Over the last decade, chronic inflammatory enteropathies (CIE) in dogs have received great attention in the basic and clinical research arena. The 2010 ACVIM Consensus Statement, including guidelines for the diagnostic criteria for canine and feline CIE, was an important milestone to a more standardized approach to patients suspected of a CIE diagnosis. Great strides have been made since understanding the pathogenesis and classification of CIE in dogs, and novel diagnostic and treatment options have evolved. New concepts in the microbiome-host-interaction, metabolic pathways, crosstalk within the mucosal immune system, and extension to the gut-brain axis have emerged. Novel diagnostics have been developed, the clinical utility of which remains to be critically evaluated in the next coming years. New directions are also expected to lead to a larger spectrum of treatment options tailored to the individual patient. This review offers insights into emerging concepts and future directions proposed for further CIE research in dogs for the next decade to come. CIE characterizes an exaggerated immune response and has a multifactorial pathophysiology . The immChronic inflammatory enteropathies are diagnosed based on the presence of chronic gastrointestinal signs ≥3 weeks), histopathologic evidence of intestinal mucosal inflammation, and the exclusion of other underlying causes –3. The t weeks, hCanine CIE is currently further subclassified  based on the response to treatment. Dogs are categorized as food-responsive enteropathy (FRE) if the clinical signs resolve or significantly improve within 2–4 weeks after starting an elimination diet trial with either a limited-ingredient novel protein and carbohydrate source  or a hydrolyzed protein diet. FRE is the most prevalent group 50–65%) of CIE in dogs , and aff5% of CIEExcellent long-term responses in dogs with FRE can be achieved with commercial hydrolyzed protein or limited-ingredient novel protein diets , 9, 10. Dogs that show a marked and long-lasting improvement or resolution of their clinical signs after administering an antibiotic (metronidazole and/or tylosin) for 2 weeks were previously classified as ARE . HoweverDogs that require treatment with glucocorticoids or other immunosuppressive drugs are diagnosed as steroid-responsive (SRE) or immunosuppressant-responsive enteropathy (IRE) , 3, 21, Enterococcus faecium or the highly concentrated probiotic strains found in Visbiome), prebiotics , or synbiotics , carrying a worse long-term prognosis and a high rate of euthanasia , 26. Furnbiotics –34, fecanbiotics –37, or snbiotics .Protein-losing enteropathy (PLE), as a special form of chronic enteropathies in dogs, can be secondary to a severe diffuse infiltrative intestinal disease process causing lymphatic vascular obstruction  or due to dysfunction of the intestinal lymphatics with primary intestinal lymphangiectasia , 40. BasCanine CIE is a multifactorial disease likely caused by complex interactions between mucosal immunity and the environment  in genetically susceptible hosts, like human IBD , 44. GenTLR4 gene and three non-synonymous SNPs in the TLR5 gene that are significantly associated with IBD pathogenesis in GSD ] and their clinical response to antibiotics points to a potential interaction between host immunity and the microbiota . Previous in GSD . Interess in GSD . Boxer d ileitis , 53. Whi ileitis .+ T cells as the HLA molecules associated with celiac disease bind specific gluten peptides that activate proinflammatory T cells  component . This st T cells . Soft-co T cells . Pedigre T cells . Perinuc T cells  and in d T cells . In a se T cells . Express T cells . These s T cells  and Iris T cells .While the role of genetics in CIE is currently being unraveled, there is evidence in humans and dogs that early life exposures can impact the intestinal microbiome and host response to influence the risk of disease later in life. After birth, the colonizing microbiota promotes development of mucosal immunity (a “weaning reaction”), which imprints the immune system to maintain its responsiveness after weaning and into adulthood. When host-microbe crosstalk is perturbed in early life, inhibition of the weaning reaction leads to pathological imprinting, which causes increased susceptibility to inflammatory pathologies in individuals that have not been exposed to a microbiota, or possibly a dysbiotic microbiota , 66. BotTLR4 and TLR5) of the innate immune system are associated with a breed-specific form of CIE in GSDs , IgG+ plasma cells, macrophages, and granulocytes, but fewer mast cells  cells in dogs with IBD compared to controls. However, there are differences in the phenotype of LP immune cell subsets with increased numbers of T cells  in dogs with idiopathic IBD  and incr with GC . Other s with GC . A study with GC . Few gen with GC . Dogs wiMicroRNAs (miRNAs) are small, non-encoding RNAs that regulate gene and protein expression. As such, they play a crucial role in the development of cells involved in innate and adaptive immunity, and in regulating the immune response . Studiesin vivo in humans include orally ingested probes  assessed in urine and measurement of fatty acid bindings proteins, glucagon-like peptide (GLP)-2, and LPS-binding protein (LBP) concentrations in serum  status as a marker of intestinal absorptive capacity is of diagnostic and therapeutic value in small animal medicine -cobalamin complex in the distal small intestine , for which several commercial tests are available. An automated chemiluminescence test is routinely used in Europe and North America, and the reference intervals for serum cobalamin concentrations in dogs used by the various veterinary diagnostic laboratories are comparable  is a metabolite that accumulates when the activity of cytosolic methylmalonyl-CoA mutase is decreased because of intracellular cobalamin deficiency . Thus, pHypocobalaminemia or cobalamin deficiency can have several causes, the most common in dogs being chronic gastrointestinal diseases and exocrine pancreatic insufficiency . CommerSubnormal or low normal serum cobalamin concentrations  are often associated with increased serum MMA concentrations . These iFusobacterium spp., and Firmicutes, and an increased abundance of Proteobacteria in fecal samples of dogs with CIE (in situ hybridization (FISH) to characterize the mucosal biogeography in colonic biopsies of dogs with CIE showed affected dogs to have decreased numbers of total bacteria within colonic crypts but no difference in total bacterial counts on the mucosal surface as compared to healthy control dogs  residing within the colonic mucosa. A causal role for these bacteria in GC pathogenesis is supported by the association of remission with eradicating invasive E. coli when enrofloxacin was used  and the total bacterial abundance in canine feces. The DI calculates a single number that expresses the extent of intestinal dysbiosis present: a DI < 0 indicates a normal microbiota while a DI >2 indicates dysbiosis  hiranonis, a bacterium associated with normal BA metabolism, causes increased concentrations of primary BAs in the colon, which are pro-inflammatory and may cause secretory diarrhea  dysmetabolism . Luminaldiarrhea . Bile acdiarrhea . A recendiarrhea . Taken tdiarrhea .The diagnostic work-up of dogs with suspected CIE remains complex  and compThe decision to perform GI endoscopy in dogs with CIE is best determined on a case-by-case basis. Treatment trials are the best tools to differentiate the varied forms of CIE as clinical history, laboratory findings, biomarkers of inflammation, and histologic abnormalities have not proven reliable to distinguish one form of CIE from another. While many dogs will quickly respond to elimination dietary trials, non-responders will require reassessment and further diagnostic testing including GI endoscopy to rule out other causes for chronic GI signs . Endoscohttps://wsava.org/global-guidelines/gastrointestinal-guidelines/)  guidelines recommend using a standardized endoscopic report form with check boxes to identify the presence/severity of mucosal lesions when endoscopy is performed (elines/) . Determielines/) , 147. Muelines/) , 148. BrThere are few studies evaluating the GI mucosal appearance of dogs with CIE following treatment. One prospective single-center study evaluated the sequential treatment response of 70 dogs with CIE placing them into FRE, ST (steroid-treated), and PLE cohorts . GastrodThe WSAVA endoscopic examination reporting forms for upper and lower GI endoscopy include various descriptors  and subjective gradations  that may make it difficult to accurately quantitate mucosal inflammation. Furthermore, studies evaluating the interobserver agreement for grading mucosal lesions amongst endoscopists using the WSAVA endoscopic score are lacking. One recent study investigated the interobserver agreement in the endoscopic assessment of mucosal appearance of the GI tract to design and validate a simple and easy-to-use endoscopic scoring system for dogs with idiopathic IBD . ValidatDiagnosis of intestinal inflammation in dogs with CIE has historically posed great challenges for clinicians and veterinary pathologists. While intestinal biopsies can be acquired endoscopically or surgically, GI endoscopy is most often utilized. Several fundamental issues in the diagnostic process have included: (1) How can small and fragile endoscopic specimens of variable quantity from different regions of the GI tract be used for histologic assessment? (2) What constitutes an acceptable biopsy with regards to size and diagnostic quality? (3) Are standardized grading schemes of value in defining histologic inflammation? If so, what are the key criteria used for assessment  and current recommendations for their use? (4) Is there a relationship between histologic changes and GI signs? and (5) Is there a relationship between histologic changes and response to treatment? To address these concerns, numerous histologic grading schemes characterizing the nature and severity of GI inflammation have been designed. Within most of these model systems, there is an emphasis on the type and severity of lamina propria cellularity that is variable and subjectively graded as normal, mild, moderate, or severe. It is now recognized that different types of cellular populations  may overlap and occur in different types of GI diseases. For example, lymphoplasmacytic enteritis is a common feature in dogs with idiopathic IBD , 152 andA limitation of the WSAVA score is that it did not consider goblet cells as a morphologic parameter, where their numbers decrease with inflammation but increase in response to successful treatment , 154. UsEndoscopic biopsy is often performed in dogs with chronic or intermittent GI signs that fail to respond to sequential treatment interventions. In these instances, it is expected that the histologic findings contained in tissue samples will confirm a diagnosis and guide treatment decisions in these patients. However, separate trials performed by different investigators have failed to show a convincing association of histopathologic findings in endoscopic specimens with