Aberrant methylated genes (DMGs) play an important role in the etiology and pathogenesis of esophageal squamous cell carcinoma (ESCC). In this study, we aimed to integrate three cohorts profile datasets to ascertain aberrant methylated-differentially expressed genes and pathways associated with ESCC by comprehensive bioinformatics analysis. We downloaded data of gene expression microarrays  and gene methylation microarrays (GSE52826) from the Gene Expression Omnibus (GEO) database. Aberrantly differentially expressed genes (DEGs) were obtained by GEO2R tool. The David database was then used to perform Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome pathway enrichment analyses on selected genes. STRING and Cytoscape software were used to construct a protein-protein interaction (PPI) network, then the modules in the PPI networks were analyzed with MCODE and the hub genes chose from the PPI networks were verified by Oncomine and TCGA database. In total, 291 hypomethylation-high expression genes and 168 hypermethylation-low expression genes were identified at the screening step, and finally found six mostly changed hub genes including KIF14, CDK1, AURKA, LCN2, TGM1, and DSG1. Pathway analysis indicated that aberrantly methylated DEGs mainly associated with the P13K-AKT signaling, cAMP signaling and cell cycle process. After validation in multiple databases, most hub genes remained significant. Patients with high expression of AURKA were associated with shorter overall survival. To summarize, we have identified six feasible aberrant methylated-differentially expressed genes and pathways in ESCC by bioinformatics analysis, potentially providing valuable information for the molecular mechanisms of ESCC. Our data combined the analysis of gene expression profiling microarrays and gene methylation profiling microarrays, simultaneously, and in this way, it can shed a light for screening and diagnosis of ESCC in future. ESCC is a serious threat to human health, and its five-year survival rate is less than 10%, making it a leading cause of cancer-related death3. So far, the molecular mechanism of the occurrence and development of ESCC has not been fully clarified. Currently, one of the main directions of ESCC research is searching for key genes or specific biomarkers that affect the occurrence and development of ESCC to determine genetic susceptibility factors in ESCC and clarify their molecular mechanisms4. Therefore, the cloning and identification of new genes related to ESCC are of great significance in terms of early warning signs, early clinical diagnosis, treatment, prognosis monitoring and the development of new anticancer drugs for susceptible individuals.Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, with incidence rates ranking fourth and eighth in China and worldwide, respectively6. DNA methylation is the most common epigenetic change, and it is closely related to gene expression regulation7, embryonic development8, X chromosome inactivation9, genomic stability10 and genomic imprinting11, and plays a significant role in cancer progression. DNA methylation of gene promoters is associated with silencing of key genes, especially oncogenes and tumor suppressors and is considered to be a hallmark in many carcinomas12. Recently, some studies have confirmed that certain genes with abnormal DNA hypermethylation or hypomethylation are involved in various processes of ESCC development14, but it is still difficult to determine the comprehensive profile and pathways of the interaction network.Epigenetics refers to the heritable alteration of gene expression unrelated to changes in DNA sequenceComprehensive analysis of multiple datasets provides the capabilities to properly identify and assess the pathways and genes that mediate the biological processes associated with ESCC. In the present research, we used publicly available datasets of gene expression  and gene methylation (GSE52826) to identify genes and pathways that were both abnormally methylated and differentially expressed between tumor tissues and normal esophagus from patients with ESCC. The selected genes were then subjected to hierarchical clustering, and functional enrichment analyses of GO and KEGG pathways. Then we used STRING to create protein-protein interaction networks and identify hub genes that were key to these signaling events. Furthermore, we validated the results using the Cancer Genome Atlas (TCGA) data to identify DMGs involved in the pathogenesis of ESCC. Finally, the GEPIA database was utilized to perform survival analysis. We believe our data provide comprehensive biological information about novel differentially methylated genes and enhance the level of understanding regarding the development and progression of ESCC.The flowchart of our study is demonstrated in Fig. GO annotation and pathway enrichment analyses of the 291 mutually inclusive hypomethylated/high-expression and 168 hypermethylated/low-expression genes were implemented using the online tool DAVID. Genes that were hypomethylated and highly expressed were enriched in extracellular matrix organization, extracellular structure organization, mitotic cell cycle process, cell cycle process, mitotic cell cycle phase transition, collagen catabolic process, nuclear division and multicellular organism catabolic process Fig. , while tThe STRING database was applied for PPI network construction, with MCODE applied for module analysis. Hub genes were identified using the cytoHubbaCytoscape software. The PPI network for genes that were hypomethylated and highly expressed is demonstrated in Fig. Hypermethylated/low-expression hub genes and hypomethylated/high-expression hub genes were then validated in another database TCGA to confirm the outcomes. The outcomes were summarized in Table According to previous research, the expression of the six hub genes, namely, DSG1, AURKA, CDK1, LCN2, KIF14 and TGM1 were demonstrated in normal esophageal tissue and ESCC. The prognostic value of six hub genes was assessed via the GEPIA database. In addition, we made box plots to visualize the relationships of the expression levels of the 6 key DMGs. Patients with high expression of AURKA were associated with shorter overall survival Fig. . AlthougAlthough surgery, chemotherapy and radiation therapy techniques have been greatly improved, the prognosis and survival rate of ESCC are still poor, given the challenges and difficulties in the screening and diagnosis of ESCC. Recently, molecularly targeted therapeutics has been carried out, but they have not exhibited beneficial effects on the long term prognosis of ESCC. Exploring the potential mechanisms of ESCC initiation and development will greatly benefit the diagnosis, treatment and prognosis assessment. In this study, we identified 291 hypomethylated/ high-expression genes and 168 hypermethylated/low-expression genes via multiple bioinformatics tools. Enrichment of these genes demonstrated that aberrant methylation indeed affects certain pathways and hub genes. Our findings can provide novel insight into the explanation of ESCC pathogenesis.17. Our results agree with the fact that carcinoma cell metastasis and invasion are closely associated with abnormal cell adhesion, endodermal cell division and nuclear division20. Extracellular matrix (ECM), is a critical component of the cancer cell niche, it can provide the tissue with the mechanical support and also mediate the cell-microenvironment interactions22. It is noteworthy that collagens are one of the major proteins found within the ECM and are associated with many aspects of tumorigenesis23. Therefore, this is consistent with the research results that the activation of these cellular processes through ECM is a major cause of tumorigenesis, progression and metastasis. The structure of cytoskeleton is regulated by many cytokines, and thecytoskeleton are closely related to the occurrence and development of tumors24. Furthermore, the enriched KEGG pathways of hypomethylated highly expressed genes were mainly linked to ECM-receptor interaction, Focal adhesion, Amoebiasis, PI3K-Akt signaling pathway and Cell cycle. ECM is the first barrier that prevents tumor metastasis26. Tumor