Based on similar textures and major element mica composition, neither textural differences, prograde growth zoning, diffusion nor a retrograde overprint can explain this correlation. Modelling shows that B devolatilization during metamorphism can explain the general trend, but fails to account for the wide compositional and isotopic variability in a single, well-equilibrated sample. We, therefore, argue that this trend represents fluid–rock interaction during peak metamorphic conditions. This interpretation is supported by fluid–rock interaction modelling of boron leaching and boron addition that can successfully reproduce the observed spread in δ11B and [B]. Taking into account the local availability of serpentinites as potential source rocks of the fluids, the temperatures reached during peak metamorphism that allow for serpentine dehydration, and the high positive δ11B values (δ11B = 20 ± 5) modelled for the fluids, an influx of serpentinite-derived fluid appears likely. Paragonite in lawsonite pseudomorphs in an eclogite and phengite from a retrogressed metabasite have B contents between 12 and 68 μg/g and δ11B values that cluster around 0% (δ11B = − 5.0 to + 3.5). White mica in both samples is related to distinct stages of retrograde metamorphism during exhumation of the rocks. The variable B geochemistry can be successfully modelled as fluid–rock interaction with low-to-moderate (< 3) fluid/rock ratios, where mica equilibrates with a fluid into which B preferentially partitions, causing leaching of B from the rock. The metamorphic rocks from Lago di Cignana show variable retention of B in white mica during subduction-related metamorphism and exhumation. The variability in the B geochemical signature in white mica is significant and enhances our understanding of metamorphic processes and their role in element transfer in subduction zones.This study presents boron (B) concentration and isotope data for white mica from (ultra)high-pressure (UHP), subduction-related metamorphic rocks from Lago di Cignana . These rocks are of specific geological interest, because they comprise the most deeply subducted rocks of oceanic origin worldwide. Boron geochemistry can track fluid–rock interaction during their metamorphic evolution and provide important insights into mass transfer processes in subduction zones. The highest B contents (up to 345 μg/g B) occur in peak metamorphic phengite from a garnet–phengite quartzite. The B isotopic composition is variable (δThe online version of this article (10.1007/s00410-020-1661-8) contains supplementary material, which is available to authorized users. Boron elemental and isotopic data can be used to track water cycling and the origin of fluid sources in subduction zone magmatism high-pressure (UHP) metamorphic rocks from Lago di Cignana . We investigate three different lithologies—a garnet–phengite quartzite, an eclogite and a retrogressed metabasite—that have experienced a similar P/T evolution in a subduction zone setting. Fluid–rock interaction models successfully match the data for both peak metamorphic and retrogressed samples, demonstrating that boron measurements in white mica are a sensitive probe into fluid–rock interaction processes and emphasizes the role of intergranular fluids for element mobility and transfer even at ultrahigh-pressure metamorphic conditions.A belt of subduction-related (U)HP metamorphic rocks in the Western Alps stretches from the Mediterranean Sea to Switzerland Fig. . These rAt the Lago di Cignana in the Valtournenche, NW Italy Fig. , tectoni40Ar/39Ar dating of phengite from the LCU gave ages around 44 Ma  a fWavelength-dispersive quantitative mineral chemical analyses were performed at the University of Kiel (Germany) using a JEOL JXA 8900R electron microprobe. Elements were measured 15 s on peak and 7 s on background with a beam diameter of 5 μm, a beam current of 15 nA and an acceleration voltage of 15 kV. Natural standards were used for calibration and a CITZAF matrix correction was applied. Secondary standards used for quality control were garnet , plagioclase  and olivine  from the Smithsonian Institution, Washington DC  was ~ 2400 to avoid interferences from 9BeH+ and 10BH+. At the start of each analysis, the analytical area was pre-sputtered for 60 s using the same beam conditions as during the measurement itself. Subsequently, using an automated routine, the secondary beam was centred relative to the field aperture and the mass centre of the 11B peak was determined. Each analysis consisted of 50 cycles during which 10B and 11B signals were counted sequentially by an electron multiplier detector with counting times of 8 s and 2 s per cycle, respectively. 11B signals were divided by time-averaged 10B signals of the cycles before and after, resulting in 49 11B/10B ratios per analysis. The 11B count rate was ca. 180 cps na−1 μg/g−1. One second uncertainties are reported in per mil as relative standard error, i.e., the relative standard deviation of all analytical cycles divided by the square root of the total number of cycles. Raw 11B/10B ratios were converted to true 11B/10B ratios based on reference standards measured at regular intervals throughout the analytical session (see details below) and converted to delta notation  using a value of 4.04362  in Edinburgh using a Cameca IMS-1270 ion microprobe. Gold-coated thin sections were sputtered by an 8 nA 11B of − 1.2% for phengite 80-3 was observed compared to the glasses, in agreement with offset of micas relative to anhydrous glasses reported by Pabst et al. , Na/(Na+K) and Si contents , Na/(NIn the garnet–phengite quartzite (LC-3), the compositional variability of phengite is small with Mg# covering a range from 0.59 to 0.63. Na/(Na+K) ranges from 0.03 to 0.06 and Si per formula unit (p.f.u.) varies between 6.89 and 7.01. Mg# and Si p.f.u. are positively correlated in phengite. Individual phengite grains are homogeneous and do not show any visible zonation in major elements . Adjacent but distinct phengite grains also lack any significant differences in major element composition.The eclogite (sample LC-1b) contains only paragonite but no phengite, displaying small variations in Na/(Na+K) (0.94–0.97) and Si p.f.u. (6.05–6.10). Mg# in paragonite is variable, covering a range from 0.54 to 0.75. However, concentrations in both MgO (0.16–0.54 wt%) and FeO (0.18–0.40 wt%) are low, so that small variations in the concentrations have relatively large effects on Mg#. Overall, variations in major element contents are small and element distribution maps show compositionally homogeneous grains .Phengite in the retrogressed metabasite (sample LC-2) is chemically more variable than white mica in the other two samples for both Si p.f.u. (6.67–7.03) and Na/(Na+K) (0.04–0.12). Mg# varies from 0.76 to 0.90 and correlates positively with Si p.f.u., similar to phengite in the garnet–phengite quartzite. Element distribution maps reveal a patchy zonation in Si, Al and Mg . Areas of higher Mg# coincide with elevated Si contents, but the spatial distribution of these areas relative to the grain boundaries is not well defined.11B versus B content  exhibits a range in [B] from 12 to 68 μg/g, with six out of seven analyses yielding < 25 μg/g B. δ11B varies from − 5.0 to + 2.8%, but a clear correlation between [B] and δ11B is lacking. Phengite in the retrogressed metabasite  has B contents in the range 29–57 μg/g and δ11B from − 4.1 to + 3.5. These values show considerable overlap with the B geochemistry of the eclogite (sample LC-1b).The diagram δent Fig.  illustraent Fig. , 3, 4. B2O  and the metasedimentary garnet–phengite quartzite (113–345 μg/g B) may reflect the relative B enrichment of sedimentary compared to mafic igneous protoliths. For the metabasic rocks, mafic rocks of the oceanic crust represent protolith lithologies. Boron contents in MORBs range from 0.4 to 2.5 μg/g  inhomogeneities of the protolith, (2) retrograde overprint, (3) prograde growth zoning, (4) diffusion, (5) prograde devolatilization with loss of volatile elements, (6) peak metamorphic fluid–rock interaction  do not support a significant retrograde overprint. The Si content of potassic white mica (muscovite/phengite) is positively correlated with pressure and indicates whether the mineral equilibrated at peak metamorphic conditions or partially recrystallized during a retrograde overprint , trace elements may be less homogeneous and can reflect distinct stages of growth zoning. Compositional zoning in trace elements has been shown to reveal complexities in the formation of minerals that were previously undetectable with petrography or major element data, since some trace elements are generally less vulnerable to diffusive resetting and can track the mineral reaction history that excludes the major elements phe = (B/K2O)WR, where [K2O]phe = 10 wt% and [K2O]WR is assumed to be 1.55 wt%  are rare and