clinical disease activity, biomarkers of inflammation, or response to therapy and outcome in dogs with idiopathic IBD , 161. ThOther factors contributing to potential discordance between histologic findings and clinical signs include the quality of endoscopic specimens and failure to biopsy the intestinal compartment most affected by a pathologic process. The effect of sample quality in detecting select histologic lesions in gastric and duodenal biopsies of dogs was investigated in one study showing that endoscopic biopsy quality  significantly affects the sensitivity to detect certain histologic lesions . As the Changes in the ultrastructural lesions of duodenal biopsies  have been reported in dogs with diet-responsive CIE before and after dietary treatment . Using bClinically useful biomarkers help detect disease, determine the severity of the disease process, and monitor the response to treatment. Several inflammatory biomarkers have been researched during the last decade and hold significant promise as diagnostic and/or monitoring tools in canine CIE . These iSerum C-reactive protein (CRP) concentration is a non-specific marker of inflammation  is another sensitive and specific marker of localized inflammatory processes in dogs  can be measured in serum, urine, and fecal samples , a stable metabolite of eosinophil peroxidase, is a biomarker of eosinophilic inflammation  as an anti-inflammatory decoy receptor can sequester ligands such as DAMP molecules  and abrogate cellular proinflammatory RAGE responses  emphasizes the importance of selecting an optimal dietary strategy for dogs with CIE.For some CIE subgroups, research over the last decade led to a change in the role of diet and recommended dietary strategies. Demonstrating the existence of food-responsive PLE (FR-PLE) and several effects of diet on the intestinal microbiome and metabolome . It requires sequential laboratory tests, diagnostic imaging, and often more invasive diagnostics . Increasic signs . Hypoalbic signs , 158, anic signs .The cornerstone of PLE treatment is lifelong dietary management with a high-protein, low-fat diet  –203, whiResponse rates with the dietary management of non-infectious, non-neoplastic PLE are high (73–85%) –203, and2 PO q24h for 1–3 weeks, then taper) might be better first-line steroid-sparing drug choices. Mycophenolate carries the risk of significant GI adverse effects  should receive supplemental cobalamin (50 μg/kg SC weekly for ≥6 weeks or 50 μg/kg PO q24h for ≥12 weeks)  . Moderat2 weeks)  but furtAs microbial imbalances are associated with intestinal inflammation in dogs with CIE, treatments attempting to reduce mucosal inflammation by normalizing dysbiotic states are a rapidly growing research area . Probiotex vivo experiments. Clinical studies have included three randomized controlled trials for dogs with FRE    or a laclus spp.  failed tlus spp. . In dogsontinued .+ and TGF-β+ regulatory T cells) and increased mucosal expression of the tight junction protein occludin. In a separate trial, probiotic mixture VSL#3 and hydrolyzed diet was administered to dogs with idiopathic IBD and compared to a treatment regimen using hydrolyzed diet and prednisone. While both treatment groups saw remission, probiotic therapy was associated with increased beneficial mucosal bacteria and up-regulated expression of several tight junction proteins suggesting improved intestinal barrier function (Saccharomyces boulardii (as part of a synbiotic preparation) improved clinical severity and stool frequency at days 45 and 60 when administered to IBD dogs with and without PLE  was investigated in comparison to combination drug therapy . Dogs trfunction . Finallyhout PLE . In the Evidence-based data regarding probiotic use for canine CIE is limited and larger clinical trials are needed. The significant decrease in clinical scores of most dogs with CIE treated with probiotics is best explained by a positive response to dietary intervention. It remains possible that additional health benefits beyond the clinical effects of probiotics might be realized, including systemic immune modulation  and poteThe efforts to promote recovery of dysbiosis and remission in dogs with chronic GI disease by administration of FMT have been investigated only to a limited extent. Guidelines regarding optimal donor screening and clinical indications, as well as the method of delivery  and frequency of administration of FMT, are currently lacking . StudiesNew concepts that have emerged from recent studies on the microbiome-host-interaction, metabolic pathways, and crosstalk within the mucosal immune system are expected to pave the way to a large spectrum of novel treatment strategies that are tailored to the individual patient. Digital health options and artificial intelligence algorithms will likely also play a role in the management of dogs with CIE. Novel diagnostics have been developed, but the clinical utility of these tests and their utility, when included in a panel of tests, remains to be critically evaluated in the future. A new direction is also the further evaluation of gastrointestinal endocrine pathways and the gut-brain axis in canine CIE.ex vivo human 3D enteroid/colonoid structures, derived from human adult intestinal stem cells (ISCs), have tremendous potential to recapitulate in vivo physiologic conditions used for fundamental research, precision medicine, regenerative medicine, and drug discovery. Under appropriate culture conditions, enteroids/colonoids produce all cell types normally found within the intestinal epithelium. Dogs represent a unique large animal model that naturally develops CIE, sharing remarkable similarities in etiology, clinical course, histologic lesions, and interventional strategies to human IBD  dysfunction plays a crucial role in the pathogenesis and progression of IBD , 228. Diuman IBD . The advin situ hybridization (RNA-ISH), and transmission electron microscopy (TEM) for comparison to whole jejunal tissue. Moreover, functional assays evaluating cellular metabolic changes , transmembrane chloride ion conductance via activation of chloride channels in intestinal epithelial cells (cystic fibrosis transmembrane conductance [CFTR] assay), and uptake and transport of parasitic exosomes in response to exposure to the swine nematode, Ascaris suum, were performed. There were no differences in morphology of organoids between healthy dogs and dogs with idiopathic IBD visualized by bright-field microscopy. Ultrastructurally, TEM identified features of differentiated cell types, microvilli, and intercellular structures  between epithelial cells in both enteroids and native jejunum. Characterization of molecular markers and IEC subtypes in enteroids and tissues using RNA-ISH/IHC showed that canine enteroids and intestinal tissue share similar phenotypic expression and cell types  describes a variety of digital applications where human tasks  are performed computer-based (machine learning (ML) algorithms can be developed to predict data  or outcomes based on previously generated patient data , dogs with CIE and their pet owners are expected to benefit tremendously from such services. The enormous potential of these tools (like AI) is likely to unfold in supporting pet owners throughout disease management for improved patient monitoring, owner compliance, and patient outcomes (via a web-based diary function) would be very valuable additional tools in canine CIE management. An online survey at the University of Leipzig, Germany that included a cross-sectional questionnaire Disease tracker app for the management of chronic gastrointestinal disease in dogs and cats—a pet owner's perspective and yielded 2,416 completed votes  revealed those aims to be aligned with most pet owners' expectations. Top-ranking net promoter scores (NPS) in this questionnaire were obtained for the possibility to be reminded of recurring treatments at home , exchange of medical data with the veterinarian, and receiving individualized treatment recommendations and monitoring instructions. The motivation for most pet owners to use this digital health app was a simple and intuitive design, effective integration of this tool into the daily routine, and improved health management of their pet.nologies . The usenitoring . Becauseoutcomes . Patient1PI concentrations) are clinically routinely used in dogs with suspected CIE. Genomic biomarkers are currently not readily available but hold great promise for an individualized approach to risk assessment, treatment, and monitoring in canine CIE. Of the inflammatory biomarkers, only serum CRP is currently widely offered, but a human fecal calprotectin assay holds promise for use with specimens from dogs   models of canine CIE with complete cytodifferentiation from intestinal stem cells have been developed and are currently being refined allowing to reduce the complexity of the intestinal mucosa to modular functioning microphysiological systems , 236. Thesponse)  and to aesponse) . These iExtraintestinal manifestations of IBD in humans include the joints (polyarthritis), skin (erythema nodosum), or eyes , but can also involve other organs, such as the liver , pancreas (pancreatitis), and lung –248. In 12 and D, and several biomarkers of inflammation are an essential basis for future routes aiming for an individualized medical approach to canine CIE. Refinement of previously established endoscopic and histologic evaluation and scoring criteria has further advanced the standardized assessment and improved the diagnostic algorithms in dogs suspected of CIE. Future investigations in this area need to validate these scoring systems and develop a consensus definition for the assessment of disease remission. More work is also needed to determine the potential benefits of using probiotics, synbiotics, and FMT in dogs with CIE. Subcategorization of food-responsiveness in PLE has changed the approach to affected dogs, and novel dietary strategies and treatment options for canine PLE are to be explored. Future avenues to further evaluate pathogenetic aspects with a focus on drug discovery will benefit from established canine organoid models. AI algorithms are an avenue to improved patient diagnosis and individualized treatment approaches, and digital health tools will likely play a key role in the future of canine CIE management. Important milestones are thus expected in the next decade of canine CIE research.Tremendous research efforts over the last decade resulted in a better understanding of the multifactorial disease complex of canine CIE and the differentiation of breed-specific enteropathies. Milestones reached in researching aspects of the immunopathogenesis, microbiome, metabolome, hypovitaminoses BAEJ and RMH contributed equally to the manuscript design, writing, generation of figures/tables, editing, and proofing. Both 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Accurate molecular subtypes prediction of cancer patients is significant for personalized cancer diagnosis and treatments. Large amount of multi-omics data and the advancement of data-driven methods are expected to facilitate molecular subtyping of cancer. Most