The relationship between tumour progression and the nervous system is still poorly understood and is likely to involve a multitude of factors. It is therefore relevant to study tumour-nerve interactions through mathematical modelling: this may reveal the most significant factors of the plethora of interacting elements regulating neoneurogenesis. The present work is a first attempt to model the neurobiological aspect of cancer development through a system of differential equations. The model confirms the experimental observations that a tumour is able to promote nerve formation/elongation around itself, and that high levels of nerve growth factor and axon guidance molecules are recorded in the presence of a tumour. Our results also reflect the observation that high stress levels (represented by higher norepinephrine release by sympathetic nerves) contribute to tumour development and spread, indicating a mutually beneficial relationship between tumour cells and neurons. The model predictions suggest novel therapeutic strategies, aimed at blocking the stress effects on tumour growth and dissemination.It is well-known that tumours induce the formation of a lymphatic and a blood vasculature around themselves. A similar but far less studied process occurs in relation to the nervous system and is referred to as  A relationship between tumours and the nervous system has been suspected since the second century AD with the work of the Greek physician Galen2neoneurogenesis, in analogy to lymphangiogenesis and (blood) angiogenesis.Significant progress has also been made in understanding the effects of stress- and depression-mediated release of chemicals by the nervous system on tumour cell dissemination46et al.in vitro experiments with autopsy analysis of prostate cancer patients; Magnon and collaboratorset al.et al.The present model aims to investigate how solid tumours induce peripheral nerve proliferation and how different types of nerves affect tumour growth and metastasis by releasing substances such as neurotransmitters. Also, we address the question of which role stress plays in cancer progression. This model was mainly inspired by recent works that focus predominantly on prostate cancer27nerve cells) are the core components of the nervous system. The electrical signals travelling inside a neuron are converted into signals transmitted by certain chemicals (neurotransmitters); these are then passed to another neuron across a synapse. A neurotransmitter released by a nerve binds to a receptor on another cell and, according to the receptor type, induces a certain action. The collection of all the neuronal structures that together control body functions below the level of consciousness  constitute the Autonomic Nervous System (ANS). The ANS is in turn made of three sub-systems; here we will focus only on two of them: the Sympathetic Nervous System , which is responsible for quick response processes such as raising blood pressure or accelerating heart rate, and the Parasympathetic Nervous System , which governs slower responses such as gastrointestinal functions.Neurons  is equipped with receptors that can sense (gradients of) chemicals, called guidance cues, which “tell” them where to expandet al.Tumours also release Axon Guidance Molecules (AGMs). These molecules were originally considered only for their role in the nervous system as guidance cues for axons. The term direct link between nerves and tumour progression. One theory for the spread of metastases from a primary tumour to a certain organ claims that circulating cancer cells are attracted and settle in a specific region of the body due to the presence of factors such as chemokines or AGMs16It was originally believed that the nervous system only indirectly affected cancer development, through perineural invasion , are average concentrations/densities over this domain, which vary in time. We develop a compartmental model in which an extra domain is considered for the tumour cells which leave the main domain. A schematic of the model, showing the variables and their interactions, can be found in We define the primary tumour cells (Tp) and migrating tumour cells (Tm). The former are those that constitute the original tumour mass; when they detach and leave the orthotopic site of the tumour they are then designated migrating. The migrating cells are particularly dangerous because they have the potential to form metastases. Herein we do not explicitly account for the further development of the migrated tumour cells: our variable Tm represents an indication of potential metastasis formation.We distinguish between G) is a neurotrophin (a kind of protein) which stimulates the growth and enhances the survival of both Sympathetic Nerve Cells (SNCs) and Parasympathetic Nerve Cells (PNCs). It has been found to be secreted by tumour cells. AGMs (A) also affect the survival and moreover the growth of both SNCs and PNCs. In reality, there are many kinds of AGMs, which can have completely different effects on nerve and tumour development. Here, for simplicity, we consider them as a single variable; taking into account the different types of AGMs would be one step towards improving the model in future.NGF (S) and PNCs (P) is enhanced by NGF and AGMs. In addition, both types of nerve cell respond to a neurotransmitter called acetylcholine (Na), but only PNCs produce it; SNCs instead secrete epinephrine  and norepinephrine . Furthermore, norepinephrine enhances tumour cell survival, growth and chemotaxis whereas acetylcholine seems to stimulate tumour cell invasion and migrationThe growth of both SNCs  are also thought to control tumour cell apoptosisA to the death term. Finally, another relevant aspect of tumour cell dynamics is migration. Tumour cells can spontaneously disaggregate and move away from their original site. This process is enhanced by substances produced by nerve cells and distant organs, including AGMs6It is well documented that tumour cells naturally undergo mitosis . The evolution equations describing NGF and AGM dynamics in the domain are therefore given byWe are interested in the effects that tumour-secreted NGF and AGMs have on the system; here the tumour secretion rate of these two growth factors is assumed to be constant8kS and kP, which are equal to their normal equilibrium values. However, when tumour cells are present nerve growth is enhanced by the secreted NGFWe assume that in a normal (i.e. tumour-free) setting both SNCs and PNCs grow in a logistic manner and tend to their carrying capacities sn and sa that we assume to be constant. However, these two neurotransmitters are also released by other cell types37cn, ca in their equations. As chemicals, they decay at constant rates dn and da, respectively. Finally, they are absorbed by tumour cells6γ5 and γ6, respectively. The evolution equations for the neurotransmitters are then expressed byNorepinephrine and acetylcholine are produced by SNCs and PNCs, respectivelyin vivo and others in vitro; some refer to human cell lines, others to rodents), we stress that, to the authors’ knowledge, no entirely homogeneous and complete experiment related to neoneurogenesis has been performed to date; thus a consistent estimation of the parameters is not possible. To test the robustness of the model to parameter alterations, we performed a parameter sensitivity analysis (see below).et al.et al.Tm, NGF and AGMs, because we are interested in the growth factors produced by the tumour (see above section). All the other values are assumed to be at their normal (tumour-free) level when the model simulation starts. A list of the initial values for all the model variables can be found in In order to explore model predictions in different scenarios we will run simulations under different initial conditions on the primary tumour cells. In particular, et al.et al.in vitro experiments  and data from human patients for the nerve density analysis; Magnon and co-workers implanted human prostate cancer cells into mice to collect most of their data . Therefore, the only possible comparison between the model results and the experimental observations is of a qualitative, rather than a quantitative, nature.A simulation of the system of qualitative agreement with the experimental observations associated with aggressive human prostate tumour as reported by Ayala and collaborators2quantitative agreement with Magnon et al.et al.et al.Overall, the output is in good An interesting feature of the model is that a smaller initial condition for primary tumour cells, for instance  to zero . This bekT has a similar effect on primary tumour cells.To test the robustness of the model, we performed a parameter sensitivity analysis by observing the effect that a 10% increase/reduction of each parameter value has on tumour cell densities at day 15. The model appears to be robust in the sense that final tumour cell densities are not greatly affected by perturbations in the parameter values. The only parameters that generate a change in the density of migrating tumour cells of 2% or more are reported in sn by sympathetic nerves. sn is multiplied by 10 for initial condition sn is increased, the primary tumour cell density settles quickly to a higher equilibrium, while tumour cell migration is enhanced. This is in accordance with the experimental observation that stress is related to higher cancer metastasis and perhaps higher mortality44Many cancer patients exhibit stress and depression, which are known to have an effect on the immune system and consequently tumour growth5sn is taken to be its baseline value, recall the primary tumour tends to zero (sn by 10) the same initial condition leads to primary tumour growth and a constant increase of migrating tumour cells  that the number of migrated tumour cells after 15 days is reduced by about 0.5% and a similar reduction is also observed in primary tumour cell density. Thus, the model corroborates the findings of Magnon et al.Regarding parasympathetic neural activity, Magnon and collaboratorsThis work is the first mathematical confirmation of the major role played by the autonomic nervous system in promoting tumour development and progression of prostate cancer and highlights neoneurogenesis as a target for cancer drug development. In the present paper we develop a simple mathematical model for tumour neoneurogenesis and cancer progression based on recent experimental evidence; it results that, regardless of the presence of angiogenesis and lymphangiogensis, tumour-induced neoneurogenesis represents a symbiotic factor for prostate tumour. This work further expands our understanding of the process by which stress can regulate cancer aetiopathogenesis: previous research predominantly emphasised the role of the immune system in mediating stress effects on tumour growth and metastasis, while our model predicts that stress can directly affect primary tumour growth through the release of neurotransmitters. In addition, the effect of parasympathetic nerves is also captured by the model through the acetylcholine-induced tumour migration.This model, though quite simple, gives good insights into tumour neoneurogenesis and offers many possibilities for expansion and improvement. First of all, the introduction of a spatial variable and thus the use of partial differential equations would allow a more precise description of the processes occurring during tumour neoneurogenesis. In particular, a spatial approach may be able to explain why sympathetic nerves tend to accumulate in normal tissues and only penetrate tumour edges, while parasympathetic nerves infiltrate tumour tissuesThe model could be further improved by considering different variables for different kinds of AGMs, which are known to have diverse effects on tumour cellsOne could also take into account the blood and lymphatic vasculatures. Guidance cues for axons also have a function in (lymph)angiogenesis47indirect connections between the two .


