Extracellular vesicles (EVs)– cell derived organic, nano-sized particles that are more then only cell dust. Nowadays, EVs are a scientifically accepted novel route for cells, including tumour cells, to communicate and influence their host, orchestrating cellular processes from simple tumour growth to complex mechanism of tumour tolerance. Here we show the current understanding of those EVs in gastrointestinal cancer and hepatocellular carcinoma (HCC). The review emphasizes the potential of EVs as a new therapeutic tool and drug target. It critically evaluates their role, in an informative manner using cutting edge, current publications. Many of the cited work are regarded as milestones in the EV field of research drawing a promising perspective for future EV studies.For more than a decade, extracellular vesicles (EVs) have been in focus of science. Once thought to be an efficient way to eliminate undesirable cell content, EVs are now well-accepted as being an important alternative to cytokines and chemokines in cell-to-cell communication route. With their cargos, mainly consisting of functional proteins, lipids and nucleic acids, they can activate signalling cascades and thus change the phenotype of recipient cells at local and systemic levels. Their substantial role as modulators of various physiological and pathological processes is acknowledged. Importantly, more and more evidence arises that EVs play a pivotal role in many stages of carcinogenesis. Via EV-mediated communication, tumour cells can manipulate cells from host immune system or from the tumour microenvironment, and, ultimately, they promote tumour progression and modulate host immunity towards tumour’s favour. Additionally, the role of EVs in modulating resistance to pharmacological and radiological therapy of many cancer types has become evident lately. Our understanding of EV biology and their role in cancer promotion and drug resistance has evolved considerably in recent years. In this review, we specifically discuss the current knowledge on the association between EVs and gastrointestinal (GI) and liver cancers, including their potential for diagnosis and treatment. Extracellular vesicles (EVs), first described using the term “platelet dust” , can be Studies performed over the past decades proved that EVs have important biological relevance. This concept is substantiated by the observations that EVs harbour functional proteins, lipids and nucleic acids, which may be transferred from cell-to-cell  Figure . ImportaOur understanding of EV biology, although not yet complete, has evolved considerably in recent years, and, to date, many excellent reviews describing the biogenesis and techniques used to evaluate EVs have been published ,26,27,28Even though cancers have been some of the most widely studied human disorders, their pathophysiology remains unknown, mostly because the mechanisms of disease are highly heterogeneous . ClearlyConsiderations of the usefulness of EVs as a candidate biomarker in GI and liver cancers should begin with a concise description of properties that characterise a good biological marker. The most frequently used definition in the literature came from a report by the U.S. Food and Drug Administration (FDA) and the National Institutes of Health (NIH) and describes biomarkers as “a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention” . To put This part of our review summarises previous studies that have described findings concerning EVs as a possible biomarker in GI and liver cancers. Intentionally, this section is divided into subsections depending on the anatomical location of the cancer.One of the initial pieces of evidence implicating small EVs (exosomes) as biomarkers was provided by Matsumotoet et al. , who shoPrevious work done by Zhou et al. showed similar expression of five miRNAs  in blood small EVs in GC patients and healthy subjects . In turnAnalysis of exosomes from patients with GC showed that long non-coding RNA (lncRNA) can become a new biomarker of GC ,55,56,57Of note, the quantification of small EVs in peritoneal lavage fluid (PLF) may have advantages over blood levels of exosomes. Indeed, Tokuhisa et al. demonstrated that exosomal miR-21 and miR-1225-5p expression in PLF can indicate the metastatic stage of GC . They foCurrently, there are studies underway to determine a potential diagnostic value of proteins associated with EVs in GC ,62. DecrIndeed, as shown above, the vast majority of studies examined blood small EVs. Previously, large EVs (microvesicles) were also suggested as a blood-based marker for GC . In a siTo the best of our knowledge, a potential diagnostic and prognostic value of EVs for CRC was first shown in a study by Choi et al. . A varieAnother feature that distinguishes CRC subjects and healthy population is significantly higher levels of blood exosomal miRNAs, including miR-let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, miR-23a , miR-19aMore recently, studies found that lncRNAs can be used as biomarkers for diagnosis of CRC ,78. CompIt should be emphasised that most authors in the cited publications state an increase in expression of various exosomal antigens in blood collected from CRC patients. A common conclusion of the majority of studies is also that increased expression of these antigens is highly correlated with the severity of diseases and the reduction of patient survival.High protein and RNA expression was observed on the surface of EVs in the majority of studies involving HCC cases. The most thoroughly investigated marker was miRNAs associated with small EVs ,83,84,85As reviewed by us, studies confirmed that exosomal miRNAs are strongly associated with clinicopathological variables of HCC patients ,86,87,88Previous studies showed that detection of exosomal miRNAs is an attractive biomarker that could serve as an indicator that differentiates HCC from other liver diseases. Sohn et al.  examinedOf note, a large body of evidence suggests that increased levels of several exosomal miRNAs were associated with a high probability of HCC recurrence ,90,91. UIt is important to note that clinical observations have confirmed that long noncoding RNAs are now recognised as an important biomarker for HCC. Subjects with HCC show increased exosomal levels of lncRNA LINC00161 compared with healthy controls and ROC curve analyses established that it could differentiate HCC from controls, with an acceptable AUC (0.79) . Xu and Limited reports have demonstrated that messenger RNA (mRNA)  and circThe molecular nature of circulating small EVs in HCC patients allows for the identification of proteins associated with these EVs that could serve as biomarkers in HCC ,100. For+ and CD133+CD44v6+ large EVs could distinguish between HCC and CCA. Unfortunately, they could not differentiate between intrahepatic and extrahepatic CCA [+l EVs to predict HCC recurrence is better than that of endothelial large EVs [Collectively, these data support the idea that small EVs are the most commonly researched among all EVs in HCC ,104,105.atic CCA . Furtheratic CCA . As susparge EVs .EVs may be characterised by vesicular tetraspanins, proteoglycans, lectins, integrins and other biomolecules, being typically present on their surface or in some cases in the EV lumen. Receptors and ligands as located on the EV surface allow them to play a regulatory part in human immune response, either via signal transduction by ligand/receptor-mediated signalling, or possibly of bigger ramification, through their content comprising mRNA/miRNA or cytosolic proteins of their parental cell ,112. Thereg cells and M2 macrophages are positively related to each other, which might result into a worsen outcome. Therefore, exploiting this axis may give us a potential therapeutic target to prevent disease deterioration [Conversely, it was discussed that miR-145 positive EVs derived from CRC (CRC-EVs) were most likely polarising surrounding macrophages into the M2 phenotype and thus supporting tumour enlargement . It was ioration .+ T cell function [In the case of immunity supercharging, VPS4A, an important tumour suppressor, maintains the expression of miRNAs in hepatoma cells by regulating EV communication . HCC celfunction . Here, afunction .+ T cells also carry out anticancer tasks by direct cytotoxicity [+ T cell EVs, and miR-298–5p in CD8+ T cell-derived EVs were naturally orchestrating the populations of mesenchymal stem cells and cancer-associated fibroblasts to prevent cancer progression or, on the contrary, to promote tumour growth [Immune competent cells of the adaptive immune response, such as various T cells, are naturally a source of EVs and are prone to be modulated by non-T cell-derived EVs. Exemplarily, it was reported that, in general, CD8toxicity . Additior growth .reg-like cells to tame immune cells for tumour prosperity by enhancing TGF-β/Smad signalling and blocking SAPK signalling [regs, consumption of CD8+ T Killer cells, cut down on NK cells and destruction of extracellular matrix to achieve phenotype changes [Particularly in gastric cancer and CRC, EVs potentially regulate PI3K/Akt and mitogen-activated protein kinase pathways to proliferate tumour cells . In CRC,gnalling . Furthergnalling . Lately,gnalling . CRC-EVs changes .However, paradoxically, some studies mentioned that tumour cells secrete EVs to induce anti-tumoural immune responses by carrying the tumour antigens , leadingFrom a clinical perspective, the following findings might be of interest. CD39 and CD73, both present on some tumour-derived EVs, are involved in inhibiting the immune response in the inflammatory tumour environment, promoting tumour immune escape and affecting the chemotherapeutic effect to a great extent ,130. In During the last decades, the idea that cancer growth is just an uncontrolled proliferation event of tumour cells has changed massively. Tumour growth and spread is actually orchestrated by many cells including tumour surrounding parenchymal, stromal cells and other cells. Communication is key between those cells . One wayDuring cancer initiation, there is a conflict between aberrant cells and non-aberrant cells. The bidirectional communication between normal cells and newly transformed cells will ultimately dictate the fate of the microenvironment towards tumour initiation or tumour inhibition. On the one hand, the transfer of EVs containing miR-122 from normal hepatocytes to tumour initiating cells (T-ICs) inhibits tumour progression. On the other hand, T-ICs subsequently release IGF-1, which prevents miR-122 production in normal hepatocytes leading to tumour initiation . Further+ hepatoma cells are capable of producing EVs rich in lncRNA H19, which are subsequently internalised by endothelial cells, changing their phenotype and increasing VEGF production [+ EVs, released by HepG2 cells upon fusion with human umbilical vein endothelial cells, accelerated the migration of vasorin+ EVs recipient cells [After tumour initiation, another important step in tumour development is tumour growth. It was nicely demonstrated that HCC cell-derived EVs contain TUC339 and that these EVs can be taken up by other HCC cells, resulting in enhanced proliferation and subsequent tumour growth . Cancer oduction . Others oduction . Moreovent cells . Eukaryont cells . Once tunt cells .Tumour development is a highly dynamic and complex process. One cancer cell subpopulation might be responsible for the initial growth; other subpopulation might be more involved in angiogenesis, while others might change the phenotype to a migratory behaviour. Both migration and invasion are essential for further tumour growth on one hand and for tumour metastasis on the other hand. It was recently demonstrated that MET protein, caveolins and S100 packed in EVs from motile HCC cells are taken up by non-motile HCC cells, which in turn acquire migratory and invasive abilities. Moreover, HCC-derived EVs triggered PI3K/AKT and MAPK signalling pathways and increased the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-P, thereby facilitating the invasion potential of HCC cells . It is n6β4 and α6β1 specifically bind to lung-resident fibroblasts and epithelial cells mediating lung tropism [Metastasis is