existing machine learning–based methods usually classify samples according to single omics data, fail to integrate multi-omics data to learn comprehensive representations of the samples, and ignore that information transfer and aggregation among samples can better represent them and ultimately help in classification. We propose a novel framework named multi-omics graph convolutional network (M-GCN) for molecular subtyping based on robust graph convolutional networks integrating multi-omics data. We first apply the Hilbert–Schmidt independence criterion least absolute shrinkage and selection operator (HSIC Lasso) to select the molecular subtype-related transcriptomic features and then construct a sample–sample similarity graph with low noise by using these features. Next, we take the selected gene expression, single nucleotide variants (SNV), and copy number variation (CNV) data as input and learn the multi-view representations of samples. On this basis, a robust variant of graph convolutional network (GCN) model is finally developed to obtain samples’ new representations by aggregating their subgraphs. Experimental results of breast and stomach cancer demonstrate that the classification performance of M-GCN is superior to other existing methods. Moreover, the identified subtype-specific biomarkers are highly consistent with current clinical understanding and promising to assist accurate diagnosis and targeted drug development. Cancer is a complex and highly individualized disease with diverse subtypes, and molecular heterogeneity exists among different subtypes of the same cancer type . As cancHigh-throughput sequencing technologies generate a large amount of multi-omics data , which pWith the strong representation ability of graph-structured data, graph neural networks (GNNs) have achieved great success and are gradually used in a node classification task. It provides one way to obtain new representations of nodes by combining the connectivity and features of its local neighborhood. Recently, some GNN-based methods have been proposed to predict molecular subtyping of cancer. Rhee et al. developed a GCN-based model to explore the gene–gene association and information passing for cancer subtyping . Lee et It is well known that abnormal behaviors of cancer cells are the result of a series of gene mutations, gene copy number variation, and gene transcription level changes in key regulatory pathways . A singlIn this study, we propose a novel and general framework M-GCN  for moleWe collect gene expression, SNV and CNV data, and clinical information of breast cancer (BRCA) and stomach adenocarcinoma (STAD) patients from The Cancer Genome Atlas (TCGA) database . As showr (TNBC) , and STAr (TNBC) , respectGenes whose expression values are lower than 10 and 3 are considered as not expressed in BRCA and STAD and then deleted, respectively. As RNA-seq data of these two cancer types are obtained from different platforms in TCGA, we set different cutoffs according to their data distributions. Then, fragments per kilobase of exon per million fragments mapped (FPKM) values of gene expression are normalized with log2-transformation. In each cancer type, a gene’s mutation frequency is defined as the number of samples with this mutation divided by the total number of samples. Genes with mutation frequency greater than To obtain molecular subtype-related transcriptome features with low noise for constructing a purified sample–sample similarity graph and effective message passing, we apply a supervised non-linear feature selection method HSIC Lasso , which c1n is the Let Since samples with similar features are more likely to fall into the same category, we first construct a sample–sample graph based on similarity of each samples pair. From the perspective of the whole biological system, compared with SNV and CNV, gene expression is the most fundamental level at which the genotype gives rise to the phenotype. So we only use transcriptomic data and apply spearman’s correlation to calculate the similarity of each two samples. We select sample pairs whose correlation coefficient  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis on top 40 subtype-specific genes. The R package “clusterProfiler” is used. K-nearest neighbor classifier (KNN), random forest (RF), support vector machine classifier (SVM), and Gaussian naive Bayes (GNB) are traditional machine learning methods and we utilize gene expression, SNV, and CNV data as features. DeepCC is a neural network-based method which utilizes transcriptomic data and leverages feedforward neural networks to classify molecular subtypes. Li’s method is a GCN-based molecular subtyping method which integrates CNV data and gene expression data.To demonstrate the performance of our method, we compare the performance of M-GCN with six commonly used or advanced methods on STAD and BRCA molecular subtyping including traditional machine learning–based methods, neural network–based method, and a GCN-based method:For BRCA, our framework M-GCN achieves best performance . M-GCN aFurthermore, we analyze the detailed results of subtype classification. As shown in For the subtype classification task with more classes and smaller sample size, our method still performs best on STAD than other methods in all metrics . The perlidation , M-GCN hOur framework M-GCN achieves best performance in BRCA and STAD molecular subtypes. The number of samples in BRCA is greater than that in STAD, and all of these methods in BRCA have good accuracy. The machine learning–based methods such as RF, SVM, and GNB have a significant difference between BRCA and STAD tasks. In addition, these methods in traditional machine learning–based methods are shallow and cannot learn the deep and complex representations of sample nodes. The performance of the neural network–based method DeepCC is higher than most of these machine learning–based methods, which shows the deep and non-linear representation are important. Li’s method may suitable for the task with more samples. M-GCN still has the better scores in evaluation metrics than the multiple omic-based methods by utilizing the cleaned structure information and message passing of sample nodes.After assessing the performance compared with other methods, we conduct three ablation experiments to evaluate the contributions of feature selection step, SNV data, and CNV data in STAD, respectively . The basOverall, the results of ablation experiments in STAD demonstrate that feature selection, SNV data, and CNV data are essential. Especially, feature selection makes a more significant contribution. One possible reason for this is that selected subtype-related features can help learn good representations of sample and reduce noise of the sample–sample graph.Similarly, to explore contributions of feature selection, SNV, and CNV data for molecular subtyping of BRCA, we also perform ablation experiments. The results of three ablation experiments are shown in On the basis of selected transcriptomic features that could accurately classify the breast cancer samples into various molecular subtypes, we further obtained the subtype-specific genes.We identify ten genes with highest specificity score of each subtype, the gene lists are shown in Moreover, specific genes of ER+, HER2+, and TNBC are significantly enriched in biological processes of response to estradiol, ERBB2 signaling pathway, and sequestering of actin monomers, respectively. Some of the findings are also highly consistent with current understandings. Daniel et al. have found that estrogen were important drivers of breast cancer proliferation and PR-B expression increased breast cancer cell growth in response to estradiol . Shah etCompared with BRCA, the current understanding of subtype markers and biological mechanisms of STAD is much less and our analysis is expected to provide more insight. From the gene expression heatmap across all the samples, it can be concluded that biomarkers of STAD perform well in distinguishing EBV, GS, and MSI . ThroughThe generation of large amounts of multi-omics data and development of deep learning methods offer a more effective mean to study the personalized diagnosis and treatment options of complex diseases, such as cancer . In thisAblation experiments demonstrate that subtype-dependent feature selection contributes most to the improvement of classification performance of cancer molecular subtypes. Furthermore, we verify the stability of the feature selection process to ensure that obtained features are reliable. When shuffling the samples and using 90% of them to perform feature selection, we find the intersection of features picked out by the 10 rounds of feature selection processes are very large for both BRCA and STAD. This is extremely beneficial to train a stable GCN-based model.On the basis of subtype-related features, we further identify a few subtype-specific features which can potentially be used for diagnostic biomarkers. In our study, ESR1, ERBB2, and MFI2 are predicted as the subtype-specific biomarkers because they have the highest specificity scores for ER+, HER2+, and TNBC samples, respectively. It is worth noting that ESR1 and ERBB2 are well accepted markers for ER+ and HER2+ breast subtype, indicating our prediction is highly consistent with current understanding. Although MFI2 has not been demonstrated as biomarkers of TNBC subtype by wet lab and clinical experiments, its encoding protein shares sequence similarity and iron-binding properties with members of the transferrin superfamily. Public studies have demonstrated these iron-binding properties serve iron uptake and promote cell proliferation, and high expression of these proteins are associated with the decreased overall survival of patients in many cancer types . As we kLarge amount of multi-omics data generated by rapid development of high-throughput technologies has enabled data-driven methods to apply in molecular subtyping of cancer. We proposed a robust GCN-based framework M-GCN for molecular subtyping of cancer by integrating gene expression, SNV, and CNV data. In addition to comprehensive information of individual samples, M-GCN fully considers message aggregation among samples for subtype classification. Compared with other six advanced computational methods, M-GCN achieves the best classification performance for molecular subtyping of breast and stomach cancer. Through ablation experiments, we demonstrate subtype-related transcriptomics features obtained by HSIC Lasso method highly contribute to sample classification, which is probably because the selected features eliminate data noise and facilitate the construction of purified graph. On the basis of the graph structure constructed by HSIC Lasso, M-GCN further strengthens connections between new features and the graph by assigning weights. By assigning higher weights, M-GCN aims to successfully pass message in GCN. Furthermore, the identified molecular subtype-specific marker of breast cancer is highly consistent with clinical cognition, so the predicted biomarkers of stomach cancer are promising to be used for molecular typing diagnosis of patients, filling in the current gap.


