Genetic and phenotypic tumour heterogeneity is an important cause of therapy resistance. Moreover, non-uniform spatial drug distribution in cancer treatment may cause pseudo-resistance, meaning that a treatment is ineffective because the drug does not reach its target at sufficient concentrations. Together with tumour heterogeneity, non-uniform drug distribution causes “therapy heterogeneity”: a spatially heterogeneous treatment effect. Spatial heterogeneity in drug distribution occurs on all scales ranging from interpatient differences to intratumour differences on tissue or cellular scale. Nanomedicine aims to improve the balance between efficacy and safety of drugs by targeting drug-loaded nanoparticles specifically to tumours. Spatial heterogeneity in nanoparticle and payload distribution could be an important factor that limits their efficacy in patients. Therefore, imaging spatial nanoparticle distribution and imaging the tumour environment giving rise to this distribution could help understand (lack of) clinical success of nanomedicine. Imaging the nanoparticle, drug and tumour environment can lead to improvements of new nanotherapies, increase understanding of underlying mechanisms of heterogeneous distribution, facilitate patient selection for nanotherapies and help assess the effect of treatments that aim to reduce heterogeneity in nanoparticle distribution.In this review, we discuss three groups of imaging modalities applied in nanomedicine research: non-invasive clinical imaging methods , optical imaging and mass spectrometry imaging. Because each imaging modality provides information at a different scale and has its own strengths and weaknesses, choosing wisely and combining modalities will lead to a wealth of information that will help bring nanomedicine forward. In 2015, 17.5 million people were diagnosed with cancer globally and its incidence is increasing. Although the prognosis of most cancer types has improved, still 8.7 million people died of cancer in that year Therapy heterogeneity, a spatially heterogeneous treatment effect, is another important source of variability between patients and between tumours within an individual. Spatial heterogeneity in drug distribution contributes to therapy heterogeneity and can lead to pseudoresistance: the treatment does not have the desired effect, not because of cellular or genetic mechanisms of resistance, but because the drug simply does not reach all tumour cells at a high enough concentration Heterogeneous drug distribution occurs for drugs of all sizes. In this review we will focus on its impact in the field of nanomedicine. Nanomedicines are sub-micron size drug delivery systems, which are designed to improve the drug delivery to tumours while reducing systemic side effects As reviewed by Garattini et al., heterogeneous drug distribution leading to therapy heterogeneity can occur on many scales On patient scale, the inter-patient variability of nanomedicine pharmacokinetics (PK) is influenced by many factors such as age, gender, body composition, prior treatments, and drug-drug interactions Also on the organ and tumour scale, many factors can contribute to heterogeneity in nanoparticle and drug distribution between different tumours in an individual patient. Accumulation of nanoparticles is tumour type and organ dependent Finally, on tissue and cellular scale, there are numerous causes for heterogeneity in drug and nanoparticle distribution within a single tumour. These intratumour differences relate among other things to endothelial cell gaps across the vessel wall, perfusion, extracellular matrix composition and immune cell presence  Some nanomedicine formulations are currently used in the clinic Many imaging methods are available to visualize at a variety of scales the three main factors  that influence spatial therapy heterogeneity and thus efficacy. These methods can be used to:1. Evaluate the effect of the therapy in a preclinical setting, to facilitate the development of new treatments.2. Improve understanding of the underlying mechanisms that lead to heterogeneous distribution of nanoparticles and drugs.3. Select patients and predict their treatment response for a personalized treatment plan: which patient likely benefits from a certain therapy and in which patient is adaptation of therapy necessary?4. Evaluate the effect of methods that aim to address therapy heterogeneity, such as modulating the tumour microenvironment, hyperthermia or sonopermeation Over the last years, several excellent review papers on imaging in nanomedicine were published focusing on imaging the biodistribution of nanoparticles Nuclear imaging techniques, namely scintigraphy, Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) provide highly sensitive quantitative information about the distribution of an administered radiopharmaceutical. They are often combined with CT (SPECT/CT or PET/CT) to add anatomical information and perform attenuation correction. Since the spatial resolution is lower than that of MRI, CT and US imaging, clinical PET and SPECT are mainly informative on patient and organ scale. In the preclinical setting, high-resolution PET and SPECT techniques demonstrate expansion to the tissue scale 18F-fluorodeoxyglucose (FDG-) PET/CT is widely used in the clinic for diagnosis and monitoring therapy response. Traditionally several parameters are analysed for diagnosis and prognosis, including maximum standardized uptake values (SUVmax), peak standardized uptake values (SUVpeak), metabolic tumour volume (MTV), and total lesion glycolysis (TLG). Recently, intratumoural heterogeneity of baseline 18F-FDG uptake measured by PET texture analysis has been introduced as new predictive and prognostic factor for neoadjuvant chemotherapy Currently, the metabolic activity measured with 64Cu-labelled tracer dose of the same liposomes and their distribution was imaged by PET/CT. This study was able to demonstrate and quantify the EPR effect in patients and found that tumour liposome concentrations were similar to those found preclinically. Heterogeneity in nanoparticle distribution on the patient and tumour scale was observed and an association was found between the amount of 64Cu-labelled liposome uptake in the tumour and the overall tumour response and progression free survival 89Zr-labelled docetaxel nanoparticles in five patients with various tumour types and observed uptake of the nanoparticles in 35% of the tumours, which they attributed to the EPR effect. Heterogeneous patterns of accumulation were seen on patient and organ scale Nuclear imaging has already gained widespread acceptance in the management of cancer using standard chemotherapeutic agents, but may also play an important role in the advancement of nanomedicine towards clinical practice. Historically, nuclear imaging is used to image the distribution of radiolabelled nanoparticles on the patient and organ/tumour scale 18F-fluorothymidine (FLT-PET) 18F-fluoromisonidazole (F-MISO)-PET Furthermore, clinical studies with different tracers are being conducted to extend the use of nuclear imaging to gaining insight into the different aspects of tumour physiology, such as proliferation (e.g. 18F-fluoroestradiol (FES-) PET Other tracers visualize expression of receptors that can be targeted by drugs (e.g. 89Zr-trastuzumab and FDG-PET/CT after just one treatment cycle. These two types of PET imaging where used to characterize distribution of the nanoparticle (trastuzumab in T-DM1) and the tumour environment (FDG uptake). In metastatic breast cancer patients, who all had HER2 positive disease on biopsy, the combination of early metabolic response on FDG-PET (significant reduction in FDG uptake in >50% of the tumour load) and positive 89Zr-trastuzumab uptake (>50% of the tumour load) on the HER2-PET could predict treatment response to the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1)  is widely used in daily clinical practice for tumour diagnosis, characterisation and response evaluation. MRI is increasingly being investigated in image-guided therapy such as MR-guided radiotherapy (MR-LINAC) Because of the lack of radiation, its high spatial resolution and excellent soft tissue contrastTumour vascular development and density, as well as perfusion and hypoxia, are key regulators of nanoparticle distribution and nanotherapy effect. Dynamic Contrast Enhanced (DCE-) MRI has been used in clinical trials to evaluate the effect of antivascular treatment on perfusion and vascular permeability Iron-oxide nanoparticles have been approved as MRI contrast agent for clinical use. However, they can also function as companion diagnostic or as (imageable) drug delivery systems co-glycolide) nano/microspheres, encapsulated with gadolinium-DTPA, SPIONs and the chemotherapeutic drugs 5-fluorouracil. In vivo, release of gadolinium-DTPA was seen from 30 minutes after intravenous injection, in the same tumour regions where most of the nanospheres had accumulated in vivo drug release and spatial distribution at tumour and tissue scale. However, these MR contrast agents may influence the stability of nanoparticle Moreover, MRI contrast agents have been encapsulated in nanoparticles as model drugs, to visualize and quantify drug release triggered by temperature, pH or ultrasound sensitive nanoparticles using MRI Computed tomography (CT) imaging is very commonly used in the clinic for diagnostic purposes and response evaluation after treatment. More recently, it was shown that CT could derive tumour transport properties in patients with pancreatic cancer that correlated with gemcitabine incorporation, pathological response, and oncologic outcome The added value of CT imaging for nanomedicine through identifying tumour transport properties was already shown in the preclinical setting. Dynamic Contrast Enhanced (DCE) CT has been used in several studies to measure the intratumoural perfusion, permeability and the accumulation of CT contrast agent-containing nanoparticles in mice In addition to imaging a contrast agent encapsulated in a nanoparticle, CT can also be utilized to image metallic nanoparticles with a high attenuation of x-rays Ultrasound (US) is a low-cost, radiation-free and patient-friendly clinical imaging method that is mostly used for tumour diagnosis and image-guided biopsies. The introduction of contrast-enhanced ultrasound (CEUS), i.e. using microbubbles as ultrasonographic contrast agents, has extended the application of ultrasound in many fields due to improved image quality and new information that cannot be obtained with standard US CEUS can provide anatomical as well as functional information about the vasculature of the tumour (micro-) environment Tumour environment modelling treatments to improve nanoparticle delivery have been investigated using CEUS. Changes in tumour physiology (i.e. vessel fraction and blood flow) measured by US imaging after collagenase treatment corresponded with changes in IFP, therefore US imaging can be used as an earlier marker of tumour response Recent advances in US imaging such as ultrafast ultrasound and super-resolution techniques provide also information on microvascular properties In addition to imaging the tumour environment, the distribution of nanoparticles can be imaged by US, using echogenic nanoparticles Although drugs cannot be imaged directly with ultrasound, drug distribution can be visualized. To achieve this, Ektate et al. developed low-temperature sensitive echogenic liposomes, loaded with doxorubicin and perfluoropentane. Tumour hyperthermia led to increased US contrast in mice, which was correlated with increased doxorubicin delivery in vivo optical imaging modalities are steadily substituting “old-school” ex vivo methodology - “dead mice tell too few tales” ex vivo / in vitro optical imaging techniques can provide supportive structural and functional information (immunohistochemistry (IHC) on tissue slices, electron microscopy (EM), flow cytometry imaging). Optical imaging is widely used in the development and evaluation of nanotherapies: molecular imaging helps unravel nanoparticles' complex in vivo fate Optical imaging modalities are used to collect a variety of information on various spatial and temporal scales in vivo release profile Traditionally, macroscopic optical imaging modalities are used in preclinical small animal experimental procedures as alternatives to conventional non-terminal / non-invasive whole-body imaging modalities . BLI, one of the most commonly used optical imaging techniques, allows real-time detection of protein-derived native light emission Fig. A. Even tin vivo is highly complex. Therefore, a deeper understanding of in vivo behaviour, targeting mechanisms and nanoparticles' specific engagement with