most values fall between − 8 and + 4%, suggesting that an initial starting point of δ11B =  + 4% serves as a reasonable upper limit. Model curves based on more extreme initial mica compositions (e.g. B = 1400 μg/g and δ11B =  + 10%) that also fit the data are, therefore, excluded.The modelled fraction of B lost during devolatilization depends on the initial B content and δ11B = 0 ± 2%, and a loss of 55–85% B if initial values of 750 μg/g B and δ11B =  + 4% are assumed . This equation is solved for For the boron partition coefficient between fluid and white mica, we use a value of 1.4 based on the work by Marschall et al. . The parm Taylor :\documen11B of − 8.4% for neutral and acidic fluids  are sufficient to cause significant variation in B concentration and B isotopic composition. Importantly, a scenario of small amount of intergranular fluid interaction with the rocks at (ultra)high pressures is consistent with the petrographic and textural evidence.Two distinct scenarios of fluid–rock interaction are considered, assuming a temperature of 600 °C that is appropriate for peak metamorphic conditions Fig. . In the g N Fig. a. In theg N Fig. b. Fluid–11Bfluid = − 7 ± 3% and [B] = 10–40 μg/g, using initial values for white mica of [B] = 500–750 μg/g and δ11Bmica =  + 2 ± 4%  from which these fluids were derived. Such low δ11B values are at the lower end of δ11B values that have been observed in high-pressure metamorphic rocks, which typically range from − 15 to + 5%  can constrain fluid–rock interaction models that encompass the data points. All of these boron addition models require only small fluid/rock ratios (≤ 0.6). High positive δ11B values are typical for slab serpentinites that formed by subduction fluid infiltration, covering a wide range in δ11B from + 7 to + 24  were reported from fluids interpreted to reflect slab dehydration . A more variable chemical composition in secondary white mica that formed due to retrograde reactions compared to homogeneous primary, peak metamorphic white mica has been also observed in a subduction-related HP metagabbro that preserves an eclogite-facies assemblage  over a small interval in [B] are difficult to reconcile with devolatilization trends or prograde zonation. Instead, we will test and model the effects of fluid–rock interaction on the B systematic of these rocks, using the equations presented earlier in the text.Both rocks had a mafic igneous rock as protolith. They also reached similar peak P–T conditions, but given the effects of retrograde metamorphism it is unlikely that effects of prograde and peak metamorphic processes are recorded in the [B]-δ11B Fig.  and the 11B of − 12.0% for neutral and acidic fluids  and ~ 22% for clinopyroxene/paragonite , respectively. Assuming an overall relative uncertainty of 40% for fluid/mica partition coefficients, values for fluid/phengite and fluid/paragonite overlap and for clarity all models were calculated using the fluid/phengite partition coefficient. The model curves shown  with high B contents of 200–400 μg/g .The initial fluid composition in the first set of models, reflecting boron leaching Fig. a, can beron Fig. b, constrg/g Fig. b. All da11B systematics in white mica from the Catalina Schist were linked to fluids from highly devolatilized (low δ11B) and less devolatilized (moderate δ11B of − 2 to + 8%) rocks HP metamorphic rocks and consistent with the petrographic and petrologic constraints. We, therefore, suggest that retrograde white mica and tourmaline rims record a similar overprint by the same kind of fluids.The key observation derived from the modelling is that small to moderate fluid/rock ratios are sufficient to cause significant shifts in [B] and the moderate decrease in δ2O contents in the siliceous protolith. The decrease in δ11B with decreasing [B] cannot be explained by textural differences between grains, prograde growth zoning, diffusion or retrogression. The trend can be successfully modelled using a Rayleigh distillation equation, simulating devolatilization of B during prograde metamorphism, but this process fails to explain the wide range in [B] and δ11B in well-equilibrated rocks with a population of petrographically and texturally indistinguishable phengite. Alternatively, we modelled fluid–rock interaction at peak metamorphic conditions and several models successfully mirror the arrangement of data points. Our preferred model invokes fluid–rock interaction with a serpentinite-derived, high-B, high-δ11B (δ11B =  + 20 ± 5) fluid at or near peak metamorphic conditions. The [B]-δ11B variations in two samples that show a clear retrograde metamorphic overprint can be modelled by fluid–rock interaction at low to moderate (< 3) fluid/rock ratios during retrogression. The modelled fluid composition yields a moderately positive δ11B of + 6 ± 6, which agrees well with slab-derived fluids generally and those for Lago di Cignana in particular. Our observations underline the value of in situ observations of fluid–rock interaction processes in HP metamorphic rocks, which in turn is crucial for our understanding of the links between metamorphic processes in subduction zones and the geochemistry of arc magmatic rocks.The white mica B concentration and isotopic data of the three Lago di Cignana (U)HP samples that have experienced a similar P/T evolution in a subduction zone setting reveal significant variability both between and within the samples. Phengite in a metasedimentary garnet–phengite quartzite has the highest B contents, reflecting B enrichment coupled too low to moderate KSupplementary file1 (PDF 12266 kb)Below is the link to the electronic supplementary material.


The utility of intratumour heterogeneity as a prognostic biomarker is the subject of ongoing clinical investigation. However, the relationship between this marker and its clinical impact is mediated by an evolutionary process that is not well understood. Here, we employ a spatial computational model of tumour evolution to assess when, why and how intratumour heterogeneity can be used to forecast tumour growth rate and progression‐free survival. We identify three conditions that can lead to a positive correlation between clonal diversity and subsequent growth rate: diversity is measured early in tumour development; selective sweeps are rare; and/or tumours vary in the rate at which they acquire driver mutations. Opposite conditions typically lead to negative correlation. In cohorts of tumours with diverse evolutionary parameters, we find that clonal diversity is a reliable predictor of both growth rate and progression‐free survival. We thus offer explanations—grounded in evolutionary theory—for empirical findings in various cancers, including survival analyses reported in the recent TRACERx Renal study of clear‐cell renal cell carcinoma. Our work informs the search for new prognostic biomarkers and contributes to the development of predictive oncology. We restrict our analysis to mutations that either increase tumour cell fitness (termed driver mutations) or confer resistance to treatment. The distribution of fitness effects and the nature of epistasis in tumour evolution are only poorly understood but it is reasonable to assume that biological constraints impose diminishing returns , where X is drawn from an exponential distribution with mean value s, and m is an upper bound on the cell division rate. Because we set m to be much larger than the initial value of r, the combined effect of drivers is approximately multiplicative. In simulations of response to treatment, we additionally model the acquisition of resistance mutations. Treatment is applied at the endpoint size (one million cells) and immediately eliminates all cells that lack resistance mutations. The tumour's subsequent growth (if any) is tracked until it again attains the endpoint size. This choice of survival outcome takes advantage of the fact that, in a simulation study such as ours, the observation time is essentially unlimited and we can look at endpoints that would be infeasible or unethical in a real clinical trial. We use Gillespie's exact stochastic simulation algorithm  when the deme population size exceeds carrying capacity 2.2K), driver mutation rate (μ) and mean driver fitness effect (s)—influence the coefficient of correlation between a predictor variable , measured at a particular tumour size, and the rate at which the tumour grows to a larger endpoint size , where t is time measured in days (results are insensitive to the choice of the constant in this term). We conducted analyses in R using the “survival” package . If a variable's effects significantly grow or diminish with time then we include a term for the interaction between that variable and log2.4n equally abundant clones then the diversity index is 1/(n × (1/n)2) = n.We measure clonal diversity using the inverse Simpson index, defined as one divided by the sum of the