in different age categories; to assess the suitability of using the past history of varicella as a predictor of immunity against VZV infection in the study sample.This cross-sectional study was conducted from December 2015 to February 2016. The sample size for the study was determined using the 92.0% prevalence rate for anti-VZV IgG observed for a group of antenatal women in the tropical Australia . Based oThe information sheet and consent form were given to the participants prior to filling the pre-tested questionnaire and obtaining the blood sample. Blood samples were kept in a cool box and transported to the Virology Laboratory, Faculty of Medicine, University of Peradeniya. The samples were centrifuged at 3000 rpm for 20 min at room temperature to separate the serum. Sera were separated into properly labeled eppendorf tubes and stored at −20 °C freezer until tested for anti-VZV IgG. All the serum samples of individual patients were tested for the presence of anti-VZV IgG using a standard commercially available VZV IgG ELISA  with a sensitivity and specificity of more than 90.0% for detecting anti-VZV IgG. During the laboratory testing of the serum samples for anti-VZV IgG, no equivocal results were obtained for any of the 181 sera tested.Data was entered into SPSS, a statistical software for Windows, Version 12.0 for analysis. Results were described through measures of central tendencies including mean, standard deviations and proportions. The relationship between categorical variables such as past history of varicella andVZV immune status and, age and VZV immune status was checked using the Chi square test.Ethical clearance (PGIS/2016/EC22) for the study was obtained from the Ethical Review Committee of the Post Graduate Institute of Science (PGIS), University of Peradeniya.The antenatal attendees, who took part in this study were from diverse socio-economic conditions and religiously diverse backgrounds. Most of them were unemployedand these women were mostly from middle class and from the estate sector families of Sri Lanka. Of the 181 antenatal women tested in the present study, 141 (77.9%) showed anti- VZV IgG positivity. Of the 141 anti-VZV IgG positive antenatal women, 61 (43.3%) were from urban, 59 (41.8%) were from rural and 21 (14.9%) were from estate populations.From the 88 antenatal women with a positive past history for varicella, 85 were positive for anti-VZV IgG, which is 96.6% from the total number of antenatal women, who reported a past history of varicella. Of the 52 antenatal women with no a past history of varicella, 28 (53.8%) showed antibodies against VZV. Of the 41 antenatal women who were unaware of their past history for varicella, 28 (68.3%) were positive for anti-VZV IgG  included for the current study, the majority were positive for anti-VZV IgG, making more than ¾-VZV IgG . On the -VZV IgG  and a gr-VZV IgG . Thus, t-VZV IgG , 19, 20,n = 61) were from urban, 41.8% (n = 59) were from rural and 14.9% (n = 21) were from estate populations and these proportions when compared to a previous study in a group of adolescents in the Kandy District, sero-prevalence of anti-VZV IgG in the urban population (53.0%) had similarity to that noted in the current study. However, rural (17.0%) and estate communities (31.0%) had differences in sero-prevalence of anti-VZV IgG between the two studies [Of the 141 antiVZV IgG positive antenatal women, 43.3%  who had a past history for varicella were positive for anti-VZV IgG. These women (age = 18–45 years) had a better understanding and recalling power of the clinical history of varicella than those recruited for the previous studies in Sri Lanka, making a positive past history a more reliable indicator to predict immunity against varicella. Of the 52 antenatal women without a past history forvaricella, 28 (53.8%) were positive for antibodies against VZV and this might be due to subclinical VZV infection causing the anti-VZV IgG positivity. Similar findings have been reported by a Canadian study where 62.0% of population with negative or unknown history turn out to be anti-VZV IgG positive claiming that in the temperate climates the anti-VZV IgG sero-positivity is high and thus that acquired through subclinical infections would also be high . Of the When considering the relationship between anti-VZV IgG sero-prevalence and age of the antenatal women, the highest number of positive individuals was recorded in the 31–35 age group, which is 85.0% of the antenatal attendeesincluded for that age category. An increase in the antiVZV IgG sero-prevalence with the age was noted among the antenatal women. However, the study population was not evenly distributed among the age categories and this might be the reason for the slight drop in the anti-VZV IgG positivity in women agedbetween 36–40 years. Due to limited funds this study was carried out using a sample size of 181 antenatal attendees but further studies should be carried out to confirm the consistency as well as changes in the anti-VZV IgG data among antenatal women in Sri Lanka.The current study identifieda relatively higher past exposure rate of ~ 78.0% to VZV in a vaccine naïve group of tropical antenatal women. This finding explains the limited number of neonatal chicken pox cases in a country with lesser exposure rates recorded for adolescents and adults in the last decade. Furthermore, age specific, population based sero-prevalence studies should be conducted in Sri Lanka to understand the anti-VZV IgG status in the country to incorporate VZV immunization to avoid the severity of adulthood varicellaand its complications.


