Despite the fact that different genetic programmes drive metastasis of solid tumours, the ultimate outcome is the same: tumour cells are empowered to pass a series of physical hurdles to escape the primary tumour and disseminate to other organs. Epithelial-to-mesenchymal transition (EMT) has been proposed to drive the detachment of individual cells from primary tumour masses and facilitate the subsequent establishment of metastases in distant organs. However, this concept has been challenged by observations from pathologists and from studies in animal models, in which partial and transient acquisition of mesenchymal traits is seen but tumour cells travel collectively rather than as individuals. In this review, we discuss how crosstalk between a hybrid E/M state and variations in the mechanical aspects of the tumour microenvironment can provide tumour cells with the plasticity required for strategies to navigate surrounding tissues en route to dissemination. Targeting such plasticity provides therapeutic opportunities to combat metastasis. Metastasis is the major cause of mortality associated with solid tumours. Tumour cells escape from the primary tumour mass, move through surrounding tissues, enter the circulation, and colonise distant organs to form secondary tumours. During this process, tumour cells have to navigate mechanical hurdles consisting of various extracellular matrix (ECM) structures and layers of cells. Cross talk between intrinsic properties of the tumour cells and mechanical aspects of their surroundings drives cellular plasticity that enables tumour cells to make this journey.2 The TME comprises cancer-associated fibroblasts (CAFs), blood vessels and lymphatic vessels, immune-inflammatory cells, and neuroendocrine and adipose cells, all of which are embedded in an ECM, a structural network that sustains and shapes the three-dimensional architecture of tissues and organs. Within the TME, tumour cells are subjected to chemical  and physical cues that originate from the cellular elements as well as from the ECM. Together, these cues impinge on cellular signalling cascades in tumour cells thereby promoting tumour development and metastasis.The cells of solid tumours are typically surrounded by a dense fibrotic tissue composed of cellular and acellular elements—the tumour microenvironment (TME)—which plays an active role in the aggressive metastatic behaviour of cancer.3 This process is orchestrated by signalling molecules such as transforming growth factor (TGF)-β and Wnt, which induce downstream pathways that regulate a network of transcription factors to control the balance between key epithelial proteins  and mesenchymal proteins (such as vimentin).5 Transcription factors such as TWIST, SNAIL and ZEB induce EMT whereas GRHL2 and OVOL2 suppress EMT.7 EMT is important in embryonic development for cell migration and regulation of tissue differentiation and homoeostasis,9 but has also been associated with cancer initiation, development, and progression.11 However, the idea that a full transition from an epithelial to a mesenchymal state is required for metastasis has been challenged by observations from pathologists and studies using genetically modified mouse models.14What triggers a cluster of tumour cells to transit to a motile state, crawl through surrounding tissues, and start the metastatic process? One concept is that this involves an epithelial-to-mesenchymal transition  cells may initiate movement is derived from active matter physics. It describes how changes in mechanical and geometric parameters such as extracellular pressure, cell density, and cortical tension, can trigger a shift from solid to fluid-like behaviour in cell clusters, without the need for transcriptional alterations such as those underlying EMT15 Fig. . This shHere, we focus on the early stage of the metastatic cascade where tumour cells leave the primary tumour, invade surrounding tissues, and enter the circulation. We present an overview of mechanical properties of the TME and discuss roles for  EMT and unjamming in tumour cell migration strategies. We then explore bidirectional cross talk between the TME and partial EMT and discuss how this may contribute to plasticity and unjamming. While a detailed description of underlying molecular pathways is beyond the scope of this review, we discuss candidate therapeutic opportunities for targeting the TME and the hybrid E/M state to break crosstalk and plasticity in order to interfere with metastatic strategies.2 Increased stiffness and pressure, both solid and fluid, are the main macroscopic mechanical alterations that can be observed in the tumour bulk.Tumour cells are subjected to multifaceted physical cues within the TME.2 This process triggers a series of intercellular feedback loops: tumour cells recruit and activate stromal cells; these stromal cells contribute to the increased production and secretion of ECM, which, in turn, stimulates tumour progression. Ultimately, these events result in a stiffer TME, which confers increased resistance to physical deformation. This alteration in tissue tensional homoeostasis has been reported to enhance cancerous transformation.17 The dysregulation of ECM deposition, named desmoplasia, involves not only changes in terms of ECM quantity, but also its architecture and organisation.18 In particular, the main components of ECM that are dysregulated and associated with cancer progression are fibrillar collagens, fibronectin and hyaluronic acid (HA).19 These alterations in ECM contribute to the increased stiffness of the TME, which has been associated with increased malignancy and invasiveness in pancreatic ductal adenocarcinoma, breast cancer, colorectal cancer and prostate cancer.24The components of the TME are not malignant per se—in fact, they are an important source of support for tissues in physiological conditions. However, as cancer progresses, many of these components are exploited by the tumour cells, causing a change in the mechanical properties of the TME. For example, CAFs can arise from resident fibroblasts and become activated in response to the release of growth factors such as TGF-β to acquire a tumour-promoting function.25 Expansion of the tumour bulk compresses tumour-associated blood and lymphatic vasculature, which, in turn, can affect the vascular integrity, ultimately leading to leaks and impaired drainage of lymphatic vessels. This impairment of the normal function of vessels leads to an increase in interstitial fluid pressure, which contributes to therapy resistance by inhibiting drug delivery to the tumour.26 In addition, impaired vascular integrity creates hypoxic regions, which induce activation of the transcription factor hypoxia-inducible factor (HIF)-1α, leading to tumour invasion and promotion of angiogenesis.27Besides alterations in stiffness, the mechanical TME is affected by increased solid and interstitial pressure as the tumour increases in size. ECM components such as HA and proteoglycans absorb water, which leads to an increase in solid pressure due to the resistance conferred by the surrounding tissue. In addition, proliferation of tumour cells generates solid pressure, as an increased uptake of soluble factors results in enhanced conversion into insoluble biomass.25 In a positive-feedback loop, the stiffer environment triggers an increase in actomyosin contractility and force application by tumour cells, causing further ECM stiffening.28 The tensile forces on the ECM also lead to the unmasking of new binding sites for integrins, further promoting cell–ECM interactions.29 In addition, tumour cells and CAFs remodel the ECM by enhancing collagen alignment through a process that requires contractility mediated by the GTPase Rho and its downstream effector Rho-associated kinase (ROCK), which has been associated with tumour invasion and attraction of vascular endothelial cells.32 Moreover, tumour cells can enhance crosslinking of collagen fibres in the ECM, which further augments stiffness of the tumour stroma. The main enzymes responsible for this crosslinking are tissue transglutaminase 2 and lysyl oxidases (LOXs), the expression of which is upregulated in several solid tumours. LOX enzymes, in particular LOX2, are upregulated in response to hypoxia and high levels of TGF-β, both of which are characteristic of the TME and associated with tumour progression and metastasis.34The physical alterations that occur within the tumour stroma are not just passive consequences of tumour growth. Tumour cells and CAFs actively change the mechanical properties of the TME through their interaction with the ECM. They adhere to ECM components through integrin receptors and use contractility mediated by the actin cytoskeleton and myosin motors to apply force onto these adhesions, causing cell-mediated deformation of the ECM proteins (termed strain stiffening), which contributes to the stiffening of tumour stroma.35 A force-transmitting cytoskeleton is essential for cells to sense the mechanical properties of the environment and several signal transducers have been implicated in this process, including ion channels, cell matrix adhesion complexes and membrane-associated phospholipases. Within cell matrix adhesion complexes, mechanoresponsive elements including integrin receptors and associated cytoplasmic proteins such as focal adhesion kinase (FAK)36 couple the ECM to the cytoskeleton across the plasma membrane, providing mechanical homoeostasis between cells and the ECM.37 In conjunction with chemosensory signalling pathways (such as those activated by TGF-β and hypoxia mentioned earlier), this bidirectional signalling controls cell shape and migratory and invasive behaviour, as well as cell survival and proliferation.39The altered mechanical cues in the TME help to create a niche that supports tumour growth, invasion of surrounding tissues, and therapy evasion. Tumour cells sense the above-mentioned mechanical changes and transduce the mechanical input into intracellular biochemical signalling.41 This ‘intra-tumour heterogeneity’ provides plasticity and confers a survival advantage on tumour cells to migrate, invade and reach distant organs.43 The conversion from a localised tumour into a full blown, disseminated cancer requires that tumour cells activate migration. EMT and unjamming represent two concepts explaining the acquisition of migratory capacity in tumours.Changes in the TME induce adaptive mechanisms, such as metabolic reprogramming in tumour cells, that, in addition to the intrinsic lack of homogeneity within tumours, contribute to the generation of tumour cell populations with diverse gene expression patterns and phenotypic features within a tumour mass.45 For example, ErbB2 is a metastasis-promoting oncogene that is frequently overexpressed in non-invasive ductal carcinoma in situ. However, only a subset of ErbB2-overexpressing cells progressed to invasive breast cancer in animal models and patient tumours and in this subpopulation ErbB2 was accompanied by overexpression of 14-3-3ζ, which led to EMT.46 The notion that EMT represents a critical step for the initiation of metastasis is challenged by the lack of evidence for EMT in the histopathology of metastatic tumour tissues as well as in several studies using animal models.48 For example, depletion of the key EMT-promoting factors SNAIL or TWIST in a mouse model for pancreatic cancer or lineage-tracing using Fsp1 as an EMT marker in a mouse model for breast cancer failed to support a role for EMT in metastasis.47 On the other hand, a study using loss of E-cadherin as an EMT marker in a mouse model for breast cancer, associated the occurrence of spontaneous EMT in a small subpopulation of tumour cells with