cell populations  is essential. Extensive use of real-time imaging techniques like IVM, could potentiate our efforts to characterize the tumour microenvironment on tissue and cellular scale  PEG surface density proved to contribute to a higher targeting specificity of arginylglycylaspartic acid (RGD) nanoparticles, than high (50 mol %) surface PEG density Interestingly, IVM has been used to identify differences in nanoparticle diffusion to tumour sites or nanoparticle tumour targeting, and to correlate them to different physicochemical properties, providing an excellent tool for head-to-head nanoparticle comparisons. The size-dependent diffusion of nanoparticles was studied after administration of a library of small fluorescent quantum dot nanoparticles with diameters of 12, 60 and 125 nm, revealing that 12 nm nanoparticles diffused twice as far in comparison to the largest size particles In addition to addressing nanoparticle tumour targeting and accumulation profiles, IVM has been used to unravel more intricate interactions between nanoparticles and immune cells; an interesting option given the increasing appeal of cancer immunotherapy. One of the first studies providing real-time insight in the behaviour of TAM, developed magneto-fluorescent nanoparticles enabling the visualization of nanoparticle-labelled TAM on tumour/organ scale  and on tissue and cellular scale (IVM). Among other findings, TAM phagocytosed more nanoparticles than other myeloid cells, they were situated in close proximity to tumour cells and displayed low motility in vivo behaviour in a similar manner regardless of the selected nanomaterial, i.e., nanoemulsions versus quantum dots. The multimodal nature of nanoparticles allows for visualization of angiogenic endothelium and neovasculature on tumour/organ scale , tissue scale (IHC and IVM), and cellular scale (IVM) Given that angiogenesis is a hallmark of cancer, the attention of intravital microscopy users has been directed particularly to nanoparticle-mediated vessel wall visualization and targeting. Already in the mid '00s, a successful attempt using vascular cell adhesion molecule 1 (VCAM-1) decorated nanoparticles paved the way for future success The new mechanistic and molecular insights that were obtained through IVM procedures inspired researchers to develop IVM-specialized imaging agents. Biocompatible organic dots for MPM in vivo imaging of inherently fluorescent drugs is hampered by their relatively low fluorescence quantum yield, which limits their detectability at therapeutic concentrations. Another approach could be the conjugation of fluorescent molecules to the drugs that are carried by nanoparticles, despite the fact that this could result into alternation of their properties. The conjugation of fluorescent dyes to macromolecular drugs has been successfully applied before Visualization of fluorescent-labelled nanoparticles and related aspects of the tumour microenvironment provide valuable information on therapy heterogeneity in nanomedicine. Imaging the administered drug itself would complete the picture. However, direct imaging of drugs remains elusive. In this respect, inherently fluorescent drugs are convenient ex vivo techniques like the tissue optical clearing strategies Utilization of fluorescent molecules is often restricted by factors such limited imaging depth. Therefore, more sophisticated in vivo optical imaging is mainly constricted to preclinical applications. Clinically, optical imaging is of course widely used on ex vivo biopsy or surgical samples. Although this consists mostly of immune histochemistry, some work has been done to complement this with fluorescent imaging Due to the limitations in tissue penetration and size of the imageable subject, Optoacoustic (photoacoustic) imaging is an emerging hybrid technique that combines the benefits of US and optical techniques, i.e. deep imaging depth, high spatial resolution and high contrast in vivo spatiotemporal changes of macrophage activity In the oncology domain the endogenous contrast is typically used to study tumour vasculature in vivoNanocarriers not only serve as optoacoustic contrast agent, but can also act as vehicles for drugs. Several nanoparticles have been loaded with drugs and combined with optoacoustic imaging for non-invasive and real-time monitoring of biodistribution and pharmacokinetics In order to monitor the drugs themselves using optoacoustic imaging they should exhibit NIR-absorbing properties. Unfortunately, few if any clinically prescribed drugs have strong intrinsic absorption in the NIR. As an alternative, small molecule NIR dyes are co-loaded with drugs of interest to monitor drug release and distribution 284Optical and optoacoustic imaging have frequently been combined with photothermal therapy PTT , 285 andMass Spectrometry Imaging (MSI) is a label free, multiplex technique that is used to visualize the molecular distribution of endogenous compounds such as metabolitesIn MSI spatially defined desorption/ionization methods are used to collect sequentially mass spectra from a small region (pixel) of a tissue sample. Among the multitude of surface sampling techniques, matrix-assisted laser desorption/ionisation (MALDI) uses a laser beam for desorption/ionization of tissue-representative molecules co-crystalized in a solidified matrix; while desorption electrospray ionization (DESI) makes use of an electrically charged solvent spray and in secondary ion mass spectrometry (SIMS), a beam of high energy primary ions  is used to release secondary ions from the sample surface. In contrast to MALDI and SIMS where the sample is analysed under vacuum, DESI is non-destructive and performed at atmospheric pressure, which renders the technique more user friendly and through appropriate solvent selection, more tuneable to increase selectivity and/or sensitivity. Depending on the ionization method used spatial resolution, sensitivity, and the molecules that can be analysed change. Mostly, MALDI is used at 10 to 20 µm spatial resolution, whilst DESI resolution spans 50 µm till 200 µm and SIMS allows for sub-µm resolution. Sensitivity wise DESI outperforms MALDI and SIMS, partly due to increased pixel size  and partly due to improved ionization efficiency. Although sensitivity is determined by e.g. physiochemical properties of the compound, typically one requires low µg/g concentrations in the case of DESI and approx. 20 µg/g tissue for detection by MALDI and SIMS.The number of drugs that have been detected using MSI is extensive, ranging from anti-cancer drugs , was introduced in the life sciences by Giessen et al. in 2014 in vivo fate after administration. However, this requires additional chemical development and the introduction of the probe may influence the biodistribution of the nanoparticle. Recently, Zandanel et al. showed that MALDI-MSI allows for the simultaneous visualization of the polymeric nanoparticle, the encapsulated drug (doxorubicin) and its metabolite (doxorubicinol) in treated mouse liver MSI also provides the opportunity to image nanocarriers, such as lipid- and metal-based nanoparticles. Typically nanoparticles are labelled or loaded with a radioactive or fluorescent probe in order to follow the in situ drug (doxorubicin) release from molybdenum disulphide (MoS2) nanosheets Xue et al. developed an MSI method that enabled not only the visualization, but also the quantification of the As indicated earlier, mass spectrometry offers unique capabilities for untargeted exploration of biological samples and provides simultaneous information of the distribution of the drug, the nanoparticle and endogenous compounds such as metabolites, lipids and proteins. Therefore, MSI can be used to detect biomarkers associated with disease, molecular changes due to drug treatment and tumour components limiting nanomedicine distribution and effect or augmenting off-target effects.Several studies report that the heterogeneous distribution of lipids and proteins could reflect the effect of therapy and/or could be used as prognostic/predictive marker for outcome. A very nice example of identifying and using endogenous proteomic profiles for distinguishing between responders and non-responders to chemotherapy for oesophageal adenocarcinoma is given by Aichler et al. 18F-fluoromisonidazole (FMISO), a widely used PET hypoxia imaging probe Also tumour hypoxia is investigated by many groups since it is associated with tumour aggressiveness and resistance to cancer treatment. Manscini et al. used MALDI-MSI to simultaneously detect pimonidazole, a clinically used hypoxia marker, its metabolites and associated biomolecules in a single experiment Interestingly, most MSI studies that investigate drug distributions do not yet exploit the multiplex capabilities of MSI. Instead of using the wealth of information on endogenous molecule distributions  already available in the acquired MSI image they superimpose the drug MSI images with standard H&E stained tissue images and/or various immunohistochemistry (IHC) images. As most of these IHC images are specific to a certain protein, this can be a laborious and time consuming effort. Moreover, correlating drug distribution with the distribution of these endogenous molecules would allow for non-supervised investigations to discover new factors that impair drug transport in tumour tissue and could be used as biomarker for prognosis and therapy response prediction.Spatial heterogeneity in nanoparticle distribution occurs at all scales and can reduce nanotherapy efficacy. A wide range of imaging modalities help visualize nanoparticle distribution or factors contributing to heterogeneous distribution, by imaging the drug, the nanoparticle and the tumour environment.in vivo applications and intravital techniques offer the possibility to visualize dynamic processes. Clinical use of optical imaging is still hampered by the limited penetration depth and therefore requires tissue sampling or intraoperative use. MSI provides information on tissue and even cellular scale. As it requires a tissue specimen it is inherently an invasive method, regardless of preclinical or clinical use. Due to this limitation, the technique is most suitable and currently most used in the preclinical setting, where an entire tumour or even the whole animal can be analysed at once. In contrast, in the clinic MSI approaches will most likely follow a workflow similar to standard histopathology. Emerging technologies allow for more accurate tissue sampling using intra-procedural multimodality imaging during biopsies When selecting imaging modalities, researchers need to take into account the desired imaging scale. Non-invasive clinical imaging methods provide three-dimensional information of the intact body on patient, organ and tumour scale, and recent developments, such as super-resolution ultrasound, have broadened their application to provide even tissue scale information. Optical imaging modalities have a superior spatial resolution, procuring images on the tissue and cellular scale, and in the preclinical setting they can provide (sometimes non-invasive) organism and organ scale information as well. Besides that, Nuclear imaging methods and most optical imaging methods require labelling of nanoparticles, drugs or aspects of the tumour environment, which has some disadvantages. First, the stability of the link between a label and a nanoparticle determines its usefulness in tracking the nanoparticle, for one could be imaging the label on itself after disconnection from the nanoparticle. Second, attaching a label could change the pharmacokinetic and drug release properties of the nanoparticle leading to erroneous prediction of the distribution of an unlabelled equivalent. Third, administering a labelled version of a previously approved therapeutic nanoparticle or drug could cause additional toxicity and obtaining clinical approval is cumbersome and costly. Fourth, administration of radiolabelled theranostic nanoparticles may decrease the effect of subsequent therapeutic administration through the ABC phenomenon The integration of imaging data collected from multimodality techniques offers a unique opportunity to combine information related to drug and nanoparticle distribution and tumour environment on multiple scales and provide synergistic advantages over the use of a single modality.in situ hybridization (FISH), creates a new dimension to molecular pathology. Now, drug levels within tumour regions can be correlated to different degrees of vascularization of the tumour - highlighted with specific vasculature staining Clinical imaging modalities are generally combined to merge functional  with anatomical information  collected on the same scale. A range of nanoparticles