Objectives. Tumour heterogeneity represents a key issue in CT perfusion (CTp), where all studies are usually based on global mean or median values of perfusion maps, often computed on whole tumour. We sought to determine whether, and to what extent, such global values can be representative of tumour heterogeneity, with respect to single slices, and could be used for therapy assessment. Materials and Methods. Twelve patients with one primary non-small cell lung cancer lesion were enrolled in this study, for a total amount of 26 CTp examinations and 118 slices. Mean and median blood flow (BF) values, calculated voxel-based, were computed on each slice and the whole tumour. To measure functional heterogeneity, entropy was calculated on BF values as well. Results. Most of the slices were not represented by the global BF values computed on the whole tumour. In addition, there are a number of lesions having equivalent global BF values, but they are composed of slices having very different heterogeneity distributions, that is, entropy values. Conclusions. Global mean/median BF values of the single slices separately should be considered for clinical assessment, only if interpreted through entropy computed on BF values. The numerical equivalence between global BF values of different lesions may correspond to different clinical status, thus inducing possible errors in choice of therapy when considering global values only. The introduction of antiangiogenic therapies aiming at preventing or regularizing the growth of vascularization in cancer tissues has aroused lively interest around promising imaging techniques capable of detecting vascular changes, thus providing fundamental information about treatments effectiveness before the appearance of morphological changes. In the last few years, computed tomography perfusion (CTp) has gained a large consensus among researchers, thanks to its capability of providing both high morphological resolution images and functional information about the investigated tissues . This no heterogeneity is an intrinsic characteristic of all tumours [Several sources of variability are known to affect the reliability of final perfusion parameters  that fro tumours , 10, at  tumours , and thi tumours . representative one for clinical assessments has been widely debated. Traditionally, the first CTp examinations were performed on one slice, due to technology limitation of data acquisition and processing apparatuses [As a consequence, which part of the tumour could be the mostaratuses . Afterwaaratuses , or grouaratuses . Howeveraratuses , chosen aratuses , or bettaratuses , or elsearatuses –21. Nonearatuses , such asaratuses , or the aratuses , jointlyaratuses . reproducibility of reliable results is an essential requirement, but it must be coupled to the clinical representativeness of numerical results. In the literature, it has been widely stated that by considering the whole tumour [Undoubtedly, the most important issue to make CTp entering the clinical practice is the possibility of achieving between-patient and among-patients standardization. To this purpose, thee tumour , or evene tumour , perfusie tumour , againste tumour  and on te tumour . The ovee tumour , where tAll the studies considered refer to global perfusion parameters, whether they are mean or median values, encompassing all the tumour characteristics. However, in this way, besides the uncertainty intrinsic to the CTp acquisition and processing procedure, global parameters also reduce the variability due to tumour heterogeneity. This is acknowledged by several authors, which recognize that global values only provide an overall measure of variability  and thatWhile it is widely agreed that considering tumour volumes give more information than using a single slice, usually single slices are merged together to provide global parameters. The aim of this work is to investigate the clinical representativeness of global perfusion values and to assess their capability to deal with tumour heterogeneity. To this purpose, an extensive analysis is carried out on a wide set of 118 tumour slices, and on corresponding whole tumours, referring to 12 baseline and 14 follow-up (FU) examinations. BF values for each voxel of the various tumour sections were calculated and showed through the use of colorimetric maps. Global mean and median BF values of each slice and of the whole tumour were then computed. In order to measure hemodynamic heterogeneity, entropy was computed on the BF values of each colorimetric map and of each lesion. The entropy is a well-known measure of the information content  and, to The first statistical analysis was carried out to exclude that the groups of single slices composing the respective whole tumours had the same mean or median value. Then, a second analysis was executed to assess whether some slices exist with the same mean or median global BF values as the whole tumour, so as to find out which perfusion pattern, at several levels, is represented by the global values of whole tumours. In addition, being each slice endowed with its own entropy value, it is also possible to assess the heterogeneity those selected slices own. To complete the analyses, a comparison between tumours with same global mean or median BF values was performed, so as to verify whether, and to what extent, a statistical equivalence of global perfusion values hints at similar perfusion patterns and heterogeneity features.During data analysis, the results achieved for baseline and FU examinations are kept separate in order to allow detecting possible differences between untreated lesions, preserving their natural vascular structure, and lesions whose vascular network has been modified by the action of antiangiogenic treatments.2 (range 433–1995 mm2) were enrolled. Five of them underwent at least one FU, for a total amount of 26 CTp examinations.This retrospective study was approved by the Institutional Review Board. Twenty-two patients  with one primary NSCLC lesion underwent CTp. Patients over eighteen, with lesions having the longest axial