increased migration capacity.48 The interpretation of studies in favour of- and arguing against a critical role for EMT remains an ongoing debate.50 Importantly, defining EMT based on the expression of a single marker underestimates the dynamic nature of EMT as this process is likely to be a transient event in cancer.51 Moreover, EMT is a non-linear programme that can be defined and controlled by distinct gene networks in a cancer-type specific manner.53 It has been shown that a pro-metastatic effect of EMT depends not only on the final state but on the molecular route that leads tumour cells to that state.54 The reverse process, mesenchymal-to-epithelial transition (MET), occurs as tumour cells arrive at distant organs, and might be important for the formation of metastatic lesions, as disseminated tumour cells locked in a mesenchymal state fail to effectively colonise these organs.57EMT can contribute significantly to tumour heterogeneity and plasticity and has been proposed to drive the initiation of metastasis.58 or mesenchymal stem cells.59 In addition, CAFs can arise from epithelial cells through EMT or from endothelial cells through endothelial-to-mesenchymal transition (EndMT) and both conversions are induced by TGF-β.61 It is largely unknown how these CAF populations differ in functionality, but they are all characterised by a myofibroblast phenotype that drives stiffening of the TME as described above.Notably, EMT also plays a role in other cell types in the TME including the generation of CAFs. CAFs can originate from normal resident tissue fibroblasts65 One advantage of maintaining an epithelial phenotype, such as expression of E-cadherin in a hybrid E/M state is an increased survival fitness through cell–cell contacts in tumour clusters in the circulation.66 Hybrid E/M is also associated with increased stemness, which, in turn, is linked to elevated plasticity and self-renewal capacities as compared with completely E or M states in breast cancer.68 Additionally, a tumour that harbours subpopulations of cells residing at different stages of a fluid, cancer-associated hybrid E/M state might have an optimal capacity to cope with variations in the TME and progress towards metastasis. A hybrid E/M state confers phenotypic and molecular diversity, which provides cellular plasticity, empowering tumour cells to navigate various physical hurdles during their journey to metastatic sites while maintaining expression of epithelial markers and intercellular adhesion.72 Indeed, in a mouse model for breast cancer, a hybrid E/M state induced the formation of tumour cell subpopulations with varying degrees of invasiveness and metastatic potential.63 The existence of hybrid E/M cell populations and their association with enhanced metastatic features including migration and intravasation, were corroborated by studies on ovarian and pancreatic cancers.74 A biophysical model also showed that hybrid E/M states give rise to heterogeneous clusters migrating collectively and leading to the circulating tumour cell clusters as observed in animal models and patients.75Rather than a complete EMT, transient subtle changes in the balance between pro- and anti-EMT transcription factors that result in a partial EMT or ‘hybrid E/M’ state might be more relevant in the context of cancer Fig. . Indeed,15 In this case, changes in mechanical and geometric parameters in the tissue trigger fluidisation in absence of EMT15  and moving states (“unjammed”) in cell aggregates.T15 Fig. . In epit83 Likewise, circulating tumour cell clusters can arise from collective cell migration and intravasation in renal cell carcinoma, lung cancer and invasive melanoma.86 Whether cluster invasion in the complete absence of a partial EMT fully explains these findings is unresolved. EMT-like changes have been detected in circulating tumour cells.87 Yet, clusters of circulating tumour cells are largely epithelial and evidence in favour of E/M hybrid clusters is still scarce, suggesting that unjamming of fully epithelial tumour tissues may occur.If and how the early steps of metastasis follow similar principles, represents an urgent, unresolved issue. In breast cancer, clusters of invading tumour cells are more prone than individual cells to survive. These clusters promote metastasis formation in mouse models and give rise to oligoclonal clusters in the circulation that are associated with poor prognosis in patients.88 Such movement has been suggested to be critical for cell mixing inside the tumour, which allows the most aggressive clones to effectively replace all other cells.89 In the outer regions, tumour cells are prone to mechanical stress due to a high abundance of ECM, which results in further unjamming.15Tumour cells in the centre of a tumour mass are likely to be jammed but increased pressure might drive a switch from a solid to a fluid-like state. Indeed, multiphoton microscopy in a spontaneous mouse model for intestinal cancer has shown coordinated migratory patterns in the tumour core that are indicative of a fluid-like behaviour.91 Likewise, unjamming of breast cancer cells triggered by a cascade of growth factor receptor internalisation, activation of extracellular signal-regulated kinase/mitogen-activated protein kinase and cytoskeletal remodelling, induces collective migration.81 Glioma cells infiltrate the brain as multicellular networks and breaking cell–cell interactions by downregulating p120-catenin was found to decrease infiltration capacity, again indicating that the ability to maintain cell–cell contacts is important.92 It is likely that the interaction between molecular programmes induced by hybrid E/M and local, physical cues in the TME creates routes for subpopulations of tumour cells to unjam and start disseminating.93Unjamming, as well as a hybrid E/M state, leads to a fluid-like migration of clusters of tumour cells that maintain cell–cell contacts. It has been reported that high expression of EMT-promoting transcription factors such as Snail and Twist leads to the collective migration of tumour cells that exhibit epithelial and incomplete mesenchymal features.94 Single cells can move through ECM networks by adopting amoeboid or spindle-like mesenchymal shapes:95 amoeboid cells generate few ECM adhesions and stress fibres whereas mesenchymal migration is associated with strong ECM interaction and actomyosin contractility.93 Collectively migrating cells adopt different morphologies such as sheets, strands, multicellular tubes and masses with irregular forms  cooperates with ECM confinement to drive unjamming, fluidisation and, ultimately, cell individualisation.101 Thus, the interaction between molecular features of tumour cells and local properties of the TME can drive metastasis by mediating interconversions between collective and individual behaviour , which facilitates ECM remodelling and regulates canonical signal transduction pathways that control cell fate.102 Mechanical cues from the TME, such as increased ECM density and stiffness, can stimulate EMT106 and act in concert with soluble EMT-stimulating factors, such as TGF-β.108 Important mediators of mechanically-induced EMT are the transcription factors TWIST1 and YAP/TAZ,110 which, upon matrix stiffening and subsequent intracellular transduction of mechanical signals, are induced to translocate to the nucleus to influence the expression of several genes that promote EMT  leading to the up-regulation of hyaluronic acid synthase 2 (HAS2) and increased HA production.113 Thus, the chemical composition and stiffening of the TME can promote  EMT in tumour cells. Notably, cells appear to possess a “mechanical memory” i.e., prolonged exposure to a stiff ECM causes EMT-like behaviour with nuclear localisation of YAP, high actomyosin contractility, and large cell matrix adhesions and this phenotype is maintained when the cells move to a soft environment for as long as the factors mediating the mechanical memory suppress a transcriptional switch.115Tumour cells sense and respond to mechanical stimuli from the TME.EMT Fig. .103,110–116 tumour cells themselves have been observed to be “more deformable” or “softer”.117 EMT might play a role in such softening of tumour cells. Cells undergoing EMT change their morphology, lose adhesive properties and undergo actin cytoskeletal rearrangement, which all influence cell stiffness and tension with neighbouring cells and the ECM.118 Mesenchymal-like cells tend to reduce their stiffness and become softer in response to force application, while epithelial cells are more likely to stiffen in response to the same force application.119 Accordingly, EMT-promoting transcription factors such as SNAIL and TWIST1 promote increased cellular deformability,120 which facilitates migration through ECM networks and intravasation.119Whereas stiffening of the TME drives EMT and the aggressive behaviour of tumours,121 This interaction is important for tuning the mechanical properties of the nucleus during migration in confined spaces. Indeed, nuclear deformability is a rate-limiting step for cell migration and some level of nuclear rupture has been observed during the migration of tumour cells in a confined space.124 The nucleoskeletal lamins regulate stiffness of the nuclear envelope and thereby determine a cell’s migratory capacity in confinement.125 How  EMT affects nuclear mechanics remains to be elucidated but a hybrid E/M will increase cellular and, perhaps, nuclear deformability to increase plasticity, allowing tumour cells to adapt to confinement and enhance migratory potential.Actin fibres connect integrin-containing adhesions with the nuclear envelope through the linker of nucleoskeleton and cytoskeleton (LINC) complex, thereby creating a physical connection between the ECM and the nucleus.127 The number of invadopodia—specialised actin-based membrane protrusions in which localised proteolytic activity degrades ECM—is also increased in tumour cells that are subjected to a stiffer environment or dense fibrillar collagen structures.129 Likewise, EMT induced by transcription factors including TWIST1 and ZEB1, promotes the formation of invadopodia in tumour cells.131 Thus, the interconnection between stiffening of the TME and EMT discussed above might enhance the ability of tumour cells and tumour-cell clusters to proteolytically degrade the ECM and break through tissue barriers. The importance of proteolytic ECM degradation, however, depends on the migratory strategy. While enzymatic breakdown of ECM is necessary for collective migration, individually migrating cells can either proteolytically remodel their surrounding ECM or adapt their shape to the already existing gaps.93 EMT driven by ZEB1 also leads to increased expression of LOXL2,132 which not only causes enhanced collagen crosslinking and TME stiffening but has been found to stimulate an EMT-associated transcription network,133 providing yet another positive feedback loop between EMT and the TME.As tumour cells undergo EMT, they also increase the production of soluble proteases or membrane-anchored MMPs, which allows invading tumour cells or tumour cell clusters to remove barriers or create tracks.135 vimentin,136 Rho/ROCK and actomyosin contractility137 and FAK.138 Notably, however, interfering with tumour–TME interactions can also have unexpected and undesirable effects. For example, whereas inhibition of FAK in a mouse model for pancreatic cancer attenuated the cancer-promoting activity of the fibrotic stroma, limited tumour progression and enhanced survival,138 depletion of CAFs, which might be expected to have a similar effect, actually led to more aggressive tumours and reduced survival.139 One explanation is the heterogeneity of CAFs in pancreatic and