the final act in liver cancer development. However, a receptive microenvironment is required for the development of malignant tumours at distant tissues. It is now well accepted that the microenvironment of the metastatic niche will ultimately decide whether disseminated tumour cells survive, remain dormant or form macrometastases . Recent  tropism , unreveaMesenchymal stem cells (MSC) are multipotent cells that reside in many adult tissues such as bone marrow, cord blood, adipose tissue, placenta, liver, lung and skeletal-muscle ,154,155.In a small fraction MSCs can also reside in normal tissues, where then can have various roles depending on the signals from surrounding cells. Studies have demonstrated that at least 20% of cancer associated fibroblasts (CAF) originate from bone marrow and are derived from MSCs . When exIn addition to their role in modulating tumour development, MSC-derived EVs might also influence the tumour response to chemotherapy or targeted therapies. In a BALB/c nu/nu mice subcutaneous xenograft tumour model, it was demonstrated that EVs  significantly induced the resistance of gastric cancer to 5-fluorouracil by antagonising apoptosis and enhancing the expression of multidrug resistance-associated proteins . AnotherApart from the well-known abilities of MSC-derived EVs in tumour growth or tumour inhibition, many other processes of carcinogenesis might also be influenced by MSC-derived EVs. MSC-derived EVs induced the epithelial mesenchymal transition (EMT) in HGC-27 cells . The EMTThe different effects of MSC-derived EVs on tumour development might be explained by the heterogeneity of MSCs. Because MSCs are heterogeneous, the EVs isolated from them are also heterogeneous and this heterogeneity may promote or prevent tumour development. Moreover, the different tumour types and models studied and the variations in methods and time EVs were administered may also account for the apparently paradoxical effects on tumour progression, as reviewed in depth by Parfejevs and colleagues .Intriguing evidence for a crucial role of EVs in resistance to pharmacological and radiological therapy of many cancer types has emerged ,175. ThiSeveral mechanisms, including increased expression of drug resistance proteins and decreased apoptosis of cells, have been purported to limit chemotherapy effectiveness in GI and liver cancers. In vivo and ex vivo analyses of GC cells found that mesenchymal stem cell (MSC)-derived exosomes induce GC resistance to 5-FU by activating the CaM-Ks/Raf/MEK/ERK pathway . As a coTo understand the nature of drug resistance in oesophageal cancer, two studies were performed ,180. IniA major advance toward the understanding of CRC drug resistance was achieved recently by several groups of researchers ,186,187.Many of the molecular experiments designed to probe the nature of drug resistance in liver cancers showed a crucial role of EVs in this phenomenon ,189. TheTaken together, these studies suggest that EVs are involved in resistance to anti-tumour agents in GI and HCC. This function is mainly related to the ability of exosomes and MVs to reduce chemo-sensitivity. Of note, exosomes are also involved in reversing the drug resistance. Despite the multiple lines of evidence supporting the existence of EV-mediated drug responses, a debate remains as to whether this phenomenon is clinically relevant. In https://clinicaltrials.gov/if applying search key words as cancer and exosomes. Most of them are not related to gastrointestinal cancers including HCC. To our knowledge, 2019 the U.S. Food and Drug Administration (FDA) acknowledged by granting Breakthrough Device Designation to Bio-Techne ExoDx Prostate IntelliScore (EPI) test, making it the first exosome-based liquid biopsy test to receive a Breakthrough Device Designation [https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/public-safety-notification-exosome-products). Therefore, it is still questionable whether EVs will make the breakthrough and be used in clinics. What are the pitfalls—the sensitivity or the specificity? A possible way out of this dilemma might be to take advantage of artificial intelligence (AI). AI might give the final needed boost to our research field, as lately published in CELL [EV research started more than two decades ago and was considered to play a role in haemostasis and disease conditions. Since then, one of the major breakthroughs in the field was, in 2007, the discovery that EVs are not empty organic structures but harbour mRNAs and microRNAs and certainly, and they are not just a cellular trash dump mechanism but rather a fine-tuned machine and a highly sophisticated extracellular cell-to-cell communication route . Up-to-dignation . Overall in CELL .


TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in  Many tumours exhibit hypoxia (low oxygen) and hypoxic tumours often respond poorly to therapy. Here, the authors quantify hypoxia in 1188 tumours from 27 cancer types, showing elevated hypoxia links to increased mutational load, directing evolutionary trajectories. Sub-regions of hypoxia can result from disrupted oxygen supply: irregular and disorganized tumour vasculature can reduce oxygen availability5. Hypoxia can also be caused by changes in oxygen demand: altered tumour metabolism7 can increase intra-cellular demand for oxygen, potentially extending hypoxia signalling to liquid tumours. The adaptation of tumour cells to this imbalance in oxygen supply and demand is associated with poor clinical prognosis in several cancer types, attributed at least in part to hypoxia-associated genomic instability and clonal selection16.Approximately half of all solid tumours are characterized by low levels of molecular oxygen (hypoxia)TP53 mutations and for those that are apoptosis-deficient18. Indeed mutations in TP53 occur at a higher frequency in hypoxic primary tumours of at least 9 types16. The abundance of proteins involved in homologous recombination (e.g. RAD51) and non-homologous end joining (e.g. Ku70) are reduced under hypoxia, and these changes can persist for 2 days after reoxygenation21. Genes central to efficient mismatch repair (e.g. MLH1 and MSH2) are also downregulated under hypoxia23. Further, co-presence of tumour hypoxia and high genomic instability15, specific cellular morphologies like intraductal and cribriform carcinoma24 or specific mutations like loss of PTEN16, synergistically predict for rapid relapse after definitive local therapy in some tumour types, particularly prostate cancer. These data underscore the relationship between hypoxia and DNA repair defects, and suggest the tumour microenvironment applies a selective pressure leading to the development of specific genomic profiles.Previous work has provided insight into the molecular origins and consequences of tumour hypoxia and genomic instability. Dynamic cycling of hypoxia can select for cells with 16. However, the influence of tumour hypoxia on pan-cancer driver alterations, mutational signatures, and subclonal architectures remains unclear. To fill this gap, we calculated tumour hypoxia scores for 1188 tumours with whole-genome sequencing (WGS) and RNA sequencing, spanning 27 cancer types. Genome sequencing data was aggregated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium and generated by the ICGC and TCGA projects. These sequencing data were re-analyzed with standardized, high-accuracy pipelines to align to the human genome (reference build hs37d5) and identify germline variants and somatic mutations, as described previously25. This sequencing data together with our high-quality hypoxia quantitation represents a powerful hypothesis-generating mechanism to suggest useful back-translational in vitro experiments and better define the hypoxia-associated mutator phenotype across cancers. We associated hypoxia with key driver alterations in coding and non-coding regions of the genome, and find hypoxia is associated with specific mutational signatures of unknown aetiology. We illustrate the joint impact of PTEN and the tumour microenvironment in influencing the evolutionary trajectory of tumours. Overall, these data highlight the genomic changes through which hypoxia drives aggressive cancers.We previously evaluated the exomic and copy-number alteration (CNA) consequences of tumour hypoxia across 19 cancer types25 and RNA-sequencing26 analyses were systematically carried out by centralized teams with consistent and high-accuracy bioinformatics pipelines. Normal reference samples had a mean WGS coverage of 39 reads per base-pair while coverage for tumour samples had a bimodal distribution with modes at 38 and 60 reads per base-pair25. All samples underwent an extensive and systematic quality assurance process25.We compiled a cohort of 1188 tumours from 27 cancer types via the PCAWG Consortium. All samples had matched tumour and reference normal WGS and tumour RNA sequencing data generated by the ICGC and TCGA projects. WGS28. Cancer type was further incorporated as a random effect in every model, allowing us to consider a different baseline value for the feature of interest for each cancer type. As a measure of effect size we report conditional R2 values, denoted as 29. We also report marginal R2 values, denoted as 29.We used linear mixed-effect models to associate hypoxia with features of interest across cancers while adjusting for tumour purity, age, and sex30, Winter31 and Ragnum32 , mature B-cell lymphomas , lung adenocarcinomas  and breast adenocarcinomas . This was in contrast to chronic lymphocytic leukaemias  and prostate adenocarcinomas  where little inter-tumoural variability in hypoxia was observed. The variability in hypoxia score was not significantly associated with the median hypoxia score within cancer types , an engineered feature that is a surrogate for genomic instability and is associated with hypoxia across several tumour types16 . Overall, hypoxia is associated with increased numbers of most types of somatic mutations.We then considered the association of hypoxia scores with 14 other metrics of the mutation density of CNAs, structural variants (SVs) and single nucleotide variants (SNVs) using linear mixed-effect models Fig. , 3. The 25, with CNA, SV and SNV data available for 1096 patients. In cases where a patient had multiple mutations in the same gene (e.g. a CNA and an SNV) we denoted these as compound events. We again used linear-mixed effect models to associate hypoxia with each driver feature across cancers  signatures tested, nine showed differential activity in hypoxic tumours compared to non-hypoxic ones, while controlling for cancer type, tumour purity, age and sex  signatures illustrated a similar story. Of the 17 ID signatures analyzed, the activity of 5 were associated with tumour hypoxia scores while controlling for cancer type, tumour purity, age and sex . This supports previous in vitro work that demonstrated the contextual synthetic lethality of PARP inhibition in cells with defective DNA repair due to hypoxia43. While we have not specifically included in vitro experimental validation data in this report, we and others have previously validated associations first revealed by analysis of mRNA-based hypoxia signatures. For example, our group previously described microRNA-133a-3p as a hypoxia-associated miRNA in prostate cancer based on mRNA signature-based associations across multiple independent datasets16. We went on to validate that microRNA-133a-3p was indeed induced under hypoxia in multiple prostate cancer cell lines and confirmed its capacity to modulate cell proliferation and invasion. Similarly, Ye et al. applied hypoxia signatures to 10 independent datasets of cell lines and primary tumour fragments under hypoxia and normoxia44. These 10 datasets represented seven cancer types and within each dataset samples under hypoxia showed higher hypoxia scores compared to normoxic samples. Further, they generated predictions of drugs that would be more or less potent under hypoxia and validated four drug–hypoxia interactions in vitro. These data illustrate that hypoxia signatures applied to large cohorts of primary tumours can generate reliable hypotheses, many of which have been validated in controlled systems. However, some