squares of all clone frequencies. This index can be straightforwardly interpreted as an effective number of distinct clones. For example, if a tumour contains 2.5t≥τ, we calculate Θ(t)=∑i(fi(t)‐fi(t‐τ)2, where fi(t) is the frequency of clone i at time t, and τ is 10% of the total simulation time (the time from when the simulation is initiated with a single cell until the endpoint time). The clonal turnover index is then the mean value of Θ(t). Time is measured in cell generations  and the time points are evenly spaced.Our clonal turnover index quantifies the average rate of change in clone frequencies. Specifically, for each time point 2.6K .We variously measure clonal diversity among all cells in the simulated tumour, all cells at the tumour edge, a small random sample of cells, or one or more small localized samples of cells (simulated biopsy). We sample the edge of the tumour by selecting all demes that are directly “visible” from one of the four sides of the square lattice and, from each of these edge demes, we sample a number of cells equal to the square root of the deme carrying capacity, Biopsies are taken from small disc‐shaped regions, centred at a given proportion of the distance from the tumour's edge to its centre of mass. When four biopsies are taken from the same tumour, they are located on perpendicular transects through the tumour's centre of mass Figure . The num2.7–6 and 10–2 per cell division  vary in their underlying biological parameters because of intrinsic and microenvironmental factors. In particular, genomic instability and mutation burden are highly variable within cancer types  but not in driver mutation rate (μ), diversity‐growth correlations instead follow the same pattern as when parameter values do not vary  and among those with low driver mutation rates . However, if driver mutation rate is also included as a continuous variable then the effect of clonal diversity disappears , the effect of clonal diversity disappears when mean cell division rate is included in the statistical model  then we find that, although both pretreatment clonal diversity and driver mutation rate predict progression‐free survival  mutations—for example due to variation in genomic instability—because higher diversity then correlates with higher driver mutation rate, which in turn predicts faster tumour growth. In the absence of all three of these factors, we find that the correlation between clonal diversity and future tumour growth rate is typically negative. These results are robust to biopsy sampling error and readily extend to forecasting progression‐free survival after treatment.Although the influence of stochastic factors fundamentally limits predictability in our model—as is the case in real cancers (Lipinski et al., In their own study cohort and in the larger TCGA kidney cancer cohort, the TRACERx Renal consortium found that low intratumour heterogeneity correlates with longer progression‐free and overall survival times when tumours have low genomic instability, but not when tumours have high genomic instability (Turajlic et al., Our work contributes to a growing body of work on predicting evolution in complex systems, which is gaining increasing attention because of its potential to provide mechanistic explanations for patterns observed in clinical data and to guide therapeutic interventions (reviewed in Lässig, Mustonen, & Walczak, We have kept our model simple so as to yield the most general insights. Our work thus has several limitations that motivate further investigation. Even allowing for the fact that we simulate a two‐dimensional slice of a larger three‐dimensional tumour, the endpoint size of one million cells is unrealistically small. This discrepancy might be relatively inconsequential, however, given our finding that forecast accuracy is robust to increasing endpoint size. The essential principles that underlie our results apply in three dimensions just as in two dimensions, but outcomes might differ quantitatively due to reduced clonal interference in the former case. In common with previous computational modelling studies (Bozic et al., An especially promising direction for future research is in combining clonal diversity measurements with information about evolutionary processes, such as the pervasiveness of selective sweeps in a given tumour type across individuals or in a particular tumour within an individual (Maley et al., None declared.MEH and RN conceived the research question and designed the study. RN designed and created the computational modelling framework. JTB, CLS and RN wrote analysis code, ran simulations and analysed output data. RN wrote the manuscript with contributions from CLS and MEH and drawing from the related master's thesis of JTB.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Figure S7Click here for additional data file.Figure S8Click here for additional data file.


Unlike traditional cancer therapies, such as surgery, radiation and chemotherapy that are typically non-specific, cancer immunotherapy harnesses the high specificity of a patient’s own immune system to selectively kill cancer cells. The immune system is the body’s main cancer surveillance system, but cancers may evade destruction thanks to various immune-suppressing mechanisms. We therefore need to deploy various immunotherapy-based strategies to help bolster the anti-tumour immune responses. These include engineering T cells to express chimeric antigen receptors (CARs) to specifically recognise tumour neoantigens, inactivating immune checkpoints, oncolytic viruses and dendritic cell (DC) vaccines, which have all shown clinical benefit in certain cancers. However, treatment efficacy remains poor due to drug-induced adverse events and immunosuppressive tendencies of the tumour microenvironment. Recent preclinical studies have unveiled novel therapies such as anti-cathepsin antibodies, galectin-1 blockade and anti-OX40 agonistic antibodies, which may be utilised as adjuvant therapies to modulate the tumour microenvironment and permit more ferocious anti-tumour immune response.  Cancer remains a main global challenge due to the lack of early diagnosis, the inherent biological complexity of the tumour microenvironment (TME) and the unavailability of highly efficacious treatment strategies. In year 2015 to 2017, there were 367,167 new cases of cancers, with 165,000 deaths in the UK alone . T. T157]. Tumour tropism represents an important aspect in engineering these viruses and it can be either a native feature of the virus or gained through the insertion of specific genes, expressing ligands that can bind to receptors ubiquitously present in cancers such as adenovirus type 3 (Ad3) or herpes virus entry mediator (HVEM) ,158. CanAnother benefit of these agents is insertion of therapeutic genes in their viral genome, so that an anti-tumour immunity can be strongly induced . NeverthThese features have been utilised in designing OVs as a form of therapeutic agents; some of which have already been approved by the FDA and other regulatory authorities. This development process however faces a number of challenges which are addressed by continued research in this area.Although the concept of using viruses to destroy cancer cells arose in the 19th century, limited success and low profiling of this virotherapy strategy meant that very little research was conducted. However, it was the discovery of genetic engineering that brought about a renewal of interest in virotherapy that allowed the generation of more potent, tumour-specific oncolytics. This initiated active research in the discovery of safer, more selective virotherapies, such as herpes simplex virus (HSV) and adeno virus-based therapies in cancer, leading to successful clinical trials and approvals by health regulatory bodies worldwide.dlsptk), a gene involved in the regulation of thymidine levels and is crucial for virus replication [dlsptk established perfect features for targeting brain cancers, but its advancement into clinical trials was stopped due to the resistance to antiviral drugs gained through dlsptk deletion and undesirable toxicities at high titres [dlsptk modifications have been implemented through genetic engineering to lower neurotoxicity of HSV-1 based OVs while still maintaining their ability to target actively dividing cells. Deletion of both HSV-1 is a large double-stranded DNA pathogen, known for its neurotropism and the capability of causing lifelong infection . Its tumlication . The remlication . The innh titres . Since dlishment ,166. G20lishment . An altelishment . G47Δ isequences . HSV1716equences .interleukin-12 (IL-12). M032 is a second-generation OV that bears the IL-12 gene, inducing T cells immunologic effects in murine models and is currently tested in a phase I clinical trial (NCT02062827) [Improvements in initiating anti-tumour effects were achieved through the insertion of