Angiogenesis is a major requirement for tumour formation and development. Anti-angiogenic treatments aim to starve the tumour of nutrients and oxygen and also guard against metastasis. The main anti-angiogenic agents to date have focused on blocking the pro-angiogenic vascular endothelial growth factors (VEGFs). While this approach has seen some success and has provided a proof of principle that such anti-angiogenic agents can be used as treatment, the overall outcome of VEGF blockade has been somewhat disappointing. There is a current need for new strategies in inhibiting tumour angiogenesis; this article will review current and historical examples in blocking various membrane receptors and components of the extracellular matrix important in angiogenesis. Targeting these newly discovered pro-angiogenic proteins could provide novel strategies for cancer therapy. Angiogenesis is the growth of new blood vessels from existing ones, it is an integral part of tumour progression and metastasis and is one of the original proposed hallmarks of cancer . The maiMany problems exist with VEGF inhibition therapy such as acquired resistance, due to the tumour microenvironment switching to utilise other pro-angiogenic growth factors such as fibroblast growth factor-2 (FGF2) . AnotherThere is therefore a growing need for alternative strategies to halt the angiogenic process; one strategy is by inhibiting key protein–protein interactions other than VEGF that are important in angiogenesis. Anti-angiogenic agents that inhibit enzyme function such as receptor tyrosine kinase inhibitors will not be discussed here but have been extensively reviewed elsewhere . This arFGFs are a family of growth factors that bind to membrane bound tyrosine kinase FGF receptors. FGF1, FGF2, and their receptors FGFR1 and FGFR2 are the main FGF molecules involved in angiogenesis, resulting in endothelial proliferation, migration and differentiation . As mentFGFs were first shown to be a targetable component of tumour angiogenesis in a study which utilised adenoviral mediated expression of a soluble form of the extracellular domain (ECD) of FGFR2 fused to an Fc tag. This acts as an FGF trap inhibiting the growth factor binding to cell bound FGF receptors, leading to reductions in pancreatic tumour formation in Rip1Tag2 mice . A more The discovery of a natural FGF2 antagonist named pentraxin-related protein 3 (PTX3) which inhibits FGF2-FGFR interactions has been utilised to create PTX3 derived peptides which can inhibit FGF2 dependent angiogenesis in vitro and in vivo . RecombiFGF-BP resulting in anti-tumour effects in colon carcinoma; this provides another possible target for anti-angiogenic therapy . W. W131]. Anti-angiogenic therapy was originally hailed as a blanket approach that could be used against any solid tumour; however evidence suggests that the expression profile of vasculature between different tumour types can be diverse. The differential expression of novel proteins has been shown in tumour endothelium from lung and colorectal cancer , 134. Di


CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In conclusion, ve was the most reliable radiological parameter for detecting response to intralesional NK cellular therapy.There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/ Glioblastoma (GBM) is a highly aggressive brain tumour where the patients' median survival is only 14.6 months in vitro activated natural killer cells (NK) in an intralesional adoptive cellular immunotherapy approach We previously demonstrated that GBMs express high levels of Neuron-glia 2 (NG2), a cell surface chondroitin sulphate proteoglycan (CSPG4), that confers proliferative NK cells are large granular lymphocytes that are involved in both innate and adaptive immune responses and are highly cytotoxic against tumour and virus infected cells. Among cytotoxic lymphocytes, NK cells are the most efficient effectors against tumours, and are capable of direct killing without prior immunization in situ. Moreover, the tumour's response to therapy in many cases is transient. Therefore, quantitative measures to characterize cancer progression are required for differential diagnosis and therapeutic monitoring. Imaging techniques, such as MRI, permit diagnosis, non-invasive, longitudinal monitoring of progression and potential responses to anti-cancer treatment. Discrimination of the boundaries between malignant and normal tissue and the evaluation of cellular heterogeneity in lesions has proven to be a limitation in conventional MRI. The use of multiparametric, longitudinal MRI imaging and subsequent data analyses may provide more accurate information and hence, improved tumour lesion characterisation trans), extracellular extravascular volume fraction (ve) and blood plasma volume fraction (vp). Diffusion weighted MRI (DWI) detects changes in water mobility and has been applied to deduce the cellularity of the tumour and thus of treatment response e since both parameters may provide information about the tumour matrix and cell density. In this study, ve proved to be the most reliable parameter for detection of early treatment effects.One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess the tumour's physiological responses to treatment The U87MG cell line was purchased from the American Type Culture Collection,  and was propagated and fingerprinted as previously described The cells were immunolabelled in a final volume of 100 µl of FACS buffer  with PCPCY5.5-conjugated anti-nestin , PE-conjugated anti-vimentin , FITC-conjugated anti-GFAP , ligands for NK cell receptors were detected using AlexaFluor488-conjugated anti-MICA , APC-conjugated anti-MICB , unconjugated anti-ULBP-1 , unconjugated anti-ULBP-2-5-6  and unconjugated anti-ULBP-3  stained with Pacific Orange- conjugated Fragment of goat anti-mouse-IgG  and HLA molecules were immunolabelled with APC-conjugated anti-HLA-A,B,C , PE-conjugated anti-HLA-E , PE-conjugated anti-HLA-G  and FITC-conjugated anti-HLA-DR,DP,DQ . V450 Horizon-conjugated IgG1 , FITC-conjugated anti-IgG2a  and FITC-conjugated9 anti-IgG2b  were used as isotype controls. All samples were stained with Live/Dead Fixable Near-IR Dead Cell Stain Kit (Invitrogen) in order to gate out dead cells. Data analysis was performed in FACSDiva Software (BD Biosciences).8–10 week old immunodeficient nude rats (Han: rnu/rnu Rowett Nude), of both sexes were included in the study. The athymic nude rat is deficient of some T-cell subtypes, but has normal complement and B-cell function Rat NK cells were purified from spleens of littermates by negative selection and cultured for 4–5 days in the presence of 1000 U/ml human recombinant IL-2 (R&D Systems) as previously described 1 or 2 million NK cells were suspended in 10 µl of PBS and infused into the same coordinates as the tumour using a stereotactic frame prior to infusion of mAb9.2.27. A glass syringe  secured in a microprocessor-controlled infusion pump  slowly delivered the NK cells over 5 minutes. After infusion, the needle was left in place for approximately 3 min and thereafter slowly retracted and the skinfolds closed with polyamide surgical thread (monotherapy NK cells). Otherwise, azide free mAb9.2.27 antibodies (4 mg/ml in 24% polyethylene glycol) were thereafter administered through a 26-gauge cannula connected to an osmotic mini pump 2/N2, and positioned prone in a dedicated animal bed heated with recirculating warm water to keep the rat's body temperature stable at 37°C. Respiration and body temperature was monitored throughout the scanning session using BioTrig Life Monitoring System . The multiparametric MR imaging protocol included the following scans: (1) T2-weighted RARE (rapid acquisition relaxation enhancement) sequence with repetition time (TR)  = 2400 ms, echo time (TE)  = 40 ms, rare factor  = 8, field of view (FOV)  = 35×35 mm2, matrix size  = 256×256, resolution  = 137×137 µm2/pixel, slice thickness of 1.5 mm  and 12 averages, (2) diffusion weighted EPI (echo planar imaging) sequence with TR = 1500 ms, TE = 35 ms, Δ = 15 ms, δ = 7 ms, b-values in the range of 300–1200 s/mm2  and three orthogonal gradient diffusion directions, FOV = 35×35 mm2, matrix size  = 128×128, resolution  = 273×273 µm2/pixel, slice thickness of 1.5 mm  and 10 averages; (3) RARE T1-map with TR = 125, 250, 500, 1200, 2500, 5500 and 10 000 ms, TE = 12.5 ms, rare factor  = 2, FOV = 35×35 mm2, matrix size  = 128×128, resolution  = 273×273 µm2/pixel, a slice thickness of 1.5 mm , (4) RARE T1-weighted pre-contrast with TR = 1300 ms, TE = 8.86 ms, rare factor  = 4, FOV = 35×35 mm2, matrix size  = 256×256, resolution  = 137×137 µm2/pixel, a slice thickness of 1.5 mm  and 6 averages; (5) RARE T1-weighted dynamic contrast enhanced (DCE) series with 206 repetitions, TR = 200 ms, TE = 8.86 ms, rare factor  = 4, FOV = 35×35 mm2, matrix size  = 128×128, resolution  = 273×273 µm2/pixel and a slice thickness of 1.5 mm , scan time 16 min . In the dynamic scan, 0.2 mmol/kg Omniscan  was injected through the tail vein after acquisition of 20 baseline images. Another dose of gadodiamide was injected after the dynamic scan and prior to the acquisition of RARE T1-weighted post-contrast images with imaging parameters as described for the RARE T1-weighted pre-contrast sequence above. The total scan time was approximately 90 min including pilot scans. An additional follow-up MRI was performed on surviving animals 3 months after treatment start (RARE T2- and T1-weighted pre- and post-contrast images only).Pre-treatment T1-weighted and T2-weighted scans were performed 3 weeks after tumour implantation to verify equal tumour load in the different groups. Post-treatment multiparametric MRI was performed at two time points; 7±2 and 17±1 days after treatment on a 7T Bruker Pharmascan , using a 38 mm rat head transmit and receive volume coil. The rats were anesthetized with 1–2% isoflurane in 1∶2 Otrans), extracellular extravascular volume fraction (ve) and blood plasma volume fraction (vp) were estimated. We also estimated the area under the curve at 1 min (AUC1min), which is a model independent semi-quantitative measure that reflects contrast agent kinetics determined by a combination of blood flow, volume and permeability MR data analysis was performed using in-house programs written in Matlab7.8.0 . The data analysis included only data from animals with successful MRI scans . At 3 months, only structural MRI was performed on N = 3 surviving animals in cohort 2 in the NK+mAb9.2.27 combination therapy group. MRI data were excluded if unsuccessful contrast agent injections or pronounced movement artefacts were noted. Tumour size was measured in post-contrast T1-weighted images by manually drawing regions of interest (ROIs) of the entire volume of the tumour based on the signal intensity changes after contrast agent administration. By implementing a two-compartment model as described by Tofts 2O2 . The fraction of Ki67 positive against Ki67 negative tumour cells (Ki67 labelling index) was quantified in five manually and randomly selected hot spots in microscopic high power fields (400× magnification) in all animals in the study group using the NIS-Elements™ BR 3.1 software, . Detection of apoptotic cells was performed with the terminal deoxynucleotide nick end-labelling (TUNEL) assay according to the manufacturer's instructions . TUNEL-positive cells were visualised with DAB as chromogen, and the signal intensity of TUNEL-positive cells was assessed from the whole tumour section in 100× magnification in all the animals. The threshold level for pixel intensity was determined on the basis of four TUNEL stained tumour sections using a Nikon Eclipse 600 microscope. This threshold level was stored and subsequently applied with identical microscope settings for scoring of all tumour sections. Immune positive elements (pixels above threshold) were measured and expressed as area fraction of the visual field for each tumour section. The Growth Index (GI) was determined as the mean ratio of proliferative to apoptotic cells for all tumours in the groups.Brains were formalin fixed, paraffin embedded, and every 20th 3–5 µm thick tissue section was collected for further histological analysis. These sections were stained with haematoxylin and eosin, and examined under a light microscope. Representative formalin-fixed, paraffin-embedded sections from each specimen were deparaffinised, epitope unmasked using the appropriate method for each antibody and immunohistochemically stained with various antibodies; anti-MIB-1 , using the avidin-biotin-peroxidase complex  method with 3, 3″-Diaminobenzidine (DAB) and Htrans, ve, vp, AUC1 min, ADC and tumour size) were analysed in Stata v13.1  using linear models with robust variance estimates adjusted for clustering of rats. Post hoc analysis and correction for