The conventional mean-field kinetic models describing the interplay of cancer and the immune system are temporal and predict exponential growth or elimination of the population of tumour cells provided their number is small and their effect on the immune system is negligible. More complex kinetics are associated with non-linear features of the response of the immune system. The generic model presented in this communication takes into account that the rates of the birth and death of tumour cells inside a tumour spheroid can significantly depend on the radial coordinate due to diffusion limitations in the supply of nutrients and/or transport of the species (cells and proteins) belonging to the immune system. In this case, non-trivial kinetic regimes are shown to be possible even without appreciable perturbation of the immune system. Cancer occurs via initiation, tumour growth, and propagation of metastases. During these stages, its development depends on the response of the immune system  and nt), effector cells (ne) and interleukin-2 (np) are as follows [k, we, wp, κe and κp are the birth and death rate constants and rates, n∗ is the maximum population of tumour cells, γ is the rate constant of elimination of tumour cells and v1, v2, v3, m1, m2 and m3 are the other rate constants and parameters characterizing the function of the immune system. The analysis of Eqs. dne/dt = dnp/dt = 0. Describing the initial phase of the growth of a tumour, one can in addition neglect the effect of tumour cells on the immune system. Mathematically, this means that the third term on the right-hand part in Eq. nt/n∗ in the first term and nt in the denominator of the second term in Eq. ne, Eq. To illustrate the general introduction above and to articulate the novelty of my report, I recall one of the conventional temporal models  is represented as:D is the nutrient diffusion coefficient, and η is the rate constant associated with the nutrient consumption by tumour cells. The boundary condition for this equation is:c∘ is the nutrient concentration outside the tumour. Taking into account that on the time scale of the tumour growth the nutrient diffusion inside the tumour is rapid, Eq. ∂c/∂t = 0. The corresponding textbook solution of Eq. λ ≡ (D/η)1/2.The growth of the population of tumour cells is considered to be determined by the nutrient distribution inside a tumour spheroid. The corresponding equation for the nutrient concentration at c(r). In this approximation, the total birth rate can be represented as:k is the birth rate constant (c∘ is considered to be included into this rate constant), knt is the birth rate calculated assuming the diffusion limitations to be negligible, andR/λ for the convenience of its derivation as it is usually done in the literature focused on the kinetics of catalytic reactions occurring inside grains 1/3λ.The local birth rate of tumour cells can be considered to be proportional to y Thiele ). In therelation  into accc, D, η and λ by the corresponding parameters, c∗, D∗, η∗ and λ∗≡ (D∗/η∗)1/2. Then, by analogy with Eq. γ is the death rate constant, and χ∗≡ (4π/3v)1/3λ∗.The elimination of tumour cells is considered controlled by the species (cells or proteins) belonging to the immune system. Mathematically, this process can be described in analogy with Eqs. k > γ and k < γ, respectively.If the population of tumour cells is small 16), one Thus, the model clearly shows that, with inclusion of spatial features, non-trivial kinetic regimes of tumour growth may be possible even at relatively small populations of tumour cells in the situations when the effect of tumour cells on the population of the species (cells and proteins) belonging to the immune system is negligible. Physically, this is related to the transition from the kinetically limited birth and death to diffusion-limited birth and death with increasing population of tumour cells. For birth and death, this transition can easily take place at different populations of cells, and it can result in the appearance of a non-trivial stable or unstable steady state.Finally, I can add that the model under consideration can be extended in different directions. For example, its current version implies that the tumour cells are of one type. In reality, the population of tumour cells is well known to be heterogeneous , 20, and


Streptococcus mutans on fabricated discs for 24 h. When S. mutans was cultivated on the discs under no sucrose conditions, the loaded protamine was not released, and the ratio of dead bacteria increased on the surface of P (125) DCPA  was loaded). Aside from P (500) DCPA (saturated level of protamine was loaded), some protamine was released, and the number of planktonic bacteria in the supernatant decreased. Using medium containing 1% sucrose, the release of protamine was promoted from P (125) DCPA due to lowered pH. However, lowering of the pH decreased the antimicrobial activity of protamine. On the other hand, P (500) DCPA released protamine before the pH was lowered, and biofilm formation was inhibited. The loaded protamine expressed antimicrobial activity, both on the surface of the materials and in the surrounding environment. The interaction of loaded protamine with calcium phosphates could promote the application of protamine in the dental field. Protamine is an antimicrobial peptide extracted from fish. In this study, we loaded protamine onto dicalcium phosphate anhydride (DCPA), a dental material. Protamine was loaded by stirring DCPA into a protamine solution. To explore the antimicrobial activity of the materials, we cultivated  Protamine is an arginine-rich polycationic protein extracted from the sperm cells of vertebrates, including fish such as salmon . It has 4), which is generally mixed with toothpaste for cleaning teeth. DCPA is used, not only as a cleaning agent, but also as a tubular occluding material to suppress dentin hypersensitivity (DH). In fact, DCPA has been used in some desensitizers, showing great potential for reducing dentin permeability [Due to the antimicrobial activity and low toxicity of protamine, we applied protamine to dental materials. Among these materials, we focused on dicalcium phosphate anhydride  in vitro. S. mutans is one of the many etiological factors of dental caries and can rapidly form cariogenic biofilms via dietary sucrose [The aim of this study is to evaluate the efficacy of protamine-loaded DCPA (P-DCPA) on the growth and biofilm formation of  sucrose . Studies2 pot using ZrO2 beads with 10 mm diameter in 40 cm3 of ultrapure water. After ball-milling, the slurry was filtered and freeze-dried for 24 h. Ten grams of commercially-available DCPA powders  was ground using a planetary mill  for 180 min at a rotation rate of 300 rpm in a ZrO−3) were diluted by ultrapure water. Freeze-dried DCPA powders (1.5 g) was added into a protamine solution (45 cm3) and rotated with a constant speed of 60 rpm for 48 h at room temperature. The samples were centrifuged for 15 min at 8000 rpm, and supernatants were removed. Powders were washed with ultrapure water 3 times and freeze-dried. The resulting powders were uniaxially compressed at 20 MPa to form compacts  for antimicrobial evaluation. Hereafter, powders loaded with protamine are denoted by P (x) DCPA; x represents the concentration of protamine. Protamine hydrochloride was kindly supplied by Maruha Nichiro Corporation. Protamine solutions , and the filtrates were assayed for HPLC analysis. The amounts of loaded-protamine were measured by HPLC analysis . In brief, 100 mg of powder was added to 0.45 cmThe particle size distribution was measured using a laser diffraction particle size analyzer  in distilled water at a range of 0.01–300 μm. Before measurement, samples were homogenized for 15 min.The zeta potential of powders was measured using a laser-doppler velocimeter  in 10 mM NaCl solution at 25 °C. The zeta potential for each sample was calculated from the measured value of the electrophoretic mobility. Streptococcus mutans (S. mutans) was used for the evaluation of antimicrobial testing. S. mutans is one of the main etiological agents of dental caries and is a well known biofilm-forming oral bacterium. Strain of S. mutans, purchased from NBRC , was used in the study. The strains were grown in a Trypticase Soy Yeast Extract Medium (TSYE media) in an anaerobic jar  at 37 °C. The cultures were stored at −80 °C in medium containing 25% glycerol.S. mutans. In the liquid culture media, protamine reacts with the components [In this study, we used TSYE media prepared to 1/10 concentration to cultivate mponents ,10. S. mutans were grown on each disc (P (0) DCPA, P (125) DCPA, P (500) DCPA) set on a 24 well plate. Each well in the plate was inoculated with 1.5 cm3 of bacterial suspension (4 × 106 CFU·cm−3) in 1/10 concentration TSYE media. After incubation anaerobically at 37 °C for 24 h, supernatants were removed and the discs were washed twice with Phosphate Buffered Saline (PBS). ® BacLightTM Bacterial Viability Kit  for 15 min in the dark. Propidium iodide (PI) specifically labels the dead cells with damaged cytoplasmic membranes in red, whereas live cells are labeled in green by SYTO®9. The stained bacteria were observed by fluorescence microscope .The pieces of each disc were stained using the LIVE/DEADTo observe the morphology of bacteria attached to the discs, other pieces of each disc were fixed with 2.5% glutaraldehyde overnight at 4 °C. Fixed discs were freeze-dried overnight and coated with gold. Each sample was observed with SEM  at 10 kV. As for the removed supernatants, planktonic bacteria were quantified by colony count, and free protamine concentration was determined by Bradford assay kit (Fujifilmwako). pH value was measured by pH meter . Further calcium concentration was measured by Inductively Coupled Plasma-Atomic Emission Spectrometry . 