for multimodality imaging have been developed To achieve multiscale information, optical imaging and MSI have been paired with clinical imaging ex vivo MSI or optical imaging with 3D / in vivo images, such as those generated with SPECT, PET, CT or MRI. This challenge is inherent to the fact that the images are acquired at different scales, can be subjected to different deformations, and lack common fiducial markers. A first step towards combining two-dimensional MSI with three-dimensional MRI is the ability to monitor MRI contrast agents with MSI, which was demonstrated by Tata et al. A challenge remains the integration of molecular information provided by 2D / in vivo behaviour of nanoparticles. Additionally, it has led to valuable information on how specific characteristics within the tumour environment can affect nanotherapy outcomes. Continued application of these imaging approaches, and especially the combination of different techniques, will further strengthen our understanding of therapy heterogeneity on organism, tissue, cellular and even subcellular scale. Meanwhile the role of imaging for monitoring PK and biodistribution is well established in research and development stage; however in the clinical setting it is not yet adopted. Clinically, spatial heterogeneity distribution of therapeutic nanoparticles, poses the threats of under- and overtreatment. Despite the fact that many studies have shown that drug concentration does not correspond with tissue drug levels, phase I and II clinical trials mainly rely on blood samples and spatially sparse biopsies to measure PK on patient scale and biodistribution on organ scale. Imaging drugs or nanoparticles in clinical studies is still rare and often no information is acquired about spatial heterogeneity of nanomedicine on organ, tumour or tissue scale. However imaging drug/nanoparticle distribution can help predict treatment effect and therefore select which patients will benefit most and in which patients a therapy adjustment or a combination therapy that reduces heterogeneity are warranted. Combining imaging of nanoparticle and drug distribution with imaging of tumour environment characteristics or early response indicators promises to help personalize treatment further. Currently, only optical imaging is commonly used to investigate, in the preclinical setting, the interplay between environment, nanoparticle and drug. It is our hope and recommendation that non-invasive clinical imaging and MSI will play a central role in future preclinical and clinical research on the interaction of drug, nanoparticle and environment. Choosing and combining imaging modalities wisely will lay the foundation for successful future nanotherapies.Integration of imaging techniques in preclinical lab practice has contributed to an improved understanding of the


This article has been corrected: The Acknowledgments information has been updated. The proper Acknowledgments section is shown below:This work was supported in part by NCI 5T32CA009599, CPRIT RP 150535, the Sheikh Khalifa Al Nahyan Ben Zayed Institute for Personalized Cancer Therapy, NCI U01 CA180964, NCATS grant UL1 TR000371 , the Nellie B. Connally Breast Cancer Research Endowment, and the MD Anderson Cancer Center Support Grant (CCSG) (P30 CA016672) and the CCSG supported Sequencing and Microarray Facility (SMF).41806-41814 . https://doi.org/10.18632/oncotarget.16018Original article: Oncotarget. 2017; 8:41806–41814.


CNS) tumours, molecular profiling is particularly relevant for the proper characterization of primary neoplasms, while CNS metastases can significantly diverge from primary disease or extra‐CNS metastases, thus compelling a dedicated assessment. Based on these considerations, effective liquid biopsy tools for CNS tumours are highly warranted and a significant amount of data have been accrued over the last few years. These results have shown that liquid biopsy can provide clinically meaningful information about both primary and metastatic CNS tumours, but specific considerations must be taken into account, for example, when choosing the source of liquid biopsy. Nevertheless, this approach is especially attractive for CNS tumours, as repeated tumour sampling is not feasible. The aim of our review was to thoroughly report the state‐of‐the‐art regarding the opportunities and challenges posed by liquid biopsy in both primary and secondary CNS tumours.Tumour molecular profiling by liquid biopsy is being investigated for a wide range of research and clinical purposes. The possibility of repeatedly interrogating the tumour profile using minimally invasive procedures is helping to understand spatial and temporal tumour heterogeneity, and to shed a light on mechanisms of resistance to targeted therapies. Moreover, this approach has been already implemented in clinical practice to address specific decisions regarding patients’ follow‐up and therapeutic management. For central nervous system ( The possibility of detecting circulating tumour biomarkers in body fluids, including cerebrospinal fluid (CSF), has been exploited in routine clinical practice for tumour diagnosis, treatment and follow up for a long time Central nervous system (CNS) tumours are an unmet need: outcome is often dismal, and therapies offer limited efficacy. Tumour molecular profiling is especially relevant in this setting, but the CNS is a peculiar anatomic/functional compartment and even a biopsy can carry significant risks. Thus, liquid biopsy could fulfil its potential in this setting, although CNS specificities apply to this tool too. For example, the blood brain barrier (BBB) hinders the circulation of tumour components from the CSF to the blood, thus potentially impairing CNS tumour sampling through the commonly used blood‐based approaches.Over the last few years, a significant amount of data have been accrued regarding liquid biopsy in both primary and secondary CNS tumours. The aim of our review is to thoroughly report the state‐of‐the‐art regarding the opportunities and challenges posed by liquid biopsy in this setting.Although blood is an ideal, easily accessible source for liquid biopsy sampling and is routinely used to identify many tumour types, data show that its sensitivity can be severely limited when dealing with CNS neoplasms. The main factor hampering the detection of CNS tumour components in blood is the BBB. The latter is a peculiar anatomic and functional structure aimed at regulating the traffic of molecules and cells into and outside of the CNS. Its functions rely on the synergic activity of a wide range of partners including endothelial cells, pericytes and astrocytes IDH1 mutations However, as will be discussed, in specific situations it seems to be possible to detect the molecular signatures of CNS tumours in blood. For example, larger tumour volumes and the presence of contrast enhancement were correlated with an increased sensitivity for To overcome this limitation, CSF seems the best alternative source for sampling of CNS neoplasms considering its direct contact with CNS structures. Present data suggest a higher sensitivity of CSF compared with blood Figure . NeverthA different strategy could be to alter the BBB permeability to allow the transfer of tumour‐derived markers into the blood. Until recently, this research topic was mostly aimed at increasing drug penetration into the CNS Last, it should be remembered that the BBB plays an active role in facilitating or preventing brain metastases (BM) from systemic tumours. Primary tumour cells can impair the BBB by releasing specific molecules such as nucleic acids or proteins, thus increasing the risk of BM development. Improved understanding of these mechanisms could pave the way not only to early detection of BM, but could also allow BM risk prediction or prevention The number of potential technical approaches is both extremely high and rapidly evolving Another important choice regarding genomic studies is whether to use a candidate gene strategy or larger‐scale approaches like whole exome sequencing. In the diagnostic clinical setting, the first is usually advised: (i) it allows reliable assessment of a specific set of genes with known diagnostic and/or predictive value; (ii) it enables very low limits of detection; (iii) it provides a faster, clinically suitable, turnaround time with lower costs; (iv) bioinformatic support is usually not required; (v) germline DNA analysis and its ethical/legal implications can usually be avoided. Conversely, for exploratory/research studies, extensive approaches, also including deep sequencing, can be considered. Among candidate gene analysis techniques, droplet digital PCR allows an extremely low limit of detection (0.001%) and provides precise DNA copy number quantification For CTCs and EVs analysis, different strategies have to be used for primary and secondary CNS tumours as these tumour components have to be selectively captured and enriched; however, the proposed approaches will be discussed in the relevant paragraphs. A further informative component worthy of mention is circulating platelets. Analysis of tumour‐educated platelets by RNA sequencing has been proven to be highly informative in both primary and secondary CNS neoplasms Molecular profiling is of paramount importance for the characterization of primary CNS tumours. For this reason, the latest WHO classification of these tumours introduced the concept of ‘integrated diagnosis’: a diagnosis based upon the integration of both morphological and molecular findings. This approach is necessary to distinguish between entities with significant biological and clinical differences, but overlapping histological features IDH1/2, ATRX, TP53, TERT promoter and histone H3‐coding genes mutations, 1p/19q chromosomal arms codeletion, EGFR alterations and MGMT promoter methylation Even considering just the group of diffuse gliomas, a wide range of molecular alterations is relevant for their proper classification and treatment: Analysis of the circulating cfNA shed by tumour cells allows detection of gene alterations, including mutations or fusions, changes in methylation profile, copy number variations and to quantify tumour burden; moreover, overall cfDNA (i.e. tumour and non‐tumour derived) concentration has been suggested to play a prognostic role in primary CNS tumours et al. evaluated the detection of IDH1 mutations in plasma cfDNA of patients with histologically proven glioma with known tumour IDH‐status:et al. investigated the possibility of detecting a wide range of alterations in a large series of tumour types by digital PCR analysis of plasma cfDNA Boisselier et al. who identified EGFR amplification and a TP53 mutation in glioblastoma patients  in a series of primary CNS tumours, mostly including grade III–IV (WHO) diffuse gliomas The possibility of using CSF as a source for cfDNA analysis in primary CNS tumours was suggested more than 20 years ago by Rhodes ts Table  1, 2. Deet al. who found that tumours closer to CSF spaces have a higher probability of being detected by CSF cfDNA analysis compared to tumours not abutting the ventricles et al. analysed CSF and blood samples of three cases of grade II intramedullary ependymomas without identifying any alteration in blood or in CSF Tumour variables, such as histological type, grade and site, could influence the efficacy of cfDNA analysis. Medulloblastoma, for example, a malignant cerebellar tumour with high cellularity could be expected to more easily shed cfDNA into CSF compared to hemispheric diffuse low‐grade gliomas with an infiltrative growth pattern. This hypothesis is supported by Wang n = 85), including 46/85 (54%) glioblastomas and 39/85 (46%) lower grade gliomas (grade II–III), using a targeted next‐generation sequencing‐based assay A very recent study specifically investigated a large series of diffuse gliomas , making this marker potentially assessable through liquid biopsy Regarding the diagnostic potential of CSF cfDNA analysis, evaluation of a selected panel of molecular alterations in CSF cfDNA allowed the identification of tumour molecular alterations in 17/20 (85%) cases of diffuse gliomas, allowing their classification according to the latest WHO criteria et al. evaluated a series of 57 patients and were able to detect at least one tumour‐specific mutation in 82.5% (47/57) of cases et al. focused on midline gliomas and evaluated H3 K27M mutations and typical partner alterations in cfDNA by droplet digital PCR Recent data further support the sensitivity of liquid biopsy for brainstem gliomas: Pan A concluding remark regarding the use of liquid biopsy as a diagnostic tool for gliomas is that particular care should be applied when selecting the gene panel to be assessed, as dramatic