multiple testing was performed using Scheffe's adjustment based on the linear model, using the svy-module in Stata for the analysis. The figures were also based on the marginal means from the linear model clustered by rat. Statistical analyses were performed separately on the two cohorts and by combining the two first time points. Ordinary linear regression was performed to test for any associations between the MRI derived parameters. P-values less than 0.05 were considered statistically significant.Immunohistochemistry data for the treatment groups was analysed using One way analysis of variance (ANOVA) with Bonferroni post hoc analysis correction for multiple testing in GraphPad Prism Statistical Software, version 5.0 . MRI derived parameters , 96.5% expressed nestin, 88% were vimentin positive and 10% expressed A2B5 . PreviouT1-weighted post-contrast MR images of rats bearing U87MG tumours demonstrated contrast enhancement within the tumour mass in all tumours one week prior to treatment, indicating high angiogenic capacity and tumour progression (data not shown). Post-treatment images at 7±1 and 17±2 days revealed increased tumour volume in all groups in both cohorts, but with a large intra-group heterogeneity . No signe, was significantly increased in the NK+mAb9.2.27 combination treated animals compared to NK cells monotherapy  and mAb9.2.27 monotherapy  in cohort 1 animals that were treated with 1 million NK cells, indicating reduction in cellular burden induced by combination treatment . These effects were reproducible in treatments with 2 million NK cells, where NK+mAb9.2.27 combination treatment significantly increased ve compared to NK cells monotherapy . NK cell monotherapy treatment decreased ve compared to mAb9.2.29  and compared to untreated controls   . The difNK cells . Parametasis see , visualitrans describes the flux from vessels to interstitium but no effect of the treatment was found on Ktrans in any of the cohorts. Furthermore, the model-independent parameter AUC1 min was not significantly changed in any of the groups at any time point (data not shown). Also, the parameter reflecting the blood plasma fraction, vp, did not change significantly between groups. Correspondingly, at end-stage histology, there was no significant difference in the angiogenic capacity of the tumours (data not shown).MRI derived parameters reflecting the vessel function and vessel wall integrity revealed less treatment dependent changes than the parameter denoting the interstitial volume fraction. The transfer constant Ktrans, ve or AUC1 min. However, for animals treated with the NK+mAb9.2.27 combination treatment and NK cell monotherapy, there was a significant association between ADC and ve  in the NK+mAb9.2.27 combination therapy group were alive at 3 months follow-up scans, whereas none were alive from the other treatment groups ex vivoe, indicated that NK cell monotherapy failed to reduce the tumour cellular burden in the tumour microenvironment and this was corroborated by histological measures of decreased apoptosis, increased cell proliferation and subsequently, the poor survival outcomes of these animals The strategy of targeting the NG2/T1-weighted post-contrast images at 3 months and histological analyses revealed a regression of the NK+mAb9.2.27 combination treated tumours. Lack of contrast enhancement at the tumour region at this time point demonstrated treatment efficacy as confirmed by the diminished tumour tissue on histological staining. In our separate study, survival analyses demonstrated a correlation of NK+mAb9.2.27 treatment with prolonged survival in several tumour models so-called flare effect, previously reported following immunotherapy with intralesionally infused lymphocytes in patients with recurrent high grade astrocytomas There is currently a pressing need for imaging biomarkers that provide information about the tumour phenotypes in response to immunotherapy when it comes to tumour cell density, cell death and tumour-stromal tissue architecture. Changes in physiological parameters that correlate with therapeutic efficacy may be detected earlier in the treatment process than gross morphological changes such as tumour size. The findings reported in this study supported this hypothesis. We observed larger tumour lesions on contrast enhanced T1-weighted structural MR images after immunotherapy despite prolonged survival. This phenomenon may reflect genuine tumour progression or alternatively, it may be due to recruitment of immune cells into the tumour from the peripheral circulation. Indeed we recorded greater tumour infiltration by microglia/macrophages with pro-inflammatory phenotypes after combination NK+mAb9.2.27 compared to monotherapy and control animals at early stages e, that describes the volume fraction of the interstitium, proved to change earlier in the treatment process than the tumour volume, and may provide important information about the treatment course when traditional measures such as tumour volume fail. Others have also suggested this parameter as a candidate biomarker for the EES e in the combination treated animals thus correlated with efficient killing effect. Animals treated with NK cell monotherapy exhibited the greatest growth index, and ve was decreased. The reduction was more pronounced in cohort 2 treated animal that received 2 million NK cells, indicating a dose dependent response. This can most likely be attributed to the presence of NK cells, in addition to the highly cellular tumour mass contributing to a tightly packed tumour cell mass, indicated also by histology. Thus, the volume of the interstitial space was reduced. The killing efficacy of NK cells alone was not sufficient to counteract the increase in cell numbers in the solid tumour mass, leading to reduced volume of interstitial space. This illustrates that mAb9.2.27 bound to the NG2/CSPG4 positive tumour cells was required to trigger ADCC mediated cytotoxicity and facilitate the efficacy of NK cells in this GBM model. The reduced interstitial volume fraction in the NK cell monotherapy group was accompanied by a tendency of reduction in the transvascular transport (Ktrans) and initial contrast uptake (AUC1 min). However, mechanical forces within the solid tumour play a role. Proliferating tumour cells induce physical stress that compresses blood vessels and therefore reduce the blood flow. Also, as a result of vessel leakiness and lack of functional lymphatics in most solid tumours, interstitial fluid pressure is significantly elevated, which is more profound in the inferior of the tumour trans and AUC1 min. The estimated Ktrans may also be influenced by blood flow and not exclusively by permeability when using low-molecular weight contrast agents trans did not give any indication of treatment course in any of the groups.Here we have demonstrated that longitudinal physiological MR imaging provided more sensitive quantitative measures that allowed us to characterize tumour progression longitudinally. The derived parameter ve, in the control tumours may be due to de novo necrosis caused by reduced blood supply, followed by hypoxia and cell death in the central parts of the tumour e and similar to other studies, where increasing ve with increasing brain tumour grade has been reported p might be due to the fact that we used a literature based arterial input function (AIF) as baseline for the analyses. Ideally, AIF should be individually measured since vp is highly dependent on the chosen AIF. However, all animals received a dose of the contrast agent dependent on the individual body weight, and the same injection time and speed were used, so inter-individual differences in the AIF were kept at the minimum. Also, inflow effects and slice positioning may influence the measured individual AIF and interfere with the result. An alternative explanation in the lack of differences in the plasma volume, is that the mAb9.2.27 is highly specific for the human CSPG4 epitope. Thus mAb9.2.27 monotherapy did not exhibit anti-angiogenic effects against the rat NG2 expressing tumour vessels. Indeed, no difference in microvascular densities as a measure of angiogenesis was demonstrated on immunohistochemistry.The increased extracellular volume fraction expressed as vADC represents the random diffusion of water molecules within tissues and has been shown to be inversely correlated with tumour cell density e in the NK+mAb9.2.27 combination therapy and NK cell monotherapy group, where high ve values corresponded with low ADC values. This indicates that the ADC value and ve may reflect different aspects of the tumour microenvironment and the ADC value may be more complex than simply reflecting the volume of the interstitial space. Our findings are supported by others e was found and the authors suggested that the interpretation of these parameters is over simplified e in breast tumour patients was reported e in these patients. The ADC value is not only dependent on the volume of the EES but also the composition of the EES. Tumours are composed of the malignant cell compartment and the “benign” stromal cell compartment that includes blood vessels, infiltrating inflammatory cells, extracellular matrix, and stromal fibroblasts. The precise composition of the tumour microenvironment varies depending on the tumour site and histology. The brain, in particular, consists of numerous specialized cell types such as glia, brain endothelial cells and a relative paucity of extracellular matrix components. Therefore, the biological interpretation of the estimated MR-based parameters may vary between tumours of different histological origin, thus the tumour specific and treatment dependent physiology must always be taken into consideration. Other groups also failed to find a correlation between cell density and ADC in gliomas e and “tumour size” at these early time points (3 weeks post treatment). This may be attributed to the high intra-tumour heterogeneity of GBMs with areas of oedema and cyst formation, vascular proliferation and necrosis, that all influence the estimated ADC value in numerous ways 2) weight the intracellular part of the ADC value more than lower b-values. The magnitude, duration, and temporal duration of the diffusion gradients determine the b-value and influence the signal observed in the DW images 2) were more useful in discriminating low-grade and high-grade gliomas Consistent with this, we found a statistically significant negative association between the ADC value and ve in GBMs in the same animals. Being aware of the limited number of animals included in the study, our results indicate that ve might be a reliable parameter for detecting early treatment response of targeted immunotherapy in GBMs using combined NK +mAb9.2.27. The ve parameter is often overseen and not reported in many studies but may prove to be a candidate for the interstitial volume as it is a direct measurement of the EES To our knowledge, we are the first to report longitudinal and simultaneous measurements of both ADC and vAlthough, standard morphological MR imaging has played a pivotal role in defining landmarks used to manage GBM clinically, physiological MR imaging may hold the potential to improve diagnosis, prognosis, and prediction for individual patients' outcome in the long term. We have demonstrated that multiparametric longitudinal imaging provided a more comprehensive portrait of the tumour's microenvironment. Each MR sequence added additional knowledge about the tumour's response to treatment and allowed studies of the inter-related physiological parameters, which is a prerequisite for individualised treatment. Targeted immunotherapy against the NG2/CSPG4 proteoglycan in combination with the killing and cytokine modulating effect of NK cells improved therapeutic effects as determined by MRI.Figure S1Comparison of tumour infiltrating immune cells after various treatments. (A) Cellular composites of resident lymphocytes (CD45highCD11b/c low); macrophage/microglia (CD45+CD11b/c+) in control, mAb9.2.27 only, NK cell only and NK+mAb9.2.27 treated representative animals. (B) NK+mAb9.2.27 treated animals had greatest proportions of microglia that were similar to those in normal brain. The microglia were ED1+CD8+ and expressed MHC-class II molecules indicating an activated capable of antigen presentation. (C) Double staining for CD8 (brown) and ED1 (red) in histological tissues showing tumour infiltrating ED1/CD8+ macrophage/microglia, greatest infiltration in NK+mAb9.2.27 treated tumours. Arrows in panel C indicate tumour region, Magnification 200X, scale bar 100 µm. Data corroborates flow cytometric findings presented in panels A and B.(TIF)Click here for additional data file.Materials and Methods S1Supporting Materials and Methods.(DOCX)Click here for additional data file.