diameter larger than 15 mm in at least three sections, were considered. Lesions whose boundaries could not be accurately identified, such as in case of highly inflamed tissues surrounding the tumour, were excluded from the study. Finally, twelve patients  with a target lesion having mean longest axial diameter of 43.5 mm (range 25.3–75.2 mm) and a mean area of 1625 mmz-coverage of 55 mm , and rearranged into 220 cine images . For each slice, the longest axial diameter was computed, using a digital calliper. Due to the inclusion criteria, the number of slices where tumours are visible changes for each examination, but they are almost always visible for at least five slices. For this reason, the set of the five central slices was selected [Axial CTp examinations were carried out using a 256-slice CT system . Patients were instructed for breath-hold and laid in the supine (feet first) position. An initial full-body, unenhanced and low-dose, CT scan was performed to identify the target lesion at the baseline condition. Then, 50 mL of intravenous bolus of contrast agent  was administered to the patients at 5 mL/s. Five seconds later, a CTp scan of 25-second duration was performed at fixed tube voltage (80 kV), current (250 mA), and exposure (100 mAs). The protocol yielded 20 scans, centred on the target lesion, with a selected , achieviμs) and of the whole lesion (μw) were computed for each examination. Median values were also computed for each slice (Ms) and the whole lesion (Mw). The ranges (r) between minimum and maximum of μs and Ms, rμ and rM, respectively, were computed as a variability measure referred to as the whole volume.Two 25-year experienced radiologists analysed the whole sequences in cine-mode fashion and in agreement, for each examination, placed a circular region of interest (ROI) within the aorta to extract the arterial input and, for each slice, drew a ROI following the margin of tumours. Voxel-based BF values, expressed in mL/min/100 g, were computed for each slice, using an in-house algorithm developed in Matlab , implementing the maximum slope method  and reprE, was computed for the first time on the BF maps of the whole tumour (Ew) and of each slice (Es), with the purpose to get a measure of the hemodynamic heterogeneity. The range rE between minimum and maximum Ew values is also considered as a measure of the heterogeneity variability in the whole tumour. E measures are reported in arbitrary units (a.u.). More details are given in the Appendix.The entropy is a measure often used in texture analysis, also applied to the oncologic field, for instance, to get a measure of texture irregularities . BesidesEs values.p values ≤0.05 were considered for statistical significance. Kendall-τ coefficient was used to assess any possible correlation between measurement errors and their magnitude: in case of concordance, data were log-transformed. Three groups of statistical tests were performed.Statistical analysis was performed by using statistical software . t-test and the Wilcoxon rank sum test, which were utilized for three different purposes. In fact, they were applied to test, for each lesion, the difference of means and medians, respectively, between each slice and the whole tumour, with the purpose to check whether a slice exists which can represent the whole tumour . The same tests were also carried out to check for μs or Ms differences between couples of slices, whether they belonged to same tumour or different ones. Finally, they were employed to select which tumours have the same statistical μw or Mw values, to further compare their perfusion patterns . In fact, computing and using a global mean, or median, perfusion value for CTp studies implicitly means that sets of BF values  with same μs (or Ms) as μw (or Mw) are clinically equivalent. The third group of tests is composed by the one-tail t-test only, which was performed to assess the differences between the means of Es for baseline and FU examinations.First, the one-way analysis of variance (ANOVA) was performed to check whether all slices (the “groups”) of the same tumour have the same mean value, that is, whether they can represent the same population, in terms of BF values. An analogous assessment was carried out for medians, through the Chi-squared test of independence. The second group consists in the two-tail The goal of this section is assessing the capability of global values, computed on the whole tumour, to represent the clinically relevant perfusion features of a tumour, assuming that the heterogeneity is among the most important ones . To thisAs the first outcome, it is worth reporting that the hypotheses that means or medians of slices were all equivalent were rejected, for each examination. Actually, this finding was expected and suggests that the variability between slices is significantly greater than the variability within slices .Tables μw and Mw values. μw and Mw values never selected the slice with maximum Es and one time selected the slice with minimum Es . μw = 125.0) corresponds to that of the first slice , having in its upper part a hyperperfused region , and a lower hypoperfused region with BF values nearly 40.st slice  shows a μw = 44.4 and Mw = 37.8) and ID6-FU4 (μw = 39.2 and Mw = 33.2), the same slices  were those with maximum E , probably due to these examinations being subsequent FUs of the same lesion. In addition, this is the only ID where mean and median select the highest E. As regards ID6-FU4, it shows limited BF ranges  and μs and Ms are substantially equivalent for the three central slices. This consideration regarding mean range also holds for ID6-FU3, where rμ = 13.8 is a little higher, but still among the lowest ones. As for median, in ID6-FU3 it also selects slice 2 (Ms = 36.9) that has the lowest Es = 6.26. On the other hand, in ID11-FU4 (Mw = 63.3 selects slice 1 (Ms = 64.3) which is the one with the lowest Es = 7.05.ID11-FU4 , Mw = 63μw = 80.0, first row) and ID6-FU1 . Although these lesions have statistically equivalent μw, the respective composing slices have a different heterogeneity distribution. In