angiogenesis and destruction of the extracellular matrix are two important conditions for tumor invasion and metastasis27. It has been reported that the expression of Focal adhesion protein is related to the occurrence, cell differentiation, invasion, lymph node metastasis and prognosis of esophageal cancer and the prognosis of patients with positive expression of Focal adhesion protein is worse than that of patients with negative expression28. The study showed that the related protein factors in thePI3K/Akt signaling pathway are highly expressed in esophageal squamous cell carcinoma, and that the pathway is associated with the microvascular formation of esophageal squamous cell carcinoma, and the invasion and metastasis of cancer cells29. In contrast, hypermethylated/low-expression genes were related to Pancreatic secretion, Renin secretion, Amphetamine addiction, Salivary secretion and cAMP signaling pathway. Abnormal pancreatic secretion plays an important role in the occurrence and development of reflux esophagitis. Eliminating reflux of pancreatic and bile in Barrett’s esophagus patients can inhibit the progression from atypical hyperplasia to adenocarcinoma31. Renin can be converted to angiotensin under certain conditions. Studies have found that angiotensin II receptor 1 is related to the differentiation of esophageal squamous cell carcinoma32. Angiotensin II and angiotensin II receptor 1 may participate in the occurrence and development of esophageal squamous cell carcinoma, but not via the invasion of esophageal squamous cell carcinoma and lymph node metastasis33. As a second messenger in cells, cAMP plays an important role in cell growth, proliferation and differentiation34. cAMP is closely related to the occurrence and development of tumors. It has been reported that the growth of tumor cells has a certain relationship with the decrease in intracellular cAMP content35. Increasing the content of cAMP can inhibit the growth of tumors and induce the apoptosis of tumors. The content of cAMP was positively correlated with the treatment of tumors. Therefore, cAMP can be used in the treatment and prevention of tumors in the clinic, and has high clinical value36.Together, these data suggested that detecting these aberrant signaling pathways could precisely predict tumor progression.The GO enrichment analysis revealed that the primary biological processes of the hypomethylated/high-expression genes were involved in the regulation of extracellular matrix organization, extracellular structure organization, mitotic cell cycle process, cell cycle process, collagen catabolic process and nuclear division, while the hypermethylated/low-expression genes were primarily linked to response to cAMP, purine-containing compound, oxygen-containing compound, muscle system process, inorganic substance. Previous research has shown that altered expression of particular genes may affect ESCC cell metastasis, invasion, apoptosis and proliferation37. The dysregulation of KIF14 has been confirmed in many human malignancies. Huang et al. found that KIF14 was highly expressed in glioma samples and positively correlated with its pathological grade and proliferative activity38. Knockdownof KIF14 by siRNA can inhibit the growth of independent anchorage and induce G2/M arrest, which leads to a decrease in the phosphorylation and activity of Akt, thus inducing apoptosis and cytokinesis failure of glioma cell lines and inhibiting the growth of tumors. Osako et al. validated that the overexpression of KIF14 in ESCC clinical specimens39. It has also been found that downregulation of KIF14 expression can significantly inhibit the proliferation of esophageal squamous cell carcinoma cells and arrest the cell cycle in the G0/G1 phase. In addition, the absence of KIF14 may enhance the sensitivity of tumors to chemotherapy40.Therefore, the study of the relationship between KIF14 and cancer and the related mechanism will be a powerful basis for early diagnosis, targeted therapy and prognosis prediction in cancer.The PPI network of hypomethylated/high-expression genes provides an approach to identifying the functional associations between them and the top three hub genes were selected from this: KIF14, CDK1 and AURKA. The kinesin family member 14 (KIF14) gene, a member of the kinesin superfamily (KIFS), promotes tumorigenesis and progression by regulating the formation of mitotic spindles, chromosome segregation, and cytokinesis41. Previous studies have found that CDK1 expression was higher in breast cance42, oral squamous cell carcinoma43, cervical cancer44 and gastric cancer45 compared to normal tissues, and positively correlated with lymph node metastasis, differentiation, clinical stage and histopathological stage. In esophageal tumors, Expression of CDK1 in ESCC was significantly higher than that in norm esophageal46. CDK1 may be a biomarker that could be used for screening tumors, early prognosis and molecular targeted therapy.CDK1 (Cyclin-dependent kinase 1) is a serine/threonine protein kinase, that plays a key role in cell proliferation at the G2/M point of the cell cycle. At present, CDK1 is a necessary condition for cell division of all eukaryotic cells. Errors in the regulation mechanism of CDK1 directly lead to cell differentiation disorders, cell cycle disorders, malignant cell proliferation and abnormal transformation, and ultimately malignant tumor formation47. The mutation or overexpression of AURKA may also cause carcinogenesis. AURKA is highly expressed in many tumors, such as breast48, ovarian49, gastric50 and colon cancers51. The abnormal expression of AURKA in various solid tumors is closely related to tumor occurrence and development. Importantly, some reports indicate that high expression of AURKA contributes to the development of ESCC52. Therefore, exploring the relationship between AURKA and esophageal cancer and its mechanism might help to provide a new target for the treatment of esophageal cancer.Aurora kinases, including Aurora-A, Aurora-B and Aurora-C are a new serine/threonine kinase found in recent years that are involved in mitosis, Aurora-A, or AURKA, regulates the functions of centrosomes and microtubules to ensure the correct separation of centrosomes and the complete division of the cytoplasmKIF14, CDK1 and AURKA may all be abnormally methylated genes that modulate the cell cycle and proliferation in ESCC. Regarding the hypermethylated/low-expression genes, the most prominent hub genes were LCN2, TGM1 and DSG1.53. The presence of this protein in blood and urine is an early biomarker of acute kidney injury54. This protein is thought to be involved in a variety of cellular processes, including maintaining the balance of the skin’s internal environment and inhibiting invasion and metastasis. Mice lacking this gene are more susceptible to bacterial infections than wild-type mice. In addition, it was found that the expression of LCN2 was different in tumors, but it was confirmed that LCN2 played a key role in the differentiation, proliferation, angiogenesis, invasion and metastasis of tumors55. HPA database analysis revealed thatLCN2 proteins were medium expressed in normal esophageal tissues, while no expression was observed in ESCC tissues. In contrast to our finding, Zhao et al. found that endogenous LCN2 expression was increased by activation of the ERK signaling pathway, which promoted the invasion and migration of ESCC56.The gene lipocalin 2 (LCN2) encodes a protein belonging to the lipocalin family. Members of this family transport small hydrophobic molecules such as lipids, steroids and retinols. The protein encoded by this gene is a neutrophil gelatinase-associated lipocalin that acts by injecting iron-containing carriers to limit bacterial growth and plays a role in innate immunity57. Current studies have shown that the mutation of the TGM1 gene is closely related to lamella stratified ichthyosis58 and may also affect the proliferation and differentiation of breast cancer cells59. Other studies have found that high expression of TGM1 in non-small-cell lung cancer may be conducive to stable adhesion between cancer cells60. In addition, researchers found that the mechanism by which TGM1 regulates