In this study, we evaluated whether 213Bi--octreotate (213Bi-DOTATATE), a 213Bi-labelled somatostatin analogue with high affinity for somatostatin receptor subtype 2 (SSTR2), is suitable for the treatment of larger neuroendocrine tumours overexpressing SSTR2 in comparison to its effectiveness for smaller tumours. We performed a preclinical targeted radionuclide therapy study with 213Bi-DOTATATE in animals bearing tumours of different sizes (50 and 200 mm3) using two tumour models: H69  and CA20948 (rat pancreatic tumour).Targeted alpha therapy has been postulated to have great potential for the treatment of small clusters of tumour cells as well as small metastases. 3 were euthanized at 10, 30, 60 and 120 min post injection of a single dose of 213Bi-DOTATATE (1.5–4.8 MBq). To investigate the therapeutic efficacy of 213Bi-DOTATATE, xenografted H69 and CA20948 tumour-bearing mice with tumour sizes of 50 and 200 mm3 were administered daily with a therapeutic dose of 213Bi-DOTATATE  for three consecutive days. The animals were followed for 90 days after treatment. At day 90, mice were injected with 25 MBq 99mTc-DMSA and imaged by SPECT/CT to investigate possible renal dysfunction due to 213Bi-DOTATATE treatment.Pharmacokinetics was determined for calculation of dosimetry in organs and tumours. H69- or CA20948-xenografted mice with tumour volumes of approximately 120 mm213Bi-DOTATATE-treated mice after the doses were applied.Higher tumour uptakes were found in CA20948 tumour-bearing animals compared to those in H69 tumour-bearing mice with the highest tumour uptake of 19.6 ± 6.6 %IA/g in CA20948 tumour-bearing animals, while for H69 tumour-bearing mice, the highest tumour uptake was found to be 9.8 ± 2.4 %IA/g. Nevertheless, as the anti-tumour effect was more pronounced in H69 tumour-bearing mice, the survival rate was higher. Furthermore, in the small tumour groups, no regrowth of tumour was found in two H69 tumour-bearing mice and in one of the CA20948 tumour-bearing mice. No renal dysfunction was observed in 213Bi-DOTATATE demonstrated a great therapeutic effect in both small and larger tumour lesions. Higher probability for stable disease was found in animals with small tumours. 213Bi-DOTATATE was effective in different neuroendocrine (H69 and CA20948) tumour models with overexpression of SSTR2 in mice.The online version of this article (doi:10.1186/s13550-016-0162-2) contains supplementary material, which is available to authorized users. For treatment of small tumour clusters and metastases, β-emitters may lack efficacy due to the relatively large tissue penetration and low linear energy transfer (LET). Targeted alpha therapy (TAT) with α-emitters has been described to be more suitable for these purposes, since the path length in tissue of α-particles is relatively short (in tissue 50–100 μm) compared to that of β-particles. Moreover, α-particles have a much higher LET (~100 keV/μm) and are therefore more powerful than β-particles (0.2 keV/μm) for therapeutic applications [213Bi), an α-emitter with a T1/2 of 45.6 min, has already been proven to be effective in TAT [90Y-DOTATOC or 177Lu-DOTATOC treatment after treatment with 213Bi-DOTATOC [213Bi-DOTATOC was able to overcome the resistance against β radiation in patients with different neuroendocrine tumours, in metastasis and in primary tumours, resulting in a high number of long-lasting anti-tumour responses.Peptide receptor radionuclide therapy (PRRT) is an effective treatment option for patients with an inoperable neuroendocrine tumour (NET) or with metastatic disease , 2. Appr-DOTATOC . TAT wit213Bi-DOTATATE in tumours of different sizes in mice . Two different tumour models with somatostatin receptor subtype 2 (SSTR2) expression were used: CA20948 (rat pancreatic tumour) and H69 . Both tumours have relatively high SSTR2 expression, and both have often been applied as a model in preclinical studies of PRRT in NET [α/β = 8.3 Gy [α/β = 2.3 Gy [213Bi is expected to follow a linear  curve which was comparable for both cell lines.The objective of this study was to investigate the therapeutic efficacy of T in NET . The rad= 8.3 Gy . The H69213Bi-DOTATATE shows accumulation in the kidneys, resulting in high radiation doses to the kidneys; in this study, we therefore determined the safe dose of radioactivity administered for TAT in this mouse model. Renal function was evaluated after TAT by SPECT/CT at different time points post treatment using 99mTc-DMSA (dimercaptosuccinic acid), a renal marker for tubular damage.For PRRT, the dose-limiting organs are bone marrow and kidneys. 213Bi/225Ac generator (222 MBq) was provided by the Institute for Transuranium Elements . Prior to elution, the generator was rinsed using 0.01 M HCl (3 mL) and eluted using 0.1 M/0.1 M HCl/NaI (0.6 mL). After elution, the generator was rinsed with 0.01 M HCl and stored in 0.01 M HCl [213Bi was determined using a germanium detector  [A 01 M HCl . The actanberra) .213Bi (0.1 M/0.1 M in HCl/NaI) was added to a solution containing 7.0 nmol DOTATATE, 0.15 M TRIS buffer and 2.6 mM ascorbic acid in a total reaction volume of 800 μL. The reaction mixture was incubated for 5 min at 95 °C followed by 5 min at room temperature. To chelate any unbound/free 213Bi, 50 nmol DTPA  was added (manuscript submitted). Analysis by instant thin-layer chromatography  and reverse-phase high-performance liquid chromatography  of the labelled peptide was performed to determine the incorporation yield (%) and radiochemical purity (RCP) expressed as percentage of labelled peptide of interest compared to other detectable compounds [Eluate containing 99mTc-DMSA kit was purchased from Mallinckrodt  and labelled according to the indicated procedure.The 2 expression was cultured in DMEM  supplemented with 10 % heat-inactivated fetal bovine serum. H69 tumour cells, human small cell lung carcinoma cells with SSTR2 expression , were cultured in RPMI 1640  supplemented with 10 % heat-inactivated fetal bovine serum and 50 IU/mL penicillin/streptomycin. All cells were cultured at 37 °C in a 5 % CO2/humidified air atmosphere.The rat pancreatic tumour cell line CA20948 (derived from a rat pancreas tumour at our institute) with high SSTR6 cells (CA20948 or H69), suspended in 200 μL sterile culture medium Hanks’ balanced salt solution (HBSS) in the case of CA20948 cells. Three days after inoculation of CA20948 cells, a tumour with a tumour size ~50 mm3  was reached, and 2 weeks after inoculation, a tumour size ~200 mm3 (used for the “larger” tumour-size group) was reached. For H69 cells, 200 μL, containing 1/3 Matrigel and 2/3 HBSS medium , was used. The cells were injected subcutaneously (s.c.) into the right flank of the mice. One week after inoculation of tumour cells, tumours with a volume of ~50 mm3  could be detected, and after 3 weeks, tumour volumes reached ~200 mm3 (used for the larger tumour-size group). Tumour volumes were estimated using a calliper.All animal studies were in agreement with the Animal Welfare Committee requirements of Erasmus MC and conducted in accordance with accepted guidelines. Male 6–8-week-old BALB/c (nu/nu) nude mice were obtained from Charles River . Subcutaneous tumours were established in mice by inoculation of approximately 5 × 103, animals were divided into five groups with seven animals in each group for both H69 and CA20948 tumour-bearing animals. Four groups of mice received a single intravenous (i.v.) injection of 213Bi-DOTATATE (200 μL) via the tail vein, 3.8 ± 0.6 MBq/0.3 nmol/200 μL for H69 tumour-bearing animals and 2.1 ± 0.2 MBq/0.3 nmol/200 μL for CA20948 tumour-bearing animals. At 10, 30, 60 and 120 min post injection (p.i.), animals in the four groups were euthanized and tissues and organs of interest  were collected and counted using a gamma counter . Control animals received a single injection of 213Bi-DOTATATE plus an excess of DOTATATE (10−6 M) i.v. via the tail vein (blocking dose) to determine the non-receptor-specific binding of 213Bi-DOTATATE. The animals of the control group were euthanized 60 min p.i.; tissues and organs of interest were harvested and counted using a gamma counter. Data was expressed in mean percentage of total injected radioactivity per gram tissue (%IA/g) ± SD.When tumour volumes reached ~120 mm3 were reached, respectively. On the day of treatment (T = 0 days), the animals  were injected i.v. via the tail vein with 213Bi-DOTATATE (2–4 MBq/0.3 nmol/200 μL) for three consecutive days. The control group  received i.v. via the tail vein three times 0.3 nmol/200 μL of unlabelled DOTATATE for three consecutive days.Tumour cells were inoculated into the right flank of the mice, and the tumours were allowed to grow until tumour volumes of approximately 50 and 200 mmAll animals were followed for 90 days after treatment. The behaviour and the health status of the animals were observed daily, and the weight of the animals and the tumour volumes were monitored twice a week.99mTc imaging, an energy peak at 140 keV was selected with an energy window width ± 10 %. A nine-pinhole aperture (diameter of each pinhole 1.4 mm) was used on each camera head. For the CT topogram, an axial length of 27 mm was used to cover the renal region. Animals were imaged under anaesthesia by isoflurane and O2. The body temperature of the animals was maintained using a heated bed. Renal imaging of H69  and CA20948 tumour-bearing animals  was performed 2 h after i.p. administration of 99mTc-DMSA (25 MBq). 