Causal relationship between cathepsins and esophageal adenocarcinoma: Mendelian randomization and single-cell RNA sequencing analysis

Causal relationship between cathepsins and esophageal adenocarcinoma: Mendelian randomization and single-cell RNA sequencing analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Causal relationship between cathepsins and esophageal adenocarcinoma: Mendelian randomization and single-cell RNA sequencing analysis Suyan Tian, Jialin Li, Mingbo Tang, Xinliang Gao, Wei Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3859370/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The incidence of esophageal adenocarcinoma (EAC) has significantly increased, particularly in Western countries. Cathepsins are a group of lysosomal proteolytic enzymes; they are associated with the occurrence and progression of various tumors. However, the causal relationship between the cathepsin family and EAC remains unelucidated. To investigate this association, Mendelian randomization (MR) and bioinformatics analyses of single-cell RNA sequencing (scRNA-seq) data were performed. MR analyses revealed that high cathepsin B (CTSB) levels decreased EAC risk. Furthermore, scRNA-seq revealed that CTSB expression was primarily distributed in macrophages. In addition, MR analysis of CTSB and macrophage scavenger receptor types I and II verified their interrelationship; CTSB primarily affects the proinflammatory phenotype of macrophages. Our findings suggest that CTSB levels affect EAC progression by regulating the expression of macrophage scavenger receptor types I and II, which induce the proinflammatory phenotypes of macrophages. Therefore, targeting CTSB may provide avenues for EAC diagnosis and treatment. Health sciences/Oncology/Cancer/Cancer epidemiology Health sciences/Oncology/Cancer/Cancer genomics Esophageal adenocarcinoma Mendelian randomization single-cell RNA sequencing cathepsins microphage Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Esophageal cancer is considered a health concern worldwide and is the sixth most common reason for cancer-related deaths 1 . Pathologically esophageal cancer is divided into two major subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma (EAC) 2 . Over the past decades, the incidence of EAC has rapidly increased compared with any other cancer, particularly in developed countries 3 . Owing to the absence of an effective and noninvasive early screening option, most patients present with advanced-stage disease at diagnosis; such patients have a 5-year survival rate of < 20% 4 . EAC primarily originates from the Barrett’s mucosa in the lower esophagus, with characteristically occurring intestinal metaplasia forming Barrett’s esophagus (BE), an EAC precursor 5 . Although BE is the predominant pathological type of esophageal cancer in Western countries, explaining the process from reflux esophagitis to BE to dysplasia and finally to adenocarcinoma using the Western model of EAC formation is difficult 6 . Cathepsins are a group of lysosomal proteolytic enzymes that are involved in the autophagy–lysosome pathway and ubiquitin-conjugating pathway, driving proteolytic degradation in the lysosomes 7 . Dysregulation of the expression or activity of cathepsins plays a role in the development of cancer, neurodegeneration, and autoimmune diseases 8 . Previous study has revealed that the autophagy–lysosome proteolytic system and the enzymatic activity of these systems are modulated in patients with esophageal cancer 9 . Therefore, some researchers have begun to elucidate the relationship between cathepsins and esophageal cancer. High cathepsin D levels have been observed in EAC cell lines 10 . Furthermore, cathepsin E mRNA expression was found to be higher in EAC tissues; however, this was associated with a reduced risk of death 11 . Meanwhile, previous studies have revealed the unregular activities of cathepsin B (CTSB), C, and S in EAC/BE tissues 12 , 13 . However, prospective observational studies and clinical trials on the causal association between various cathepsin types and EAC are limited. Mendelian randomization (MR) is an efficient method to elucidate the causal effects of exposure on an outcome using the genetic variants from genome-wide association studies (GWAS), particularly at the phenotype level. Furthermore, single-cell RNA sequencing (scRNA-seq) technology is a powerful tool to determine the microscopic pathogenic mechanisms of risk factors at the individual cell level. In the present study, we integrated MR and scRNA-seq analyses to comprehensively