Endothelial progenitor cells (EPC) may influence the integrity and stability of the vascular endothelium. The association of an altered total EPC number and function with cardiovascular diseases (CVD) and risk factors (CVF) was discussed; however, their role and applicability as biomarkers for clinical purposes have not yet been defined. Endothelial dysfunction is one of the key mechanisms in CVD. The assessment of endothelial dysfunction in vivo remains a major challenge, especially for a clinical evaluation of the need for therapeutic interventions or for primary prevention of CVD. One of the main challenges is the heterogeneity of this particular cell population. Endothelial cells (EC) can become senescent, and the majority of circulating endothelial cells (CEC) show evidence of apoptosis or necrosis. There are a few viable CECs that have properties similar to those of an endothelial progenitor cell. To use EPC levels as a biomarker for vascular function and cumulative cardiovascular risk, a correct definition of their phenotype, as well as an update on the clinical application and practicability of current isolation methods, are an urgent priority. Cardiovascular diseases (CVD) such as myocardial infarction (MI), cerebral and peripheral arterial disease (PAD) and arterial hypertension are the leading causes of globalmortality ,2.Coronary artery disease (CAD), leading to narrowing or complete blockage of arterial blood supply to the myocardium, is the most prevalent heart disease . PharmacKnown cardiovascular risk factors (CVF) contribute to the cascade of atherogenesis especially by inducing injury and dysfunction in endothelial cells. Endothelial integrity is highly reliable due to repair and renewal by endothelial progenitor cells (EPC) derived from different sources, e.g., bone marrow (BM), circulating endothelial progenitor cells (CEPC) or adventitial residents . The impRoss’ classic paradigm already stated that endothelial cells (EC) injury is one of the most important stimuli for the development of atherosclerotic plaque . In 1997Triggers for EPC recruitment in neo-angiogenesis or vascular injury include the increased availability of angiogenic growth factors or chemokines, such as the vascular endothelial growth factor (VEGF), as well as angiopoietin or stromal cell-derived factor (SDF)-1 bonding with the chemokine receptor (CXCR-4), thereby expanding its expression on EPCs ,11,12,13Adiponectin, adipocyte fatty acid-binding protein, heart-type fatty acid-binding protein, lipocalin-2, fibroblast growth factor 19 and 21, retinol-binding protein 4, plasminogen activator inhibitor-1, and 25-hydroxyvitamin D are just a few of the already known biomarkers linked to cardiovascular and metabolic diseases. These biomarkers may help predict CVD risk. More research is needed to assess biomarkers’ validity and their potential to improve clinical decision-making and therapy management. The endothelium has a unique position as both a sensor and participant in the atherosclerotic process . BiomarkIn this review, we focus on current definitions of EPCs, discuss the individual relevance of circulating and adventitial resident progenitors in endothelial and vascular integrity, function, rejuvenation and restoration and address some promising therapeutic approaches and remaining questions.A consensus on the ideal marker for the identification of EPC cell types is lacking. This marker may originate from multiple precursors, such as a haemangioblast (HPC), BM progenitors, tissue-resident mesenchymal stem cells (MSC), and especially, from adipose tissue , impedinThe three existing techniques used to isolate and cultivate human EPCs ,22 lead The use of density barrier centrifugation is one method that can be utilised in order to separate mononuclear cells from peripheral blood mononuclear cells (PBMNCs). In most cases, cells are sown onto plates that have been coated with fibronectin and then grown using endothelial growth factors. The remaining spindle-shaped cells not only endocytose acetylated low-density lipoprotein (LDL) but also express EC markers and possess other characteristics of ECs. This is, in addition to the fact that EC indicators are present in these cells , acquireUsing markers such as CD133, combined with CD34 and VEGFR2, ensured that only progenitor cells with vasculogenic properties were identified ,27. CellThereafter, in vitro colony-forming cell assays allowed for the isolation of two cell types: colony-forming unit (CFU)-Hill cells and endothelial colony-forming cells (ECFC). In the ‘Hill assay’ and the ECFC, or ‘Ingram protocol’, monocytic cells isolated from blood samples were cultured for two days on fibronectin-coated dishes and then replated for further cultivation ,27. CFU-In summary, EPCs seem to represent two distinct populations with overlapping antigen expressions : hematopoietic-derived spindle-shaped cells from isolation method I/II, also referred to as circulating angiogenic cells or early EPCs (CFU-Hill colonies), and ECFCs, or late EPCs . Late EPAt present, there is a lack of information or knowledge regarding cell phenotypes in different diseases as the cells originate from different vascular beds and sources . Half ofInvestigating the role of CECs in endothelial injury with regard to plasma markers of endothelial injury  led to a correlation between CECs and vWf in heart failure .Almost all types of CVD were associated with hypertension, diabetes, smoking and high cholesterol. These CVFs can contribute to endothelial dysfunction ,46,47,48+CD133+CD309+ and CD34+CD133+ cells suggested the progression of cerebral small vessel disease [Physical activity at a moderate level was identified to be potentially beneficial for preventing CVD. The increase in EPC count was found to be mediated by eNOS and VEGF, and apoptosis was reduced in the cells . Physica disease . EPC cou disease . EPC cou disease .Some data still suggest that those “monitoring effects” are mostly seen in the young population. Aging by itself is another depriving factor of EPC ,63. Age Using EPC as a therapeutic target for CVD may therefore underlay an age-related effect. Early EPC implantation increased neovascularisation in young mice, but not in older mice with elevated cholesterol or other CVD risk factors .Mechanisms of heart failure in adult heart disease recently received a lot of research attention, but its pathogenic and prognostic significance in single-ventricle physiology is still unknown ,80,81. CHowever, early phase 1/2 clinical trials utilizing the intracoronary delivery of derived progenitor cells demonstrated dependable and safe outcomes in patients with single ventricle physiology. Except for all-cause mortality after staged procedures, derived cardiac progenitors cells administration improved ventricular function and was linked with fewer late problems in patients with single ventricles. Patients treated with cardiac progenitors cells and those who suffered from heart failure with reduced EF but not heart failure with intact EF may experience a substantial improvement in clinical outcomes ,87.Nonetheless, increasing the EPC count is a promising strategy. Early information  on EPC’sYet, there are dangers associated with the direct implantation of EPCs mainly triggered by the high number of cells with inflammatory potential that could be coinjected . DespiteAdditionally, the transfer of plasmid DNA for VEGF to young and senescent EPCs via ultrasonic microbubble transfection could enhance the angiogenic effects of both older and young cells, thereby leading to better outcomes and solving two problems at once Other new therapeutic options with EPC as a target include the treatment of ischemic stroke in patients with pre-existent CVD .2, the human vascular system constantly maintains a balance between coagulation and bleeding, inflammation and immuno-protection rendering difficult the identification of one single area of activation or damage contributing to the release of a specific set of ECs [There is a high risk of postoperative morbidity for patients following major surgery. Endothelial dysfunction in the perioperative period may increase the risk of surgical complications through altered vascular homeostasis and, consequently, decrease tissue perfusion. Targeting the endothelium and optimising natural physiological function in the perioperative phase is becoming a more popular as a way to improve postoperative outcomes. With an estimated surface area of more than 1000 mt of ECs . MSCs ant of ECs . EPCs wit of ECs . Their rt of ECs ,32.Differentiating EPCs from CECs remains difficult . EndotheWhile the identification of resident-tissue EPCs is advancing somewhat, the mechanism of progenitor cell release and the distinct role of different EPC types are still not understood. Differentiating between the role of early and late EPCs in CVD is important as, in CAD patients, a decreased amount of early EPCs was found , accompaMost studies to date describe factors which promote/inhibit either total (CD34+ or CD34/VEGFR2) or early EPC (CD34+/VEGFR2 +/CD133+ or VEGFR2+/CD133+) mobilisation and homing from the BM under physiological and pathological drug therapy conditions . ChangesWithin the identification of all of these influencing factors, a single clear mechanistic explanation to understand EPC function, mobilisation and endothelial integrity is lacking. Improvements in risk classification and the ability to regulate thrombotic and inflammatory cascades should enhance perioperative outcomes. The underlying subclinical microvascular endothelial dysfunction seems to have a greater impact on perioperative morbidity, contributing more to complications, such as impaired wound healing and end organ dysfunction, than the less common but more devastating macrovascular endothelial dysfunction.Ongoing studies address sheer stress as another key factor since low or disturbed shear stress, which occurs in vessel branch points, the outer wall of bifurcations, and the inner wall of curvatures are considered pro-atherogenic . Cell tuEC architecture and mechanisms of endothelial homeostasis are potential sensors and target therapeutic interventions in the atherosclerotic process. While current studies assessing EC subunits, phenotype and function enabled a general understanding of the pathophysiology of atherosclerosis, an ideal test of endothelial function for use in a clinical setting has yet to be established. A secure method for counting cells and differentiating cell kinds and compositions is urgently required, as is a deeper understanding of other modulators such as flow pattern, growth and transcription factors, and gene expression. CECs may serve as sensitive indicators of pre-existing damage and disease development, whereas EPCs may act as a biomarker of repair and a promising therapeutic target. Regenerative medicine has the potential to expand the therapeutic window for a variety of diseases, where surgical and medicamentous alternatives have run out. In CVD, safe cell replacement with new ones could usher in a new era of therapy.