3 of bacterial suspension (4 × 106 CFU·cm−3) in 1/10 concentration TSYE media including 1% sucrose. After incubation anaerobically at 37 °C for 24 h, supernatants were removed, and the discs were washed twice with PBS. Biofilms were formed on each disc (P (0) DCPA, P (125) DCPA, P (500) DCPA) set on a 24 well plate. Each well in the plate was inoculated with 1.5 cm®9 and exopolysaccharides (EPS) were labeled with 1.0 μM Alexa Fluor 594 dextran conjugate . Fluorescently labeled dextran serves as a primer for Gtfs and can be incorporated during EPS matrix synthesis [The bacterial cells were labeled with 5.0 μM SYTOynthesis . PlanktoS. mutans was diluted to 4 × 106 CFU·cm−3 using TSYE media  prepared to 1/10 concentration. Protamine solution (0.15 cm3) , diluted by ultrapure water, and 1.35 cm3 of bacterial suspension were added to a 24 well plate. After incubation for 24 h at 37 °C, the number of bacteria in the suspensions was quantified by colony count. The antimicrobial effects of protamine in different pH conditions was tested by colony count. The pH of each media was adjusted  using 1M HCl. An overnight culture of t-test was performed with a significant level of p < 0.05. All the data were statistically analyzed to determine the mean and the standard deviation of the mean. The Student’s −3  DCPA, P (125) DCPA, and P (500) DCPA) are shown in ®9 showed green fluorescence, whereas dead bacteria stained with propidium iodide (PI) showed red fluorescence. Compared with the control disc (P (0) DCPA), both protamine-loaded DCPA discs affected cell viability. The ratio of dead bacteria/live bacteria increased with an increase in the amount of loaded protamine  DCPA disc, whereas we could not detect protamine in the supernatant of the P (125) DCPA disc  and P (500) DCPA discs, and that the pH of the supernatants was sustained at around 6 when We could not observe the release of protamine from P (125) DCPA nor a significant reduction of planktonic bacteria in the supernatants. In contrast with the planktonic bacteria in the supernatants, the surface of the P (125) DCPA disc exhibited antimicrobial activity. As for P (500) DCPA, about 10% of the loaded protamine was released from the disc, and significant antimicrobial effects were observed in the supernatant. Furthermore, antimicrobial activity on the disc surface was confirmed. S. mutans synthesize adhesive glucans from sucrose, and glucans mediate the firm adherence of its cells to the tooth surface [S. mutans in normal medium with 1% sucrose and examined the antimicrobial activity of protamine-loaded DCPA. The three-dimensional images of the biofilm stained by SYTO®9 and dextran conjugates are shown in ®9 showed green fluorescence, whereas extracellular polysaccharides (EPS) stained by dextran conjugates showed red fluorescence. Compared with the P (0) DCPA and P (125) DCPA discs, there was no difference in biofilm thickness and EPS production. On the other hand, P (500) DCPA inhibited biofilm formation, particularly EPS production  DCPA and P (500) DCPA disc surfaces  DCPA disc was lower than other samples A. −3 protamine in the supernatant  DCPA disc (pH 5.9) were significantly higher than those of the P (0) DCPA (pH 4.2) and P (125) DCPA (pH 4.3) discs B. Due toernatant C. In a cultivation environment containing 1% sucrose, the pH of the supernatants dropped to around 4, with biofilm formation. For the P (500) DCPA sample, the disc released protamine before lowering the pH, and the released protamine expressed antimicrobial activity in the supernatant. In the case of P (125) DCPA, the lowering of the pH with biofilm formation promoted the release of protamine from the disc with dissolution of the DCPA powders. However, there was no significant decrease in the number of planktonic bacteria when protamine was released. This result suggests that the change in pH influenced the antimicrobial activity of protamine in the sample supernatants. S. mutans with 250 μg·cm−3 protamine under different pH conditions . After incubation for 24 h, no bacteria were detected when treated with protamine in a culture environment of pH 7, 6, and 5 (2 in protamine compared with the control (no protamine). These results suggest that a decrease in pH reduces the antimicrobial activity of protamine.To investigate the effect of pH against the antimicrobial activity of protamine, we incubated 6, and 5 . HoweverS. mutans dissolves hydroxyapatite [Several desensitizers have been introduced for hypersensitivity treatment. These treatments are mainly categorized as tubular occlusion and blockage of nerve activity . Above ayapatite . BacteriIn this study, we have prepared DCPA powders by loading protamine as an antimicrobial agent. Protamine generally consists of 20 arginine molecules from a total of 30 amino acids . Due to S. mutans on the fabricated discs. In the case of P (125) DCPA, which loaded half of the saturated level of protamine, the ratio of dead bacteria on the surface of the disc increased, and protamine was not released from the materials  DCPA, which loaded the saturated level of protamine, released approximately 10% of the protamine from the disc into supernatants, which had a pH of around 6. A previous study demonstrated that protein adsorption to calcium phosphates, such as hydroxyapatite, employs multilayer adsorption. As a result of the increased distance as the number of layers increases, the electrostatic interaction between the protein molecules and the material surfaces became weaker . TherefoS. mutans. The mechanism of its antimicrobial activity followed two patterns: i) direct contact with bacteria and the material surfaces; and ii) releasing protamine to the surrounding environment. Additionally, the pH value affected the release of protamine and its antimicrobial activity. In particular, in conditions of low pH, more protamine was released from the materials at a higher rate. However, how protamine was released from materials in oral saliva, which contains high levels of ions and other proteins, is not clear. The mechanism of the adsorption and release of protamine therefore requires further investigation. Additionally, toxicity testing of protamine in oral tissue is also necessary.In conclusion, this study demonstrated that protamine-adsorbed DCPA powders expressed antimicrobial activity against Many kinds of dental materials, such as resin composites, contain calcium phosphates ,29. It i


Data presented in this article focused on the application of Methyl Ester Sulphonate (MES) surfactant and nanopolystyrene in water based drilling fluid. Data from rheology study using Bingham and Power law models showed that the synergy of MES and nanopolystyrene improved the formulated drilling fluid. Filtration study under LPLT and HPHT conditions showed that MES and nanopolystyrene drilling fluid reduced filtration loss by 50.7% at LPLT and 61.1% at HPHT conditions. These filtration data were validated by filter cake permeability and scanning electron microscope images. Specifications tableValue of data•Rheology result shows that methyl ester sulphonate an anionic surfactant affect water based drilling fluid properties: Yield Point (YP), Plastic Viscosity (PV) and flow behaviour characteristics.•Synergy of methyl ester sulphonate and nanopolystyrene was effective to control fluid loss at low and high temperature environment.•Filter cake formed after LPLT and HPHT filtration, showed a thin non-erodible low permeability filter cake which supports low fluid loss data obtained. This is further validated by Scanning Electron Microscope measurement.•The overall result obtained from this work encourages the usage of environmentally friendly additives and environmental waste material as drilling fluid rheology and fluid loss control additives.1Recently nanotechnology has seen many applications in oil and gas operations, from drilling to enhance oil recovery. Nanoparticles application in drilling fluid design has been reported in 2Methodology for testing drilling fluid as recommended by American Petroleum Institute for water based drilling fluid (API 13B-1) was used for all rheology and filtration test.2.1Methyl Ester Sulphonate (MES) surfactant (279.7 g/mol), nano-polystyrene (25 nm) and bentonite. Other drilling fluid additives includes: sodium hydroxide (NaOH), barite and poly anionic cellulose (PAC).2.2Base fluid was prepared by the addition of 15 g of bentonite to 350 cc of fresh water and stirred continuously for 15 minutes using 11,000 rpm mixer. 1 g of PAC was added and stirred for 5 minutes and 150 g of barite was added and stirred for another 10 minutes to prepare sample C. Sample D drilling fluid was prepared by dissolving 0.1 g of MES in deionize water and prepare 0.01 wt% nanopolystyrene with the MES solution. The final solution was ultrasonicated for 15 minutes to achieve good dispersion of nanopolystyrene in MES solution. Zeta potential measurement of dispersed nanopolystyrene in MES solution and polydispersity index are -41 mV and 0.15 respectively, which is indicative of stable nanofluid and well dispersed. 50 cc of prepared nanofluid was added to 300cc of sample C drilling fluid and mixed for 10 minutes. Two other drilling fluid was prepared: one with MES alone and base fluid (sample A) and the other is nanopolystyrene and base fluid (sample B). 2.3Rheology of designed drilling fluid was measured with a viscometer following API standard procedure recommended for water based drilling fluid (API 13B-1). Rheology measurement readings were carried out three times to ensure consistency and repeatability of the results. Rheological parameters of Bingham and Power law models including shear stress-shear rate relationship, plastic viscosity, yield point, gel strength, consistency and flow behavior index are determined. These rheology parameters were determined using the following:2.4Low Pressure Low Temperature (LPLT) filtration loss measurement conducted at operating condition of 100 psi and 24 °C. Filtrate volume was collected every 5 minutes for total filtration time of 30 minutes after which thickness of filter cake was measured. High Pressure High Temperature (HPHT) filtration loss was conducted at operating condition of differential pressure of 500 psi (600 psi of top regulator pressure and 100 psi of bottom backpressure) and temperature of 150 °C. Filtrate volume was monitored for every 5 minutes and accumulated filtrate volume after 30 minutes was recorded. Thickness of filter cake was also measured afterwards. Permeability of the filter cake was determined by the rate of filtration through the filter cake as described by Darcy׳s law. The Darcy׳s law calculation is as follows:2.5Filter cake obtained after HPHT filtration was collected and dried in an oven at 100 °C for 24 hours. After which the samples were collected and carefully packaged to be sent for SEM measurement.32. Sample D yield point satisfies the operating requirement. 2 values obtained indicate that PV and YP values obtained are good fit.Bingham plastic model and power law model were used to evaluate rheology properties of five drilling fluid samples. 2 values is indicative that K and n values are a good fit. Gel strength describes behavior of drilling fluid when the pump is inactive and its ability to suspend drilling fluid particles from settling down. Excessive high gel strength will require high pump pressure to re-start circulation. Drilling fluid without nanoparticles, has 10-minute gel strength of 6–8 lb/100 ft2Power law model shows that all drilling fluid formulations showed a shear thinning behavior as indicated by flow behavior index (n) values. The values of flow behavior index are less than unit, which implies that as active additives such as MES and nanopolystyrene was included in the formulation of sample D, its plastic viscosity increased. Consistency index (K) has to do with hole cleaning capacity, as the values of K increases the annular viscosity increases. Sample D has the highest K value which implies that addition of MES and nanopolystyrene is effective for wellbore hole cleaning. Power law model RFiltration property of drilling fluid is dependent on the colloidal material additive included in the drilling fluid. It can be concluded, based on the data presented that methyl ester sulphonate (MES) which dispersed the drilling fluid additives improved the rheology of sample D formulation. Meanwhile, addition of nanopolystyrene effectively reduced drilling fluid filtrate loss and resulted to a thin non-erodible and low permeability filter cake. This filtration data was validated by SEM images of filter cake.