differences exist between the molecular landscapes of adult and paediatric tumours. Moreover, reliance on single markers should be avoided. For instance, the H3 K27M mutation, which was initially considered as a potential pathognomonic alteration of malignant diffuse brainstem glioma, has been reported in a wider range of tumours et al. demonstrated the possibility of detecting MYD88 mutations, a common alteration of primary central nervous system lymphomas (PCNSL)  Table  37. ThisMGMT promoter methylation in blood‐derived cfDNA was first reported by Weaver et al. in a small case series of high‐grade diffuse gliomas, achieving a 50% sensitivity et al. and further studies confirmed this possibility and also explored the correlations with outcome and longitudinal evolution during disease course CDKN2A promoter methylation assessment Epigenetic alterations are also relevant biomarkers in primary brain tumours. The possibility of assessing Analysis of circulating micro RNAs (miRNAs) is another potential strategy. miRNAs are noncoding short RNA regulatory molecules, which can widely affect transcription and translation. Most available data relate to diffuse glioma, in particular glioblastoma, and a broad range of miRNAs have been investigated, alone or in specific combinations, as possible diagnostic or prognostic biomarkers Overall, the main limitation of this approach is the heterogeneity of the reported data. Although many different candidate miRNAs have been proposed, their validation in larger cohorts is lacking, hampering translation into clinical practice.Last, the possibility of exploiting other nucleic acids such as long noncoding RNAs or mitochondrial DNA should also be taken into consideration Circulating tumour cells (CTCs) analysis is another potential approach for liquid biopsy and allows the assessment of nucleic acids, including mRNAs, which are usually quickly degraded in the circulation, and other tumour cell components such as cytoplasmic proteins. Detection and capture of CTCs are challenging due to their extremely low concentration in blood, thus many different techniques have been investigated et al. observed a sensitivity of 8/11 (72%) in a series of grade III and IV gliomas sampled before radiotherapy et al. were able to detect circulating neoplastic cells in 21% (29/141) of patients with glioblastoma by GFAP immunostaining of mononuclear circulating cells vs. radiation necrosis) in an additional series of five cases The presence of blood CTCs in primary CNS tumours was debated due to the extremely low incidence of extra‐CNS metastases et al.: CTCs can show a different gene expression profile compared to the previously resected tumour sample. Specifically, increased mesenchymal differentiation and a reduced neural‐like profile were observed. These data suggest that a single approach or a single set of markers may be unable to fully sample tumour heterogeneity Last, an interesting observation regarding tumour heterogeneity was reported by Sullivan et al., platelets can sequester tumour‐shed extracellular vesicles which allow detecting of tumour‐derived biomarkers, including proteins and nucleic acids like the EGFRvIII mRNA, with high sensitivity and specificity et al., showed that analysis of RNA splicing profiles of tumour‐educated platelets was able to identify patients with tumours and further distinguish those with localized or metastatic disease. In particular, this approach correctly classified 85% (33/39) of individuals as patients with a neoplasm in a series of 39 glioblastomas. Regarding the specific diagnostic potential, 78% of glioblastoma patients were correctly classified when compared with controls and five other tumour types in a training series of 175 samples. Similar results were observed in a validation set of 108 cases. Furthermore, in a series of 114 samples (62 patients) with brain lesions, tumour‐educated platelet analysis allowed correct identification of cancer patients in 91%, discrimination between primary and secondary tumours in 93% and discerned between the mutational subtypes in 82% Analysis of circulating platelets is another intriguing approach to achieve liquid biopsy of CNS tumours Extracellular vesicles (EVs) are a heterogeneous range of membrane‐bound carriers secreted or shed by both normal and neoplastic cells. Many studies have investigated their physiological role in intercellular communication, which can occur between different tissues or compartments et al. et al. were able to detect EGFRvIII in EVs of 7/25 (28%) patients with glioblastoma Similar to CTC, liquid biopsy by EVs analysis offers some advantages over cfNAs, allowing mRNAs characterization and evaluation of cytoplasmic proteins et al. detected IDH1 mutations in CSF‐derived EVs of patients with IDH‐mutant gliomas using highly sensitive techniques; conversely, serum‐derived EVs of the same patients were negative et al. EGFR alterations can also be assessed in EVs. More recently, Chen Concerning immune system evasion, a recent study identified PD‐L1 in glioblastoma‐derived EVs and the presence of this ligand was associated with impaired T‐cell activation miRNAs are another cargo of EVs through which they can exert their regulatory functions on distant tissues et alLast, circulating proteins and metabolites can be also exploited as biomarkers of neoplastic diseases, including brain tumours. The improved knowledge of tumour molecular profiles and their correlation with cellular metabolism could enable new avenues for diagnosis and monitoring of CNS neoplasms, as recently shown by Ballester Brain metastases are the most frequent intracranial tumours. They develop in 9–17% of patients with solid neoplasms  and their incidence is increasing et al., showed that more than 50% of BMs harbour private molecular alterations (compared to the primary tumour or extra‐CNS lesions) which are potentially actionable Brain metastases from solid tumours often show a divergent molecular profile compared to the primary lesion. Spatial and temporal heterogeneity is an intrinsic characteristic of malignant tumours and this hallmark is strictly intertwined with the continuous evolutionary pressure which selects the neoplastic cells that best fit in a specific anatomic compartment or timepoint during the course of disease, also because of external variables, such as ongoing treatments. Brastianos Neoplastic meningitis (NM) is a specific type of secondary CNS involvement which is characterized by the spread of neoplastic cells across the leptomeninges and their circulation through the CSF. Definitive diagnosis is based upon cytological demonstration of malignant cells in the CSF, but sensitivity is limited and thus diagnosis usually relies on supporting clinical and neuroimaging findings The possibility of detecting tumour‐derived cfDNA in patients with secondary CNS involvement has long been reported cfDNA analysis proved a quite high overall sensitivity as a diagnostic tool for BMs or NM from solid tumours, ranging from 40% to 100%, but sample sizes of the available studies are still limited. Nevertheless, if we compare this tool with traditional cytology (the gold‐standard for NM diagnosis at present) et al. et al. et al. et al. With respect to the variables potentially affecting liquid biopsy yield, the type of CNS involvement should be considered. BMs may have limited contact with CSF, especially when dealing with a single or few lesions in a supratentorial intraparenchymal location. Conversely, tumour cells circulation in the CSF is a hallmark of NM, and thus a higher sensitivity can be expected in the latter condition. The results of Yang As for primary CNS tumours, the most pressing question is choice of the source for cfNAs . Blood or other easily accessible body fluids  would obviously be preferable, but they may not be representative of CNS‐restricted lesions Table . Some imet al., the specific detection of the EGFR T790M mutation in CSF cfDNA was associated with clinical efficacy of treatment with osimertinib, a third‐generation EGFR tyrosine kinase inhibitor developed to overcome T790M‐induced resistance EGFR T790M detection, no response to treatment was observed.Within the possible practical applications of liquid biopsy in this setting, CSF testing in patients with CNS involvement by NSCLC seems one of the most promising, mirroring the already widespread practice of blood‐based testing to promptly detect emerging resistance‐associated mutations in extra‐CNS NSCLC. As recently shown by Nanjo Last, it is worth pointing out that the significant differences observed between studies may depend on the wide range of techniques that have been tested so far. This variability should be addressed in future studies.et al. et al. The presence of common membrane markers, like EpCAM, significantly helps CTCs collection in secondary CNS tumours, but alternative strategies are also possible et al. showed that analysis of tumour‐educated platelets in patients with BMs allows identification of primary tumour type in 70% of samples Best Finally, EVs analysis in secondary CNS tumours is especially intriguing not only as a diagnostic/profiling tool, but also as a prognostic assay. Multiple studies suggested that tumour‐derived EVs may play an active role in creating a pro‐metastatic niche in distant tissues The recent technological advancements paired with the growing importance of molecular profiling in CNS neoplasms can explain the far‐reaching results achieved in just the last few years regarding liquid biopsy use in CNS tumours The ever‐increasing reliance on molecular traits for proper classification of primary CNS tumours will favour the clinical adoption of liquid biopsy as a routine diagnostic tool in selected cases, for example, when surgical resection is not indicated. Conversely, resection and histological examination will probably remain the cornerstone diagnostic approach if feasible, considered its therapeutic relevance and the risk of diagnostic pitfalls due to overlapping molecular features even between significantly different tumour entities . Moreover, a tissue sample is needed for whole genome or proteomic studies which are now increasingly warranted in translational/clinical research protocols.Liquid biopsy could instead represent a game‐changing development for disease follow up considering that repeated surgical sampling is not feasible in CNS tumours. Its first implementation, in this setting, could probably be as a companion tool for disease monitoring and tumour burden quantification: liquid biopsy data could help resolve conflicting clinical and radiological findings, for instance, when dealing with a suspected pseudo‐progression. The following step could be the longitudinal assessment of tumour heterogeneity: for example, patients with IDH‐mutant diffuse gliomas can have very‐long disease courses characterized by a progressive increase of tumour malignancy. Liquid biopsy could enable prompt detection and molecular characterization of disease progression, allowing optimization of clinical management. Nevertheless, the relevance of liquid biopsy in this setting will ultimately depend on the identification of significant, actionable, prognostic and/or predictive markers.The same considerations apply to secondary neoplasms, but special caution should be applied when dealing with synchronous intra‐ and extra‐CNS disease progression, as blood‐derived results could be uninformative of the CNS disease. Nevertheless, the outcome of patients with BMs has dramatically changed in the last few years, thanks to the newly available targeted treatments In the coming years, liquid biopsy will probably become a common tool for the diagnosis and follow up of both primary and secondary CNS tumours. For research purposes, liquid biopsy will be a cornerstone to determine longitudinal changes in the molecular profile of tumours, thus improving our knowledge of tumour resistance/progression mechanisms. Further studies, evaluating larger, prospective series, are needed to evaluate technical variabilities, validate its use in specific disease settings and, most importantly, to assess which is the ultimate clinical benefit for patients with CNS tumours.This work was supported by the Rete Oncologica del Piemonte e della Valle d'Aosta (no specific grant number applicable) and received funding specifically dedicated to the Department of Medical Sciences from Italian Ministry for Education, University and Research  under the programme ‘Dipartimenti di Eccellenza 2018–2022’. Project n° D15D18000410001. G.S. was supported by a 3‐year FIRC‐AIRC fellowship and by ‘Roche per la ricerca’ ‐ Grant 2017. A.B. was supported by IMI contract n. 115749 CANCER‐ID.The authors declare they have no conflicts of interest.