the development of gastric cancer may be related to the Wnt signaling pathway. When the expression of TGM1 is inhibited, Wnt signaling pathway activity is significantly reduced61. Similar to our research, Zhong et al. found that the level of TGM1 was increased in ESCC56. Recent studies have shown that the expression of transglutaminase 3 (TGM3) in normal esophageal squamous epithelium is significantly reduced than cancer tissue62. TGM3, mainly expressed by the superficial epithelial layers of normal head and neck mucosa, is a key factor in the terminal differentiation of keratinocytes. Analysis of the changes and mechanisms of TGM3 in the occurrence and development of ESCC is expected to reveal a molecular marker for the early detection of ESCC.Transglutaminase 1 (TGM1), the main subtype of three TGM genes expressed in the epidermis and stratified squamous epithelium, encodes for calcium-dependent mercaptase TGM1. TGM1 has two forms in the cytoplasm and membrane junction and has the ability to transfer amino acids to glutamic acid residues in proteins to form isopeptide bonds63. At the same time, Krunicl et al. observed by electron microscopy that the number and distribution density of desmosomes on the surface of squamous cell carcinoma cells decreased or even disappeared64. These results fully reflect that the decrease in cell adhesion molecules is closely related to the biological malignant behavior of squamous cell carcinoma. Other studies have shown that the expression of DSG1 in the esophageal mucosa of patients with eosinophilic esophagitis is significantly decreased compared to that in normal controls56.Desmoglein 1 (DSG1) is a Ca2 + -dependent adhesin desmosomal cadherin that is an important component of desmoglein. DSG1 widely exists in the epithelium, myocardium and other tissues and plays an important role in cell-to-cell junctions. In recent years, DSG1 was been found to have abnormal expression or function in skin, mucosa and tumors. It was found that the expression of Dsg1 in cancer cell lines decreased or even disappeared compared to that in normal control cells65. Our study combined the analysis of gene expression profiling microarrays and gene methylation profiling microarrays via bioinformatics analysis of available microarray data. In this way, it is possible to develop more precise and reliable screening results. However, we only validated candidate abnormally methylated genes that were differentially expressed using the Oncomine and TCGA databases. Further experiments will be necessary to confirm that these genes and pathways are linked to ESCC.Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, however the molecular mechanism of the occurrence and development of ESCC has not been fully studied.Previous reports have mostly analysed only methylation or only gene expression data and have not been sufficiently powered to discover hub gene expression data, and have not been sufficiently powered to discover hub genes and pathwaysIn conclusion, our research offers a comprehensive bioinformatics analysis of aberrantly methylated DEGs associated with multiple signaling pathways that may be involved in the occurrence and development of ESCC.In addition, the six most modified hub genes were identified, includingKIF14, CDK1, AURKA, LCN2, TGM1, and DSG1.These new discoveries may provide insights for unraveling the pathogenesis of ESCC, and these candidate genes may be optimal abnormal methylation-based biomarkers for the precise diagnosis and treatment of ESCC.Further molecular biological experiments are required to verify the function of the identified candidate genes in ESCC.https://www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information (NCBI)67. In total, 17 ESCC and 17 normal specimens were obtained from GSE20347, while 30 ESCC and 30 normal samples were obtained from GSE38129. Both expression microarrays used the platform GPL571: [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array. For the gene methylation profiling microarray, GSE52826 included a total of 4 ESCC tumour samples and 4 adjacent normal surrounding tissue samples. The platform of this methylation microarray was GPL13534 (Illumina Human Methylation Bead Chip).The gene expression microarray of GSE20347 and GSE38129, and the gene methylation microarray GSE52826 were downloaded from the Gene Expression Omnibus  is an interactive web toll that allows users to compare different groups of samples in a GEO series to examine differentially expressed genes according to experimental conditions. | log FC| > 1 and P < 0.05 were used as the cut-off standards to obtain DEGs, and | t| > 2 and P  <  0.05 were used as the cut-off standards to find DMGs. Eventually, a Venn diagram was used to identify hypermethylated/low-expression genes and hypomethylated/high-expression genes.We used the GEO2R online tool to analyze the raw data of microarrays and identify DMGs and DEGs between the tumor tissues and normal tissues. GEO2R (https://david.ncifcrf.gov/). DAVID is the Database for Annotation, Visualization and Integrated Discovery and is often used to perform functional and pathway enrichment analysis, which offers systematic and integrative functional annotation tools for investigators to unravel biological meaning behind a large list of genes68. P < 0.05 was regarded as statistically significant.Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on selected genes that were hypomethylated/high-expression and hypermethylated/low-expression via DAVID  to construct a PPI network of hypomethylated/high-expression genes and hypermethylated/low-expression genes70. An interaction score of 0.4 was regarded as the cut-off criterion and the PPI was visualized. Then, MCODE was conducted to screen modules of the PPI network with MCODE score>4 and number of nodes >4 in Cytoscape software. The top 3 hub genes were selected by the CytoHubba app in Cytoscape software. To determine the expression pattern of six hub genes in ESCC, the datasets were analyzed in Oncomine (https://www.oncomine.org). Oncomine is an online database consisting of previously published and open-access microarray data71. The analysis enables multiple comparisons of gene expression between different studies, the importance of the gene expression across the available studies was also taken into account. The consequences were filtered by selecting ESCC vs. normal tissue.PPI analysis is essential for illustrating the molecular mechanisms of key cellular activities in carcinogenesis. We used the STRING database (72. MEXPRESS (http://mexpress.be/) is a data visualization tool designed for the easy visualization of TCGA expression73, DNA methylation and clinical data, as well as the relationships between them. In TCGA set validation, the top 20 genes that with high DMGs were selected. To confirm the results, we used the MEXPRESS to validate hypermethylated/low-expression hub genes and hypomethylated/ high-expression hub genes in the TCGA database. The human Protein Atlas is a website that involves immunohistochemistry-base expression data for distribution and expression of 20 tumor tissues, 47 cell lines, 48 human normal tissues and 12 blood cells. In our study, direct contrast of protein expression of different hub genes between normal and HNSCC tissues was used by immunohistochemistry image. The probability of survival and significance were calculated using the GEPIA database. GEPIA is a newly created online interactive web server which enables users to explore the RNA sequencing expression information of tumors/normal tissues or samples from the Genotype Tissue Expression (GTEx) projects and The Cancer Genome Atlas (TCGA), based on a criterion processing pipeline. GEPIA offer customizable functions such as profiling regarding to pathological stages, cancer types, differential expression analysis, survival analysis, correlation analysis and similar gene detection.The Cancer Genome Atlas (TCGA) database, collaboration between the National Cancer Institute and National Human Genome Research Institute, has generated comprehensive, multi-dimensional maps of the key genomic changes in various types of cancersSupplementary information.Supplementary material 2.