CD36 has been repeatedly proposed as a prognostic marker in various cancers, mostly of epithelial origin  and also for hepatic carcinoma and gliomas. Data gathered in preclinical models of various cancers have shown that blocking CD36 might prove beneficial in stopping metastasis spread. However, targeting the receptor in clinical trials with thrombospondin mimetic peptides has proven ineffective, and monoclonal antibodies are not yet available for patient use. This review presents data to support CD36 as a potential prognostic biomarker in cancer, its current stage towards achieving bona fide biomarker status, and knowledge gaps that must be filled before further advancement towards clinical practice.Metastasis requires cellular changes related to cell-to-cell and cell-to-matrix adhesion, immune surveillance, activation of growth and survival signalling pathways, and epigenetic modifications. In addition to tumour cells, tumour stroma is also modified in relationship to the primary tumour as well as to distant metastatic sites (forming a metastatic niche). A common denominator of most stromal partners in tumour progression is CD36, a scavenger receptor for fatty acid uptake that modulates cell-to-extracellular matrix attachment, stromal cell fate , TGF Metastasis is a rather inefficient process if the number of circulating tumour cells is to be compared with the number of clinically overt metastatic sites . From vaPerhaps surprisingly, scrutinizing the less investigated stromal tumour tissue—or the modern and reinvented “stromal metastatic niche”—could provide some answers. The metastatic niche has been defined as “extracellular matrix, nonmalignant cells, and the signalling molecules they produce” . Differeβ activation, and immune signalling [A common denominator of most stromal partners playing a role in tumour progression is expression of CD36—a scavenger receptor for fatty acid (FA) uptake that modulates cell-to- extracellular matrix attachment, stromal cell fate , TGFgnalling . Unlike Several gaps remain in CD36-related knowledge. Learning from paths travelled in understanding other receptors as cancer-related biomarkers, one can argue that there are still plenty of blank spots on the CD36 cancer-related map. In cancer studies, CD36 is investigated mostly in relationship with TSPs, a family of matrix proteins acting as ligands, and most data are related to TSP interactions and, to a lesser extent, TGFß. Only a handful of papers have been published directly addressing CD36, almost all measuring levels of expression in relationship to tumour growth and metastasis. Furthermore, lessons from myeloproliferative diseases and gliomas suggest that mutated receptors do not require a ligand to be active. In the case of CD36, constitutive activation, regardless of TSP expression within the tumour or tumour niche, could be largely overlooked if the main focus falls on the ligand rather than the receptor.In addition, a parallel analysis of stromal CD36 versus tumour CD36 is usually missing from the scientific argument in most original articles. Last, but not least, studies in animal models using athymic mice remove a key player from the stroma, the immune cell, yielding an incomplete tumour–stroma interaction panel.What is the ultimate goal in CD36 research? One size fits all? Probably not. That pattern has not yet been the case for any molecular intervention in diagnostics, prognostics, or therapy. However, the chase for a single but efficient antimetastatic molecular target is justifiable from several perspectives. First and foremost, time is gained for diagnostic and therapeutic intervention. Second, absence of metastases will translate into an absence of cellular polymorphisms derived from environmental change. Thus, molecular therapies addressing the primary tumour could have a greater impact on relapse and survival rates.How far is CD36 from validated status as a biomarker and what type of biomarker would best fit are the questions the present review will attempt to answer, after analysing the evidence tying CD36 to the metastatic process and the translation into knowledge towards clinical practice.β1, β2, and β5) and tetraspanins  [β amyloid species [CD36, also known as platelet glycoprotein (GP) 4/, FA translocase (FAT)/, scavenger receptor class B member 3 (SCARB3)/, GP88, GPIIIB, or GPIV , is an ipanins CD, CD81 [99, CD81) . The ext9, CD81) ; (ii) co species . Plasmodium falciparum–infected erythrocytes.As a surface protein, CD36 is widely distributed. Found on platelets and monocytes/macrophages, it is involved in cellular activation. It not only mediates the initial binding of platelets on extracellular matrix protein like collagen I  and TSP- In vitro studies proved that CD36 is located in lipid rafts, along with caveolin-1, mediating FA uptake [CD36 expression was also detected in differentiated adipocytes . In vitrA uptake . Both fuA uptake .In skeletal muscle, CD36 expression on the cell surface is an important mechanism for FA uptake and short-term regulation through subcellular redistribution . HoweverCD36 also has been described on endothelial cells of human dermis, but only in the microvasculature and not in the large vessels  and in cAnother type of epithelial cell expressing CD36 is the taste receptor cell within lingual taste buds in tongue of rodents , pigs, a In vitro studies showed that caveolin-1 is required for the transport of CD36 to the apical membrane, thus regulating its surface availability for FA uptake [CD36 is expressed in the brush border membrane of duodenal and jejunal enterocytes  in both A uptake . MoreoveA uptake .Also at the intestinal level, CD36 was detected on enteroendocrine cells secreting secretin and cholecystokinin in the mucosa of the duodenum, jejunum, and proximal ileum on both apical membranes and cytosolic granules .γ, an upstream regulator of CD36 expression. Enhanced expression and subsequent fat uptake and triglyceride (TG) accumulation may accelerate progression of nonalcoholic fatty liver disease [In mouse liver, CD36 is expressed on hepatocytes, endothelial cells, and Kupffer cells  and the  disease , insulin disease .In the normal brain, CD36 expression is low, but it is upregulated upon stroke due to monocyte-macrophage infiltration. It appears that CD36 contributes to acute ischemic brain injury during the inflammatory phase and is involved in phagocytosis during the recovery phase .In ovary, CD36 is found on serous ovarian epithelial cells , contribCD36 was first identified as platelet GPIV, due to thrombocytes' ability to bind TSP . In the  in vitro and in vivo; induction of apoptosis by TSP-1 via activation of CD36, p59fyn, caspase- 3–like proteases, and p38 mitogen-activated protein kinases [Febbraio and collaborators have played an important role in understanding CD36 functions by creating mice with a null mutation in the CD36 gene through targeted homologous recombination . The ani kinases ; underst kinases , 45 cont kinases  diet res kinases , serving kinases  orosenso kinases . Convers kinases .More recent mouse experimental models include double-knockout animals for CD36 and other genes such as leptin , tyrosinThe CD36 molecular structure includes two transmembrane domains located near both ends of the molecule, joined by a large extracellular region ; the traOther studies have contributed to identifying and understanding the role of CD36 association with other membrane or intracellular molecules. An interesting example is the discovery of CD36 as a regulator of Toll-like receptors 4 and 6 heterodimer assembly that can subsequently trigger inflammatory signalling in microglia . Such daβ-catenin [Interesting avenues of research might also be opened by a few recent studies that identified a role for noncoding RNA molecules in CD36 expression regulation with functional consequences. miR-758-5p decreases lipid accumulation in foam cells via regulating CD36-mediated cholesterol uptake , long no-catenin , and uc.-catenin .Following data gathered from these models and others not mentioned here, the involvement in lipid metabolism and cell-to-matrix adhesion has been confirmed for various cell types, and other functions that are site-specific have been identified and are presented briefly below. in vivo by increasing prostaglandins D2, F2, and E2 synthesis in the liver [CD36 has long been known as a scavenger receptor able to bind oxidized LDL (oxLDL) and HDL (oxHDL) , 73 but he liver .2+ dynamics, phospholipid composition, and cAMP levels and associated conduction anomalies with a high incidence of sudden death [β-oxidation [CD36 is a key factor in acute and adaptive regulation of muscle FA oxidation in response to a chronic metabolic stimulus and for the selection of skeletal muscle fuel under basal conditions, during acute exercise, or after muscle training . In hearen death . Moreovexidation .Pathology Impact. Following the interaction of oxLDL with CD36 on intimal transmigrated macrophages, oxLDLs are internalized. They bind to the nuclear hormone receptor PPARɣ followed by the upregulation of CD36, which amplifies oxLDL uptake and foam cell formation [ormation . Moreoveormation .CD36 can bind to extracellular matrix proteins, such as collagen  and throThe term “biomarker” came into frequent use from the 1970s  and is cCorrelated with invasion of tumours and metastasis, CD36 has been repeatedly proposed as a prognostic marker in various types of cancers, mostly of epithelial origin. In the following section, we discuss the results that build up the case for CD36 as an “early prognostic marker in metastasis” and review its progress towards clinical validation. increased in human tumour cell lines, human biopsies from ovarian tumours [ decreased in endothelial cells, as a bypass program of its antiangiogenic effect [To be considered as a potential biomarker of metastatic cancer, CD36 must respect the first rule of biomarkers—to exhibit a constant change in disease versus health, in this case, in metastatic cancer versus normal paired control tissue. Indeed, the expression of CD36 has been demonstrated to be tumours , gastric tumours , gliobla tumours , and ora tumours . In contc effect .