Skin and oral mucosa are continuously exposed to potential metal sensitizers while hosting abundant microbes, which may influence the host response to sensitizers. This host response may also be influenced by the route of exposure that is skin or oral mucosa, due to their different immune properties.Streptococcus mitis influences the host response to nickel sulfate (sensitizer) and titanium(IV) bis(ammonium lactato)dihydroxide (questionable sensitizer) in reconstructed human skin (RHS) and gingiva (RHG).Determine how commensal S. mitis for 24 hours. Histology, cytokine secretion, and Toll‐like receptors (TLRs) expression were assessed.RHS/RHG was exposed to nickel or titanium, in the presence or absence of S. mitis increased interleukin (IL)‐6, CXCL8, CCL2, CCL5, and CCL20 secretion in RHS but not in RHG; co‐application with nickel further increased cytokine secretion. In contrast, titanium suppressed S. mitis–induced cytokine secretion in RHS and had no influence on RHG. S. mitis and metals differentially regulated TLR1 and TLR4 in RHS, and predominantly TLR4 in RHG.S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG, whereas titanium remained inert. These results indicate the important influence of commensal microbes and the route of exposure on the host's response to metals.Co‐exposure of  S. mitis and metals.Host response to  They are both capable of hosting abundant microbes, responding to environmental assault, and maintaining immune homeostasis.Nickel is classically regarded as a contact sensitizer because nickel allergy on the skin and oral mucosa is frequently reported.Allergic manifestations to titanium‐based implants after orthopedic or dental surgery are also being increasingly reported.Staphylococcus aureus.Because microbes trigger innate immune responses similar to those triggered by metals, it can be expected that microbes will also influence sensitization to metals. Furthermore, emerging evidence suggests that microbes maintain a symbiotic relationship with the host and influence both physiological and pathological host events via a group of host receptors known as the toll‐like receptors (TLRs). TLRs have been repeatedly shown to participate in a broad range of host events: nickel‐induced allergy,Streptococcus mitis, a facultative commensal bacteria found on skin and on oral mucosa.The aim of this study is to determine how commensal bacteria influence the response of skin and oral mucosa to potential metal sensitizers in vitro. We exposed RHS and RHG, consisting of a stratified differentiated epithelium on a fibroblast‐populated dermis/laminal propria (collagen hydrogel), to a mixture of metal and bacteria: nickel (II) sulfate hexahydrate or titanium(IV) bis(ammonium lactato)dihydroxide was co‐applied with 22.1Human neonatal foreskin was obtained after informed consent from patients undergoing routine surgical procedures. Human non‐inflamed gingival tissue was obtained from healthy donors undergoing dental implant surgery or wisdom tooth extraction. Skin and gingiva were used anonymously and in accordance with the “Code for Proper Use of Human Tissue” as formulated by the Dutch Federation of Medical Scientific Organizations. Procedures are approved by the local medical research ethics committee of the Amsterdam UMC.6 cells) were seeded onto fibroblast‐populated collagen hydrogels in a 24 mm diameter transwell  and cultured submerged for 3 days. To induce epithelial differentiation, the cultures were then lifted to an air‐liquid interface and cultured for an additional 10 days. Twenty‐four hours before exposure and also at the time of exposure, the cultures were refreshed with medium without penicillin‐streptomycin or hydrocortisone. Cultures were incubated at 37°C, 7.5% CO2, and culture medium was refreshed twice a week.RHS and RHG were constructed exactly as described previously.2.2S. mitis LMG 14557 was cultured anaerobically at 37°C in a modified semi‐defined medium (pH 7.0) prepared exactly as described previously, in the presence of 1% glucose.S. mitis was cultured overnight for 24 hours, and then the pre‐culture was diluted into 1:1000 for another 16 hours growth until within the exponential phase (OD600 around 0.5). OD600 value was measured using SpectraMax Plus 384 . The number of colony forming units (CFUs) of S. mitis at the time of exposure was determined by viable bacterial cell counting (CFU/mL): A sample was taken from the prepared S. mitis exposure, serial dilutions were made and plated on the brain hart infusion (BHI) agar plates, and colonies were counted after 96 hours of anaerobic incubation at 37°C.2.3Titanium(IV) bis(ammonium lactato)dihydroxide solution  and nickel (II) sulfate hexahydrate  were used at the following concentrations: titanium: 20 mg/mL (68 mM) and 40 mg/mL (136 mM); nickel: 3 mg/mL (20 mM) and 15 mg/mL (97 mM). These concentrations were selected because they showed no more than a 5% decrease in metabolic activity ‐2,5‐diphenyltetrazolium bromide assay) in reconstructed human epidermis, compared to unexposed cultures.S. mitis was performed as follows: sterile gauze filters  were placed on top of the RHS or RHG cultures. Then 25 μL of prepared mixture of the following four combinations were applied onto the center of the filters: (a) Hank's Balanced Salt Solution  as control; (b) 109 CFU/mL of S. mitis in HBSS; (c) nickel or titanium at two different concentrations as indicated above; (d) 109 CFU/mL of S. mitis mixed with nickel or titanium at two concentrations. The impregnated filters were kept on the cultures for 24 hours at 37°C, 7.5% CO2, and 95% humidity. To control the amount of S. mitis in the mixtures, CFU counting was performed as described above (section: S. mitis growth condition).Exposure to chemicals and S. mitis (by CFU counting) and RHS/RHG (by MTT assay) was determined. S. mitis samples were taken from the surface of the RHS or RHG using a sterile microbrush. Together with the filter, the brushed samples were sonicated and plated out on BHI agar plates for CFU counting as described above. The viability of RHS and RHG was measured by MTT assay as described previously.After 24 hours exposure, the viability of the applied 2.4RHS and RHG samples were fixed in 4% paraformaldehyde and embedded in paraffin. As previously described,2.5Culture supernatants were collected at the time of harvesting and secretion of proteins determined by commercially available sandwich enzyme‐linked immunosorbent assays (ELISAs). For IL‐6, IL‐10, CCL2, CCL5, and CCL20, antibodies and recombinant proteins were purchased from R&D Systems . For CXCL8, CXCL12, and IL‐18, ELISA kits were used .2.6RHS or RHG epithelium were carefully removed from the fibroblast‐populated collagen hydrogel and washed with PBS. Next, total RNA and protein were isolated from the epithelium using a AllPrep RNA/Protein Kit  following the manufacturer's instructions. Isolated protein was further precipitated using methanol and resuspended in complete lysis‐M buffer (Sigma‐Aldrich). Thereafter, the amount of protein was measured using the Pierce BCA Protein Assay Kit , and RNA was measured using Nanodrop ND‐1000 Spectrophotometer (Thermo Fisher Scientific). The RNA and protein samples were stored at −80°C before further process.2.72 First Strand Kit (Qiagen). Quantitative Reverse Transcriptase PCR (qRT‐PCR) reactions were performed on a ViiA 7 Real‐Time PCR System  using Fast SYBR Green Master Mix (Applied Biosystems) and paired primers : HP205798, OriGene Technologies, Rockville). Individual gene expression levels were normalized to GAPDH expression levels, and shown in fold‐change compared to the RHS or RHG control.Genomic DNA elimination and complementary DNA (cDNA) synthesis were performed using RT2.8As described previously, the isolated proteins were prepared and separated on 4%‐12% Bis‐Tris Plus Gel (Invitrogen) and transferred to a Polyvinylidene fluoride (PVDF) membrane . The membranes were blocked with 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline with Tween 20 (PBST) for 1 hour and incubated with antibodies against TLR1, 2, 3, 5, and 6 ; TLR4 ; and tubulin  overnight at 4°C. Thereafter, membranes were washed three times in PBST and further incubated with infrared dye‐conjugated secondary antibodies against mouse  or against rabbit . After washing, the blots were visualized using Sapphire Biomolecular Imager  according to the manufacturer's instruction.2.9S. mitis unexposed and exposed RHS and RHG were compared using unpaired t‐tests. For the dose‐dependent effect of nickel or titanium, differences were compared using one‐way analysis of variance (ANOVA) followed by Bonferroni's multiple‐comparisons test. Statistics were performed using GraphPad Prism version 7.0. Data represent the mean ± standard error of the mean (SEM) of three independent experiments, each performed in duplicate, and consisted of a different skin or mucosa donor (not patient patched) and a different S. mitis inoculum. Differences were considered significant when the P‐value was < .05; *denoting P < .05, **P < .01, and ***P < .001. Data are represented as mean ± SEM.Differences between 33.1Exposure conditions have no detrimental effect on RHS or RHG histology. Both the RHS and RHG featured a stratified, differentiated epithelium on a fibroblast‐populated collagen hydrogel Figure . SimilarS. mitis rRNA (in red) present in the form of biofilms on top of both RHS and RHG and also sparingly within the epithelium (DAPI‐stained keratinocyte nuclei and S. mitis DNA in blue). As examples, RHS exposed to S. mitis  showed the s Figure , RHS expn Figure . After 2s Figure . No signe Figure , or whenm Figure .3.2S. mitis on RHS and RHG, pro‐inflammatory and antimicrobial cytokine secretion was determined  from RHS. Titanium, in the presence or absence of S. mitis, did not influence cytokine secretion from RHG. IL‐1α, IL‐1β, IL‐10, and CCL28 levels were below the detection limit of the ELISA in both RHS and RHG in all experimental conditions (data not shown). Taken together, these results indicate that nickel induces innate cytokine release, which is further enhanced by S. mitis in RHS but not in RHG. In contrast, titanium is inert and even suppresses