other cancers that may have diverse impacts on tumour growth and progression within the TME, including immune-modulation.141Interfering with the metastatic process remains a major challenge. Crosstalk between tumour cells and the TME is complex and dynamic and provides plasticity that allows tumour cells to adapt to different environments and escape therapy. We have discussed the mechanical interplay between the TME and tumour cells and a role for partial EMT in this process. Several candidate targets exist, which, when inhibited, might block this mechanical interaction and prevent tumour cell plasticity, including integrins,142 A network topology-based modelling approach has been applied to identify approaches for interfering with feedback loops in EMT networks, which may point to new strategies to interfere with plasticity and, hence with metastasis.143 Signal transduction cascades and transcription factors promoting a stable hybrid E/M state might serve as promising therapeutic targets, including GRHL2, OVOL2, NUMB and NRF2.145 Such a strategy has been successfully explored in breast cancer cells, in which the expression of SNAIL is associated with the hybrid E/M state. Deletion of SNAIL or either deletion or overexpression of ZEB1 pushed cells either in a complete E or in an M state, in each case resulting in attenuated capacity to form tumours.146 Despite these promising results, strategies that drive hybrid E/M cells into MET pose the risk of driving metastatic outgrowth of already disseminated tumour cells.57 On the other hand, strategies that lock cells in the M state might attenuate the outgrowth of primary and secondary tumours but drive the dissemination of individual tumour cells.56 An alternative promising strategy that exploits the highly plastic hybrid E/M state has made use of a combination of peroxisome proliferator-activated receptor γ (PPARγ) activation and MEK inhibition to enforce transdifferentiation of the tumour cells into post-mitotic adipocytes.147 This points to an exciting possibility that while plasticity allows tumour cells to adapt to different environments during metastasis it also represents a state that is vulnerable to differentiation therapy.Strategies that simultaneously target different mechanisms of tumour cell plasticity, including the hybrid E/M state, might prevent tumour cells from adapting to changes in the TME.In this review, we have discussed the dynamic interactions of tumour cells with the TME. In particular, we highlighted the importance of tissue mechanics and the role of  EMT in the early steps of the metastatic cascade. The TME provides a pathological mechanical environment that tumour cells sense and respond to. The initiation of the metastatic cascade requires acquisition of a migratory phenotype that is influenced by this environment. The role of EMT in this process is likely different in different tumour types and in most cases involves a partial EMT or hybrid E/M state. EMT and unjamming provide distinct mechanisms to initiate movement and to what extent hybrid E/M sets the stage for unjamming of epithelial tumour cell clusters is poorly understood. The hybrid E/M state provides tumour cells with plasticity affecting stemness, tumour growth, and migration, allowing them to navigate variations in the mechanical TME as they use collective strategies to invade local surrounding tissues and enter the circulation. It is the bidirectional cross talk between partial EMT-driving molecular programmes in the tumour cells and the heterogeneous local mechanical properties of the environment that drive the early stages of the metastatic cascade. Further insight into the dynamic nature of this process at different stages of the metastatic cascade is required. This will depend on integration of multiscale theoretical models, in vitro models incorporating tumour heterogeneity and relevant mechanical variations in the TME, and in vivo models that capture the full complexity of the metastatic process. Disrupting mechanical tumour–TME interactions and/or tumour plasticity at the level of the hybrid E/M state offers promising avenues for therapeutic strategies. In this area, we have only just begun to scratch the surface of what might be possible.


Although there are effective nucleoside analogs to treat HSV, VZV, and HCMV disease, herpesvirus infections continue to contribute to significant morbidity and mortality. Acyclovir is the drug of choice for HSV encephalopathy, yet there is an estimated 6–19% mortality rate with half of the survivors experiencing moderate to severe chronic neurological deficits. For VZV, current treatments are inadequate to prevent acute and persistent pain due to zoster. Treatment of HCMV with GCV requires close monitoring particularly in patients with impaired renal function and there are no approved treatments for congenital HCMV infections. New therapeutic options to control cytomegalovirus reactivation in bone marrow and stem cell transplant patients are needed to improve patient outcome. No successful chemotherapeutic options are available for EBV, HHV-6, 7, and 8. Drug resistance is a concern for HCMV, HSV, and VZV since approved drugs share common mechanisms of action. Targeting DNA encapsidation or capsid assembly provide additional options for the development of non-nucleoside, small molecule anti-herpesviral drugs. Herpesviridae) are classified as either alpha, beta or gamma herpesviruses depending on their genetic relatedness, host range, replication cycle and latency properties  or genital lesions. Worldwide, 3.7 billion people under age 50 (67%) and 417 million people aged 15–49 (11%) are estimated to be infected with HSV-1 and HSV-2, respectively ,b. GenitVaricellovirus contains the species human alphaherpesvirus 3, commonly known as varicella zoster virus (VZV or HHV-3). VZV infection typically occurs at a young age resulting in varicella (chickenpox). Varicella occurs primarily in unvaccinated children and young adults and can lead to complications such as encephalitis, pneumonia, or bronchitis  and human herpesviruses type 6A (HHV-6A), 6B (HHV-6B), and 7 (HHV-7). Shared characteristics include infection and establishment of latency in lymphocytes and monocytes/macrophages. Like the other herpesviruses, most people are infected with the Betaherpesvirinae by adulthood, and many do not present with symptoms. Primary infections are typically asymptomatic in immunocompetent individuals, however, HCMV infection during pregnancy can significantly impair fetal development (Within the elopment . HCMV-aselopment ]. Congenelopment . Howeverelopment . Currentelopment . Solid oelopment . The recelopment .The gammaherpesviruses include Epstein-Barr virus (EBV) and HHV-8. Infectious mononucleosis and Burkitt’s lymphoma result from infection of B-cells with EBV . The prein vitro activity against HCMV with novel mechanism of action. The compound was active against laboratory and clinical isolates (EC50 ∼4 nM with CC50 90 μM). Due to letermovir’s distinct mechanism of action, cross resistance to cidofovir, foscarnet and/or ganciclovir was absent.Toxicity is an issue among approved HCMV therapies including ganciclovir (GCV), foscarnet and cidofovir. These drugs have complex administration and monitoring requirements and are associated with several toxicities including myelosuppression. For example, GCV, the preferred drug of the three is not recommended for patients when the absolute neutrophil count is under 500 cells/ml or when the platelet count is under 25,000/ml . HematolIn murine studies of HCMV infected xenografts, AIC246 had favorable toxicological and pharmacological profiles and proved more effective against HCMV than valganciclovir . Phase Iin vitro for GCV and letermovir showed that mutations conferring resistance to letermovir developed more rapidly than gangciclovir  but no in vivo studies were reported for the compound series. The VZV compound series had excellent in vitro activity (IC50 10 nM – 1 μM), showed no cellular cytotoxicity (CC50 > 37 μM) and was effective against a panel of clinical isolates and ACV-resistant VZV strains. Only empty capsids were observed in the nuclei of VZV infected cells In the presence of compound.In 2000, the thiourea compound its N--thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative . Below, we provide the first on model . The resin vitro, was evaluated in a SCID-Hu thymus/liver VZV infection model (p = 0.0037). The treatment phase was extended beyond the typical 7 days to observe any potential side effects of the compound. Only the vehicle group lost weight whereas the compound treatment groups showed positive weight gain and the mice appeared well-hydrated with smooth fur indicating that Comp 1 was well-tolerated. Previous studies showed that compounds from the α-methylbenzyl series were not toxic in multiple cell lines  evaluates the animal care and use program at SUNY Upstate Medical University and facilitates the conduct of meaningful scientific research that: (1) avoids or minimizes discomfort, distress, and pain in experimental animals consistent with sound scientific practices, (2) uses the minimum number of animals necessary to obtain valid results, and (3) considers non-animal models wherever possible. OLAW PHS Assurance: D16-00318; USDA Certificate: 21-R-0037; NYSDOH Unit: A073; AAALAC: Full Accreditation (accredited since 7/31/1999).TK, JM, and RV wrote the manuscript. JM and RV designed the experiments and analyzed the data. DL, ML, WC, and JM performed the animal studies. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diffusion-weighted magnetic resonance imaging (DW-MRI) potentially interrogates site-specific response to neoadjuvant chemotherapy (NAC) in epithelial ovarian cancer (EOC).n = 47 participants). Apparent diffusion coefficient (ADC) and solid tumour volume (up to 10 lesions per participant) were obtained from DW-MRI before and after NAC (including double-baseline for repeatability assessment in n = 19). Anatomically matched lesions were analysed after surgical excision (65 lesions obtained from 25 participants). A trained algorithm determined tumour cell fraction, percentage tumour and percentage necrosis on histology. Whole-lesion post-NAC ADC and pre/post-NAC ADC changes were compared with histological metrics  for each tumour site .Participants with newly diagnosed EOC due for platinum-based chemotherapy and interval debulking surgery were recruited prospectively in a multicentre study  increases differentially at disease sites despite similar tumour shrinkage, making its utility site-specific. After NAC, ADC correlates negatively with tumour cell fraction; change in ADC correlates positively with percentage necrosis.ClinicalTrials.gov NCT01505829. Also, digital analysis of the extent of necrosis13 has not been widely available. In this study, we aimed to measure the change in ADC metrics in EOC treated with platinum-based chemotherapy in a quality-assured/controlled multicentre trial,14 compare the changes in ADC between anatomic disease sites and relate them to histological measures of response  as assessed by digital pathology.Traditionally, response in EOC has been assessed with unidimensional size measurements, which have been shown to be robust across tumour types and observer assessments.14). Participants were enrolled at four hospitals . All participants gave written informed consent. Inclusion criteria were histology/cytology-confirmed high-grade serous, endometrioid or clear cell histology, at least one solid mass >2 cm in long axis on CT or MRI and scheduled for platinum-based NAC with IDS after three or four cycles. Exclusion criteria were abdomino-pelvic radiotherapy within six months of screening, contra-indications