determine the causal effects of different cathepsin types on EAC risk and explored the possible mechanisms. Results Defining the causal relationship between various cathepsins and EAC/BE The causal relationship between nine cathepsins (CTSB and cathepsin E, F, G, H, L2, O, S, and Z) and EAC/BE was determined using two-sample MR analysis. The IVW method was used as a primary method to determine the effect of various cathepsins on EAC/BE risk. CTSB levels affected EAC and BE risk (p = 0.001, odds ratio [OR] = 0.890, 95% confidence interval [CI] = 0.833–0.951); the p-value of the MR-Egger intercept test was 0.02 (Fig. 1 ). Furthermore, the MR-PRESSO test was performed, and the p-value of the MR-PRESSO global test was 0.243 (Table 1 ). Table 1 Univariable MR analysis results of the sensitivity analyses between cathepsins and EAC/BE, EAC, and BE exposure outcome MR-Egger intercept MR-PRESSO global test MR-IVW MR-Egger Egger intercept p_value p_value Q Q_df Q_pval Q Q_df Q_pval cathepisn B esophageal adenocarcinoma/ Barrett’s oesophagus 0.051 0.020 0.243 16.613 16 0.411 9.859 15 0.829 esophageal adenocarcinoma 0.016 0.580 0.954 5.655 16 0.991 5.335 15 0.989 Barrett’s oesophagus 0.064 0.016 0.067 24.472 16 0.080 16.459 15 0.352 cathepisn E esophageal adenocarcinoma/ Barrett’s oesophagus 0.000 0.991 0.499 8.980 9 0.439 8.980 8 0.344 esophageal adenocarcinoma 0.021 0.499 0.481 9.225 9 0.417 8.680 8 0.370 Barrett’s oesophagus 0.017 0.476 0.661 7.049 9 0.632 6.490 8 0.593 cathepisn F esophageal adenocarcinoma/ Barrett’s oesophagus -0.014 0.005 0.006 26.575 10 0.003 10.736 9 0.294 esophageal adenocarcinoma -0.026 0.020 0.042 22.556 11 0.020 12.774 10 0.237 Barrett’s oesophagus -0.005 0.014 0.032 22.427 10 0.013 11.123 9 0.267 cathepisn G esophageal adenocarcinoma/ Barrett’s oesophagus -0.001 0.609 0.178 8.966 8 0.345 8.613 7 0.282 esophageal adenocarcinoma -0.019 0.509 0.801 6.742 10 0.750 5.837 9 0.756 Barrett’s oesophagus -0.016 0.669 0.245 10.010 8 0.264 9.734 7 0.204 cathepisn H esophageal adenocarcinoma/ Barrett’s oesophagus 0.008 0.669 0.646 8.601 7 0.283 8.321 6 0.216 esophageal adenocarcinoma 0.017 0.591 0.354 12.686 7 0.080 12.040 6 0.061 Barrett’s oesophagus 0.001 0.968 0.629 8.230 7 0.313 8.227 6 0.222 cathepisn L2 esophageal adenocarcinoma/ Barrett’s oesophagus -0.009 0.169 0.739 5.111 7 0.646 4.989 6 0.545 esophageal adenocarcinoma -0.002 0.955 0.971 2.699 7 0.911 2.696 6 0.846 Barrett’s oesophagus -0.017 0.581 0.861 4.481 7 0.723 4.141 6 0.658 cathepisn O esophageal adenocarcinoma/ Barrett’s oesophagus 0.016 0.227 0.693 7.079 10 0.718 6.988 9 0.638 esophageal adenocarcinoma -0.042 0.197 0.750 7.617 10 0.666 5.675 9 0.772 Barrett’s oesophagus 0.013 0.613 0.370 9.188 10 0.514 8.913 9 0.445 cathepisn S esophageal adenocarcinoma/ Barrett’s oesophagus 0.021 0.080 0.588 17.997 19 0.523 14.553 18 0.692 esophageal adenocarcinoma 0.030 0.077 0.578 16.966 20 0.655 13.482 19 0.813 Barrett’s oesophagus 0.014 0.354 0.294 24.222 19 0.188 23.062 18 0.188 cathepisn Z esophageal adenocarcinoma/ Barrett’s oesophagus -0.013 0.336 0.970 3.559 10 0.965 2.528 9 0.980 esophageal adenocarcinoma 0.347 0.347 0.508 9.104 10 0.522 8.119 9 0.522 Barrett’s oesophagus -0.009 0.562 0.576 8.496 10 0.581 8.132 9 0.521 After the combined EAC and BE cases were analyzed as a whole, subgroup analyses were performed for EAC and BE, respectively. For patients with EAC, univariable MR analysis revealed that both CTSB (p = 0.004, OR = 0.866, 95% CI = 0.786–0.954) and cathepsin S (p = 0.007, OR = 0.897, 95% CI = 0.829–0.971) decreased EAC risk. The causal association between CTSB and cathepsin S and OCA risk was tested using the MR-Egger intercept and MR-PRESSO global tests; neither horizontal pleiotropy nor outliers were observed (p > 0.05). Table 1 summarizes the results. Furthermore, univariable MR analysis of cathepsins and BE revealed a causal relationship between CTSB and BE (p = 0.017, OR = 0.910, 95% CI = 0.843–0.983) (Fig. 1 ); the p-values of the MR-Egger intercept and MR-PRESSO global tests were 0.016 and 0.67, respectively (Table 1 ). However, cathepsin Z was associated with high BE risk (p = 0.030, OR = 1.086, 95% CI = 1.008–1.170). Subsequently, reverse MR analysis (supplement Table 1 ) was performed. No reverse causality was observed between all cathepsin types and the entire EAC/BE cohort or EAC and BE subgroups. Next, multivariable MR analysis was performed to analyze the genetic predisposition for multiple cathepsin types in relation to EAC/BE risk. After controlling for other cathepsin types, CTSB levels were still