S. mitis–mediated cytokine release from RHS.In contrast to nickel, exposure of titanium to RHS or RHG did not result in a dose‐dependent increase in cytokine secretion. Notably, titanium even suppressed the 3.4S. mitis and metal exposure. The mRNA expression of TLR2, 4, and 6 was undetectable in all conditions (data not shown). In RHS, TLR1, 3, and 5 mRNA was not regulated (neither increased nor decreased) under any of the experimental conditions investigated  resulted in increased TLR1 mRNA expression  followed by a decrease at the higher concentration (15 mg/mL)  in the complex organotypic model. TLR2, 4, and 6 mRNA levels were not regulated in RHG under any of the experimental conditions studied (data not shown).Exposure of RHG to n Figure . When S.) Figure . In contS. mitis alone did not influence this TLR1 expression, whereas nickel greatly reduced TLR1 expression. When co‐exposed with S. mitis, high nickel concentrations also reduced TLR1 expression. Titanium, either alone or co‐exposure with S. mitis resulted in pronounced clear decrease in TLR1 expression. In contrast, TLR4 expression was suppressed in RHS after S. mitis exposure, as well as metal exposure (concentration dependent). However, co‐exposure of S. mitis with nickel or titanium resulted in TLR4 protein being detectable again. Taken together, we show that for RHS, TLR1 and TLR4 protein was decreased or unaltered by S. mitis and/or metal exposure compared to unexposed RHS  increased TLR4 expression.In contrast to RHS, TLR1 protein levels were negligible in RHG with only a slight band being visible when RHG were exposed to nickel Figure . HoweverS. mitis and metal exposure are able to differentially regulate TLR1 and 4 expression in RHS and RHG, with TLR1 and 4 being involved in RHS, and predominantly TLR4 being involved in RHG.Taken together, these results indicate that 4S. mitis, influence the host response to metals in reconstructed human skin (or RHS) and gingiva (or RHG). Nickel and titanium were chosen for our study because nickel is a well‐characterized contact sensitizer,S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG. In comparison, titanium remained inert  to commensal microbes, and none have shown such well‐preserved tissue integrity after 24 hours of co‐culture. For example, a recent study described gene regulation in RHG after Streptococcus oralis biofilm exposure via a titanium implant inserted into the RHG for 24 hours; however, no data on tissue viability and limited data on tissue integrity were shown.Candida albicans for 24 hours. However, this study also showed an increased number of caspase‐3 positive cells, strongly suggesting a significant decrease in tissue viability.S. mitis increased the cytokine release of IL‐6, CXCL8, CCL5, and CCL20 in RHS but not in RHG. Such relative inertness of RHG to S. mitis is supported by findings of others describing the regulatory role of S. mitis as an oral commensal on immune balance, where it inhibits CD4+ T cell proliferation, promotes Th17 responses, and induces the secretion of IL‐10 in human monocytes.S. mitis affects the skin. Taken together, our results and those of others support the characteristic of oral mucosa in balancing a potential overactivated immune response induced by commensals. This is necessary for maintaining oral health because the oral cavity is continuously exposed to food antigens, sensitizers, and abundant microorganisms, for example, viruses and fungiPreviously we have described intrinsic differences between skin and gingiva with regard to innate immunology, wound healing, and the response to contact sensitizers.This article describes in detail the results obtained from foreskin keratinocytes. Our previous study using adult abdominal skin showed secreted protein concentrations falling within the ranges observed for the foreskin donors in all cases.S. mitis strongly enhanced the innate immune response to nickel in RHS, especially the release of CXCL8. In contrast, the S. mitis– and nickel‐mediated cytokine response in RHG was considerably lower than in RHS, in line with a more tolerogenic environment  may2NP is also inert for THP‐1 macrophages because it does not influence genes involved in modulating macrophage maturation, inflammatory responses, chemotaxis, and leukocyte migration.2 cannot induce skin sensitization in LLNA in mice.2NPs were found to act as an adjuvant when co‐applied with the bacterial fragments LPS and peptidoglycan;In contrast to nickel, titanium suppressed t Figure . Indeed S. mitis and metals differentially regulate TLR1 and 4 in RHS, and predominantly TLR4 in RHG  have important roles in sensitization and in host–microbe interactions; however, they are absent in the present study. Furthermore, we used only a single bacteria species for the exposure, whereas in vivo, a broad variety of other microbial species  are also in contact with the skin and the oral mucosa. The influence from these on host tissues, especially the pathogens, may provide more information to interpret conflicting results from in vitro and in vivo studies. In addition, sensitization occurs on host tissues, which are already influenced and primed by local commensal microbes, whereas in this study Lin Shang: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; writing‐original draft; writing‐review and editing. Dongmei Deng: Conceptualization; data curation; formal analysis; methodology; supervision; writing‐review and editing. Sanne Roffel: Data curation; formal analysis; investigation; methodology; writing‐review and editing. Susan Gibbs: Conceptualization , funding acquisition , project administration (lead), resources (lead), supervision (lead), validation , writing‐review & editing .Susan Gibbs is scientific advisor for Geneus BioTech CRO (SME). The other authors have no interests to declare.


All prediction models are made publicly available at predictdb.org.The integration of transcriptomic studies and genome‐wide association studies (GWAS) via imputed expression has seen extensive application in recent years, enabling the functional characterization and causal gene prioritization of GWAS loci. However, the techniques for imputing transcriptomic traits from DNA variation remain underdeveloped. Furthermore, associations found when linking eQTL studies to complex traits through methods like PrediXcan can lead to false positives due to linkage disequilibrium between distinct causal variants. Therefore, the best prediction performance models may not necessarily lead to more reliable causal gene discovery. With the goal of improving discoveries without increasing false positives, we develop and compare multiple transcriptomic imputation approaches using the most recent GTEx release of expression and splicing data on 17,382 RNA‐sequencing samples from 948 post‐mortem donors in 54 tissues. We find that informing prediction models with posterior causal probability from fine‐mapping ( These mechanisms help elucidate the function of loci identified in genome‐wide association studies (GWAS) by identifying potential causal genes that link genetic variation with complex traits  Project  between distinct causal variants can lead to noncausal associations between predicted expression and complex traits . This data arises from 17,382 RNA‐seq samples from 54 tissues of 948 post‐mortem subjects, aligned to the GRCh38 assembly. Primary and extended results generated by consortium members are available on the Google Cloud Platform storage accessible via the GTEx Portal (see URLs).Eight hundred and ninety‐nine whole‐genome sequencing samples were analyzed, 68 of them at an average coverage of 30x on HiSeq200, and the rest on HiSeqX. Eight hundred and sixty‐six GTEx donors' samples were included in the downstream variant call files, after excluding one each from 30 duplicate samples and 3 donors. Among these, 838 subjects with RNA‐seq data were included for QTL mapping and analysis.N < 150 per tissue; 30 factors for 150 ≤ N < 250; 45 factors for 250 ≤ N < 350; and 60 factors for N ≥ 350. Expression phenotypes were adjusted for unwanted variation using covariates such as gender, sequencing platform, and PCR protocol, the top five principal components from genotype data, and said PEER factors. Finally, fastQTL  results published in Aguet et al.  < 0.05 in these samples were included. We used 49 tissues with sample sizes ranging from 65 (Kidney Cortex) to 602 .This ancestry restriction mitigated problems due to LD mismatch when integrating with most publicly available GWAS summary statistics, which are conducted on predominantly European populations. Prediction models in other ancestries are important, and we are currently dedicating substantial effort to creating and analyzing such models. However, non‐European models are beyond the scope of this paper.We only generated models for genes annotated in GENCODE v26 as protein‐coding, long noncoding RNA (lncRNA), or pseudogenes.2.3We fitted an Elastic Net model for each gene–tissue pair with available adjusted expression data. We restricted the set of variants to those present in the HapMap 3 CEU track  of a variant affecting gene expression to select explanatory variables, without restricting to variants in the HapMap CEU track. For every gene, we used all variants in the gene's cis‐window with MAF > 0.01 and PIP > 0.01. Since dap‐g groups variants in clusters according to LD, we kept the top variant (by PIP) per cluster to avert variable redundancy. Since we reasoned that more probable variants should bear more impact in the model's outcome, we multiplied each variant's penalty term in the Elastic Net regularization by a factor of 1 − PIP. We used the same thresholds from the previous subsections (ρ > .1 and p < .05) to select models with acceptable prediction performance.We also trained models via the Elastic Net algorithm using fine‐mapping information to refine the list of variants to be used as explanatory variables and lent more weight to variants with higher chances of affecting expression phenotypes. To this aim, we used We refer to these models as DAPGW‐M.2.6mashr; Urbut et al., mashr on the same set of variants from DAPGW‐M models. We kept models only for eGenes and