the therapeutic gene 2062827) .More recently, in October 2015, FDA and EMA approved the use of Talimogene Iaherparepvec (T-VEC) in the treatment of advanced melanoma . T-VEC iThis family of double-stranded DNA viruses causes upper-respiratory tract and gastrointestinal infections in humans . HoweverAnother genetically engineered oncolytic virus, DNX2401, uses the genome of an adenovirus-gamma 24 with a cyclic arginine/glycine/aspartic acid (RGD-4C) peptide motif inserted in it and a Δ-24 mutation . DNX2401Currently there are numerous virotherapies that are being tested in clinics and the most promising ones are focused on brain cancer. Central nervous system (CNS) malignancies usually show a dim prognosis with a median survival rate of just 14.6 months in the case of glioblastoma according to WHO . The intWhen compared with other immunotherapies OVs seem to be more potent anti-tumour immunity inducers . The issAltering the tumour microenvironment is the most attractive feature for combination strategies with other immunotherapies such as checkpoint inhibitors, dendritic vaccines and CAR T-cells, where the entrance problem is often encountered . CombinaMelanoma is susceptible to immunotherapies and targeting it with both OVs and immune checkpoint inhibitors that were specifically designed to target this cancer and have previously been approved for clinical use was thought to have a better outcome, compared with monotherapies . HoweverIn preclinical assays made on murine models, many OVs have shown better results in the overall survival rate and cancerous cell destruction when combined with immune checkpoint inhibitors . The insThe potential of combining checkpoint inhibitors with OV therapy is currently being tested in a phase II clinical study (NCT02798406) where the adenovirus DNX-2401 with pembrolizumab are used in the treatment of glioblastoma (GBM) and gliosarcoma. Interim results of this phase II revealed that there was no dose-limiting toxicity or unexpected safety issues with 47% of the patients experienced clinical benefit and two patients with >94% tumour regression . Followireg cells, etc., to develop new therapies and to achieve greater treatment efficacy. Such recent and ongoing efforts are discussed below.Advances in immunotherapies against both solid tumours and haematological malignancies have shown promise and an increase in clinical benefits in recent years. However, the complex dynamic of the TME, the heterogeneity of tumours and their histological features pose everlasting challenges in achieving improved treatment efficacy. Despite the successes in targeting non-tumour components, including immunosuppressive mechanisms, our lack of understanding of the cellular and molecular crosstalk in the tumour niche results in only a few patients exhibiting objective control of tumour progression. Therefore, numerous investigations are being carried out to identify potential targets such as galectin-1, cancer associated cathepsins, OX40+Foxp3+ TGalectin-1  is a key pro-tumourigenic player with multiple roles in the TME. This glycoprotein is secreted by tumour cells and induces tumour cell proliferation, migration and invasion of local tissue, reviewed elsewhere . Tumour-Earlier studies from both in vitro and in vivo experiments demonstrated an increase in extracellular Gal1-induced apoptosis in Th1 cells via its interaction with CD43, CD45 and CD7 receptors, resulting in tumour-induced immune evasion ,200. MorIt was thought that Gal1 secreted from large tumours induces T cell apoptosis and switches to Th2 cytokine production. However, recent data suggests that Gal1 conditions the tumour endothelial cells by inducing expressions of PD-L1 and Gal9 and suppresses anti-tumour immunity by causing T cell exclusion, which in fact takes place relatively earlier during tumour growth . Thus, aAnother evolving anticancer treatment is a combination of radiation therapy and anti-PD-1 therapy. Early clinical data (phase I) showed an improved outcome of the combination of photon radiation with anti-PD-1 immunotherapies in patients with non-squamous non-small cell lung carcinoma (NSCLC) , which hCathepsins are lysosomal enzymes which are highly expressed in tumour cells in response to the hypoxic and slightly acid microenvironment. A number of these cathepsin enzymes has been implicated in the progression of tumour growth and metastasis in the last two decades ,212,213.regs has been known to suppress effective anti-tumour immunity [regs infiltrate into tumour hosting tissues, which is often associated with poor prognosis in cancer patients [Although they are indispensable at preventing autoimmunity, Foxp3-expressing Timmunity . Tregs ipatients ,228,229.regs in TME  [regs that under certain conditions can inhibit the generation of Foxp3 Tregs [n = 30), with 12 patients having regression of at least one metastatic lesion after a single course of treatment [reg ratio, leading to rapid tumour shrinkage [A number of strategies have been tested in an attempt to dampen down the immunosuppressive effects of Tiewed in ), such ag., OX40 ), select2646748) , etc. Am2646748) , has shop3 Tregs . Severalp3 Tregs . A phasereatment . Anotherreatment . Currentreatment ,237. Thihrinkage ,237.regs in combination with other anti-cancer treatments may enhance anti-tumour immune responses and clinical benefit.More recently, OX40-positive plasmacytoid DCs (major producers of IFN-I) were discovered to promote anti-tumour immunity in the TME . pDCs arAnti-cancer immunotherapy as monotherapy or in combination has shown promising results in several cancers. Although earlier successes were more evident in haematological malignancies, more recent reports show increased clinical benefits in patients with solid tumours; in particular, oncolytic virotherapy, which is able to cross the blood brain barrier and kill cancer cells directly but also indirectly by enhancing T cell trafficking in the TME. However, resistance to therapies via mechanisms such as tumour-induced immune evasion, may lessen the therapeutic efficacy. Therefore, further research efforts should focus on rational combinations of immunotherapies to help overcome the anti-tumour immune resistance and help drive clinical benefit.


Current trends concerning hydraulic cylinder sealing systems are aimed at decreasing energy consumption which can be materialized by minimizing leaks and reducing friction. The latest developments in the field of materials and sealing system geometries as well as modern simulation possibilities allow maximum performance levels of hydraulic cylinders. Reducing friction is possible by hydro-dynamic separation of the sliding and sealing points already at very low velocities and by using materials, such as plastomers, from polytetrafluoroethylene (PTFE) (virgin PTFE and filled PTFE). It is within this context that this paper discusses a theoretical and experimental study focused on the tribological behavior of coaxial sealing systems mounted on the pistons of hydraulic cylinders. It presents a methodology for the theoretical determination of the lubricant film thickness between the cylinder piston and the seal. The experimental installation used for measuring fluid film thickness is presented, and the results obtained under various working conditions are compared to the theoretical ones. For the analyzed working conditions related to pressure, speed, and temperature, the paper concludes with a set of criteria for the selection of the optimum seal material so as to maximize energy efficiency. An important characteristic of hydraulic motors, whether linear or rotary, is their high energy density. Hence, a small-sized motor is capable of generating high power outputs. This benefit is due to the deployment working fluids at increasingly higher pressures (hundreds of bar) which requires rethinking the constructive and functional aspects of such motors. Thus, an important role is assigned to the sealing system.A sealing system is defined as the assembly of elements designed to create the complete separation of two different media. The main component of such a system is the seal itself, a deformable or non-deformable element placed in a specially conceived seat. As the seal is pushed onto the sealed surface by a pre-tensioning force applied at mounting and/or by the fluid pressure, it achieves its function of rendering a hermetic system .Conceiving an adequate sealing system requires information about seal geometry, the utilized materials, material–fluid compatibility, the quality of the contacting surfaces, sealing dynamics, etc. All these contribute to the energy efficiency of a sealing system, considering that high friction forces, fluid leaks or heat generation are responsible for the loss of energy that affects hydraulic systems. Studies conducted by ORNL/NFPA   have revThe energy efficiency of a linear hydraulic motor is increased