Cardiac rhythm abnormalities, such as atrial fibrillation (AF) and ventricular tachycardia (VT)/ventricular fibrillation (VF), account for a significant proportion of adverse cardiac events and mortality . In factSaadeh and Fazmin review the mechanisms by which age-related mitochondrial dysfunction promotes arrhythmic triggers and substrate. This provided insights into novel potential anti-arrhythmic pharmacological interventions that specifically target upstream mitochondrial function, and hence ameliorates the need for therapies targeting downstream changes which have constituted traditional antiarrhythmic therapy. Deng et al. reviews the key role of uric acid in mediating oxidative stress and inflammation, which underlie fibrotic change of the atria predisposing to AF. Fong et al. conducted a meta-analysis into modulated calcium homeostasis and calcium release events in AF, demonstrating higher sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression in the between primary diseased AF group, but lower expression in the secondary AF groups. Costa et al. describe in-depth the molecular interactions between sex hormones and the cardiac ion channels, as well as the clinical implications of these interactions on the cardiac conduction system, in order to understand the link between these hormones and the susceptibility to cardiac arrhythmias.Cellular, animal and computational models have been used to study the molecular and electrophysiological mechanisms underlying cardiac arrhythmias . Saadeh Avula et al. presented APD dispersion data from two-dimensional optical mapping in a mouse model with F1759A SCN5A overexpression modeling long QT syndrome type 3. They then presented theoretical models for APD dispersion, methods for analysis and calculation of APD dispersion, and showed that APD dispersion in clustered patterns is the predominant configuration for spontaneous occurrence and persistence of AF and VF. Liu et al. conducted experiments to examine the effects of autonomic input in vein of Marshall-mediated AF. They found that electrical stimulation of the left superior ganglionated plexi shortened atrial refractoriness, increased APD dispersion and the vulnerability window. These effects were prevented by low level stimulation or ethanol ablation of the vein of Marshall, or muscarinic blockade by atropine. Acquired causes of VT/VF may arise from remodeling after myocardial infarction. In a mouse model with knockout of the CC chemokine receptor 9 (CCR9), abnormalities in ion currents, calcium handling, gap junction and action potential conduction were prevented, suggesting that the CCR9 can be a promising therapeutic target for reducing the likelihood of myocardial infarction-related arrhythmias .via the development of ischemic stroke .AF is the commonest cardiac rhythm abnormality observed in clinical practice and accounts for significant morbidity and mortality c stroke , dementic stroke , 8 and hc stroke . Shang eKim, Yu, Kim, Lee et al.), demonstrating that in patients with normal left ventricular ejection fraction, high H2FPEF score and increasing age were independently associated with the development of ischemic stroke. Song et al. found that a comprehensive evaluation of serum uric acid and B-type natriuretic peptide levels, left atrial diameter and left ventricular ejection fraction can stratify the risk of stroke in patients with non-valvular AF. A large retrospective cohort study conducted propensity score matching between with atrial flutter and AF . Among patients without history of stroke, the risk of dementia was higher in patients with AF than in patients with AFL.One of the most devastating outcomes of AF is stroke for which the CHA2DS2-VASc score has been used for its risk stratification . A recenPeng et al.). patients with short episodes of AF <24 h were compared to those with 24 h or longer in a propensity score-matched analysis for cryoablation of pulmonary veins isolation . Higher success rate and lower incidence of stroke/transient ischaemic attacks were observed for the <24 h group compared to the ≥24 h group during follow-up. Recurrence in AF remains a problem despite improvement in ablation technology. A prospective cohort study found that leukocyte telomere length predicted disease progression from paroxysmal to persistent AF following catheter ablation . Soluble Suppression of Tumorigenicity 2, was shown to be predictive of AF recurrence after radiofrequency ablation in patients with persistent AF . A systematic review and meta-analysis found that the HAS-BLED score has moderate predictive abilities for bleeding risks in patients with AF regardless of type of oral anticoagulants . For medical treatment of AF, beta blockers are frequently used. Interestingly, beta blockers consistently decreased long-term mortality in patients with high-burden and low-burden of premature atrial complexes, and this effect appears not to be mediated through a reduction in AF or new onset stroke . Recent work has explored traditional Chinese medicine as an adjunct therapy .The following studies investigated the impact of interventional and medical treatment on outcomes in patients with AF. Balloon-based catheter ablations, including hot balloon ablation and cryoballoon ablation, have rapidly emerged as alternative modalities to conventional catheter ablation owing to their procedural advantages and better clinical outcomes and safety profiles. A recent study found that patients undergoing hot balloon ablation had a higher incidence of touch-up ablation and longer procedural time, but with comparable clinical outcomes on mid-term follow-up . In a propensity score-matched study between patients undergoing transcatheter and surgical aortic valve replacement, post-operative AF had a worse impact on heart failure-related hospital admissions and composite outcome of mortality, stroke and heart failure-related admissions . In a single center prospective study, machine learning models were developed using least absolute shrinkage and selection operator (LASSO) and random forest (RF) algorithms for variable selection . Their LASSO-Cox model showed an area under the curve of 0.84 for predicting 1-year mortality.A registry study from Shanghai, China investigated the impact of new onset AF in patients with acute myocardial infarction. Interestingly, the authors found that asymptomatic rather than symptomatic new onset AF was significantly predictive of cardiovascular mortality and all-cause mortality . Another study from the same group examined a large cohort of 4,873 patients undergoing the head-up tilt test, finding that direct drug potentiation is safe and sensitive for diagnosing vasovagal syncope . Patients with an overlap syndrome between chronic obstructive pulmonary disorder and obstructive sleep apnoea showed higher rates of cardiac arrhythmias  compared to those with either condition alone . Heart rate was shown to be predictive of all-cause mortality in patients suffering from multiple myeloma, but further work is needed to explore the prognostic role of cardiac arrhythmias in this condition . Li and Zhang developed an autodetection algorithm using neural network combined with the attention-based bidirectional long-short term memory model for classifying nine different types of ECG patterns, including normal electrocardiogram, atrial fibrillation, atrioventricular block, left bundle branch block, right bundle branch block, premature atrial complexes, premature ventricular complexes, ST depression and ST elevation (Li and Zhang). Regarding AF treatment, the H2FPEF Score, which reflects the degree of left ventricular diastolic dysfunction, was improved following catheter ablation . Zhang et al. compared the catheter ablation to pacing in patients with tachycardia-bradycardia syndrome, reporting lower rates of the composite endpoint of cardiovascular-related hospitalization and thromboembolic events, as well as the progression of atrial fibrillation and heart failure.Age was identified as a risk factor of syncope recurrence in elderly patients with vasovagal syncope and positive head-up tilt test . Another study from the same group investigated whether the obesity paradox in all-cause mortality is present among the Chinese population with an ICD but did not demonstrate its presence . Sun et al. found that compared to night-time heart rate of ≤ 50 or ≥70 bpm, heart rate between 50 and 70 bpm was associated with lower risks of ventricular tachyarrhythmias, appropriate ICD shocks, inappropriate ICD shocks, and all-cause mortality. A retrospective study from Hong Kong, China investigated Chinese patients who were hospitalized for acute heart failure, demonstrating that fragmented QRS was a significant predictor of VT/VF, sudden cardiac death and cardiovascular mortality .VT/VF can arise from acquired causes, such as ischaemic heart disease, electrolyte disturbances and drugs. van der Voorn et al.). The commonest ion channelopathy globally is long QT syndrome, which is more common in Western compared to Asian populations  has aided risk stratification although its sensitivity, specificity, positive predictive value and negative predictive value can differ depending on the protocols employed (Brugada syndrome (BrS) is characterized by coved or saddle-shaped ST segment elevation in the right precordial leads . It has employed . Recent employed .Monasky et al. evaluated the role of genetic testing for risk stratification in BrS. They recommend that whole exome or whole genome testing and family segregation analysis should always be performed. Chen X. et al. studied the clinical and genetic characteristics of 104 probands with early repolarization syndrome, reporting its association with loss-of-function genetic defects in genes encoding the cardiac calcium channel. They identified a unique clinical entity characterized by decreased heart rate and QTc, as well as increased transmural dispersion of repolarization. In the case of the CACNA1C-P817S variant, impaired trafficking of the channel to the membrane contributes to the loss-of-function in the calcium channel.Nevertheless, recent available evidence indicates that risk prediction is more accurate when a multi-parametric approach rather than rely on a single investigative method . MonaskyWang Y. et al.). The pre-treatment baseline QTcd were significantly longer in responders treated with metoprolol compared to non-responders and that it was negatively correlated with SS after metoprolol treatment. The prenatal management for immune-associated congenital heart block in fetuses was reviewed, in particular issues pertaining to clinical management, including the roles of autoantibodies in its pathophysiology, diagnosis and prognosis . A study of multiple myeloma patients from Xi'an, China found that approximately half of the patients suffered from cardiac arrhythmias . In particular, those with sinus bradycardia had lower incidences of all-cause mortality compared to those without it.Finally, several studies examined risk stratification strategies for other tachycardias and also bradycardias. For example, the role of baseline-corrected QT interval dispersion (QTcd) in predicting the effectiveness of metoprolol in pediatric postural tachycardia syndrome was examined  and Tianjin Key Medical Discipline  Construction Project.