effect sizes only for variants with PIP > 0.01 (from dap‐g) at each gene–tissue pair. Unfortunately, there is no natural prediction performance measure in this scenario as cross‐validation was not performed.Finally, we explored an entirely different algorithm to determine the prediction models. We executed multivariate adaptive shrinkage in R  expression study for independent validation of prediction performance. We obtained GEUVADIS expression data and sample information from the European Bioinformatics Institute web portal at For each one of the four previous model training schemes  we predicted expression through PrediXcan  and keeping only variants with matching alleles. Then, GTEx variants with missing summary statistics for any GWAS were imputed with the best linear unbiased prediction method, a standard in the field  using 49 tissues, for a total of 17,052  tuples. We integrated with t et al. .When analyzing the versatility of the models and GWAS preprocessing schemes, we used GWAS studies not belonging to the rapid GWAS study. This was decided because the rapid GWAS project has a common, homogeneous variant set that could dominate comparisons.2.9SEs) of the estimated AUC were estimated by a bootstrap approach using 2,000 replicates, as implemented by Robin et al. . To reduce LD misspecification problems, most apparent when applying summary statistics‐based versions of PrediXcan on GWAS of European populations, we used only European samples.We restricted the analysis to genes that are annotated as protein‐coding, lncRNA, and pseudogenes in GENCODE version 26  and prediction performance p < .05 were kept. We will refer to this family as the EN‐M models.The first strategy used the Elastic Net  and cross‐validated prediction performance threshold (p < .05) to accept models. We will refer to this family as the CTIMP‐M family. We verified that this method's performance is not significantly improved by using all available GTEx variants, as explained in the Supporting Information Materials.The second strategy used CTIMP . Only those models achieving good enough cross‐validated prediction performance (p < .05) and correlation (ρ > .1) were kept. We will refer to this family as DAPGW‐M (dap‐g weighted). As discussed later, the cross‐validated prediction performance of this approach cannot be fairly compared to EN‐M and CTIMP‐M because the preselection of fine‐mapped variants is based on the same underlying data.The third strategy used the PIP of a variant being causal for gene expression as estimated by the Bayesian fine‐mapping method mashr  and 686,241 from MASHR‐M (143% more than EN‐M). Please note that given the different criteria used to accept a model as valid, simple counts of available models should not be considered a measure of performance.We show the distribution of cross‐validated prediction performances in Figure 3.2Next, we sought to validate the models' predictions in an independent RNA‐seq data set. We analyzed data from the GEUVADIS project  for genes present in all four methods on the LCL tissue. We observed that all four families achieved similar levels of performance, with MASHR‐M, DAPGW‐M, and CTIMP‐M faring slightly but consistently better than EN‐M. Mean correlations were .028 (SE = 0.006), .027 (SE = 0.006), and .018 (SE = 0.006) points larger for MASHR‐M, DAPGW‐M, and CTIMP‐M, respectively compared to EN‐M.To compare prediction performances, we used Spearman's rank correlation coefficient n = 115 individuals, much lower than the 341 available in GEUVADIS.We attribute the smaller performance differences to low power, since GTEx LCL tissue has a sample size of 3.3Next, we assessed whether any of these models perform better at identifying causal genes. We considered the number and proportion of colocalized genes among the significant ones as measures of association quality.p‐value below the Bonferroni‐corrected threshold (0.05/number of gene–tissue pairs) within each trait.We used the four families of models to correlate predicted expression with 87 phenotypes through 49 tissues using the summary version of PrediXcan. Results of applying the EN‐M models to GWAS summary statistics, harmonized and imputed to GRCh38  that a trait shares causal variants with a gene's expression (or an intron's splicing quantification), within a GWAS region and the overlapping gene's cis‐window. We say that a gene–tissue pair is “colocalized” with a trait if it achieves an enloc rcp > 0.5. Note that rcp ≤ 0.5 should not be interpreted as a false association; rather, it only means that there is not enough evidence of colocalization. See Section We used We say that a gene–tissue pair that is both significant and colocalized is a “prioritized” detection or candidate. To simplify the interpretation of results across multiple tissues, we count the number of unique genes among the prioritized gene–tissue pairs for each trait.SD] = 44.4), 25.5 (SD = 40.3), 36.7 (SD = 57.8), 36.6 (SD = 57.0) genes were identified with EN‐M, CTIMP‐M, DAPGW‐M, and MASHR‐M, respectively. We display the numbers of detections for each trait in Figure SE = 0.013), 8.88% (SE = 0.013), 9.01% (SE = 0.013), 11.7% (SE = 0.013) of identified genes with EN‐M, CTIMP‐M, DAPGW‐M, and MASHR‐M, respectively, were colocalized. In general, we observed that associations obtained through both DAPGW‐M and MASHR‐M models tend to agree .We found that MASHR‐M typically yields more candidate genes. On average 28.3  and 0.0682 (SE = 0.0287), respectively, providing evidence that MASHR‐M models are better equipped for detecting known genes and reinforcing our choice of MASHR‐M as the best option.When considering the AUC for the combined OMIM and rare‐variant‐based silver standards, we computed the point estimate and estimated the 3.5The prediction models' usefulness depends on the availability of their variants in the GWAS of interest. Publicly available GWAS use different sequencing and genotyping techniques, based on different genotype imputation panels and human genome release versions so that the lists of available variants vary wildly across traits. Thus, a GWAS might lack particular variants from a prediction model, so that the model cannot properly infer variation patterns as shown in Barbeira, Pividori et al. . Since m1.Harmonization only (no imputation): Simple harmonization of variants by lifting over genomic coordinates from the GWAS to match the GRCh38‐based GTEx prediction models, and then filtering for matching alleles (“Harmonization” for short)2.Harmonization and imputation of missing summary statistics (“Imputation” for short) on harmonized GWAS.We compared S‐PrediXcan results from MASHR‐M models on 69 publicly available GWAS with two preprocessing schemes:The 69 traits included in this analysis are those among the 87 traits not belonging to the Rapid GWAS project, to prevent the highly homogeneous Rapid GWAS datasets from dominating comparisons.We show in Figure Figure Figure Figure Therefore, we recommend to always perform variant harmonization due to its low complexity and time requirements, followed by summary‐statistics imputation if possible. For newer GWAS with modern sequencing and genotyping, summary‐statistics imputation may not be as critical depending on their intersection with model variants.4dap‐g) and cross‐tissue patterns (from mashr) improve the reliability of causal gene detection. These models (MASHR‐M) yield more detections when integrating GWAS and eQTL studies and show improved performance when validating results in a silver standard of known gene‐to‐trait associations (OMIM database). We make all prediction models and results publicly available.Through extensive analysis of different model training schemes, we conclude that using fine‐mapping information . EN‐M additionally features some “built‐in” redundancy: for a set of variants in LD among each other, they all tend to be included in a model with the effect spread between them.While our recommended MASHR‐M method offers several benefits compared to existing approaches, there is still room for improvement. Potential developments could rely on fine‐mapping methods that jointly incorporate cross‐tissue patterns or consensus between different fine‐mapping approaches. Also, epigenetic information has been shown to improve transcriptome prediction (Zhang et al., Our validation in silver standards, especially our difficulty interpreting the results from the rare‐variant‐based silver standard, also illustrates the need for well‐curated, large databases of known gene‐to‐phenotype associations to assess the performance of either new or improved methods.In conclusion, we present here a method for predicting the genetically regulated component of transcriptomic traits with a superior performance both in terms of prediction performance and gene–trait association detection.F. A. is an inventor on a patent application related to TensorQTL. S. E. C. is a cofounder, chief technology officer, and stock owner at Variant Bio. E. T. D. is chairman and member of the board of Hybridstat Ltd. B. E. E. is on the scientific advisory boards of Celsius Therapeutics and Freenome. G. G. receives research funds from IBM and Pharmacyclics, and is an inventor on patent applications related to MuTect, ABSOLUTE, MutSig, POLYSOLVER, and TensorQTL. S. B. M. is on the scientific advisory board of Prime Genomics Inc. D. G. M. is a cofounder with equity in Goldfinch Bio, and has received research support from AbbVie, Astellas, Biogen, BioMarin, Eisai, Merck, Pfizer, and Sanofi‐Genzyme. H. K. I. has received speaker honoraria from GSK and AbbVie. T. L. is a scientific advisory board member of Variant Bio with equity and Goldfinch Bio. P. F. is a member of the scientific advisory boards of Fabric Genomics Inc., and Eagle Genomes Ltd. P. G. F. is a partner of Bioinf2Bio. E. R. G. receives an honorarium from Circulation Research, the official journal of the American Heart Association, as a member of the Editorial Board, and has performed consulting for the City of Hope/Beckman Research Institute. R. D. has received research support from AstraZeneca and Goldfinch Bio, not related to this study.Supplementary InformationClick here for additional data file.