Gil José María's affiliation appeared incorrectly and should be Anesthesiology Service, Hospital de la Santa Creu i Sant Pau, IIB Sant Pau, Spain.In the article, “Evaluation of a strategy to shorten the time to surgery in patients on antiplatelet therapy with a proximal femur fracture (AFFEcT Study): Study protocol for a multicenter randomized controlled clinical trial”Dr. Delgado Claudia Erika's name appeared incorrectly as Claudia Erica.


Neuroblastoma (NB) is a solid childhood cancer originating from the sympathetic nervous system. Most NBs arise from the adrenal gland but metastases to distant sites such as bone marrow, liver and lungs are not uncommon. Despite intense treatment, including high-dose chemotherapy, many high-risk NBs relapse and treatment resistance is a significant clinical problem.NB is a heterogeneous disease, i.e., tumours display high inter-tumour heterogeneity. Recent findings also point to genetic intratumour heterogeneity (ITH) in NB, albeit lower than has been observed in other adult tumours. This can be a result of clonal evolution due to Darwinian selection among tumour cells . It can ALK, NRAS genes) predicted to activate the RAS-MAPK pathway, Hippo-YAP pathway and epithelial-mesenchymal processes [per se frequently exhibit spatial genetic variation [There are different dimensions of genetic ITH in NB. Temporal genetic ITH has been observed when comparing treatment naïve and matched relapsed NBs. Relapsed NBs are enriched in mutations  regional fluctuations of subclones, (2) subclones that coexist over vast anatomic space, (3) clonal sweeps, where one clone expands to outgrow all other clones in a region, and (4) so-called evolutionary explosions, a type of massive regional chromosomal instability triggered by inactivation of the p53 pathway. Of these, the presence in the primary tumour of the two latter trajectories are strong predictors of high-risk disease and inferior outcome in NB as well as other paediatric cancers . This inversus orthotopically display non-invasive localized tumour expansion versus metastatic spreading to distant organs, respectively [ITH is often described as genetic and functional differences across the neoplastic cells themselves, however heterogeneity of the tumour microenvironment is overlooked. Xenograft experiments using the same cell populations implanted in different environments display highly varying growth patterns. The same cells injected subcutaneously ectively . This suectively . Deliverectively .mesenchymal (“undifferentiated”) and adrenergic, that exist within the same tumour. These cells are genetically identical however differ in their transcriptional and epigenetic landscape [In line with this, recent studies have also described multiple cell-type states of NB cells, termed andscape . In a siandscape . Similarandscape . Mechaniin vivo. This is despite the tumours being genetically identical. Furthermore, ontology analysis revealed differences in genes regulating neuronal/mesenchymal phenotypes as well as stromal and angiogenic processes, again indicating the involvement of tumour-stromal interactions in the ITH observed in NB [Using patient-derived orthotopic xenograft (PDOX) models of NB, we recently described additional evidence of functional ITH in NB. By implanting multiple NB samples derived from a single tumour into ten mice, we uncovered clear differences in transcriptional profiles and divergent tumour growth rates ed in NB .In summary, there are several dimensions of ITH in NB, including genetic and functional diversity of NB cells, as well as microenvironmental tumour heterogeneity, all of which can relate to progression and treatment response Figure . Disenta


Variation of the western North Pacific subtropical high (WNPSH) is an important meteorological factor for determining summertime rainfall and temperature over East Asia. Here, three major modes of summertime WNPSH variability are identified and corresponding environmental changes are investigated using cyclostationary empirical orthogonal function analysis. The leading mode exhibits a clear reinforcement of WNPSH associated with global warming. The second and third modes are characterized by intra-seasonal variation of the WNPSH intensity related to sea surface temperature variability in the central and eastern equatorial Pacific. Although WNPSH variability is regarded as a local manifestation, it reflects much wider changes in the entire North Pacific. The three modes exert seasonally and geographically distinct impacts on the East Asian weather by setting anomalous atmospheric circulation and altering the direction of moisture and heat transport. As such, the leading WNPSH modes are an important indicator of summertime weathers in countries neighboring the western North Pacific. This study also shows that extreme weather events are likely to increase as global warming intensifies. The sinking branch of the Hadley circulation makes large and semi-permanent anticyclones over the mid-latitude Pacific5. Variation of this subtropical high is caused by changes in both tropical and mid-latitude environments, including tropical convective activity7, meridional circulations9, zonally asymmetric diabatic heating10, and synoptic wave activity in the extratropical region12. Generally, this anticyclone system intensifies in boreal summer, and it expands westward to the western North Pacific14. According to the extent to which the western North Pacific subtropical high (WNPSH) expands toward the East China Sea, significant difference is observed in monsoon circulation, typhoon tracks, and moisture transport over East Asia, shaping distinctive summer weathers in the coastal regions17.Among the global-scale circulations, the Hadley circulation over the Pacific is the most dominant meridional air movement caused by strong upward motion with low-level convergence in the tropics due to tropical heat surplus18. However, investigation of long-term WNPSH variation on the basis of this empirically determined criterion is difficult, because the thickness of atmospheric column keeps on increasing due to continued tropospheric warming. A robust rising trend of geopotential height makes it ambiguous to examine seasonal and inter-annual WNPSH variability based on the location of the 5870 m contour alone. As a simple alternative, zonal mean of geopotential height at a corresponding pressure level is extracted from the raw data, called eddy geopotential height24. This method can partially make up for the increasing trend of the atmospheric column thickness, but it would not accurately delineate WNPSH variability25. Not only is global warming signal itself an important climate factor for WNPSH variability, but also global warming effect may not necessarily be zonally uniform. Thus, dubiety is inexorable in conclusions based on a crude exclusion of global warming effect.Due to its importance in East Asian weathers, several studies have attempted to investigate WNPSH variability. Some studies used a critical value such as 5870 m geopotential height contour on the 500-hPa pressure surface as the location of the WNPSH boundary30. In the present study, however, WNPSH variability over the subtropical region is a focus of investigation so that East Asian weathers are understood directly in terms of WNPSH-induced atmospheric circulation changes rather than an indirect implication of ocean-atmosphere coupling processes. Distinct sources of WNPSH variability are separated and physically consistent atmospheric and oceanic patterns associated with them are identified via cyclostationary empirical orthogonal function (CSEOF) analysis32. By using this technique, key physical relationships between Pacific climate variability and WNPSH variation can be clearly identified. We, therefore, expect that this study would enable us to better understand WNPSH variability on various time scales and contribute to more accurate seasonal predictions of weather extreme events such as heavy rainfalls and heat waves during summer over East Asia.The objective of this study is to define major WNPSH variability without ambiguity and understand its physical linkage to East Asian summertime weathers in the context of large-scale climate dynamics. Previous studies have investigated how slowly-varying oceanic variability such as El Niño–southern oscillation or Pacific decadal oscillation affects East Asian weathers35. Therefore, WNPSH variability is analyzed by excluding the mid-latitude region.Three major modes of WNPSH variability during July through September are identified from 500-hPa geopotential height via CSEOF analysis Fig. ; the sea36, suggesting that the two variables have a common source of variability.The spatial patterns of the loading vector and corresponding time series showing an increasing trend indicate that the first mode is associated with widespread warming Fig. . Among m39. In September, the anticyclonic circulation in the subtropics and the cyclonic circulation in the mid-latitude region are strengthened over the western Pacific40. There is no long-term trend in the second and the third CSEOF modes . Daily datasets, unless otherwise stated, are used in order to address both the mean and variance of physical changes in each month. Figure Precipitation associated with the first WNPSH mode exhibits distinct behavior for each month Fig. . In July44. In general, temperature response is more obvious over the continent due to the lower heat capacity whereas precipitation changes are more notable over the ocean.The first WNPSH mode represents consistent warming over China and the high-latitude region during the whole summer Fig. . Major sFor the second mode, precipitation changes match well with the WNPSH variations and are largely explainable by vertical moisture convection Fig. . In JulyThe third mode shows a notable increase of precipitation in the coastal region of East Asia in July, when WNPSH is weaker and further away from the continent Figs  and S1d.Regression analysis in CSEOF space explains well the physical relationship between the East Asian summer weathers and the WNPSH variability. As can be seen in Fig. 47. Specifically, the regressed patterns of SST suggest that the second and the third WNPSH modes are linked with the central Pacific (CP) and the eastern Pacific (EP) El Niño events, respectively. It is of interest to note that the intraseasonal WNPSH variability as represented by these modes is intimately connected with the seasonally persistent SST variability in the equatorial Pacific. Despite the seemingly similar forcing  patterns in summer, seasonal evolution of WNPSH varies significantly in terms of its intensity and location from one mode to another. In future study, it would be important to delineate physical linkage between WNPSH variability and SST variability such as El Niño-southern oscillation and Pacific decadal oscillation.Variation of WNPSH is an important modulator of East Asian weathers on daily and intra-seasonal time scales via a direct control of the atmospheric circulation in the North Pacific and East Asia. The distinct sources and physical mechanisms of the three leading modes of WNPSH variability are investigated in the present study. The three leading modes all seem physically related with large-scale sea surface temperature (SST) variability in the Pacific Fig. . It seemThis study shows that the