to MRI, or receipt of an investigational compound or device within 30 days of starting treatment.Participants with newly diagnosed stage III or IV ovarian, fallopian tube or primary peritoneal cancer were recruited prospectively in a multicentre trial with multicentre research ethics committee approval  MRI examinations before starting chemotherapy. Two pre-NAC MRI examinations were conducted up to 7 days apart if participants were able to tolerate both examinations; only one pre-NAC examination was conducted if participants were unable to tolerate the second pre-NAC examination. Post-NAC MRI examinations were conducted after three or four cycles of chemotherapy within 8 days prior to IDS.1-weighted, and T2-weighted imaging standardised between centres  covered the abdomen and pelvis in three stations . For each examination, regions-of-interest (ROIs) encompassing the whole solid lesion on all slices were drawn by region growing on computed b = 1000 smm−2 images16 by JCW (2 years’ experience with pelvic MRI) and checked by NMdS (20 years’ experience). Cystic areas were excluded by visual matching with T2-weighted images. Up to five target and five nontarget largest lesions per participant were analysed. Lesions were selected on pre-NAC MRI examinations and the same lesions were followed-up on post-NAC MRI examinations. ADCs were estimated by mono-exponential fitting of signal intensity at b-values 100, 500 and 900 smm−2. The median ADC, 25th and 75th centile  were estimated from all fitted voxels in the ROIs for each lesion. The volume of each solid lesion was obtained by multiplying the number of voxels in the ROIs by voxel volume (range 0.013–0.016 cm3). For each lesion, the change in solid tumour volume and ADC after three or four cycles of NAC was expressed as percentage change from pre-NAC measurements.Images were analysed at the lead centre using in-house software . Intermediate signal intensity masses on TAnatomical localisation diagrams provided to participating surgeons with detailed annotations on radiologist-selected imaged target lesion location enabled matching of lesions with those identified at surgery. These matched lesions were marked with sutures at excision to identify them to the pathologist.Formalin fixed tissue specimens were sectioned at three to four-millimetre intervals, embedded in paraffin and 2–3-micron sections mounted on glass slides. Haematoxylin and eosin (H&E) stained sections were reviewed by two gynaecological-oncology histopathologists in consensus . From each selected lesion, after review of the entire lesion, they chose a single index slide that most closely represented the residual viable tumour and necrosis across the whole lesion.17 The proportion of viable tumour cells to total cells in the sample (tumour cell fraction) was recorded. Areas of viable tumour and necrosis outlined by AS on 20 slides were used to train a modified algorithm (MicroNet) to segment tumour and necrosis regions on the whole study sample.18 Algorithm training was deemed acceptable on achieving 90% validation accuracy. A pretrained H&E tissue segmentation algorithm removed background noise and artefacts.17 The ratios of segmented tumour or necrosis area to the whole-slide segmented tissue area were recorded as %residual tumour and %necrosis, respectively.Whole H&E stained slides were digitised to a resolution of 0.26 µm per pixel . An algorithm previously trained to 92.61% accuracy on a lung model was used to identify tumour cells, differentiating them from stromal, lymphocytes and other cells such as macrophages.P values < .05 were considered statistically significant. Median, lower and upper quartiles were used to summarise imaging and histology parameters.Statistical analysis (NP) used commercially available software  and GraphPad Prism for Windows, . 19 were used to assess repeatability of solid tumour volume and ADCmedian for each disease site .95% limits of agreement (LoA)Probability density functions for voxel-wise ADC estimates were determined for pre-NAC and post-NAC measurements; the first pre-NAC examination was used in those with two examinations . Analysis was done on a per lesion basis rather than a cumulative voxel analysis to remove bias towards larger volumes, and the sum of probability density estimates calculated for each anatomic location.median before neoadjuvant chemotherapy (pre-NAC) between lesions that remained measurable after neoadjuvant chemotherapy (post-NAC), and those that became nonmeasurable were compared using linear mixed-effects regression models to each pre-NAC parameter, including status of lesion (measurable/nonmeasurable) as a fixed effect and per-participant random intercept effects to account for clustering within participants. For those lesions that remained measurable post-NAC, further linear mixed models were used to compare percentage change between pre- and post-NAC in solid lesion volume and ADCmedian across disease sites (fixed effect), adjusting by baseline pre-NAC values and including per-participant random intercept. Models were fitted to logarithm-transformed data when normality assumption did not hold . Pairwise comparisons between disease sites are presented with adjusted differences and p-values corrected for multiplicity by Bonferroni.The ADCmedian and tumour cell fraction; (ii) post-NAC preoperative ADCmedian and %residual tumour; (iii) pre/post-NAC change in ADCmedian, ADC25 and ADC75 and %necrosis were assessed using Spearman’s correlation.The relationships between (i) post-NAC preoperative ADCFifty-two participants were enrolled. All participants were newly diagnosed and chemo naïve. Five participants were excluded, leaving 47 participants  57–70 years) with pre-NAC DW-MRI . 28 of 40 ovarian, 35 of 50 peritoneal, 19 of 27 omental and 14 of 22 lymph node lesions reduced in volume below the lower LoA.Site-specific repeatability for solid tumour volume was assessed in 123 lesions  from 19 participants. 95% LoA were −19.2 to 17.9 cmmedian were −10 to 9 × 10−5 mm2s−1 for solid elements of ovary, −13 to 16 × 10−5 mm2s−1 for peritoneum, −17 to 17 × 10−5 mm2s−1 for omentum and −27 to 21 × 10−5 mm2s−1 for lymph nodes , and between omental lesions and lymph node lesions , but no differences between peritoneal and omental  or ovarian lesions , nor between ovarian and nodal lesions  or ovarian and omental . 28 of 40 ovarian, 24 of 50 peritoneal, 8 of 27 omental and 17 of 22 lymph node lesions increased in ADCmedian above the upper LoA. The pre-NAC ADCmedian of lesions that became nonmeasurable on post-NAC scans was not significantly different from those that remained measurable , peritoneal , omental  and lymph nodes .Site-specific 95% LoA for ADCIn total, 99 sections  from 93 lesions in 25 participants were assessed on digital pathology . When considered by disease site, this held true for the peritoneum  only. The change in ADCmedian for all sites considered together showed positive correlation with %necrosis  for the peritoneum . Illustration  from 65 lesions obtained from 25 participants . Table median measurement is a clinically useful method of detecting the presence or absence of viable tumour for peritoneal deposits, but not at other sites. This is useful in assessing relapsed disease, which is predominantly peritoneal. These multicentre findings also confirm pilot data where a negative correlation between epithelial cell density and diffusivity was demonstrated in 15 lesions excised at IDS in participants with primary ovarian or peritoneal cancer,4 and in 24 participants with prostate cancer.20 In orthotopic preclinical models of solid ovarian tumours, the change in ADC following docetaxel also was shown to correlate negatively with Ki67, CA125 and Bcl-2, all of which predict residual tumour burden.21This study shows that in EOC/fallopian tube/primary peritoneal cancer lesions responding to NAC, there is a differential increase in ADC by anatomic site of the lesion despite a similar volume reduction of >80%. This study also confirms a site-specific correlation between the ADC changes and histological metrics . Thus, post-NAC preoperative ADC23 the literature is inconclusive about the effect of stroma, fibrosis and inflammatory infiltrate on ADC values indicating that they are influenced by a complex interplay of biophysical processes.25 In a tissue comprised primarily of fat, such as the omentum, the return of normal fatty stroma interspersed with residual tumour may obscure a post-treatment ADC rise. This phenomenon is well-described in adult bone marrow.26 This underlines the important contribution of the surrounding normal tissue to the ADC and is borne out by the omental data in this study, where ADC increases in responding lesions were lower than other sites. The differences between ADC response between peritoneal deposits and lymph nodes is of interest, as both are densely cellular tissues. It may well be that swelling and inflammatory response in lymph nodes causes a much greater ADC rise than in the fibrotic peritoneum, where inflammation and oedema is less. This requires further investigation. The study was not powered to assess the relationship of site-specific ADC change with progression-free survival (PFS) in these treatment-naïve patients, although we have previously shown that an increase in ADC of peritoneal lesions after one cycle of chemotherapy indicates improved PFS in relapsed disease.14Although negative correlation between cell density and diffusivity is widely accepted,28 where abnormal nodal architecture results in variable increases in ADC; imaging-pathology studies have not been conducted as surgery is not part of the patient management. In other cancer types involving lymph nodes, treatment is usually chemoradiotherapy, where radiotherapy influences ADC rises30 due to early acute tissue injury and swelling,31 and cannot be easily compared to our findings.Only six lymph node lesions were analysable for imaging-pathology correlations, which limits imaging-pathology comparisons for lymph nodes alone, but we have included these for completeness in reporting this study. ADC changes in lymph nodes following chemotherapy have been reported in lymphomas,21 A multicentre clinical trial in lung cancer concluded that presurgical ADC or change in ADC did not correlate with pathologist-assessed necrosis of resection specimens but assessment was dependent on a single pathologist reading.32 Clinical evidence relating ADC change following chemotherapy to subsequent necrosis may be confounded by pre-existing microscopic necrosis within the tumour; ADC change has been linked to necrosis in tumour types without much pre-existing necrosis, but not in others.33ADC increases post-treatment have been shown to correlate with percentage necrosis in preclinical studies.34 To minimise error, this was done by an experienced specialist gynaecological pathologist at a national cancer centre. However, selection of a single histological slide per lesion also represented a fourth limitation. Although analysis of the entire lesion may be ideal, it was impractical to digitise and analyse large numbers of sections in each lesion. Also, the resource to do this was limited and could not be accommodated.Our study has several limitations. First, lesions that became unmeasurable, and therefore showed the greatest response, did not contribute to the ADC measurements. Second, lesions that could not be matched between pre-IDS MRI and histology samples were excluded, which reduced the sample size. Moreover, matching between the imaging and histology was done on the larger resectable lesions that were easily identified at surgery; smaller lesions, which may represent a bigger response, were not available for histological correlation, possibly introducing bias towards more slowly responding lesions. The selection bias was minimised by choosing up to ten lesions per participant on the baseline MRI from different anatomical sites that were representative of the participant’s disease. Third, the direction of pathological sectioning did not always exactly match the axial imaging plane. We addressed this by selecting a histological slice that most accurately represented the proportions of residual tumour and necrosis within each lesion, but this depended on detailed pathologist review, not 3D molds.In conclusion, in this relatively small study in EOC, the ADC repeatability and extent of increase following treatment is anatomic site dependent. The post-NAC preoperative ADC and change in ADC is an indicator of residual viable tumour and percentage of necrosis, respectively, primarily within peritoneal deposits. When using ADC as a response indicator in EOC lesions, therefore, consideration must be given to the ADC increase compared with measurement repeatability and to the anatomic site.Supplementary Material