by diminishing the friction forces in the sealing systems. Small friction forces can be ensured, however, only within certain limits, as reducing the contact pressure between the seal and the sealed surface automatically causes fluid leakage. The relationship between friction and pressure contact is influenced by the geometry and the material of the seal which entails the necessity of identifying a compromise solution between obtaining a reduced friction force and reduced leakage.Most seals used in hydraulic drives are made from polymers, in particular from elastomers, plastomers or thermoplastic elastomers. The type of material that is utilized depends on the specific working conditions and its compatibility with the sealed fluid, fluid pressure, fluid temperature, etc. In order to ensure a long service life for sealing systems, the properties of seal materials need to be known in detail. The fact that, in many cases, contradicting characteristics are required, makes it difficult to identify and select the most adequate material.Research concerning the construction and functioning of sealing system dates back over 80 years ago . ReferenThe last years of the 20th century produced studies concerning the operational behavior of sealing systems made from elastomers. Thus, in 1987, Nau  presente2S), and mixtures of these [2S or carbon, added into the composition of PTFE influence seal behavior in different ways. Thus, for example, while bronze improves wear resistance at the temperature of the surrounding environment, its effect is opposite at high temperatures.As to the use of plastomers, materials which combine the qualities of elastomers and plastics, such as rubber-like properties with the processing ability of plastic , studiesof these . FillersOver the last years, many studies have presented results on the regime of friction in the sealing tribological system. It was revealed that, in the case of certain dynamic sealing systems, the occurrence of a relative motion between the seal and its contact surface causes a thin fluid film to be built up between the two elements. Müller  addresseThe results of other research concerning the character of friction in the sealing tribological system are described in the works of Kanzaki et al. , StupkieThe present paper analyzed the mechanism and character of friction in coaxial sealing systems made from PTFE and its compounds. The use of these materials was justified by their low friction properties that ensure a high level of energy efficiency. The study’s focus on sealing systems was determined by their widespread use, constructive simplicity, and the polymers they are made from—materials with pronounced low friction properties.The paper is structured as follows: Coaxial sealing systems are assemblies consisting of a seal made from a material with advanced low friction properties and an O-ring that ensures the pre-tensioning of the entire package. Generally, the seal is made of polytetrafluoroethylene (virgin or filled PTFE) and the O-ring of elastomers of various types: nitrile butadiene rubber (NBR), fluorocarbon (FKM), ethylene propylene diene monomer (EPDM), hydrogenated nitrile butadiene rubber (HNBR), fluorosilicone (FVMQ), and silicone rubber (Q). rε0 of 10% to 25%. Upon the onset of fluid pressure inside the hydraulic motor, the O-ring is deformed additionally and exerts a greater radial pressure.In order to achieve the sealing effect, a radial pressure has to be exerted upon the seal  by means of the O-ring. The O-ring is placed into its seat in a pre-tensioned state with an initial specific radial deformation v) of the piston, the dynamic viscosity (η) of the working fluid, and the compressibility of the seal material, the friction between the seal and the cylinder surface can be of dry, fluid or mixed type. Fluid friction ensures a good energy efficiency due to the diminished friction forces, a situation nevertheless conflicting with the phenomenon of sealing. The hydrodynamic separation between the seal and the surface of the cylinder that is typical for fluid friction causes increased fluid loss by leakage, as the fluid flows towards the lower pressure side.A relative motion occurring between the seal and the inner surface of the cylinder generates, according to the laws of hydrodynamic lubrication, a dynamic pressure and a fluid film of variable thickness. Depending on the velocity (g) has the beneficial effect of diminishing the friction forces, its inconvenience is leakage. The effect of inadequate sealing is a certain fluid loss that can manifest as leakage (the Poiseuille component of flow) or drag (the Couette component of flow). Leakage means fluid loss, even at rest, caused by the pressure drop between the two sealed chambers. Fluid drag is determined by the existence on the moving component of a fluid film that is necessary for ensuring minimum friction forces [While the presence of a gap (n forces . Evidentdp/dx): a large gradient means a thin fluid film in the sealed area, while a small gradient determines a thicker fluid film. dp/dx) causes a smaller quantity of dragged fluid.The thickness of the fluid film between the seal and the cylinder surface is determined by the evolution of the pressure gradient in the gap (dp/dx) and the dimension of the gap, g(x), in the direction of motion x while neglecting inertial forces is [g* is the dimension of the gap at the point of zero pressure gradient (dp/dx = 0).For a viscous flow, the basic relationship between the pressure gradient (orces is :(1)dpdx=g) formed between the seal and its contact surface.Crucial for assessing the type of friction in a coaxial sealing system is determining the magnitude and evolution of the gap  on the sealed surface is a parabola described by Equation (2) [b0 is the width of the contact surface between the O-ring and the seal, and rpmax0 is the maximum contact pressure given by Equation (3) [At rest and in the absence of the sealed pressure a, the dition (2) :(2)pr0=ption (3) :(3)pr0marm is Poisson’s ratio for the ring material (NBR), rε0—is its initial specific radial deformation, and H is the Shore A hardness of the material.All notations used in Equation (3) refer to the O-ring: p1, in which case, in the absence of a relative velocity v, the distribution of pressure on the sealed surface is given by Equation (4) [β = Ar/An = the real non-dimensional contact area; An, Ar = the nominal, real contact area, respectively; v = gliding velocity; L = width of the seal; g0 = fluid film thickness at zero pressure gradient.In tion (4) :(4)p(x)=rε0 of the O-ring, and the pressure of the working fluid.The real non-dimensional contact area is less than unity and is a quantity that accounts for the materials of the two elements of the friction pair , the initial specific radial deformation While at rest, the seal and the cylinder are in direct contact; upon onset of motion, the two elements will be completely separated by a “wedge”-shaped gap .It is the radial elastic deformation of the seal that allows the generation between the two surfaces of a thin fluid film. The separation of the two surfaces is caused by the dynamic component of the developed pressure given by Equation (5) :(5)pd(x)The deformation of the seal (the magnitude of the gap) is small compared to the pre-tensioning of the O-ring at mounting;The thickness of the fluid film in the gap is small compared to the radius of the seal;It is admitted that the pressure distribution in the gap is identical to that determined for the O-ring;It is admitted that, on radial direction, a balance appears between the pressure created by the compression of the O-ring and the dynamic component of the pressure in the gap. The two pressures are of equal magnitude and the form of the gap is determined by the distribution of pressure generated by the O-ring.The fluid flow through the thus formed gap can be studied starting from several hypotheses:g0 is [pm and pE are Poisson’s ratio and Young’s modulus of the seal material, respectively; h = thickness of the seal.The calculation of the magnitude of the gap formed between the seal and the cylinder surface is based on the hypothesis that the seal is a cylinder with thin walls subjected to a pressure given by Equation (5). Thus, at zero pressure gradient, the gap g0 is ,26:(6)g0Equation (6) highlights the direct dependency of the fluid film thickness on the velocity, the viscosity of the working fluid, and the seal width. Thus, the velocity influences decisively the type of friction in the coaxial sealing system. In the absence of motion among the components of the tribosystem (at the debut of motion), friction is dry due to the adhesion forces among the contacting materials. Upon the onset of the motion, the adhesion forces, the internal friction forces as well as the forces caused by the asperities of the two surfaces clinging one to another determine mixed