aerobic cancer cells may also mimic the biological state of hypoxia  and this may affect signature-derived hypoxia estimates. Further, while pimonidazole (which was used to develop the Ragnum hypoxia signature32) reflects oxygen tensions below 10 mmHg (1.3% O2), it is difficult to directly relate hypoxia signature scores with oxygen tension45. Overall, hypoxia signalling can be distinct from microenvironmental hypoxia and this remains a critical caveat of this study.Our study cannot conclusively say whether hypoxia exerts a selective pressure that enriches for specific genomic alterations or if these genomic changes directly result in hypoxia. Experimental studies of single genes support that both effects may contribute to the associations we describeDiving into the mutational processes related to hypoxia, we confirmed that several SBS and small indel signatures related to impaired DNA repair were associated with hypoxia. This raises the potential confounder that because hypoxic tumours have more mutations, we have more power to detect related mutational signatures. However, we demonstrated that hypoxia is indeed strongly associated with many mutational signatures with unknown aetiology, particularly SBS5, which is found in nearly all cancer types. Modelling these associations in vitro is particularly difficult and these data provide a high confidence measure of the mutational signatures that may be directly or indirectly driven by tumour oxygen levels. It is difficult to disentangle the timing of these events: whether a specific driver mutation gives rise to a specific mutational signature or if these are separate processes. Better mapping of the evolutionary timing of hypoxia will be particularly important in addressing this question and the advent of hypoxia signatures may facilitate future studies in this area.16, and other models that link hypoxia to genomic instability and downstream clonal selection42. Previous work has also demonstrated that patients with allelic loss of PTEN and elevated hypoxia rapidly relapse after definitive treatment for localized prostate cancer16. Here, we showed that tumours with alterations in PTEN and elevated hypoxia are enriched for a polyclonal tumour architecture. This illustrates the joint influence of the tumour mutational landscape and microenvironment in guiding evolutionary trajectories across cancers. Further, these data suggest that increased subclonal diversification may be a novel route via which PTEN drives aggressive tumour phenotypes, in concert with tumour hypoxia, and this can be better defined with future back-translational in vitro experiments. The PCAWG dataset is the largest publicly available pan-cancer dataset to date and this limits our ability to validate our discoveries in independent datasets. Ultimately, it will be necessary to validate our findings in large, independent cohorts. Hypotheses generated here, particularly those around hypoxia and tumour evolution, will require long term, systematic in vitro modelling and will be the subject of future studies. Overall, this work shows that a hypoxic tumour microenvironment is associated with specific mutational processes and distinct somatic mutational profiles, and may direct the subclonal architecture of cancers.We observed a significant association between elevated hypoxia and the number of clonal mutations. This supports the idea that hypoxia is an early event in cancer, as we have suggested previously31, Buffa et al. 30 and Ragnum et al. 32, as described previously16  using mRNA-abundance-based signatures of tumour hypoxia developed previously by Winter et al.16 TCGA data between comparable cancer groups. The groups compared are as follows (PCAWG cancer type and TCGA cancer type): Bladder-TCC and BLCA; Breast-AdenoCA and BRCA; Cervix-SCC and CESC; CNS-GBM and GBM; ColoRect-AdenoCA and COADREAD; Head-SCC and HNSC; Kidney-RCC and KIRC; Liver-HCC and LIHC; Lung-AdenoCA and LUAD; Lung-SCC and LUSC; Ovary-AdenoCA and OV; Panc-AdenoCA and PAAD; Prost-AdenoCA and PRAD; Skin-Melanoma and SKCM; Thy-AdenoCA and THCA; Uterus-Adeno and UCEC. Of the 27 cancer types in PCAWG, 11  did not have hypoxia data from comparable cancers in TCGA and were not used for the comparison16. For Spearman’s correlations, p-values were calculated using algorithm AS89.To compare hypoxia scores generated by the different signatures, the median hypoxia score was calculated for each of the PCAWG cancer types based on each signature. The median hypoxia scores from each signature were then scaled from +1 to −1 using the plotrix package (v3.7). Scaled median hypoxia values for the PCAWG cancer types were also compared to scaled median hypoxia values from previously published16. Hypoxia scores were then calculated for all normal tissue samples related to the 19 cancer types with hypoxia scores . Tumour hypoxia scores were compared to normal tissue hypoxia scores for each tissue type where at least 15 normal tissue samples were available (Mann–Whitney U-test). A total of 5649 independent tumours and 625 independent normal tissue samples were evaluated in the comparisons.Previously calculated pan-cancer tumour hypoxia scores were gathered for 7791 independent tumours from 19 cancer types based on the Buffa hypoxia signatureTP53 mutational status, etc.) across cancers using the lme4 package (v1.1-17). For each feature of interest, we compared a full model  to a null model (i.e. a model without the feature of interest) using an ANOVA to determine if hypoxia was significantly associated with the feature of interest across cancers. A generic example of this is shown below with Eqs.  which uses a simulation-based approach to create standardized residuals33. We used linear mixed-effect models to associate each feature with hypoxia score across cancers and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while tumour purity, age and sex were incorporated as fixed effects. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. A Bonferroni p-value adjustment was applied to the p-values from linear mixed-effect modelling since fewer than 20 tests were conducted. All models were adjusted for tumour purity based on previously published purity data33. The full model for evaluating PGA is shown below as an example as follows:Previously published data for 15 mutational density and summary features were downloaded for 1188 tumoursTo assess if SNVs per megabase were independently associated with hypoxia after adjusting for the total number of deletions we created two linear mixed-effect models. The full model associated hypoxia with SNVs per megabase while adjusting for cancer type, age, sex, tumour purity and the number of deletions (Eq. ). For co25. For each of the 653 driver features, we summarized if a patient had an SNV, CNA or SV. Some tumours had more than one type of event in a gene (e.g. a CNA and an SNV) and these events were classified as compound events. We then used linear mixed-effect models to associate the mutational status of each gene with hypoxia score and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while tumour purity, age and sex were incorporated as fixed effects. The driver mutation analysis did not specifically consider the type of mutation in the gene and only considered if the gene had a mutation or was wildtype. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. An FDR adjustment was applied to the p-values from linear mixed-effect modelling. The full model for evaluating PTEN is shown below as an example as follows:Data for driver mutations was first summarized at the gene level for 1096 tumours with previously published driver mutation data35. For each tumour, we calculated the proportion of total mutations attributed to each mutational signature. The proportion of mutations attributed to each signature were calculated by dividing the number of mutations attributed to each signature by the total number of mutations in the tumour. We used linear mixed-effect models to associate the proportion of mutations attributed to each signature with hypoxia score and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while purity, age and sex were incorporated as fixed effects. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. An FDR adjustment was applied to the p-values from linear mixed-effect modelling. The full model for SBS1 is shown below as an example as follows:Previously published data for mutations attributed to various specific signatures was downloaded for 1188 tumours38 subclonal reconstruction data was used to summarize the number of clonal and subclonal mutations in all 1188 tumours. We used linear mixed-effect models to associate the number of these timed mutations with hypoxia score and compared each full model with a null model. Cancer type was incorporated as a random effect in each model while purity, age and sex were incorporated as fixed effects. Tumours belonging to cancer types with fewer than 15 samples were excluded from the analysis. A Bonferroni adjustment was applied to the p-values from linear mixed-effect modelling since fewer than 20 tests were conducted.Previously reported37. Hypoxia scores were median dichotomized to classify patients as hypoxic or normoxic. To test for an interaction between tumour hypoxia and PTEN mutational status in selecting for a particular subclonal architecture, we used linear mixed-effect models together with an ANOVA. An interaction model was first created where the relationship between the hypoxia scores and PTEN mutational status was modelled as an interaction :9\docum47 (v5.9.1). Figures were compiled using Inkscape (v0.91).All data analysis was performed in the R statistical environment (v3.4.3). Data visualization was performed using the BPG packageFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The Brain Tumor Social Media (#BTSM) Twitter hashtag was founded in February 2012 as a disease-specific hashtag for patients with brain tumor.To understand #BTSM’s role as a patient support system, we describe user descriptors, growth, interaction, and content sharing.We analyzed all tweets containing #BTSM from 2012 to 2018 using the Symplur Signals platform to obtain data and to describe Symplur-defined user categories, tweet content, and trends in use over time. We created a network plot with all publicly available retweets involving #BTSM in 2018 to visualize key stakeholders and their connections to other users.From 2012 to 2018, 59,764 unique users participated in #BTSM, amassing 298,904 tweets. The yearly volume of #BTSM tweets increased by 264.57% from 16,394 in 2012 to 43,373 in 2018 with #BTSM constantly trending in the top 15 list of disease hashtags, as well the top 15 list of tweet chats. Patient advocates generated the most #BTSM tweets (33.13%), while advocacy groups, caregivers, doctors, and researchers generated 7.01%, 4.63%, 3.86%, and 3.37%, respectively. Physician use, although still low, has increased over time. The 2018 network plot of retweets including #BTSM identifies a number of key stakeholders from the patient advocate, patient organization, and medical researcher domains and reveals the extent of their reach to other users.From its start in 2012, #BTSM has grown exponentially over time. We believe its growth suggests its potential as a global source of brain tumor information on Twitter for patients, advocates, patient organizations as well as health care professionals and researchers. Social media acts as one of the greatest facilitators of information, ideas, and discussions by creating a borderless global platform. As of April 2019, there are an estimated 3.5 billion active social media users who connect with people around the world at near-instantaneous speed .tweets—short messages limited to 280 characters [Twitter makes it possible for unique Twitter users to follow each other, create unique posts, and interact through aracters . Tweets aracters . On Twitaracters ,6. Twittaracters ,8. In moaracters .patient-run Twitter community not owned by any organization [The #BTSM was founded in February 2011 by authors LS and CB —2 brain LS and CB started a monthly tweet chat  operating under the Twitter account @BTSMchat in 2013. The #BTSM chat occurs on the first Sunday