AbstractTo analyze the use of serum cancer antigen 19‐9 (CA19‐9), cancer antigen 125 (CA‐125) and carcinoembryogenic antigen (CEA) in predicting the malignant potential of mucinous ovarian tumor, and to assess the clinical factors associated with these tumors.This retrospective study collected the data from 314 patients who were diagnosed with mucinous ovarian tumor. These patients had preoperative serum CA19‐9, CA‐125, CEA available and underwent surgery at Ramathibodi Hospital between January 2010 and December 2016. The diagnostic performance of CA19‐9, CA‐125 and CEA was analyzed using the receiver operator characteristic curve. The associations between clinicopathological factors and serum CA19‐9, CA‐125 and CEA level were also analyzed.P = 0.005), 1.74 , 1.90 , 1.58 , respectively. CA‐125 provided the highest diagnostic performance, with an area under receiver operator characteristic curve of 0.745, to differentiate between borderline, malignant or benign mucinous ovarian tumor.A total of 314 patients were recruited in this study. They consisted of 221 patients with benign mucinous ovarian tumor (70.38%), 65 patients with borderline mucinous ovarian tumor (20.70%) and 28 patients with mucinous ovarian carcinoma (8.92%). Multivariate analysis revealed that the tumor size, elevated serum CA19‐9, CA‐125 and CEA influenced the tumor pathology. The mucinous ovarian tumor with large tumor size, elevated serum CA19‐9, CA‐125 and CEA more than the cut off values showed a positive correlation with the risk ratio of 1.60 (95% CI = 1.13–2.28; Preoperative elevation of the serum CA19‐9, CA‐125, CEA and tumor size are useful predictors to differentiate between benign, borderline and malignant mucinous ovarian tumor. The best predictor is CA‐125, followed by CA19‐9 and CEA. Mucinous ovarian tumors account for 15% of epithelial ovarian tumors.Management of mucinous ovarian tumors is not different from other histopathologic cell types of ovarian tumor. Factors to be considered include age, fertility‐desire, unilateral or bilateral involvement, chances of mBOT or malignancy, patients' health conditions and surgeons' experiences. In the benign condition, if the patients are young and desire fertility, the unilateral salpingooophorectomy or ovarian cystectomy is the treatment of choice depending on the intraoperative findings. If the bilaterality is present, unilateral salpingooophorectomy with unilateral ovarian cystectomy, or bilateral ovarian cystectomy can be considered. If mBOT is suspected, the surgery adopted can be similar to that as for a benign case; however the excision of suspected peritoneal lesions, omentectomy or appendectomy if the appendix appears abnormal should also be performed. The surgery for a malignant mucinous ovarian tumor is the same as other epithelial ovarian malignancies; which can be fertility‐sparing for stage I. The roles of intraoperative frozen section for mucinous ovarian tumors are limited. The accuracy of the frozen section when compared to the final pathological diagnosis has been reported at 65–70% for benign and mBOT because of the large diameter and high heterogeneity. This may be due to the enormous size of mucinous tumors that require sampling.Cancer antigen 19‐9 (CA19‐9) is elevated mostly in gastrointestinal, biliary tract or ovarian tumors, particularly in mucinous type.t‐test or Wilcoxon rank‐sum (Mann–Whitney U) test for continuous data, depending on the distribution of the data. A P‐value of 0.10 was used as a cutoff threshold to select variables for multivariate analyses. Taken into account of the outcome of interest, which was the borderline or malignant pathological results, multiple logistic regression analysis was performed by forward selection method. A P‐value of 0.05 was considered as the level of statistical significance. The diagnostic performance of these tumor markers was obtained by the receiver operating characteristic (ROC) curve, from which the area under the curve was used to compare each tumor marker in the discrimination between benign, borderline or malignant mucinous ovarian tumor. All analyses were performed using stata version 14.2 statistical software (StataCorp).After approval from the Ramathibodi Institutional Review Board, this retrospective study collected the data from 314 patients who presented with ovarian mass (es). They were admitted for surgery under the Obstetrics and Gynecology Department, Faculty of Medicine Ramathibodi Hospital, Mahidol University between January 2010 and December 2016. The inclusion criteria included those who were diagnosed with mucinous ovarian tumor by pathological confirmation with available information of preoperative serum CA19‐9, CA‐125 and CEA. The exclusion criteria included those with incomplete medical records and those who were diagnosed with other malignancies. All the pathological slides were reviewed by a gynecologic pathologist to confirm the final diagnosis based on 2014 WHO classification and rule out metastatic mucinous tumor. The demographic data included age, parity, menopausal status, intraoperative tumor size, tumors' complication , pathological results  and serum tumor marker before the surgery . Descriptive statistics were summarized by frequency and percentage, mean and standard deviation (SD), or median and interquartile range, according to the distribution of data. The univariate analysis was applied by Pearson's chi‐squared or Fisher's exact test for categorical data and Student P = 0.005), 1.74 , 1.90 , 1.58 , respectively. The sensitivity and specificity of each tumor marker  in borderline or malignant mucinous ovarian tumor for CA19‐9 were 52.7% and 83.8%, CA‐125 were 68.2% and 83.9%, CEA were 31.9% and 90.8%, respectively.The data of mucinous ovarian tumor patients who underwent surgery and preoperative examination for serum CA‐19‐9, CA‐125, and CEA at Ramathidodi Hospital during January 2010 until December 2016 revealed 314 records. There were 221 patients with benign mucinous ovarian tumor (70.38%), 65 patients with borderline mucinous ovarian tumor (20.70%) and 28 patients with mucinous ovarian carcinoma (8.92%). The mean age was 47.3 years old. The majority of the patients were multiparous (61.5%) and in premenopausal status (51.13%). There were 220 patients (72.1%) with serum CA19‐9 ≤ 39 U/mL, 212 patients (67.7%) with serum CA‐125 ≤ 35 U/mL and 250 patients (83.9%) with serum CEA ≤ 3.8 U/mL. For the tumors' characteristic, the median of tumor size was 12 cm (range 2–60 cm). Twenty‐six patients presented with bilateral tumor (8.3%) and 22 patients (7.0%) had complications . The patients' and tumors' characteristics by pathological classification are demonstrated in Table Table In predicting malignancy in ovarian tumor patients, besides history taking and physical examination, the use of tumor markers as a part of evaluation is also important. In this study, we focused on mucinous ovarian tumor, which is a subtype of epithelial ovarian tumor. The well‐known tumor marker used in the preoperative evaluation of epithelial ovarian tumor is CA‐125This retrospective study collected data of 314 patients, which was a larger sample size than that in previous studies. In addition to these serum tumor markers, tumor size is also related to the association of the malignant or borderline pathological diagnosis of mucinous ovarian tumor in our findings. In our study, larger tumor size correlated with borderline or malignant tumors with the median of 18 cm (range 3–40 cm), while that in the benign mucinous tumor group was 10 cm (range 2–60 cm). This result was similar to the study by Cho H.Y. and colleagues published in 2014,All of the three serum tumor markers  had strong association with the tumor pathology. The elevation above the widely used cut‐off levels of the serum CA19‐9 (>39 U/mL), CA‐125 (>35 U/mL) and CEA (>3.8 U/mL) was significantly associated with the borderline or malignant mucinous tumor with the risk ratio of 1.74 (95% CI = 1.22–2.47), 1.90 (95% CI = 1.34–2.70) and 1.58 (95% CI = 1.10–2.29), respectively. Moreover, the serum CA‐125 demonstrated the strongest association with the diagnosis of borderline or malignancy among those mucinous ovarian tumors, followed by CA19‐9 and CEA. The results correspond to the studies by Tamakoshi,Our study is a study that evaluated the diagnostic performance of serum CA‐125, CA19‐9 and CEA in mucinous ovarian tumor. Although CA‐125 is the best serum marker in our results, CA19‐9 and CEA were comparable. The results not only answered our research questions, but also could be applied in clinical practice as one of the preoperative tools for the differential diagnosis between borderline or malignant and benign mucinous ovarian tumors. The limitations of this study were the retrospective nature of this research, selection and information bias and incomplete patient's data. In addition, routine preoperative evaluation of these serum tumor markers has yet to be implemented.Preoperative elevation of the serum CA19‐9, CA‐125 and CEA was a useful predictor to differentiate between benign and borderline or malignant mucinous ovarian tumor. In our findings, serum CA‐125 had the best diagnostic performance, followed by serum CA19‐9 and CEA, respectively. Tumor size was also a significant associating factor. We suggest that the patients with mucinous ovarian tumor who were suspicious for borderline pathology or malignancy from the clinical manifestation, should undergo preoperative imaging for evaluation of the tumor size. In addition, these three serum tumor markers should also be preoperatively obtained in order to make a proper surgical plan.None declared.


To assess whether vaginal secretions and breast milk of women with coronavirus disease 2019 (COVID‐19) contain severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2).Single centre cohort study.Renmin Hospital of Wuhan University, Wuhan, Hubei province, China.We studied 13 SARS‐CoV‐2‐infected pregnant women diagnosed between 31 January and 9 March 2020.We collected clinical data, vaginal secretions, stool specimens and breast milk from SARS‐CoV‐2‐infected women during different stages of pregnancy and collected neonatal throat and anal swabs.We assessed viral presence in different biosamples.n = 2) and neonatal pneumonia (n = 2). One of nine stool samples was positive; all 13 vaginal secretion samples, and five throat swabs and four anal swabs collected from neonates, were negative for the novel coronavirus. However, one of three samples of breast milk was positive by viral nucleic acid testing.Of the 13 women with COVID‐19, five were in their first trimester, three in their second trimester and five in their third trimester. Of the five women in their third trimester who gave birth, all delivered live newborns. Among these five deliveries, the primary adverse perinatal outcomes included premature delivery (In this case series of 13 pregnant women with COVID‐19, we observed negative viral test results in vaginal secretion specimens, suggesting that a vaginal delivery may be a safe delivery option. However, additional research is urgently needed to examine breast milk and the potential risk for viral contamination.New evidence for the safety of vaginal delivery and breastfeeding in pregnant women infected with SARS‐CoV‐2, positive viral result in a breast‐milk sample. New evidence for the safety of vaginal delivery and breastfeeding in pregnant women infected with SARS‐CoV‐2, positive viral result in a breast‐milk sample. In December of 2019, Wuhan, in Hubei Province, China, experienced an outbreak of pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). On 11 March the World Health Organization declared that the spread of coronavirus disease 2019 (COVID‐19) was a pandemic, which