fact, the heterogeneity in all slices (except for slice 3) of ID11 is quite comparable, as it can be seen from Es values of μs = 150.8 and Es = 7.75. In addition, here the heterogeneity is made of local homogeneities, with a hyperperfused upper region and a hypoperfusion in the lower one.In this section, we extend the analysis over the whole dataset, by considering all the CTp examinations together. As regards the whole sets of slices, the most meaningful result is that on the whole 93 slices were not represented by the global BF values computed on the whole tumour. As for the sets of whole tumours, here we analyse the sets of slices referring to two couples of meaningful lesions. μw = 111.8) shows a heterogeneity that keeps quite “homogeneous” within all slices , also in terms of mean (rμ = 15.2) and median (rM = 10.2) BF , with all μs around μw = 111.8. On the other hand, the heterogeneity in ID2-FU1 (μw = 112.8) is made of well-defined hyper- and hypoperfused regions, mostly evident in the upper and lower part, respectively, of slices 2 and 3.Similar comments can be done for ID3 and ID2-FU1, made of five slices each, whose BF maps are shown in Es for baseline and FU examinations, separately. Related histograms are reported in Figures p values ≤10−4).Finally, we also analysed the distribution of all slice entropies Quantitative imaging has gained an increasing interest in these last years, as the need of personalized therapies progresses , deepeniIn this work, we have deeply analysed the representativeness of global mean and median values, as far as the heterogeneity is concerned, starting from the assumption that computing and using a global mean, or median, perfusion value for clinical purposes means accepting that the characteristics of the tumour are represented by that value alone. Accordingly, this implies that sets of BF values , with statistically equivalent mean or median values, are equivalently representative. The outcome of this work proves that these are numerical equivalences only, not clinical ones. In fact, we have discussed lesions with same global mean or median BF values, which showed a very different heterogeneity. In addition, we analysed tumour slices having the same global values as the whole tumour and we realized that, when those slices existed, for baseline examinations they were never those with the highest information content. Rather, it happened that in two examinations the whole tumour had mean and median BF values corresponding to the slice with the lowest heterogeneity , while the remaining slices showed relevant clinical signs of different heterogeneities.E, in two subsequent FU examinations. In addition, results prove that using the median as a more robust estimator is not so effective as expected, since mean and median practically select the same slices. Rather, in ID6-FU3, the median operator selects the slices with maximum and minimum Es, at the same time. This behaviour has relevantly misleading properties, all the more so because median is considered a powerful outlier removal. Actually, this is true, if erroneous values lay in the extreme of values domain, but median cannot work in case that discriminating BF outliers strongly depends on spatial displacement and arrangement of BF values themselves.For FU examinations, there was only the wide, and highly necrotic, uniform lesion ID6 where mean and median selected the region with the highest Global perfusion values computed on the whole tumour cannot be appropriate for therapy assessment and cannot improve the reproducibility of heterogeneity, accordingly. In addition, we have shown that the global values computed on the whole tumour have a correspondence with parts of tumour  that, just occasionally, could have either maximum or minimum entropy. In the remaining cases, they have not any correspondence with any real parts of the tumour and just represent a generic tumour BF averaged behaviour, which is far from representing its real clinical features. And perhaps, this happens much more times than expected. As far as single slices are concerned, although preserving more details, they may be not representative of the clinical status of the whole lesion and this could severely mislead clinical considerations.The solution is not at hand, but the research carried out in this work suggests that the first step is being very prudent in considering the global (mean or median) BF values as useful indicators for therapy assessment. On the other hand, measuring heterogeneity is a key issue to achieve useful information to assess the effectiveness of antiangiogenic therapies that cannot be left out of consideration. This is confirmed by the comparison between the average BF entropy of all slices before (baseline) and after (FU) treatment, proving the effectiveness of treatments themselves, expectedly reducing the overall BF heterogeneity of tumours. We have also seen that the single slices of a tumour are widely varying from each other and can represent different BF heterogeneity patterns that, on the whole, could provide a radiologist with an overall view of the whole tumour. Indeed, using all the single slices of a tumour, endowed with global BF values and a BF heterogeneity measures, would represent a step forwards, useful to help radiologists to draw more reliable clinical considerations.As concluding notes, we believe that improving the reliability of voxel-based perfusion values has to be coupled with the reproducibility assessment of heterogeneity measurements. To this purpose, a deeper application of the bioengineering and computer science techniques to CTp data processing, in a multidisciplinary team, will play a key role in the next future to help translation of CTp into clinics.The achievements of this work could be also assessed using other perfusion parameters, such as the blood volume and the mean transit time. In addition, a study dedicated to validate the BF entropy as a surrogate biomarker for the overall survival is being carried out, based on the existing correlation between BF entropy and tumour grading.