of every month and discusses broad topics of interest to the brain tumor community  ,12. As oIn this study, we examine #BTSM as a hashtag, and its role as an aggregator and technical facilitator of brain tumor conversations by identifying its key users and their interactions, and the type of information shared within this community since 2012. Furthermore, we report recent trends and developments within #BTSM.passive consumption of tweets featuring the #BTSM hashtag , we report total impressions, which is the number of times a tweet has appeared in any user timeline or search result on Twitter [The data are all publicly available tweets with the #BTSM hashtag from January 1, 2012, to December 31, 2018, obtained via the Symplur Signals platform . Informa Twitter . AggregaTo visualize the interactions between key #BTSM stakeholders, a network plot was created to visualize content sharing via retweets in 2018. Tweet transcripts from Symplur were first downloaded and merged to ensure that all nodes can be plotted and represented in the network. To create the retweet network plot, the open source software Gephi   to celebrate the Brain Tumor Awareness Month (BTAM) .In the second half of 2018, our exploratory data analysis found #SNO2018 linked to a mid-November surge in #BTSM activity. The annual meeting of the Society of Neuro-Oncology (SNO) was held in New Orleans, Louisiana, from November 15 to 18, 2018 [The 2018 network plot of retweets including #BTSM  identifiThis study finds that tweets including the #BTSM hashtag between 2012 and 2018 contained high degrees of Twitter user-to-user engagement where the vast majority of the tweets mentioned at least one other user on Twitter . Although we did not analyze the quality of information shared by users, the study found that sharing content such as website links and media  versus text alone corresponds to increased engagement with #BTSM tweets, with 27.66% and 42.00% of the tweets containing visual media (photos/videos) and website links, respectively. Twitter users do not just create unique messages, but also engage with other Twitter users by way of the #BTSM hashtag. As demonstrated in the network plot, major health care organizations  now play a central role in #BTSM communication by including the hashtag in their tweets. For future studies, different ways to weigh volume and unique connections, along with user clustering across the network, should be considered to give a more nuanced depiction of the network involved.A limitation to this study is the focus on metrics that captures participation within #BTSM based on content generation and interaction, which ignores passive Twitter users who might otherwise rely on #BTSM for information without actively engaging through likes and retweets. Twitter users might be searching #BTSM for information without instigating any sort of active and thus trackable method of engagement . The @BTSMchat community organizers have attempted to track passive viewers during live tweet chats by prompting self-identification through this statement, “If you are here tonight to just listen, please tweet ‘#BTSM’ so we know you’re in the audience” . To the Throughout the year, major brain tumor events and medical conferences play valuable roles in expanding #BTSM’s reach by introducing the brain tumor community on Twitter to the hashtag. In 2018, attendees and supporters of both BTAM and the Annual Meeting of the SNO heavily utilized #BTSM in their tweets along with other branded hashtags such as #BTAM and #SNO2018. Although it is unclear whether new Twitter users were converted into active #BTSM participants, tweets from any casual user may still be impactful for the awareness they bring to the larger brain tumor community.One potential limitation for #BTSM growth is the somewhat older age noted for many persons diagnosed with brain tumors. For 2012-2016, the median age at diagnosis for brain and other central nervous cancers was 59 years in the United States , althougAs #BTSM and other disease-specific hashtags continue to expand, it is increasingly important to address the accuracy of health information shared on Twitter as it affects the public and may have patient safety implications. One way to address these issues is to increase the number of brain tumor stakeholders with a blue Twitter-verified profile badge, which Twitter describes as an authenticity indicator as a public interest account. As of April 1, 2020, the majority of #BTSM influencers identified by our network plot lacks this important badge. By increasing the number of verified accounts, it would be easier for the general public to vet individual accounts they might encounter, thereby increasing the likelihood of accurate information consumption among patients and survivors.A key feature of #BTSM is the high involvement of national organizations and groups such @BTSMchat in prompting conversations within the hashtag. By hosting a live, facilitated discussion on the first Sunday of each month, @BTSMchat creates an interactive space for various stakeholders within the brain tumor community  and forms a structure for sustained engagement. The @BTSMchat chat themes in 2018 ranged from large systemic issues  to more personal topics . @BTSMchat’s identified future goals include (1) widening the lived experiences of tweet chat participants with a focus on social diversity, geographic diversity , and diversity of central nervous system tumor experience; (2) partnering with moderators of brain tumor Facebook Groups by providing Twitter education and cross promoting the tweet chat; and (3) creating and curating resources for patients and care partners who want to pursue or deepen their participation in peer-to-peer support opportunities, including chat moderation, and through community education, conference attendance, public speaking, and research collaboration through traditional medical research pathways.When social media platforms first entered the national consciousness, health care professionals could not grasp how these new forms of communication could apply to their work. It is now clear that the larger platforms such as Facebook, WeChat, and Twitter are here to stay. Twitter is a 14-year-old platform that continues to connect people not by personal relationship but rather by shared interests. It is only natural that patients, caregivers, clinicians, and researchers all with a shared interest in neuro-oncology would ultimately converge, share, and form connections, because Twitter was designed to facilitate these forms of information exchange. It is interesting to note that #BTSM was created by patients who saw a needs gap and filled it, without the intervention of a health care structure and is sustained without formal institutional support.We believe that the growth of the #BTSM hashtag highlights the potential of patient innovation through social media platforms, and serves as a global source of brain tumor information for all brain tumor constituents.


Chylothorax is the occurrence of chyle (lymph) in the pleural cavity secondary to damage of the thoracic duct. It is a rare form of pleural effusion which appears as a milky white turbid fluid. Malignancy is the leading cause of nontraumatic chylothorax while inadvertent surgical injury to the thoracic duct is the major cause of traumatic chylothorax. We report a case of spontaneous left-side chylothorax following septic pulmonary embolization (SPE) with Methicillin-Resistant Staphylococcus aureus (MRSA). This is a rare case of a nonmalignant, nontraumatic, and nontuberculous spontaneous chylothorax which was conservatively treated with fibrinolysis and diet modification. The accumulation of lymph in the pleural space due to damage or obstruction of the thoracic duct results in chylothorax. It is an uncommon cause of pleural effusion with high content of triglycerides. The presence of chylomicrons in the pleural fluid is the definitive diagnostic criterion of chylothorax . AlthougμL, WBC of 444 cells/μL with 65% segmenters, pleural fluid pH of 8, LDH of 659 IU/L, pleural fluid protein of 6 g/dL, and glucose of 28 mg/dL. The pleural fluid/serum LDH ratio is 4.2 and the fluid/serum protein ratio is 1, which is thus reflective of exudative effusion. Chylothorax was confirmed with pleural fluid triglyceride of 319 mg/dL and cholesterol of 84 mg/dL. There was no microbiologic growth on the pleural fluid. The possibility of an infected chylothorax was considered given the bacteremia and exudative nature of pleural fluid. The team decided for fibrinolysis with two (2) doses of 4 mg Alteplase instilled intrapleurally due to the loculated nature of the effusion. In addition to fibrinolytic treatment, the patient was placed on low-fat with medium-chain triglyceride and high-protein diet. Fluid cytology was negative for malignancy, and all serologies to rule out connective tissue disease were negative. Strongyloides IgG was negative. Over time, pleural drainage decreased and the chest tube was removed after 5 days with no pleurodesis required. There was interval improvement of chest imaging ten (10) days postfibrinolytics   showed bnolytics .The accumulation of lymph in the pleural space due to damage or obstruction of the thoracic duct results in chylothorax. It is an uncommon cause of pleural effusion with high content of triglycerides, and the presence of chylomicrons is the definitive diagnostic criterion of chylothorax . In a laChylothorax resulting from septic pulmonary embolism has not been reported in the literature. In our case, MRSA bacteremia led to septic pulmonary embolism. Pulmonary effusion and pulmonary abscess may be seen in cases of septic pulmonary embolization (SPE). In a systematic review of patients with SPE, pleural effusion is seen on 32% by X-ray and 29% by chest computed tomography (CT) imaging . We hypoChylous pleural effusions are typically described exudative lymphocytic pleural effusions with milky appearance. In a retrospective study, chylothoraces may present with variable pleural fluid appearance and biochemical characteristics . The chyEmpyema was considered a differential diagnosis in our case given the loculated appearance of fluid on chest imaging. The pleural fluid's milky appearance, elevated fluid triglyceride, unremarkable pleural fluid pH, and WBC made us consider chylothorax the predominant pathology. The rare coexistence of empyema and chylothorax, known as empyematous chylothorax, is possible in this case given the overlapping biochemistry of the pleural fluid. In small case series, pleural fluid triglycerides in the chyliform range in the setting of acute bacterial parapneumonic effusion and empyema may indicate severity of the disease .The treatment approach to chylothorax varies in that some clinicians adopt early surgical intervention while others adopt a conservative approach. Though the conservative approach may have a role to play in small chylothoraces, therapeutic thoracentesis or chest tube drainage is the initial step in large symptomatic chylothoraces. The initial output for this case was about 300 mL only; thus, our team has elected to drain the tube and modify the diet. Conservative treatment includes the use of a low-fat diet supplemented with medium-chain triglycerides (MCT) and/or total parenteral nutrition (TPN) .Since our repeat CT of the chest showed evidence of loculated pleural fluid, two (2) doses of Alteplase were instilled intrapleurally. The role of fibrinolytics is not established but has been proposed for patients with idiopathic chylothorax who failed conservative therapy but refused surgery in one case report . There w


The wrong grant number was erroneously entered in the original manuscript and needs to be changed from NRF-2017R1D1A1B03035677 to NRF-2019R1I1A3A01060073 in [Funding: This work was supported by a grant from the National Research Foundation (NRF-2019R1I1A3A01060073) and from Stem Centric Co. Ltd. in the Republic of Korea.The authors would like to apologize for any inconvenience caused to the readers by this change. The changes do not affect the scientific results. The manuscript will be updated and the original will remain online on the article webpage, with a reference to this Erratum.