is now disrupting health and public life around the world.SARS‐CoV‐2 shares 79% nucleotide sequence identity with SARS‐CoV and 50% with Middle‐East respiratory syndrome coronavirus (MERS‐CoV).Human transmission is through droplets and direct contact, though recent investigations have demonstrated that SARS‐CoV‐2 is also detectable in stool and blood.All pregnant women with SARS‐CoV‐2 admitted to Renmin Hospital of Wuhan University in China between 31 January and 9 March 2020, and their babies admitted to Wuhan Children’s Hospital , Tongji Medical College, Huazhong University of Science & Technology, were included. The diagnostic criteria for COVID‐19 were based on the Diagnosis and Treatment Protocol for the 2019 Novel Coronavirus Pneumonia published by the National Health Commission of China.We extracted demographic information, clinical course, laboratory indices and imaging results of infected pregnant women from the medical records, maternal throat swabs were collected upon admission. Samples of vaginal secretions from nine women were collected during pregnancy and samples from four women were collected after delivery. Neonatal throat and anal swabs were collected on the 1st and 3rd days after birth. Breast milk samples from three women were collected on the 1st, 6th and 27th days after delivery and stool specimens were taken from nine women between 5 and 8 March. The details of sample collection are shown in the Supplementary material Tables . We collWe reviewed the medical records of 13 pregnant women with SARS‐CoV‐2 infection; where five were infected in the first trimester, three in the second trimester and five in the third trimester. One of the infants included here has been previously reported.n = 8); followed by cough (n = 5), dyspnoea (n = 1), myalgia (n = 1) and diarrhoea (n = 1). Three women demonstrated an increased leucocyte count, two women had lymphopenia and five women had elevated C‐reactive protein levels. Impaired liver function was observed in three women , and two women had increased aspartate aminotransferase levels. Eight women manifested patchy ground‐glass opacities or consolidation shadows, with peripherally distributed computed tomography imaging features.In terms of clinical manifestations, fever was the most common symptom (+5 to 38+4 weeks. Two neonates (babies of patients 1 and 2) were born prematurely (at 35+5 and 35+6 weeks), with birthweights of 2830 g and 2300 g, respectively. The pregnancy of patient 1 was ended prematurely because of fetal distress, and patient 2 underwent a spontaneous preterm birth. The neonate of patient 5 was large for gestational age (at 3910 g)  had a biochemical pregnancy with serum human chorionic gonadotrophin concentrations dropping from 25.9 IU/l to <2 IU/l. The other four women  had ongoing pregnancies. All five women in their third trimester (patients 1–5) delivered, with only one (patient 5) undergoing vaginal birth. While patient 1 experienced dyspnoea – with fetal heart rate monitoring that suggested fetal distress – the other three women exhibited no clear medical indication for a caesarean section. Lack of knowledge regarding COVID‐19 and concerns about potential mother‐to‐child transmission as a ‘social factor’ became an indication for caesarean section in these last three pregnant women  were born prematurely and diagnosed with neonatal pneumonia by chest X‐ray  . BWM reports consultancies for ObsEva, Merck KGaA, iGenomix and Guerbet, with no potential conflicts of interest. The other 13 authors have no conflicts of interest. Completed disclosure of interests forms are available to view online as supporting information.HH, JY and TY contributed to concept and design. YW, CJZ, CZ, CD, HZ, BWM and CLD drafted the manuscript. CZ and CD performed the statistical analyses and CL, LD, YC and JL collected the data.This study was approved by the Medical Ethical Committee of Renmin Hospital of Wuhan University , Wuhan Children’s Hospital , Tongji Medical College, Huazhong University of Science & Technology  and the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University .This work was supported by the National Key Research and Development Programme of China . The funders had no role in the design or conduct of this study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit this manuscript for publication.We wish to thank the women, researchers and clinical staff who provided significant contributions to this study.Figure S1. Imaging examination of the four delivered women and their neonates.Click here for additional data file.Table S1. Data of maternal sample collection.Table S2. Data of neonatal sample collection.Table S3. Samples of clinical and laboratory characteristics.Table S4. Neonatal outcomes.Click here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disorder induced by antiphospholipid antibodies, which occurs exceedingly rarely in pediatric population and even more rarely reported in HIV positive children.A case of 11 years old boy had a sudden onset of swelling in his left lower leg along with pain which were worsening gradually. Initially, topical ointment was applied for 1 month which were ineffective in reducing pain and swelling. Instead, the symptoms were aggravated and suddenly spread to the proximal thigh, accompanied by dyskinesia of left lower leg. Both color doppler ultrasonography and vascular CT scan of left lower leg revealed deep venous thrombosis. His serum anti-phospholipid antibodies (aPLs) were tested positive. He was a known case of HIV virological failure with substantial HIV viral load (VL) despite receiving regular antiretroviral therapy (ART). His symptoms improved after giving aggressive antithrombotic and high dose corticosteroid treatments.When pediatric patients develop thrombotic disease, APS also needs to be ruled out. The autoantibodies levels should be routinely tested to look for recurrent thrombosis in children with HIV/AIDS. Antiphospholipid syndrome (APS) which is also named as Hughes Syndrome, is an autoantibodies-induced autoimmune disease characterized by recurrent arterial or venous thrombosis and/or fetal loss associated with thrombocytopenia and persistently seropositive  for anti-phospholipid antibodies (aPLs) . The pre+ T lymphocyte cell count was 522 cells/μL and plasma VL was 943 copies/mL with a blood pressure around 97/69 mmHg at that time. Initially, topical ointment was applied for 1 month which were ineffective in reducing pain and swelling. Instead, the symptoms were aggravated and suddenly spread to the proximal thigh, accompanied by dyskinesia of left lower leg. The child was a known case of HIV positive since the age of two and was believed to be infected through mother to fetus transmission. He has been taking first-line regimen of ART (AZT + 3TC + NVP) since last 4 years when his CD4+ T lymphocyte cell count had reached 346 cells/μL at that time. The plasma HIV RNA was undetectable after half a year of ART.An 11 years old boy had a sudden onset of swelling in his left lower extremity along with pain which were worsening gradually for past 1 month duration. He had no history of fever, headache, cough, chest tightness, chest pain, abdominal pain and diarrhea. His CD4His mother was HIV seropositive person. The child had no personal and/or family history of thrombotic diseases. There was no history of any prior surgery, trauma, prolonged bed rest, obesity, smoking and any other common risk factors of thromboembolic events.On physical examination during hospital admission, the maximum circumference of his left thigh was 38 cm and the right thigh was 34 cm. The maximum circumference of his left calf was 27 cm and the right calf was 24 cm. His left lower thigh was inflamed and swollen, accompanied by tenderness and presence of varicose veins.9/L with a CD4+ T lymphocyte cell count of 430 cells/μL, hemoglobin (Hb) was 126 g/L and platelets was decreased to 66 × 109/L. On virology test, HIV RNA was found to be 580 copies/mL. The coagulation function test showed PT was reported to be 14.0 s and activated partial thromboplastin time (APTT) was prolonged to 44.3 s. The international normalized ratio (INR) and Fibrinogen (FIB) were 1.16 and 15.1 mg/L respectively. D-dimer was 6.26 mg/L. C-Reactive Protein (CRP) was 32.14 mg/L, procalcitonin (PCT) was 0.029 ng/ml and erythrocyte sedimentation rate (ESR) was 41 mm/h. The aCLs IgG was at 57 , IgM was at 24 . Anti-neutrophil cytoplasmic antibody (pANCA) and anti-β2GPI antibody were tested positive. In addition, some other autoimmune antibodies were also tested positive: ANA ; dsDNA: +; nucleosome: ++; histone: +; mitochondrial M2: +. Other examinations including liver and kidney function tests were unremarkable.On hematological tests, his white blood cell (WBC) count was 3.28 × 10Color doppler ultrasonography of lower extremity vessels revealed deep venous thrombosis in his left lower leg, accompanied by soft tissue edema and superficial lymphangiectasia. Vascular CT scan of left lower extremity showed extensive mural thrombosis Fig. .Fig. 1THe was then diagnosed HIV complicated with thrombotic antiphospholipid syndrome. He was initially treated with low molecular weight heparin (LMWH) calcium 3000 IU every 12 h for 6 weeks followed by warfarin (1.25 mg/day) for long-term anticoagulant therapy at a target INR of 2.0–3.0. Besides, methylprednisolone (40 mg/day) treatment was also given to the patient for ten days which was substituted with 30 mg/day prednisone after that. The prednisone dosage was gradually reduced by 5 mg each time every 2 weeks until reached 5 mg/d and maintained for another 2 months. Additionally, antiviral regiment was adjusted to ABC + 3TC + LPV/r due to his persistent high HIV viremia.+ T lymphocyte cell count was 633 cells/μL and HIV RNA was undetectable. His left lower extremity blood vessels were returned to normal. (Table After one month of treatment, the swelling and pain were gradually relieved and vascular ultrasonography on follow up visit had showed the blocked vessel was partial recanalized and platelets count had returned to normal. After 3 months of treatment, his symptoms continued to be ameliorated. However, the aCLs and β2-GP1 antibody titers were still tested positive. After 6 months of treatment, his CD4The etiology of APS is complex and has not been well delineated. The most popular hypothesis suggested a “two hit “ mechanism in pathogenesis of APS. The first hit is the presence of aPLs antibodies and interaction with vascular endothelial cells, neutrophils, platelets and monocytes resulting in endothelial dysfunction and injury. The second hit is the facilitation from predisposing factors like infections, contraceptives, malignancies and genetic susceptibility etc. which eventually leading to thrombotic events , 5. CommThe HIV virus, as a superantigen that may even resemble some autoantibodies, can induce APS through possible “molecular mimicry” mechanisms. Moreover, HIV virus can directly damage vascular endothelial cells and subsequent generation of autoimmune antibodies. Thus, participating the “double hit” pathway in the development and progress of APS , 10. TheThe superimposed HIV infection might even further increase the risk of thrombosis in APS. The existing studies have showed that when compared with general population, HIV infected patients have 2–10-folds increased risk of developing thrombosis . FirstlyAPS is the principal cause of thrombotic disease in the pediatric population . When thHIV may induce the occurrence of APS and promote life threatening thrombotic events through several mechanisms. Therefore, the autoantibodies levels should be routinely tested in HIV/AIDS patients, especially in patients with virological failure with high levels of HIV viremia.