β activation [β activation [Most preclinical studies address CD36 indirectly, in the context of TSP binding. These studies exploit the antiangiogenic effect of TSP 1 and 2 via CD36 signalling, by using recombinant proteins, or TSR peptides, to compensate for loss of TSP in tumour cells. Controversies arose when migration and invasion of cancer cells seemed to be promoted although the primary tumour responded to treatment. A TSP-1 null/breast cancer mouse model demonstrated reduced pulmonary metastases, although there was no impact on primary tumour growth, indicative of effects on the metastatic . The samtivation , and thativation .Results with another mouse model of metastatic breast cancer indicated that although CD36 expression in the whole primary tumour was downregulated, this alteration was related to loss of stromal receptor. This hypothesis was confirmed by normal expression of CD36 on isolated tumour cells .In metastatic prostate cancer, CD36 was activated in tumour cells, which led to increased cell migration and invasion, linked to downstream activation of MAPK . versus primary human ovarian tumours; moreover, blocking CD36 with monoclonal antibodies resulted in reduced tumour burden in a mouse xenograft model [Ovarian tumour cells harvested from ascites of patients also express CD36, which was used by Wang et al. as a target for TSP-1-induced apoptosis and subsequent tumour shrinkage in a mouse xenograft model . A recenft model . Furtherft model . These rCD36 has been reported on glioblastoma cells as well, in a specific subset of stem-like cells, with role in stemness preservation and tumour initiation .Recently, a study addressed directly the involvement of CD36 in tumour growth and metastasis, by overexpressing CD36 in oral squamous carcinoma cell lines. Tumour cells were then transplanted into immune-competent mice, showing significantly increased metastatic potential over their wild-type counterparts. Conversely, knocking CD36 down led to zero lymph node invasions. The same antimetastatic effect was obtained with CD36- targeting antibodies. While metastasis was prevented, or if already present, significantly reduced, primary lesions were not affected by the treatment . Unlike The model that can be delineated so far is that a high CD36 level correlates with metastatic cancers and thus is poorly prognostic. However, blocking CD36 in a tumour system composed of tumour cells and stromal niche would equally affect both populations. An earlier work of deFillipis et al.  proposesThese results highlight the importance of integration of models in a correct spatial context, in which stromal niches and tumour cells interact. From this perspective, which part is more important for metastasis? And, in consequence, which population of cells should be targeted in studies aiming at validation of CD36 as a prognostic biomarker see ? Which pUnfortunately, these aspects have not yet been covered by clinical studies, as discussed in the next section, although CD36 is emerging as a candidate prognostic biomarker in different types of epithelial cancers, alone or in panels with other proteins. in vitro human tumour cell lines point to CD36 as a metastasis-related indicator, prompting investigations on a larger scale in human tumour samples  in which genes involved in metabolic rewiring towards aerobic glycolysis andpatients .Direct targeting of CD36 in tumour pathology has not yet been addressed in cancer-related clinical trials. Rather, its ability to bind TSP1 and modulate antiangiogenic responses was exploited therapeutically. Several clinical trials tested TSP-1 peptidomimetics specifically binding to CD36, but they were discontinued for lack of response or severe adverse effects . Data gaContinuing the pipeline of TSP peptidomimetics, in line with the lipid scavenger function of CD36, apolipoprotein A-I–mimetic peptides are being tested in preclinical trials, but mostly for noncancerous pathologies –104.CD36 has been and continues to be investigated as a possible biomarker in metabolic diseases , cardiovascular diseases, and autoimmune/inflammatory conditions .The hope is that data gathered from these clinical trials will be highly informative about the pharmacological profile and side-effects of various types of CD-36 related compounds, for further repurposing in cancer therapy.Plenty of data have been gathered to demonstrate CD36 involvement in metastasis spreading and, yet, novel and exciting avenues are still opening. Along with new reports of CD36 involvement in normal mitochondrial function , one couFurther on, based on tumour animal models, the next step would be translation to human pathology. This process has already started, at least at the bioinformatics level, contributing to acknowledgment of CD36 as a possible prognostic biomarker for metastatic cancer, by compiling data from repositories and meta-analyses. So far, high levels of CD36 have been proposed as a poor prognostic marker for colon and ovarian cancer  as well If the molecule is to be included in further clinical trials, validation in large cohorts remains to be accomplished, along with clarifications in some grey areas, such as site of detection of CD36  and proper quantification.


Digitoxin also significantly reduces HSPB1 mRNA and the HSPB1/RBFOX2 mRNA ratio in PC3 cells. In vivo, in a syngeneic, immune competent rat model of metastatic CRPC, we show that digitoxin also suppresses Tgfbr2 expression, as well as expression of other genes classically driven by NFκB, and of multiple EMT genes associated with metastasis. Concurrently, digitoxin suppresses tumor growth and metastasis in these animals, and prolongs survival. Gross tumor recurrence following tumor resection also appears prevented in ca 30% of cases. While the existence of a collaboration between NFκB and TGFβ to drive EMT and metastasis has previously been appreciated, we show here, for the first time, that chronic, low concentrations of digitoxin are able to block CRPC tumor progression, EMT and the ensuing metastatic disease.Castration Resistant Prostate Cancer (CRPC) is thought to be driven by a collaborative mechanism between TNFα/NFκB and TGFβ signaling, leading to inflammation, Epithelial-to-Mesenchymal-Transition (EMT), and metastasis. Initially, TGFβ is a tumor suppressor, but in advanced metastatic disease it switches to being a tumor promoter. TGFBR2 may play a critical role in this collaboration, as its expression is driven by NFκB and it is the primary receptor for TGFβ. We have previously reported that the cardenolide drug digitoxin blocks TNFα/NFκB-driven proinflammatory signaling. We therefore hypothesized that digitoxin might break the collaborative process between NFκB and TGFβ by also inhibiting expression of TGFBR2. We therefore tested whether TGFβ-driven EMT and resulting metastases would be suppressed. Here we show,  EMT ion (EMT) . TGFβ haon (EMT) , 9. In ton (EMT) , 11. EMTon (EMT) , and in on (EMT) , 14. Theon (EMT) . For exaon (EMT) .Historically, the TNFα/NFκB pathway has been considered to be the “major culprit” contributing to the proinflammatory condition in prostate and other cancers , 17, 18.In vitro studies shown below support this hypothesis. As an in vivo platform to test this hypothesis we deployed the Prostate Adenocarcinoma III (PA-III) rat model, a hereditary, spontaneous/autochthonous, transplantable, immunologically intact, castration resistant prostate cancer (CRPC) model in the Lobund-Wistar (LW) rat  rat –29. The 2, at 37°C.The PAIII tumor has been maintained since 1976 by serially passaging freshly isolated tumor cells in naïve Lobund-Wistar (LW) rats. The tumor was originally isolated from a spontaneous (autochthonous), hormone resistant prostate tumor growing in an immunologically intact CRPC-prone Lobund-Wistar (LW) rat –30. Prio6 cells) from an existing PAIII tumor, as determined by a trypan blue exclusion test, were injected subcutaneously (s.c.) into the right flank of thirty 2-month old LW rats (ca. 250 g body weight). After ten (10) days, 15 of the rats began to receive daily doses of digitoxin (0.03 mg/kg). Digitoxin (Sigma) was prepared as follows: (i) a stock solution was prepared by dissolving 300 mg digitoxin in 10 ml of 95% ethanol. (ii) two serial 1:20 dilutions were prepared by mixing, in order, 1 ml of the stock solution with 19 ml PBS. (iii) prorated volumes of the last dilution, calculated according to the specific weight of each rat, were then administered intraperitoneally (i.p.) each day in the 9–10 AM time interval. On the morning of day 40, all animals were sacrificed. The weight of each primary tumor was recorded. The number and diameter of pleural metastases were determined using a digital calipers . Primary tumors, and selected lung metastases, were immersed in liquid nitrogen, and stored for later analysis at minus 80°C. Over the course of this protocol, rats were observed daily by a veterinarian, board certified in laboratory and animal medicine, and no differences in body weight, appetite or physical appearance were noted either during or at the end of the experiment.An equal number of viable PAIII cells (1 × 106 cells), as determined by a trypan blue exclusion test, were injected intravenously through the tail vein of thirty 2-month old LW rats (250 g body weight). Fifteen of the rats immediately began to receive daily injections (i.p) of digitoxin (0.03 mg/kg), as described and justified for Protocol #1. Lungs were harvested for analysis on day 10.An equal number of viable PAIII cells  metastatic sites on the pleural surfaces were measured with digital calipers . Diameters of pleural metastases were measured in millimeter mode. The largest linear dimension was recorded as the diameter of the metastatic site.Rat serum samples were analyzed on duosets from R&D Systems for rat serum IL-6 and CINC-1 . Human cytokines and chemokines in serum or culture fluids were measured by multiplexed electro-chemiluminescence on the MSD Sector 6000 platform , as described .PC3 cells were obtained from ATCC, and were cultured in Hams F-12K nutrient mixture medium with 10% FBS and Pen-strep. HeLa cells were obtained from the ATCC, and cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml). Digitoxin was obtained from Sigma (>95% purity). Oleandrin was obtained from Indofine Chemical . Drugs were solubilized as stock solutions in 95% ethanol and diluted for experiments to a final ethanol concentration of 0.01%.PC3 cells and HeLa cells were seeded in 6 well plates overnight, then cotransfected overnight (16 h) with NFkB-luc and LacZ plasmids using the Fugene HD transfection reagent (Roche). The cells were then treated with either digitoxin or oleandrin at different concentrations, for 12 h. Then cells were treated with TNFα, and the digitoxin and other cardiac glycosides, at different concentrations, for another 10 h. Cells were harvested and lysed with 1x passive lysis buffer. Luciferase assays were performed with the Promega Luciferase Assay System. The luciferase values were normalized to β-galactosidase activity.PC3 cells and HeLa cells were treated with digitoxin at different concentrations and/or 20 ng/ml TNFα for 24 h. The medium was then replaced, including with fresh drugs and TNFα, and continuously treated for 3 days. Cells were then lysed in M2 buffer . Twenty microgram of protein from the cell lysate of each sample was fractionated by SDS-PAGE and immunoblotted. The blots were visualized with chemiluminescent substrate (Pierce). The antibodies against Vimentin (code: sc-7870) and E-cadherin (code: sc-5565) were obtained from Santa Cruz Biotechnology, CA. Alternatively, cells were analyzed by capillary western blot , as described .ca. 104/ml, and then