99mTc-DMSA images were acquired for 12 min [Renal function imaging was performed by a four-headed multi-pinhole SPECT/CT camera . For jection) .7 was used for simulation to reduce the variation (<5 %) in the data. The mean absorbed energies for the 10 MBq 213Bi-DOTATATE injection were calculated for spheres of 1, 10, 100, 200, 300 and 500 mg containing tissue with a mass density of 1 g/mL to determine the spherical node S-values. A homogeneous activity distribution over the sphere was assumed. The absorbed dose Di was calculated for each volume i according to the MIRD schema:The absorbed radiation dose (defined as the mean absorbed dose in Gy) in organs and tumours was estimated from the results obtained in the biodistribution studies as described by Konijnenberg et al.  in a sphÃi the time-integrated activity (TIA) in organ i and S(i ← i) the absorbed dose rate per unit activity . Only the self-dose S-values were considered and were obtained by interpolation between the sphere S-values for the measured organs or tumour mass. For small tumours, a minimum tumour mass of 50 mg was used for the absorbed dose calculation. The Ãi in each organ was determined for 213Bi and its daughters taking the biodistribution into account according to the methods shown in Additional file S-values and with the nephron model by Hobbs et al. [with t test and using the nonparametric Mann-Whitney test. P values <0.05 were considered statistically significant. For statistical significances in tumour growth patterns, one-way ANOVA was used. Values are presented as the mean and the standard deviation (SD).Statistical analysis was performed using An incorporation yield of ≥99 % and a RCP ≥ 85 % were found for the radiopeptide, as determined by ITLC and HPLC, respectively.213Bi-DOTATATE in H69 tumour-bearing mice revealed relatively high tumour uptake. The highest tumour uptake was reached at 30 min p.i., 9.8 ± 2.4 %IA/g. Tumour uptake T1/2 was 7 ± 2 min, and tumour clearance T1/2 was 2.3 ± 1.1 h. An even higher renal activity compared to tumour uptake was found at all time points, the highest renal activity being found at 10 min p.i., 44 ± 10 %IA/g. At 120 min p.i., the renal activity still was 25 ± 3 %IA/g. Blood clearance in H69 tumour-bearing animals was fast, and clearance T1/2 was 11 ± 2 min, see Fig. Biodistribution studies using 213Bi-DOTATATE in CA20948 tumour-bearing mice was already highest at 10 min p.i., 19.6 ± 6.6 %IA/g, and the tumour uptake T1/2 was therefore unable to estimate. A clearance T1/2 of 2.2± 0.9 h was found for CA20948 tumours. Similar renal activity was found in CA20948 tumour-bearing animals. Renal activity was highest at 10 min p.i., 52 ± 17 %IA/g. Renal clearance T1/2 was 10 ± 3 min, whereas blood clearance T1/2 was 14 ± 1 min, see Fig. Tumour uptake of 213Bi-DOTATATE calculated for 100 mg tumour. The renal-absorbed dose was four times as high as the tumour-absorbed radiation dose: 2 Gy/MBq injected 213Bi-DOTATATE. The absorbed radiation dose of 213Bi-DOTATATE in the pancreas, spleen and liver was low compared to those in the tumour and kidney, 0.15, 0.06 and 0.06 Gy/MBq, respectively. Similar results were observed in CA20948 tumour-bearing animals. A lower tumour-absorbed radiation dose was found than that in the kidneys: 0.78 Gy/MBq in the tumour vs 1.6 Gy/MBq. Absorbed radiation doses in the pancreas, spleen and liver were 0.13, 0.05 and 0.06 Gy/MBq, respectively. For both tumour-bearing animal models, an absorbed dose of < 0.02 Gy/MBq was found in the bone marrow.Dosimetry calculations in both tumour models were based on the data obtained from the biodistribution studies at 10, 30, 60 and 120 min p.i. In H69 tumour-bearing mice, a relatively high tumour-absorbed radiation dose was estimated: 0.45 Gy/MBq injected Tumour- and kidney-absorbed radiation doses in H69 or CA20948 tumour-bearing animals were mainly caused by the α-particles emitted. The absorbed dose rate was higher in CA20948 than in H69 tumours, and the kidney-absorbed radiation dose was similar; see Fig. 213Bi-DOTATATE, see Fig. 3 of the control and treated animals of both tumour models are indicated in Table 3 could not be measured accurately; therefore, 50 mm3 was used as the minimum tumour sizes in our calculation and this was in three cases of H69 tumour-bearing animals with a small tumour and in all cases of CA20948 tumour-bearing animals with a small tumour. Overall calculated cumulative tumour-absorbed dose in small tumours was higher than large tumours. In CA20948 tumours, the cumulative tumour-absorbed dose was higher than in H69 tumours.Therapeutic anti-tumour effects were observed in all animals treated with 213Bi-DOTATATE (cumulative injected activity of 9.4 ± 0.7 MBq) treatment. In CA20948 tumour-bearing animals with large tumours, the inhibition of tumour growth was initiated around 30 days after therapy (cumulative injected activity 10.2 ± 0.4 MBq 213Bi-DOTATATE), leading to a tumour growth delay time of 50 days. Similar results were found in CA20948 tumour-bearing animals with small tumours after therapy (cumulative injected activity 7.7 ± 0.4 MBq); a delay in tumour growth of around 30 days was observed, and in one of the animals, no tumour regrowth was observed. The delay time was estimated excluding the animals with cure or stable disease. In H69 tumour-bearing animals with small tumours, a delay of tumour growth was found for around 30 days after therapy (cumulative injected activity 7.4 ± 0.5 MBq), and in two animals, no tumour regrowth was observed until the end of the study at day 90. Here, the two animals with cure/stable disease were excluded from the estimated delay time.Shrinkage of tumours was observed in H69 tumour-bearing animals with large tumours and a delay of tumour growth around 70 days after 213Bi-DOTATATE showed a higher survival rate than the control animals, see Fig. 213Bi-DOTATATE therapy . Similar results were obtained in H69 tumour-bearing animals with large tumours after TAT; a median survival > 90 days was found . In the CA20948 tumour-bearing animals without TAT, a median survival of 29 days was found for animals bearing large tumours, whereas for animals bearing small tumours, this was 35 days. After 213Bi-DOTATATE, a median survival of 70 days was found in the large-tumour-animal group  and > 90 days in the small-tumour-animal group .All animals treated with 213Bi-DOTATATE therapy or unlabelled DOTATATE treatment. Renal function was determined by renal uptake of 99mTc-DMSA (25 MBq). No significant differences in 99mTc-DMSA uptake in the kidneys were observed in 213Bi-DOTATATE-treated mice and control animals for both tumour models. In H69 tumour-bearing mice, a mean renal uptake of 2.6 ± 0.8 MBq (mean concentration of 2.9 ± 0.6 kBq/mm3) in the control group (n = 4) vs 2.1 ± 0.9 MBq (mean concentration of 2.9 ± 0.8 kBq/mm3) in 213Bi-DOTATATE-treated animals (n = 8) was found. Similar results were obtained in CA20948 tumour-bearing animals: mean uptake of 2.1 ± 0.2 MBq (mean concentration of 3.1 ± 0.4 kBq/mm3) in 213Bi-DOTATATE-treated animals (n = 4) and 2.1 ± 0.3 MBq (mean concentration 3.2 ± 0.3 kBq/mm3) in control (n = 2) were found. Figure 99mTc-DMSA uptake in kidneys of both tumour models.Renal function was investigated in H69 and CA20948 tumour-bearing animals at day 90 after 213Bi-DOTATATE TAT in tumours of different sizes in two different tumour models, both with expression of SSTR2. 213Bi-DOTATATE showed a very good therapeutic effect in both small and large tumours in the two different tumour models. Tumour-absorbed doses (Gy) in CA20948 tumour-bearing animals were higher compared to those in H69 tumour-bearing mice. Despite these findings, 213Bi-DOTATATE showed greater therapeutic effects in H69 tumour-bearing animals than in CA20948 tumour-bearing animals; a higher survival rate was observed in H69 tumour-bearing animals at the end of the study. However, no significant difference in tumour (re)growth inhibition time between the two tumour models was found, even though the absorbed dose in the tumour was higher in CA20948 tumour-bearing animals. In vitro, CA20948 cells appeared also more radioresistant than H69 cells; at external beam irradiation with 2 Gy, a survival fraction of H69 was 37 % [In this study, we compared the efficacy of was 37 % .213Bi-DOTATATE therapy. In H69 and CA20948 tumour-bearing animals with large tumours, a partial response was found. A longer tumour growth delay was found in animals with large tumours of both tumour models compared to small tumours. The time a tumour takes to reach 2000 mm3 in small tumours is longer than in large tumours; therefore, Δ delay time for tumour growth was used. Nevertheless, in animals showing regrowth, the small tumours were less responsive than the large tumours in both models.In this study, we observed a higher anti-tumour effect in small tumours. In H69 tumour-bearing mice with small tumours, two animals showed stable disease and in eight animals, a partial response was found after treatment. In the case of CA20948 tumour-bearing animals with small tumours, one animal showed stable disease till the end of the study and partial response was found in eight of the animals after 213Bi-DOTATATE TAT [213Bi-DOTATATE therapies in both tumour models. However, no complete remissions were observed in these animals. Besides tumour perfusion [2, tumour size [The reason for partial remission can be explained as the distribution of the radiation-absorbed dose depends on the tumour size, the physical properties of the radionuclide and its possible inhomogeneous intratumoural distribution. In those cases where the tumour dimensions are smaller than the range of the ionizing particles, a large proportion of the absorbed energy can “escape”, resulting