The most common cause of ascites is liver cirrhosis. Additional causes such as heart failure, cancer, and pancreatitis among others can also precipitate this abnormality. Spontaneous bacterial peritonitis (SBP) is an infection of ascitic fluid that happens without any evidence of an intra-abdominal surgically-treatable cause. Ascites of cardiac origin can also be complicated by SBP. Here we present a case of a 62-year-old male with extensive cardiac history who presented to our service with ongoing dyspnea and orthopnea. He also had significant abdominal distention and pitting edema. The patient was found to have constrictive pericarditis and was admitted for pericardiectomy. Ascitic fluid was consistent with a transudative process. Lab and imaging did not show evidence of liver or kidney disease. Ascitic fluid was indicative of ascites of cardiac origin. Postoperatively patient developed intermittent fevers initially thought to be due to pericarditis but later found to be due to SBP complicating his recurrent ascites. Such a temporal association of SBP that complicates ascites after pericardiectomy has not been discussed frequently in literature. Ascites is defined as a collection of more than 25 mL of fluid in the peritoneal cavity. The most common cause is liver cirrhosis. Additional causes include heart failure, cancer, pancreatitis, tuberculosis, and hepatic vein obstruction . Of thesThe patient was a 62-year-old male with a past medical history of coronary artery disease, chronic heart failure, paroxysmal atrial fibrillation on warfarin, recurrent pericarditis, idiopathic constrictive pericarditis, and chronic obstructive pulmonary disease (COPD) that presented to us for evaluation for pericardiectomy. He was asymptomatic on arrival, though he previously had shortness of breath and orthopnea that resolved with outpatient diuretic treatment just before admission. He denied fever, chest pain, further shortness of breathing, or cough. Physical exam was significant for an irregular heart rate, significant lower extremity pitting edema, and abdominal distension with fluid wave consistent with ascites. Consequently, he was admitted to internal medicine service, and cardiothoracic surgery was consulted. Laboratory studies revealed a hemoglobin/hematocrit of 10.8/32 and total iron binding capacity (TIBC) suggestive of anemia of chronic inflammation, and mild elevation of aspartate aminotransferase (AST) at 53 (Ref <41 U/L) with all other liver enzymes within the normal range.Preoperative testing focused on assessing the possibility of hepatic cirrhosis as an etiology of his ascites, and confirmation of his constrictive pericarditis. Preoperative paracentesis fluid analysis revealed fluid albumin of 2.0 g/dL, a fluid protein of 4.3 g/dL, absolute neutrophil count (ANC) of 13 /cmm, and the serum ascites albumin gradient (SAAG) of 1.4 g/dL . These results revealed a transudative etiology consistent with cardiogenic ascites from heart failure described by SAAG >1.1 and ascitic fluid protein >2.5 g/dL (ascitic fluid protein in cirrhosis is <2.5 g/dL). Subsequent ultrasound of the liver revealed a homogenous liver without nodularity or evidence of cirrhosis. Preoperative right heart catheterization demonstrated features consistent with constrictive pericarditis, including Kussmaul’s sign , a “square root sign” on the left ventricular (LV) diastolic tracing, and equalization of LV and right ventricular (RV) diastolic pressures. He then underwent a successful pericardiectomy.Perioperatively patient developed intermittent fevers ranging from 100 to 101.2 that resolved without any intervention. These fevers were initially attributed to pericarditis. The patient had no leukocytosis and cultures were negative. Postoperatively, the patient developed recurrent ascites requiring a repeat diagnostic/therapeutic paracentesis. Albumin, fluid was 1.3 g/dL, protein, fluid was 3.0 g/dL, serum albumin was 2.1 and ANC was 499 /cmm. Doppler ultrasound did not show any evidence of portal or hepatic vein thrombosis. These findings were consistent with spontaneous bacterial peritonitis and the patient was treated empirically with a five-day course of IV ceftriaxone. The patient remained afebrile and asymptomatic throughout three days of antibiotic therapy, so another paracentesis was performed on day three. This revealed a decrease in ANC to 132 /cmm, demonstrating a therapeutic response. Ascitic fluid cultures were negative for aerobic or anaerobic bacteria, likely due to sterilization. The patient completed his five-day course of antibiotics and did not reaccumulate further ascitic fluid while on furosemide and spironolactone. He was subsequently discharged to a rehabilitation facility.SBP is a common and life-threatening infection of ascitic fluid first described by Krencker in 1907  and furtSBP is most often seen in patients with liver cirrhosis, though rare reports have described SBP occurring secondary to cardiogenic ascites -11, renaCardiogenic ascites is an uncommon cause of ascites, arising secondary to congestive hepatopathy. Bacterial infection of this fluid is exceedingly uncommon but may occur due to changes arising from intestinal hypoperfusion exacerbated by hemodynamic instability. To our knowledge, this case of SBP of cardiogenic ascites following pericardiectomy in a patient with a history of chronic constrictive pericarditis is the eighth such reported case of SBP in literature. Careful diagnosis with a high clinical suspicion is essential for the timely management of this potentially fatal entity.


Here, we report a rare case of recurrent GCT of the thoracic vertebra, which was managed by three-level total A 50-year-old woman presented with a 2-month history of progressive lower back pain. Magnetic resonance imaging revealed destruction of the T11 vertebra and a soft tissue mass. The patient underwent tumour resection. Computed tomography at the 2-year follow-up revealed relapse of the resected tumour, which had spread to the T12 vertebral body. Subsequently, denosumab therapy was administered to the patient for 1 year. The growth of the tumour was controlled, and its boundary line was clear. Thereafter, TES for the T10-T12 vertebrae was performed, and spinal reconstruction was completed through a one-stage single posterior approach. The patient’s condition improved postoperatively, and no evidence of recurrence of GCT of the bone or spinal deformity was observed at the 32-month follow-up.Denosumab therapy contributed to tumour regression. Three-level TES may be an effective and feasible strategy for managing large recurrent GCTs of the spine after denosumab therapy. Giant cell tumour (GCT) of the bone is a rare, invasive benign bone tumour, accounting for approximately 5% of primary bone tumours. It typically originates in the metaphyseal ends of long bones and rarely in the spine –3. Approen bloc spondylectomy (TES) generally reduces local tumour recurrence and is currently a widely accepted surgical procedure for spinal tumours [Surgical treatment is the foundational treatment strategy for spinal GCT of the bone with the aim of preserving functionality, relieving pain, controlling local recurrence, and promoting prolonged survival . Althoug tumours , 10.Spinal GCT has a high recurrence rate of approximately 25–50% after surgical treatment. Therefore, reducing recurrence is the key to treatment , 11. DenA 50-year-old woman was admitted to our hospital in June 2013 owing to back pain radiating to the lower left abdomen for 2 months with progressive exacerbation. Physical examination revealed a tender point on the back, paraparesis with motor strength of 4/5 in both lower limbs, and decreased left inferior abdominal wall reflex. Magnetic resonance imaging (MRI) revealed destruction of the T11 vertebra and a soft tissue mass. To prevent rapid neurological deterioration owing to tumour growth, local curettage was planned using the posterior approach. However, pathologic examination of the neoplastic specimen using instant frozen section showed that the tumour was more likely to be malignant; thus, the involved vertebral body and upper and lower intervertebral discs were completely resected. Thereafter, spinal reconstruction was performed with a screw system and titanium mesh.Follow-up computed tomography (CT) showed tumour recurrence at 14 months after surgery , the patient had recovered well and could work normally. Moreover, no local tumour recurrence was observed on MRI and radiography, and three-dimensional CT showed successful biological reconstruction of the spine Fig. a-d.Fig.GCT was first described by Cooper and Travers in 1818, and it mainly occurs in the femur, tibia, and radius, accounting for 55% of the lesions, but rarely originates from the vertebra above the sacrum . Spinal GCTs of the spine are reported to be expansile lytic lesions, with pain caused by a stretched periosteum being the most common manifestation, followed by pathological fracture (41%) and neurologic symptoms 32%) –17. Diag2% –17. DHowever, most of these tumours are benign, and only a small number of GCTs 1–2%) may undergo malignant transformation, leading to a poor prognosis. According to the previous reports, GCTs can transform into fibrosarcoma, osteosarcoma, malignant fibrous histiocytoma, undifferentiated high-grade pleomorphic sarcoma, and undifferentiated sarcoma , 18, 19.% may unden bloc resection are the most commonly used surgical methods; the former causes minor trauma with a high recurrence rate (27–65%), while the latter causes major trauma, often resulting in permanent nerve injury, with a low recurrence rate (0–12%) [en bloc resection of GCT of the spine was technically feasible, this recommendation was based on some very low-quality evidence and consensus among some experts [Owing to the complexity of the spinal anatomy, the treatment of GCT of the spine has become a huge challenge. Intralesional curettage and  0–12%) , 7, 12. % , 7, 12 experts . The metHistologically, GCT of the bone contains osteoclast-like giant cells that express RANK and stromal cells that express RANKL, a key mediator of osteoclast formation, activation, function, and survival. Excessive secretion of RANKL causes an imbalance in bone remodelling in favour of bone breakdown . DenosumIn conclusion, the present report highlights a rare case of a large recurrent GCT in the thoracic spine, which was managed using three-level TES and denosumab therapy. Denosumab therapy contributes to tumour regression. TES may be an effective and feasible strategy for managing huge recurrent GCTs of the spine after denosumab therapy.