POT1 is recurrently mutated in about 3.5% of CLL. In addition, cases with short telomeres have higher telomerase (TERT) expression and activity. TERT activation and shelterin deregulation thus may be pivotal in maintaining the minimal telomere length necessary to sustain survival and proliferation of CLL cells. On the other hand, activation of DNA damage response and repair signaling at dysfunctional telomeres coupled with checkpoint deregulation, leads to terminal fusions and genomic complexity. In summary, multiple components of the telomere system are affected and they play an important role in CLL pathogenesis, progression, and clonal evolution. However, processes leading to shelterin deregulation as well as cell intrinsic and microenvironmental factors underlying TERT activation are poorly understood. The present review comprehensively summarizes the complex interplay of telomere dysfunction in CLL and underline the mechanisms that are yet to be deciphered.Telomeres are nucleprotein structures that cap the chromosomal ends, conferring genomic stability. Alterations in telomere maintenance and function are associated with tumorigenesis. In chronic lymphocytic leukemia (CLL), telomere length is an independent prognostic factor and short telomeres are associated with adverse outcome. Though telomere length associations have been suggested to be only a passive reflection of the cell’s replication history, here, based on published findings, we suggest a more dynamic role of telomere dysfunction in shaping the disease course. Different members of the shelterin complex, which form the telomere structure have deregulated expression and  Telomeres are repetitive DNA sequences at the ends of the chromosomes that play a pivotal role in maintaining genomic stability by capping and protecting the ends from degradation and fusions. Maintenance of telomere length is a key for immortalization in cancers. In chronic lymphocytic leukemia (CLL), telomere length has been identified as an independent prognostic factor in various studies. In addition, the deregulation of different telomere components has a profound influence on the CLL pathomechanisms. The present review is thus aimed at summarizing the clinical and biological aspects of telomere shortening, mutations and deregulated expression of telomere associated genes, and mechanisms that are important for telomerase activation in CLL, to pave way for a deeper understanding of telomere dysfunction in CLL pathogenesis.All eukaryotic chromosomes have specialized nucleo-protein structures called telomeres which cap the ends. The nucleic acid component of the telomeres comprises of long tracts of DNA repeat sequences, ending with a 3’ single stranded DNA overhang. In mammals, the telomere sequences consist of TTAGGG hexamers, repeated many kilo bases in length . In somaPOT1 mutations point to a global telomere dysfunction that plays an important role in disease pathogenesis and evolution.In stem cells, germ cells, and in various cancers, the telomere length is maintained, most commonly by the reverse transcriptase enzyme, telomerase (TERT). It is an RNA-dependent DNA polymerase that uses the telomerase RNA component (TERC) as a template to synthesize the telomeric DNA . Thus inThe primary role of telomeres is to confer genomic stability. The shelterin complex shields the telomeres from activation of the DNA damage response signaling at the telomeres. In particular, TRF2 of the sheltein complex is important to prevent activation of the ATM  and subsTerc and crossing through generations G1 to G6 by knocking out Terc led to increased incidences of spontaneous malignancies and decreased stress response and survival  analysis is considered to be the gold standard. The method includes the process of using a restriction enzyme that does not detect the telomere repeat sequence to digest the non-telomeric DNA, followed by resolution on a gel and southern hybridization , 20. Evein situ hybridization (FISH) using fluorescence labelled (CCCTAA)n telomere binding probes are used for analyzing telomere length, where the intensity of the signal directly corresponds to the length of the telomere sequence in a given sample. The method when coupled with chromosomal banding is a valuable tool for analyzing telomere length of individual chromosomes. FISH based telomere length measurements could be made high-throughput by using flow cytometry (flow-FISH) (Fluorescence ow-FISH) . AnotherHowever, the most widely used technique for telomere length measurement is by qPCR, based on a method devised by Cawthon et al. . In brieMseI and NdeI that do not digest the telomeric repeats, followed by ligating adapters complementary to the overhangs created by these enzymes. The non-telomeric parts of the genome that have these adapters on both the ends form a pan-handle like structure due to complementarity between the ends, suppressing PCR amplification. On the other hand, the telomeic sequences have a digested 5’ end and a 3’ G rich overhang that is not processed by the enzymes. Ligation of telorette to the 3’ overhang allows specific amplification of telomeres of all chromosomes. This method is useful for genome-wide analysis of the distribution of critically short telomeres.In addition to the above methods that were used for telomere length analysis in CLL, newer techniques have been developed for analyzing different aspects of telomere length. The STELA PCR is capable of analyzing critically short telomeres only on a subset of chromosomes such as XpYp that have unique subtelomeric sequences suitable for designing chromosome specific primers. This limitation was overcome by the universal STELA method (U-STELA) . The tecBfaI, CviAII, MseI, NdeI to digest the non-telomeric DNA as well as the non-canonical sub-telomeric DNA and to generate 5’ AT and TA overhangs. The 5’ ends of the digested DNA are then dephosphorylated to prevent re-ligation of the ends. Double stranded DNA adapters with phosphorylated 5’ AT and TA overhangs containing C3 spacers are tagged to the digested ends. Telomeres are then amplified using a primer pair specific for the TeSLA-T and 5’ AT/TA adapters. TeSLA allows high resolution analysis of the distribution of <1 to 18 kb long telomeres.The STELA and U-STELA, though highly sensitive, they are biased towards detection of short telomeres (<8kb). The method was further improved and the telomere shortest length assay (TeSLA) was developed . In TeSLNovel approaches for telomere assessment such as using CRISPR/Cas9 RNA-directed nickase system to specifically label telomeres followed by high throughput imaging using nano channel array have also been developed. This technique permits mapping and analysis of individual telomeres based on subtelomere repeat elements (SRE) and unique sequences in the chromosomes. Recently, another method for telomere length measurement by molecular combing or DNA fiber analysis was reported  where ceDue to the wide range of methods used for telomere length analysis, the comparability of telomere lengths analyzed in different CLL studies are limited. Moreover, while TRF and STELA based methods have greater reproducibility, qPCR and FISH based methods need to be very carefully and extensively optimized to limit batch effects . One of TP53 and ATM which are critical checkpoint genes activated upon telomere shortening and dysfunction are found in the minimally deleted regions of 17p- and 11q-, respectively. Deletion of these genes therefore permits these tumor cell clones to undergo further telomere shortening compared to non-17p-/11q-, without activating cell death pathways. In line with this, short telomere length was found to be associated with the presence of mutations in TP53  , 31 sugg in TP53 , 41, 43 P53  , 38, 51 TP53/ATM mutations, 17p-, 11q- could be explained as a direct outcome or “consequence” of increased proliferation of the cells harboring these high risk features , CD27(+), post-germinal center (GC) B-cell subsets , where aTP53 or ATM which would eventually allow for further telomere shortening and cell proliferation. This notion is supported by study on temporal association of genomic alterations in CLL, where 17p-/TP53 mutations and 11q-/ATM mutations were found to be later events in CLL pathogenesis  and telomeric repeat binding factor 2   . These t B-cells . Anotherfunction . Also, d outcome . The sheSince telomere maintenance is one of the key features of cancers, the telomere system has been considered an attractive target for cancer therapy. Accordingly, therapeutic agents targeting various components of telomeres and the different maintenance mechanisms have been developed and studied across cancers. One of the first inhibitors of telomerase to have progressed to clinical trials is imetelstat. It is a synthetic lipid conjugated 13-mer oligonucleotide that competitively binds to hTR, thereby inhibiting telomerase function . In vitrAnother class of molecules that affect telomerase activity include nucleoside analogs such as 6-thio-2’-deoxyguanosine (6dG), didanosine (ddITP), azidothymidine (AZT-TP), and 5-fluro-2’deoxyuridine (5-FdU). These compounds when incorporated at the telomeric ends by telomerase leads to chain termination and uncapping of the telomeric ends . UncappiThough limited clinical progress has been achieved with inhibitors of telomerase, various telomere based immunotherapies are successfully being evaluated in clinical trials for different malignancies. Since telomerase is one of the most commonly expressed tumor associated antigen, different methods are being employed to activate adaptive immune responses against telomerase. TERT peptide vaccines such as INO-1400 (NCT02960594—solid tumors), GV1001 (NCT04032067—Benign Prostatic Hyperplasia), UCPVax , and GX301  are currIn summary, though the direct inhibition of telomerase has shown limited success, hTERT based immunotherapy are rapidly gaining importance for the treatment of a wide range of tumor entities. In CLL, the novel agents such as ibrutinib and venetoclax have achieved tremendous clinical success however, treatment of Richter transformation has still proved to be challenging. Since Richter syndrome is a highly proliferative tumor type, they might have a greater dependency on telomerase than CLL and hence the novel TERT based immunotherapies either as single agents or in combination with checkpoint inhibitors maybe of interest.The relation between telomeres and CLL is complex. Though a large amount of effort has been put forward in understanding the prognostic relevance of telomere length and telomerase, various other aspects such as mechanisms underlying telomerase activation and molecular alterations leading to deregulation of telomere maintenance system still needs to be understood. In summary, deregulations of the different components of the telomere system play important roles at specific phases of CLL pathogenesis and progression. A deeper understanding of these mechanisms is vital for the development of therapeutics options for targeting these disease features, especially in patients that turn refractory to novel agents, or as combination treatments to improve efficacy or in the treatment of Richter transformation.The authors BMCJ and SS wrote the manuscript. Both authors contributed to the article and approved the submitted version.This study is supported by Deutsche Forschungsgemeinschaft (DFG) (SFB 1074 projects B1 and B2).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.