in a suboptimal effect of TATE TAT . In largerfusion  and inhoour size , 22, vesour size  is obserour size , especiaFor somatostatin receptor-targeted PRRT, nephrotoxicity is often the dose-limiting factor, since reabsorption of radiolabelled somatostatin analogues by cells in the proximal tubule of the nephron occurs .In this study, the calculated renal-absorbed dose using our simplified model was compared to the model described by Hobbs et al. . We assu99mTc-DSMA can provide information on renal function after renal tubular damage and has been used as renal marker in PRRT [213Bi-DOTATATE). Kidney histology was performed in CA20948-bearing animals (data not shown), and no significant differences were observed in control vs treated animals. However, 213Bi-DOTATATE is known to accumulate in the kidney, leading to a high renal-absorbed dose. In patients, the maximum tolerated dose in kidney is postulated to be 23–28 Gy [l-lysine as kidney protectant prior to the 213Bi-DOTATATE treatment; in the absence of l-lysine, the maximum tolerated activity was <13.1 ± 1.2 MBq (manuscript in preparation). in PRRT . In this23–28 Gy . Kidney 23–28 Gy , 32, esp177Lu and 90Y. We therefore believe that TAT is most promising for the treatment of NET. In combination with PRRT using 90Y and 177Lu peptides [Even though we have not been able to achieve complete remission of all tumours, this study demonstrated that TAT can be effective for the treatment of both large and small tumours. These findings are in line with those of Kratochwil et al.  who demopeptides , 34, eve213Bi-DOTATATE shows very good therapeutic efficacy for the treatment of both large and small tumours in these mouse models. In small tumours, 213Bi-DOTATATE demonstrated greater anti-tumour effect by its higher probability for inducing stable disease. Furthermore, TAT using 213Bi-DOTATATE demonstrated to be safe in our experimental setting, with kidneys as dose-limiting organs. 213Bi-DOTATATE therefore held great promise for targeted therapy for neuroendocrine tumours that express SSTR2.TAT with All applicable international, national and/or institutional guidelines for the care and use of animals were followed.


High tumour stromal content has been found to predict adverse clinical outcome in a range of epithelial tumours. The aim of this study was to assess the prognostic significance of tumour-stroma ratio (TSR) in endometrial adenocarcinomas and investigate its relationship with other clinicopathological parameters.n = 400). TSR was measured using a morphometric approach (point counting) on digitised histologic hysterectomy specimens. Inter-observer agreement was determined using Cohen’s Kappa statistic. TSR cut-offs were optimised using log-rank functions and prognostic significance of TSR on overall survival (OS) and disease-free survival (DFS) were determined using Cox Proportional Hazards regression analysis and Kaplan-Meier curves generated. Associations of TSR with other clinicopathological parameters were determined using non-parametric tests followed by Holm-Bonferroni correction for multiple comparisons.Clinicopathological and 5-year follow-up data were obtained for a retrospective series of endometrial adenocarcinoma patients (P = 0.034) in univariable Cox-regression analysis. Using the optimal cut-off TSR value of 1.3, TSR-high (i.e. low stroma) was associated with worse OS  and DFS  in univariable analysis. However, TSR did not have independent prognostic significance in multivariable analysis, when adjusted for known prognostic variables. A highly significant association was found between TSR and tumour grade (P < 0.001) and lymphovascular space invasion (P < 0.001), both of which had independent prognostic significance in this study population.TSR as a continuous variable associated with worse OS (Low tumour stromal content associates with both poor outcome and with other adverse prognostic indicators in endometrial cancer, although it is not independently prognostic. These findings contrast with studies on many - although not all - cancers and suggest that the biology of tumour-stroma interactions may differ amongst cancer types. Endometrial cancer (EC) is the most prevalent gynaecological malignancy in the Western world and ranks as the ninth commonest cause of cancer-related mortality in women in the UK . MoreoveMalignant epithelial tumours are composed of carcinoma cells, together with stromal fibroblasts, immune effector cells, microvasculature and the extracellular matrix, which are collectively referred to as the tumour microenvironment. The dynamic interplay between cancer cells and stromal components within the tumour microenvironment contributes to malignant progression and metastasis . As suchThis study received ethical approval from Leeds NRES committee (Ref: 05/Q1107/41). Patients gave their written informed consent for their tissue samples to be used in research. Clinicopathological and follow-up data were collected for a retrospective series of 400 women in the Yorkshire area (UK) diagnosed with endometrial adenocarcinoma between 2005 and 2007 who had undergone a hysterectomy at our tertiary referral centre . Median follow-up was 79.7 months (reverse Kaplan-Meier method). For both overall survival (OS) and disease-free survival (DFS), patients were censored at end of follow-up. OS was defined as time from diagnosis to death and DFS was defined as time from diagnosis to recurrence or death. Staging data were converted from the International Federation of Gynaecology and Obstetrics (FIGO) 1988 to the FIGO 2009 staging system  accordin2 (±0.25 mm2) was sampled from one slide for each patient using a digital slide viewer . In each instance, the slide that most accurately represented the tumour mass was used; in cases where more than one histological type was observed, multiple areas were marked up and sampled in order to obtain representative measures of tumour heterogeneity. Similarly, in large tumour masses where there could be variation in proportion of tumour and stroma, at least three 9 mm2 areas were defined. Virtual graticule software (RandomSpot) [For each patient, 2 representative slides of 4 μm haematoxylin and eosin-stained tissue sections were selected and subjected to mark-up by a histopathologist (NMO). Areas selected for mark-up were from the superficial region, as in , in ordedomSpot)  was useddomSpot) , 30. ThedomSpot) , were: uCases were marked up and scored with the observer blinded to the outcome data. Totals for each scoring category were generated and TSR was calculated by dividing the total tumour count over the total stroma count for each case.coxph and survfit functions in the R package Survival, whereby the optimal cut-off gave the lowest log-rank P value. The prognostic significance of TSR, both as a continuous variable and using the TSR cut-off, on OS and DFS was determined using Cox Proportional Hazards regression analysis in R [U  tests or Kruskal-Wallis tests followed by Mann–Whitney-U post-hoc tests, as appropriate. Corrections for multiple comparisons were performed using Holm’s sequential Bonferroni method.Inter-observer agreement on point classification  was assessed using Cohen’s Kappa statistic. TSR was log-transformed prior to analysis to allow identical inferences for both tumour and stroma content to be made. Time-dependent survival analysis was used to optimise the TSR cut-off using sis in R . Kaplan-Patient characteristics are summarised in Table Including all histological types, the median percentage fraction of tumour was 66.0 % range 12.7–92.2 %) whilst the median percentage fraction of stroma was 20.1 % (range 2.0–81.2 %). The median TSR was 3.3 (range 0.16–45.20). TSR cut-off optimisation identified a TSR cut-off of 1.3 for OS which, in an idealised sample with only tumour and stroma scores, would correspond to a tumour-stroma ratio of 56.5 %:43.5 %. Representative images of TSR low and TSR high tumours are depicted in Fig. .7–92.2 %P = 0.032) and showed a trend towards associating with poorer DFS (P = 0.058) (Table P = 0.009) and DFS (P = 0.015) . TSR was also higher in the tumours of patients aged ≥75 years compared with patients aged <55 years, although this association was weaker (P = 0.019). TSR was not significantly associated with any other clinicopathological variable, including stage, histopathological subtype, depth of myometrial invasion, lymph node status or cervical involvement  as well as in tumours with lymphovascular invasion , which have been shown to be active players in tumour progression and metastasis through their bidirectional interactions with cancer cells (as well as other cells within the tumour microenvironment) via cytokine/growth factor mediated signalling and extracellular matrix remodelling –39. Howefeatures . As yet,The advantages of this study are its large cohort size and comparatively long follow-up period. Another advantage is the use of a digitised scoring method, which provides a framework for the objective measurement of TSR. One potential limitation is the heterogeneity of EC subtypes included in the study. However, TSR was not found to differ significantly between endometrioid/non-endometrioid/mixed histology subtypes, thus justifying such an inclusive approach.In summary, this study shows that low tumour stromal content associates both with poor outcome and adverse prognostic features endometrial cancer, although it is not independently prognostic. These findings are consistent with the idea that the biology of tumour-stroma interactions and their prognostic influence are not universal amongst epithelial tumours.