treated with digitoxin at the different concentrations for 24 h. Fresh medium and drug were added every day. Cells were continuously treated in this manner for up to 3 days (HeLa cells) or up to 6 days (PC3 cells). Then 1/10 volume 12 mM MTT stock solution was added. After 4 h incubation at 37°C, an equal volume of 10% SDS solution was added to the wells. Absorbance was read at 570 nm after another 4 h incubation.Cells were seeded in 24 well plates at densities of Samples of grossly dissected, frozen (−80°C) primary tumors were mounted on a cryostat chuck. Thin sections (20 micron) were cut and stained with Hematoxylin and Eosin (H& E) to distinguish between vital from non-vital areas of the tumors. Serial thick sections (300 micron) were then cut and mounted on sub-0°C slides. Using the 20 micron stained slides as a “stencil,” ten random samples were micro-dissected from viable regions of the tumor in 300 micron thick sections with a 1 mm diameter micropunch device. The tumor samples were then soaked in RNA Later at 4°C overnight.GEO133696.Total RNA isolated from micro dissected tissues was validated for integrity using the Experion Automated Electrophoresis System (BioRad) before library preparation. An amount of 1 μg total RNA was input for library preparation using the TruSeq RNA Sample Prep Kit v2 (Illumina). Sequencing libraries were validated for size and integrity using an Experion Automated Electrophoresis System (BioRad), and library concentration was determined using the KAPA Library Quantification Kits for Illumina (KAPA Biosystems). Twelve to fifteen pmole of Library were used as input for clustering by a c-Bot (Illumina) before sequencing on a Genome Analyzer IIx (Illumina). Sequencing was performed by single read of 101 bp length with an indexing read of 7 bp. FASTQ data were generated using CASAVA, and raw reads were analyzed and groomed using FASTQC and FASTQ Groomer, respectively. RPKM values for transcriptome expression levels were generated using Bowtie 2, TopHat 2, and Cufflinks 2. RPKM values were used for Comparative Marker Selection and differential gene analysis by Cuffdiff. Raw FASTQ files for each profiled sample and processed data of FPKM values for transcript expression levels are available at the NCBI Gene Expression Omnibus at the accession number The tissues were homogenized with RNA lysis buffer (Ambion) according to the manufacturer's protocol. Multiplex Reverse Transcription was performed with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). For microRNA analysis, the miRNA expression profiles were analyzed by the TaqMan Low Density Rodent MicroRNA Panel v2, as described . Brieflyt-test) and non-parametric (Mann-Whitney) tests were used to compare tumor weights, number and sizes of classical metastases, following subcutaneous flank injection of cells. Identical tests were performed to analyze number and size of metastasis-like lung foci following intravenous cell administration. Histograms were prepared on the basis of log distributions of diameters of tumor-seeded metastases, and metastasis-like lung foci following intravenous administration. Unbiased averages, means and medians of distributions were prepared using total data. Analyses of cytokine and chemokine data were analyzed using GraphPad, Version 6. In all cases, differences were taken as significant if P < 0.05.MicroRNA and mRNA relationship analysis was generated using overlap between the TargetScan  predicted miR targets and the RNA seq data (Illumina GIIx). The IPA algorithm was also used to generate networks focusing on prostate cancer, and metastases. Parametric  are dose dependent and statistically significant at a digitoxin concentration of 25 nM. These data indicate that concurrent TNFα treatment is needed for optimal activation of EMT activity. Consistently, digitoxin also reduces the viability  proteins, we tested PC-3 and HeLa cells for digitoxin-dependent decreases in the EMT effector protein Vimentin and concomitant increases in the EMT effector protein E-Cadherin. The western blots for HeLa cells  and for pression . Summaryiability  and growiability  of PC3 cHSPB1 and RBFOX2. HSPB1 is a positive biomarker for EMT in prostate cancer (ca 30% reduction in HSPB1 mRNA (P = 0.0239) compared to control conditions. HSPB1/RBFOX2 is significantly reduced by ca. 41% (P = 0.0179). The digitoxin-dependent reduction of both HSPB1 and the HSPB1/RBFOX2 ratio can be interpreted as supporting the concept that digitoxin suppresses EMT in human PC3 cells.To further test the ability of digitoxin to suppress EMT, we used RNA-seq to examine the effect of digitoxin on the expression and ratio of mRNAs for e cancer , while Re cancer . Figure in vivo, we subcutaneously injected passaged rat tumor cells into syngeneic rats. Then, 10 days later we treating the rats with digitoxin or vehicle. After a total of 40 days we measured both growth of the primary tumor and metastases to the lung. As shown in unimodal distribution of sizes. These data suggested that digitoxin might arrest seeding of metastases upon administration on day 10, and permit survival and limited growth only for those metastases that had already been established.To test for the ability of digitoxin to suppress castration resistant prostate cancer number of implanted/metastatic sites were now virtually the same, with or without digitoxin. However, sizes of intravenous-administered metastases are now significantly lower in the drug-treated group (P = 0.004). These data suggest that once in circulation, the cancer cells have equivalent access to the pleural niche. However, digitoxin also reduced the growth rate for the implanted metastasis, whether it has been seeded directly from the primary tumor, or by intravenous injection.To further test this hypothesis, we bypassed biological metastasis by administering tumor cells through intravenous tail vein injection. Digitoxin treatment was then initiated immediately, and continued until animals were sacrificed on day 10. ca day 50. By contrast, digitoxin treated rats survived significantly longer, with 30% of the animals surviving up to day 80. Post-mortem analysis on day 80 of these drug-treated survivors showed no evidence of primary tumor recurrence after resection. Survival curves of treated vs. untreated were significantly different based on a log rank Mantel-Cox Chi-Square test . Thus, the effect of the drug treatment was to extend survival for digitoxin- treated rats following resection, and to confer long term resistance to tumor recurrence on a significant fraction of the digitoxin-treated animals following tumor resection.Finally, to test whether treatment with digitoxin provided any survival benefits to the rat under a clinically relevant condition, we again implanted tumor cells subcutaneously into 30 rats. Ten days later, we began treating half the rats with digitoxin. Eight days later (day 18), we resected all primary tumors from all the animals. Digitoxin- treated rats which survived resection surgery continued to receive a daily injection of digitoxin for the duration of the experiment. lower levels of both IL-6 and CINC-1 in their sera. Thus, the composite reductions in IL-6 and CINC-1 correlate closely with the digitoxin-dependent reductions in primary tumor weights shown in picked randomly from the lower and upper extremes, respectively, from the IL-6 vs. CINC-1 graph . ca 53%) are biomarkers for highly aggressive, poor outcome human CRPC.To aid in grouping and selecting tumors and tumor regions suitable for RNA-seq analysis, we first stratified animals on the basis of serum IL-6 and serum CINC-1 see , 1–4. CI-1 graph . Frozen Inflammatory Response (P = ca. 10 −8). Inflammatory Response. In ascending order of P-value, these include granulocyte adhesion and diapedesis ; agranulocyte adhesion and diapedesis (movement of lymphocytes and monocytes from blood into tissue); leukocyte extravasation signaling ; circadian rhythm signaling; tight junction signaling ; and cAMP-mediated signaling. Thus, this set of molecular characteristics identifies both cancer cells and their tumor microenvironment as gene expression targets for digitoxin.To test for common targets of these digitoxin-dependent genes, we subjected the set of 101  mRNAs to Gene Ontology (GO) analysis. P < 0.05 level. Based on the presence of at least one functional Nfkb sequence, and supporting data from the literature, we identified 17 Nfkb-driven genes. Tgfbr2 expression is among those mRNAs that were significantly reduced by digitoxin in rat tumors. Thus, there appears to be a functional parallel, marked by digitoxin inhibition, between NFκB signaling to TGFBR2 in human prostate PC-3 cancer cells, and Nfkb signaling to Tgfbr2 in rat prostate cancer cells. In addition to Tgfbr2, increased expression of 9 of the reduced mRNAs in To test whether the mechanism of digitoxin action in these CRPC tumors in the rat model might involve suppression of Nfkb-driven genes in primary tumors, we surveyed the set of all significantly affected mRNAs at the HSPB1/Rbfox2 ratio. The observed reductions, comparing 8 digitoxin-treated tumors and 8 control tumors, did not approach significance. However, in general, the ability of digitoxin to reduce EMT signaling observed in human prostate cancer PC3 cells would appear to have been replicated in this rat prostate cancer model.To test for effects of chronic digitoxin therapy on EMT gene expression, we identified mRNAs associated with EMT in the entire significant dataset of 101 significantly modified mRNAs see . Figure elevation of miRs is associated with reduction of target mRNAs. Thus, the IPA data in ZEB2, a classical EMT gene. The microRNA miR-204-5p targets TGFBR2, the hub receptor for TGFβ. The sequence relationships between microRNAs identified as either rat (rno) or mouse (mmu) and their human (hsa) counterparts are further delineated in Finally, we also tested for possible associations between digitoxin-affected microRNAs (miRs) and EMT by screening 250 miRs in primary rat tumors for digitoxin effects. in vitro and in vivo, and that an important consequence is significant suppression of EMT, tumor progression and tumor recurrence. At the in vitro protein level in the human PC-3 cell system, digitoxin is not only able to suppress Vimentin expression while enhancing E-Cadherin, but is also able to block TGFBR2 expression. This inverse Vimentin and E-Cadherin response is a traditional signature for suppressing EMT. Furthermore, because TGFBR2 expression is driven by NFκB, and digitoxin blocks NFκB signaling, the suppression of TGFBR2 expression by digitoxin was expected and found. Consistently, digitoxin is also able to significantly suppress mRNA expression for both HSPB1, a biomarker for EMT in prostate cancer, and the HSPB1/RBFOX2 ratio. Finally, using RNA-seq to probe the in vivo rat prostate cancer system, digitoxin was found to suppress Nfkb signaling to multiple mRNAs, including Tgfbr2, as well as multiple EMT mRNAs. Unexpectedly, these include Wt-1 and Axl; both are important as drivers of human metastatic disease. These apparent parallels between digitoxin action in both systems therefore make it likely, as hypothesized, that digitoxin may work by breaking the collaborating process linking inflammation, TGFβ, and EMT.These data show that digitoxin suppresses signaling by both TNFα/NFκB and TGFβ/TGFBR2 pathways in castration resistant prostate cancer cells, both TGFBR2 mRNA and to reduce TGFBR2 protein expression , and were conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).BP contributed by conceiving the project, managing the research, and writing the paper. MAS contributed by managing the research and writing the paper. WW, PK, MES, and QY contributed by performing experiments. JS, OE, and SB contributed by analyzing the data. CD contributed by performing experiments, analyzing data, and writing the paper. RB contributed by performing experiments and writing the paper. HP contributed by designing experiments, managing the research, and writing the paper. XZ and MW performed bioinformatic analyses.BP is the sole invertor on an issued patent regarding digitoxin. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Primary varicella-zoster virus (VZV) infection causes varicella (chickenpox) and the establishment of a lifelong latent infection in ganglionic neurons. VZV reactivates in about one-third of infected individuals to cause herpes zoster, often accompanied by neurological complications. The restricted host range of VZV and, until recently, a lack of suitable in vitro models have seriously hampered molecular studies of VZV latency. Nevertheless, recent technological advances facilitated a series of exciting studies that resulted in the discovery of a VZV latency-associated transcript (VLT) and provide novel insights into our understanding of VZV latency and factors that may initiate reactivation. Deducing the function(s) of VLT and the molecular mechanisms involved should now be considered a priority to improve our understanding of factors that govern VZV latency and reactivation. In this review, we summarize the implications of recent discoveries in the VZV latency field from both a virus and host perspective and provide a roadmap for future studies. This observation was based on a careful examination of ~3500 patients, including 192 HZ cases, who visited his practice over a 16-year period, combined with cautious reading of the available anatomical and epidemiological literature regarding HZ. This led to his famous hypothesis that: “Following the primary infection (chickenpox), virus becomes latent in the sensory ganglia, where it can be reactivated from time to time (herpes zoster)” [Most adults worldwide are infected with the neurotropic human alphaherpesvirus, varicella-zoster virus (VZV) . VZV is zoster)” . EighteeL) and unique short (US), that are flanked by inverted terminal repeat (TR) and internal repeat (IR) structures with high G + C contents (68% for the TRL/IRL and 59% for the IRS/TRS)  IRS and TRS sequences flank the US region. The composite structure generally allows for two isomeric configurations [S region is inverted. While up to 5% of virions are reported to contain structural isoforms with an inverted UL region [L region that is required for viral DNA cleavage during packaging [S/TRS. All except R5 are G + C rich, and all are subject to length and structural polymorphisms that vary both within and between strains [open reading frame (ORF) 11, ORF14, and ORF20, respectively) and may therefore exert an effect on protein function. Two copies of R4 are present in the IR and TR, neighboring the origin of replication (OriS), while R5 is located between ORF60 and ORF61.VZV particles are pleomorphic to spherical in shape, ~150–200 nm in diameter and are composed of three proteinous layers: a nucleocapsid containing the viral double stranded DNA (dsDNA) genome, a tegument layer, consisting of numerous proteins of both viral and host origin surrounding the nucleocapsid, and an envelope comprising a host-derived lipid bilayer inserted with viral glycoproteins facing outwards A,B 6,7,,7,6,7,8.IRS/TRS) C [10,11,urations  that difackaging  i.e., th strains ,24,25,26S/TRS region [ORF0 [ORF9A [ORF33.5 [VLT) [ORF0, ORF42/45, ORF50, and VLT, require the host-splicing machinery to remove introns from pre-mRNA and have also shown evidence of alternative splicing, resulting in the synthesis of alternative proteins [The VZV genome was originally reported to encode 65 unique viral genes, three of which are located in the duplicated IRS region . Four adon [ORF0 , ORF9A [0 [ORF9A , ORF33.5[ORF33.5 , and the.5 [VLT)   will yiproteins ,35,36. Halphaherpesviruses, VZV genes have been loosely designated as immediate-early (IE) genes, early (E) genes, and late (L) genes, with each ordered wave of expression thought to be dependent on the protein products of previous classes. Proteins encoded by IE genes act as transcriptional regulators, while those produced by E genes are mainly involved in DNA replication, and L genes encode structural proteins that are crucial for virion formation and egress. VZV encodes at least five transcriptional regulatory proteins specified by four putative IE genes, ORF4, ORF61, ORF62 and ORF63, and one L gene, ORF10. All except the ORF61 protein, IE61, are part of the VZV virion [Varicellovirus is homologous to VZV IE62 [ORF4, ORF61, ORF62, and ORF63, while IE4, IE61 and IE63 either do not or minimally stimulate the ORF61 promoter  and trigeminal ganglia (TG). While VZV DNA has been detected in various other sensory  ,66,67,68Two non-mutually exclusive routes by which VZV may infect sensory ganglion neurons have been proposed A. First,In the absence of robust animal models, VZV latency studies have been dominated by the use of naturally VZV-infected cadaveric human ganglia, either snap-frozen at autopsy  or explaVLT, frequently accompanied by co-expression of ORF63 RNA, albeit at lower quantities than VLT [VLT  and ORF63 (0.36%) by in situ hybridization than the reported 2–5% of neurons harboring latent VZV DNA [VLT and ORF63 RNA retain the potential to be translated during latency, these viral proteins could not be detected in latently infected human ganglia by immunohistochemistry [VLT and ORF63 RNAs, alternative approaches with higher sensitivity, such as screening for the loading of VLT and ORF63 RNA onto polysomes combined with targeted enrichment for viral RNAs could provide conclusive data. Thus, VZV latency in naturally infected human ganglia is characterized by the exclusive expression of VLT and often, ORF63.The difficulty of obtaining and working with post-mortem human ganglia has resulted in discrepant numbers and identities of viral transcripts, and in some cases, the corresponding proteins have been detected ,101,102.than VLT ,73,74,75 VZV DNA , suggesthemistry . Howeverin vivo VZV latency, ganglia from these individuals are likely to have been latently infected for many decades. In contrast, when using in vitro hESC-derived neuronal models, latency is usually profiled within 14 days. Although transcriptome-wide profiling by RNA-sequencing of in vitro “latent” VZV infections has revealed a marked reduction in viral gene expression, all viral genes are expressed in the absence of detectable levels of viral proteins [ORF63 RNA has been observed, it is not known whether these systems express detectable VLT and if so, whether the latent VLT isoform is transcribed [Although naturally VZV-infected, short-PMI, human cadaveric ganglia most closely resemble proteins ,97. Whilnscribed ,56,97. TVLT and frequently, ORF63 RNA). Either way, the remarkable switch between lytic and latent transcriptional states is best explained by the assembly of repressive chromatins upon the viral episome. Indeed, studies using late PMI (>9 h post mortem) VZV-latently infected human ganglia have demonstrated euchromatic (H3K9ac) chromatin modifications on promoters of latency-associated ORF63 and ORF62, the latter of which is frequently expressed in late PMI deregulated ganglia [ORF14 and ORF36, which are not expressed during latency, nor detected in late-PMI ganglia, have not been associated with H3K9ac [VLT and ORF63 loci would be bound by repressive chromatin, and it will be important to determine whether the VLT locus remains active due to the presence of predicted sequence motifs for flanking chromatin insulators such as CCCTC-binding factor (CTCF), as has been observed for HSV-1 [The molecular mechanisms by which VZV latency is established are largely unknown and much of what is assumed is actually informed by studies of the related herpes simplex virus type 1 (HSV-1). Here, the key concept is that during the traversal of neuronal axons, the genome-containing nucleocapsid is delivered to the nucleus in the absence of functional tegument proteins for IE gene transcription , and this allows loading and maintenance of repressive chromatin upon the viral episome which inhibits viral transcription , their size, coding potential, functions, and mechanisms of action may vary.While all VLT represents a major advance in studies of VZV latency. VLT is encoded antisense to the ORF61, and it is remarkable to note that all known alphaherpesvirus latency-associated transcripts (LATs) originate from genomic regions encoding infected cell polypeptide 0 (ICP0) homologs . During latency, a single VLT isoform is expressed in the neurons of virtually all analysed VZV-infected human TGs [VLT is expressed in all VZV-infected neurons. VLT appears to be structurally complex compared to the LATs of HSV-1, pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1), which are composed of only two exons and a single intron  specific promoter. During productive infections, lyVLT is transcribed with true-late kinetics, is defined as being absolutely dependent on viral replication, and is translated into proteins (pVLT) in cell cultures and zoster skin lesions [LATs during lytic alphaherpesvirus infection is incompletely understood, the observed features of VLT are consistent with the general pattern of LATs. During lytic infection, LATs appear to be expressed with late kinetics [LAT isoforms are produced using different transcription start sites or alternative splicing [LATs encode proteins [VLT in the same cell type.Although entified . Termed  lesions . Althougkinetics ,117. Altsplicing ,118,119,proteins ,120. Givproteins ,97, thesVLT during lytic and latent stages is now a priority. Early investigations using transfection assays showed that VLT selectively represses ORF61 transcription when co-expressed with multiple VZV coding IE genes  in ARPE-19 epithelial cell cultures, resulting in diminished expression of the ORF61 protein, IE61 [ATG start codon (to ATA) showed a similar effect—suggesting that it is mediated by the RNA itself. While the specific functions and requirements of VLT and lyVLT