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Heart failure with preserved ejection fraction (HFpEF) is a multi-organ disorder that represents about 50% of total heart failure (HF) cases and is the most common form of HF in the elderly. Because of its increasing prevalence caused by the aging population, high mortality and morbidity, and very limited therapeutic options, HFpEF is considered as one of the greatest unmet medical needs in cardiovascular medicine. Despite its complex pathophysiology, numerous preclinical models have been established in rodents and in large animals to study HFpEF pathophysiology. Although age and sex differences are well described in HFpEF population, there are knowledge gaps in sex- and age-specific differences in established preclinical models. In this review, we summarize various strategies that have been used to develop HFpEF models and discuss the knowledge gaps in sex and age differences in HFpEF. Heart failure (HF) is a leading cause of death worldwide and its prevalence is estimated to be 1%–2% of the adult population in developed countries, rising to ≥10% among people over 70 years of age . HF is aHFpEF consists of about 50% of total HF cases , and itsExtensive clinical and preclinical studies have resulted in the development of efficient therapies for HFrEF . HoweverHFpEF is recognized to be a multi-systemic disorder with broad phenotypic heterogeneity . PrognosAnimal models of HFpEF should mimic the phenotypes and symptoms observed in patients with HFpEF. Diastolic dysfunction as well as concentric hypertrophy and fibrosis are the main cardiac characteristics for HFpEF. Patients with HFpEF also develop systemic derangements such as exercise intolerance and pulmonary congestion/edema. Different strategies for induction of HFpEF risk factors, such as aging, hypertension, and metabolic stress, have been used alone or in combination to reproduce diastolic dysfunction and other phenotypes of HFpEF in animal models.The aging heart shows phenotypic changes such as cardiac hypertrophy, diastolic dysfunction, worsened myocardial performance, and preserved systolic function at rest but impaired while performing exercise . The oveConsistent with the observations in humans, studies on C57BL/6 mice  showed that aging results in left ventricular and left atrial hypertrophy, diastolic dysfunction, worsened myocardial performance, and a modest decline in systolic function . In addiSenescence-accelerated mouse (SAM) is a model of accelerated aging. Untreated male senescence-prone (SAMP8) mice develop diastolic dysfunction at 6 months of age , whereasAlthough aging animal models and patients with HFpEF share some common features such as cardiac hypertrophy, diastolic dysfunction, and exercise intolerance, aging alone does not result in HF in most animal models . HoweverHypertension is one of the major comorbidities contributing to the development of HFpEF. Because of its structural and functional consequences on the cardiovascular system, hypertensive stress is widely used in different animal models to study HFpEF.Dahl salt-sensitive (SS) rat model is a commonly used HFpEF model . TreatmeDeoxycorticosterone acetate (DOCA)–salt hypertension leads to hypervolemia due to an impairment of renal sodium handling where the kidneys reabsorb more water and sodium . When yoSpontaneously hypertensive rat (SHR) is an inbred rat strain that has a predisposition to the spontaneous development of hypertension . At arouAngiotensin II plays a major role in cardiac homeostasis and regulates such processes as afterload, preload, cardiac hypertrophy, and fibrosis . Young mWhile animal models of hypertension display cardiac features of HFpEF at an early stage, extracardiac features of HFpEF are not well represented and some hypertensive models progress to HFrEF at a later stage . TherefoObesity and diabetes are two of the major comorbidities in HFpEF . PatientLepr gene, which encodes the leptin receptor and that leads to the worsen affinity of leptin to its receptor  develop insulin resistance and dysfunctional leptin receptors due to a mutation in receptor . This mureceptor . Howeverreceptor .Ob/ob mouse is a leptin-deficient model in C57BL/6J background that spontaneously develops obesity and type 2 diabetes. Concentric hypertrophy, diastolic dysfunction, and preserved LVEF have been observed in ob/ob female and male mice . HoweverDb/db leptin receptor-deficient mouse model is characterized by a point mutation in the gene that encodes the leptin receptor in C57BL/6J or C57BL/6KsJ background. This mutation leads to obesity and results in type 2 diabetes . At 6 moThese models of metabolic stress have marked obesity with (ob/ob and db/db mice) or without diabetes (Zucker rats) and develop cardiac features of HFpEF in both sexes. Sex differences in cardiac HFpEF features have been observed, but age differences have not been determined in these models.Early attempts on the development of HFpEF animal models mainly focused on the use of single perturbations  to induce the clinical features of HFpEF. However, because HFpEF is a systemic disorder with multiple comorbidities, these single-hit models typically do not mimic certain symptoms of patients with HFpEF. Very recently, researchers established several novel models that utilize perturbations on two or more risk factors of HFpEF to recapitulate major clinical features of this complex syndrome .Obese ZSF1 rat is one of the first models that combine more than one perturbation to induce HFpEF phenotype in rodents. This model established by crossing female Zucker diabetic rat and male spontaneously hypertensive HF rat develops both cardiac and extracardiac HFpEF phenotype . Nguyen l-arginine methyl ester (l-NAME), respectively. After 15-week treatment, two-hit stress–treated mice display preserved EF and significant diastolic dysfunction accompanied by extracardiac HFpEF phenotypes including pulmonary congestion and exercise intolerance (l-NAME-only– and HFD-only–treated mice, two-hit stress–treated mice exhibit more severe HFpEF phenotypes and developed impaired cardiomyocyte contraction and relaxation kinetics (Recently, olerance . This moolerance . Comparekinetics . Using tkinetics .l-NAME and HFD would present exacerbated HFpEF progression, including the development of significant HFpEF phenotypes in old females. This two-hit model was later used to demonstrate the roles of NAD metabolism and Xbp1s-FoxO1–regulated lipid accumulation in the pathogenesis of HFpEF (L-NAME and HFD on female mice resulted in significantly attenuated phenotype as compared to male counterparts . Female of HFpEF .A recent study by Withaar et al.  combinedA more recent study by The aforementioned two- and multi-hit strategies generate novel animal models that display typical cardiac and extracardiac features of human HFpEF patients. Sex differences in HFpEF phenotypes were observed in two of these models , and advThe aforementioned murine and rat models have some limitations due to their sizes and different cardiac structure and function as compared to large animals and humans . Large aIn recent years, multi-hit swine HFpEF models were also established. Using streptozotocin injection, HFD and renal artery embolization, Despite sharing more similarities in their cardiovascular physiology with humans, large animal preclinical models have their limitations. Primary limitation of large animal preclinical models is the high general cost compared to rodent models . This ofMost of the pharmacological and device therapies for HFpEF investigated in completed clinical trials have been shown to be ineffective. So far, only the recent EMPEROR-Preserved clinical trial showed that empagliflozin reduced the combined risk of cardiovascular death or hospitalization for HF in patients with HFpEF . It is iIt is highly unlikely that a single animal model will fully recapitulate the phenotypes of the heterogenous HFpEF population. For example, a limitation of HFpEF models induced by hypertension is the fact that, in the majority of patients with HFpEF, it continues to display HFpEF symptoms even when their blood pressure is under control . Some moMany preclinical HFpEF studies used animals of one sex or mixed sex, limiting the interpretation of sex-specific differences in HFpEF pathogenic mechanisms. Because of the sex disparities in HFpEF, it is important to include both sexes in HFpEF research and analyze the outcomes separately to determine any potential sex-specific mechanisms. While it is well established that the prevalence of HFpEF sharply increases with age , the maj