Recent studies suggest that host innate immunity is regulated by the enzymes controlling m6A epitranscriptomic changes. Here, we aim to explore the associations between the levels of METTL3 and CD33+ myeloid-derived suppressor cells (MDSCs) in tumour tissues and the survival of patients with cervical cancer (CC).Methyltransferase-like 3 (METTL3) is a member of the m+ cells or CC-derived cells was knocked down by METTL3-specific siRNA, and MDSC induction in vitro was performed in a co-culture system in the presence of METTL3-siRNA and METTL3-knockdown-CC-derived cells compared with that of the corresponding controls.Specimens of paraffin embedded tumour from 197 CC patients were collected. The expression levels of METTL3 and CD33 were measured by immunohistochemical (IHC) staining. The clinical associations of the IHC variants were analysed by Pearson’s or Spearman’s chi-square tests. Overall survival (OS) and disease-free survival (DFS) were estimated by the Kaplan–Meier method and log-rank test. Hazard ratios (HRs) and independent significance were obtained via Cox proportional hazards models for multivariate analyses. METTL3 in CD33+ MDSCs compared with tumour-adjacent tissues from the same CC patients. Importantly, METTL3 expression was positively related to the density of CD33+ cells in tumour tissues (P = 0.011). We further found that the direct CD33+CD11b+HLA-DR− MDSC induction and tumour-derived MDSC induction in vitro were decreased in the absence of METTL3. The level of METTL3 in tumour microenvironments was significantly related to advanced tumour stage. The levels of METTL3 and CD33+ MDSCs in tumour tissues were notably associated with reduced DFS or OS. Cox model analysis revealed that the level of METTL3 in tumour cells was an independent factor for patient survival, specifically for DFS  and OS , while the CD33+ MDSC number was an independent predictor for DFS . Interestingly, in patients with advanced-disease stages (II–IV), METTL3 in tumour cells was an independent factor for DFS  and OS , while CD33+ MDSC density was an independent factor for OS .We found that tumour tissues displayed increased levels of METTL3 and CD33+ MDSC expansion is linked to high levels of METTL3 and that METTL3 and CD33+ MDSCs are independent prognostic factors in CC.Our findings suggest that CD33 Cervical cancer (CC) is one of the most common tumours, ranking fourth for both incidence and mortality in women worldwide –3. CC is6-methyladenosine (m6A) RNA methylation, which is the most abundant modification ubiquitously occurring in eukaryotic mRNAs  and OS . In the patients with early-stage disease, we found that the combination of high METTL3 levels and intratumoural CD33+ MDSCs was negatively correlated with OS , but there was no significant association with DFS. Importantly, we found that the combination of high tumour METTL3 levels and intratumoural CD33+ MDSCs was significantly correlated with poor DFS  and OS  in patients with advanced disease stages ; after the methylation of m6A, RNAs will nucleate and transport to the cytoplasm faster and then produce more proteins for function and proliferation. Some studies have shown that METTL3 expression can promote tumour cell proliferation, leading to poor patient prognosis. The tumour-infiltrated MDSC population usually induces antitumour immunity tolerance by inhibiting the proliferation and function of T cells, such as hindering antigen presentation by antigen-presenting cells [+ MDSCs have been found in tumour microenvironments and lead to a poor prognosis [+ MDSCs in the tumour microenvironment of 197 patients with CC. The positive association between METTL3 levels and CD33+ MDSCs and the prognostic value of these two variants in CC patients were demonstrated. Importantly, we demonstrated that knockdown of METTL3 in CD33+ cells or HeLa cells could attenuate MDSC or tumour-associated MDSC differentiation in vitro.The development of tumour cells depends on the tumour microenvironment, which includes tumour cells, various other cells and extracellular components . The immng cells . Increasrognosis –40. In t6A methyltransferases, especially METTL3, can affect many physiological and pathological diseases through p53 and other genes [6A methyltransferase significantly affects gene expression and mRNA splicing patterns, leading to changes in normal cell signalling pathways and apoptosis [6A-modified direct targets IKBKB and RELA (two key regulators of the NF-κB pathway) mediated by METTL3 become factors that promote tumour development [6A-enriched mRNA and alter the mRNA expression of genes with key biological functions in GSCs (such as ADAM1937) [+ MDSC density. The results indicate that METTL3 could directly induce CD33+CD11b + HLA-DR− MDSC differentiation or tumour-associated MDSC differentiation in vitro. Moreover, both METTL3 and CD33+ MDSCs were independent factors for the prognosis of CC patients, and the combination of METTL3 levels and CD33+ MDSC density displayed prognostic value for CC patients, including patients at early or late disease stages. The function, distribution and clinical relevance of the proportion of tumour-derived CD33+ MDSCs have been explored in recent years. MDSCs are generally elevated in tumour tissues and in the peripheral blood of cancer patients and are linked to antitumour immunity suppression, resulting in tumour growth and metastasis [6A RNA modification is positively associated with the increase in MDSC expansion and affects tumour development and prognosis in CC and induces CD33 + cells to differentiate into MDSCs in the tumour microenvironment. We further demonstrate the prognostic value of the combination of the METTL3 level in tumour-infiltrating immune cells and CD33+ MDSC density in CC patients, especially for those in advanced disease stages. A mechanistic study to support the role of METTL3 in the regulation of tumour-derived MDSC differentiation is currently underway, and the underlying mechanisms will be clarified in the near future.Mer genes . At the poptosis . In bladelopment ; in glioDAM1937) . In receDAM1937) , 42. OurDAM1937) . Therefotastasis , 34, 44.tastasis . Here, oOur study demonstrated a comprehensive result of the relationship between METTL3 and CD33 in CC and revealed that METTL3 could induce direct MDSC and tumour-associated MDSC differentiation in vitro. The results showed that both biomarkers were adverse indicators for prognosis and may have significant relationships in the microenvironment of CC. Our research may offer clues for further research into the mechanism behind METTL3 in the regulation of MDSC-mediated immune suppression in the CC microenvironment.Additional file 1: Figure S1. The DFS and OS curves of 197 CC patients, and expression of METTL3 and CD33 in tumours or in tumour-adjacent tissues. (A and B) Disease-free survival  and overall survival  curves of 197 CC patients in this study. (C and D) Statistical analysis showing the comparison between the expression of METTL3  and the number of CD33+ MDSCs  in tumour-adjacent tissues (Non-tumour) and tumour tissues (Tumour). (E and F) The association between METTL3 expression in tumour cells and the expression of METTL3 in TILs in early stage  or in advanced stage . (G and H) The association between METTL3 expression in tumour cells and intratumoural CD33+ MDSC number in early stage  or in advanced stage . (I and J) The association between METTL3 expression in TILs and intratumoural CD33+ MDSC number in early stage  or in advanced stage . Statistics were conducted with a paired Student’s t test in C and D. The correlation statistical analysis was performed using Spearman’s correlation and linear regression. R, Spearman’s correlation, is the correlation coefficient.Additional file 2: Figure S2. Kaplan–Meier curves of DFS and OS according to METTL3 expression in different cell populations and intratumoural CD33+ cells of early-stage patients. (A and B) Kaplan–Meier curves showing the relationship of DFS  and OS  of CC patients and METTL3 expression in tumour cells. (C and D) Kaplan–Meier curves showing the relationship of DFS  and OS  of patients and METTL3 expression in TILs. (E and F) Kaplan–Meier curves showing the relationship of DFS  and OS  of CC patients and intratumoural CD33+ MDSC density. The percentages of DFS and OS were calculated by the Kaplan–Meier method, and P values were calculated by the log-rank statistic. Cut-off selection was based on X-tile.


