The study explores [18F]FMISO production failed on 2 occasions. Static PET images at 4 h were analysed using tumour-to-muscle (T:M) SUVmax and tumour-to-blood (T:B) SUVmax. The 0–45 min dynamic PET scans were analysed using Casciari model to report hypoxia and perfusion. Akaike information criteria (AIC) were used to compare data fittings for different pharmacokinetic models. Pathological tumour regression grade was scored using American Joint Committee on Cancer (AJCC) 7.0. Shapiro-Wilk test was used to evaluate the normality of the data.Patients with biopsy-proven rectal adenocarcinoma were imaged at 0–45 min, 2 and 4 h, at baseline and after 8–10 fractions of CRT (week 2). The first 6 patients did not receive an enema (the non-enema group) and the last 4 patients received an enema before PET-CT scan (the enema group). FMISO PET scans of rectal cancer. There are two principal problems namely spill-in from non-tumour tracer activity from rectal and bladder contents. Emphasis should be made on reducing spill-in effects from the bladder to improve data quality. This preliminary study has shown fundamental difficulties in the interpretation of [18F]FMISO PET scans for rectal cancer, limiting its clinical applicability.This pilot study with small number of datasets revealed significant challenges in delivery and interpretation of [The online version of this article (10.1186/s13550-017-0324-x) contains supplementary material, which is available to authorized users. Hypoxia in cancer cells can lead to radioresistance and influence radiotherapy (RT) response –3. Strat2) less than 80 mmHg is considered hypoxemic hypoxia [Tumour hypoxia is a consequence of imbalance between oxygen consumption and supply  due to f hypoxia .The Eppendorf electrode is considered the gold standard for measuring oxygen distribution in tumours  and has 18F]fluoromisonidazole ([18F]FMISO) in tumour cells pre-treatment is suggestive of hypoxia [2 polarography defined tumour hypoxia in head and neck (H&N) cancer [18F]FMISO uptake spatially corresponded with exogenous and endogenous biomarkers of tissue hypoxia in animal models [18F]FMISO uptake was observed in positron emission tomography (PET) images with reduced hypoxia in animal models [18F]FMISO PET showed contradictory results in detecting tumour hypoxia in human soft tissue sarcomas FMISO PET parameters in human rectal tumours before and after 8–10 fractions of CRT to predict clinical response.There is an increasing interest in developing predictive biomarkers of tissue hypoxia using functional imaging for personalised RT in patients with rectal cancer that are considered for neoadjuvant chemoradiotherapy (CRT). Therefore, the aim was to explore changes in FMISO PET at baseline (scan 1) and after receiving 8–10 fractions of CRT within approximately 2–3 weeks of the baseline scan (scan 2). [18F]FMISO was provided by the Radiochemistry Laboratory, Wolfson Brain imaging Centre, University of Cambridge, and produced based on the synthesis methods developed by Oh et al. [18F]FMISO with a prescribed dose of 370 (range 333–397) MBq. Dynamic PET images were acquired at 0–45 min in list-mode and static images at 2 and 4 h. Each PET scan was preceded by a CT scan, which resulted in 512 × 512 × 61 voxels of size 0.98 × 0.98 × 2.53 mm at all stages of scanning . PET datasets were reconstructed using time-of-flight ordered subset expectation maximisation (OSEM) protocol (GE’s VPFX) in 3D mode with two iterations, 24 subsets and 6.4 mm Gaussian filter  in accosolution . Dynamic70 range 3–397 MBqThe data split into two cohorts; the first six patients did not receive enema, and the last five were planned to receive MICROLAX® micro-enema before the 4 h PET-CT scan. The former will be referred to as the non-enema group and the latter as the enema group throughout this manuscript.muscle) were drawn over the gluteus maximus muscle  were manually delineated on MR scans by JF , JW and TG and transferred to the PET-CT using rigid registration in Eclipse radiation treatment planning software , Inc., Palo Alto, CA). The ROItumour were transferred to PMOD for analysis , tumour-to-blood standardised uptake value maximum (T:B SUVmax), tumour hypoxic volume based on the threshold value of T:M SUVmax > 1.3 and T:B SUVmax > 1.3 and the corresponding percentage (%) of the total tumour volume that is hypoxic based on the corresponding threshold values.The 4 h static FMISO PET scans. It was assumed that the calculation of % changes pre- and during CRT may reduce systematic bias by cancelling some of the systematic errors on two occasions. The Shapiro-Wilk test FMISO PET, with a range of 0–13.16%. It increased in 3 patients and reduced in 5 by week 2 CRT.(b)T:B SUVmax: Tumour hypoxic volume was defined using a threshold of T:B SUVmax ratio > 1.3 and the corresponding percentage of tumour volume that is hypoxic are outlined in Table 3 and decreased by 56% to 0.76 (IQR 0.78) cm3 by week 2. All patients scan demonstrated a hypoxic volume at baseline (range 0.48–36.25%), which fell in 6 patients (including the 5 with a reduction in T:M SUVmax -defined hypoxic volume) at week 2.(None of the changes in PET parameters obtained from static scans at 4 h between baseline and week 2 (from Tables a) Tumour: The most meaningful parameters, hypoxia (Ka) and perfusion (F), from the Casciari model are presented in Fig. Ka was 0.92 (IQR 0.41) min-1 at baseline and decreased by 24% to 0.70 (0.10) min-1 by week 2. The median F was 4.10 (IQR 1.71) millilitres/gram/minute ( ml g−1 min−1) at baseline and decreased by 29% to 2.48 (IQR 3.62) ml g−1 min−1 by week 2 ). In 9/11 patients that were scanned twice, the tumour perfusion decreased in non-responders and increased in responders except in one patient. None of the other changes in PET parameters between baseline and week 2 showed any clear trend with clinical outcome.(b) Muscle: The Ka and F from Casciari model in muscle are reported in Fig. F was 0.12 (IQR 0.04) ml g−1 min−1 at baseline and increased by 376% to 0.60 (0.12) ml g−1 min−1 by week 2. The median Ka was 0.02 (IQR 0.03) min-1 at baseline and increased by 3073% to 0.86 (0.76) min-1 by week 2. These values were higher than those reported by Schwartz et al. [.It is impossible to avoid bladder emptying at some point prior to the 4 h scan. [udy Fig. .Fig. 3TThe tumour ROI can be seen in close proximity to the bladder in patient 1 as shown in Fig. The trial protocol was changed following the first 6 participants to mandate use of a MICROLAX® enema to be administered and the rectum emptied prior to the 4 h scan to reduce error due to excreted tracer in the rectal lumen. Four patients scanned at baseline and three scanned at week 2 underwent a microenema.18F]FMISO in hypoxic cells reached an equilibrium state at 4 h post injection, and the increase in tumour activity could be purely an effect of increased bladder activity.Figures 18F]FMISO PET has been a valuable research tool in assessing hypoxia in various cancer types for predicting response to radiotherapy treatment in H&N and lung cancer [18F]FMISO PET parameters from static and dynamic imaging of rectal tumours before and after 8–10 fractions of CRT and reported the associated challenges.Imaging with [g cancer –40. In t18F]FMISO in hypoxic cells and slow cellular washout from non-hypoxic tissue limits the contrast between the tissue of interest and the background at early time points post injection FMISO concentrations in blood and muscle are nearly identical by a few minutes after tracer injection, T:M ratios were considered equivalent to T:B ratios with the benefit that no blood sampling is required. Since T:M SUVmax represented SUVmax of tumour divided by SUVmax of muscle, and since the normalisation factors in numerator and denominator cancel each other, the numerical outcome of T:M SUVmax is mathematically equivalent to the T:M ratio which is approximately equivalent to the T:B ratio  ratio ≥ 1.2 was considered to indicate significant hypoxia as first described by Rajendran et al. . Thereaf18F]FAZA PET at 2 h. Tumour hypoxic volumes obtained in this study were difficult to compare with those shown by Roels et al. [18F]FDG PET. Our T:M ratio values are however similar to those obtained in other solid tumour types such as head and neck squamous cell carcinoma FMISO PET data allows the measurement of hypoxic cells and blood perfusion simultaneously [Dynamic [aneously  which isaneously .18F]FMISO to reach equilibrium within hypoxic cells [Imaging at 2 h may not be sufficient for [ic cells . The tra18F]FMISO in plasma has been shown to be intact by 90 min post-injection FMISO pharmacokinetic model or due to inconsistencies in the TACs. Therefore, fitting gluteus muscle data successfully to the Casciari model suggested that the tumour data needed further analysis and it was absolutely necessary to seek a pharmacokinetic model that fitted the tumour data successfully. Even though more parameters may be required to fit more complex data successfully, this does not mean that the outcome is more meaningful just because the data has been fitted by a more complex model FMISO reduction to nitro radical is reversible in the presence of oxygen. It is possible that the prolonged duration of increased blood perfusion beyond 45 min increased the accumulation of tracer in muscle during the first 2 h post tracer injection which then reversed between 2 and 4 h when we observed a large washout of tracer suggesting that the trapping mechanism is rather time dependent and fluctuating. The pattern of blood perfusion in acute hypoxia can alter on a 20 min time scale [Ka and F showed weak relationship suggesting that the muscle [18F]FMISO uptake was not affected by the changes in muscle perfusion FMISO PET can be employed for the quantification of rectal cancer.Catheterization of the bladder has been suggested as an invasive alternative to this problem, but it is a potential source of infection and uncomfortable for patients . In addi18F]FMISO is excreted through the biliary tract into the gastrointestinal tract as well as through the urine. When rectal lumen contains high [18F]FMISO activity and it is within close proximity of the tumour, the spill-in of activity from rectal lumen into the tumour is unavoidable due to the limited resolution of the PET scanner. The use of an enema shortly prior to the 4 h scan may reduce this non-tumour luminal activity as shown in Figs. 1FFMISO is). Since the tracer uptake in tumour changes over time, the data may benefit from careful delineation of tumour region at each time point, albeit controversial due to the time and cost involved. In the case of Additional file Tumours are generally adjacent to normal rectal wall and therefore difficult to outline on CT. Therefore, tumours were first outlined on MR scans and then modified to match 4 h PET uptake since there is inevitable time difference between the PET-CT and MR scans obtained on the same day. Rigid registration was used to transfer the ROI between