isoforms during lytic and latent infections remain unknown, these data allow us to speculate that VLT may function to maintain latency by repressing the transcription of ORF61, a promiscuous transcriptional regulator, during lytic infections. However, it will be pivotal to confirm these observations in other experimental systems, especially since not all functions attributed to LATs of related herpesviruses in vitro can subsequently be confirmed in vivo [The functional characterization of in, IE61 . This ef in vivo ,122.VLT and ORF63 RNA, the expression of which correlates significantly in human TG, independent of the latent viral DNA load [VLT and ORF63 RNA are produced by the same or by distinct populations of neurons, and how this may influence the ability of the virus to reactivate. ORF63 is essential for VZV replication [ORF63, once translated (IE63), could be crucial for the initiation of reactivation, supported by the correlation between ORF63 RNA abundance and PMI [VLT-mediated repression of ORF61 transcription and translation may also function to retain IE63 in the cytoplasm and prevent the transactivation of lytic viral promoters , and provide swift protective immunity upon pathogen re-exposure [RM are expected to home to sites of latency and reactivation in ganglia and skin. Indeed, recent studies showed that VZV-specific TRM cells persist in the healthy skin of latently VZV-infected adults decades after primary infection [RM in ganglia [RM in latently VZV-infected human ganglia in regard to their contribution to VZV latency and reactivation control. This will provide more insight into the inverse relationship between VZV-specific T-cell immunity and the risk of HZ development.VZV reactivation, clinically presenting as HZ, is associated with the waning of systemic VZV-specific T-cell immunity, but not humoral immunity, suggesting a critical role of VZV-specific T-cell memory in preventing virus reactivation ,148. Howexposure ,150. In exposure ,153,154 exposure , VZV-spenfection . Profoun ganglia ,158. Whi ganglia . More deVLT and the latent VZV transcriptome in naturally-infected, short-PMI TG has provided us with an new perspective on VZV latency [VLT and/or ORF63 RNA.VZV has evolved with the ability to establish lifelong latent infection in neurons, enabling the virus to reactivate later in life and cause recrudescent disease accompanied by VZV spread to naïve individuals. Throughout the decades of latency, the virus must ensure survival of the infected neuron, avoid irreversible silencing of the viral episome, and prevent elimination by host immune responses. Although the mechanisms that coordinate the interplay between the virus and the host remain largely unknown, the many recent exciting advances in the field provide us with the questions to be addressed and tools (ex vivo and in vitro latency models) that can be used to answer them. Thirty-five years on from the discovery of latent VZV residing in sensory ganglia , the ide latency . With thVLT and ORF63 RNA in latently infected short-PMI human TG [VLT and ORF63 RNA are co-expressed by the same or distinct neurons and to define the subtypes of neurons harbouring the latent VZV genome. Recent developments in the generation of recombinant VZV  human TG  and to ahuman TG ,160,161 human TG . Human Thuman TG , and nothuman TG , as has human TG ,164. The in ref. ) will fa ganglia ,103,104.[VLT RNA , no funcVLT and ORF63 RNA, and IE63 and IE61 during the initiation of virus reactivation.Transcriptional regulation of VZV gene expression during lytic and latent infection has been poorly defined. In analogy to HSV-1 , the reaRM in skin [The factors triggering and barriers restricting VZV reactivation are poorly understood and are of great interest for future research. The infrequent clinical reactivation of latent VZV suggests that either virus reactivation is rare or that most reactivation events are cleared before HZ can develop. Local immune responses mediated by VZV-specific T in skin ,166 or g in skin ,144 are  in skin . The rec in skin ,160,161 Post-mortem acquired human samples are extremely informative if handled carefully, especially for investigating human-restricted pathogens like VZV, but they are also restrictive in that the resulting datasets only represent a “snapshot” of latency. To overcome this, we hope that technological advances in specific subtypic differentiation methodology for human sensory/sympathetic neurons or organoid culture system from hPSC combined with single cell sequencing methodologies targeting transcriptomes and epigenomes will provide novel ways to examine the dynamics of VZV latency and provide new insight into the molecular mechanisms that control this.VLT and its potential role in governing VZV latency and/or reactivation also opens up the tantalising possibility of re-engineering current VZV vaccines not to establish latency or reactivate. While a significant amount of work is required to get from here to there, not least determining whether VLT is truly required for latency and/or reactivation, pursuing this long term goal gives new impetus to the VZV research field.Finally, the discovery of


Immunotherapeutics are increasingly recognized as a key tool in the armamentarium against malignancy. The success of immune checkpoint-targeting drugs and adoptive cell therapy has refocused attention on the potential anti-cancer effect of eliciting a tumour-specific immunological response. Sarcomas are a rare and diverse group of tumours with a limited prognosis in advanced disease despite systemic therapeutics. Various vaccine strategies including peptide vaccines against cancer testis antigens, dendritic cell vaccines, and viral vectors have been trialled in sarcoma with growing evidence of efficacy. Here, we review the principles of successful vaccine development and how these have been applied thus far to the treatment of sarcoma. KIT and PDGFRα mutations and are sensitive to imatinib [Sarcomas are a diverse group of aggressive mesenchymal tumours that represent around 1% of solid adult malignancies. Ten percent of sarcomas are bone sarcomas and 15% of sarcomas are gastrointestinal stromal tumours (GIST). GISTs are usually associated with imatinib . Localisimatinib ,4,5. In-imatinib . Some suimatinib .Despite the origin of a malignant clone of cells from self, certain features of cancer cells mark them out as unique from the host . These fThe discovery of tumour antigen specific T cells in melanoma patients with progressive metastatic disease encouraged investigation into why the immune system becomes tolerant of cancer-associated antigens . TherapeMycobacterium tuberculosis. Intravesical BCG treatment reduces the risk of superficial bladder tumour recurrence and progression to invasive disease as compared with transurethral bladder tumour resection alone and other chemotherapeutics [Sipeleucel-T is an autologous vaccine composed of dendritic cells exposed ex vivo to a fusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostatic acid phosphatase . In a plapeutics ,16.A successful cancer vaccine incorporates three key steps; selection of a tumour-specific or tumour-associated molecule, presentation of the tumour-specific molecule to the immune system, and stimulation of an adequate immune response. In this review, we will discuss how the principles of a successful vaccine have been applied thus far to the development of vaccines in the treatment of sarcomas and other strategies to alter the immune response to sarcoma which may work synergistically with vaccines.A critical step in vaccine development is selection of the appropriate tumour-associated molecule to elicit an anti-tumour immune response. The ideal candidate is a molecule that is exclusively expressed by the tumour. This can encompass self-antigens, differentiation antigens specific to that cell type, abnormally or overexpressed antigens, or antigens present due to a mutated protein, also known as neoantigens ,18,19,20Cancer testis antigens (CTAs) are germline antigens which are expressed exclusively in germ cells and in several cancer types. The expression in these cell types is 1000 fold higher than in other cells, making them good targets for cancer vaccines . The absAberrant ganglioside antigen expression is also a rational tumour-specific target and vaccines have been tested in different tumour types including melanoma and sarcoma ,35. A biUnique chromosomal translocation events that are ubiquitous within a sarcoma subtype such as the t translocation in synovial sarcoma or the t in myxoid/round cell liposarcoma are attractive vaccine targets as the newly-formed peptide will potentially create a tumour-specific neoantigen ,38. A frTumour cells as a whole can potentially provide a variety of tumour-associated antigens and vaccination with patient-derived tumour cells and laboratory established cancer cell lines have been attempted . In one To elicit a host cellular immune response, a tumour-associated antigen is presented by major histocompatibility (MHC) molecules . One metDirect delivery of antigen-presenting cells within the vaccine can be a method of presenting the tumour-associated antigen to the immune system. Autologous dendritic cells that have been exposed to tumour-associated antigens or whole tumour cell lysates are strategies that have been trialled in sarcoma ,49,50,51Dendritic cell vaccines exposed to tumour-associated antigens have been combined with other therapeutics in sarcoma. In an early phase trial, 10 children with relapsed cancer including one with Ewing sarcoma and one with rhabdomyosarcoma received four cycles of the hypomethylating agent decitabine, followed by two vaccinations with dendritic cells exposed to MAGE-A1, MAGE-A3, and NY-ESO-1 peptides . The ratp = 0.0172) [Maturing dendritic cells in the presence of tumour cell lysate can expose the dendritic cell to a greater number of tumour-associated antigens. A phase 1 study trialled a vaccine of autologous immature dendritic cells pulsed with irradiated tumour lysate from the patient and KLH . One chi 0.0172) .+ T cells infiltrating the tumour and non-responders had a higher level of myeloid derived suppressor cells. Overall clinical outcomes were similar to that expected in this population with a 12-month disease free survival of 70.6%. A second phase 1 trial studied the combination of radiation with intratumoural dendritic cell injection in a similar population [Delivery of the antigen presenting cell directly into the tumour could induce a tumour-specific immune response. The combination of radiation and tumour-specific vaccines has been shown to increase anti-tumour immune responses in an animal model, possibly due to the upregulation of Fas ligand on tumour cells . Tumourapulation . EighteeAnother mechanism of antigen delivery is using recombinant viral vectors expressing the tumour-associated antigen which are taken up by dendritic cells . This apA modified integration-deficient lentivirus vaccine encoding full-length NY-ESO-1, LV305, has a Sindbis virus envelope which binds DC-SIGN cell surface markers specific to immature dendritic cells . SubcutaDendritic cells can also be targeted using the DEC-205 receptor. Antigen-antibody conjugates result in durable antigen-specific cellular responses and this approach has been incorporated into NY-ESO-1 vaccine development ,71.