Further analyses showed a reduction in adenosine-recruitable coronary flow reserve, but this was exclusively the result of an increase in basal coronary blood flow, while maximal coronary flow per gram of myocardium was maintained; the latter was consistent with the unchanged arteriolar wall/lumen ratio, arteriolar density and peri-arteriolar collagen content. However, isolated small arteries displayed selective blunting of endothelium-dependent vasodilation in response to bradykinin in DM + HC + CKD swine, suggesting that changes in coronary microvascular function rather than in structure contributed to the perturbations in myocardial oxygen delivery. In conclusion, common comorbidities in swine result in CMD, in the absence of appreciable atherosclerosis, which is severe enough to produce perturbations in myocardial oxygen balance, particularly during exercise, resembling key features of INOCA.Comorbidities of ischemic heart disease, including diabetes mellitus (DM), hypercholesterolemia (HC) and chronic kidney disease (CKD), are associated with coronary microvascular dysfunction (CMD). Increasing evidence suggests that CMD may contribute to myocardial ‘Ischemia and No Obstructive Coronary Artery disease’ (INOCA). In the present study, we tested the hypothesis that CMD results in perturbations in myocardial perfusion and oxygen delivery using a novel swine model with multiple comorbidities. DM (streptozotocin), HC (high-fat diet) and CKD  were induced in 10 female swine (DM + HC + CKD), while 12 healthy female swine on a normal diet served as controls . After 5 months, at a time when coronary atherosclerosis was still negligible, myocardial perfusion, metabolism, and function were studied at rest and during treadmill exercise. DM + HC + CKD animals showed hyperglycemia, hypercholesterolemia, and impaired kidney function. During exercise, DM + HC + CKD swine demonstrated perturbations in myocardial blood flow and oxygen delivery, necessitating a higher myocardial oxygen extraction—achieved despite reduced capillary density—resulting in lower coronary venous oxygen levels. Moreover, myocardial efficiency was lower, requiring higher oxygen consumption for a given level of myocardial work. These perturbations in myocardial oxygen balance were associated with lower myocardial lactate consumption, stroke volume, and LVdP/dThe online version of this article (10.1007/s00395-020-0778-2) contains supplementary material, which is available to authorized users. Common comorbidities of cardiovascular disease, including diabetes mellitus (DM), hypercholesterolemia (HC) and chronic kidney disease (CKD), are well-known risk factors for the development of coronary artery disease of both large epicardial arteries and smaller coronary arteries , 18, 31.Although the mechanisms underlying INOCA remain incompletely understood, there is increasing evidence that CMD, in particular impaired endothelium-dependent vasodilation, plays an important role , 13, 66.All animal experiments were approved by the Animal Care Committee at the Erasmus University Medical Center  and performed in accordance with the “Guiding Principles in the Care and Use of Laboratory Animals” as approved by the Council of the American Physiological Society. Ten female Yorkshire × Landrace swine 25 ± 1 kg) were included in the DM + HC + CKD group while 12 healthy female Yorkshire × Landrace swine of similar age and weight were used as controls . Two swine assigned to the DM + HC + CKD group died prematurely, one animal died 2 weeks after CKD induction and one animal died during chronic instrumentation due to surgical complications. In the Normal group, also two swine were lost. One animal died during chronic instrumentation, while another animal experienced severe lameness prior to chronic instrumentation and was excluded from the study. Four additional swine  were included for measuring coronary flow reserve (CFR) in vivo and small artery function ex vivo. An overview of the experimental design and technical procedures is presented in Fig.  kg were −1 day−1 i.v. for 3 consecutive days. The severity and stability of DM was monitored bi-weekly by measuring blood glucose and ketone levels.The induction of comorbidities in the DM + HC + CKD group has been described in detail elsewhere . Briefly−1), Sedazine  and atropine (2 mg) and artificially ventilated  isoflurane was added for anesthesia). CKD was produced by micro-embolization of the global right kidney as well as the lower pole of the left kidney. For this purpose, the renal arteries were catheterized under fluoroscopy guidance  with a Swan–Ganz catheter, inserted through a 9F sheath in the right common carotid artery. Following inflation of the balloon to prevent back-flow into the aorta, 75 mg of polyethylene microspheres with a diameter of 38–42 μm  were infused into each kidney via the distal port of the catheter. The wound was closed and the animals were allowed to recover.Two weeks after DM induction, animals were sedated with intramuscular injection of a cocktail of Zoletil  was gradually introduced. The Normal group continued to receive regular swine chow. Animals were housed in pairs but were fed separately and had ad libitum access to drinking water.One week after CKD induction, a high-fat and high-sugar diet containing 10% sucrose, 15% fructose, 25% saturated fats and 1% cholesterol  supplemented with sodium chloride (20 g day−1), Sedazine  and atropine (2 mg), and artificially ventilated , to which 2–2.5% (vol/vol) isoflurane was added. As described in detail elsewhere [−1) for 6 days, and antibiotic prophylaxis (25 mg kg−1 amoxicillin i.v.) for 7 days. All catheters were flushed daily with heparinized saline  to prevent the formation of blood clots and maintain catheter patency. One DM + HC + CKD swine had a malfunctioning coronary flow probe.After an overnight fast, Normal and DM + HC + CKD swine (5 months after CKD induction) were sedated with an intramuscular injection of a cocktail of Zoletil . Hemodynamic variables were continuously recorded digitally on a Codas workstation , with blood samples collected during the final 30 s of each exercise stage when steady-state hemodynamics had been achieved. Blood samples were analyzed for pO2, pCO2, pH, bicarbonate, O2 saturation (SaO2), lactate and hemoglobin concentration .Experiments started 1 week after surgery. First, the glomerular filtration rate (GFR) was measured at rest, using continuous inulin infusion . On a su−1 min−1) in combination with phenylephrine (5–7.5 µg kg−1 min−1) to maintain mean arterial pressure at baseline levels [In a seperate experiment, coronary blood flow was measured in awake resting swine  under basal conditions and during maximal coronary vasodilation using intravenous infusion of adenosine (0.5 mg kge levels . CFR was−1), Sedazine  and atropine (2 mg) and anesthetized with pentobarbital (9 mg kg−1 h−1 i.v.). Subsequently, a sternotomy was performed and ventricular fibrillation was induced using a 9 V battery, and immediately the heart, kidneys, liver and pancreas were excised, weighed, prepared, and stored for later biochemical, molecular, and histological analyses.At the termination, swine were sedated by intramuscular injection with a cocktail of Zoletil  and the nitric oxide donor and endothelium-independent vasodilator S-nitroso-N-acetylpenicillamine  were measured following preconstriction with 10−6 mol L−1 of the thromboxane-A2 analogue U46619 (Sigma-Aldrich).In a subgroup of animals , coronary small arteries (∼ 300 µm diameter) were isolated from the epicardial surface of the left ventricular apex and studied in vitro using a Mulvany wire myograph . Vasodilation to the endothelium-dependent vasodilator bradykinin , high-density lipoprotein (HDL), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), albumin, sodium, and creatinine, as previously described . ArteriaSamples of the pancreas, liver, kidney, left anterior descending artery, right coronary artery, left circumflex artery, and left ventricular anterior free wall were excised, cryo-embedded in Tissue-Tek or fixed in 4% buffered formaldehyde, and embedded in paraffin for histological analyses. 4.5-μm-thick slides of the pancreas were stained for insulin . Six to eight fields at 200 × magnification were analyzed and data were averaged per animal. Cryosections of the liver (4 μm thick) were stained with Oil Red O (Sigma-Aldrich) for quantification of liver fat deposition. Five to seven liver lobes were analyzed and data were averaged per animal.From formaldehyde-fixed, paraffin-embedded kidneys, 3-μm sections were sliced and stained. For the analysis, the upper pole of the left kidney, which was not embolized, was used. Tubulo-interstitial damage and glomerulosclerosis were scored on periodic acid–Schiff-stained sections in a blinded manner. The tubulo-interstitial damage was scored in at least 20 different non-overlapping fields per animal at a magnification of 200×. The amount of inflammatory infiltrate between tubuli, interstitial fibrosis, tubular atrophy, and dilatation were scored on a scale of 0–5, with 0 indicating not present, and 5 indicating that > 75% of all tubuli were affected. A total tubulo-interstitial damage score was calculated by summing the scores for the four variables. Glomerular score was performed at a magnification of 400× on 50 separate glomeruli. The analysis was done by quadrants, with the score of 0 indicating that no quadrant was affected and 4 indicating that the whole glomerulus was affected. Scored variables were matrix expansion, sclerosis, adhesion of Bowman’s capsule and dilation, from which a total glomerulosclerosis score was then calculated. All renal histological analyses were performed by observers blinded to the treatment of the animal.2) and expressed as ratio between the perivascular area and the lumen diameter. The latter was defined as the area encompassed by two times the distance from the lumen center to the external elastic lamina in all directions. Cross-sectional areas of cardiomyocytes with clearly visible nuclei were measured for each slide, using a Gomori silver stain. Capillary density was quantified using an endothelial cells staining with biotin-labeled lectin . All vessels smaller than 10 μm in diameter and without vascular smooth muscle cells were counted. Capillary to fiber ratio was calculated by dividing capillary density by the total number of cardiomyocytes per mm2.Left ventricular anterior wall sections (4.5 μm thick) were stained for the quantification of myocardial collagen deposition, myocyte size, and capillary density. Six to eight fields were examined in the endocardial part of each slide, at 200 × magnification. Collagen deposition was assessed using Picrosirius red staining, with interstitial and perivascular collagen deposition being analyzed separately. Using light microscopy, interstitial collagen was measured as area occupied by all