dynamic and static scans. This should be done with caution as the internal organ motion due to air, rectal peristalsis or other reasons may not allow accurate delineation of the tumour (Additional file 18F]FMISO has been shown to have lower tumour uptake compared to other hypoxia tracers [18F]HX4 for imaging liver and [xCu]Cu-ATSM for imaging bladder was also recommended even though the retention of radio-copper in animal models at early time post-injection has been shown to partially reflect processing of copper rather than a sole hypoxia marker [18F]FAZA and [xCu]Cu-ATSM for colorectal tumours for visualisation purposes, though semi-quantitative methods were attempted [18F]FMISO in colorectal was not recommended and our finding suggest the same [18F]FDG PET has been useful in the management of rectal cancer as a marker of glucose metabolism but not hypoxia directly and therefore is not discussed here.FD18F]FMISO was improved using enema, while other problems may not be currently solvable, for example, the spill-in from bladder. Given the high activity regions within the pelvis, similar challenges in analysing and interpreting [18F]FMISO uptake may exist for other tumours, such as cervix and prostate cancer. Frusemide may be considered to aid rapid urinary excretion and dilute the residual radioactivity in the bladder. Another  solution may be to keep patients well hydrated and request bladder emptying immediately before the PET scan. However, for target delineation purposes, it is preferred that the bladder has the same size and shape as on scans acquired for radiotherapy planning i.e. ‘comfortably full’ for the registration algorithm to work well. Since CT is unable to provide a good contrast in the soft tissue within the pelvis, PET-MR may be potentially beneficial albeit controversial due to the lack of attenuation correction in the bone.Since the TRG scores were obtained in 8 patients and clinical response in 3 patients who did not undergo surgery, the conclusions drawn from this study are limited by the small number of subjects. The acceptability of procedures to patients and clinical implementation is a major challenge in clinical imaging trials. Despite these challenges, some problems are solvable, for example, the spill-in from non-tumour accumulation of [18F]FMISO PET scanning for rectal cancer. Preliminary data does not support the hypothesis that a reduction in [18F]FMISO uptake is predictive of clinical response. There are two principal problems namely spill-in from non-tumour activity in the rectum and from bladder into the surrounds that encompass tumour. Careful consideration should be given to PET acquisition and reconstruction to minimise spill-in counts from the bladder. This preliminary study has shown fundamental difficulties in interpretation of [18F]FMISO PET for rectal cancer, limiting clinical applicability.In conclusion, this pilot study with small datasets revealed significant challenges in delivery and interpretation of [


Interestingly, and somewhat counter-intuitively, anti-angiogenic therapy can promote CD8+ T-cell infiltration of tumours and increase the efficacy of adoptive CD8+ T-cell therapy. Rather than inhibit tumour angiogenesis, anti-angiogenic therapy ‘normalizes’ (matures) tumour blood vessels by promoting pericyte recruitment, increasing tumour blood vessel perfusion and sensitizing tumour blood vessels to inflammatory stimuli. A number of different approaches are currently being explored to increase recruitment by manipulating the expression of homing-associated molecules on T-cells and tumour blood vessels. Future studies should address whether these approaches improve the efficacy of adoptive T-cell therapies for solid, vascularized cancers in patients.The success of adoptive T-cell therapies for the treatment of cancer patients depends on transferred T-lymphocytes finding and infiltrating cancerous tissues. For intravenously transferred T-cells, this means leaving the bloodstream (extravasation) from tumour blood vessels. In inflamed tissues, a key event in extravasation is the capture, rolling and arrest of T-cells inside blood vessels which precedes transmigration across the vessel wall and entry into tissues. This depends on co-ordinated signalling of selectins, integrins and chemokine receptors on T-cells by their respective ligands which are up-regulated on inflamed blood vessels. Clinical data and experimental studies in mice suggest that tumour blood vessels are anergic to inflammatory stimuli and the recruitment of cytotoxic CD8 Retrospective studies of resected primary tumours from colorectal cancer patients were the first to show that the presence and location of tumour-infiltrating T-lymphocytes are prognostic for patient outcome . Specifiin vitro yet the same cells fail to eradicate cancer in the patient due to tumour-induced immunosuppression. Early attempts to overcome immunosuppression included isolating tumour infiltrating lymphocytes (TILs) from resected melanoma lesions, expanding tumour-reactive T-cells and infusing large numbers back to patients with progressive metastatic melanoma [gm) to overcome dominant immunosuppression in the cancer patient [Tumour-specific T-cells isolated from cancer patients recognize and kill tumours melanoma . These g patient . The rec patient –9. Howevgm) injected into the bloodstream are recruited into cancerous tissues to bring about cancer cell killing  surface of blood vessel endothelial cells (EC) that directs entry into tissues. Homing addresses for lymph nodes and sites of inflammation in the skin and other organs have been identified . The congm T-cells. For example, transduction of CAR T-cells with chemokine receptors for human tumour cell-derived chemokines increases homing to solid cancers and controls their growth in immunodeficient mice [Some of the adhesion molecules and chemokines that regulate T-lymphocyte homing into sites of inflammation and/or lymph nodes have been implicated in homing to cancers in experimental mice –25. Knowent mice ,27.+ T-lymphocytes do not spontaneously roll or undergo activation induced arrest in tumour blood vessels even following LPS administration [+ T-cell flux through tumour blood vessels, absence of shear stresses necessary for recruitment and/or inefficient chemokine presentation could all contribute to poor CTL recruitment. Additional mechanisms include tumour-derived products which restrict expression of homing molecules, such as ICAM-1, on tumour EC [Early studies of leucocyte (neutrophil) behaviour inside blood vessels of subcutaneous tumours in immunodeficient mice demonstrated lower levels of rolling and arrest than in normal skin, even after administration of tumour necrosis factor-α (TNF-α) or LPS, which suggested that tumour blood vessels are anergic to inflammatory stimuli ,29. Subsstration . The facstration ,30. A nuumour EC  and induumour EC . EC anerumour EC .gm T-cell therapy for solid tumours is cell dose dependent [gm T-cells recognizing relevant tumour antigens will be required before applying the rules governing tumouricidal T-cell homing in transplanted syngeneic or xenogeneic tumours to human cancers.Adoptive T-cell therapy does restrict the growth of solid vascularized cancers in experimental mice and in patients so, if tumour blood vessels are poor at recruiting injected T-cells, how does it work? Intravital imaging has demonstrated that tumour specific T-cells are recruited, not from blood vessels within the tumour nest but, from blood vessels in the peritumoural area where homing-associated adhesion molecules are preferentially expressed  Figure . Recruitependent ,38 so un111indium-labelled TILs homed to melanoma lesions and TIL homing was associated with objective clinical responses in 38% (10/26) patients [+ T-cells traffic indiscriminately to lymph nodes, liver, spleen and lungs in tumour-bearing mice while still being able to control tumour growth [There have been very few clinical studies of T-cells following injection into the venous circulation of cancer patients. However, an early study showed that patients . In a phpatients . These rpatients , which, patients . In exper growth . This figm may, however, contribute to their lack of persistence which has been repeatedly reported in patients and experimental mouse models. This may be due to lack of encounter with cognate antigen following homing to non-cancerous tissues and/or lack of homing to lymph nodes where survival and/or reactivation signals are presented. Inclusion of CD28 or other co-stimulator signalling domains in so-called second and third generation CARs prolongs the persistence of CAR T-cells but whether this is related to lymph node homing remains to be determined. Checkpoint blockade inhibitors and host-conditioning using fludarabine and cyclophosphamide increase the efficacy of TILs, CAR and TCRgm T-cells but whether this is related to altered homing is also not known [Indiscriminate homing of CAR T-cells, TILs and TCRot known –45. Howeot known .+ regulatory T-cells (Tregs) [It is over 40 years since Judah Folkman first proposed that tumour growth could be restricted by blocking the growth of blood vessels that supply the tumour . Since t (Tregs) . The use (Tregs) . Vascula (Tregs) ,51. Treg (Tregs)  but a ra (Tregs) ,54, eosi (Tregs) , irradia (Tregs)  and TLR  (Tregs) , in the  (Tregs) ,32. Ther+ T- and/or NK cell secretion of LTα3 and IFN-γ which induce expression of peripheral node addressin  and the arrest chemokine CCL21 respectively. Although comprising <10% of the tumour vascular network, newly formed HEV in mice recruit adoptively transferred naïve T-cells directly from the bloodstream into the tumour where cross-presenting APC activate naïve T-cells to kill tumour tissue [gm T-cells which are predicted to have superior efficacy in adoptive cell therapies.An exquisite example of tumour blood vessel maturation is the development (neogensis) of specialized HEV blood vessels. Studies in mice suggest that, similar to tumour blood vessel normalization, HEV neogenesis is regulated by type 1 inflammation  and can r tissue . The devr tissue . Interesr tissue , as oppor tissue  which suAlthough the promise of CAR T-cell therapy for the treatment of solid cancers has yet to be realized, it is clear that adoptive immunotherapy using T-cells expressing conventional TCRs does not work for all patients. Inefficient recruitment by immature tumour blood vessels and lack of persistence of injected T-cells are important determinants of successful outcomes in experimental mice. Manipulating homing addresses on T-cells and vascular addresses on tumour blood vessels to facilitate the recruitment of tumour-specific T-cells offer a combined approach to overcome these limitations in cancer patients. However, longitudinal imaging of transferred T-cells using non-invasive imaging using PET/SPECT tracers will be required to determine whether homing and persistence of transferred T-cells correlate with therapeutic outcomes.