We report on the preparation of a biopolymer thin film by hydroxypropyl methylcellulose (HPMC), which can be used as a dry green lubricant in sustainable manufacturing. The thin films were characterized through scanning electron microscopy, energy-dispersive spectroscopy, and Raman spectroscopy; the films showed desirable levels of thickness, controllability, and uniformity. Tribology tests also showed desirable tribological and antiwear behaviors, caused by the formation of transfer layers. Zebrafish embryo toxicity studies showed that HPMC has excellent solubility and biocompatibility, which may show outstanding potential for applications as a green lubricant. The results of the present study show that these techniques for biopolymer HPMC provide an ecologically responsible and convenient method for preparing functional thin films, which is particularly applicable to sustainable manufacturing. To meet 3R demands, manufacturers proposed concepts of sustainable manufacturing and green lubricants, in which the idea of 3R is incorporated into the research and development of processing-related technologies. Major development directions of such technologies include green lubricant materials [The United Nations Framework Convention on Climate Change signed during the 2015 United Nations Climate Change Conference in Paris has made carbon reduction and energy conservation an issue to be addressed by all countries around the globe. To facilitate the most efficient use of resources, product designs and manufacturing philosophies that focus on “reducing” the use of resources, “reusing” resources, and “recycling” resources  over a long period of use, resulting in the silicon substrate showing significant Si signals during EDS element mapping. Moreover, According to To find out where the HPMC had been removed to and the reason why the silicon substrate was able to maintain low COFs at the later phase of lubrication, this study analyzed the chrome steel ball used to scrape the silicon substrate. The results indicated that during lubrication, the HPMC was transferred from the surface of the silicon substrate to that of the chrome steel ball, forming a protective layer between the two objects and leading to an HPMC–HPMC lubricating behavior. Considering that most polymers have a self-lubricating effect , it is tA pin-on-disk tribotest was adopted to analyze the coatings synthesized using HPMC. To facilitate the analysis, the first and last 50 data cycles were averaged, as displayed in n = 5 for each treatment). This may have been caused by the shrinkage of the chorion; if the chorion shrank and then touched the surface of the embryo, then that may have caused reflection contractions [At 24 h post fertilization (hpf) after incubation in HPMC according to the guidelines of the Fish Embryo Acute Toxicity (FET) test, treated embryos did not show any phenotype differences between groups, including coagulation of the embryos, lack of somite formation, or non-detachment of the tail . Howeverractions .n = 20 for each treatment). In addition, another phenotype marker, pericardial edema, was significantly increased in the 1% HPMC-treated embryos . This suggested that the circulatory systems in the 48-hpf embryos were affected [At 48 hpf after incubation in HPMC, the hatching rate, which is one of the phenotype markers of the FET test, decreased following the increase of the concentrations of HPMC  and stirring the mixture until the latter completely dissolved. A micropipette was used to administer 150 μL of the solution slowly unto a silicon substrate that had been cleaned using alcohol, acetone, and isopropanol. The HPMC coated silicon substrate was then left to stand for 1 h in an environment with a temperature of 25 ± 2 °C and a relative humidity (RH) of 60% ± 5%.i.e., Renishaw system 2000 micro-Raman spectrometer, Renishaw, New Mills, UK). Regarding the uniformity analysis, it involved randomly selecting nine points on the film and identifying the characteristic peak intensity of each point using the Raman spectrum; the peak value intensities were then recorded for a film uniformity comparison. A 3D scanner  was applied for the thickness measurement.Film properties such as thickness and surface characteristics were analyzed using a SEM  and EDS, whereas thin film properties were assessed using the Raman spectrum  was recorded by connecting a load cell to the rotating disk. The friction coefficient was measured for further analysis of the lubrication and antiwear properties. The friction experiments were repeated to ensure the reproducibility of the results.This study investigated the lubricating properties and antiwear behavior of 3.5% HPMC by using a ball-on-disk tribometer . For testing, a ball was fixed on a stationary holder, and the bottom disk was rotated at a specified speed. Tribology tests were performed in ambient air at ambient temperatures with a sliding speed variation of 0.01–0.10 m/s and loading variation of 2–8 N. All friction and wear tests were conducted at a rotation radius of 2 mm. The steel balls were composed of AISI 52100 with a hardness of 61HRC. The tested HPMC coating was prepared on a silicon substrate and mounted onto the bottom disk of the tribometer. The resistance to the motion of the disk  were used and maintained under standard conditions, as described in Westerfield’s guidelines for the experimental use of zebrafish [Zebrafish strain and husbandry: Wild-type zebrafish (ebrafish . The preebrafish ) and 1X (1)Dry films made from biopolymers feature an adjustable thickness and favorable uniformity.(2)Compared with bare silicon frictions, silicon substrates coated with HPMC dry films demonstrate a superior lubrication result.(3)The forming of transfer layers is the primary reason that HPMC demonstrates superior lubrication properties.(4)HPMC is a biologically and environmentally friendly biopolymer material as well as a suitable and sustainable candidate for making green lubricants.