inversely associated with EAC/BE risk (p = 0.004, OR = 0.896, 95% CI = 0.831–0.966). For the separate EAC and BE analyses, similar results were obtained (EAC: p = 0.034, OR = 0.899, 95% CI = 0.814–0.992; BE: p = 0.019, OR = 0.899, 95% CI = 0.822–0.982) (Fig. 2 ). However, after adjusting for other cathepsin types, no statistically significant causal associations were observed between cathepsin S and EAC and cathepsin Z and BE (Fig. 2 ). There was no directional pleiotropy and heterogeneity in these causal associations. Overall, the results of univariable MR, multivariable MR, and sensitivity analyses suggest that high CTSB level decreases EAC risk. Defining the CTSB-related cell subpopulation To understand the pathways or molecular mechanisms affecting EAC onset and progression at the single-cell level, we analyzed the scRNA-seq data of patients with EAC to determine the types and proportion of different cell types. After filtering low-quality cells, 28,112 cells were retained. Then, UMAP plots were constructed to visualize the cells in the two-dimensional space. We annotated the cells based on canonical markers and identified eight distinct cell subtypes: T lymphocytes, B lymphocytes, epithelial cells, fibroblasts, myeloid cells, endothelial cells, mast cells, and proliferative cells (Fig. 3 a). Figure 3 b presents the signature genes and canonical markers used to annotate the clusters. UMAP analysis revealed epithelial cells and lymphocytes as the major cell subpopulations of EAC. Overall, higher immune cell infiltration was observed in EAC tissues (48%). To determine CTSB-related target cells, the distribution of CTSB genes was visualized on the UMAP figure. Figure 3 c demonstrates that CTSB was highly expressed in the myeloid subpopulation. To accurately identify the cell subpopulation associated with CTSB, the myeloid subpopulation was reclustered. Finally, three distinct cell subtypes were identified, namely, macrophages, monocytes, and dendritic cells (Fig. 3 d). Figure 3 e presents the signature genes and canonical markers used to annotate the clusters; macrophage was the main subpopulation, followed by monocytes. Thereafter, we again visualized CTSB distribution on the UMAP of the myeloid subpopulation; macrophages were identified as the CTSB-related target cells (Fig. 3 f). Because the remaining cells were few and the difference in marker gene expression between macrophage subtypes was not significant, futher division on the selected macrophages was not performed. MR analyses exploring the causal relationship between cathepsin B and macrophages As mentioned above, in patients with EAC, CTSB was highly expressed in macrophages. As a bridge between innate and adaptive immunities, the phenotypes and functions of macrophages are considerably complex 14 . To elucidate the effect of CTSB on the macrophages in EAC, we investigated the causal association between CTSB and macrophage receptor types, which is needed for various macrophage functions 15 . Supplementary table 2 presents the results of two-sample MR analysis between cathepsin B and macrophage receptors. MR analysis revealed that CTSB affected the expression of macrophage scavenger receptor types I and II, which belong to class A receptors that play a role in antigen presentation 16 (known as MSR-A) 17 (IVW method: p = 0.004, OR = 1.122, 95% CI = 1.038–1.213). The respective p-values of the MR-Egger intercept and MR-PRESSO global tests were 0.083 and 0.451, indicating no directional horizontal pleiotropy. Furthermore, reverse MR analysis revealed that the expression of macrophage scavenger receptor types I and II affected CTSB levels (IVW method: p = 5.845 × 10 − 5 , OR = 1.285, 95% CI = 1.137–1.452); and no directional pleiotropy was observed. The p-values of the MR-Egger intercept and MR-PRESSO global tests were 0.334 and 0.316, respectively. Nevertheless, no causal association was observed between CTSB and another important receptor, macrophage mannose receptor, also called CD206, the cell expression marker for M2 macrophages 15 . (IVW method: p = 0.205, OR = 1.052, 95% CI = 0.973–1.137; inverse results: OR = 0.984, p = 0.692, 95% CI = 0.911–1.064). Overall, CTSB affects the expression of macrophage scavenger receptor types I and II, and changes in the macrophage phenotype secondary to MSR-A upregulation may induce the upregulation of CTSB in patients with EAC. This possible positive feedback regulation between CTSB and macrophage scavenger receptor types I and II may accelerate the development of EAC. Discussion The incidence of EAC has