Paediatric brain tumours have a high mortality rate and are the most common solid tumour of childhood. Identification of high risk patients may allow for better treatment stratification. Magnetic Resonance Spectroscopy (MRS) provides a non-invasive measure of brain tumour metabolism and quantifies metabolite survival markers to aid in the clinical management of patients. Glycine can be identified using MRS and has been recently found to be important for cancer cell proliferation in tumours making it a valuable prognostic marker. The aims of this study were to investigate glycine and its added value to MRS as a prognostic marker for paediatric brain tumours in a clinical setting.116 children with newly diagnosed brain tumours were examined with short echo-time MRS at the Birmingham Children’s Hospital and followed up for five years. Survival analysis was performed using Cox regression on the entire metabolite basis set with focus on glycine and three other established survival markers for comparison: n-acetylaspartate, scyllo-inositol and lipids at 1.3 ppm. Multivariate Cox regression was used in conjunction with risk values to establish if glycine added prognostic power when combined to the established survival markers.p < 0.05) and correlated with tumour grade (p < 0.01). The addition of glycine improved the prognostic power of MRS compared to using the combination of established survival markers alone.Glycine was found to be a marker of poor prognosis in the cohort (Tumour glycine was found to improve the MRS prediction of reduced survival in paediatric brain tumours aiding the non-invasive assessment of these children. Paediatric brain tumours are the most common solid tumour of childhood and the largest cause of cancer death in that patient group. Identification of high risk patients may allow for better treatment stratification and more effective disease management. Treatment may include surgical resection, radiation therapy and chemotherapy, with specific protocols based on tumour diagnosis and clinical risk factors including age at diagnosis, histopathological and molecular features and radiological imaging features . DespiteMagnetic Resonance Spectroscopy (MRS) provides a non-invasive measure of brain tumour metabolism, which can identify and quantify metabolite survival markers to aid in the clinical management of patients . MRS hasGly is a conditionally essential amino acid, which has several physiological roles including as an inhibitory neurotransmitter in the central nervous system, cytoprotection, protein synthesis, extracellular structural support and an important role in the one-carbon metabolic cycle . NotablyAlthough histology remains the gold standard for diagnosis, not all brain tumours are surgically resected or biopsied and non-invasive MRS has proven valuable in the clinical management of these patients . MetabolThe brain tumour cohort consisted of 98 patients after quality control criteria were applied, with 18 removed during the quality control process. The patient cohort consisted of 81 biopsied tumours and 17 unbiopsied tumours. There were 59 survivors (60%) and 39 non-survivors by the end of the 5 year follow up period. All patient deaths were as a result of their disease. The mean age (±SD) of the patient cohort at diagnosis was 7.51 (±5.4) years, and 71% of patients were male. Age and gender were not found to be predictive of survival in this cohort with log-rank values of 1.2 and 0, respectively. The patient tumour type distribution with associated frequency and deaths within the 5 years is shown in Table p < 0.03) in the patient cohort based on a univariate Cox regression analysis and a concentration cutoff of 0.55 mM  . Example spectra from a medulloblastoma patient case highlight high Gly levels with the associated poor prognosis of the patient .Gly was found to be a predictor of poor prognosis . An example long echo spectrum and a graph of the Gly concentration correlation between echo times is shown in To validate the Gly concentrations a comparison between matched short and long echo-time MRS was conducted and a strong correlation was found . This result was also seen in the Kaplan Meier curves based on risk values, where larger separation of the high risk and low risk groups was seen in the analysis with Gly included , diffuse astrocytoma and germinoma tumour types was not well predicted through relative risk alone. Observing individual results, MRS relative risk in these tumour types only predicted the correct prognosis in 2 out of 9 DIPG patients, 2 out of 4 diffuse astrocytoma and 1 out of 3 germinoma patients. The high grade tumour group had a high MRS relative risk and low grade tumour group had a low MRS relative riskMean MRS relative risk for tumour types with This study has established that tumour Gly concentration, measured non-invasively by MRS, is a prognostic marker in a cohort of paediatric brain tumour patients. A higher concentration of Gly was significantly associated with reduced survival, indicating that Gly is a marker of poor prognosis across a wide range of paediatric brain tumours. Furthermore, the addition of Gly to the existing set of metabolites previously associated with survival in brain tumours improved the overall prognosis prediction potential of MRS. We also detected a significant correlation between tumour grade and Gly concentration in the smaller cohort of cases where the tumour was biopsied. Although these results agree with prior work of Gly concentration as a marker of malignancy , 19–23, ex vivo colorectal cancer tissue has been the only study to date to link Gly concentration measured by MRS with adverse prognostic outcome [Recent evidence suggests that Gly has a particularly important role in cancer metabolism perhaps explaining why Gly concentration is higher in more aggressive tumours. Gly is particularly linked to the one-carbon metabolic cycle whereby non-essential amino acids donate carbon units to produce complex metabolic reactions within folate, methionine and transsulfuration pathways including regulation of cell biosynthesis, redox status and epigenetic status , 24, 25. outcome .in vivo short echo-time MRS at 1.5T correlate with Gly levels determined from ex vivo tissue samples and other in vivo methods [The detection of Gly by MRS is potentially hindered owing to the spectral overlap with Myo-Insositol ( M-Ins). However, literature reports provide evidence that Gly in tumours can be reliably detected using the methods applied in this study. , 28. M-I methods , 28. The methods . Medullo methods , 11, 28.It was also important to establish the added value of Gly to the overall survival prediction of metabolite markers measured by MRS. We found that NAA, S-Ins and TLM 1.3 ppm were individual markers of prognosis in this cohort, which is consistent with prior results reported in paediatric brain tumours , 29. GluS-Ins has been previously associated with poor prognosis in paediatric brain tumours, however, its role in tumour metabolism is not well known, warranting further investigation , 31. LipThere is clinical interest in establishing non-invasive markers of prognosis in individual tumour types and the prediction of individual outcome. In this study this was preliminarily investigated through the MRS derived relative risk value. The high grade grouping of tumours had a significantly greater relative risk value than the low grade tumour group. Looking at more specific tumour types, the relative risk value was good at predicting prognosis for most tumour types, but performed the best for groups that consisted of high numbers of patients with either good or poor survival, e.g. pilocytic astrocytoma and atypical teratoid rhabdoid tumours. The exceptions to this provide an interesting insight into the information provided by MRS. Diffuse astrocytoma and DIPG tumour groups progress to a higher grade prior to death and may evolve to a more aggressive MRS profile during tumour progression . GerminoNon-invasive measures of diagnosis and prognosis in brain tumours are becoming increasingly important. This is particularly evident given that approximately a quarter of paediatric brain tumours are not biopsied and therefore receive no histological diagnosis. In this instance, metabolites can provide additional clinical information non-invasively. It is important to note that MRS has been successfully used to discriminate relapse from treatment related effects, to non-invasively identify tumour type and to provide additional molecular genetic information to aid in clinical management , 37, 38.In conclusion, Gly was found to be a marker of poor overall survival using non-invasive MRS in paediatric brain tumours. This is an important finding as Gly has been recently found to be vital for cancer cell proliferation in tumours. The addition of Gly to other reported metabolite survival markers improved the prognosis prediction potential of MRS, thus, improving MRS as a tool for aiding in the clinical management of brain tumour patients.116 children with newly diagnosed brain tumours were examined with MRS prior to treatment as part of their clinical investigations at the Birmingham Children’s Hospital. The accrual period was between September 2003 and July 2009, with all patients followed up for five years. The cohort overlaps with that from a previously reported study and so should not be considered a an independent validation of the biomarkers reported in that study . Rather MRI and MRS were acquired pre-treatment on a 1.5T Siemens Symphony Magnetom with a single-channel head coil and 1.5T GE Signa Excite scanner with an 8-channel head coil. MRI included standard T1- and T2-weighted images of the brain, diffusion-weighted imaging followed by gadolinium contrast administration and then further T1-weighted imaging. MRI was utilised to provide anatomical reference to the primary tumour and measure contrast enhancement within the tumour. The MRS voxel was placed within the solid component of the primary tumour measuring 1.5 or 2 cm sided cube depending on the size of the tumour. PRESS (Point Resolved Single Voxel Spectroscopy) localisation was used with a repetition time of 1500 ms and an echo-time of 30 ms. Fifteen patients had matching long echo-time (135 ms) MRS data, which was analysed as a validation method for the Gly fit. Water suppressed data were acquired with 256 or 126 repetitions, depending on voxel size for signal averaging. Water unsuppressed data were additionally taken as a metabolite concentration reference. MRS data were transferred from the scanner to a PUKKAJ PACs system (version 6.6.1) used for storage of research and advanced MRI data.Unprocessed MRS signals were analysed using the TARQUIN  softwareIndividual spectra were inspected by two expert spectroscopists blinded to the survival data and excluded if they did not meet one or more of the quality control criteria. These were displaced voxel location (too close to lipid containing structures), unstable spectral baseline, presence of spectral artifacts, signal-to-noise ratio (SNR) <5, overall metabolite linewidths >0.15 ppm and water linewidth >10 Hz. Voxels containing more than 10% of uninvolved brain as assessed by the conventional MRI were excluded from the analysis.p < 0.05) had their metabolite concentration cutoff optimised iteratively based on the lowest p-value. Gly and established metabolite survival markers, TLM 1.3 ppm, NAA and S-Ins were additionally optimised in the same manner. Metabolite concentrations for all tumours were calculated, and mean values for each tumour diagnostic group where n > 2 are shown in Table n < 2, were not shown in Table All metabolites in the basis set were utilised to test whether they were markers of prognosis through a univariate Cox regression, log-rank test and Kaplan–Meier analysis using a median metabolite concentration cutoff. Metabolites that were found to be significant (MRS provides multiple markers of tumour prognosis, hence while establishing new individual markers is of clinical interest the combination of markers may provide more predicting power. Therefore, the added value of Gly to the survival prediction of MRS was assessed by combining established survival markers with Gly included or excluded from a multivariate Cox regression analysis. Risk prediction analysis, using the multivariate Cox regression model, was performed to assess the MRS prognosis prediction, with and without Gly included. This was visualised through Kaplan–Meier survival curves using median risk score cutoffs.p-value of < 0.05 was deemed significant in all tests.Risk analysis, was also performed to assess the ability of MRS as a whole to predict individual tumour type prognosis Table . Surviva