Ameloblastomas are epithelial odontogenic tumours that, although benign, are locally invasive and may exhibit aggressive behaviour. In the tumour microenvironment, the concentration of oxygen is reduced, which leads to intratumoral hypoxia. Under hypoxia, the crosstalk between the HIF-1α, MMP-2, VEGF, and VEGFR-2 proteins has been associated with hypoxia-induced angiogenesis, leading to tumour progression and increased invasiveness. This work showcases 24 ameloblastoma cases, 10 calcifying odontogenic cysts, and 9 dental follicles, used to investigate the expression of these proteins by immunohistochemistry. The anti-HIF-1α, anti-MMP-2, anti-VEGF, and anti-VEGFR-2 primary antibodies are used in this work. The results have been expressed by the mean grey value after immunostaining in images acquired with an objective of 40×. The ameloblastoma samples showed higher immunoexpression of HIF-1α, MMP-2, VEGF, and VEGFR-2 when compared to the dental follicles and calcifying odontogenic cysts. Ameloblastomas show a higher degree of expression of proteins associated with intratumoral hypoxia and proangiogenic proteins, which indicates the possible role of these proteins in the biological behaviour of this tumour. Odontogenic cysts and tumours comprise a large part of oral and maxillofacial pathologies, and, therefore, there is a need to explore their biological behaviour, deepening knowledge about their development and progression.1. Although surgery is the most acceptable treatment modality so far, it produces severe aesthetic and functional sequelae that lead to a loss of life quality2.Ameloblastomas (AMEs) are one of the most common odontogenic tumours. They are characterized by a slow growth, high recurrence and morbidity rates, and local invasiveness3.A calcifying odontogenic cyst (COC) is an odontogenic cystic lesion that has a less aggressive behaviour than an AME. The prognosis of a patient with COC is favourable, with few recurrences reported after simple enucleation5.During tumour progression, the concentration of oxygen in the microenvironment around the tumour cells is reduced, resulting in intratumoral hypoxia, characterized by reduced oxygen pressure in the cells, which leads to various biochemical responses and may result in a number of compensatory cellular mechanisms that allow the continuation of neoplastic development. A hypoxic microenvironment is characteristic of many solid tumours. Hypoxia is also associated with a more aggressive phenotype, which affects angiogenesis and cellular invasiveness9, which stimulate the release of angiogenic and growth factors in the basement membrane, such as the vascular endothelial growth factor (VEGF/VEGFA)10.Angiogenesis and cellular invasion are complex multistage processes that begin with extracellular matrix (ECM) degradation caused by proteolytic enzymes, matrix metalloproteinases (MMPs), and, especially, cancer-associated MMP-2 and -915. Among VEGF receptors (VEGFRs), VEGFR-2 is overexpressed under hypoxia15. VEGFR-2 plays an important role in activating the components responsible for proliferation, including endothelial cell invasion, migration, differentiation, and angiogenesis16.The signalling pathway involving VEGF/VEGFA and VEGFR is a promising target for cancer treatment, as it has been identified as the main regulator of tumour angiogenesis19. Three studies have verified the expression of VEGF and found a higher expression of this protein in AME when compared to odontogenic keratocyst, dentigerous cyst or tooth germ, suggesting that the up-regulation of this protein in AME might be associated with neoplastic or malignant changes of odontogenic epithelial cells22. There are no studies on the expression of VEGFR-2 in these pathologies or on the relationship between all these proteins.Some of these proteins have already been studied in AME, COC and DF. Three studies verified the expression of HIF-1 α and found a higher expression of this protein in AME when compared to COC and DF, and suggested that may be associated with its aggressive biological behaviour31. In addition, HIF-1 may act as a direct transcriptional activator for the VEGF gene promoter33.Studies in skin, breast, gastric, brain, salivary gland, and pulmonary cancer have shown the role of these proteins in promoting tumour progression, where the hypoxia-inducible factor 1 alpha (HIF-1α) plays a regulatory role. When HIF-1α expression is stabilized, through hypoxia, it can induce MMP-2 expression, which degrades ECM, stimulating the expression of growth factors such as VEGF, which binds to its receptor VEGFR-2, initiating a series of processes, including angiogenesis, proliferation, and invasion, which will contribute to tumour growthTherefore, the objective of this study was to analyse the expression of these proteins in AME, as this biological approach may be useful for better understanding the behaviour of this neoplasm.All AME samples expressed HIF-1α, MMP-2, VEGF/VEGFA, and VEGFR-2. There was a strong immunoexpression of HIF-1α, predominantly in the nucleus of tumour cells Fig. A,B. VEGFThe COC samples showed low or no immunoexpression of HIF-1α Fig. E,F, VEGFThere was a statistically significant difference in the immunoexpression of HIF-1α, MMP-2, VEGF/VEGFA, and VEGFR-2 proteins between the parenchyma of tumour cells and the stroma of tumour cells Fig. A–D, wherIt was also observed, significant difference in the immunoexpression of HIF-1α, VEGF/VEGFA, and VEGFR-2 between AME and COC samples and between AME and DF samples, where the expression of these proteins was higher in AME than in COC and DF Fig. A,B,D, an34. Solid tumours often contain regions with low oxygen concentrations around tumour cells, due to insufficient blood supply, resulting in intratumoral hypoxia5. Hypoxia-induced factor 1 (HIF-1) is a transcriptional activator responsible for the regulation of elements responsive to this phenomenon. In a hypoxic condition, HIF-1α binds with the HIF-1β subunit, becoming active and migrating to the cell nucleus in order to regulate cell survival mechanisms35, this corroborates with our findings, suggesting that the HIF-1α found was active. Its activation plays an essential role in the invasive process, being fundamental for tumour growth and aggressiveness, with overexpression observed in many human tumours38. Immunohistochemistry studies have observed the overexpression of HIF-1α in AME and have discovered that this is associated with its aggressive biological behaviour19.In our study, AME samples presented strong HIF-1α immunoexpression, predominantly in the nuclei of tumour parenchyma cells, where the transcription factor is active and triggers the transcription of several genes, initiating several mechanisms, such as angiogenesis, cell proliferation, and tumour invasion39. Pinheiro et al.39 also observed, through immunohistochemistry, the cytoplasmic expression of MMP-2 in the tumour parenchyma of AMEs and implied the high expression of this protein with the most aggressive infiltrative behaviour of this neoplasm.Our results show a strong cytoplasmic immunostaining of MMP-2 in the central cells of the islands formed by the AME tumour epithelium, a region most likely to suffer intratumoral hypoxia. MMPs are secreted by stromal cells, endothelial cells, or by tumour cells themselves10. Hypoxia may also induce macrophages to produce more VEGF and suppress immune response40. Tumours induce neovascularization in order to acquire nutrients for continuous growth and metastatic spread. This “angiogenic switch” is induced by VEGF, which in turn can be produced by cancer cells and host stromal cells in a tumour41, which contributes to the disruption of the balance between angiogenic promoters and inhibitors42. Experimental and clinical reports have confirmed that VEGF plays a central role in regulating angiogenesis and vasculogenesis in solid tumours44. Its signalling may affect several significant tumour functions in addition to vascular permeability and neovascularization45, such as tumour cell proliferation, migration, and autocrine invasion47. Kumamoto et al.20 and Dineshkumar et al.21 demonstrated a strong cytoplasmic expression of VEGF in AME, however, the immunoexpression was concentrated in the peripheral cells of the tumour epithelium, indicating the proangiogenic role of this protein.ECM degradation by these MMPs stimulates the release of angiogenic and growth factors, such as VEGF, which is considered to be the most important inducer of angiogenesis and vascular permeability45 suggested that the VEGF expression in a head and neck carcinoma cell line may play two different roles in tumorigenesis: (1) through its paracrine function, essential for tumour-associated angiogenesis, and (2) through its autocrine function, where VEGF plays an important role by directly enhancing mitogenesis and invasiveness by maintaining proliferation, enhancing survival, and increasing the invasion of carcinomas. Therefore, VEGF may serve a proangiogenic and protumorigenic role in the pathogenesis of neoplasms. In addition, Tong et al.45 stated that VEGF-targeted therapy has the potential to fulfil both anti-angiogenic and anti-tumorigenic functions. Higher VEGF expression in tumour parenchyma, compared to tumour stroma, suggests a pro-tumorigenic role instead of a pro-angiogenic role, where it would be possible to observe a higher expression of VEGF in the stromal microenvironment. This hypothesis has been reinforced by several authors47.In our study, VEGF showed strong cytoplasmic immunoexpression in the central cells of the islands formed by the tumour parenchyma, farthest region from the stroma, the supporting tissue, indicating the possible activity of this protein in intratumoral hypoxia area, suggesting an alternative role to its angiogenic function. Tong et al.28. Altered Wnt pathway signalling has been identified in COCs35 as well as ameloblastomas36, and may be one of the factors involved in expression of VEGF in these lesions22. In the study by Dineshkumar et al.21, there was no statistically significant difference in VEGF expression between AME and COC. In our study, VEGF had a higher expression in AME than in COC; however, there was no statistical difference. This finding reinforces the fact that COC, despite being a cystic lesion of odontogenic origin and having a milder clinical behaviour when compared to AME, still has aggressive characteristics1.Cystic lesions showed an increased concentration of VEGF in the cystic fluid that was produced by parenchymal cells, which can induce proliferation in cyst lining epithelial cells15. In our study, VEGFR-2 immunoexpression was observed in the cell membrane of tumour cells. VEGFR-2, localized on endothelial cell surfaces, appears to regulate VEGF endothelial cell permeability and proliferation48. VEGF receptors were originally believed to be expressed only in endothelial cells, however, it was later shown that they could also be expressed in tumour cells, including head and neck cancers49. This is the first study to evaluate the expression of this receptor in AME and COC.Among VEGF receptors (VEGFRs), VEGFR-2 is overexpressed under hypoxiaOur results show higher immunoexpression of HIF-1α, MMP-2, VEGF, and VEGFR-2 in ameloblastomas when compared to calcifying odontogenic cysts and dental follicles, suggesting that the relationship between these proteins may contribute to the behaviour of this neoplasm. As the centre of the tumour islands showing higher expression of HIF-1α curiously promote higher VEGF expression in the same region, as well as a higher expression of VEGFR-2 in tumour cells. From this, we can extrapolate that VEGF in the central cells of islands formed by tumour parenchyma may signal peripheral cells for greater proliferation and invasion, which is likely to be