We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration  It is nlignancy , 4. We hlignancy . Thus, sSolid tumours can be considered as abnormal organs composed of neoplastic and non-neoplastic stromal cells embedded in a complex extracellular matrix (ECM) . Each tuvia the corpus callosum  confirmed the histological analysis, showing a brighter, homogeneous brain tumour area in T2 weighted images of the invasive phenotype Figure  and no cin vivo and raises the intriguing question as to why patient glioblastomas unfold into different phenotypes when transplanted into the rodent brain.In summary, glioblastoma patient-derived xenografts based on organotypic spheroids can be reliably established in mice and maintain typical glioblastoma characteristics. Notably, depending on the parental tumour, they develop according to three distinct phenotypes that we classified as ‘invasive’, ‘intermediate’ and ‘angiogenic’. The separation of key glioblastoma features into different phenotypes provides an opportunity to study their development in vivo phenotypes as PDX models, is also important in view of the fact that these are more amenable to genetic manipulation and in vitro studies.To exclude that glioblastoma phenotypes are a result of the experimental procedure of spheroid generation, we analysed xenografts derived from five patient-derived glioblastoma stem-like cell lines. Interestingly, we observed three very similar phenotypes to those obtained with organotypic spheroids. Three cell lines  gave rise to an invasive phenotype, one cell line (NCH421k) to an intermediate phenotype and one (NCH644) to a full-blown angiogenic phenotype with microvascular proliferation . This suTumour angiogenesis is normally expected to arise in solid tumours when a distinct size or distance from blood vessels has been reached , 14. To To assess to what extent aggressiveness was associated with the tumour phenotypes, we correlated the mouse survival with the phenotype. We found that the angiogenic phenotype was most aggressive  . This was exemplified by the observation that animals with invasive tumours could display a relatively high proliferation index and short survival, (e.g. P8), or a low proliferation index and long survival (e.g. T101)  Figure . Howeverg. T101) .In summary, histopathological tumour phenotypes are correlated with mouse survival, with the angiogenic tumours being the most aggressive. Aggressiveness is correlated with high proliferation, microvascular proliferation, necrosis and low invasiveness, where only the proliferation index is independent of the phenotype.In view of the differences in the vessel structure observed by histology and MRI, we performed a detailed analysis of the vessels in the three phenotypes. Immunostaining with a mouse-specific CD31 antibody confirmed a close to normal vessel morphology in invasive tumours, and a grossly aberrant morphology in the angiogenic phenotype Figure . QuantifIt has previously been suggested that tumour stem cells contribute to vessel formation through a transdifferentiation process, although these data have been questioned by others . By immuIn conclusion, pathological tumour vessels are found in angiogenic and intermediate phenotypes, while invasive tumours retain a largely normal vessel morphology. Furthermore we show that ECs in glioblastoma are stroma-derived and do not detect any transdifferentiation of tumour cells into functional endothelium.in vivo growth, stromal-free spheroids of invasive tumours (P8T) showed little proliferation in vitro and remained similar in size, while spheroids of intermediate tumours (P3T) proliferated in vitro resulting in larger spheroids  between angiogenic vs. invasive; angiogenic vs. intermediate and intermediate vs. invasive tumour cells respectively were identified Figure . ClusterALDOC, HK2, PGAM1 and LDHA were significantly up-regulated in angiogenic tumour cells  was also up-regulated at the transcriptional level in angiogenic and intermediate tumours  , proteases , integrins , laminins , Fn1, Hspg2, CD93 and Bmp1  of NOD/Scid mice. Cluster analysis showed that ECs from tumours displayed genome-wide transcriptomic changes compared to normal brain ECs Figure . The mos) Figure . Similar1 Figure . Genes d1 Figure .Angpt2 which was up-regulated in intermediate and angiogenic ECs , a regulator of the cell cycle, and FOXO1, responsible for gluconeogenesis and angiogenesis, as the most prominent regulators of EC activation  whereas FN1, NID1, NID2, LAMB1, LAMC1, LGALS1, TNC) and their modulators , cell adhesion molecules/cell surface receptors  and ligands    and 11. versus EC genes up-regulated in the angiogenic phenotype and representing cell membrane, extracellular matrix and secreted molecules (resulting in 1004 proteins and 3240 PPIs)  databases. We focused on tumour DEGs ) Figure . This anBy further focussing on the direct connections between the two cell types, we identified numerous interactions within extracellular matrix and plasma membrane proteins associated with angiogenesis . SeveralIn summary, we show that through autocrine and paracrine interactions tumour and ECs cooperate to create a unique angiogenic, tumour promoting microenvironment and we identify key molecules that represent attractive targets to modulate essential steps of this tumour-microenvironment crosstalk.Tumours leverage their surroundings to progress and evade therapy. Thus, a better understanding of the interactions between tumour and microenvironment may improve our ability to provide effective treatment. Here, we provide a detailed histological and molecular characterization of glioblastomas and their surrounding brain parenchyma, with an emphasis on the crosstalk between tumour and endothelial cells. We used human-to-mouse patient-derived glioblastoma xenografts, which similar to our previously described rat models , 6, wereThe origin of the three phenotypes is currently not fully understood. It should be noted that the here described histological features are present at variable intensities in all human glioblastomas (hence the diagnosis), sometimes juxtaposed in distinct patterns within the same tumour (intra-tumour heterogeneity). The PDX model may therefore reflect the spatial differences of the original tumour. Although we cannot rule out this hypothesis, it should be noted that all biopsies were derived from the contrast-enhancing tumour core and their spatial origin can thus not simply be correlated to the PDX phenotype. Alternatively, the phenotype may result from the patient-specific genetic profile of the