Scaffolds for regenerative medicine applications should instruct cells with the appropriate signals, including biophysical stimuli such as stress and strain, to form the desired tissue. Apart from that, scaffolds, especially for load-bearing applications, should be capable of providing mechanical stability. Since both scaffold strength and stress–strain distributions throughout the scaffold depend on the scaffold’s internal architecture, it is important to understand how changes in architecture influence these parameters. In this study, four scaffold designs with different architectures were produced using additive manufacturing. The designs varied in fiber orientation, while fiber diameter, spacing, and layer height remained constant. Based on micro-CT (μCT) scans, finite element models (FEMs) were derived for finite element analysis (FEA) and computational fluid dynamics (CFD). FEA of scaffold compression was validated using μCT scan data of compressed scaffolds. Results of the FEA and CFD showed a significant impact of scaffold architecture on fluid shear stress and mechanical strain distribution. The average fluid shear stress ranged from 3.6 mPa for a 0/90 architecture to 6.8 mPa for a 0/90 offset architecture, and the surface shear strain from 0.0096 for a 0/90 offset architecture to 0.0214 for a 0/90 architecture. This subsequently resulted in variations of the predicted cell differentiation stimulus values on the scaffold surface. Fluid shear stress was mainly influenced by pore shape and size, while mechanical strain distribution depended mainly on the presence or absence of supportive columns in the scaffold architecture. Together, these results corroborate that scaffold architecture can be exploited to design scaffolds with regions that guide specific tissue development under compression and perfusion. In conjunction with optimization of stimulation regimes during bioreactor cultures, scaffold architecture optimization can be used to improve scaffold design for tissue engineering purposes. In the field of tissue engineering, a scaffold can be used to provide a temporary or permanent support structure for cells to form a tissue. In the specific case of musculoskeletal regeneration, a general tenet is the need for a scaffold design capable of withstanding load-bearing forces. From literature, it is known that scaffold architecture influences the apparent stiffness of the scaffold  is generally not able to capture the stress and strain distribution in actual scaffolds due to variations in scaffold architecture inherent to most fabrication processes  was used to produce scaffolds with four different scaffold designs. AM ensures a high controllability and reproducibility of the scaffold architecture, so that fiber diameter, fiber spacing, and layer thickness were constant across the different designs. By changing the angle of layer deposition and shifting layers laterally, different architectures were obtained. Most notably, designs where the layers were not shifted laterally contained crossing fibers at the same position in each layer. This created vertical supporting columns for the load. In designs where layers were shifted laterally, the locations of crossing fibers in successive layers varied, resulting in the absence of supporting columns. In order to investigate the effect of scaffold architecture on stress and strain distribution and subsequently cell differentiation stimulus prediction, μCT-based models of the scaffolds were prepared, and stress and strain distributions within the scaffolds were predicted using computational fluid dynamics (CFD) and FEA.a priori in silico could accelerate the design of scaffolds and optimize the loading regimes for tissue engineering purposes.The goal of this study was to investigate the influence of additive manufactured scaffold architecture on the distribution of surface strains and fluid flow shear stresses within the scaffold, and subsequently the expected cell differentiation. The results show a distinct effect of the scaffold architecture on surface strains and fluid flow shear stresses under mechanical compression and imposed fluid flow. As reported before, the octahedral shear strain magnitudes exceed the surface shear strain magnitudes, which are reflected in the cell differentiation stimulus values on the scaffold surface. The results of the study show that regions of the scaffold could be designed favoring specific cell differentiation stimuli. Coupling with biophysical loading regimes Scaffolds were fabricated by a bioscaffolder , as previously described  in a custom designed holder with which a fixed compression can be applied, as previously described  and segmented with the material as scaffold and the void as fluid volume. Remeshing was applied to improve the quality of the meshes.X-direction or the Y-direction along the Y-axis or X-axis, respectively, to prevent rotation of the FEM while allowing for lateral expansion.Linear elastic and isotropic material properties of 300PEOT55PBT45 were taken from literature, for which a bulk Young’s modulus of 88 MPa  with a fluid velocity of 100 µm/s. The fluid was modeled as an incompressible Newtonian fluid based on culture medium properties, with a density of 1,000 kg/mFor the validation between the scanned and simulated compression of the scaffold, the deformed mesh of the FEM from the complete scaffold was exported. In Mimics, the meshes were superimposed for their fit and visually assessed for their similarities.A custom-made MATLAB script identified the shared surface faces of the CFD and FEA models. A Fortran subroutine with user code was written and executed during an FEA simulation by Marc Mentat to calculate the surface strains; the strains of the element face lying on the surface of the material FEM  thresholds used for cell differentiation were 0 ≤ S < 0.001 for resorption, 0.001 ≤ S < 1 for bone, 1 ≤ S < 3 for cartilage, 3 ≤ S < 6 for fibrous tissue (FBT), and S ≥ 6 for necrosis . However, with the octahedral shear strain, besides more cartilage (51%), also necrotic tissue was predicted (2%) Figure . The 0/4In tissue engineering, scaffolds designed for load-bearing applications should provide an environment that is capable of withstanding the forces that arise during the daily activities of a patient. In addition, the scaffold should provide differentiation stimuli to cells to ensure optimal tissue development. An important stimulus in this aspect is the dynamic mechanical environment that a scaffold can offer to the cells residing on the scaffold (Martin et al., Using a device previously reported by Baas and Kuiper , scaffolUpon mechanical compression of the scaffolds, strains mainly developed at the crossing of fibers. Both the magnitude of the strain and the surface area affected by the mechanical compression were influenced by the scaffold architecture Figure . In the In addition to strain values, the results of fluid flow analysis also depended on the pore shape Figure . In all In order to determine the effect of different stress and strain distributions on the potential cell differentiation in the initial stage of cell attachment and differentiation on the surface of a tissue engineering scaffold, an adaptation of the mechano-regulation theory of Prendergast was used. The original theory combines octahedral shear strains with fluid flow shear stresses to predict cell differentiation (Lacroix et al., The higher shear strain values seen from the mechanical compression at the crossing of the fibers resulted in the prediction of more cartilage formation and in some cases FBT at these locations. Large differences could be seen between the different scaffold architectures, depending on the strain magnitudes that were developed in the scaffolds. The influence of fluid flow on cell differentiation prediction correlated with fluid shear stress distributions. For the 0/90 offset architecture, the higher fluid shear stresses resulted in a larger amount of cartilage prediction, while predicted cartilage formation was lowest for the 0/90 architecture. The results from this study correlate well with a study by Olivares et al. . At an iThe combination of fluid flow and mechanical compression on cell differentiation stimulus prediction showed that the scaffold architecture had an effect on the relative contribution of both mechanical stimuli on cell differentiation stimulus values. Even though Figure In this study, different software packages were used for the CFD and FEA simulations. Therefore, the effect of compression-induced fluid flow, as well as possible scaffold deformation due to fluid flow, cannot be taken into account. Although the fluid flow is low and can probably be neglected as a factor for scaffold deformation, the compression-induced fluid flow is likely to have a significant effect on cell differentiation predictions. When a total compression of 10% is applied, it is not unrealistic to assume that a significant fluid flow will occur due to this compression. Ideally, a coupled fluid-structure interaction model should be developed in order to address this aspect. However, these models are computationally very demanding, and it may be difficult to devise such a model for the complex geometries used in this study.Finally, it should be noted that the scaffold architecture, and specifically the orientation of subsequent layers in scaffolds that are acquired using 3D fiber deposition, can have an effect on MSC cell differentiation even without external compression or fluid perfusion. A recent study by Di Luca et al.  shows thThe results of this study show that the scaffold architecture has a significant influence on stress and strain distributions. In particular, removing the possibility of load bearing with solid structures spanning from the top to the bottom of the scaffold, or even reducing the amount of these structures in a scaffold, has a great effect on the strain distribution. Additionally, the different architectures result in differences in the effective pore size and shape, which subsequently influence the fluid shear stress distribution. Therefore, by only changing the angle of orientation, varying stress and strain distributions can be exploited to design scaffolds for specific applications. Although in this study the material properties are based on the polymer PEOT/PBT, this methodology can be used for different biomaterials with different mechanical properties. The possibilities to design a scaffold with desired stress and strain distribution are therefore vast. Coupling these models with bioreactor studies to experimentally determine cellular differentiation within scaffolds subjected to specific fluid perfusion and compression regimes can provide valuable insights in the design of scaffolds and culture conditions for osteochondral, bone and cartilage replacements.WH, YY, LM, and JR designed the experiments. WH and AD conducted the experiments. WH, LM, and JR analyzed data and wrote the manuscript. All the authors discussed the results and revised the manuscript.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.