collagen fibers and expressed as a percentage of the myocardial area, perivascular collagen being excluded from this analysis. Using a linear polarization filter, the percentage area of the myocardium occupied specifically by collagen type I and III fibers was measured . PerivasRight coronary artery, left circumflex artery, left anterior descending artery, and left ventricular sections were stained with resorcin–fuchsin solution to assess media-to-lumen ratios of large coronary arteries and coronary arterioles . Smooth muscle actin was stained  in left ventricular sections to determine coronary arteriolar densities, according to an earlier described protocol ; in shor2 consumption (BVO2) was calculated as the product of cardiac output and the difference in O2 content between arterial and mixed venous blood. Systemic vascular conductance (SVC) was computed as the ratio of cardiac output and mean arterial pressure. Myocardial O2 and lactate delivery were computed as the product of coronary blood flow and arterial blood O2 content or arterial lactate concentration. Myocardial O2 (MVO2) and lactate consumption were calculated as the product of coronary blood flow and the difference in O2 content or lactate concentration between arterial and coronary venous blood. Myocardial oxygen extraction was computed as 100% MVO2/Myocardial O2 delivery. Myocardial work was computed as the product of cardiac output and systolic arterial pressure. Systemic parameters, including cardiac output, BVO2, SVC, cardiac work, and stroke volume, were normalized for body weight in kilograms (kg). Myocardial parameters including MVO2, coronary blood flow, myocardial oxygen and lactate delivery, and myocardial lactate consumption were normalized per gram (g) of myocardium perfused by the left anterior descending coronary artery, which was estimated to be 40% of the left ventricle [Data are presented as mean ± SEM. Digital recording and offline analysis of hemodynamic data obtained at rest and during exercise was performed. Body Oentricle , 30.t test. Correlations were calculated by Pearson’s correlation. Statistical significance was accepted when p < 0.05 (two tailed).Statistical analysis was performed in SPSS Statistics 21.0 . Two-way ANOVA for repeated measures was used to analyze tabular in vivo hemodynamic and myocardial oxygen balance responses to graded treadmill exercise, as well as the ex vivo microvascular function, with Bonferroni post hoc testing when appropriate. A two-way ANCOVA was used to analyze hemodynamic and myocardial oxygen balance data expressed as a function of whole body—or myocardial—oxygen consumption. Comparison of other variables between the two groups was performed by unpaired Student’s p = 0.10) as compared to healthy Normal swine. Pancreas staining demonstrated an ~ 80% reduction of the insulin-producing ß-cells in the islets of Langerhans in DM + HC + CKD swine, but insulin plasma levels were maintained. There was no difference in ASAT plasma levels between groups, while ALAT levels even decreased in DM + HC + CKD compared to Normal swine. In addition, DM + HC + CKD showed a trend towards an increase in liver fat deposition (p = 0.09), which correlated positively with plasma levels of total cholesterol  and correlated inversely with ASAT . Although kidney weights were not different from Normal swine, renal dysfunction was present in DM + HC + CKD swine reflected in increased creatinine plasma levels and a significantly impaired GFR, as measured by inulin clearance, increased histological scores of tubulo-interstitial injury, and glomerular sclerosis. Metabolic and renal dysfunction resulted in elevated TNF-α plasma levels. Absolute and relative left ventricular weights were not different between groups. Similarly, cardiomyocyte cross-sectional area showed no significant differences between groups. In contrast, total collagen deposition (collagen type I and III) was elevated in DM + HC + CKD compared to Normal swine. Interestingly, this elevation was due to increased deposition of collagen type I fibers, known to form thick and stiff bundles, in DM + HC + CKD compared to Normal, while deposition of the compliant thin collagen fibers (collagen type III) was unaltered . Furthermore, significant decreases in left ventricular subendocardial capillary density as well as capillary-to-fiber ratios were observed in DM + HC + CKD swine. Macroscopic and microscopic examination of the large coronary arteries, showed no signs of atherosclerosis and no changes in media-to-lumen ratio in DM + HC + CKD swine.Metabolic, renal, inflammatory, and cardiac characteristics of the DM + HC + CKD swine model at 5-month follow-up are presented in Table VO2 was blunted in DM + HC + CKD animals, which was associated with increased circulating levels of lactate  were lower in DM + HC + CKD swine at rest and during low-intensity exercise, while arterial pH was not different between groups , which appeared to be principally due to a small increase in basal flow while maximal flow was unaltered  Adenosine-recruitable coronary flow reserve was reduced, which was due to an increase in basal resting coronary blood flow per gram of myocardium. In contrast, maximal coronary blood flow per gram of myocardium was not altered, consistent with maintained arteriolar densities and wall/lumen ratios and maintained perivascular collagen content. (vi) Coronary small arteries demonstrated selective blunting of endothelium-dependent vasodilation. The implications of these findings will be discussed.The present study tested the hypothesis that prolonged exposure to comorbidities results in perturbations in myocardial blood flow and oxygen delivery, leading to a shift towards anaerobic metabolism and cardiac dysfunction in exercising swine. The main findings were that (i) the combination of diabetes mellitus, hypercholesterolemia and chronic kidney disease resulted in lower cardiac output at rest and during exercise, which was accompanied by impaired systemic vasodilatation and increased circulating levels of lactate. (ii) Exposure to these comorbidities resulted in increased levels of oxygen consumption at similar levels of cardiac work, indicating reduced myocardial efficiency. (iii) The comorbidities also resulted in perturbations in myocardial perfusion and oxygen delivery, at a time when coronary atherosclerosis was negligible. (iv) The perturbations in myocardial oxygen balance were associated with lower lactate consumption and reductions in stroke volume and LVdP/dThe presence of risk factors, including DM, HC and CKD, has been associated with CMD and INOCA in both experimental , 57, 63 The present study in chronically instrumented swine demonstrates that comorbidities can cause significant perturbations in myocardial oxygen balance both at rest and particularly during exercise. Thus, DM + HC + CKD animals demonstrated increased myocardial oxygen consumption at a given level of cardiac work, particularly during exercise, causing a counterclockwise rotation in the relation between cardiac work and oxygen consumption Fig. a. This rAlthough mitochondrial function was not measured in the present study, a direct link between cardiac mitochondrial dysfunction and microvascular dysfunction in animal models of metabolic disease was recently proposed , 23. In The present study further shows that the basal higher oxygen consumption in DM + HC + CKD was not fully met by a commensurate increase in myocardial oxygen delivery, which necessitated an increase in myocardial oxygen extraction, resulting in lower levels of coronary venous oxygen content. Cardiovascular comorbidities can cause perturbations in myocardial oxygen delivery by affecting the coronary circulation at different levels, including proximal obstructive CAD, distal small artery and arteriolar dysfunction and remodeling, and alterations in capillary structure and function . In the The perturbations in myocardial oxygen delivery were accompanied by a reduction in lactate consumption—particularly during exercise—indicating a shift towards anaerobic metabolism, suggestive of myocardial ischemia . Althougtmin and body O2 consumption, we observed in our previous study , an increase in left ventricular passive stiffness evidenced by an increase in the slope of the end-diastolic pressure volume relation, in the presence of a maintained ejection fraction [There is increasing evidence that comorbidities such as DM, dyslipidemia and CKD are linked not only to INOCA , 8, 13 bfraction . The prefraction , 41, 60.fraction , 58, 60.fraction , 39, 45.Our DM + HC + CKD swine model represents a model of early diastolic dysfunction (or pre-HFpEF), as left atrial pressures were not elevated either at rest or during exercise. Nevertheless, cardiac output was decreased which was due to a decrease in stroke volume as well as to chronotropic incompetence, i.e., limited capacity of the DM + HC + CKD swine to increase their heart rate during exercise. The latter is a common feature seen in HFpEF patients with CKD , as wellAlthough several large and small animal models (for CMD) have been described, no single animal model perfectly emulates the human disease . In the It is increasingly recognized that CKD is an important risk factor for the development of CMD , 49. TheThe present study further demonstrates that functional changes in the coronary microvasculature are already present well before overt plaque formation occurs. These early changes result in an impaired myocardial oxygen balance and reduced cardiac efficiency. The observation in a small group of animals that endothelial function in isolated coronary small arteries was perturbed, suggests a role for the loss of nitric oxide in mediating the impairments in myocardial oxygenation in DM + HC + CKD animals. Future studies should focus in more detail on the mechanisms underlying the increased myocardial oxygen consumption as well as the perturbations in oxygen delivery.Although our data provide valuable information regarding the mechanisms of CMD at this early stage, without the influence of proximal obstructive CAD possibly affecting the distal microvasculature , it is iThe present study is the first to investigate the effects of three common risk factors on myocardial oxygen balance in swine at rest and during graded treadmill exercise. Our findings demonstrate that, in the absence of coronary atherosclerosis, comorbidities can result in CMD that is severe enough to critically impair myocardial oxygenation, thereby resulting in anaerobic metabolism. Thus, our DM + HC + CKD swine model represents a bona fide large animal model of INOCA. A link between CMD and left ventricular diastolic dysfunction has recently been shown in clinical studies. In our model, overt CMD is present at a time when diastolic dysfunction is still modest . These fSupplementary material 1 (DOCX 27 kb)Below is the link to the electronic supplementary material.