effect of global warming and El Niño, although it is prominent in winter, is not limited to winter. Global warming and El Niño appear to affect the location and intensity of WNPSH significantly in summer by altering the distribution of energy and subsequent circulation in the tropics. The summertime precipitation and surface temperature in East Asia, in turn, are affected considerably by the variation of WNPSH. It is remarkable that the variation of the semi-permanent subtropical Pacific anticyclone in a small area of the western Pacific  and the coastal regions experience drier condition in August in East Asia. The present study strongly suggests a possibility that inclement weathers will become more frequent in the East Asian countries unless global warming is restrained by policy efforts.48. Target variable is chosen to be the monthly 500-hPa geopotential height to diagnose the multi-time scale variability of the WNPSH. The daily mean oceanic and atmospheric variables such as SST, 850-hPa geopotential height, surface (2 m) temperature, 3-dimensional wind, net radiation at the surface, precipitation, specific humidity, total cloud cover, total column water, and relative humidity were used to investigate the synoptic influences of WNPSH to East Asian weather.We used 39-year (1979–2017) European Center for Medium-Range Weather Forecast ERA-interim reanalysis datasets at a horizontal resolution of 1.5° × 1.5°32. In this analysis, meteorological datasets which have a dimension of both space and time, T, can be written asn is the CSEOF mode number, LVn is called the nth CSEOF loading vector, and PCn(t) is corresponding PC time series, r, and t stand for space and time, and D and T denote the spatial domain and the record period for CSEOF analysis, respectively. A major difference from the traditional EOF analysis is that CSEOF analysis captures temporally varying spatial patterns. CSEOF loading vectors are temporally evolving but repeating spatial patterns, i.e.,d is called the nested period. In the present study, nested period is set to three months because we focus on the boreal summer ; we chose JAS since 500-hPa geopotential height over the target (WNPSH) area are maximized in these three months. CSEOF loading vectors are mutually orthogonal in space and time, and PC time series are mutually uncorrelated.The CSEOF analysis technique is used to extract major modes of the summertime WNPSH variability49. The evolutions of atmospheric and oceanic variables including SST, precipitation, 2 m temperature, and all other analysis variables are decomposed to be physically consistent with those of 500-hPa geopotential height (target variable in this study) via regression analysis in CSEOF space. The key idea of regression in CSEOF analysis is that all the variables are decomposed to have the identical PC time series of the target variable. To accomplish this goal, PC time series of the predictor variable, PCPm(t), is regressed onto the target PC time series, PCn(t), as in (3):M(=20), εn) should be interpolated to match the temporal resolution of the other variable in (3). Then, regressed CSEOF loading vectors for the predictor variable, LVPRn, are obtained as in (4):LVPm are the CSEOF loading vectors derived from the predictor variable. Note that the regressed patterns in (4) are defined over the spatial domain R, which may not necessarily be the same as the target domain D in (1). Further, the temporal resolutions of the target and predictor variables differ in this study. For example, the patterns in Fig. nth mode of the target variable, Tn.To understand the environmental responses to the variation of 500-hPa geopotential height, regression analysis is conducted in CSEOF space), as in :3\documeSupplementary information


Time curves for 5-FU signals from the liver and bladder were also acquired. We observed significant differences (p < 0.01) in 5-FU signal time dependencies for the HT-29 and H-508 tumours. Retention of 5-FU occurred in the H-508 tumour, whereas the HT-29 tumour is not expected to retain 5FU due to the observation of the negative b time constant indicating a decline in 5FU within the tumour. This study successfully demonstrates that CSI may be a useful tool for early identification of 5-FU responsive tumours based on observed tumour retention of the 5-FU.Colorectal cancer is the third leading cause of cancer death worldwide. 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapies for treatment of solid tumours, including colorectal cancer. The efficacy of treatment is dependent on tumour type and can only be determined six weeks after beginning chemotherapy, with only 40–50% of patients responding positively to the 5-FU therapy. In this paper, we demonstrate the potential of using Magnetic Resonance (MR) Chemical Shift Imaging (CSI) for  Colorectal cancer is responsible for approximately 14% of new cancer cases and 12% of cancer deaths in Canada3. According to Canadian Cancer Statistics (2016), 7% of men and 6% of women are expected to develop colorectal cancer during their lifetimes2. Moreover, an increasing incidence of colorectal cancer among young adults in Canada has been observed4.Colorectal cancer is the third most common cause of cancer death worldwide according to the World Healthcare Organization9. However, the treatment response rate (percentage of patients whose tumour shrinks or disappeared after treatment) for 5-FU based chemotherapy are relatively low10. Biochemical modulation of 5-FU has increased response rates up to 40–50%12, while the influence on overall survival has been limited8. The improvement in overall survival was observed when 5-FU was modulated with irinotecan14. The current colorectal cancer mortality rates reflect – at least in part – resistance of individual tumours to 5-FU treatment. It has been clinically demonstrated that the “trapping phenomenon”  correlates with the clinical effectiveness of 5-FU chemotherapy15. Thus, a method to detect the responsiveness of a tumor to 5-FU in the earliest stages of treatment  may enable effective targeting of 5-FU therapy to responsive tumours and also may prevent unnecessary exposure to cytotoxic chemotherapy in patients with 5-FU resistant tumours.5-Fluorouracil (5-FU) is one of the most widely used cytotoxic chemotherapies for treatment of a variety of solid tumours, including colorectal and breast cancerIt is hoped that early identification of 5-FU resistant colorectal tumours will enable oncologists to choose treatment strategies more likely to improve patient survival and minimize unnecessary morbidity.18. It was found that the increase in Apparent Diffusion Coefficient (ADC) of tissue water within the tumour was significantly higher in responders compared to non-responders16. Marugami et al. was able to distinguish responders from non-responders 9 days after the initiation of chemotherapy based on the ADC changes in liver methastasis17. However, patients already received two infusions of 5-FU. Similar result was obtained by Lavdas et al.18. Although these results can be considered as an early detection of the tumour response, there are several limitations associated with DWI. Due to power requirements, hardware limitations and other external factors DWI accuracy is limited and the image quality is low19. Furthermore, DWI image are often susceptible to various artifacts like ghosting, blurring, ringing, distortions etc20.Several previous studies using MRI were conducted using Diffusion-Weighted Imaging (DWI) for early detection of the treatment response in patients with colorectal cancer19F magnetic resonance (MR) spectrum28. All of them can be visualized using Magnetic Resonance Imaging (MRI). Chemical-Shift Selective images of 5-FU and its metabolites have been previously acquired in a rat model25 and in a mouse model28. The high natural abundance (approximately 100%) and large gyromagnetic ratio of Fluorine-19 (19F) lead to a strong observed signal of 5-FU. 19F MRI is a non-invasive and non-ionizing imaging technique. Another significant benefit of using 19F MRI is the absence of fluorinated compounds in the human body, thus there is no natural background signal. These characteristics together, make 19F MRI a promising method for monitoring 5-FU retention immediately following a single chemotherapy treatment. This would be a significant improvement to the current method for evaluating the efficacy of chemotherapy based on the observation of reduced tumour size after prescribed chemotherapy treatment.It is hypothesized that the early monitoring of 5-FU retention at the site of colorectal tumours can indicate the tumour responsiveness to 5-FU chemotherapy. 5-FU metabolizes into fluorinated nucleotides (Fnuc) and α-fluoro-β-alanine  that each display a different chemical shift  is an extension of MR spectroscopy (MRS), allowing metabolite information to be measured. This technique has already been used for imaging the small intestines of mice with orally administrated 5-FU34 and sensitive (H-508)35 to 5-FU administration as a function of time. Significant differences between SNR values allow us to infer the potential utility of 19F CSI to detect resistance of colorectal cancer to 5-FU treatment and potentially guide clinical personalized medicine.The purpose of this study was to determine if there were observable differences between the signal to noise ratio (SNR) from colorectal tumours that are insensitive (HT-29)A total of 23 mice were imaged: 9 mice with the HT-29 tumour (non-responder), 9 mice with the H-508 tumour (responder), and 5 mice with both tumour types. For each animal, the SNR from the tumour, bladder and liver voxels were calculated.The obtained values were used for plotting the SNR time-dependence curves to detect the presence or absence of 5-FU retention in given tumours. If any organ or tumor was bigger than a single voxel, a mean value of the SNR from all voxels containing the organ or tumor was calculated and used in the subsequent analysis.19F CSI images acquired at 7.5(a & b) and 40 minutes (c & d) after bolus injection, superimposed on the 1H scans for two representative mice. HT-29 tumour cells were injected into mouse 1  were thresholded to create necessary contrast in tumour voxels between the images acquired at 7.5 and 40 minutes after bolus injection.Figure e 1 Fig. , wherease 1 Fig.  receivedThe SNR of the HT-29 tumor was 9 at 7.5 minutes after injection Fig. . The SNRThe liver SNR from the H-508 mouse decreased gradually from 17.8 to 8.3 throughout time. However, the SNR from the liver voxels in mouse 1 was equal to approximately 11 at 7.5 minutes after bolus and 10 at 40 minutes after injection. Bladder SNR was equal to 24.7 and 25.7 for mouse 1 and mouse 2 respectively at 7.5 minutes after injection. At the end of the time period, bladder SNR was equal to 86 for the HT-29 mouse and 58 for the H-508 mouse.Figure 19F CSIs of mouse 3 superimposed on proton scans. This mouse had both tumour types. The HT-29 tumour was injected into the left flank of the animal whereas the H-508 colorectal adenocarcinoma was injected into the right flank. There are no liver voxels on these CSI images. 