Although midurethral mesh tape slings are considered the standard of care in the treatment of female stress urinary incontinence (SUI), complications such as pain, dyspareunia, or erosion are known to occur in addition to persistent incontinence. The management of these types of mesh sling complications can be very complex, especially when the pain is not just isolated to the vagina but extends into other areas, such as the abdomen which requires a much more extensive dissection. Additionally, if a mesh sling needs to be removed, the patient will most likely have a return of her SUI that often necessitates subsequent treatment. Vaginal and/or laparoscopic removal or revision of mesh tape slings should be considered in patients presenting with complications such as vaginal pain, abdominal pain, dyspareunia, or urinary obstructive symptoms. In those patients who demonstrate persistent SUI, concomitant laparoscopic Burch urethropexy can be considered and can safely be performed at the time mesh removal. In this case report we present a patient who required a dual-approach removal of two painful midurethral slings in addition to concomitant treatment of persistent SUI with a laparoscopic Burch urethropexy procedure. This is due in large part to their ease of application, minimally invasive approach, and comparable outcomes to other more involved procedures such as Burch urethropexy , 2. Howelved SUI , 7. Addilved SUI . Often wApproximately 50% of women who present with symptomatic sling complaints require surgical management after failure of less invasive methods –10. WhenIn the present case we describe the successful treatment of recurrent SUI in a patient requiring vaginal/laparoscopic removal of two midurethral slings with concomitant laparoscopic Burch urethropexy.A 50-year-old postmenopausal para 3 woman with a prior single-incision minisling  and subsequent retropubic  sling presented with complaints of severe dyspareunia and chronic pain. On initial interview, she also reported persistent refractory stress urinary incontinence (SUI). Her past medical history was significant for depression, anxiety, and hypertension for which she was taking a low dose of hydrochlorothiazide. Her other prior surgeries included a cholecystectomy and tonsillectomy. She admitted to a 32-pack-year smoking history and had a BMI of 34. She had never been on hormone-replacement therapy and was not currently sexually active secondary to pain.Her prior clinical records and operative reports were obtained. Six years before, she had a total abdominal hysterectomy, posterior colporrhaphy, and single-incision minisling for SUI using the standard technique. However, she continued to report persistent SUI and five years after her initial sling placement she received a retropubic (RP) TVT sling placed via the standard technique. Postoperatively, she complained of urinary retention and obstructive voiding, and the RP sling was transected in the midline with relief of some of her obstructive symptoms one month after placement.Approximately 4 months following her RP sling insertion, she began to experience notable diffuse pelvic/perineal pain localized to the periurethra, groin, right and left lower abdominal quadrants, and suprapubic area. She also experienced dyspareunia as well as persistent mixed urinary complaints. Her specific urinary symptoms included urinary urgency/frequency, nocturia, difficulty emptying her bladder, and stress urinary incontinence. She also reported anal incontinence with need for splinting for defecation. Her preoperative IIQ-7 score was 52.3 and the UDI-6 score was 54.2.Physical examination revealed contraction and banding of the mesh tape sling(s) bilaterally in the periurethral area. Palpation of the mesh reproduced her pain, with radiation to her groin and up into her lower abdomen, right greater than left. There was no evidence of mesh extrusion. Her exam also diagnosed a stage II anterior and posterior wall prolapse without significant tissue atrophy. She had pronounced urethral hypermobility and a positive standing cough stress test. Urinalysis was negative with prior urodynamic testing showing urodynamic SUI without significant detrusor overactivity or impaired voiding and a normal bladder capacity. Her ICS-IUGA mesh complication score was 1Be/T4/S2, S4 .Based on the patient's history and physical examination it was recommended that she undergo vaginal removal of the single-incision sling and vaginal and laparoscopic removal of the RP mesh tape sling with concomitant laparoscopic paravaginal repair, posterior repair, and Burch urethropexy. All procedures risks, benefits, cure rates, complications, and alternatives to surgery were discussed in detail with the patient. She agreed to the plan of care and informed consent was obtained.Under general anesthesia, a suburethral vertical incision was made and the vaginal epithelium was dissected laterally off the underlying urethra and periurethral tissues. The arms of the RP sling had retracted bilaterally; therefore the SIS was immediately visible. It was carefully dissected away from the urethra, cut in the midline, and then dissected out laterally to its entry into the obturator internus muscle bilaterally. The entire SIS sling was removed intact including its self-fixating tips. The defects in the muscle and fascia were then closed with absorbable polyglycolic acid suture. The edges of both arms of the retropubic sling were identified bilaterally and then dissected up towards the pubocervical fascia and the retropubic space without entry into the space. They were tagged for later identification and laparoscopic extraction .Attention was turned to the abdominal portion of the procedure and open laparoscopy was completed through the umbilicus followed by the addition to two lateral ports and a suprapubic port. The abdomen was inspected and adhesiolysis of rectosigmoid adhesions was performed without traumatic injury to the bowel. The bladder was retrograde filled in order to visualize the superior edge of the bladder and reduce risk of injury. The vesicoperitoneal reflection was identified and the peritoneum was incised 3 cm above the border extending between the obliterated umbilical ligaments. The space of Retzius was bluntly developed until reaching the pubic symphysis. The bladder was then drained and blunt dissection was continued down into the retropubic space until reaching the pubocervical fascia. The arms of the RP sling entering through the pubocervical fascia were identified and were followed to the anterior abdominal wall on each side of the pubic symphysis . Using tHer anterior wall prolapse and paravaginal defects were then addressed using interrupted placement of 2-0 nonabsorbable multifilament sutures for a bilateral paravaginal defect repair, which secured the pubocervical fascia back to the arcus tendineus fascia pelvis bilaterally with the intent to restore her anterior anatomy prior to the Burch urethropexy.©) figure-of-eight sutures, placed bilaterally 1 cm lateral to the midurethra and at the level of the urethrovesical junction. The sutures were placed through the pubocervical fascia and carried up to Cooper's Ligament. They were tied to create appropriate bridging and tension without kinking the urethra (Figures Burch urethropexy was then performed laparoscopically using CV-2 nonabsorbable polytetrafluoroethylene (Gore-Tex Figures  and 5. CThe patient was discharged on postoperative day one after successfully completing a voiding trial following removal of the Foley catheter and vaginal packing. At the time of discharge, she was given instructions for regular voiding and adequate fluid intake to prevent urinary retention. She was seen three months after surgery in the clinic and denied any further abdominal/pelvic/groin pain. In the interim, she had been treated for candida vaginitis and started on estradiol vaginal cream. She denied any symptoms of vaginal bulge, obstructive voiding, or SUI. Her postoperative IIQ-7 score was 9.5 and UDI-6 score was 25, meeting the clinically important difference cutoff or MCID . The patMidurethral mesh tape slings have become a popular surgical option for stress urinary incontinence, due in large part to their ease of placement and comparable success rates compared to other anti-incontinence procedures such as the Burch urethropexy and the fascia lata slings. Even with recent reports of complications and some of the concern over the vaginal use of mesh, mesh tape slings are still considered the standard of care; they have been found to have an overall 7- to 10-year cure rate of 80% to 95%. However, given the recent public media attention concerning mesh complications, as well as the fact that if a patient has a complication with a mesh tape sling or other previous placed vaginal mesh, she may be hesitant to have more synthetic mesh placed. Unfortunately, for many of these patients, their SUI will persist or worsen after sling removal. As was performed with this patient, one option to treat refractory SUI without utilizing a mesh tape sling is via concomitant laparoscopic Burch urethropexy at the time of sling removal. As we found with this patient and subsequent other cases, performing Burch urethropexy does not increase blood loss or intraoperative complication risk. This is unlike our previous published experience with repeat laparoscopic Burch procedures following previous Burch or MMK. Although successful, we found those procedures to be extremely difficult, with increased risks of complications and blood loss secondary to the increased scar tissue found in the space , 15. BasOur case has a few unique components to highlight. Firstly, she was offered Burch urethropexy due to the persistence of her SUI that had been refractory to two mesh slings. The decision was made to proceed with Burch urethropexy rather than other nonmesh anti-incontinence procedures such as the Marshall-Marchetti-Krantz urethropexy or the fascia lata sling as the Burch has comparatively better success rates and lower risk for complications and postoperative sequelae. Furthermore, as our standard technique to midurethral sling removal involves a combined vaginal and laparoscopic approach with dissection of the retropubic space, the inclusion of a laparoscopic Burch urethropexy at time of surgery would not entail any further dissection given that the space was already open , 16, 17.Although there have been no studies exploring a single-stage sling removal and Burch urethropexy or any other combined anti-incontinence procedure, the general opinion has been to wait at least 6 months following sling removal to allow full healing, restoration of anatomy, and resolution of inflammation. However, this is a theoretical concern without any definitive evidence showing a difference in results. In our clinical practice, the degree of scarring in the RP space has never been severe enough to dramatically distort the anatomy or to cause any acute reactive process to degrade the urethropexy. Additionally, any defects in anterior wall support can be corrected with concomitant paravaginal defect if necessary. Once the paravaginal defect is corrected, the Burch urethropexy can be performed without concern for overcorrection of the paraurethral tissue.Within our practice, indications for removal of a mesh sling include complaints of vaginal or pelvic pain that is reproduced with palpation of the sling, dyspareunia, dysuria, mesh erosion, obstructive voiding, or recurrent urinary tract infections. For retropubic slings, we typically offer to remove the entirety of the sling via a dual-approach surgery. For women with a symptomatic retropubic sling who opt for removal and present with refractory stress leakage without a concomitant voiding dysfunction, we offer a laparoscopic Burch urethropexy at the time of sling excision immediately following laparoscopic removal of the abdominal portion of mesh. However, for patients with transobturator slings, a laparoscopic approach is rarely indicated. Patients with a symptomatic transobturator sling and stress leakage are also offered laparoscopic Burch urethropexy at the time of sling excision. However, these patients are counseled that this would involve a separate laparoscopic procedure following removal of the vaginal portion of the transobturator mesh.In general, prior to removing a symptomatic mesh sling, options should be provided to patients to manage stress urinary incontinence either at time of removal or at a later date. Although a repeat operation with another midurethral sling has a higher complication rate, it is commonly performed in the setting of refractory SUI and should be provided as an option to patient who would like surgical management. However, the provider must expect many patients to be hesitant to agree to this and therefore must be prepared to discuss other alternatives as well as potential complications of any approach. Laparoscopic Burch urethropexy has been an effective treatment for refractory SUI in the setting of previous failed midurethral slings that have not required removal. Based on this knowledge and reassuring results as found in this case and others like it, providers should consider concomitant Burch urethropexy for persistent SUI as a possible option for anti-incontinence at the time of sling removal.