The release kinetic of SRL from the tablets was demonstrated to be erosion of HPMC. Pharmacokinetic study on beagle dogs showed that the SRL-SMEDDS tablets were bio-equivalent to the commercial tablets but lower Cmax and larger Tmax were achieved. In conclusion, the SMEDDS tablets were presented as promising delivery system for sustained release of SRL.The clinical application of sirolimus (SRL) as an immunosuppressive agent is largely hampered by its narrow therapeutic range. This study focused on developing SRL tablets with a sustained release profile for better safety. SRL was highly water insoluble and its solubility has been efficiently enhanced by preparing self-microemulsifying drug delivery system (SMEDDS). The SRL-SMEDDS was physically adsorbed by microcrystalline cellulose (MCC). The sustained release of SRL was achieved by addition of hydroxypropyl methylcellulose (HPMC) to prepare tablets. The formulation of the tablets was optimized by single factor test and orthogonal design. The optimal formulation was composed of 10% of HPMC 100lv and 5% of HPMC K4M. The  To keep the blood concentration of SRL in the therapeutic range, a sustained release behavior is desired. Sirolimus (SRL) has been widely used as an immunosuppressive agent for organ transplantation . But the®. But the SRL nanocrystals produced by ball milling are time-consuming, inefficient, and expensive. The SRL belongs to the biopharmaceutics classification system (BCS) class II drug category, showing poor solubility and good permeability. Nanocrystal technology is applied to improve the solubility of SRL in the Rapamune®), ritonavir (Norvir®), and saquinavir (Fortovase®). The self-microemulsifying drug delivery system (SMEDDS) presents as an excellent carrier for hydrophobic drugs . CompareThe SMEDDS is usually composed of oil, emulsifier, and co-emulsifier . After oIn our previous study, solid SRL-SMEDDS was developed and optimized to improve the stability of SRL during storage , 10. ThiMaterialsSRL was purchased from Kerui . Transcutol HP and Labrafil M 1944CS, Cremophor EL were gifted by Gattefossé . Hydroxypropyl methylcellulose  was purchased from Colorcon . MCC (Avicel PH-101) was obtained from FMC . Lactose (Foremost 315WG) was obtained from Kerry Group . All other reagents were of analytic grade. Preparation of SMEDDSThe SMEDDS was prepared based on our previous study . BrieflyCharacterization of the SMEDDSThe emulsion was prepared by addition of 0.5 mL of SMEDDS into 50 mL of water under magnetic stirring at 37 °C for 10 min. The average diameter of emulsion droplets was measured by the Nicomp 380 . In-vitro dissolution of SRL-SMEDDS was carried out using the paddle method. Briefly, the SMEDDS containing 1 mg of SRL was filled into a hard gelatin capsule. Then 6 capsules were added into 250 mL of media stirring at 100 rpm. At each time point , 3 mL of media was collected and equal volume of fresh media was added. The concentration of SRL in the media was analyzed by the high performance liquid chromatography . Media of water, 0.4% SDS water solution, pH 1.2 hydrochloric acid solution (HCl), pH 4.5 and pH 6.8 phosphate buffer solution (PBS) were used, respectively. Preparation of the SMEDDS-tabletsTo develop the SRL sustained release tablets, solid SMEDDS was first prepared. The SRL-SMEDDS was mixed with the powders of MCC and lactose by continuous grinding. The solid SMEDDS was obtained after the mixture had been incubated in a dryer overnight for complete adsorption. Tablets of the solid SMEDDS was produced using a single punch tablet machine . The percentage of the MCC and lactose in the solid SMEDDS was regulated to test the adsorption efficacy based on the morphology of the tablets. To achieve a sustained release behavior, HPMC was mixed with solid SMEDDS for tableting. The weight and hardness of the tablet were fixed at 560 mg and 60 N, respectively. Each tablet contained 1 mg of SRL. The single factor test was performed by regulating the type  and amount of HPMC. In-vitro release testin-vitro release of SRL from the tablets was carried out. Six tablets were immersed in 250 mL of water (0.4% SDS) at 37 °C. The stirring speed was 100 rpm. At each time point , 3 mL of media was collected and equal volume of fresh media was added. The concentration of SRL was determined by HPLC.The Optimization of the tablets2 h = 20%, Q6 h = 65% and Q10 h = 90% (Q represents the cumulative release) to avoid burst release and incomplete release in the initial and last stage, respectively. The release profile of the tablets was scored according to Equation 1. The tablets were optimized by an orthogonal experiment to achieve a proper sustained release profile. The amount of HPMC, ratio of HPMC 100lv: HPMC K4M, and amount of lactose were investigated as independent variables. The selected levels were presented in 2 h| + |65 - Q6 h | + |90 - Q10 h|                               Equation 1 K = |20 - QCharacterization of the optimal tabletsThe release behavior of the SRL-SMEDDS tablets optimized by the orthogonal experiment was further evaluated. The tablets with different hardness , shape (round and irregular-shaped) and preparing method (direct powder compression and wet granulation) were prepared and tested. In addition, the dissolution method (basket apparatus and paddle apparatus), stirring speed  and dissolution medium  were also investigated. f2). When 50 < f2 < 100 was achieved, the release profiles of the three batches of tablets would be regarded as being similar. Finally, to investigate the release kinetics, the data was plotted with the kinetic models of zero order, first order, Higuchi, and Ritger-Peppas models. Three batches of the optimized tablets were prepared and the release profiles were evaluated using the similarity factor  were fasted but had free access to water overnight prior to the experiment. The animals were randomly divided into two groups. Each animal was orally administered with two pieces of the RapamuneQuantitative analysis of SRL in bloodThe rapid resolution liquid chromatography tandem mass spectrometry  was used to determine the SRL blood concentration. 500 μL of blood was mixed with 500 μL of sodium acetate buffer solution (pH 4.6) and 10 μL of ascomycin (FK520) acetonitrile solution (1000 ng/mL) as internal standard. The mixture was vortexed for 30 sec and 2 mL of tert-butyl methylether was added. After being mixed in a swing mixer for 10 min, the mixture was centrifuged at 5000 rpm for 3 min. The organic layer was collected and dried under nitrogen gas at 40 °C. The mobile phase (100 μL) was added to dissolve the residues. The samples (10 μL) were then injected and separated by a ZOBAX C18 column . The column temperature was 50 °C, and water/acetonitrile (1 : 9) were used as the mobile phase at a flow rate of 0.3 mL/min. The mass spectrometer was performed using an electrospray ionization (ESI) interface in positive ionization mode and multiple reaction monitoring mode. The selected reaction monitoring of SRL and FK520 were m/z 937.2→409.3 and m/z 814.4→604.2, respectively. A standard linear calibration curve in the range of 0.5-32 ng/mL was used to determine the SRL concentration.Pharmacokinetic data analysismax), time (Tmax) to Cmax, and area under the concentration-time curve (AUC0−t) were calculated based on the blood concentration versus time profiles. The relative bioavailability (Fr) of SRL-SMEDDS tablets to Rapamune® was calculated using Equation 2. The maximum blood concentration (C0→t) SRL - SMEDDS tablet/(AUC0→t)Rapamune × 100%                               Equation 2 Fr = . Characterization of the SRL-SMEDDSThe average diameter of the emulsion droplets formed by the SMEDDS was 23.27 ± 0.60 nm  with PDIThe dissolution of SRL-SMEDDS was shown in Preparation of tabletsSMEDDS was solidified by adsorption in MCC and lactose. As shown in The MCC and lactose had been commonly used as fillers in tablet preparation. In this study, they were also used as solid carriers of the liquid SMEDDS. Most of the SMEDDS were distributed on the surface of the solid particles, which would hamper the flow properties of the particles , 13.Single factor testThe hydrophilic matrix system based on HPMC had been extensively explored for controlled drug delivery. In this study, HPMC 100lv, K4M, K15M, and K100M were used to obtain a sustained release of SRL. It was observed that the release speed of SRL was in the range of 100lv > K4M > K15M > K100M . The amounts of HPMC 100lv and K4M were further investigated because of more complete release at 12 h than HPMC K15M and K100M. For both HPMC 100lv and K4M prepared tablets, the SRL release was remarkably decreased with the increase of HPMC amount . When HPSimilar results were found in the tablets with HPMC K4M. Differently, addition of 5% HPMC K4M had no influence on the SRL release as compared with tablets without HPMC. Further increase of HPMC reduced the release rate and 20% HPMC tablets had incomplete release (<80%) at 12 h. The release of SRL was largely influenced by the viscosity and amount of HPMC. The hydration of the hydrophilic HPMC polymer contributed to a viscous gel layer. The release of the drugs was realized by dissolution and diffusion through the gel network. Gradual erosion of the gel layer also contributed drug release . Higher The HPMC 100lv showed more satisfying release profiles than HPMC K4M. However, it was also found that the reproducibility was poor when the amount of HPMC 100lv was less than 15% due to rapid swelling and erosion of the tablets.Combination of two viscosity grades of HPMC could get a desired apparent viscosity . Thus, tOptimization of the tablets4) orthogonal experiment was used to optimize the release profile of the tablets (p = 0.041) was found among the three levels of variable A while variable B and C showed no significant difference (p > 0.05). Since the MCC had a porous structure and more SMEDDS could be adsorbed than the lactose, lactose was removed from the formulation. The optimal combination would be A2B1C1, with 15% HPMC (100lv : K4M = 1 : 2). The rest of the tablets was filled with MCC as filling agent. The tablets based on the optimal formulation had smooth surface and complete shape  was 0.8932, indicating that the main mechanism of the drug release was erosion.Modeling of the release profile of the optimal tablets was investigated. The linear correlation Pharmacokinetic study ®, the self-prepared tablets had decreased Cmax . Thus, the SRL-SMEDDS tablets reduced the