+ T cell response [Prior vaccine studies have shown that presentation of a tumour neoantigen can often only lead to a minimal immune response . Adjuvanresponse . MN59 isresponse . Saponinresponse ,75. Tollresponse . TLR agoresponse . Combinaresponse .Using different types of vaccine with the same tumour-associated antigen also improves the immune response. This strategy has been successful in synovial sarcoma and myxoid liposarcoma with a CMB305 vaccine regimen targeting NY-ESO-1 . The hosA different vaccine strategy with other adjuvants also focuses on the NY-ESO-1 antigen in sarcomas . The CDXThe tumour microenvironment is critical to a strong immune response after vaccination. Removing inhibitory signals at the immune checkpoint may play a key role in successful vaccination. Immunosuppression within the tumour microenvironment, characterised by lymphocyte depletion, macrophage infiltration and TGF-B signalling, correlates with worse prognosis across tumour types . TumoursDirect therapeutic approaches to release an immune response are the administration of immune checkpoint inhibitors such as CTLA-4, PD-1, and PD-L1 inhibitors and adoptive cell therapies. An 86 patient single arm phase 2 trial of pembrolizumab in the seven commonest forms of bone and STS showed activity in UPS and dedifferentiated liposarcoma . PatientSingle-agent nivolumab and combination immunotherapy with ipilimumab and nivolumab have been trialled in metastatic sarcoma patients recruited to parallel phase 2 trials . The resAdoptive cell therapy is currently being investigated in many tumour types including sarcoma . T cellsThe addition of vaccines to immune checkpoint therapies may be a potent combination. Following the promising phase 1 trial results with the CMB305 vaccine and the observation that an extreme responder to the vaccine had a greater percentage of PD-1 positive NY-ESO-1 positive T cells, the CMB305 regimen has been combined with atezolizumab in a randomised 88-patient phase 2 study in locally advanced or metastatic NY-ESO-1 positive sarcoma ,87. The The clinical outlook of locally advanced and metastatic sarcoma is limited with a median overall survival, even with salvage second-or third line therapies, in the order of 12–18 months ,88,89. T


VV has been also financially supported by an EPSRC-funded CMIC Platform Grant . This work was partly supported by H2020-WIDESPREAD-04-2017-Teaming Phase 1, Grant Agreement 763781, Integrated Precision Medicine Technologies. However, the funders had no role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. PAW has received no specific funding for this work.”There is a funder missing from the Funding statement. The full statement should read: “VV has been partially supported by an FP7-PEOPLE project (Project ID: 627025; URL:


Glioblastoma (GB) contains diverse histologic regions. Apparent diffusion coefficient (ADC) values are surrogates for the degree of number of cells within the tumour regions. Because an assessment of ADC values and volumes within tumour sub-compartments of GB is missing in the literature, we aimed to evaluate these associations.A retrospective cohort of 48 patients with GB underwent segmentation to calculate tumour region volumes (in cubic centimetre) and ADC values in tumour regions: normal tissue, enhancing tumour, proximal oedema, distal oedema, and necrosis. Correlation, Kaplan-Meier, and Cox hazard regression analyses were performed.F  = 166.71 and p ≤ .001 and tumour region volumes : F  = 136.3 and p ≤ .001. Post hoc comparisons indicated that the only significantly different mean score was the peritumoural volume in oedema region (p < .001). We observed a positive significant correlation between ADC of distal oedema and peritumoural volume, r = .418, df = 34, and p = .011. Cox proportional hazards regression analysis considering only tumour region volumes provided an almost significant model: − 2 log-likelihood = 146.066, χ2 (4) = 9.303, and p = .054 with a trend towards significance of the hazard function: p = .067 and HR = 1.077 for the non-enhancing tumour volume.We found a statistically significant difference among ADC values for tumour regions: ADC values together with volumes of oedema region might have a role as predictors of progression-free survival (PFS) in patients with GB; we recommend a routine MRI assessment with the calculation of these biomarkers in GB. In GBM, there is a statistically significant difference among ADC values for tumour regions.In GBM, there is a statistically significant difference among tumour region volumes .ADC of distal oedema and peritumoural volume depict a positive significant correlation.p = .067, HR = 1.077) for progression-free survival.The non-enhancing tumour volume depicts a trend towards significance  methylation status ), range 18–70 years and 12 females: 50.5 ± 12.6, range 18–66 years. Exclusion criteria considered surgical resection, radiotherapy, or chemotherapy previous to the inclusion in the study, absent histopathologic diagnoses, missing imaging data, and presence of artefacts. After resection, they received chemotherapy including temozolomide  and radiation therapy (60 Gy), the Stupp protocol . Table The mixed between-within subjects ANOVA conducted to assess the impact of .765, F 1, 77 = .6F  = 136.3, p ≤ .001, and η2 = .014, meaning a moderate effect. Post hoc comparisons using the Tukey HSD indicated that the only significantly different mean score was peritumoural oedema (p < .001), with no significant differences between necrosis and enhancing tumour volumes. Table 3) observed in GB.Assessment of volumes across tumour regions showed similar results; one-way between-groups ANOVA showed a statistically significant difference for the three evaluated tumour region volumes : F  = 1.584, p = .161, and η2 = .10 (moderate effect size). There was a substantial main effect for tumour regions, Wilks’ Lambda = .505, F  = 21.055, p < .001, and η2 = .495 (a large effect size), with a gradual increment of volumes between regions. The main effect of MRI-based involvement of SVZ and cortex was not significant, F  = 1.075, p = .370, and η2 = .068 , meaning no influence of the MRI-based classification in tumour volumes. Table 3) grouped by tumour regions and across SVZ and cortex involvement (MRI-based classification).A mixed between-within subjects ANOVA conducted to assess the impact of MRI-based involvement of SVZ and cortex on patients’ volumes across three tumour regions showed no significant interaction between variables, Wilks’ Lambda = .811, r = −.045, df = 45, and p = .767; a fair, positive, partial correlation was depicted between ADC of the proximal oedema and peritumoural volume, r = .347, df = 43, and p = .019; this fair, positive, association was more significant between ADC of distal oedema and peritumoural volume, r = .418, df = 34, and p = .011; and a positive, poor, partial correlation was observed between ADC of necrosis and necrosis volume, r = .263, df = 4540, and p = .093. An inspection of the zero order correlation suggested that controlling for age and gender increased the significance of correlations between ADC values in oedema and peritumoural volumes. Figure Partial correlation analyses between ADC values and their corresponding tumour region volumes were controlled for age and gender. There was a poor, negative, partial correlation between ADC of the enhancing tumour and its corresponding volume, χ2 (3) = .673 and p = .879; Fig. χ2 (4) = 4.166, and p = .384. A second Cox proportional hazards regression analysis considering only tumour region volumes provided an almost significant model: − 2 log-likelihood = 146.066, χ2 (4) = 9.303, and p = .054. Only the non-enhancing tumour volume depicted a trend towards significance of the hazard function: p = .067 and HR = 1.077. This value indicated that, for every additional unit increase in cubic centimetres of a non-enhancing tumour, patients increased 7.7% of the risk to report a tumour progression, controlling for all other factors in the model.The median PFS time was 10 months . Second, we manifest that there is not a substantial effect of the MRI-based involvement of SVZ and cortex by GB in ADC measurements. Third, our multivariate assessment of volumes showed a statistically significant difference among tumour regions , with significant post hoc difference only for peritumoural oedema. We also found a substantial correlation between proximal and distal ADC values with the peritumoural volumes; these facts agree with recent studies that accept pre-treatment diffusion MRI as a predictive imaging biomarker (specific diffusion signature) associated with particular survival  and singDespite that there are dozens of recent studies showing the usefulness of several ADC-derived parameters for tumour differentiation and survival in GB , 31, an Report of ADC values is not standard, but can have a relevant meaning in the follow-up of treatment response in GB; recent studies have proved a significant association of ADC in subgroups stratification , the met2-weighted imaging (T2-w) is often assumed to signal penetration into the brain tissue [Gliomas produce microscopic invasion to surrounding tissues, especially white matter (WM) tracts , from thn tissue . We founn tissue .Some limitations of this study need to be mentioned: the data were analysed retrospectively, so a control group was not available. Several clinical and surgical variables were intentionally out of the scope of this study . As part of a research line investigation, the potential of quantitative MR biomarkers in brain tumours, this study focused on selected variables, and we acknowledge this study deserves a sequel of additional findings. Also, for this group of patients, we did not have access to advanced biomarkers such as spectroscopy, perfusion, and diffusion tensor imaging techniques. We would have liked to use MRI perfusion for the differentiation of true progression/recurrence from pseudoprogression, considering that many other studies proposed this technique as an affordable and valuable diagnostic tool. Future prospective studies could regard perfusion as an additional method to be performed. However, none of these advanced MRI biomarkers is used to anticipate tumour progression on a day-to-day basis , 42. InsIn conclusion, specific ADC values at selected tumour regions  is a quantifiable predictive biomarker in the PFS of patients with GB. This parameter might offer clinical applicability to clinicians in the short term and routine assessment in GB.