A recent review article suggests that elimination of infused crystalloid fluid might occur faster in females than in males. To study this question, a population kinetic analysis was performed to compare the turnover of buffered Ringer’s solution when infused at different rates in males and females.Data were retrieved from seven series of experiments where 44 intravenous infusions of Ringer’s acetate had been given to female volunteers and 67 to male volunteers. Frequent measurements of the blood hemoglobin and the urinary excretion were used as input in a kinetic two-volume model with micro-constants and covariates, using a nonlinear mixed effects software. The key outcome measure was the rate of irreversible elimination of infused fluid, which was expressed as the half-life, obtained as the excreted urine divided by the modeled plasma volume expansion over time.The half-life amounted to 24 min  in the females and 38 min (33–42) in the males. The urinary excretion differed somewhat less than suggested by these figures during the experimental period (3–4 h) because the plasma volume became less expanded in the females. This was due to that fluid that had been distributed peripheral tissues (the interstitium) returned slightly more slowly to the central fluid space (the plasma) in the females. Gender did not serve as a statistically significant covariate to other rate constants in the kinetic model.The half-life of infused Ringer’s acetate was 60 % longer in healthy male volunteers than in female volunteers.The online version of this article (doi:10.1186/s13293-016-0105-7) contains supplementary material, which is available to authorized users. Males and females have always been assumed to handle infusion fluids in the same way. However, males have a 5–10 % higher content of body water than females –4. PooliTo explore this issue in better detail, the present study estimated the rates of distribution and elimination of infused Ringer’s acetate in both male and female volunteers by a population kinetic analysis that allowed gender to be included as a covariate . PopulatThis study was based on 111 strictly controlled infusion experiments from 7 studies in which Ringer’s acetate had been administered by intravenous infusion to 70 healthy volunteers with normal serum creatinine. Details on the cohorts are given in Table All subjects gave their approval for participation after being informed about each study’s purpose. The experiments started between 7 and 9 AM in an appropriate facility at Karolinska University Hospital at Huddinge, Söder Hospital in Stockholm, or the University Hospital in Linköping, Sweden. The subjects had been allowed to ingest one glass of fluid and one sandwich before coming to the hospital; exceptions were one study in which the females were in the fasting state  and anotThe volunteers rested for 30 min on a bed to reach a hemodynamic steady state. A cannula was placed in the cubital vein of each arm, one for blood sampling and the other for infusion of Ringer’s acetate. Volunteers were covered in blankets to ensure good thermal comfort. The arm used for blood sampling placed on a body-warm heating pad.The plasma volume was expanded infusing Ringer’s acetate solution intravenously, using infusion pumps . The fluid had the following ionic composition (in mmol/l): sodium 130, potassium 4, calcium 2, magnesium 1, chloride 110, and acetate 30 . During and after these infusions, venous blood (4 ml) was withdrawn to measure the hemoglobin (Hb) concentration and the hematocrit (Hct) on the apparatus used for routine measurements in the hospital’s Clinical Chemistry Laboratory, which was either a Technicon H2  or a Cell-Dyn Sapphire . The samples were withdrawn in a standardized manner, and duplicate samples collected at baseline ensured a coefficient of variation (CV) of about 1 %. A small discard volume of blood was drawn before each blood collection to preclude an admixture of rinsing solution, and 2 ml of 0.9 % saline was then injected to prevent clotting. Sampling was always performed every 5 min during the infusion and up to at least 30 min afterward, and then, the sampling was spaced at 10–15-min intervals. The baseline sample was drawn in duplicate, and the mean of the two concentrations was used in subsequent calculations. The subjects voided just before the experiments, and this urine was discarded. The volume of the urine excreted during the study was recorded.The volume kinetic method used is based on repeated measurement of the blood Hb concentration, which is the inverse of the blood water concentration . InfusioThe dependent variable in all experiments was simultaneously fitted to two-volume kinetic model with micro-constants after which the influence of covariates was tested sequentially as guided by a reduction of the residual error.Ro to expand the volume of central body fluid space Vc to vc. The fluid distribution to the peripheral body fluid space Vt was governed by k12 and its return from vt to vc by rate constant k21. The elimination was given by two flows: the urinary excretion was set to the product of the volume expansion of Vc over time and an elimination rate constant, k10, and represents irreversible elimination. In contrast, the second flow representing elimination of fluid from the kinetic system is not irreversible as it probably represents filtration of fluid in continuous (non-fenestrated) capillaries that later returned to the plasma via the lymphatic flow [Vc and another micro-constant, kb  [The Hb-derived fractional plasma dilution was used to indicate the volume expansion of  plasma) . Hence,\Symbols in capital letters denote baseline values. A minor correction was made for the effects of blood sampling on the plasma dilution .Vc, k12, k21, kb, and k10) were estimated by using the Phoenix software for nonlinear mixed effects (NLME), version 1.3 . The sandwich method was employed as the variance estimator.The primary parameters in the model  search routine and the additive error model for the within-subject residual error.The kinetic analysis was performed in two parts. The first part consisted in the development of a P < 0.01) than another curve fit was a reduction of the −2(LL) (log likelihood) by 6.64 points. The base model with the significant covariates added constituted the final model and had the lowest residual error of all runs [The second part of the kinetic analysis consisted in the addition of covariates. Gender, body weight, and rate of infusion per kilo body weight were sequentially tested as covariates to all five parameters in the model. The threshold for accepting the one curve fit as significantly better  reported here was based on the urinary excretion and derived from the following two equations:T1/2 becomes the same between two groups if the urinary excretion and the AUC for the volume expansion are the same or if relative differences between them cancel out so that their ratio is equal.Previous work shows that the model-predicted irreversible elimination correlates closely with the measured urinary excretion, even when examined over 15-min intervals , 14. TheSimulations were performed with Matlab R2012b .Demographic data was reported as the mean (standard deviation), and the kinetic data are reported as the mean . The residual error between the measured values and the computer-simulated (predicted) values were used to describe how well the kinetic model fitted the data. These results were expressed as the median residual error, which shows whether the measured data are systematically higher or lower than the predicted ones , and the median absolute residual error, which reflects the degree of deviation of measured from predicted data .Most volunteers were aged 20–30 years and received Ringer’s acetate at rates ranging between 0.33 and 1.66 ml/kg/min  in the females and 38 min (33–42) in the males.The program derived the half-life of the infused fluid volume as a secondary parameter by using the equation P < 0.001 by Wilcoxon’s matched-paired test).Inclusion of the covariates significantly improved the goodness-of-fit. For the plasma dilution, the mean residual error decreased from −0.007 to −0.001 dilution units and the median absolute residual error from 0.032 to 0.011 dilution units cf. Fig. . The corThe results of the covariate analysis are further illustrated in Fig. The robustness of the model was evaluated by bootstrapping, which result is given in Additional file Simulations based on the optimal estimates of the kinetic parameters Table  were useThe present study was based on more than 100 infusion experiments with Ringer’s acetate in healthy volunteers and allowed a comparison between the rates of irreversible elimination of crystalloid fluid depending on gender. The calculations show that half-life of the fluid was almost 60 % longer in the males. This difference would be reflected directly in the urinary excretion if the plasma volume expansion during the experiments had been the same. However, the plasma volume expansion was less pronounced in the females, which somewhat decreased the difference in eliminated fluid volume within the 3-h study period. The reason was that the females retained distributed fluid in peripheral tissues for a longer period of time  but also on the volume of distribution (Vc) and the rate constants for distribution (k12) and redistribution (k21). Overall, the inclusion of gender as a covariate greatly improved the ability of the kinetic model to predict the input variables, which were plasma dilution and urinary excretion  is a proportionality factor between plasma dilution and volume expansion and would represent the plasma volume if the Hb molecules were evenly distributed in the blood volume.Volume kinetics illustrates how the body handles the infused fluid in a descriptive manner that assumes nonlinear processes. The distribution of infused fluid between body fluid compartments is determined by the four micro-constants, which are inversely proportional to the half-lives of distribution and elimination functions Fig. . The estVt, can be estimated as Vck12/k21 and would then average 3.9 l in the females and 6.3 l in the males. These volumes probably reflect mostly the difference in body weight