tumour (inter-tumour heterogeneity), which induces specific changes to its microenvironment. In agreement with this, the characteristics of each phenotype were detectable at early stages of tumour growth. We have recently shown in the rat model that invasive tumour growth is strongly associated with EGFR gene amplification and receptor activation . This teA limitation of current PDX models for the study of tumour-host interactions is the requirement of immunodeficient animals. In this regard it is worthy to note that nude mouse strains still carry active microglia/macrophage populations, which are the main immune component in the brain. Of note we did not observe any difference between xenografts generated in nude or NOD/Scid mice, while macrophages are largely defective in the latter.Using cell type-specific transcriptomic analyses, we find that the histopathological features are clearly reflected at the molecular level. Tumour cells of the angiogenic phenotype displayed higher levels of hypoxic  and angiogenic markers . Of note, ANGPT2, generally thought to be only expressed in ECs, was also up-regulated in tumour cells. As pathological vessels were observed early during tumor development, vascular modifications seems to develop before the onset of hypoxia, possibly as a result of genetic determinants of the tumour leading to a pseudo-hypoxic state e.g. a constitutively activated HIF1α pathway in non-hypoxic conditions could induce angiogenesis, although other genetic factors may be involved. Collectively these data suggest that tumour angiogenesis is likely the combined result of genetic determinants of the tumour and the response to specific microenvironmental conditions. We have previously shown that ‘intermediate’ (P3) and ‘angiogenic’ (P13) PDX tumors adapt to anti-angiogenic treatment by switching to a glycolytic metabolism and increasing invasion , 21. InteGFP expression in the host microenvironment allowed us to unequivocally distinguish host endothelial cells from neoplastic human cells. Based on combined GFP/CD31 staining and functional EC properties (SP phenotype), we could not find any evidence for transdifferentiation of tumor cells into endothelium indicating that blood vessels in glioblastoma originate from the stroma. This does not exclude the existence of vascular mimicry, where tumor cells without EC properties align to form blood cell-containing vascular channels , which wANGPT2, CD93, FN1, ITGA5, NID1, NID2 and G2 checkpoint kinase: WEE1 . FN1, a downstream target and regulator of TGFB1, which plays a key role in matrix assembly [in vitro: activated stromal cells retained their glioblastoma specific phenotype and were able to influence tumour cell properties in vitro. In future studies it will be interesting to address the role of inter-cellular exchange of genetic and/or protein material between tumour and stromal cells via vesicles or tunneling nanotubes as has recently been reported for other cancers [In line with a co-evolutionary model of neoplastic and non-neoplastic stromal cells, we identified many genes that were concomitantly induced in ECs and tumour cells in the angiogenic phenotype, and some of these showed the highest number of putative protein to protein interactions  and the Haukeland University Hospital  from patients who gave informed consent. All studies were conducted according to the Declaration of Helsinki, and with approval from the local ethics committees  Luxembourg, local ethics committee Haukeland University Hospital, Bergen). All tumorectomies were from grade IV glioblastomas  on fibronectin pre-coated surface.Five glioblastoma stem-like cell lines  were generated from glioblastoma patient material  by enzym2, and placed lying prone in a cradle equipped with a heating pad set to 37°C. MRI sequences used included T2-weighted , and T1-weighted  after injection of contrast agent . In plane resolution was 0.078mm * 0.078mm and slice thickness 1mm.MR images were acquired on a 7T Pharmascan small animal MR scanner (Bruker Biospin GmbH) with a quadratic mouse head transmitter/receiver coil. Animals were anesthetized with 1-2% isoflurane mixed with 50% air and 50% OCoronal sections (8μm) from paraffin-embedded brains were stained with hematoxylin (Dako) and 1% eosin (Sigma). For immunostaining, sections were pre-treated for 5min with Proteinase K (Dako) followed by 30min incubation at 95°C in retrieval solution (Dako). The Dako Envision+System-HRP was used following the manufacturer's instructions. Primary and secondary antibodies were incubated for 1h. Signal was developed with 3,3′-diaminobenzidine chromogen in 5-20 min. IHC of mouse endothelial cells was performed on isopentane flash-frozen tissues and cryostat sections (10μm) were fixed with acetone and chloroform. Nonspecific binding was blocked with 2% FBS in TBS and antibodies were incubated for 1 hr at RT. Pictures were acquired with a Leica DMI 6000B microscope. Vessel quantification was done using ImageJ software. Additional IHC preparations were performed using a Discovery XT automated staining module (Ventana) and standard protocols (list of antibodies in vs. no), vascular proliferations (yes vs. no) and the degree of invasiveness was assessed on the basis of hematoxylin-eosin (H&E) stained sections . Additionally, we analysed the proliferative capacity (Ki67 positive cells / whole cell population (in %)) and Angpt2-positive vessels per mm2. A significance level was set for p < 0.05. Kaplan-Meier survival analyses were performed after median split and dichotomized in high or low (for Angpt2 and Ki67) or dichotomized for: necrosis: yes vs. no; vascular proliferations: yes vs. no; invasion: high > 1 vs. low < 1. All statistical analyses were performed using JMP 8 and JMP 11 . Kaplan-Meier survival analyses including Log-Rank and Wilcoxon testings were performed to assess the influence of neuropathological features on mice survival. Pearson's and likelihood ratio chi-squared tests were performed to analyze neuropathological differences between the groups. Multivariate analyses for independence were performed with the Ch2 test including the following parameters: necrosis, vascular proliferation, invasion score, proliferation index. Other analyses were performed with the two-tail Student's t-test. Significance was set for: *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.Neuropathological analysis and quantification was performed independently by two experienced neuropathologists . The existence of necrosis  or 1500 (‘75% control’) cells per well of 96 well plates pre-coated with agar. To obtain stroma-containing spheroids 1500 sorted tumour cells (75%) were premixed with 500 sorted eGFP-positive stromal cells (25%). The growth of spheroids was monitored during a 2-week period and pictures (Leica DMI 6000B microscope) were taken after 1, 7 and 14 days of culture. Spheroid area was calculated with Image J and the growth ratio was obtained by comparing the spheroid areas at day14 to day 7.