tendency of SRL blood concentration exceeding the therapeutic range, contributing to better safety. The SRL-SMEDDS tablets also showed increased Tmax  and t1/2 , suggesting that the SRL-SMEDDS tablets maintained more steady blood concentration than the commercial tablets.The blood concentration-time curve of SRL was shown in ® was 94.35 ± 21.76 ng·h/mL and 88.01 ± 18.65 ng·h/mL (p > 0.05), respectively. The relative bioavailability was 107.20%, indicating that the self-prepared tablets were bio-equivalent to the commercial tablets. In-vitro degradation of SRL was rapid in the strong acid media while SMEDDS improved the stability of SRL, and the gastric emptying guaranteed a relatively short incubating period. Though degradation of SRL was also found in pH 6.8 with less than 80% left at 12 h, the controlled release property and fast absorption of SRL-SMEDDS minimized the percentage of degraded SRL. The AUC of the SRL-SMEDDS tablets and Rapamune® (9). Preparation of sustained release system, however, might decrease the bioavailability because of incomplete release in-vivo and diverse absorption efficacy in different section of the intestinal (Our previous study showed that the SRL-SMEDDS had improved bioavailability upon Rapamunetestinal . in-vitro release were investigated. The optimal tablets were further tested for release method, stirring speed, and medium. The release kinetic was demonstrated to be the best fit of the Ritger-Peppas model with a mechanism of erosion. The SRL-SMEDDS tablet was bio-equivalent to the commercial tablet but it had decreased Cmax and more steady blood concentration. In conclusion, this study provided a promising candidate of SRL with a sustained release behavior and better safety. In this study the SRL-SMEDDS tablets were developed. HPMC 100lv and HPMC K4M were combined to achieve a sustained release of SRL. The release behavior was optimized by an orthogonal experiment. The influences of hardness, shape, and preparing method of tablets towards the


Based on several novel concepts that are integral to the revised Starling model, we review the clinical features of DSS and discuss a number of implications that are relevant for fluid management. We also highlight the need for context-specific clinical trials that address crucially important questions around the management of DSS.Dengue is the most common mosquito-borne viral infection in the world. The most feared complication is a poorly understood vasculopathy that occurs in only a small minority of symptomatic individuals, especially children and young adults, but can result in potentially fatal dengue shock syndrome (DSS). Based mainly on expert opinion, WHO management guidelines for DSS recommend prompt infusion of a crystalloid fluid bolus followed by a tapering crystalloid fluid regimen, supplemented if necessary by boluses of synthetic colloid solutions. However, following publication of a number of major trials undertaken in other, primarily adult, critical care scenarios, use of both synthetic colloid solutions and of fluid boluses for volume expansion have become controversial. Synthetic colloids tend to be used for severe DSS cases in order to boost intravascular oncotic pressure, based on the classic Starling hypothesis in which opposing hydrostatic and oncotic forces determine fluid flow across the microvascular barrier. However, the revised Starling model emphasizes the critical contribution of the endothelial glycocalyx layer (EGL), indicating that it is the effective oncotic pressure gradient across the EGL not endothelial cells  Dengue is increasingly being recognized as a major threat to global health, especially among people living in endemic areas who are exposed to multiple infections with different serotypes. Although the majority of symptomatic infections manifest as a self-limited illness characterized by fever and systemic symptoms, life-threatening complications can develop. The most feared complication, particularly prevalent in children and young adults experiencing secondary infections, is a poorly understood vasculopathy that presents as a systemic vascular leak syndrome . The vasSevere leakage resulting in DSS is seen less frequently in adults than children , 5, likeIn this review we highlight some of the current controversies relating to DSS fluid resuscitation and consider how evolving knowledge of the physiological principles governing fluid dynamics at the microvascular level can help us to understand the clinical features observed in DSS and potentially be of use in guiding fluid management of shock.In the 1960s, when DSS was first described mortality rates were high, but recognition of the marked intravascular volume depletion and the urgent need for fluid resuscitation led to a notable improvement in outcomes. Great care is needed however to limit iatrogenic complications, in particular development of respiratory compromise due to fluid overload, especially in settings where HDU/ICU facilities with the capacity to provide advanced respiratory support are scarce. The World Health Organization (WHO) dengue management guidelines recommend prompt infusion of 10–20 ml/kg crystalloid fluid boluses for children with established DSS, followed by a tapering crystalloid fluid regimen supplemented by boluses of synthetic colloid solutions as necessary ] or to bThe evidence for the current WHO management guidelines is limited and the recommendations are based primarily on expert opinion and some observational data , 9, althAnother controversial issue is the use of fluid boluses for resuscitation. Rapid correction of profound hypovolemia has always been accepted as an important feature of shock resuscitation, but there is very little formal evidence available in this regard. A large trial in African children (FEAST) with impaired perfusion associated with severe febrile illness of mixed etiology yielded unexpected but unambiguous results, demonstrating that fluid boluses were associated with significantly increased 48-h mortality . HoweverFinally, although blood transfusion is considered life-saving for individuals with DSS and severe bleeding, identification of occult bleeding can be difficult. WHO management guidelines recommend prompt administration of fresh blood as soon as significant bleeding is suspected , but eviIn health, intravascular volume is regulated within tightly circumscribed limits by complex homeostatic mechanisms. Historically, the general principles governing fluid resuscitation developed in parallel with evolving knowledge of human physiology and vascular biology. Starling originally hypothesized in 1896 that opposing hydrostatic and oncotic pressures in the capillaries of the microcirculation determined fluid flow between the intravascular and interstitial fluid compartments . In lineThe revised Starling model emphasizes the crucial role of the EGL in fluid filtration across the “surface glycocalyx endothelial complex.” Although the detailed structure remains to be elucidated, we now know that the EGL consists of an ordered matrix of glycoproteins and proteoglycans anchored in endothelial cells and extending throughout all vascular beds. Plasma proteins such as albumin adhere to and consolidate the structure, resulting in formation of a negatively-charged size-selective barrier that is semipermeable to macromolecules , 33. CelSeveral important consequences of the revised Starling model need to be acknowledged. Originally, plasma was thought to be filtered to the interstitial space at the arteriolar end of capillaries under a dominant hydrostatic pressure gradient, with the majority of the fluid later being re-absorbed at the venular end under a dominant oncotic pressure gradient. Now however, it is clear that the effective oncotic pressure gradient lies within microvessels, i.e., it is the local colloid osmotic pressure difference between free-flowing plasma and the subglycocalyx space that opposes filtration. As the subglycocalyx space is virtually protein free in comparison with interstitial fluid protein levels, the oncotic pressure gradient opposing fluid filtration is greater than expected with the original model, and thus the rate of fluid loss to the interstitial space is much lower than previously thought  31, 35), 3531, 3de novo synthesis. The revised Starling model suggests that a slow plasma leak would be balanced by a proportionate increase in lymphatic return that, together with cardiovascular, renal, and adrenal compensatory mechanisms, should initially serve to ensure that plasma volume remains close to normal. However, over time progressively worsening hypo-albuminaemia would reduce the colloid osmotic gradient between free-flowing plasma and the subglycocalyx space, thereby increasing filtration into the interstitial space to a level that outstrips the capacity of the lymphatic system to return the filtered fluid to the circulation. Eventually plasma volume becomes compromised, but by the time cardiovascular decompensation actually occurs the intrinsic homeostatic mechanisms are maximally upregulated, explaining the counterintuitive rise in diastolic pressure that is observed.Considering the observed clinical features of DSS, a slow sustained plasma leak evolving over several days is consistent with the revised Starling model. Characteristically shock occurs late in the illness episode (day 4–6), heralded by increasing hemoconcentration that is followed by a rise in diastolic pressure with concomitant narrowing of the pulse pressure (PP). Secondly plasma albumin concentrations are often markedly reduced (<20 gm/L) immediately after initial volume replacement counterbalances the effect of hemoconcentration, but rapidly return to normal within 24–48 h as the vasculopathy resolves—i.e., within a timeframe consistent with redistribution rather than consumption and in vivo, but increased levels of several glycocalyx constituents have been documented, with some evidence indicating associations with the timing and severity of leakage  can disrupt the surface glycocalyx both directly and/or through inflammatory-dependent pathways  in the light of new knowledge; what we understand to be the best approach today may not remain so in the future.DT: topic conceptualization for this review, literature review, writing – original draft preparation, writing – review and editing. HT and BW: literature review, writing – original draft preparation, writing – review and editing. 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.