Proper development and function of the vertebrate heart is vital for embryonic and postnatal life. Many congenital heart defects in humans are associated with disruption of genes that direct the formation or maintenance of atrial and pacemaker cardiomyocytes at the venous pole of the heart. Zebrafish are an outstanding model for studying vertebrate cardiogenesis, due to the conservation of molecular mechanisms underlying early heart development, external development, and ease of genetic manipulation. Here, we discuss early developmental mechanisms that instruct appropriate formation of the venous pole in zebrafish embryos. We primarily focus on signals that determine atrial chamber size and the specialized pacemaker cells of the sinoatrial node through directing proper specification and differentiation, as well as contemporary insights into the plasticity and maintenance of cardiomyocyte identity in embryonic zebrafish hearts. Finally, we integrate how these insights into zebrafish cardiogenesis can serve as models for human atrial defects and arrhythmias. All vertebrate hearts comprise the same fundamental structural units: the atrial and ventricular chambers, which are specialized to reflect their functions receiving and expelling blood, respectively. The coordinated assembly of these chambers into a functional organ is an intricate process that is critical for normal embryonic development and throughout life of all vertebrates. In humans, mutations in genes that disrupt chamber formation and maintenance are associated with congenital heart defects (CHDs), which are the most common type of birth defect ,7,8,9,10While proper blood circulation is necessary in all vertebrate organisms, the number of atrial and ventricular chambers within vertebrate hearts can vary. Mammals have a four-chambered heart with two atria and two ventricles, which are necessary for circulation to the lungs and the rest of the body. However, teleosts, such as the common model zebrafish, have a relatively simple two-chambered heart consisting of only a single atrium and ventricle. Despite these differences, the underlying molecular signals and morphogenetic processes, particularly those that govern the specification and maintenance of cardiac chambers during heart development, are conserved across species. Moreover, due to their external development, the ease of genetic manipulation, and their ability to survive the first week post fertilization without a functional cardiovascular system, zebrafish have become an excellent tool for understanding the early processes underlying vertebrate cardiogenesis ,18,19,20Proper heart function relies on the coordinated contraction of the atrial and ventricular chambers. Importantly, zebrafish cardiomyocytes share key electrophysiological properties with mammalian cardiomyocytes, allowing them to also serve as a model for human conduction defects ,21. Cardventricular myosin heavy chain (vmhc), also referred to as myosin heavy chain 7 (myh7), can be detected in the medial ventricular progenitors as early as the 13 somite stage [atrial myosin heavy chain (amhc), also named myosin heavy chain 6 (myh6), begins slightly later at 19 somites [pitx2c and meis2b are upregulated on the left and downregulated on the right in the zebrafish atrium, consistent with the left atrial expression pattern of Pitx2 in mammals [Like all vertebrates, the zebrafish heart is the first organ to form and function during embryogenesis. The initial stages of zebrafish heart development involve the specification, migration, and differentiation of cardiomyocyte progenitor cells as they are integrated into the forming heart tube ,32,33,34te stage . Express somites . It was  mammals ,38. As tImportantly, vertebrate hearts are formed from the progressive differentiation of progenitor populations that grow the heart at the poles. Thus, the zebrafish cardiac disc then elongates over the yolk to form the linear heart tube E. The easonic hedgehog mRNA promotes atrial and ventricular specification and yields hearts with increased cardiomyocytes, while loss of HH signaling through the genetic and pharmacological inhibition of the Smoothened receptor reduces cardiac progenitor specification, consequently resulting in hearts with decreased atrial and ventricular cardiomyocytes [Numerous early signaling pathways are involved in the regulation of cardiomyocyte specification as part of their requirements in the establishment of the body plan, which factors into determination of the number of atrial cardiomyocytes within the zebrafish heart. Signals including Hedgehog (HH), canonical Wnt/β-catenin (Wnt), and Nodal exert similar effects promoting the specification of both atrial and ventricular cardiomyocytes. Increasing HH signaling through the injection of myocytes . Similarmyocytes ,52, due myocytes ,54  mutant, which harbors a mutation in the major embryonic producer of RA aldh1a2 , and treatment with RA signaling antagonists results in enlarged hearts with an increase in the number of atrial and ventricular cardiomyocytes [Although initial studies in mice and zebrafish concluded that retinoic acid (RA) signaling promotes atrial cardiomyocyte specification ,56, pivomyocytes ,58. Excemyocytes ,60. Thusalk8/lost-a-fin (laf) mutants, treatment with pharmacological BMP signaling inhibitors, and an inducible dominant-negative Alk8 transgene all demonstrated that BMP signaling is required early for atrial progenitor specification during gastrulation and results in hearts with decreased atrial cardiomyocytes. Consistently, increasing BMP signaling through the injection of constitutively-active alk8 mRNA results in increased atrial cardiomyocytes, reinforcing the role of BMP in atrial specification [In contrast to signals that have similar consequences on the specification of atrial and ventricular cardiomyocytes, early signals that affect dorsal–ventral patterning have differential effects on chamber specification. The dorsalizing pathway fibroblast growth factor (FGF) signaling preferentially affects the specification of ventricular progenitors with minimal effect on the atrium . Conversfication ,63. Thushoxb5b, an RA-responsive gene within the ALPM which is posterior to the cardiac progenitors, has been shown to result in excess atrial cardiomyocytes, suggesting it may function downstream of RA to restrict the number of atrial cardiomyocytes [cloche mutants, which we now understand are caused by mutation of the transcription factor Npas4l [Scl  and Etv2 (Etsrp) function to restrict heart size through promoting hematopoietic and vascular lineages within the ALPM. Cloche mutants or concurrent depletion of Scl and Etv2 with morpholinos both produce enlarged hearts due to an increase in atrial cardiomyocytes [Noggin3 transgene at 16 hpf causes a specific decrease in atrial cardiomyocytes that is independent of its earlier role ventralizing embryos [lats1; lats2 mutants, it was shown that decreased Hippo signaling results in an increased number of atrial cardiomyocytes, supporting that Hippo signaling restricts atrial size. Subsequent analysis revealed that Hippo functions upstream of BMP signaling to restrict the number of SHF-derived cardiomyocytes that arise from the ALPM and contribute to the venous pole of the heart [Subsequent to the signals that modulate atrial cardiomyocyte number and chamber size through establishment of cardiomyocyte progenitor populations, signals then control atrial chamber size through influencing cardiomyocyte differentiation from within the ALPM. While RA signaling may restrict the number of both atrial and ventricular cardiomyocytes, the lineage tracing analysis suggested that signals downstream of RA may differentially affect atrial and ventricular productions . Depletimyocytes . Howeverr Npas4l , and dowmyocytes   and Nuclear receptor 2 (Nr2f/Coup-tf) transcription factor families have been shown to be necessary to promote the differentiation of atrial cardiomyocytes at the venous pole. Isl1 marks the venous SHF in zebrafish. Consistent with this expression, isl1 mutants have decreased atrial cardiomyocytes, which temporal differentiation assays have shown to be due to reduced differentiation [Isl1 is specifically required for the differentiation of cardiomyocytes at the venous pole in zebrafish embryos, contrasting with the requirement of Isl1 in mice, which is required for the differentiation of all SHF progenitors at both the posterior venous and anterior arterial poles [isl2b is required for development of the arterial pole in zebrafish, suggesting that in teleosts Islet family members have distinct roles in progenitor differentiation at the different poles of the heart [In addition to the aforementioned signaling pathways, specific members of the Islet  and atrioventricular septal defects (AVSDs). AVSDs, a subtype of ASD, account for 5–7% of