19F SNR values higher than 50 were thresholded to visually create necessary contrast in tumor voxels between the figures acquired at different times.Figure Figure p < 0.01). After 30 minutes, bladder time dependency plateaued at a value of 83 with minor oscillations.Figure All tumour SNR time curves were fitted using an exponential function with two fitting parameters – amplitude “a” and time constant “b” Eq. . One HT-−1. On the contrary, the mean time constant “b” for the H-508 tumour was equal to 0.018 ± 0.015 min−1. The results are significantly different according to two-sample unpaired t-test (p < 0.01). Furthermore, according to one-sample t-test, the mean value for the H-508 tumour is significantly greater than 0 (p < 0.01). Figure p < 0.01 between the tumour types. Based on this statistical analysis, we can conclude that 5-FU uptake of studied tumors was significantly different.The mean value + /− one standard deviation of the time constants for the HT-29 tumour in single-tumor mice was equal to -0.004 ± 0.008 min−1 and −0.001 ± 0.008 min−1 respectively. The difference in mean values was not statistically significant (p > 0.01). However, the Wilcoxon test showed that SNR curves of two different tumours from the dual-tumour animals were significantly different (p < 0.01).The mean values of the time constant “b” for the H-508 and HT-29 tumors of dual-tumour cohort were equal to 0.006 ± 0.009 min19F CSI imaging. Time curves of the 5-FU signal acquired following bolus injection can reveal the difference in uptake of 5-FU in different tumour types. Time curves of the 5-FU signal acquired right after a bolus injection of the chemotherapy drug had different dynamics for two different types of human colon adenocarcinoma. Therefore, detection of tumour resistance to chemotherapy based on a 5-FU retention is possible approximately one hour after bolus injection.The results of this study illustrate the feasibility of detecting a 5-FU retention in different types of colorectal cancers using p < 0.01) for the HT-29 and H-508 tumour types. Increasing 5-FU SNR throughout time is characteristic of the H-508 tumour, whereas the HT-29 tumour type SNR shows a tendency to decrease or to remain constant. This result was obtained for the single-tumor mice and it was statistically significant (p < 0.01). A significant difference between time constant “b” for the dual-tumour animals was not observed (p > 0.01). It could be due to a small number of samples (n = 5). These results are consistent with the literature which shows that the HT-29 tumor demonstrates resistivity to 5-FU therapy while the H-508 tumor can be treated using 5-FU35.5-FU SNR changes with time were found to be significantly different , Fnuc signal was observed 1 hour after bolus and Fbal signal was detected 10 minutes after injection32. In that study, the authors used a 7.0T animal MRI system. The absence of a 19F signal from the fluorinated metabolites could be due to the small concentration of these metabolites in the organs and tumours. Our study time interval was likely too short to allow for a high concentration of catabolites to be produced. Future imaging of the metabolites Fbal and Fnuc requires further parameter optimization due to the low SNR of these metabolites even 40 minutes after bolus. Additionally, to improve the sensitivity of the RF coil, a phased array coil could be used for better coupling with 19F nuclei in the tumor and organs.Unfortunately, we were not able to observe the signals of 5-FU metabolites such as Fbal and Fnuc. 5-FU catabolizes into Fnuc in the tumour19F CSI imaging can be used to detect retention of 5-Fluorouracil in a murine model of colorectal cancer. This technique may extend prediction of patient tumour resistivity to 5-FU chemotherapy based on the pharmacokinetics of 5-FU at an early treatment stage, yielding an improvement in personalized cancer therapy. Implementing innovative imaging strategies that identify patients who respond to 5-FU would increase the efficacy of the treatment by allowing targeted matching of patients and chemotherapy agents in the neoadjuvant setting. This MRI-based technique could be readily implemented clinically as a means of non-invasively monitoring early response to 5-FU chemotherapy. Identifying resistant tumours early in cancer treatment will enable patients unlikely to respond to 5-FU therapy to rapidly alter treatment plans. The use of 19F CSI to triage patients into different chemotherapy regimens could be a significant innovation and alter routine clinical practice.Overall, our studies demonstrate that All human colon adenocarcinoma cell lines, media, sera, and culture reagents were obtained from ATCC , Life Technologies , Becton Dickinson  or Sigma . HT-29 cells were grown in McCoy’s 5 A medium and NCI-H508 were grown in RPMI-1640 medium, with both cell lines supplemented with 10% FBS, 100U/ml Penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells were grown to 50–75% confluency in T-75 flasks prior to injection into animals.6 human colon adenocarcinoma cells were mixed with a 1:1 ratio cold Matrigel  for each 100 µl bolus injection into 36–40 day-old male (Nu/Nu) nude mice.This study was approved by Lakehead University Animal Care Committee, and all procedures were done in compliance with the regulations of the Canadian Counsel on Animal Care (CCAC). HT-29 and NCI-H508 cells were grown in T-75 flasks between 60–80% confluency. Cells were trypsinized with 0.25% (w/v) Trypsin −0.53 mM EDTA solution, resuspended in appropriate medium, and counted. 1 × 106 HT-29 or NCI-H508 cells using a BD Eclipse 27 G × 1/2 needle. Mice were injected under the skin in the left (HT-29) or right flank (NCI-H508), weighed, labelled by ear piercing, and immediately returned to cages. Mock mice were injected similarly with 100 µl of a 1:1 ratio of appropriate cold medium and Matrigel while control mice received no injection. The H-508 tumour grew more slowly than HT-29. Therefore, the dual-tumour mice were injected with HT-29 tumor 5 days post HT-508 cells injection. A total of 23 mice were injected with either HT-29 cells (9 mice), H – 508 cells (9 mice) or both adenocarcinoma types (5 mice). All animal imaging was performed 12–16 days post injection or when a caliper measurement of the volume of the tumours did not exceed either 450 ± 75 mm3 for animals with single tumours or 600 ± 75 mm3 in total for the dual-tumour mice. The mouse body weight was between 30–37 g.Briefly, nude mice were aestheticized using 3% isoflurane and injected with 100ul of a 1:1 cold Matrigel solution of either 1 × 10Prior to the MRI scanning procedure, all animals were anesthetized using 5% isoflurane oxygen mixture and anesthesia was maintained at 2% with oxygen during all subsequent experiments. Mice were catheterized using a MTV-01 tail-vein catheter (SAI Instruments). All animals received a slow bolus injection of 300 µl 5-FU (50 mg/ml) over the course of 2 minutes inside the magnet bore. During each MRI acquisition, animals were kept at 37 C° with a temperature-controlled water-filled blanket .1H/19F quadrature birdcage coil. Proton localization was performed using a multi-slice T1-weighted Turbo Spin Echo (TSE) pulse sequence with a Field of View (FOV) of 75 × 75 mm2, TR/TE = 2000/55.19 ms, slice thickness of 2 mm and Number of Signal Averages (NSA) = 3. Acquisition matrix size was equal to 256 × 256 which corresponds to the in-plane resolution of 0.29 mm. The total number of slices was equal to 16 for each mouse. Proton scans were used to determine the location of the tumour, bladder and liver.MRI was performed using a clinical Philips 3.0 T Achieva whole-body scanner, equipped with a custom-built dual-tuned 19F CSI images were acquired for up to 70 minutes with a time step of 2 minutes and 30 seconds. Mice that had one tumour type were studied using CSI with 8 × 5 resolution, FOV of 20 × 50 mm2 and NSA = 3, whereas mice with both tumour types were studied using 3 × 5 matrix, FOV equal to 31 × 18.6 mm2 and NSA = 9. All CSI images were acquired using spectral bandwidth of 32 kHz (266 ppm at 3.0 T) and TR/TE = 5000/4.27 ms. The data sampling number was 1024, yielding a spectral resolution of 0.26 ppm.After the bolus injection, Images were analyzed using a custom imaging processing program written in MATLAB R2016b .a and time constant b were fitting parameters. The time constant determines signal dynamics. If the time constant is positive, the signal will grow with time, conversely when it is negative, signal will decrease. Thus, time constant can be used as an indicator of 5-FU kinetics. A two-sample t-test was applied for b time constant to analyze the statistical significance of the fitted results for the single tumour animals. A one-sample t-test was applied to the b time constant of each group of single tumour animals to evaluate if the mean b value of each group was significantly different from 0. A Wilcoxon signed rank test has been used to evaluate the difference between SNR time curves obtained from different tumors in dual-tumor animals. OriginPro 2016 was used to conduct statistical analysis .All SNR time curves were fitted by the exponential function


Histologically, proliferation of oval to short spindle-shaped cells with fibrocollagenous stroma, abundant various-sized vessels, microcysts and thickened anastomosed bone trabeculae were seen. Mitotic figures were rarely seen. Immunohistochemically, the tumour cells were focally positive for fibroblast growth factor 23. The imaging findings in the current case are in accordance with the histological features. Among them, 68Ga-DOTATOC positron emission tomography/CT scan for somatostatin receptor imaging provides valuable information for determining PMT localization and characterization.Primary phosphaturic mesenchymal tumours (PMTs) frequently occur in the soft tissue or bone, but rarely in the spine. The majority of patients experience long-term ostalgia and recurrent fractures. Detection of PMT can be challenging, but the clinical symptoms dramatically improve after removal of the tumour.Wepresent a case of primary PMT in the lumbar spine. CT scan showed a low-density tumour with a well-defined sclerotic margin in the L5 vertebra. MRI revealed a hypointense tumour on T However, the vast majority of patients experience symptomatic osteomalacia representing long-term ostalgia and recurrent fractures with increased fibroblast growth factor (FGF) 23 secretion. Therefore, diagnostic imaging plays an important role in early detection and subsequent treatment. Imaging findings of PMT are non-specific, leading to difficulties in making a radiological diagnosis.2 In this report, we present a case of PMT of the lumbar spine. CT scan, MRI and 68Ga-labelled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-d-Phe1-Tyr3-octreotide (68Ga-DOTATOC) positron emission tomography (PET)/CT scan were performed. The resulting imaging features corresponded well with the serological and pathological characteristics of the tumour.Intraosseous primary phosphaturic mesenchymal tumours (PMT) are rare tumours that are associated with oncogenic osteomalacia.