Lubricin is an important boundary lubricant and chondroprotective glycoprotein in synovial fluid. Both increased and decreased synovial fluid lubricin concentrations have been reported in experimental post-traumatic osteoarthritis (PTOA) animal models and in naturally occurring joint injuries in humans and animals, with no consensus about how lubricin is altered in different species or injury types. Increased synovial fluid lubricin has been observed following intra-articular fracture in humans and horses and in human late-stage osteoarthritis; however, it is unknown how synovial lubricin is affected by knee-destabilizing injuries in large animals. Spontaneous rupture of cranial cruciate ligament (RCCL), the anterior cruciate ligament equivalent in quadrupeds, is a common injury in dogs often accompanied by OA. Here, clinical records, radiographs, and synovial fluid samples from 30 dogs that sustained RCCL and 9 clinically healthy dogs were analyzed. Synovial fluid lubricin concentrations were nearly 16-fold greater in RCCL joints as compared to control joints, while IL-2, IL-6, IL-8, and TNF-α concentrations did not differ between groups. Synovial fluid lubricin concentrations were correlated with the presence of radiographic OA and were elevated in three animals sustaining RCCL injury prior to the radiographic manifestation of OA, indicating that lubricin may be a potential biomarker for early joint injury. It is a major cause of morbidity in humans and domestic veterinary patients, affecting more than 12% of U.S. population between age of 25–74 years and 20% of dogs older than one year3. Although age is a major risk factor for osteoarthritis, OA can develop in patients of all ages as a result of traumatic joint injuries, genetic pre-dispositions, or other risk factors4. With limited treatment options for end-stage OA, efforts have been made to understand mechanisms of OA initiation to aid early diagnosis and to help develop therapies that can delay or halt joint degeneration.Knee osteoarthritis (OA) is a joint disease characterized by changes in articular cartilage, subchondral bone, synovial tissues, menisci and ligaments6. More than 20% of dogs over the age of one year and more than 80% of dogs over the age of eight were diagnosed with OA in one clinical study3, with an estimated $1.32 billion spent by U.S. pet owners on RCCL treatment in 20037. RCCL is the greatest risk factor for canine stifle OA, and – as in repaired ACL human joints – surgical intervention is aimed only at joint stabilization and does not prevent OA progression following cruciate ligament rupture8. Medical management alone of partial or complete CCL rupture in dogs generally consists of long-term systemic nonsteroidal anti-inflammatory drug (NSAID) administration, strict exercise restriction and is often associated with lower quality of life or return to function indices when compared to surgical treatment.Anterior cruciate ligament (ACL) injury is one of the leading causes of post-traumatic knee OA in humans. Similarly, rupture of the cranial cruciate ligament (RCCL), the ACL equivalent in quadrupeds, is one of the major causes of OA in canine patients9. Secreted by superficial zone chondrocytes and synovial fibroblasts, lubricin is found both on the articular surface and within synovial fluid10. Human patients with camptodactyly-arthropathy–coxa vara–pericarditis (CACP) syndrome who lack functional lubricin and Prg4 knockout mice develop severe, early-onset polyarthropathy, demonstrating the significance of lubricin in chondroprotection and joint homeostasis13. In addition, lubricin-deficient synovial fluid from human patients with CAPC syndrome had inferior boundary lubricating ability, and increased joint friction was observed in mice with lubricin loss-of-function mutations13. The highly O-glycosylated tandem repeats give rise to lubricin’s anti-adhesive properties, which prevent aggregation of protein deposits on cartilage and inhibit synovial membrane hyperplasia12. In addition to its lubricating and anti-adhesive functions, recent studies have shown that lubricin downregulates inflammation via binding interactions with the damage associated molecular pattern receptors toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4)15.Lubricin, hyaluronic acid (HA) and surface-active phospholipids are considered the major lubricating biomolecules in synovial fluid; however, how these molecules are altered in knee-destabilizing injuries is not completely understood. Lubricin, or proteoglycan 4 (PRG4), is a highly conserved glycoprotein that enables near-frictionless cartilage boundary lubrication via its extensive O-linked glycosylation16. However, elevated synovial fluid lubricin concentrations have been reported in humans with tibial plateau fracture and late-stage OA18. Similar increases in synovial fluid lubricin have also been observed in several studies in both experimentally-induced and naturally occurring PTOA models in horses22. However, studies evaluating synovial fluid lubricin in other large animal models, especially large animal models of knee joint instability, are lacking. To the authors’ knowledge, only one prior study has evaluated lubricin in a canine experimental ACL transection model, which revealed increased cartilage lubricin immunostaining depth in injured cartilage; however, synovial fluid lubricin was not measured23. Therefore, the objectives of this study were to measure synovial fluid lubricin concentrations in healthy canine stifle joints and in joints with spontaneous RCCL and to determine if changes in synovial fluid lubricin correlated with RCCL injury, radiographic evidence of OA, or concentrations of pro-inflammatory cytokines/chemokines in synovial fluid.Despite the significance of lubricin in joint homeostasis, there is no consensus as to how synovial fluid lubricin is altered in response to joint trauma and subsequent post-traumatic osteoarthritis (PTOA). In one study investigating human patients with ACL injury, synovial fluid lubricin concentrations were acutely decreased post-injury and gradually returned to values similar to the contralateral knee over the course of a yearDogs with RCCL had significantly higher radiographic OA scores as compared to controls, with the exception of intra-articular mineralization Fig. . ObjectiThe intra-assay coefficient of variation for the lubricin ELISA was 11.6%, and 1 sample was assigned a value of 2000 µg/mL, or the upper limit of detection of the assay due to oversaturation. The mean synovial fluid lubricin concentration in the RCCL group was 905.2 ± 91.8 µg/mL (mean ± SEM), which was approximately 16 times greater than the control group  Fig. . The eleThe intra-assay coefficient of variation for the HA ELISA was 3.17%. The mean synovial fluid HA concentration in the control group was 1.21 ± 0.08 mg/mL, and the HA concentration in the RCCL group was 0.52 ± 0.05 mg/mL (mean ± SEM)  and healthy equine synovial fluid-treated wells (3.0 × 103 ± 1.5 × 103 cells/cm2) as compared to the serum-free media wells (1.3 × 104 ± 1.5 × 103 cells/cm2) . The anti-adhesive effect of RCCL canine synovial fluid on primary canine synovial fibroblasts is comparable to that of healthy equine synovial fluid.At 24 h, synovial fibroblast adhesion was reduced in the RCCL canine synovial fluid-treated wells (370 ± 156 cells/cmp < 0.05), except for IL-2 and IL-6 (ρ = 0.31 with p = 0.11). Lubricin concentrations did not correlate with IL-2, IL-6, IL-8, or TNF-α , and all methods were carried out in accordance with approved guidelines.Knee (stifle joint) synovial fluid samples from 30 adult dogs sustaining rupture of the cranial cruciate ligament and presenting to the Cornell University Hospital for Animals from 2014 to 2018 were obtained with informed consent at the time of surgery and stored in liquid nitrogen , one veterinary surgeon (HR), and one veterinary student researcher (DG). A stifle OA scoring system adopted from Innes et al. was used, which evaluated four parameters including joint effusion (0–2), osteophytosis (0–3), intra-articular mineralization (0–2), and a global OA score (0–3) with 0 being no evidence and 2 or 3 being most severe19. Transparent 96-well plates  were coated with 10 μg/mL of peanut agglutinin  in 50 mM sodium bicarbonate buffer at pH 9.5 and incubated at 4 °C overnight. The plates were blocked with 3% bovine serum albumin  in DPBS for 1 h at room temperature. Bovine synovial fluid  was centrifuged at 4,000 × g for 1 h at 4 °C to remove red blood cells, followed by incubation with Streptomyces hyaluronidase and protease inhibitor cocktail in 50 mM NaAc overnight at 4 °C. The synovial fluid was purified using DEAE Sepharose  anion exchange fast liquid protein chromatography (FPLC) similar to previously described methods, and the 490–570 mM NaCl elution fraction was used as the standard for the lubricin ELISA19. The standard ladder of 12 concentrations of purified bovine lubricin ranged from 0.002 µg/mL to 1 µg/mL, in increments of two-fold serial dilutions. Diluted canine synovial fluid samples and FPLC-purified, bovine synovial lubricin were added to the plates in duplicate and incubated for 1 h at room temperature with gentle shaking. A 1-h incubation with anti-lubricin monoclonal primary antibody  diluted 1:5,000 in DPBS was followed by a 1-h incubation with secondary antibody  diluted 1:2,000 in DPBS28. Both incubations were done at room temperature with gentle shaking. The plates were developed at room temperature with 1-Step Ultra TMB  for 20 min or until a royal blue color appeared, at which point the reaction was stopped with 2 N H2SO4. Absorbance was measured at 450 nm with 540 nm background subtraction on a microplate reader , and concentrations were calculated using SparkControl Magellan 2.2  with a four parameter Marquardt fit. Inflammatory cytokines and chemokines, including IL-2, IL-6, IL-8, and TNF-α were measured in undiluted canine synovial fluid samples using a canine-specific electrochemiluminescent multiplex immunoassay  following the manufacturer instructions57. Synovial fluid HA concentrations were measured using a commercially available sandwich ELISA . Synovial fluid samples were diluted 1:80,000 in PBS with 5% Tween20, and HA concentrations were measured in duplicate according to manufacturer instructions. Absorbance was measured at 450 nm with 540 nm background subtraction.For the lubricin ELISA, all canine synovial fluid supernatants were diluted 1:2,000 in DPBS, aliquoted and stored at -70 °C for up to 2 weeks. A custom sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure synovial fluid lubricin, similar to previously described, but with a purified bovine synovial fluid lubricin standard4 cells/cm2. Primary canine synovial fibroblasts were culture-expanded and stored as passage 0 (P0) in liquid N2 cryotanks prior to the experiment. P0 cells were plated and grown in growth media for an additional 2 passages