CHDs, while ASDs are reported in as many as 20% of CHD patients ,7,8,9. ATBX5, HH signaling, NR2F2, and NKX2.5 are some of the specific and better studied genes associated with ASDs and AVSDs in humans. Murine models have highlighted that improper specification of the venous SHF, leading to loss of the dorsal mesenchyme protrusion, is a primary cause of ASDs [Tbx5 mutant mice develop AVSDs, which can be rescued with restoration of HH signaling [Tbx5a mutant zebrafish, called heartstrings, have distended hearts, although cardiac specification does not appear to be affected [Tbx5 expression within the posterior SHF [Tbx5 expression and repress Tbx1 expression. Blocking RA signaling at the time point when Tbx5 is required in the SHF results in AVSDs [Mutations in  of ASDs . In huma of ASDs ,111. Is ignaling ,113. Tbxaffected  , and double outlet right ventricle (DORV) [NR2F1 and NR2F2 have been associated with craniofacial defects in humans [Nr2f1a and Nr2f2 function redundantly to restrict the number of ventricular cardiomyocytes and promote pharyngeal muscle specification at earlier stages, but do not overtly function redundantly in promoting atrial cardiomyocyte differentiation [nr2f genes in zebrafish may provide an understanding of the variability of CHDs affecting both the atrium and ventricle, and craniofacial defects associated with NR2F mutations in humans.myocytes . Howeverith CHDs ,129,130 ith CHDs . Furthermyocytes . In miceucidated ,78. Nr2fnded AVC , which we (DORV) ,120. Addntiation . Thus, uNkx2.5 haploinsufficiency in humans is associated with ASDs, AVSDs, and tetralogy of Fallot (ToF), as well as conduction defects, such as atrial fibrillation, arrhythmias, and atrioventricular (AV) block [Nkx2.5 plays during cardiogenesis  block ,132,133,ogenesis . As illuelopment ,135. Imp the SAN , reminish hearts . Interese atrium ,136,137.illation ,106 (Tabstic SAN . Additiodycardia ,94.HCN4, which lies downstream of the SAN regulatory genes mentioned above and is vital for normal pacemaker function [HCN4 are associated with sick sinus syndrome in humans [Hcn4 knockout mice are embryonic lethal due to a lack of functional pacemaker cells [Hcn4 in adult mice leads to bradycardia and eventual heart block [hcn4 causes bradycardia and sinus pauses reminiscent of the sick sinus syndrome phenotypes found in patients with HCN4 mutations [Nkx2.5 and Shox2, as well as their downstream targets, such as Hcn4.An additional SAN marker that has been modeled in zebrafish is the ion channel function . Mutation humans ,104 (Taber cells , and knort block . In zebrutations . TherefoZebrafish are an excellent model for dissecting the molecular and genetic mechanisms of vertebrate heart development. Contemporary advances through the use of zebrafish have furthered our understanding of conserved factors driving the development and maintenance of the vertebrate atrium and SAN. Mutations in genes that are required for the specification, differentiation, and maintenance of venous pole cardiomyocytes are often associated with a spectrum of CHDs affecting the atria in humans. While numerous factors have been shown to be involved in the development of the vertebrate atrium and SAN, how these signaling pathways interact and are coordinated to promote proper cardiogenesis and maintain cardiomyocyte identity require further investigation. Future work in zebrafish will help to dissect the critical mechanisms and regulatory networks governing venous pole development in the vertebrate heart, providing insights in the etiology of CHDs in humans.


Tumor cells establish a complex ecosystem called the tumor microenvironment (TME) which consists of stromal cells, immune cells, extracellular matrix (ECM) macromolecules and enzymes . It is nIreland and Mielgo reviewed the literature on the roles of macrophages and fibroblasts. The authors first examined the origin of these cells and their physiological functions and discussed how they are activated in the context of malignancies. Unlike their quiescent counterparts, CAFs are activated in response to chronic tissue damage which is also called “wounds that do not heal,” leading to crosstalk between tumor cells and CAFs via a wide range of cytokines. Among these cytokines, TGFβ and IL6 play a major role in therapeutic resistance by driving an epithelial mesenchymal transition (EMT) in tumor cells. Tissue resident or bone marrow (BM) derived macrophages are recruited to, and activated at, the tumor site by tumor-derived factors. Upon failing to eradicate tumors, M1-like macrophages are polarized to M2 TAMs by tumor-secreted immunosuppressive cytokines, such as TGFβ, IL4 and IL10. The authors also discuss current efforts to target CAFs and M2 TAMs with therapeutic approaches ranging from blocking the recruitment of macrophages to the tumor site to repolarizing them into M1-like macrophages and reprogramming CAFs. Ongoing phase I/II clinical trials include the blockade of CSF1, CSF1R, CCL2, and CD40 in M2 TAMs and Smo and IGF in CAFs. Similarly, Liu et al. discussed how fibroblasts build the tumor microenvironment by secreting ECM, associated enzymes and other factors. The authors reviewed the recent literature on the critical importance of CAF-ECM and CAF-tumor cell interactions in tumor cell migration and invasion. LOX-induced ECM crosslinking has emerged as a viable molecular target and several LOX inhibitors are in preclinical development. Furthermore, Nallanthighal et al. provided a comprehensive review on the mechanistic properties of ECM that regulate the phenotype of cancer stem cells (CSC). Tumor ECM is stiffer due to overexpression of a wide range of macromolecules and ECM-modifying enzymes, which causes transmission of the signal to CSCs via focal adhesion kinase (FAK) and its downstream effectors driving the transcription of stemness genes, such as OCT4, SOX2, and NANOG. CSCs also evade immune recognition by ECM mediated activation of the PI3K/Akt pathway.The following articles provide a comprehensive review of the current understanding of TME as illustrated in Haro and Orsulic reported a favorable survival outcome that is associated with CAFs in B-cell lymphoma patients. Potential mechanisms proposed are the entrapment of malignant B cells by CAFs and associated ECM in lymph nodes and induction of apoptotic cell death by CAF-derived TGFβ.In contrast to solid tumors, Turdo et al., the authors discussed potential strategies to target CSCs by blocking the stem cell specific pathways such as Notch, Wnt, and Hedgehog or TME-produced cytokines that regulate the stemness of malignant cells. Efforts to sensitize CSCs to ICIs by either CSC-primed dendritic cells or CAR T cell therapy with CSC-specific antigens were also discussed.In Chaib et al. discussed the types of myeloid cells, their significance in tumor progression and recent strategies to target this cell population. Due to their suppressive activity, MDSCs have been implicated in the development of therapeutic resistance against ICIs. The authors discussed a comprehensive list of myeloid cell targets, such as PI3Kγ, NF-κB, CSF, CCL2, TLRs and histone deacetylases in pre-clinical and early clinical trials. A comprehensive review by Canning et al. examined the immune landscape of head and neck squamous cell carcinomas (HNSCC) according to human papilloma virus (HPV) status. While HPV(+) HNSCCs were infiltrated with lymphocytes that correlated with better clinical outcome, HPV(–) counterparts were infiltrated with highly immunosuppressive immune cells that correlated with poor outcome. In early clinical trials with ICIs, HPV(+) patients experienced better clinical outcomes compared to HPV(–) patients despite their high tumor mutation burden. These findings concur with the immunosuppressive role of TME in therapy response in solid tumors.One potential target in TME are the immune cells of myeloid origin. Mehner and Radisky reviewed the dual role of serine protease inhibitor Kazal type 1 (SPINK1) in the tumor and the TME. Aberrantly overexpressed SPINK1 in a wide range of solid tumors and the stroma renders tumor cells resistant to apoptotic cell death and increases cell proliferation, while enhanced expression in the tumor stroma contributes to therapeutic resistance.Finally, In summary, although TME is viewed as a promising therapeutic target, we still face significant challenges in developing adequate tools to effectively target these cells. Current variable successes in targeting tumor stroma highlight the need to better understand the molecular characteristics of stromal cells to develop more precise and less toxic targeted therapies.HK wrote the manuscript. SO edited the manuscript and drew the figure. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this review article, the current understanding of the interaction between the peripheral nerve and the skeleton system is summarized, with a particular focus on bone marrow mesenchymal stem cells (BMMSCs), except for osteoblasts and osteoclasts. The novel application of nerve-based bone regeneration via BMMSCs may provide a new strategy in tissue engineering and clinical treatment of osteoporosis and bone disorders.Large bone reconstruction is a major clinical issue associated with several challenges, and autograft is the main method for reconstructing large defects of maxillofacial bone. However, postoperative osteoporosis of the bone graft, even with sufficient vascularization, remains a primary problem. Therefore, better understanding of the mechanisms and clinical translation of bone homeostasis is required. Neuronal innervation of the bone is an emerging research topic, especially with regards to the role of peripheral nerves in regulating bone homeostasis. Moreover, sensory and autonomic nerves regulate this process  Large defects of the maxillofacial bone caused by tumors, trauma and congenital malformation, etc., seriously affect the appearance and function of the patient. Moreover, functional reconstruction is clinically difficult, but is highly important. Tissue engineering is a promising technology, with previous cutting-edge studies showing the importance of advanced scaffold materials with controlled release or molecularly imprinted intelligence for tissue regeneration or cancer treatment via certain neurotransmitters via sensory nerves Based on the fact that postoperative osteoporosis, despite a sufficient blood supply, inevitably develops in autografted bones indicates that there may be systemic factors other than just blood supply that control the internal environment of the bone graft. As the blood supply of bone is accompanied by innervation, it has been hypothesized that nerves play an important role in regulating the bone homeostasis. Previous studies have revealed that sympathetic nerves and sensory nerves affect bone metabolism and bone remodeling via which innervation regulates bone homeostasis may involve BMMSCs. Both sympathetic and sensory nerves play a crucial role in regulating bone remodeling via specific neurotransmitters. In particular, sensory neurotransmitters, such as NGF, Calcitonin Gene-related Peptide (CGRP), SP, and Semaphorin 3A (Sema3A) positively regulate bone formation via BMMSCs. The present review examines how peripheral nerve regulates bone homeostasis via MSCs, which may provide a basis for translational research of systemically regulating bone remodeling, regeneration and prevention of osteoporosis.Remodeling of innervation effectively prevents osteoporosis of the bone graft, suggesting that the mechanism Osteoporosis is a very common skeletal degenerative disease in the elderly and menopausal women. Moreover, it is characterized by decreased bone mineral density (BMD) and destruction of bone microarchitecture, which give rise to an increased risk of fragility fracture of by more than 40% Osteoporosis is related to 'bone homeostasis', which mainly refers to the relatively stable state of the intraosseous environment under the precise regulation of the system network, which should be the bone remodeling balance between osteogenic activity of osteoblasts and bone resorption activity of osteoclasts. In addition, osteoporosis occurs when the activity of osteoblasts decreases and that of osteoclasts increases, due to the disturbed balance in bone homeostasis.Bone homeostasis is closely related to the 'aging' of BMMSCs. Friedenstein et al. latissimus dorsi muscle to regenerate mandibular bone, and the results provide a good example of ectopic bone formation in-situ bone formation with autologous adipose stem cells and β-tricalcium phosphate granules in vitroBMMSCs, as key cells for bone regeneration and maintenance, have become a hot topic of research in recent years. Furthermore, there are ongoing clinical trials for treating non-union of the bone, osteonecrosis of the femoral head and other bone diseases by directly injecting autologous MSCs or implanting with carriers via the CD40/CD40L pathway Stem cells are controlled by a microenvironment called the 'stem cell niche', rather than existing independently in vitro model, our previous study reported that inflammatory microenvironment can cause loss of differentiation potential of normal BMMSC and apoptosis but can also lead to a reduced ability of BMMSC to induce apoptosis of osteoclasts via the NFκB and Wnt pathways, and the alteration of these pathways affects runt-related transcription factor 2 (Runx2) and peroxisome proliferator-activated receptor γ (PPARγ), which are two key transcription factors regulating osteogenesis and adipogenesis, and thus causes stem cell aging Previous studies have shown that immune cells can secrete pro-inflammatory factors that damage BMMSCs and affect tissue regeneration .As nerve fibers are involved in the formation of the bone marrow stem cell niche, the regulation of stem cells by the nervous system has become an increasing focus of research According to Hilton's rule via stimulation from the sympathetic nerves and it can inhibit bone formation via the β2 receptor expressed on osteoblasts Based on the clinical discovery that patients who are paralyzed are prone to osteoporosis, the role of the nervous system in maintaining bone mass in bone metabolism has become an important research topic via the upregulation of the Erk/ Bcl-2 pathway NGF is a member of the neurotrophic factor family and plays a critical role in the development of various types of nerve cells in the central and peripheral nervous systems via the trigeminal axons, and can perform its biological effects by binding to the main receptor transient receptor potential vanilloid 1 (TRPV1). Moreover, the effect of CGRP in bone metabolism has been demonstrated in osteoporosis-phenotype CGRP-knockout mice CGRP is a 37 amino acid peptide synthesized by the CGRP gene on the short arm of chromosome 11 and is widely found in the central and peripheral nervous systems via RANKL/OPG pathways under the activation of TRPV1, resulting in reduced bone resorption in animal models with experimental periodontitis in vivo and in vitroCGRP can act on various intraosseous cells such as osteoblasts and osteoclasts. Furthermore, it has been shown that CGRP inhibits osteoclasts via NK1R in advanced bone formation. It has also been demonstrated that in skull osteoblast SP induces increased mineralization and expression levels of the bone-related proteins Runx2 and osteocalcin + signaling accelerates NF-κB nuclear translocation in osteoclasts, and SP activates NF-κB in bone marrow macrophages (BMMs), which directly facilitates RANKL-induced macrophage osteoclastogenesis and bone resorption activity SP, a neuropeptide of the tachykinin family, is a highly conserved 11-amino acid neuropeptide involved in pain perception, and has identical protein sequences in mice, rabbits and humans in vitro+ MSCs to the wound region, which accelerates bone remodeling and bone maturation by activating the Erk1/2 signaling pathway In addition, previous studies have shown that SP can stimulate the proliferation of BMMSCs and the mineralization of differentiated BMMSCs via peripheral nerves.Sema3A, an axon guidance molecule, belongs to the semaphorin family and is an important member of the vertebrate sensory neurotransmitter microenvironment. In addition, Sema3A is a membrane-associated secretory protein in the central nervous system, which is involved in guiding axonal and neuronal migration npr1 gene, which is located at 10p11.22 and plays a broad role in angiogenesis, axon guidance, cell survival, migration and invasion. Another receptor of Sema3A Plexin A1 (PlxnA1) forms a complex with Nrp1 to protect bone Neuropilin 1 (Nrp1), the main receptor for Sema3A, is a protein encoded by the The majority of the factors regulate bone homeostasis from a single aspect, while Sema3A both inhibits bone resorption and increases bone formation to protect bone via its β2 receptor, which stimulates osteoclast formation leading to bone resorption via Stromal cell derived factor 1 (SDF-1), matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 3 (TIMP-3). Moreover, this process results in decreased bone mass via the NE/abrd3/JNK pathway The negative regulation of bone remodeling by SNS has also been previously reported via the internal oblique muscle is attached to the ilium and innervates the internal oblique muscle, but also innervates the ilium periosteum and bone marrow. Therefore, the reconstruction of sensory nerves may play an important role in maintaining the homeostasis of the bone graft. Our previous studies developed a method of using neurorrhaphy between the ilioinguinal nerve and the inferior alveolar nerve or auricular nerve during reconstruction of the mandibular bone In the reconstruction of the jaw, the commonly used vascularized