These findings indicate that cathepsin-targeted FGS has potential to improve treatment outcomes in breast cancer patients.The majority of breast cancer patients is treated with breast-conserving surgery (BCS) combined with adjuvant radiation therapy. Up to 40% of patients has a tumor-positive resection margin after BCS, which necessitates re-resection or additional boost radiation. Cathepsin-targeted near-infrared fluorescence imaging during BCS could be used to detect residual cancer in the surgical cavity and guide additional resection, thereby preventing tumor-positive resection margins and associated mutilating treatments. The cysteine cathepsins are a family of proteases that play a major role in normal cellular physiology and neoplastic transformation. In breast cancer, the increased enzymatic activity and aberrant localization of many of the cysteine cathepsins drive tumor progression, proliferation, invasion, and metastasis. The upregulation of cysteine cathepsins in breast cancer cells indicates their potential as a target for intraoperative fluorescence imaging. This review provides a summary of the current knowledge on the role and expression of the most important cysteine cathepsins in breast cancer to better understand their potential as a target for fluorescence-guided surgery (FGS). In addition, it gives an overview of the cathepsin-targeted fluorescent probes that have been investigated preclinically and in breast cancer patients. The current review underscores that cysteine cathepsins are highly suitable molecular targets for FGS because of favorable expression and activity patterns in virtually all breast cancer subtypes. This is confirmed by cathepsin-targeted fluorescent probes that have been shown to facilitate  To date, breast cancer remains the most frequently diagnosed cancer and the leading cause of cancer-related mortality in women worldwide, representing about 25% of all cancer cases and 15% of all cancer deaths . Margin BCS based on visual and tactile feedback assisted by current localization techniques, such as implanted radioactive iodine seeds, still results in tumor-positive resection margin rates up to 40% –15. A tuNumerous pathology and imaging methods for intraoperative guidance and margin assessment have been evaluated to decrease tumor-positive resection margin rates after BCS, but most have significant clinical and technical limitations that have precluded widespread adoption , 19. An Tumor-targeted NIR fluorescence-guided surgery combines the administration of a contrast agent, consisting of a fluorophore and a targeting moiety, with the use of a fluorescence-sensitive camera, matched to operate in the range of NIR fluorescence light (700–900 nm) . It alloin vivo on the resection cavity surfaces and ex vivo on the resected specimen [ex vivo detected tumor-positive margin to the in vivo location of the residual tumor that needs to be excised is extremely difficult. Therefore, detection of residual tumor tissue on the surgical cavity walls seems to be the most promising approach.In BCS, tumor-targeted NIR FI for tumor-positive resection margin detection could be used both specimen –23. Howespecimen . Consequin vivo margin assessment in breast cancer patients [Multiple contrast agents directed at different molecular targets are under extensive investigation for NIR fluorescence-guided BCS . Howeverpatients , 26–29.Cathepsins are an important group of proteolytic enzymes that play a major role in both normal cellular physiology and disease , 31. TheCysteine cathepsins are mainly, but not exclusively, located within the endo-lysosomal system, where they are crucial for lipid and protein metabolism, autophagy, and antigen presentation , 36. In Dysregulated activity of cysteine cathepsins is associated with various pathological conditions, including atherosclerosis, neurodegenerative disease, osteoporosis, arthritis, and cancer –46. In nDuring neoplastic transformation, the normally tightly regulated activity of cysteine cathepsins is altered by gene amplifications and the formation of transcript variants . AnotherOver recent decades, it has been shown that various members of the cysteine cathepsin family have a more than tenfold overexpression and a more than 50-fold increase in enzymatic activity in breast cancer compared with healthy breast tissue –58. UntiIn this review, we first discuss the expression of the individual cysteine cathepsins in breast cancer to substantiate their potential use as a target for fluorescence-guided breast cancer surgery. The scope of this review is restricted to the most extensively investigated cysteine cathepsins B, C, K, L, O, S, and V. We summarize their specific role in cancer progression, cellular sources, and correlation to clinicopathological characteristics such as molecular subtype Table . In addiCathepsin B is one of the more ubiquitously expressed members of the cysteine cathepsin family that has been a main focus of research in breast cancer. It has both carboxypeptidase and endopeptidase activity and its substrates include ECM components such as laminin, fibronectin, collagen types I and IV, and proteoglycans . Lah et in vitro and in vivo studies have demonstrated that the cellular origin of cathepsin B overexpression can change during tumor progression [The cellular sources of cathepsin B in breast cancer are predominantly tumor cells and macrophages and, to a lesser extent, stromal components such as myoepithelial cells, fibroblasts, myofibroblasts, and endothelial cells of the neo-vasculature , 62. Botgression . In the gression –70. It igression –74. ThisThe important contribution of cathepsin B to breast cancer progression becomes clear in cell line and murine model studies. Knockdown or selective inhibition of cathepsin B suppresses cancer invasion and metastasis to the lung and bone, whereas an increased cathepsin B expression and activity promotes metastatic spread –79.In vitro studies revealed a functional role of upregulated cathepsin B in pericellular proteolysis of the ECM and basement membrane components, driving tumor invasion [Several underlying molecular mechanisms of cathepsin B overexpression and its contribution to cancer progression have been elucidated, some of which seem to be breast cancer subtype-specific. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, for instance, overexpression is the result of an increased transcription of the cathepsin B gene due to an HER2-activated kinase signaling network . In horminvasion , 82. Intinvasion , 61, 62.invasion .In conclusion, cathepsin B is upregulated in various breast cancer subtypes and has a broad spectrum of cellular sources at the primary and metastatic site. It plays a prominent role in many tumor-promoting processes and seems to be specifically implicated in breast cancer metastasis to the lung and bone. Jointly, the data suggest that the upregulation and partly extracellular localization of cathepsin B may be a prime target for imaging purposes.Cathepsin C is a ubiquitously expressed lysosomal aminopeptidase required for the activation of pro-inflammatory neutrophil serine proteases such as elastase and proteinase 3, signifying its role as a mediator of inflammation . CathepsA wealth of information on the specific role of cathepsin C in breast cancer was derived from a series of experiments by Xiao et al., ascribing this enzyme a key role in lung metastasis . In humain vitro, to induction of signaling pathways that result in neutrophil recruitment and the formation of neutrophil extracellular traps (NETs)—web-like structures composed of granule proteins and decondensed chromatin that promote tumor progression and metastasis [The functional role on a molecular level of cathepsin C is related, at least tastasis . This istastasis , 89. Thetastasis . InteresTo summarize, in breast cancer, cathepsin C holds a specialized role during the early stages of pulmonary colonization. By stimulating a signaling cascade leading to the activation and exploitation of neutrophils, this enzyme promotes cancer cell proliferation in the lung. The mainly lysosomal location of cathepsin C could hinder its accessibility to probes, making it less ideal as an imaging target . On the Increasing evidence provides insight into how breast cancer cells take advantage of yet another member of the cathepsin family, cathepsin K. Cathepsin K is an endopeptidase that functions in the lysosomal and extracellular environment . As a poOne of the molecular mechanisms through which cathepsin K contributes to breast cancer progression is by activation of ECM degrading matrix metalloproteinases, which enhances the invasiveness and metastatic capacity of breast cancer cells . AdditioAs for associations with other clinicopathological characteristics, assessment of both hormonal receptor-positive and -negative cell lines and patient samples at different stages imply a stage rather than receptor-dependent expression , 95, 99.To conclude, cathepsin K plays a major role in the metastatic spread of breast cancer cells to the bone. Its overexpression and increased enzymatic activity in bone-residing breast cancer cells and its extracellular localization indicates the potential associated with this protease for the imaging of bone metastases.In vitro ribonucleic acid (RNA) interference and pharmacological inhibition studies demonstrated that breast cancer cells use cathepsin L to enhance their proliferative, invasive, and migratory capacity by degrading ECM components[in vivo, as mice injected with cathepsin L knockdown breast cancer cells or treated with a selective cathepsin L inhibitor exhibited significantly smaller tumors compared to the controls with functional cathepsin L [A considerable number of experiments have been performed to investigate the expression and role of