friction. As the velocity grows, the velocity of the fluid determines the complete separation of the tribosystem components (known as grease planning) which indicates the presence of fluid (hydrodynamic) friction .maxR of the two surfaces initially contacting. In the case of coaxial sealing systems, the conducted experimental research has revealed that fluid friction is present even when the average thickness of the fluid film falls below the sum of the roughness values maxR of the two surfaces [Generally, fluid friction appears only when the thickness of the fluid film is at least equal to the sum of the roughness values surfaces . This issurfaces .g0 and, consequently, the type of friction. The decreasing of the viscosity, due to the work temperature increase, diminishes the thickness of the fluid film which can affect the type of friction.The viscosity of the working fluid also influences the magnitude of In order to determine the type of friction in coaxial sealing systems, the test included seals made of three different polymeric materials of the category of polytetrafluoroethylenes (virgin PTFE and filled PTFE). In all cases, the O-ring was made of nitrile butadiene rubber (NBR) of 70 Shore A hardness. Polytetrafluoroethylene has one of the smallest friction coefficients ever recorded in a solid material (0.05 to 0.1). As it includes high-bonded carbon and fluorine, PTFE is almost completely inert to the substances it comes into contact with. These two properties account for the successful use of this material for tribological applications designed for reducing energy consumption in friction-intensive machinery as well as for reactive and corrosive applications.The added carbon fibers increase wear resistance, reduce the friction coefficient, and improve the thermal expansion properties. The addition of bronze to PTFE improves compression strength, thermal conductivity, and electrical conductivity. Also reduced is the tendency to extrusion while maintaining good sliding and wear properties. The PTFE-ul with added bronze is the standard material in hydraulic applications .rε0 of 15% resulted for the dimensions of the O-ring seat. The real non-dimensional area β for the friction pair consisting of the steel cylinder and the three seals made of different materials depends on the initial specific radial deformation rε0 of the O-ring and on the fluid pressure. An initial specific radial deformation A = 5.68 × 10−9 and B = 4827.627 [The fluid used for testing was anti-wear hydraulic oil ISO VG 32 which is a premium light-weight paraffinic-based hydraulic oil, ideal for industrial applications or for hydraulic systems . Equatio4827.627 .For oil temperatures between 20 °C and 60 °C (293 to 333 K), The influence of the working fluid pressure on the thickness of the fluid film was analyzed for an oil temperature of 60 °C (333 K) which implies a dynamic viscosity of 0.011 Pa∙s. The considered velocity was of 0.2 m/s. g0= f(v) diagram.For an oil pressure of 100 bar at a temperature of 60 °C (333K), g0= f(T) graph.The variation of the working fluid temperature also causes modifications of the fluid film. For a pressure of 100 bar and a velocity of 0.2 m/s, g0 can be noticed, for the analyzed interval from 0 to 200 bar, the diminishing of the fluid film was, however, rather small (of approximately 1 μm). For PTFE with carbon fibers (PTFE CF10) at high working pressures, the thickness of the fluid film decreased significantly up to 6.5 μm which worsens the conditions of friction;While with increasing pressure a decrease of The thickness of the fluid film grows with increasing velocities. The presence of a consistent fluid film at the seal–cylinder interface causes a significant decrease of friction forces and seal wear. On the other hand, high velocities carry the risk of fluid drag and, consequently, of inadequate sealing;The increase of the sealed fluid temperature causes the diminishing of its dynamic viscosity which determines a diminishing of the fluid film thickness. This triggers an unfavorable friction type;maxR = 0.63 to 2.5 μm [g0 is greater than the roughness sum of the surfaces that form the friction pair which yields the conclusion that fluid (hydrodynamic) friction is dominant. If the maximum admissible limit of the fluid film thickness is set to g0 = 10 μm, where fluid drag is within acceptable limits for pistons, The fluid film thickness has micrometric values (1 to 20 μm). As the recommended maximum roughness  of hydraulic cylinder interior surfaces is of o 2.5 μm , under cThe above figures yield a series of conclusions:As the fluid film thickness cannot be measured directly, this was achieved indirectly by the resistive method. This requires measuring the voltage drop on the resistance created by the fluid film between the seal and the surface of the hydraulic cylinder. The electrical connections to the sealing system are shown in oilR is determined by fluid film of thickness g0, and Ra is the resistance of the utilized measuring device (Ra = 10 MΩ). In the presented measuring configuration, the electrical resistance oilR is computed by Equation (9):Resistance oilR of the fluid film and its thickness g0 is linear [The dependency between resistance s linear :(10)CR= The graphs in the following figures  present The analysis of the three graphs shows that the measured values were very close to those calculated by Equation (6) with a maximum error of 3.5%. Thus, the conclusions of the theoretical research were confirmed, namely, that the thickness of the fluid film grows with the increasing velocity and is diminished as the pressure and temperature of the sealed fluid increase.The paper analyzes the operational behavior of three seals made of PTFE-based materials, one of 100% PTFE concentration (virgin PTFE), and two others with 10% carbon fibers and 46% bronze, respectively.The sealing mechanism was explained with a focus on the fact that, at the onset of a certain velocity, the elements of the friction pair are separated by a fluid film of a certain thickness. The presence of the fluid film causes hydrodynamic friction.The thickness of this film grows with increasing relative velocity;With increasing working pressure, between the seal and its adjacent surface, dry contact areas exceed the hydro-dynamically separated ones;Higher working fluid temperature and pressure cause smaller film thicknesses;Of the three tested materials, the most adequate for utilization is PTFE 46D with added bronze;Virgin PTFE and PTFE CF10 (with added carbon fibers) are adequate for small velocities and relatively high temperatures;At high working pressures, seals made of PTFE CF10 deform less, and a sudden decrease of the fluid film occurs with adverse effects on friction;The theoretically determined computational relationship for the fluid film thickness has been confirmed by the experimental results.The theoretical and experimental studies yielded a series of significant conclusions:The research results yielded the conclusion that the utilization of coaxial sealing systems with seals made of PTFE 46D is the recommended solution for the construction of linear hydraulic motors.


Tumour angiogenesis and lymphangiogenesis are hallmarks of cancer and have been associated with tumour progression, tumour metastasis and poor patient prognosis. Many factors regulate angiogenesis and lymphangiogenesis in cancer including non-coding RNAs which are a category of RNAs that do not encode proteins and have important regulatory functions at transcriptional and post-transcriptional levels. Non-coding RNAs can be encapsulated in extracellular vesicles called exosomes which are secreted by tumour cells or other cells in the tumour microenvironment and can then be taken up by the endothelial cells of blood vessels and lymphatic vessels. The “delivery” of these non-coding RNAs to endothelial cells in tumours can facilitate tumour angiogenesis and lymphangiogenesis. Here we review recent findings about exosomal non-coding RNAs, specifically microRNAs and long non-coding RNAs, which regulate tumour angiogenesis and lymphangiogenesis in cancer. We then focus on the potential use of these molecules as cancer biomarkers and opportunities for exploiting ncRNAs for the treatment of cancer. The growth of new tumour blood vessels and lymphatic vessels is a key element of cancer progression having a close association with metastatic spread, poor patient prognosis and survival ,2. TumouPrevious work has shown that a range of growth factors, cell surface receptors and a multitude of signalling molecules drives the remodelling of the blood and lymphatic vasculatures in cancer ,4. RecenLong ncRNAs (lncRNAs) exhibit a range of different regulatory functions in distinct cellular compartments  Figure . They caAn important functional feature of miRNAs and lncRNAs is that they can exert effects on the regulation of complex