drastically increased, particularly in Western countries 18 . Except for gastroesophageal reflux and obesity, which are common risk factors, studies suggest that genetic susceptibility to EAC/BE and related gene variants play a role in inflammation 19 , DNA damage and repair 20 , and metabolism 21 . In the present study, both univariable and multivariable MR analysis revealed that higher CTSB levels are associated with a decreased risk of EAC/BE. Furthermore, scRNA-seq analysis revealed that CTSB is significantly overexpressed in the macrophages of EAC tissues. Further two-sample MR analysis revealed that CTSB levels in patients with EAC are strongly associated with the expression of macrophage scavenger receptor types I and II. The human cysteine cathepsin protease family is mechanistically associated with the progression and metastasis of carcinomas. Particularly for CTSB, experimental and epidemiological studies suggest that it exerts different cancer-promoting or cancer-inhibiting effects in different tumors 22 , 23 . A recent study focused on the proteolytic activity of CTSB in the extracellular matrix and suggested that it plays an important role in tumor invasion and metastasis 24 . However, some other studies have revealed that CTSB induces lysosomal membrane permeabilization and subsequently leads to cathepsin-mediated cancer cell death, which is an important tumor-suppressor mechanism 25 , 26 . Specifically for EAC, a study by Ali et al. has revealed that the risk variant at chr8p23.1 in EAC cell lines implicates multiple gene targets, including CTSB 27 . Furthermore, the findings of this study clarified the dominant anticancer role of CTSB in EAC/BE, providing some evidence that the more effective function of CTSB in the EAC/BE course is cathepsin-mediated cancer cell death rather than its proteolytic activity in the extracellular matrix. Moreover, various stimuli can induce the CTSB present in the lysosome, leading to inflammation via the ATG7-dependent mechanism 28 ; this finding also partially explains the relevance of EAC/BE and inflammation, the third risk factor for EAC/BE. Cathepsins are secreted from tumor and immune cells, including tumor-associated macrophages 29 . In the present study, scRNA-seq analysis revealed that CTSB in patients with EAC/BE is associated with infiltrating macrophages. Furthermore, MR analysis of CTSB and various macrophage receptors revealed the mutual relationship between CTSB levels and macrophage scavenger receptor types I and II; this indicates that the effect of CTSB on macrophages is related to MSR-A upregulation. As a pattern recognition receptor, MSR plays a vital role in phagocytosis 30 and proinflammatory cytokine release of macrophages 31 . It is essential for promoting the immune responses of patients with EAC. However, no causal effect was observed between CTSB and the macrophage mannose receptor, a marker receptor of M2 macrophages, which is related to immunosuppression. Therefore, it is reasonable to assume the presence of a possible positive feedback regulation between CTSB and the proinflammatory phenotype of macrophages rather than the immunosuppressive phenotype of macrophages, which possibly exerts an important inhibitory effect on EAC/BE occurrence and development. This study has its own limitations., first, all results of this study were from European individuals, which may lead to inevitable selection bias. Besides, considering the partial overlap between macrophage-related GWAS data and cathepsin-related GWAS data, further mechanistical studies on cathepsin B and macrophage phenotypes are warranted. To the best of our knowledge, this is the first study in which MR analysis and scRNA-seq data mining were integrated to determine the causal relationship between cathepsins and EAC and the potential mechanism underlying the identified relationship. Our findings may provide avenues for the diagnosis and treatment of EAC in the future. In summary, we revealed that CTSB levels are associated with EAC risk and that infiltrating macrophages in EAC may be involved in this process. These insights imply CTSB as a potential target for EAC intervention and treatment. Methods Data collection The GWAS data of cathepsins (µg/L) were acquired from the INTERVAL study, which included 3,301 European individuals 32 . The summary data for macrophages were acquired from the INTERVAL study and FINRISK surveys, which included 8,293 European individuals 33 (accession: https://gwas.mrcieu.ac.uk ). Furthermore, the summary data for EAC/BE (confirmed by pathological