depending in gender. However, they are still smaller than the physiological size of the interstitial fluid space, which can be explained by that volume kinetics only indicates body spaces that can be expanded by fluid [Vc and Vt requires up to 30 min to be completed, as apparent in Fig. The volume of the peripheral space, by fluid . The reakb/k10 indicates that one third of the “elimination” occurs by this route. The fluid filtered via kb also amounts to one tenth of the fluid that is both distributed and redistributed during the study period (k12), which takes place via fenestrated capillaries in the kidneys and gastrointestinal tract [As with pharmacokinetics, the volume kinetic system is not a mechanistic-based model and assumes nonlinear processes that can be described by a summation of linear components. Therefore, the constants may or may not represent identifiable physiological processes. However, an interesting feature of the kinetic model is that some of the fluid that is eliminated from the kinetic model is not recovered as urine. One may speculate that this residual, or reversible, elimination reflects filtration of fluid into parts of the interstitium that do not equilibrate with the plasma. Such filtration occurs through continuous, non-fenestrated capillaries in muscle and connective tissue from where it is slowly returned to the plasma via the lymphatic system . This flal tract .Vc no longer increased with the infusion rate . A feature of the very highest infusion rates was that the size of ate Fig. , which iThe present evaluation shows that the plasma volume expansion and the rate of elimination after infusion of a standardized volume of crystalloid fluid cannot be assumed to be equal in males and females. Gender apparently constitutes a bias in mixed cohorts of volunteers where the kinetics of crystalloid fluid is studied. For good, most of the previously performed studies have included one gender only. Although less desired in the present report, only one gender was included in each of the seven studies included here. However, all experiments were planned and supervised by the author, and during the past 20 years, great care has been taken to perform them in the same highly standardized fashion. The results can therefore be taken to design and power a prospective trial to confirm the observations. The physiological responses to fluid, both with regard to central hemodynamics and water-retaining hormones, should then be patterned and used as time-varying covariates in a fluid volume kinetic analysis of the same type as the present one.Limitations of the present study include that none of the included series of volunteer experiments were intended to compare gender-dependent differences in fluid volume kinetics. All experiments induced hypervolemia as the volunteers were reasonably normovolemic when the infusions were initiated. No experiments were performed during ongoing menstruation. Notes of the phase of the menstrual cycle were not taken from the females but could, if anything, be expected to prolong the half-life of the crystalloid fluid. Finally, central hemodynamics and other physiological responses to the infusions were not monitored. They could possibly have suggested mechanisms to explain the reported differences in fluid kinetics between females and males.A population kinetic analysis based on 111 experiments on 70 volunteers showed that elimination of infused Ringer’s acetate occurs faster in females than in males.


In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation.  However, FAs, which are not converted to FA-CoA or bound to FABPs, can cause cellular stress.6 Adipocyte FABP4 binds FAs and provides feedback inhibition of lipolysis through interaction with lipases. FABP4−/− adipocytes, in which this feedback inhibition is lost, show higher lipolytic rates and higher levels of intracellular free FAs.8 In addition, FABP4 delivers FAs to the nucleus to activate peroxisome proliferator-activated nuclear receptor γ, a main transcriptional inducer of adipocyte FA storage. FA metabolism is deregulated in a number of cancers.9 Specifically, FABPs are involved in tumour biology through their function in regulation of PPAR activity and/or FA uptake and oxidation.10 Interestingly, in FABP4−/− mice, ovarian tumour xenograft growth and metastasis were reduced.11 This was linked to reduced utilization of adipocyte-derived FAs in tumour cells and to reduced FABP4 expression in adipocytes and adjacent tumour cells.11The metabolism of fatty acids (FA) has been recognized as a key process required for the formation of new blood vessels (angiogenesis).12 However, many ovarian tumours are not responsive to anti-VEGFA therapies due to upregulation of alternative pathways. As such, NOTCH1 signalling induction by its ligand Delta-like ligand 4 (DLL4), itself a target of VEGA, is associated with anti-VEGFA resistance in ovarian cancer patients.13 NOTCH1 signalling limits angiogenesis by modulating the VEGFA response, leading to larger, better perfused tumour vessels, less hypoxia and reduced response to VEGFA-targeting therapies.14 Target genes of NOTCH1 and VEGFA signalling, such as the NOTCH1 targets Jagged 1 and HESR1 and the VEGFA target matrix metallopeptidase 9, are upregulated in tumour-associated ECs from invasive ovarian carcinoma.15 Dual targeting of DLL4/NOTCH1 and VEGFA signalling is more effective than single therapy in orthotopic mouse ovarian tumour models,16 making NOTCH1 and VEGFA signalling an attractive target in ovarian tumour angiogenesis.Vascular endothelial growth factor A (VEGFA) is a major inducer of tumour blood vessel growth (tumour angiogenesis). Indeed, vessel density in ovarian tumours increases with tumour progression and correlates with expression of VEGF receptor 2.FABP4 gene transcription.17 However, tumour angiogenesis in FABP4−/− mice, and its relation to NOTCH1/ VEGFA signalling pathways and endothelial FA metabolism, has not been studied in detail.11 In this study, we aimed to investigate the role of FABP4 in ovarian tumour angiogenesis and in EC FA metabolism. We show that endothelial FABP4 expression requires NOTCH1 and VEGFA signalling, and is required for ovarian tumour angiogenesis. Furthermore, endothelial FABP4 silencing led to deregulation of enzymes governing FA storage and lipolysis, and increased the rate of FAO. FAO is required for vascular sprouting and contributes to NADPH and reactive oxygen species (ROS) scavenging in certain cell types.18 However, we found that FABP4 silencing, while increasing FAO, led to decreased sprouting. FABP4 silenced cells showed an increased mitochondrial membrane potential, which was dependent on increased FAO. We conclude that FABP4 is required for the regulation of free FA levels in the cell to protect from FA-induced ROS production in ECs, and that FABP4, a target of VEGF and NOTCH signalling, plays a significant role in the formation of tumour vasculature, controlling ROS formation and intracellular FA trafficking in FA-rich environments.We have shown that in ECs, VEGFA upregulates FABP4 expression indirectly by inducing DLL4, which activates NOTCH1 signalling and initiates 14 These xenografts were chosen to study tumour endothelial FABP4 expression in response to NOTCH1 signalling and NOTCH1/ VEGFA-targeting therapies in vivo. In these xenografts, we found that more than 50% of the vessels in DLL4-overexpressing xenografts showed a strong FABP4 expression compared with 10–20% of the vessels in the control  tumours (P<0.00001) . In tumours treated with BEV and DBZ combined, vessels with strong FABP4 staining were significantly less than in dimethylsulphoxide-treated DLL4-overexpressing tumours, and no difference to EV tumours was observed  . In tumoobserved . This sh11 NOTCH1 and VEGFA signalling are implicated in the progression of ovarian tumour angiogenesis.16 However, studies investigating the expression in tumour vessels are lacking. We obtained tumour microarrays of 16 low-grade and 51 high-grade serous ovarian carcinomas and determined expression of FABP4 protein in tumour vessels and tumour epithelial cells. Epithelial FABP4 expression in low-grade and high-grade carcinomas was similar in intensity and extent. However, vascular FABP4 expression in low-grade carcinomas was more than two-fold higher than FABP4 in the vessels of high-grade tumours  correlated with FABP4 expression, whereas in vessels within the tumour and the tumour–stroma interface this correlation was lost. Instead, components of the VEGF signalling pathway were more prominent, with DLL4 and VEGFR2 being more highly expressed .11 We utilized the chitosan-nanoparticle drug delivery system, which has a high potential for clinical application, to deliver two different small interfering RNA (siRNA) sequences targeting mouse FABP4  on tumour angiogenesis and tumour growth.se FABP4  in an orse cells .FABP4  led to a reduction of vascular sprouting. While sprout number was overall not affected or slightly increased, we observed a significant reduction of sprout length by 50%  when intracellular free FAs are abundant, and by downregulating d in ECs  we detecd in ECs , and reld in ECs , indicatd in ECs .PPARG mRNA expression, pointing to an activation of FA storage . At higher ΔΨm ROS production increases exponentially.25 We measured ΔΨm to determine mitochondrial integrity in conditions of FABP4 KD and FAO inhibition. Seventy to eighty percent of untreated control cells had a medium ΔΨm (represented by R1), which was significantly decreased by FAO inhibition and/or FABP4 KD (Ψm cells (represented by R2), likely due to activation of apoptosis (Ψm cells (represented by upper R1) . Thus increased hypoxia in combined BEV and DBZ-treated tumours in vivo may be contributing to reduced FABP4 expression. As FABP4 is a key gene downstream of DLL4/NOTCH1 and indirectly of VEGF, by targeting it we may overcome NOTCH1-mediated resistance in a less toxic way than by using γ-secretase inhibitors.27Endothelial FABP4 expression in normal and tumour tissues of ovary and brain has been reported.28 Even good prognosis groups showed 74% progression at a median time of 17 months from the start of treatment and a median survival of 27 months. In a subgroup analysis