Giant cell tumours (GCTs) of the tendon sheets in fingers are rare. We therefore find it of interest to report on 5 cases identified in the Inuit population in Greenland within 16 months prior to this study.The Inuit account for 56,000 people of the total population in Greenland. From November 2010 to 16 months prior to this study, we diagnosed 5 cases  with a GCT of the flexor tendon sheet of a finger. The patients were aged between 10 and 54 years, and 4 were women. All of them had noticed slow-growing tumours over 3 or more years and were referred for a suspected ganglion.In two cases, the tumour was located at the distal interphalangeal (DIP) joint in the thumb and in one case at the third finger. Two other patients had tumours at the metacarpophalangeal (MCP) joint of the third finger and the thumb, respectively; one of these two had a communicating tumour to the DIP joint. The last patient had two tumours on the same finger, one at the MCP joint and the other at the DIP joint. In one case, the tumour had also eroded the cortex of the first phalanx of the thumb, and the largest tumour measured 5 cm.GCTs of the flexor tendon sheets in fingers are rare. It could be a coincidence that we have seen 5 cases within a short period of time. It is not possible to identify past cases through a register. A tumour in a finger is not the most common location for a ganglion, especially not at the DIP level. Therefore, a large tumour at this location is more likely to be a GCT. Giant cell tumours (GCT) in the tendon sheets in fingers are benign, slow-growing tumours. They originate from the synovium in the tendon sheets .This type of tumour is the second most common type of tumour found in the hand. It is surpassed only by ganglion cysts ,2. The aThe treatment of choice is operative removal, but the recurrence rate is high, up to 44% has been reported  in a population of 400,000 . We therIn the period from November 2010 to 16 months prior to this study, we diagnosed 5 patients with GCTs in the tendon sheets of fingers . Patients were aged between 10 and 54 years, and 4 were woman. Two of the patients have had a previous operation more than 3 years ago in their home village in connection with the yearly visit of an orthopaedic surgeon. The operation had been performed under local anaesthesia for a suspected synovial ganglion. Microscopy was performed in one of the two cases. This confirmed it was a GCT. Both had recurrence of the tumour within months.However, all 5 patients were admitted to the capital city suspected to have synovial ganglions, and all of them had a medical history of a slow-growing tumour over several months or years.During the operation, we found the tumour adherent to the flexor tendon sheet in 2 cases; the first patient had a tumour at the metacarpophalangeal (MCP) joint of the right third finger, and the second patient had 2 tumours, one at the MCP joint and the other at the distal interphalangeal (DIP) joint, both in the left third finger. In one case, the tumour was adherent to the extensor tendon but continued around the finger to the flexor tendon sheet at proximal interphalangeal (PIP) joint level of the third finger. In the last 2 cases, the tumour was adherent to the extensor tendon, DIP joint of the left third finger and DIP joint of the right thumb.In one of the patients with recurrence of the tumour, bony destruction had appeared on the first phalanx of the thumb . The tumDuring a follow-up 3 years after surgery, 3 of the 5 patients clinically had recurrence of excised tumours, of which one has had another surgery where it was histologically confirmed to be a GCT.In the 16 months prior to this study, we have operated 5 Inuit patients in Greenland with a GCT in fingers. The aetiology of tumour development is unknown, so it may be a coincidence. A genetic disposition or exposition from the environment may be possible. All 5 cases were found among those born to Inuit parents in Greenland.It was impossible to trace other cases of GCT or ganglion cysts treated in the past. Ganglion cysts present themselves mainly at the dorsal side of the wrist 60–70%) (0% (7).Slow-growing finger tumours, especially appearing at DIP levels in fingers (which is not an uncommon place for GCTs) therefore has to be suspected to be a GCT instead of ganglion cysts, which is not an uncommon place for GCT.


It was conducted as a primary study for vaccine implementation, and to investigate the association of climatic and socioeconomic factors with the epidemiology of this infection. In this cross- sectional study, anti VZV antibodies were measured in serum samples obtained in a national school-based health survey (CASPIAN- III). Association of demographic, socio-economic, and climate of the living region with the frequency of VZV was investigated by multivariate multilevel analysis. Overall, sera of 2753 individuals aged 10–18 were tested for VZV antibodies, from those 87.4% were positive. The prevalence was statistically different in four socio-geographic regions (P<0.001), varying between 85.24% in West region (mostly mountainous areas with cold climate) to 94.59% in Southeast region . Among variables studied, only age and mean daily temperature of the living area were positively associated with the VZV seroprevalence. Our findings show that most Iranians develop immunity to VZV before the age of 10, but a substantial proportion of them are yet susceptible to the infection. Therefore, it seems that the best strategy to reduce the burden of the disease is to vaccinate high- risk adults, i.e. those without a history of varicella infection. The regional temperature might be the only determinant of VZV epidemiology in Iran.The objective of this study was to evaluate the  Varicella zoster virus (VZV). Although varicella is typically a self-limited illness, the course may infrequently be complicated by secondary bacterial infections, transient cerebellar ataxia, and disfiguring scars. Life threatening events including pneumonia, necrotizing fasciitis, encephalitis, septicemia, and disseminated varicella rarely may be observed, as well [Chickenpox (varicella) is an extremely infectious viral disease with universal distribution, which is caused by  as well . Congeni as well . After p as well . In gene as well .While VZV-specific immune globulin or antiviral medications can prevent or alleviate severe varicella infection and may reduce the risk of complications, the only way for controlling varicella in a community is extensive VZV immunization . DocumenSo far, varicella vaccine is not part of the Iranian national immunization program and its usage is not allowed in private clinics . On the Limited seroprevalence studies have been conducted in Iran about the susceptibility of adult population to varicella infection; most of them have been conducted in restricted areas of the country and have included specific groups such as health care workers, medical students, and premarital or pregnant women. Thus, such findings cannot serve as a representative sample of the whole population . A systeEpidemiology of varicella is apparently different according to the climate: while in temperate regions, up to 90% of children are infected before the age of 10, only small proportions of children are seropositive in tropical and subtropical regions. Thus, the susceptibility to varicella is more common among adults living in tropical regions than in temperate climate . Iran isThe aim of this study was to evaluate the frequency of antibodies to VZV in a large population of Iranian adolescents living in different regions of the country, as a guiding data for policy making about vaccine implementation, and to investigate the effect of climatic and socio-economic factors on epidemiology of this infection.In this multicenter cross-sectional study, serum samples obtained in a national health survey were analyzed. This national school-based health survey (CASPIAN-III study) was conducted in 2009–2010 on 10–18 year-old students. Participants were recruited through multistage, cluster sampling method from 27 provinces of Iran, as explained previously . EligiblIn the current study, residual samples from 20 provinces were tested at the laboratory of Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences. Some samples including those from remaining seven provinces were either unsuitable for testing or unidentifiable because of missing codes and were excluded from the study.Demographic data and related household risk factors were extracted from CASPIAN-III database. Using principle component analysis variables including parents’ education, parents’ job, possessing private car, school type (public/private), type of home (private/rented) and having personal computer at home were summarized in one main component named socio-economic status (SES) score. A lower score corresponds to a lower SES. The method and variables which was used for calculating SES of family was approved previously in the Progress in the International Reading Literacy Study (PIRLS) for Iran .Regional information for each province such as population density (the ratio of population to area for each province) and budget (the ratio of income to costs for each family) were obtained from 2011 national census data . ClimatiTotal antibodies to VZV (IgM/ IgG) were measured by a commercial enzyme linked immunosorbent kit  according to the kit instruction. The sensitivity and the specificity of the test were 98% and 97% respectively.The CASPIAN-III survey was approved by the institutional review boards of the Ministry of Health and Medical Education and the Ministry of Education and Training, as well as the collaborating universities at provincial level. The current study was also approved by the ethical committee of the Alborz University of Medical Sciences (project number: 2015.65.786). Written informed consent and oral assent were obtained from parents and students, respectively.Continuous and categorical variables are expressed as mean (SD) and number (percentage) respectively. Qualitative and quantitative variables across regions were assessed using Chi square and ANOVA test respectively. Considering the effect of mix of individual level and provincial level factors on the frequency of VZV, besides taking account of clustering (provinces and regions) in our study, we used hierarchical multilevel modeling. Generalized Linear Mixed Models were used to perform the univariate and multivariate multilevel analysis to evaluate association of VZV seroprevalence with independent variables. Individual- and provincial- level variables that were associated with VZV seroprevalence in univariate analysis (P<0.2) were successively included in a multivariable multilevel model. Results of univariate and multivariate multilevel analysis are presented as odds ratio (OR) and 95% confidence interval (CI). Statistical analysis was performed using R language (R Package “lme4”) , 19. P-vOverall, 2753 individuals aged 10–18 were tested for VZV antibodies, of them 2406 (87.40%) had positive test. The positive percentages for socio-geographical regions were 94.59% for Southeast, 89.94% for North-Northeast, 85.24% for West and 86.31% for Central region. Considering individual-level variables, the age and the sex had similar distribution across regions, while the number of children in a family, living area , and SES scores were statistically different between regions. Among provincial level covariates, budget and humidity were significantly heterogeneous across regions (p<0.05).Four significant variables of age, number of children in a family, mean daily temperature, and budget were obtained from univariate models; including them in a final model showed that one individual