iliac bone, the DCIA flap, often undergoes severe postoperative absorption, which affects the success of dental implants. In the preparation of a conventional iliac bone flap, the ilioinguinal nerve adjacent to the vascular pedicle is usually severed or sacrificed. The ilioinguinal nerve travelling via the stimulation of osteogenic cell recruitment to new bone formation areas As autologous bone grafts can cause damage and complications to surrounding bone and tissue in the donor site, there has been increasing research into tissue engineering of bone. Moreover, tissue-engineered bone is becoming a promising novel tool and contains four key elements: a scaffold with osteoconductivity; growth factors that induce osteogenesis; seeded cells with osteogenic potential; and tissue engineered vascularization or adequate blood supply via different types of neurotransmitters, but the specific mechanism is still elusive. Sensory nerves positively regulate bone remodeling via different sensory neurotransmitters acting on BMMSCs, such as CGRP, SP and Sema3A. Furthermore, sensory neurotransmitters can inhibit BMMSC aging and promote osteogenesis, which is of great significance for osteoporosis after clinical bone transplantation. Preliminary clinical studies have reported a positive role of innervation in maintaining the bone homeostasis in bone grafts. However, the innervation of bone graft requires further investigation. It is emphasized that there may be an important connection between the nervous system and bone remodeling, which will facilitate the development of bone grafting and tissue engineering. Furthermore, the novel application of nerve-based bone regeneration using BMMSCs provides a new insight in tissue engineering and clinical treatment of osteoporosis and other bone disorders (Figure The role of central and peripheral nervous systems in bone remodeling has been proposed in the present review, and the latter system is of particular interest. Sensory and autonomic nerves regulate the peripheral nervous system


The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis.Extracellular vesicles, small vesicles carrying in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum.Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their In contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV.In conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.The online version of this article (10.1007/s11307-020-01521-9) contains supplementary material, which is available to authorized users. Cancer cells rely on the recruitment and activation of various benign, physiological cells, most prominently immune cells, to facilitate and support growth, invasion, and finally metastasis. Mediators of the required cell-cell interaction have thus been a major focus of research, not only for understanding the process of metastatic spread but also for identification of biomarkers for disease stage, progression, and resistance mechanisms , 2.inter alia (mi)RNA, proteins, and peptides and their individual content largely reflects the parental cell  of tumor extracellular vesicles + T cells after injection of tumor cell–derived extracellular vesicles  and a higher percentage of CD8+ T cells  was observed. Furthermore, a trend towards decrease in monocytes  and an increase in mature macrophages  was detected , and cytoskeletal proteins  [Mass spectrometry revealed marked differences between extracellular vesicles from healthy mouse serum as well as tumor cell–derived extracellular vesicles. Mainly, three groups of proteins have been described in extracellular vesicles: antigen-binding proteins such as heatshock proteins and MHC proteins class I/II, proteins involved in signal transduction (tubulin) . In our tubulin) A network analysis of the abundant proteins in tumor cell extracellular vesicles Fig.  revealedin vitro and ex vivo analyses, in vivo experiments were performed to assess the kinetics of extracellular vesicle distribution and accumulation. We observed a first, unspecific pass of the extracellular vesicles through the measurable organs via the blood stream, followed by a specific accumulation of tumor cell extracellular vesicles in selected organs. The extracellular vesicles from healthy mouse serum did not show a conclusive, specific distribution, following the initial first pass and accumulated in liver and spleen, as might be expected for subcellular vesicles of a certain size [Following ain size .in vivo distribution studies, further detailed analyses were performed at 24 h after extracellular vesicle application [In vivo imaging, as well as ex vivo biodistribution analysis of selected organs revealed an increased accumulation of tumor cell–derived extracellular vesicles in the lung, the primary target organ of metastasis in the 4T1 model [To minimize the risk of dye transfer from labeled extracellular vesicles, increasing unspecific background signal and the results from lication , 27, 28.T1 model , as compT1 model ; the effThe comparison of tumor cell–derived extracellular vesicles with a heterogeneous population of extracellular vesicles from healthy mouse serum, instead of liposomes as in numerous comparable studies , takes iHowever, future studies will have to investigate the potential contribution from extracellular vesicles, shed by tumor-associated immune cells in the immediate tumor microenvironment or systemically in response to tumor-secreted factors.+ T cells, which inhibit pro-tumor regulatory T cells as well as anti-tumor natural killer cells. Regarding these seemingly contradictory effects of CD4+ T cells, their prognostic value remains unclear [+ T cells remains similarly unclear. However, as cytotoxic T cells exhibit a potent anti-tumor effect, their increase might be a response to initial changes of the immune cell composition towards a premetastatic niche [We further examined the biological effects of tumor cell–derived extracellular vesicles in the lungs of extracellular vesicle-treated animals. Twenty-four hours after a single administration of extracellular vesicles already, the immune cell composition of the host animals showed measurable changes. We observed a numerical decrease of CD4 unclear , 31. Theic niche . Our obsic niche , who didThe relative decrease of monocytes after treatment with tumor cell–derived extracellular vesicles and the simultaneous increase of macrophages might be due to promotion of an accelerated maturation of monocytes to tumor-associated macrophages. The increase of tumor-associated macrophages is a crucial step not only in the progression of the primary tumor, but also the development of distant metastases . Thus, tvia the tail vein and the consecutive, rapid distribution in the blood pool might not in all aspects represent the “natural” route, extracellular vesicles reach systemic distribution from the primary tumor.A limitation of our study is the route of extracellular vesicle application. In order to label the vesicles for subsequent tracking, isolation was necessary. The route of administration In follow-up projects, extracellular vesicles from tumor types with different metastatic patterns will be evaluated, based on the presented study to answer the question, immediately provoked by our results: does the distribution pattern fully explain organotropism of specific tumor types or are additional factors required? We believe to have given a sound basis for these future studies.in vivo using fluorescence reflectance imaging enables the in vivo visualization of their distribution and specific accumulation in target organs of subsequent metastasis. The pattern of this distribution is different between extracellular vesicles, derived from tumor cells and bulk vesicles from healthy controls and mirrors the potential pattern of metastasis. Tumor cell–derived extracellular vesicles contain a variety of proteins with the potential to establish a tumor-receptive microenvironment in potential target organs of metastasis. The observed alterations of the pulmonary immune cell population suggest that the establishment of an immunosuppressive environment as described for the premetastatic niche follows this initial phase of immune cell rearrangement. Our work further emphasizes the role of extracellular vesicles as potential mediators of tumor spread but, with a view to comparable studies, also hints towards a specific kinetic course of metastatic spread and the preceding microenvironmental changes. This time line as well as the distribution pattern as a potential factor of organotropism should be the focus of further cancer research. The suggested in vivo imaging approach, in combination with proteome and cell population analyses can serve as a tool for further research in the mechanism of systemic tumor spread.In conclusion, tracking of DiR-labeled extracellular vesicles ESM 1(DOCX 273 kb)ESM 2(XLSX 2616 kb)