prior to plating as P3 cells for adhesion assays.Primary canine synovial fibroblasts were isolated from the right hips of two dogs undergoing total hip replacement surgeries . Briefly, canine synovial membrane tissue was placed in a petri dish in Dulbecco’s Modified Eagle’s Medium with 1 g/L glucose, 4 mM L-glutamine, 110 mg/L sodium pyruvate (DMEM/Low Glucose) , supplemented with 25 mM HEPES buffer, 10% fetal bovine serum, 100 IU/mL penicillin and 100 µg/mL streptomycin. The tissue was then cut into pieces no larger than 0.5 cm. Dissected tissue was digested with 10 mL/g growth media containing 1.5 g/L collagenase Type II  and 0.15 g/L DNase I  at 37 °C for 2 h with agitation. The digested tissue was then filtered through cheesecloth and 41 μm nylon mesh  and centrifuged at 400 × g for 10 min at room temperature. Cell pellets were re-suspended in growth media and plated at 5 × 102) in 0.5 mL serum-containing medium and cultured at 37 °C and 5% CO2. Two primary canine primary synovial fibroblast cell lines were cultured in two separated plates, and data were reported as mean values for the combined two cell lines. After 24 h, the cells were imaged with an Olympus CK2 inverted microscope (150 × magnification) and Nikon DS-Fil camera. In addition, five images per well (center and four corners) were captured with a microplate reader  for cell counting in Image J. Mean cell number was calculated using 5 fields/well. After an additional 48 h incubation at 37 °C, the cells were stained with 0.4 mL of 0.5% crystal violet solution in 20% methanol for 20 min at room temperature. Phase contrast images of stained cells were captured at 150 × magnification, and the absorbance at 570 nm was measured with a microplate reader. All absorbance measurements were normalized to the average A570 of the serum-free media negative control group.Synovial fluid from the middle carpal joints of two healthy horses were used as healthy control synovial fluid due to the limited volumes of healthy canine synovial fluid available. Four canine synovial fluid samples with volumes greater than 0.3 mL were randomly selected from the RCCL group. All synovial fluid samples were diluted to 12.5% in serum-free DMEM/low glucose media. In each 24-well tissue culture plate , the bottom of duplicate wells were coated with 0.5 mL of serum-free medium (n = 4), 12.5% healthy equine synovial fluid (n = 2) or 12.5% RCCL canine synovial fluid (n = 4) in serum-free medium for 24 h at 37 °C. The wells were washed 3 times with DPBS prior to seeding with 45,000 cells per well , followed by transfer to a PVDF membrane . The membrane was blocked with 5% skim milk powder in PBS with 0.1% Tween20 for 1 h at room temperature. The anti-lubricin primary antibody  was diluted 1:1,000 in half-strength blocking buffer, and the membrane was incubated overnight at 4 °C. The secondary antibody  was diluted 1:10,000 in PBS with 0.1% Tween20 and incubated for 1 h at room temperature. The immunoblot was developed in enhanced chemiluminescence substrate  and imaged on a ChemiDoc  imaging system. The signal intensities were analyzed using ImageJ. Investigators were blinded to radiographic or sample status for all outcome measures. Normality of each sample distribution was assessed prior to statistical analysis with the Shapiro–Wilk test (α = 0.05). For radiographic data, linear weighted kappa tests were used to assess the inter-rater agreement, with kappa values < 0.20 being poor, 0.21–0.40 being fair, 0.41–0.60 being moderate, 0.61–0.80 being good, and 0.81–1.0 being very good. Control subjects with global OA scores > 1 were excluded from analysis as these were not considered healthy controls due to the radiographic presence of OA.post-hoc Dunnett’s test (α = 0.05) was used for cell count and absorbance value in the cell adhesion assay with the serum-free media treatment considered the control. Wilcoxon rank-sums tests (α = 0.05) were used to analyze differences between all other data sets, including radiographic OA grades and synovial fluid lubricin, HA, and cytokine concentrations. Spearman’s rank-order correlations were performed to determine associations between injury duration, lubricin concentration, and inflammatory cytokine/chemokine concentrations. A p value < 0.05 was considered significant. The linear weighted kappa test was performed in MedCalc 18.10 , and the remaining statistical analyses were performed in JMP Pro 13 .Thresholds to discriminate between control and RCCL groups for radiographic parameters and synovial fluid lubricin and HA concentrations were obtained from the value that maximized sensitiviy-(1-specificity), or the Youden Index, on the receiver operating characteristic curve. An ANOVA test (α = 0.05), followed by Supplementary informationSupplementary information


Consideration of heart health for cancer survivors is increasingly important, as improved cancer survivorship has resulted in a growing number of survivors affected by cardiovascular disease. However, there is limited knowledge of cardio-oncology among both patients and a variety of health professionals. Thus, efforts are needed to increase awareness about cardio-oncology. Social media represents one potential opportunity to disseminate information about cardio-oncology to a large audience. We highlight one example of a social media educational/advocacy campaign conducted on Twitter (a “Twitter Chat”) that garnered nearly 1.2 million impressions (views by Twitter users) in just 24 h. We provide both quantitative and qualitative data to support the efficacy of using Twitter for such educational/advocacy campaigns, and describe key features that contributed to its success. Twitter Chats inexpensively utilize innovative technology to provide education and foster community. Long-term studies are needed to understand whether Twitter Chats can change knowledge and behavior related to cardio-oncology. Cardio-oncology is an emerging subspecialty focusing on the prevention and management of cardiovascular injury from cancer therapies , 2. It iSocial media represents one potential opportunity to disseminate medical information to a large audience. Social media offers a way to distinguish and disseminate medical information much more rapidly and broadly than through traditional peer review and subsequent health communication efforts . In 2019http://twitter.com) has been embraced by interdisciplinary professionals in cardio-oncology around the world. Twitter provides opportunities for networking, support, collaboration, and education for individuals across specialties, including cardiology, hematology, and medical and radiation oncology. Practitioners from these specialties have pioneered the use of the hashtagsThe microblogging website Twitter  hosted a Twitter Chat focusing on cardio-oncology. SBM began hosting Twitter Chats in May 2018, utilizing the hashtag #BehavioralMedChat. Since then, there have been 20 #BehavioralMedChat events with over 8000 unique tweets. Cardio-oncology was chosen as the #BehavioralMedChat topic for February 2020, due to increasing interest in the topic within SBM. Given the interdisciplinary nature of both SBM and cardio-oncology, this Twitter Chat was designed to appeal to a diverse audience, including researchers, clinicians, cancer patients and survivors, and the lay public. As such, we invited six guests including two cardio-oncologists (Doctors of Medicine [MDs]), a registered dietitian (RD), a registered social worker (RSW), and two patient advocates. The four discussion questions for the chat were similarly developed with a diverse audience in mind, and were designed to be answerable by both experts and non-experts , 621,562 , and 308,945 . Thus, the cardio-oncology Twitter Chat generated nearly 2.5 times more impressions than the average of the three prior SBM Twitter Chats. This high number of impressions in a short amount of time is likely attributable to the cardio-oncology Twitter Chat’s interdisciplinary audience with a high number of non-overlapping followers engaging with intentionally high-quality material.Another important Twitter analytic is “engagement rate”, or the number of times that a Tweet was engaged with  divided by the number of impressions. Thus, engagement rate demonstrates the value of posted content through followers’ interactions with that content. The engagement rate for posts was obtained in one of two ways. The engagement rate for Tweets posted by the Society of Behavioral Medicine Twitter account  was obtained directly from Twitter. The engagement rate for posts from other accounts hosting comparison Twitter Chats was obtained via Popsters, a commercial social media analytics tool . EngagemOf the four questions posted during the cardio-oncology Twitter Chat, question #1 had the highest engagement rate (66 engagements/3935 impressions = 1.7%). The next highest engagement rate was for question #3 (53 engagements/3611 impressions = 1.5%), followed by questions #2 (41 engagements/3278 impressions = 1.3%) and #4 (22 engagements/2802 impressions = 0.8%). For reference, the median Twitter engagement rate across industries is 0.045% . SpecifiSeveral other factors may have contributed to the high engagement rate for the cardio-oncology chat. First, in reviewing the top tweets from the cardio-oncology Twitter Chat, tweets generally fell into four categories: 1) knowledge impartation (K), 2) advocacy awareness (A), 3) interdisciplinary collaboration (I), and 4) learning impact (L), which we have termed “KAIL”. Examples of each category are provided in Fig. The emerging role of social media in general – and Twitter in particular – in the dissemination of health information has been recognized for some time , 19. TwiHowever, less is known about the most effective ways to disseminate high-quality health information through social media. In one qualitative study of 17 physicians, participants frequently described uncertainty about strategies for social media use . FurtherWe submit that Twitter Chats help overcome many of the pitfalls of social media use for health care professionals. Specifically, Twitter Chats have three primary advantages. First, Twitter Chats are inherently structured to facilitate engagement and two-way communication. Second, the simultaneous use of a designated hashtag by all participants during the Twitter Chat helps facilitate real-time conversation about a specific topic. Finally, by bringing together a diverse group of guests and participants – each with their own followers – Twitter Chats can expand the reach of posts far beyond the audience of a single user.In sum, Twitter Chats inexpensively utilize innovative technology to provide education and foster community. Long-term studies are needed to understand whether Twitter Chats can change knowledge and behavior related to cardio-oncology. The impact of Twitter Chats on knowledge dissemination could be operationalized as an increase in altmetrics , on