level variable  and one provincial-level variable  were associated with the VZV seropositivity at the 0.05 level of significance .In this study, the overall seroprevalence of VZV in 10-18-year-old adolescents was 87.40%, ranging from 85% to 95% in different socio-geographic regions, and from 65% to 100% in different ages.This prevalence is much higher than some previous reports from Iranian teenagers. In a systematic review conducted on reports from Iran, VZV prevalence was reported as 59.44%  in 11-15-year-old children and 75.93%  in 16-20- year-old adolescents of nine provinces of Iran  and the Although in our study, significant difference was documented in the VZV seroprevalence according to the region and age groups, but the ratios were not so divergent; the findings indicate that at age of 10, about 20% of Iranian children are susceptible to this infection, and at age of 18, the susceptibility declines to less than 10% in all four socio-geographic regions. It seems that the non-representative method of sampling or inadequate number of participants in previous studies can explain the differences of the current findings with some of the previous studies in Iran.Our study revealed that although varicella is a childhood disease in Iran, but a substantial proportion of adolescents, i.e. about 20%, are at risk of developing this infection. VZV vaccination may abolish severe form of this disease in immune deficient patients, and may reduce complications as secondary bacterial infections and neurologic complications in normal children. Though, there are many concerns about introducing routine childhood vaccination at national level, particularly because there is no estimate of public health impact and socio-economic burden of the infection and cost effectiveness of universal vaccination, which need to be provided by a national surveillance system .In this situation, it seems that the best strategy to control the VZV infection is to offer the vaccine to individual adolescents and adults without a history of varicella, in particular to at- risk population including women of childbearing age, health care providers, day-care personnel, teachers, college students, and military personnel. This recommendation is in accordance to the WHO statement for VZV immunization in developing countries, which prefer the introduction of other new vaccines with much greater public health impact, such as rotavirus, and pneumococcal vaccines, prior to introducing varicella vaccine in routine childhood immunization programs .Iran is placed in the temperate zone of climate with the southern regions extending to the subtropical area and generally with an arid climate. Our results are roughly in agreement with the pattern of VZV prevalence in temperate regions such as Europe, North America, and Japan in pre-vaccination era –26. An iLimited information is available about prevalence of VZV in countries neighboring Iran. In Pakistan, a tropical pattern of VZV epidemiology was found with a seroprevalence of 42.5% in the 11-15- year age group and 46.7% in the 16-20- year age group . Our finIn our study, the Southeast region, with the subtropical climate, had the highest VZV seroprevalence, but because of diverse climates existing adjacent throughout the country, it is difficult to assign a specific climate to the mentioned socio-geographic regions. We found that regardless of age, the only risk factor associated with VZV seropositivity was the annual mean daily temperature: higher temperatures were associated with higher rates of seropositivity.Meteorological factors have been identified as the main determinants of VZV epidemiology in many parts of the world. Temperature might affect survival, transmission, and also the severity of presentation of VZV infection (mild or severe disease), and the effect of temperature might be different in tropical and temperate regions. Generally, it is known that the incidence and prevalence of VZV primary infection is higher in temperate regions than tropics , 31. SevWe found that gender and socio-economic factors including living area , ratio of income to costs, population density, number of household siblings, as well as parental literacy and job were not important risk factors for VZV infection in Iranian adolescents. Higher age group was related to increased seropositivity; however some discrepancy existed in the age- specific prevalence of VZV in different regions. This might be due to limited outbreaks in previous years or because of small sample size of some age-region subgroups.Having five or more siblings in the household and lower household budget were related to higher VZV seropositivity, but differences were not significant in multivariate model. Household size has been considered as a risk factor for VZV transmission in tropical areas like Guinea Bissau with an extremely large household sizes (mean 14.5 people) . But in Study limitations: The first limitation is that we were not able to include all age groups in our study, thus we cannot provide a complete picture of VZV epidemiology in Iran. Likewise, data from a number of provinces were not available for this study. However, this survey is the first representative nationwide seroepidemiological study about VZV in Iran and also in the Middle East and North Africa (MENA) region; studied provinces were from different socio-geographic regions, covering more than 80% of the total adolescent population. For compensating the likely occurring bias because of depletion of samples of some provinces, we assessed pooled data of four socio-geographic regions for determining risk factors of VZV infection across the country.Conclusion: Natural VZV infection immunizes most of Iranian children before the age of 10, but a substantial proportion of adults and adolescents of the country are susceptible to the infection and are at the risk of developing severe and complicated infections, as well as congenital varicella syndrome, which might have serious health effects. Therefore, it seems that the best strategy for controlling the infection is to offer the vaccine to those adolescents and adults who do not have a history of varicella, in particular to those at increased risk of contracting or spreading the infection.


Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTORhigh) or mTOR inhibitor (aH3/aH4low) alone.Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation . Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt , H3 and H4  were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTOR The development of targeted drugs has significantly enriched the armamentarium of anti-tumour agents. As the phosphatidyl-inositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is aberrantly activated in many tumour types, mTOR suppression is regarded to be an attractive approach for cancer therapy . Two mTOet al. have suggested that the therapeutic integration of a HDAC inhibitor in anti-tumour protocols might distinctly improve the strategy to handle advanced malignancy . A mouse IgG1-PE or IgG1-Alexa  was used as an isotype control. Fluorescence data were related to fluorescence values of the unstimulated controls which were set to 100%.IGFr expression and activity, which is a necessary prerequisite to transduce growth factor-related signalling to the Akt-mTOR pathway was explored by flow cytometry. DU-145 cells were kept in serum-free cell culture medium overnight and then treated with different concentrations of IGF for different time periods. Subsequently, cells were washed in blocking solution  and then incubated for 60 min. at 4°C with phycoerythrin (PE)-conjugated monoclonal antibodies directed against IGFr  or phospho-IGFr . Receptor surface expression was then measured using a FACScan [Becton Dickinson, Heidelberg, Germany; FL-2H (log) channel histogram analysis; 1 × 10To verify that IGFr-triggered Akt-mTOR activation directly contributes to histone modification, IGFr blocking studies were carried out. DU-145 cells were pre-incubated for 60 min. with a function blocking anti-IGFr monoclonal antibody  under serum-free conditions. Controls remained untreated. IGF (100 ng/ml) was then added and the following proteins analysed by western blotting: pIGFr, pRaptor, pRictor, pAkt, aH3 and aH4. In addition, IGFr protein synthesis was down-regulated by small interfering RNA (siRNA) directed against IGFr  with a siRNA/transfection reagent  ratio of 1:6. Non-treated cells and cells treated with 5 nM control siRNA  served as controls. Subsequently, the consequences of IGFr knock-down for histone H3 and H4 acetylation were analysed by the western blot assay using aH3 and aH4 antibodies, as indicated above.To explore whether histone acetylation specifically acts on the Akt-mTOR pathway, DU-145 cells were transfected with siRNA directed against HDAC1  or HDAC2 , with a siRNA/transfection reagent  ratio of 1:6. Non-treated cells and cells treated with 5 nM control siRNA  served as the controls. Thereafter, aH3, aH4, pIGFr, pRictor, pRaptor and pAkt were analysed by western blotting.U-test. Differences were considered statistically significant at a P < 0.05.All experiments were performed 3–6 times. Statistical significance of the flow cytometry experiments was determined with the Wilcoxon–Mann–Whitney IGFr surface expression is a necessary prerequisite to growth factor-evoked activation of the Akt-mTOR signalling pathway. Therefore, the content of total membranous IGFr as well as phosphorylated IGFr in the DU-145 cells was evaluated. Figure Activation of DU-145 cells with IGF caused a distinct increase in pmTOR, pRaptor, pRictor, pAkt and pp70s6k, compared to the controls Fig. A. IGF alTo investigate whether H3 and H4 acetylation is a down-stream event following IGFr-Akt-mTOR activation, tumour cells were incubated with a function blocking antibody directed against IGFr. Receptor blockade not only diminished pIGFr, pRaptor, pRictor and pAkt, but also lowered histone H3 and H4 acetylation  fashion, on pIGFr, pRictor and pAkt in a negative (tumour-promoting) fashion, whereas blocking the IGF-driven mTOR pathway induced tumour-suppressive effects on pIGFr, pRictor, and pAkt, but induced tumour-promoting action on aH3 and aH4. Therefore, the mTOR/HDAC inhibitor combination might create a molecular scenario, where particular pro-tumour effects exerted by one drug will be negated by a partner drug. This means loss of aH3 and aH4 ug alone . Combiniug alone . HDAC- pug alone . Finallyug alone . Applyin of HDAC .in vitro studies. To evaluate the therapeutic relevance, an animal model would be required. Promising results have come from a phase I study in patients with relapsed or refractory Hodgkin and non-Hodgkin lymphoma where panobinostat and everolimus in combination showed favourable clinical effects [Overall, evidence has been presented that HDAC-mTOR cross-linking exists in tumour cells, and combined application of an HDAC- and mTOR inhibitor might overcome limitations encountered with a mono-drug regime. This conclusion is drawn from  effects . It migh


Journal of Experimental Botany, Vol. 65, No. 2, pp. 755–766, 201410.1093/jxb/ert455doi:The Acknowledgements section reads that this study was financially supported by the Distinguished Scientists Program, King Saud University, Riyadh, Saudi Arabia (grants to HR and RH).In fact, this work on Rainer Hedrich’s side was funded by an ERC grant.The corrected Acknowledgements section is reproduced below.AcknowledgementsThis study was financially supported by the Distinguished Scientists Program, King Saud University, Riyadh, Saudi Arabia (grants to HR) as well as funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/20010–2015)/ERC Grant Agreement number 250194-Carnivorom (grant to RH).