mediated by MMP-2 expression. The methods used in this work, although indicating the expression of the studied proteins, limit the depth of our knowledge regarding the role of HIF-1α, MMP-2, VEGF, and VEGFR-2, as the expression of proangiogenic proteins in the centre of tumour epithelial islands suggest a secondary role of these proteins in the proliferation, survival, and invasion of ameloblastoma. In this sense, mechanistic studies must be done to answer these questions.The Human Research Ethics Committee of the Health Sciences Institute of the Federal University of Pará approved this research (protocol number: 2371410). All procedures performed were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. In addition, informed consent was obtained from all participants and/or their legal guardians.Twenty four AME samples were retrieved from the files of the Department of Oral Pathology, School of Dentistry, Universidade Federal do Pará (UFPA), Belem, PA, Brazil, and ten COC and nine dental follicle (DF) samples were retrieved from the files of the Department of Clinic, Pathology, and Dental Surgery, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil.35. Formalin-fixed, paraffin-embedded tissues were studied by immunohistochemistry. Sections of a 5-µm thickness were obtained and mounted on 3-aminopropyltriethoxysilane-coated slides (SIGMA-ALDRICH). The sections were deparaffinised in xylol and hydrated in graded ethanol solutions. The sections were immersed in 20% H2O2 and methanol in a 1:1 ratio for 20 min to inhibit endogenous peroxidase activity. A citrate buffer (pH 6.0) was used for antigen retrieval in a Pascal chamber (DAKO) after immersion for 30 s. Subsequently, non-specific biding sites were blocked with 1% bovine serum albumin  in a phosphate-buffered saline (PBS) solution for 1 h. The slides were incubated with the respective primary antibodies against HIF-1α , MMP-2 , VEGF/VEGFA  and VEGFR-2 . All primary antibodies were diluted in PBS and incubated for 1 h at room temperature. Afterwards, the sections were incubated for 30 with EnVision Plus (DAKO) detection system (DAKO). Diaminobenzidine (DAKO) was used as the chromogen, and sections were counterstained with Mayer’s haematoxylin (SIGMA-ALDRICH). For negative controls, primary antibodies were interchanged with non-immune serum.Immunostaining was performed using an immunoperoxidase assay and the EnVision technique as previously described by Costa et al.https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html) of ImageJ . The evaluation of immunostaining was performed by measuring the mean grey value, which consists of the average grey value within the selection. Both neoplastic cells and stromal cells were included in the quantification.To assess the intensity of HIF-1α, MMP-2, VEGF, and VEGFR-2 staining, brightfield images from at least five randomly selected fields from each sample were acquired using an AxioScope microscope (CARL ZEISS) equipped with a CCD colour camera . The images were acquired at the same magnification (40×). Areas of diaminobenzidine staining were analysed using the immunohistochemistry (IHC) image analysis toolbox plugin . A Mann–Whitney test was carried out to evaluate the statistical differences in protein immunoexpression between the stroma and the tumour parenchyma . The Kruskal–Wallis test and Dunn’s post-test were carried out to evaluate the statistical differences in protein immunoexpression between AME, COC, and DF.


IntroductionWe aimed to characterize the most influential radiation oncologists on Twitter, the correlation between their Twitter activity and their academic profiles as measured by the Scopus H-index as well as their activity around the American Society for Radiation Oncologists (ASTRO) 2018 meeting.MethodsWe defined radiation oncology influencers as any radiation oncologist with 500 or more followers on Twitter through the first two weeks of August 2019. We collected their available characteristics, their Scopus H-index, and Twitter metrics. We examined their general Twitter activity as well as their specific activity before, during, and after the 2018 ASTRO annual meeting. We identified the most frequent tweet content categories for each influencer.ResultsWe identified 48 radiation oncologist influencers; 79% were male, 75% were based in the United States, and 94% were affiliated with an academic center. Among them, 44% had high H-indices of ≥21, an average value in academic faculty for full professors or department heads.There were no correlations between H-index and Twitter metrics such as the number of individuals the influencer was following (p = 0.58), the number of followers (p = 0.66), the number of tweets (p = 0.88), and the number of likes (p = 0.54). During the period around ASTRO 2018, the mean number of tweets per influencer was 4437 .ConclusionCurrent radiation oncology influencers are predominantly North American males from academic institutions. A correlation between academic productivity as measured by the H-index and Twitter metrics was not demonstrated. The fact that some influencers had a low H-index supports that a high academic profile as measured by traditional metrics is not necessary to have a voice in the Twitter radiation oncology community.  This is representative of an official recognition of this platform for data sharing and dissemination of relevant information.Influencers in social media are defined as people who have built a reputation for their knowledge and expertise on a particular topic and who have a significant footprint in their community . They maNevertheless, we know very little of who the current Twitter influencers in radiation oncology are and what is their disseminated content. As radiation oncologists, we need to know who is influencing us to detect possible bias such as origin, sex, or obvious financial bias of influencers. Furthermore, we examined the academic profiles of the most influential radiation oncologists on Twitter to know whether being an influencer is associated with being an often-cited scientist. Conferences are potentially a time of increased Twitter activity to promote research and discussion. We analyzed Twitter activity around an important radiation oncology conference.Identifying radiation oncology influencers on TwitterThe main objective of this study was to analyze individual radiation oncologists that reach a large audience through Twitter. Because these data are available to everyone on Twitter, no ethics committee permission was demanded. Therefore, we did not include institutional accounts as they are managed by several people and not necessarily radiation oncologists. We realized early on in our research that it would be unrealistically time-consuming to manually gather all tweets for analysis. After identifying the seemingly topmost popular Twitter accounts, we decided to establish an arbitrary cutoff of 500 followers in order to have a sufficient number of accounts to analyze. We ended up with a total of 48 Twitter influencers. We identified influencers in radiation oncology based on having greater than 500 followers. Radiation oncologists were searched on Twitter using the search tool on Twitter, by typing in the following keywords: “radonc,” “radiation oncologist,” and “radiation.” Twitter metrics were collected on the physician’s profile including gender (based on profile picture and name), continent, and country in which the individual practices, hospital or practice affiliations, account creation date, number of followers, and number of individuals he or she was following.Data collection and analysisWe chose to analyze data regarding Twitter activity during the 2018 ASTRO annual meeting that took place between October 21 and 24 of 2018. We manually searched Twitter for self-identified radiation oncologists . All tweets were analyzed individually by the first author in August 2019. We were more specifically looking at the Twitter account activity (a) before October 1 to 20, (b) during October 21 to 24, and (c) after October 25 to November 1, the ASTRO meeting. The number of tweets were categorized as either (1) original tweets by the influencer, (2) quoted from another tweet with edit, or (3) retweets . Additionally, we analyzed the number of comments, likes, and retweets.Furthermore, we identified the content of the tweets. Thirteen different categories were identified according to the most frequent types of tweets and the main message conveyed by the tweet, as utilized by Ciprut et al. . These iTo assess the influencer’s academic productivity, we initially searched on Google Scholar for their H-index , which wSummary statistics included mean, standard deviation, median, range, and frequency of influencers’ characteristics and Twitter content. Pearson correlation was used to correlate the academic productivity (as measured by the H-index) with various Twitter metrics. A generalized linear mixed model was used to analyze the variation in Twitter activity across time; using Bonferroni correction, a p value below 0.003 was considered statistically significant.Table Table A total of four influencers (8%) had an H-index of ≤3. Indices associated with senior faculty was seen in an important minority of radiation oncologists: 21 (44%) had an H-index of ≥21, while 15 (31%) had an H-index of ≥31. Table When looking at tweet content, Table www.astro.org/membership). Men are therefore not necessarily overrepresented in this group. The majority of Twitter influencers were from well-established academic institutions, with only three influencers being from non-academic centers. This suggests a high representation of academic institutions on Twitter compared to an ASTRO survey distributed in 2017 in the United States, which found that a minority (41%) of responders were working in academic institutions. Up to 44% of the identified Twitter influencers had H-indices of ≥21, an average value in academic faculty for full professors or department heads in radiation oncology and considered representative of a mature academic position [Overall, the results show that the top radiation oncologist influencers are mainly 79%) male from the United States. This is representative of the proportion of male members (75%) of ASTRO (position ,7, while9% male fposition . On the This study provides content analysis of influencers' tweets within the field of radiation oncology. The majority of tweets were related to academic content. However, Twitter metrics were not correlated to the Scopus H-index. Previous studies analyzing the use of Twitter during medical conferences in other medical specialties have demonstrated that physician influencers may be important drivers of conference participation ,10. In aWe arbitrarily chose Twitter influencers as being those who had more than 500 followers to include about 50 of the most influential Twitter members of the radiation oncology field. This list notably included all the influencers that the 2018 ASTRO meeting recommended to follow during the conference. To classify the content of the tweets, we used the methodology described by Ciprut et al. in urology , which, In conclusion, we found that current radiation oncology influencers are predominantly North American males from academic institutions. A correlation between academic productivity as measured by the H-index and Twitter metrics was not demonstrated. The fact that some influencers had a low H-index supports that a high academic profile as measured by traditional metrics is not necessary to have a voice in the Twitter radiation oncology community. The impact of Twitter for the propagation of opinions, representation of our specialty, education of our patients, and the need for quality control are topics that will require further attention as Twitter use continues to grow in our field.