it was found that patients with low-grade carcinoma and less aggressive tumour features showed reduced benefit from BEV treatment compared with those in the more aggressive, high-grade group.28 Based on our observations, tumours of the low-grade subgroup are likely to have high endothelial FABP4 expression. This subgroup could potentially benefit from FABP4 inhibition.FABP4 was more abundant in vessels of low-grade than high-grade ovarian carcinomas. Recent studies on the effect of BEV in ovarian cancer have shown improvement of progression-free survival (39 vs 34 months), but not overall survival.29 Tumours in which nests of tumour cells are surrounded by well-developed stromal structures containing the majority of the vessels are less responsive to single therapies targeting VEGFA, compared with tumours in which the vessels are embedded in the tumour mass. Thus, the stroma separates tumour cells from the vessels, potentially creating more mature vessels.29 NOTCH1, known for its limiting and stabilizing effect on angiogenesis, was co-expressed with FABP4 in these stromal regions, potentially promoting a more mature vasculature, and resistance to VEGFA-targeting therapies.In high-grade carcinomas, FABP4 was specifically expressed in stroma-rich regions, whereas vessels directly adjacent to tumour cells did not express FABP4. The tumour architecture is a determinant of resistance to antiangiogenic therapy.11 but a role of FABP4 in tumour vessels has not been studied directly. Nieman et al.,11 who studied ovarian tumour growth in FABP4−/− mice, attribute the decreased tumour burden solely to the lack of FABP4 expression in adipocytes and decreased FA metabolism in adjacent cancer cells. The researchers observed decreased microvessel density in tumours; the importance of endothelial FABP4 was, however, not investigated in that study.11 Our results show that siRNA-mediated FABP4 silencing in the vessels, while maintaining FABP4 in adipose tissue, leads to substantial loss of vessels and reduced tumour growth. This, in addition to effects of FABP4 on FA metabolism and mitochondrial ROS in ECs in vitro, strongly supports a role of FABP4 in tumour angiogenesis.Previous work highlighted the role of FABP4 in adipocytes and adjacent ovarian cancer cells8 Endothelial FABP4 preserves the controlled metabolism of FAs in ECs, by keeping the intracellular release and degradation of FAs in balance (PPARG mRNA induction in FABP4 KD cells may have represented an attempt to (a) compensate for a lack of activating ligands or (b) channel excess free/unbound FAs into lipids.Adipocytes of FABP4−/− mice have decreased rates of lipolysis and accumulate intracellular free FAs. balance . We showΨm, indicating enhanced electron transport chain activity, electron leakage and oxygen reduction. Since total ROS production was only partially inhibited by the superoxide dismutase mimetic MnTMPyP, FABP4 loss may generate cytoplasmic ROS also by increasing the levels of unbound FAs, leading to activation of ROS generators such as NADPH oxidases in the cytoplasm.30 An increase of ROS production may reduce the ability of ECs to proliferate, sprout and form vascular networks.FABP4 silenced cells have increased FAO. This may serve to remove excess free FAs resulting from increased lipolytic activity when FABP4 is lacking. We further hypothesize that activated FAO in FABP4 KD ECs is directly contributing to increased mitochondrial ROS production. The increased oxidation of FA leads to a higher Δ31 Our study provides a rationale for testing vascular endothelium-targeted delivery platforms with FABP4 siRNA to inhibit tumour growth.Our data show that FABP4 is required for angiogenesis and an important target in tumour angiogenesis, especially in tumours that are low grade, rich in stroma and embedded in FA-rich tissues. RNA-based therapies that enable tissue-specific delivery are currently being tested in clinical trials.All cells were routinely tested for mycoplasma contamination. Primary human umbilical ECs were purchased from Lonza, Visp, Switzerland and cultured in endothelial growth medium 2 (Lonza). At least three different pools of 3–5 donors were used for experiments. SV40-transformed mouse ECs (SVEC4-10) were from ATCC . SKOV3 cells used to obtain SKOV3 ip2 cells were from ATCC and cell line authentication was routinely performed.m) was performed using Lipofectamine RNAiMax  according to the manufacturer's instructions. Two different ON-TARGETplus siRNAs targeting FABP4  were used  and the ON-TARGETplus non-targeting pool (Dharmacon).Reverse transfection of siRNA duplexes (20 nACTB as a housekeeping gene. QrtPCR primer sequences (Invitrogen) were hFABP4 ; hPPARG ; mFABP4  and ACTB .RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription (RT) was performed using the High Capacity cDNA RT kit . QrtPCR was performed using the SensiFAST SYBR No-ROX Kit . Raw data were analysed using the 2(−ΔΔct) method, using Immunoblotting was performed using the Novex NuPAGE SDS-PAGE Gel and blotting system (Invitrogen). Antibodies were anti-total HSL, anti-p565 HSL and anti-660 HSL , anti-FABP4  and anti-β-actin-peroxidase (Sigma-Aldrich).m carnitine and 500 μm bovine serum albumin-conjugated oleic acid , and 0.5 μCi/ml [U-14C]-OA . To measure FA accumulation, cells were lysed after 3 h of incubation. To measure FAO, 3 n KOH, in a separate container that was inserted into the tissue culture flask, was used to capture CO2. Sixty per cent perchloric acid (Sigma-Aldrich) was injected to capture intracellular CO2, as described previously.10 Etomoxir  was used to inhibit FAO.Briefly, FA accumulation and oxidation were measured in HUVEC in endothelial growth medium 2 containing 50 μNeutral and polar lipids in PFA-fixed cells were stained with the fluorescent dye LD540  and analysed by flow cytometry.Transfected cells were exposed to 10% Trypan blue solution . Total cell number and trypan blue positive cells were determined using the Cellometer Auto T4 cell counter and software according to the manufacturer's instructions .m) was from Santa Cruz Biotechnology .Total ROS levels were determined by 2′,7′-dichlorodihydrofluorescein diacetate ; mitochondrial ROS levels were determined by MitoSOX (Life Technologies). ROS content was measured by flow cytometry. Manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride  kit  according to the manufacturer's instructions. JC1 is a cationic dye that accumulates in the mitochondria, driven by ΔΨm. Briefly, cells were grown as indicated and incubated in 1.25 μm JC1 and analysed by flow cytometry. The gating strategy was based on the presence of three cell populations with high (upper R1) versus medium (R1) versus low (R2) emission in the PE channel.ΔHUVEC were grown to confluence on ImageLock 24-well plates . The scratch was introduced with a woundmaking tool  and imaged with the Incucyte (Essen BioScience). The percentage of wound closure relative to the control (70–80% closure) was measured.HUVEC spheroids were grown in hanging drops containing 2.5 mg/ml methylcellulose (Sigma-Aldrich), embedded in Gibco Geltrex Basement Membrane Matrix (Life Technologies) and incubated for 48 h.Paraffin-embedded cell pellets or tissue sections were retrieved, dewaxed and rehydrated according to standard procedures. m/hFABP4 was stained with anti-FABP4  , mFABP4 n=5 per group).14 Vascular FABP4 was scored and a combined intensity and extent quick score was calculated.Formaldehyde fixed-paraffin embedded sections of full sets of EV and DLL4-overexpressing U87 xenografts treated with DBZ and/or BEV were obtained as described previously  and all subjects gave informed consent as appropriate. Vascular FABP4 in tissue microarrays was scored by Dr Ioannis Roxannis, Oxford University Hospitals, and a combined intensity and extent quick score was calculated. Vascular FABP4 in paired samples was scored using Aperio ImageScope and IHC Image analysis software , reporting the normalized strong positive score.in vivo experiments and protocols were approved by MD Anderson's Institutional Animal care and Use Committee. For SKOV3 ip2 cell injections, 1 × 106 cells were injected directly into the ovary.Eight- to 12-week-old female athymic nude mice (NCr-nu) were purchased from Taconic farms  and cared for in accordance with the guidelines set up by the American association for Accreditation of Laboratory Animal care and the U.S. Public health Service Policy on Human care and Use of Laboratory Animals. All FABP4 siRNAs  were incorporated in chitosan nanoparticles as described previously.19 Briefly, chitosan was dissolved in 0.25% acetic acid. The nanoparticles were spontaneously formed after adding tripolyphosphate (0.25% w/v) and siRNA (1 μg/μl) under constant stirring at room temperature. The nanoparticles were incubated for 40 min at 4 °C, centrifuged at 12 000 r.p.m. for 40 min at 4 °C and stored at 4 °C until use.Control or mouse Twice weekly injections of 3.5 μg/100 μl control siRNA-CH, sequence #1-CH and sequence #2-CH were started after 1 week (10 mice/group). The mice were observed daily for adverse effects and killed when any of the mice seemed moribund. Mouse weight, tumour weight and number of nodules were noted. Tissue specimens were fixed with formalin or were snap frozen.In vitro experimental data are shown as mean of at least three biological replicates. For animal experiments, 10 mice were assigned per treatment group. This sample size gave 80% power to detect 50% reduction in tumour weight with 95% confidence.32 After tumour inoculation, mice were randomly allocated into treatment groups. Mice that did not develop tumours were excluded from the analyses, unless otherwise mentioned. Investigators were blinded as to on which group they were performing necropsy. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Holm–Sidak's multiple comparison test to compare groups of more than two, or two-sided Student's t-test to compare two groups . It was assumed that the sampling distribution of the mean was normal. Variances were similar between groups that were statistically compared. P<0.05 was considered statistically significant.Results are expressed as mean±standard deviation (s.d.) or standard error of the mean (s.e.m.) as indicated.