cathepsin L in breast cancer. Cathepsin L is a lysosomal and extracellular endopeptidase whose expression is upregulated in breast cancer cells and TAMs, and which increases with advancing tumor grade , 61, 67.omponents–106. Thihepsin L , 105.One of the mechanisms by which cathepsin L is upregulated is the loss of stress-induced shutdown of selective messenger RNA (mRNA) translation . Under pIn conclusion, across a variety of breast cancer subtypes significant overexpression and amplified enzymatic activity of cathepsin L is observed, which increases with advancing grade and stage and has an important role during lung metastasis. It has also been established that breast cancer cells can maintain high cathepsin L levels, prioritizing its expression during stress conditions. Both cathepsin L upregulation in breast cancer cells of different molecular subtypes and its partly extracellular localization make cathepsin L a suitable target for intraoperative fluorescence imaging of breast cancer.Currently, only a limited number of studies have been conducted to examine the expression and role of cathepsin O in breast cancer. Nevertheless, these few studies show that cathepsin O is highly expressed by breast cancer cells . The oveIts expression in ER-positive breast cancer cells suggests cathepsin O could be a suitable target for tumor imaging. However, cathepsin O expression and activity in tumor cells versus normal breast tissue, its cellular sources and localization should first be more extensively investigated for all breast cancer subtypes.Cathepsin S is a lysosomal and extracellular endopeptidase , 33. ItsEvidence for the contribution of this protease to tumor progression is reflected by the profound impact of knockdowns and pharmacological inhibition in TNBC cell lines as well as in animal models , 116. ThCathepsin S has been shown to have a prominent role in orchestrating breast to brain metastasis , 116. DuAnother mechanism by which cathepsin S contributes to breast cancer progression is the proteolytic degradation of the aforementioned BRCA1, resulting in suppressed DNA double-strand break repair activity . ContrarTaken together, these studies indicate the relevance of cathepsin S in the progression of breast cancer cells, especially TNBC, and show its contribution to brain metastasis by facilitating the crossing of the blood–brain barrier. Increased cathepsin S expression and activity in breast cancer and its part extracellular localization would make this enzyme a perfect target for the tumor imaging of TNBC and its brain metastases.Cathepsin V is a mainly lysosomal endopeptidase that shares structural similarities with cathepsin L; however, it exerts distinct functions and has a confined tissue distribution . Under pMore recently, cathepsin V expression has been associated with advancing tumor grade, distant metastasis, and breast cancer recurrence , 120. ExOn the molecular level, cathepsin V promotes the degradation of GATA binding protein 3 (GATA3) in ER-positive breast cancer cells . GATA3 iIn summary, cathepsin V seems to play an important role in the progression from DCIS towards invasive breast cancer. The available data indicates that elevated cathepsin V levels promote the progression of ER-positive cancer cells by degradation of GATA3. Cathepsin V upregulation in breast cancer cells compared to normal breast tissue indicates that this protease could be useful for targeted FI. A possible disadvantage is its mainly lysosomal localization, which could make it less accessible to an imaging probe.in vitro to study their role in cellular (patho)physiology. However, since the introduction of clinical NIR FI systems, cathepsin-targeted probes are under extensive investigation for their possible use in intraoperative tumor visualization. Because of the increased expression and proteolytic activity of cysteine cathepsins in breast tumors of virtually all molecular subtypes, these proteases show great potential as targets for fluorescence-guided breast cancer surgery in a large patient population. Moreover, since cysteine cathepsins derive from both tumor cells and tumor-stromal cells, such as TAMs, cathepsin-targeted probes will potentially result in a more homogeneous tumor signal compared to probes targeting tumor cell-specific proteins, such as epidermal growth factor receptor (EGFR), that often display high intratumor and interpatient heterogeneity [In recent decades, numerous cathepsin-targeted FI agents have been developed. Initially, cathepsin-targeted probes were used to visualize cathepsin activity ogeneity , 126.quenched) probes that only fluoresce after activation. They can either be activated by one specific cathepsin or be pan-reactive, targeting multiple cysteine cathepsins at once. The advantage of activatable agents over traditional fluorophores is the substantial reduction in off-target (or background) fluorescence and an increased speed of detection since no time is required to clear from non-target tissues [Most cysteine cathepsin-targeted contrast agents are so-called turn-ON  . The folin vivo breast cancer visualization in a syngeneic orthotopic mouse breast cancer model [in vivo. In addition, it enabled rapid imaging of enzyme activity in fresh frozen human breast cancer tissue sections [in vivo by the fluorescent signal and VGT-309 could be used for fluorescence-guided resection [in vivo in a syngeneic orthotopic mouse model. However, due to the selectivity of BMV157 for cathepsin S, its fluorescence signal in breast tumors was weaker than that of BMV109 [Various quenched substrate-based (qSBP) and activity-based probes (qABPs) have been investigated in breast cancer cell lines and mouse models. The cathepsin S-directed qABP BMV083, developed by Verdoes et al., allowed for er model . Withanasections , 131. Suesection . Bender f BMV109 .Further improvement of BMV109 by Ofori et al. resulted in the quenched substrate-based probe 6QC-NIR, designed to exploit the latent lysosomotropic effect . This caTo increase tumor selectivity, Widen et al. synthesized a so-called AND-Gate probe, DEATH-CAT-FNIR, that required activation by cathepsin L and caspase 3, a different type of protease . This ANin vivo in a syngeneic subcutaneous mouse model and was succesfully used for photodynamic therapy (PDT), a method for cancer treatment that involves the activation of a photosensitive molecule by a light source to cause selective cytotoxic damage to cancer cells [designed ankyrin repeat protein (DARPin), which is a small antibody mimetic. In both a congenic and syngeneic orthotopic breast cancer mouse model, DARPin 8h6 highlighted mammary tumors both in and ex vivo with a considerable contrast to healthy tissue [in vivo with a high tumor to background ratio [in vitro [ex vivo in breast cancer compared with normal muscle tissue in an orthotopic mouse model [Ben-Nun et al. developed a cathepsin B, L, and S targeted, photosensitized qABP. This probe, YBN14, was able to visualize breast tumors er cells . Kramer y tissue . A cathend ratio . Porrebain vitro . The PEGse model .To the best of our knowledge, LUM015 is the only cathepsin-targeted contrast agent for fluorescence-guided breast cancer surgery that has been introduced in the clinic.ex vivo imaging of resected breast cancer tissues [A first-in-human phase I clinical trial showed that preoperative intravenous LUM015 administration did not raise safety signals and resulted in tumor-specific fluorescence that could be detected upon  tissues . A subse tissues . In a ph tissues . In an a tissues . An ongoThe previously described cathepsin-targeted probe VGT-309 is currently under investigation in a phase II clinical trial in 40 patients undergoing surgery for primary lung cancer or lung metastases (NCT05400226). The preliminary results in two patients have already been published and illustrate the successful clinical translation of VGT-309 and its potential to improve surgical management of patients undergoing cancer resection . HoweverTo date, cathepsin-targeted activatable imaging probes for fluorescence-guided breast cancer surgery have been administered intravenously, necessarily days or hours prior to surgery. Alternative to systemic delivery, cathepsin-targeted turn-ON probes could be topically applied onto the breast cancer patient’s surgical cavity to rapidly differentiate between tumor and healthy tissue, as has been demonstrated in a recent preclinical study . The mai, we have discussed the expression and role of cysteine cathepsins in breast cancer and their applicability to fluorescence-guided breast cancer surgery. The cysteine cathepsins B, C, K, L, O, S and V are all highly overexpressed by and have an increased proteolytic activity in virtually all breast cancer subtypes and play major roles in tumor progression. Most of these proteases have been shown to be suitable targets for fluorescence-guided breast cancer surgery, a technique that can identify tumors, facilitate residual tumor identification and guide additional resection. The first and only cathepsin-targeted probe introduced in the clinic thus far shows promising results, possibly preventing the need for re-excision and—potentially—tumor recurrence. Various other cathepsin-targeted probes demonstrate great potential for intraoperative breast cancer imaging in preclinical studies. Due to their particular design, some of these probes offer certain advantages, such as retention of the fluorescence signal in the target cells or a lower background signal, probably making them the most preferable agents for fluorescence-guided breast cancer surgery. Clinical translation of these promising cathepsin-targeted fluorescent probes in the near future could greatly improve treatment outcomes for breast cancer patients, especially if used in a topical application protocol that is more patient friendly and easily integrated into the surgical workflow.In this review