® (BD Biosciences). Sorted eGFP-negative tumour cells were used alone (5×104 cells/well) or premixed with eGFP-positive stromal cells (5000 cells/well = 10% of tumour) before plating. After 96 hours the cells on both sides of the membrane were determined by cell labelling . Cell invasion was normalised to the total number of cells, thus correcting for cell proliferation .Invasion assays were performed in Boyden chambers as previously described  using th6 cells/ml followed by 90min incubation with Hoechst 33342  at 37°C. After washing, cells were resuspended in ice-cold HBSS 2% FBS, 10mM HEPES pH 7.4 buffer (100 μl/test). Prior to flow cytometric analysis, cells were incubated with the IR-LIVE/DEAD® Fixable Dead Cell Stains  and appropriate preconjugated antibodies for 30 min at 4°C in the dark (TM SORP cytometer (BD Biosciences) fitted with a 632nm (30mW) red laser, a 355 (60mW) UV laser, a 405nm (50mW) violet laser and a 488nm (100mW) blue laser. For cell sorting, single cell suspensions were stained with the IR-LIVE/DEAD® Fixable Dead Cell Stain and the mouse-specific CD31 antibody for 30 min at 4°C. eGFP-negative tumour cells were sorted to the cold flow cytometry buffer and used directly for RNA extraction with the QIAGEN® RNeasy Mini Kit (Qiagen) (n = 3). eGFP+CD31+ mouse endothelial cells from xenografts and control mouse brains were sorted directly to Trizol® LS (Life Technologies) .Glioblastoma xenografts and control mouse brains were dissociated with the MACS Neural Tissue Dissociation Kit (P) (Miltenyi) following the manufacturer's instructions. For phenotyping flow experiments were performed as described . Single the dark . Data ac® RNeasy Mini Kit (human-specific microarrays) or the Trizol® LS extraction kit (Life Technologies) (mouse-specific microarrays), according to the manufacturer's protocol. GeneChip® Human Gene 1.0ST Arrays and GeneChip® Mouse Gene 2.0 ST Arrays (Affymetrix) were used to determine the expression profiles. Total RNAs were processed using the Ambion WT expression kit (Life Technologies) and the Affymetrix WT Terminal & Labeling kit before being hybridized on Affymetrix arrays (protocol P/N 702808 Rev.6). Upon hybridization, microarrays were washed, stained and scanned according to manufacturer's standard procedures. CEL files containing hybridization raw signal intensities were imported into the Partek GS software for statistical analysis. First, probe intensities were summarized to gene expression signals using Partek default options . IPA was used for the identification of the DEGs with specific location: cell membrane, extracellular matrix. Microarray data are available in the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/) under accession numbers E-MTAB-3948 and E-MTAB-3949.Total RNA was extracted using the QIAGENs  and up-regulated in EC . Mouse genes were mapped to human homologues and unique and unambiguous gene symbols were considered for further analyses. Putative protein-protein interactions (PPI) were identified with the GeneMania server  from 11 in vitro cultured spheroids were used for protein extracts . Proteins were extracted in lysis buffer  pH 8.5). Protein concentration was determined with 2D Quant . The signal was visualized using the ECL™ Advance Western blot detection kit , the ImageQuant LAS4010 imaging station and software  . VEGF concentration (pg/μg of protein) was assessed for the three phenotypes using representative xenografts: (P8, T101 invasive; T16, P3 and NCH421k intermediate; P13, NCH644 angiogenic. Experiments were performed in triplicates with protein extract from 2 animals each. The results are presented per xenograft phenotype.


Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from fermented herbage. We have determined the complete genome sequence of strain L. plantarum CGMCC 8198, which consists of genes that are likely to be involved in dairy fermentation and that have probiotic qualities.We report the complete genome sequence of  Lactobacillus plantarum is a type of versatile lactobacillus that has been extensively characterized functionally to document its probiotic attributes (–L. plantarum CGMCC 8198 (formerly named L. plantarum TH1) was isolated in our previous study , of which 2,388 were functionally classified. Besides, 2,908 tRNA-coding genes and 3,086,220 rRNA-coding genes were also found  activity has been reported in Bifidobacterium (Lactobacillus (Enterococcus (Bacteroides ( L. plantarum are limited and the precise mechanism underlying the hypocholesterolemic effect of BSHs is still not clear. Our analysis found that five putative active sites, Cys1, Asp20, Tyr78, Asn171, and Arg224, were found in all three bsh genes of L. plantarum CGMCC 8198. However, these active sites were the same as in PVA but were a little different with BSHs in Lactobacillus. For example, the catalytic residue of Tyr78 was replaced by Asn78 in BSHs of L. johnsonii ATCC 33200 and L. acidophilus. These results indicate that BSHs in L. plantarum CGMCC 8198 might have individual mechanisms for the hydrolysis of bile salts, and might escape cell death by bile salts in the intestine and reduce plasma cholesterol in vivo through the hydrolysis activity of its BSHs. This work indicates that this novel probiotic stain could be used in the food or drug system to improve the health of patients suffering from cholesterol-related diseases.A comparative analysis of strains acterium , 16, Lacbacillus , Enterocrococcus , BacteroL. plantarum CGMCC 8198 has been deposited in GenBank under accession no. MEGY00000000.The complete genome of