biological responses: miRNAs by targeting mRNAs encoding multiple proteins involved in the same or a related molecular pathway, lncRNAs by controlling the remodelling of chromatin to modulate gene expression, or by targeting miRNAs. Moreover, a miRNA can act in concert with other miRNAs, lncRNAs or transcription factors to mediate gene silencing in a precise manner.It has recently been shown that ncRNAs can be encapsulated in extracellular vesicles called exosomes and thus take on a pivotal role in cell-to-cell communication which occurs in the tumour microenvironment and ultimately contributes to tumour progression, remodelling of blood vessels and lymphatics, and tumour spread ,25. In tde novo from endothelial progenitor cells or cancer stem cells in a process called vasculogenesis [Tumour cells require oxygen and nutrients to proliferate, and solid tumours therefore require blood vessels to facilitate their growth and spread . Tumour ogenesis . Tumour ogenesis . SeveralLymphatic vessels can undergo multiple changes in cancer which facilitate the spread of tumour cells to organs distant from the primary tumour, such as lymphangiogenesis and other forms of lymphatic remodelling . LymphanThe remodelling of the lymphatic vasculature in cancer is driven by a diverse range of growth factors that are produced by tumour cells and cells in the tumour microenvironment. These growth factors bind cognate receptors on LECs and activate several molecular pathways that ultimately drive lymphangiogenesis; they are described below. Some members of the vascular endothelial growth factor (VEGF) family of secreted proteins are inducers of angiogenesis and lymphangiogenesis in cancer. The human VEGF family is composed of VEGFA, VEGFB, VEGFC, VEGFD and placenta growth factor (PIGF). These growth factors bind and activate cognate receptors VEGFR1, VEGFR2 and VEGFR3 with different affinities ,39,40,41The VEGFC-VEGFR3 and VEGFD-VEGFR3 signalling pathways have been the most studied in lymphangiogenesis and lymphatic vessel remodelling in cancer and are considered major drivers of these processes . In a tuVEGFA is secreted by tumour cells and stroma and is the most studied inducer of tumour angiogenesis; its presence is correlated with tumour size, blood vessel density and metastasis . VEGFA iBesides the VEGFs, other growth factors and signalling pathways have been reported to stimulate lymphangiogenesis and angiogenesis. Angiopoietins 1 and 2 (ANGPT1 and ANGPT2) and their receptors (TIE1 and TIE2) on endothelial cells are required for both these processes . FibroblLymphangiogenesis and angiogenesis in tumours involve complex regulation by stimulatory molecules which modulate interacting signalling pathways to induce endothelial cell proliferation, sprouting, migration and eventually formation of new vessels from the pre-existing network. It is clear from the literature that the proteins which are best validated as lymphangiogenic factors in vivo, acting directly on LEC, are VEGFC and VEGFD. Likewise, VEGFA is the best validated angiogenic factor in cancer. These conclusions are based on many mouse genetic models, tumour xenograft models and clinicopathological studies ,4,57. Recently, miRNAs and lncRNAs have been found in exosomes in cancer . ExosomeExosomal miRNAs are capable of regulating lymphangiogenesis and angiogenesis within the primary tumour and at metastatic sites . Within Several research groups have also shown that tumour cells can secrete exosomal miRNAs which influence non-endothelial cell types in the tumour microenvironment such as fibroblasts and macrophages so they can, in turn, promote tumour angiogenesis and/or lymphangiogenesis. For instance, Fan and colleagues have shown that exosomal miR-210 derived from lung cancer cells induces the reprogramming of normal fibroblasts into cancer-associated fibroblasts (CAFs) by activating the JAK2/STAT3 pathway in these cells. Reprogrammed fibroblasts then upregulate the expression of several angiogenic factors, including VEGFA, which stimulate the remodelling of tumoural blood vessels . AnotherIn specific tumour settings, cells of the tumour microenvironment, including endothelial cells, can secrete exosomes that target tumour cells or other cell types and thereby indirectly regulate tumour angiogenesis. For instance, mesenchymal stem cells secrete exosomes containing miR-100 which inhibits angiogenesis by modulating the mTOR/HIF-1α/VEGFA signalling axis in breast cancer cells, thus reducing production of VEGFA by these cells . ExosomaRecently, lncRNAs have been shown to be involved in cell-to-cell communication in the context of tumour angiogenesis . lncRNAsFAM225A lncRNA has been shown to promote oesophageal squamous cell carcinoma (OSCC) progression and angiogenesis by sponging miR-206 in BECs. Specifically, OSCC cells secrete high levels of exosomal FAM225A which can then be endocytosed by BECs wherein this lncRNA inhibits miR-206 leading to upregulation of the expression of miR-206 targets . These iAnother example of how exosomal lncRNAs regulate tumour angiogenesis is that chondrosarcoma cell-derived exosomes carry lncRNA RAMP2-AS1 which is delivered to BECs. Here, RAMP2-AS1 acts as a competing endogenous RNA by sponging miR-2355-5p thereby upregulating the expression of one of its targets, the VEGFA receptor VEGFR2, leading to activation of angiogenic signalling .Some lncRNAs have been shown to directly upregulate the expression of VEGFA in endothelial cells thereby promoting angiogenesis. For example, it has been shown that exosomal lncRNA Hotair, produced by glioma cells, upregulates the expression of VEGFA in brain microvascular endothelial cells and thus stimulates their remodelling .https://mirxes.com/gastroclear/ (accessed on 9 February 2021)) [Tumour-derived exosomes are found in a variety of body fluids including blood, tears, urine, milk, saliva and ascites , and they 2021)) .We envisage a scenario in which cancer-derived exosomal ncRNAs that promote tumour angiogenesis and lymphangiogenesis (such as those discussed in previous sections of this article) are included in panels of ncRNA biomarkers for liquid biopsy to detect and stage cancers in the clinic. The purpose of monitoring ncRNAs that promote angiogenesis and lymphangiogenesis would be to provide information about the propensity of detected tumours to metastasize, via blood vessels or lymphatics, to distant sites in the body which is the most lethal aspect of cancer for most patients. We acknowledge there is considerable work that still needs to be done to clinically validate specific exosomal ncRNAs as biomarkers of cancer metastasis.In this article we have focussed on ncRNAs which promote angiogenesis, lymphangiogenesis and metastasis in cancer. In contrast, there are a range of ncRNAs which are known to be anti-angiogenic or anti-lymphangiogenic and could therefore form the basis of therapeutic strategies to restrict tumour growth and spread ,110,111.There are a range of non-cancer clinical settings in which it could be beneficial to promote angiogenesis and/or lymphangiogenesis such as wound healing, cardiovascular diseases and lymphoedema. ncRNAs which promote angiogenesis and lymphangiogenesis, such as those discussed in this article, could be exploited for these purposes by employing lipid-nanoparticle formulated, nucleoside-modified synthetic RNA. The potential of such a strategy has been exemplified by recent animal studies in which lymphangiogenesis was successfully stimulated in a therapeutic setting by delivering exosomal miRNAs, derived from adipose-derived stem cells (ADSCs), to LECs. ADSCs are multipotent cells located in adipose tissue that have been used in therapeutic settings  because While non-coding RNAs do not encode proteins they do play a critical role in regulating the levels of many cellular and extracellular proteins , and thuOur current insight into the regulation of tumour angiogenesis and lymphangiogenesis by non-coding RNAs can be exploited for development of new cancer biomarkers and therapeutics, as discussed here. However, further research is required for this to occur. In particular, specific non-coding RNAs which promote tumour angiogenesis and/or lymphangiogenesis will need to be clinically validated as useful biomarkers of metastasis before being incorporated in liquid biopsy strategies for detecting and staging cancers. The use of anti-angiogenic or anti-lymphangiogenic non-coding RNAs as therapeutics to restrict tumour growth and spread seems attractive, particularly in light of recent regulatory approvals for vaccines based on lipid-nanoparticle formulated, nucleoside-modified RNA. A key challenge for this approach will be to determine the best anti-angiogenic/anti-lymphangiogenic non-coding RNAs to use for restricting cancer metastasis, which will need to be carefully assessed in clinically relevant animal models of cancer.