−1), elevated serum alkaline phosphatase (933 IU l−1) and FGF23 (96.3 pg ml−1), and high urinary phosphorus (1.8 g day−1) levels. Based on these findings, tumour-induced osteomalacia such as PMT, which is associated with FGF23 secretion, was suspected. Systemic venous sampling for FGF23 analysis was performed. However, tumour localization was not successful.A 54-year-old male had been experiencing chronic pain in his chest, back and both legs for 3 years. He was found to have hypophosphataemia and a high serum alkaline phosphatase level, and was referred to our hospital for further examination and treatment. Laboratory tests showed low serum phosphorus   . The sca3 Although pain relief can be obtained using radiofrequency ablation, symptomatic treatment alone is usually chosen for unresectable cases. The duration of symptoms varies from 9 months to over 20 years1 and is an important factor for differentiating between benign and malignant PMTs.4 These facts suggest that diagnostic imaging plays an important role in early detection of the tumour and subsequent treatment. Venous sampling for analysis of FGF23 in the whole body is sometimes helpful for localization of the responsible tumour,5 although localization was not achieved with this method in the present case.Clinical symptoms dramatically improve after removal of PMTs, and surgical excision is the first choice of treatment.1 These tumours overexpress FGF23, which inhibits reabsorption of phosphate in the renal tubules and decreases 1,25-dihydroxy vitamin D levels. Subsequently, hypophosphataemia and osteomalacia occur.PMT is a rare tumour that is associated with oncogenic osteomalacia.8 The patients were 3 males and 6 females, ranging in age from 14 to 66 years (mean 48.0 years). The tumours were located in the cervical (3 cases), thoracic (3 cases), lumbar (1 case) and sacral (2 cases) spines. PMT often appears as a low density on non-enhanced CT images,6 and sclerosis is sometimes seen, which was consistent with our case. Microcystic changes may be present in some PMT cases,8 but they are not pathognomonic. Previous reports have shown that the differences between benign and malignant PMT are found in the infiltrative pattern of the primary tumour and the tumour size.4 Nakanishi et al9 reported that whole-body MRI using a single-shot short T1 recovery-echo planar imaging sequence could show PMT as high intensity. These results imply that CT scan and MRI might be useful for assessing the localization of PMT. However, these imaging findings are non-specific and PMTs could be missed if they are small.Primary PMT frequently occurs in the soft tissues of the extremities, but rarely occurs in the spine. To our knowledge, only nine cases have been reported in the literature.68Ga-DOTATOC imaging focuses on detecting neuroendocrine tumours and has some beneficial pharmacokinetic properties. 68Ga-DOTATOC PET/CT scan has higher spatial resolution and has been reported to better detect neuroendocrine tumours compared with single-photon emission CT.2 In addition, a 68Ga-DOTA-labelled somatostatin analogue is superior to 18F-fludeoxyglucose, which is widely used clinically for tumour imaging, as well as for the detection of neuroendocrine tumours. Histologically, most PMTs are composed of small, bland, spindled-shaped cells that produce a distinctive smudgy matrix, with a well-developed capillary network.1 The identification of FGF23 production by tumour cells has been used to confirm the diagnosis of PMT.101 In the present case, CT scan showed a low-density tumour with a well-defined sclerotic margin. On MRI, the tumour was hypointense on T2 weighted imaging and showed heterogeneous enhancement. 68Ga-DOTATOC PET/CT scan demonstrated intense focal uptake within the tumour. The hypointensity on T2 weighted imaging may be due to the histological fibrocollagenous stroma and the focal uptake may correspond to excess FGF23 production. We found no reports that compared imaging and histological findings of PMT of the spine.A subset of cases also show a prominent fibrohistiocytic reaction and woven bone production.68Ga-DOTATOC PET/CT scan for somatostatin receptor imaging is much better for the localization of PMT compared with CT scan or MRI. CT scan and MRI are useful for assessing the extent of the tumour.In conclusion, Detection of PMT can be challenging, but the clinical symptoms dramatically improve after removal of the tumour.68Ga-DOTATOC PET/CT scan for somatostatin receptor imaging is useful in the localization of PMT.CT scan and MRI should be modalities of choice in evaluating the extent of the tumour.Written informed consent was obtained.


The objective of this paper is to carefully study the performances of a new piezoelectric micropump that could be used, e.g., for drug delivery or micro-cooling systems. The proposed micropump is characterized by silicon diaphragms, with a piezoelectric actuation at a 60 V input voltage, and by two passive valves for flow input and output. By means of a 3-D Finite Element (FE) model, the fluid dynamic response during different stages of the working cycle is investigated, together with the fluid–structure interaction. The maximum predicted outflow is 1.62 μL min Pumps are devices that provide momentum to transfer fluids and can be integrated in Micro Electromechanical Systems (MEMS) to develop so-called micro-fluidic systems . As an eSeveral actuation systems have been investigated in the literature , with paIn any case, it has been clearly established that, in addition to the actuator, the presence of valves in the device can affect the performance. Despite the fact that the valveless micropumps have the benefit of no risk of wear and fatigue of the valves, it is hard to control the flow to the desired direction at inlet and outlet, causing energy loss and liquid reflux. Consequently, in order to improve the control of the fluid flow, mechanical valves (either active or passive) must be introduced in the system. The main novelty of the present paper is the comprehensive study of a complete pumping system, composed of a piezoelectric actuator and passive valves, using three-dimensional modelling and a simulation of the device performing a complete cycle of pumping with a consideration for the two-way fluid–structure interaction. An innovative layout is considered, starting from a patent  that wasThe paper is organized as follows. After the introduction, the layout of the device is shortly discussed. The subsequent section contains a thorough description of the computational model which then leads to some preliminary results. Then, we consider some modifications of the geometrical features of the pump, with the purpose of optimizing its performances.In most cases, a pumping device, composed of a micropump and valves, is of complex realization in view of the difficult integration of the different components of the actuating system on a limited number of wafers. Moreover, the integrated valves may present specific issues related to a lack of tightness, which is the cause of a leakage and backflow.In order to overcome the aforementioned limits, an innovative device based on the use of two wafers has been recently conceived and patented . In spitMultiphysics modelling encompasses two main parts: the “deformable” solid, containing the actuation portion and the valves for controlling the fluid flux, and the “fluid domain”, which interacts with the solid domain through the interface at the boundary of the solid and fluid domains. Due to the sake of symmetry, half of the micropump is modelled.In the initial design, the micropump consists of a circular pumping chamber, delimited by lower and lateral fixed surfaces and by an upper silicon diaphragm. As shown in The geometrical specifications of the fluid domain are presented in The model for the solid parts is depicted in The mechanical properties of polycrystalline silicon and PZT are given in The investigation of the interaction between the moving solid parts and the fluid is a key point, especially in mechanical micropumps. The motion of the solid, i.e., the oscillating displacement field, induces the motion in the fluid. Also, the fluctuating pressure in the fluid acts as a surface load on the fluid/solid interface. By coupling the governing equations of these two domains, the mutual interaction can be thoroughly studied.® 18.1 and 19.2, with the mechanical and CFX solvers. At each staggered loop, the mechanical solver sends the time derivative of the displacement of the interface nodes to the target nodes in the fluid domain. On the other hand, the CFX solver sends back the stress to the solid nodes based on the traction equilibrium at the interface [For the proposed micropump, a two-way fluid–Structure Interaction (FSI) is modeled with the commercial code ANSYSnterface .The valves are modelled by means of quadrilateral 8-node solid elements (SHELL182 in ANSYS nomenclature), which are suitable for modelling thin to moderately thick shell structures. The aim of choosing this kind of element is to reduce the computational cost, taking into account the specific geometrical features of the problem at hand. The 20-node brick element is chosen for the oscillating membrane (SOLID186), and the coupled-field element is chosen for the piezoelectric actuator (SOLID226). In the fluid domain, the tetrahedral elements with 10 nodes (FLUID221) are used. The number of elements for the solid and fluid domains are 33,968 and 1,425,377, respectively. Some views of the adopted mesh are reported in For the boundary conditions on the fluid domain, both the inlet and outlet have a zero relative pressure with respect to the outside of the domain. The faces attached to the silicon membrane and valves are set to the fluid–structure interaction sites as well. The other faces of the fluid domain are set to stationary walls, i.e., the velocity of the fluid is equal to zero.stroke volumeThe proposed micropump is investigated for different values of the input voltage frequency. The most important figure of merit is the outflow of the micropump that is reported in As it is shown by the chart in At the first stage of the micropump working cycle, the silicon membrane is deformed in the upward direction due to the PZT deformation. The pressure gradient causes an inlet valve opening and the fluid enters in the chamber. The computed maximum deflection of the diaphragm for different actuation voltages is demonstrated in The analysis shows that the maximum fluid velocity occurs in the channels during the first and the second half cycles see . The maxThe apparent power consumption of the actuator can be approximately computed by assuming harmonic variation :(2)PA=Iedead zones, affects the efficiency of the micropump, since a certain portion of the volume is not involved in the fluid flux. To decrease the dead zones in the pumping chamber, a geometrical optimization is carried out. The specific shape of the streamlines shown in The modified layout is shown in The same analysis has been done on similar geometries with different length/width ratios of the ellipse. As noticed in In the manufacturing process of this two-wafer micropump, wafer bonding is one important step. There are several methods for silicon wafer bonding such as anodic bonding , metal bTo validate the model, a commercial micropump designed for biomedical applications and fabricated by Debiotech  is modelA complete pumping cycle at the frequency of 1 Hz is simulated. To validate the model, the pressure profiles measured inside and outside the chamber in the Debiotech micropump is compared with the results of the numerical model and shown in In this paper, the response of a new piezoelectric micropump is investigated by means of a multiphysics computational model. The most important novelty of the proposed design, inspired by a recent patent  related min−1 . The pow