Extracellular vesicles (EVs) emerged as potential diagnostic and prognostic markers for cancer therapy. This review summarizes mechanisms of signaling specificity and cargo transfer by EVs in the oncogenic and cancer‐associated signaling cascades Wnt, TGF‐β, ErbB, VEGF, and PD1–PD‐L1. Translatable EV functions and existing knowledge gaps are discussed to possibly further exploit EVs for diagnostics and therapeutic approaches in the future. in vivo. Several signaling cascades have been found to use EVs for signaling in the tumor–stroma interaction. These include potentially oncogenic, verbatim transforming, signaling cascades such as Wnt and TGF‐β signaling, and other signaling cascades that have been tightly associated with tumor progression and metastasis, such as PD‐L1 and VEGF signaling. Multiple mechanisms of how these signaling cascades and EVs interplay to mediate these complex processes have been described, such as direct signal activation through pathway components on or in EVs or indirectly by influencing vesicle biogenesis, cargo sorting, or uptake dynamics. In this review, we summarize the current knowledge of EVs, their biogenesis, and our understanding of EV interactions with recipient cells with a focus on selected oncogenic and cancer‐associated signaling pathways. After an in‐depth look at how EVs mediate and influence signaling, we discuss potentially translatable EV functions and existing knowledge gaps.In recent years, extracellular vesicles (EVs) emerged as potential diagnostic and prognostic markers for cancer therapy. While the field of EV research is rapidly developing and their application as vehicles for therapeutic cargo is being tested, little is still known about the exact mechanisms of signaling specificity and cargo transfer by EVs, especially  EGF(R)epidermal growth factor (receptor)EMTepithelial–mesenchymal transitionEP(s)extracellular particle(s)EV(s)extracellular vesicle(s)Exo(s)exosome(s)Herhuman epidermal growth factor receptorISEVInternational Society for Extracellular VesiclesMSCmesenchymal stem cellsMV(s)microvesicle(s)MVBmultivesicular bodyPD‐Lprogrammed death‐ligandTGF‐βtransforming growth factor‐βVEGFvascular endothelial growth factorWntwingless and Int/wingless‐related integration site1Extracellular vesicles (EVs) are small, lipid bilayer components that are released from many cell types . They can transport signaling‐active molecules in the form of proteins, DNA/RNA, lipids, and metabolites between cells of different origins . In the Recent reviews have highlighted the role of EVs in cancer including on EV biogenesis and function , their r22.1Extracellular vesicles are defined as lipid bilayer‐surrounded particles, naturally released from cells . As EVs +); and (c) in‐depth description of how EVs were isolated   divide again into three functional groups: receptor‐regulated; inhibitory; or partnering Co‐SMADs. SMAD‐independent TGF‐β signaling involves, among other molecules, MAPK, JAK/STAT, Rho‐GTPases, and Akt . M. M129]. Initial or acquired resistance to PD‐L1‐directed therapeutics harbors a great burden for patients with the above‐mentioned malignancies, since they cannot benefit from these treatment options . Early p8.1The presence of PD‐L1 on the surface of EVs has been recently described not only for various solid tumors, such as breast cancer , metastaet al. [Interestingly, PD‐1 was not detected on Exos derived from chimeric antigen receptor (CAR) T cells, although the maternal cells expressed the immunosuppressive receptor . In theiet al.  comparedet al. . And sinet al. .8.2Since immune evasion is one of the major drivers of tumor progression, EVs carrying PD‐L1 and their effect on various immune cells has been extensively investigated in the last years.Stimulation with interferon‐γ increased the levels of PD‐L1 on Exos and suppressed T‐cell function in the melanoma tumor environment . In glioet al. [et al. [Focusing on myeloid‐derived cell lines, Ning et al.  showed tet al. . Fleming [et al.  investig [et al. . Exosoma [et al. .The effect of exosomal PD‐L1 on immune evasion was also shown in genetic murine models . Here, P8.3Changes in the microenvironment can interfere with EV release, such as changes in pH , 133, thWith the fast developing field of research on PD‐L1 and immune checkpoint modulation, PD‐L1 on EVs arose as potent mediators of immune evasion. At the same time, PD‐L1‐bearing EVs were successfully tested for the prediction of treatment response in various tumors  amplifications and EGFRvIII mutations are commonly detected in various cancer patients . Patientet al. [+, CD3‐ PD‐L1+, and CD3+ 15s+ (Treg‐derived) Exos [Extracellular vesicles carrying PD‐L1 might be useful as diagnostic and prognostic markers, for example in pancreatic cancer , and as et al.  establiset al. . The comet al. . In conted) Exos .Despite these recent advances in the search for diagnostic and prognostic markers, none of the above‐mentioned EV‐associated biomarkers have been approved by national or international agencies controlling the approval of new biomedical test and testing devices, such as the U.S. Food and Drug Administration (FDA). Nevertheless, for the detection of EV‐associated biomarkers in prostate cancer progress can be noted, since recently ExoDx Prostate (IntelliScore) (EPI) , 153 was9.2et al. [in vitro and in vivo. Furthermore, it was shown that cancer‐associated fibroblasts are able to promote drug resistance and stemness, epithelial–mesenchymal transition (EMT), and metastasis of colorectal cancer cells through Exos increasing miR‐92a‐3p levels [Cancer stem cells harbor certain characteristics such as chemotherapy resistance . Accordiet al.  describep levels . MiR‐92ap levels . In addip levels . These dp levels .In the context of ErbB signaling, drug resistance to trastuzumab  has been proposed by the transfer of lncRNAs , 158. Reet al. [et al. [For VEGF signaling, another mechanism of EVs mediating drug resistance has been described by Feng et al.  A VEGF vet al. . Ko et a [et al.  showed a [et al. . Neverth—two ligands activating HER2 signaling [Likewise, EGFR/Her1 and HER2 have been found on EVs from the cancer cell lines SkBr2 and BT474 that both overexpress HER2 . Interesignaling . Xenogenignaling .This neutralizing effect on commonly applied, therapeutic monoclonal antibodies by epitope presentation on EVs most likely constitutes a basic mechanism of altered drug response that can possibly be transferred into multiple other signaling contexts.in vivo is still debated [et al. who state most likely transferable mechanisms to be considered in other contexts: First of all, the presentation on EVs itself might hinder the recognition of PD‐L1 by common antibodies [As stated above, also PD‐L1‐positive EVs can be used as biomarkers for treatment resistance. Nevertheless, the question how exosomal PD‐L1 mediates resistance to anti‐PD‐L1 therapy  debated . Possibltibodies . Secondltibodies . Thirdlytibodies . Taken t9.3et al. [Wnt/β‐catenin signaling activation can contribute to immune evasion , 162. Maet al.  investiget al. . They thet al. . Reducinet al. [et al. had suggested similar effects in the context of antileukemia immunity by leukemia‐derived Exos and reduced TGF‐β1 expression [In the context of TGF‐β signaling, Rossowska et al.  tested tet al. . The deset al. . These det al. . Resultspression .etal. [et al. [+ breast cancer cells was made possible by loading Exos and expressing a chimeric LAMP2b‐DARPin protein on the surface of EVs [et al. [+ breast cancer feasible and safe administration options to specifically guide prodrug/enzyme regimens to cancer cells with minimal off‐target toxicity [+ subpopulation through a chimeric protein designed to be presented at the EV surface. Therefore, they successfully administered in vitro‐transcribed mRNA through EVs [For ErbB signaling, Ohno etal.  demonstr [et al.  engineere of EVs . Gomari  [et al.  additiontoxicity , 170. Though EVs , 170.In the future, these approaches will have to be tested in more clinically relevant model systems  to ensure lasting immunological tolerance and to investigate their therapeutic efficacy and feasibility to enable translation into the clinical settings. Also, the transfer of these established tools and methodologies into other pathological contexts, where other signaling cascades are involved, especially in pathologies currently lacking therapeutic options, will be fundamental.In vivo studies in zebrafish by Yang et al. showed the delivery of exogenous siRNA by brain endothelial cell‐derived Exos inhibiting VEGF‐ signaling [One of the benefits of using EVs as carriers for therapeutic applications is their biological properties, such as a potentially prolonged half‐life time and their ability to cross biological borders, such as the blood–brain barrier . In vivoignaling .et al. used EVs to modify the tumor microenvironment [+ dendritic cells and subsequently tumor‐specific CD8+ T cells [In a slightly different but highly innovative approach, Hong ironment . They teironment . Highly  T cells . In a mu T cells .et al. [in vitro and in vivo [The therapeutic application of MSC‐derived EVs exploiting their multiple beneficial properties has been the focus of many researchers in the last years: In 2013, Lee et al.  demonstr in vivo . The stu in vivo . For com10Signaling via EVs during tumor initiation, progression, and metastasis has become an important research area with implications for the analysis of liquid biopsies, cancer diagnostics, prognostics, and therapeutic approaches. To gain a deeper and mechanistic understanding of signaling components present in or on EVs will be one of the major challenges in the next years for correct translation into the clinical setting.Unfortunately, further oncogenic and cancer‐associated signaling cascades could not be covered in this review. However, this does not mean that they have no role in the EV–cell communication. Indicatively, oncogenic NOTCH signaling and hormone receptor‐mediated signaling are further highly relevant examples of EV‐mediated signaling cascades in the tumor–stroma interaction.To exploit EVs for early cancer detection or to interfere with EV biogenesis, spread, or signaling, it is crucial to find answers to the following questions: Which cargo is found in or on EVs and what is its role in EV biogenesis? Which cargo of EVs has functional implications and which is ‘hitch‐hiking’ because of its expression in the cell lineage or tumor context? To find answers, it will be necessary to establish readouts in order to demonstrate the specific activation of a signaling pathway by the respective signaling agent . Current studies often correlate EV uptake with a certain, rather unspecific outcome failing to answer questions of exact biological mechanisms of signaling induction . Analysein vivo [Technical improvements will be a prerequisite , 175. Lain vivo , 175. UnIn vivo studies will also help to better understand the challenges of EV‐based therapeutic approaches, such as EV half‐life time and unspecific or limited targeting. Engineering methods to incorporate various cargoes into EVs has been proven successful, and phase I clinical trials have tested the feasibility of large‐scale EV production and safety in patients with various cancers (as reviewed in Ref. [Autologous EVs are characterized by low immunogenicity and therefore remarkable biocompatibility . This in in Ref. ).The past years have shown rapid progress in identifying cargo and mechanisms of EVs in diseases; however, we believe a more detailed mechanistic and physiological understanding of signaling via EVs will be necessary to use EVs for diagnostics and therapy of cancer.The authors declare no conflict of interest.AS and MB wrote the manuscript. Both authors approved the final version.