Fiducial markers are frequently used before treatment for image‐guided patient setup in radiation therapy (RT), but can also be used during treatment for image‐guided intrafraction motion detection. This report describes our implementation of automatic marker detection with periodic kV imaging (TrueBeam v2.5) to monitor and correct intrafraction motion during prostate RT.We evaluated the reproducibility and accuracy of software fiducial detection using a phantom with 3 implanted fiducial markers. Clinical implementation for patients with intraprostatic fiducials receiving volumetric modulated arc therapy (VMAT) utilized periodic on‐board kV imaging with 10 s intervals during treatment delivery. For each image, the software automatically identified fiducial locations and determined whether their distance relative to planned locations were within a 3 mm tolerance. Motion was corrected if either ≥2 fiducials in a single image or ≥1 fiducial in sequential images were out of tolerance.Phantom studies demonstrated poorer performance of linear fiducials compared to collapsible fiducials, and wide variability to accurately detect fiducials across eight software settings. For any given setting, results were relatively reproducible and precise to ~0.5 mm. Across 17 patients treated with a median of 20 fractions, the software recommended a shift in 44% of fractions, and a shift was actually implemented after visual confirmation of movement greater than the 3 mm threshold in 20% of fractions. Adjustment of our approach led to improved accuracy for the latter (n = 7) patient subset. On average, table repositioning added 3.0 ± 0.3 min to patient time on table. Periodic kV imaging increased skin dose by an estimated 1 cGy per treatment arc.Periodic kV imaging with automatic detection of motion during VMAT prostate treatments is commercially available, and can be successfully implemented to mitigate effects of intrafraction motion with careful attention to software settings. IGRTimaged‐guided radiotherapyOBIon‐board‐imagerRTradiation therapyVMATvolumetric modulated arc therapy1Prostate setup variation over a course of radiotherapy (RT) can compromise treatment, especially for highly conformal treatments using volumetric modulated arc therapy (VMAT), in which steep dose gradients are devised to limit radiation to adjacent normal tissues. Image‐guided radiotherapy (IGRT) using implanted fiducial markers can reduce setup error, and potentially improve treatment efficacy and decrease treatment related morbidity.The Varian Truebeam v2.5  enables planar kV images to be acquired during treatment delivery using the orthogonal on‐board‐imager (OBI). The software uses a proprietary search algorithm to find the location of the fiducials in each triggered image and determines if they are within a predetermined tolerance from the expected location. In this report, we present our experience implementing this feature into our clinical workflow. We initiated a process of evaluating this software for intra‐fraction monitoring in VMAT to the prostate, including work using a phantom, a pilot experience of ten patients undergoing therapy, and analysis including seven subsequent patients with a refined approach , under approval from an institutional review board.The software package evaluated allows the user to define the frequency of triggered kVs based on various criteria: breathing motion of the patient, elapsed time, number of MU delivered, or gantry angle. The user further defines the predetermined tolerance of relating the position of the identified marker to the expected position . The search region is depicted as a colored circle and the identified marker location as a crosshair Fig. . The cir2A phantom was created to mimic patient composition and geometry with three 0.28 × 10 mm gold collapsible fiducial markers implanted using a 22G, 20 cm needle . These fiducials can be placed in either a linear or crumpled orientation Fig. , inserteThree fiducial‐type settings  were chosen for superiority in automatic detection for additional phantom studies to evaluate accuracy and reproducibility. To assess accuracy, automatic marker detection was utilized with the phantom correctly aligned (shift = 0.0 mm) as well as with offsets (~0.5 mm increments) purposefully introduced via phantom positioning shifts in the superior‐inferior direction.. With the phantom in these different positions, a single treatment arc was used with triggered kV images acquired every 20 degrees. This enabled testing of the capability to identify when the fiducials moved outside of the 3 mm tolerance. The reported positional accuracy for this treatment couch  is 0.07 ± 0.22 mm.th patient with cylindrical fiducial markers placed at an outside institution was excluded, as it was found that the selected fiducial marker settings performed poorly. All patients had Gold Anchor fiducials placed transrectally prior to RT. RT was delivered with a VMAT technique using 2‐3 arcs and 6 MV photons. Triggered kV images were acquired every 10 s using the OBI, with fiducial‐type detection settings of “GoldSeed_1_3x5_0” (n = 7) or “Clip_1_5x4_0” (n = 10). Intrafraction motion correction with a couch shift was implemented when two fiducial markers in a single kV image were outside of set 3 mm tolerance, or when the same fiducial marker in two sequential images was outside of tolerance. Intrafraction correction required a treatment pause, acquisition of new orthogonal kV images utilizing on demand kV‐kV pair imaging , registration of the kV image to CT simulation image, alignment of fiducial markers, and manual couch shift. During treatment delivery, radiation therapists visually verified the automatic fiducial marker detection before treatment was stopped, as the automated algorithm did not have perfect sensitivity or specificity. When the software suggested a shift based on predefined criteria, the therapists would qualitatively confirm or reject whether the shift was necessary. If the software was unable to identify the fiducials, then treatment continued unless the therapists noted the fiducial to be outside of tolerance.Clinical performance of the software was evaluated on a total of 17 prostate cancer patients with intra‐prostatic collapsible fiducials . The University of Chicago Institutional Review Board (IRB) approved this study (IRB14‐934A). Fourteen patients had three crumpled fiducial markers (group A), while three patients had at least one linear fiducial marker (group B). An 18Planned fiducial locations, for both phantom experiments and clinical implementation, were determined using the planning CT . The fiducials were manually contoured using the CT and the marker location was set as the software identified centroid.3The phantom analysis revealed that linear fiducials were often inaccurately identified or not found as compared to crumpled fiducials Fig. . Figure 2. Patient size was not found to be a factor in the identification of markers. For the excluded patient with cylindrical fiducials, using the same settings as in the other 17 patients, the software found that in 45% of kV images the fiducial was correctly identified and within tolerance, representing a very low rate of accurate detection.A summary of the software's performance for each patient is shown in Table 4As compared to Calypso, which can continuously monitor intrafraction motion, this approach represents a cheaper, more readily available, and more efficient method of correcting intrafraction motion.Overall, this approach carries certain advantages and disadvantages. An additional angular limitation of this approach as compared to the electromagnetic tracking approach is that, at certain angles such as 0 and 180, there are geometric limitations to evaluating motion in the anterior and posterior directions, which is commonly due to bladder and rectum motion. Furthermore, there are angles in which fiducials may overlap and limit automatic detection. If it is known for a given patient that the fiducials are in a configuration that results in certain angles being more informative, while other angles are less informative, then the appropriate choice of angular frequency can be made. The software does not allow selection of specific imaging angles, but one could change the angular frequency setting, as opposed to using temporal or MU frequency, to avoid less informative angles. In addition, it is recommended to always use three fiducials as opposed to one or two, to limit overlap complications . When fiducials did overlap, the software labeled them as “not found,” this was visually confirmed, and treatment continued. We recognize that there are intrinsic limitations to this approach and with motion management using 2D information in general; however, this represents an active area of research and development.While the number of patients included is believed to be sufficient for making our conclusions and demonstrating important considerations, when implementing this approach, there are many variables that may influence outcome, some of which include: specific patient anatomy, fiducial configuration , the arbitrary nature of categorizing a spectrum of fiducial orientations as simply crumpled or linear, and image quality (noise) as a function of patient thickness. The potential interplay of multiple factors makes it difficult to attribute variability in the system to any one cause. For example, patients 5, 7, and 11 exhibited a higher rate of “not found” fiducials. A majority of these 'not found' data points fell at imaging angles traversing the attenuating hips, which may suggest that the fiducials may be obscured by a potential decrease in image quality; however, this was not found to be a consistent issue across all patients.Periodic kV imaging during VMAT prostate treatments is a commercially available method to account for intrafraction motion. Appropriate use requires attention to software settings and fiducial type/configuration as well as user verification of software analysis. The “GoldSeed_1_3x5_0” setting was initially selected to best represent crumpled gold fiducial markers. However, it was found that the “Clip_1_5x4_0” actually performed better for such fiducial markers. For our clinic, the crumpled fiducial markers combined with the “Clip_1_5x4_0” software setting provided the best precision and accuracy on both phantom and patient studies. We used the Gold Anchor fiducial markers in this feasibility study because these markers were our standard, as they can be deployed with a smaller 22G preloaded needle compared to other markers. The ability to deploy a crumpled marker has also been suggested to improve localization stability due to irregular folding within tissue. Performance of this software with appropriate parameter settings may be different with alternative  markers. While a 10 s time interval between each kV image was used in this work, this time interval may be reduced for more precise, high‐dose treatments. However, decreasing the time interval between each kV image will increase the influence of software latencies. While these latencies are not explicitly quantified by the vendor , they are certainly non‐zero. In addition to software latencies, there are human latencies that factor into the visual assessment by treating therapists. This is challenging to quantify but judgments were consistently made for each image prior to the subsequent image, implying that a determination could be made in <10 s. During both latency periods, the treatment beam will continue to deliver radiation to the patient, arguing for ~3 markers . Our clinic currently uses placement of three crumpled gold fiducial markers, the “Clip_1_5x4_0” setting, a 10 s interval between kV images, and a 3 mm tolerance threshold. The full functionality of the 6 degree of freedom couch was not used in this work , but this would potentially allow for more accurate correction of combined shifts/rotations and might decrease the number of false‐positives and/or multi‐shift fractions. We did not evaluate the automatic beam hold feature in this software package, in which the radiation beam is gated based on automatic fiducial detection; appropriate fiducial configuration and software settings are expected to be of even greater importance in this context. The number of detected shifts was found to be surprisingly high compared to the number of shifts found to actually be required, which suggests that the algorithm is conservatively overcompensating and therefore still realistically requires human supervision. Nonetheless, by using the proper fiducial marker configuration and software settings to account for intrafraction motion, clinics may consider reduced setup margins,No actual or potential conflicts of interest exist.