diagnosis) were collected from the GWAS Catalog ( https://www.ebi.ac.uk/gwas/ ), which included 6,167 patients with BE, 4,112 individuals with EAC, and 17,159 controls 34 . The scRNA-seq data for EAC were retrieved from the Gene Expression Omnibus ( https://www.ncbi.nlm.nih.gov/geo/ ) (accession number: GSE173950). All donors were asked to complete the trial consent, related studies were reviewed and approved by institutional ethics review committees at the involved institutions. Selection of instrumental variables The following criteria were used to select cathepsin-related genetic variants: (a) r 2 measure of LD among instruments < 0·001 within a 10,000 kb window, and (b) p-value < the genome-wide significance level identified in the corresponding study, i.e., 5 × 10 − 6 . MR analyses A genetic variant was justified as a valid instrument if it satisfied the following three core assumptions: (i) highly correlated with the exposure, (ii) was independent of any confounders between the exposure and the outcome, and (iii) was not directly associated with the outcome. The inverse variance-weighted (IVW) method was used as the primary method to determine the overall effect size of an exposure on the outcome 35 . In IVW, the effect was elucidated using the Wald ratio method for each SNP. Subsequently, by using a random-effect inverse variance meta-analysis, these individual MR estimates were combined to achieve an overall summary value. The R TwoSampleMR package was used to perform MR analyses 36 . To evaluate the validity of the core instrumental variables assumptions, various sensitivity analyses and statistical tests were employed. Cochran’s Q test was performed to determine the heterogeneity of the SNPs. A p-value of < 0·05 suggested the presence of heterogeneity. When significant heterogeneity was observed among the SNPs, the random-effect model was used. Otherwise, a fixed-effect model 37 was used. MR-PRESSO global test and MR-Egger intercept were used to determine outliers and identify horizontal pleiotropy 38 . The MR-Egger intercept assessed the presence of a directional pleiotropic effect (intercept p-value < 0.05). On the other hand, the MR-PRESSO outlier test was performed to correct for horizontal pleiotropy by removing or down-weighting the outliers when horizontal pleiotropy was significant (p-value of the MR-PRESSO global test < 0.05). Furthermore, the MR-PRESSO distortion test was performed to identify significant distortion in the causal estimates before and after removing the outliers. The R MR-PRESSO package was used to perform the MR-PRESSO global, outlier, and distortion tests 39 . In reverse MRs, the GWAS datasets mentioned above were used, i.e., EAC/BE was the exposure, whereas the levels of cathepsin types were the outcomes. Lastly, when analyzing the causal effects on EAC/BE and determining the direct causal effects of each exposure in univariable analysis, multivariable MR analysis was used to consider multiple cathepsins. The “MendelianRandomization” package was used 37 . Furthermore, reverse MRs where lung cancer was regarded as the exposure and cathepsins as the outcome were used to evaluate the reverse casualties and justify the presence of bidirectional causality. scRNA-seq analysis The droplet-based sequencing (DropSeq) was used to process the scRNA-seq data. The Seurat package was used to analyze the downloaded digital gene–cell matrix of the 17 EAC cohorts 40 . Cells from different samples were filtered based on the following criteria: (a) the detected genes per cell should be > 200, and (b) the percentage of mitochondrial genes should be < 20%. A global scaling method was used to normalize the gene expression matrices of the remaining cells, with a default scale factor, followed by natural log transformation using log(1 + x). The NormalizeData function was used for this. Then, the FindVariableFeatures method was used to identify the top 2000 highly variable genes. The “RunPCA” function was used to process the scaled and normalized gene expression data via linear dimensional reduction. The inflection point of the ElbowPlot function was used to determine the final number of principal components. Based on the Euclidean distance, the “FindNeighbors” function was used to prepare a K-nearest neighbor graph of the selected principal components. Then, the Louvain algorithm was performed to optimize modularity using the “FindClusters” function. Using the “RunUMAP” function, the cluster results of EAC scRNA-seq data were visualized using uniform manifold approximation and projection (UMAP) 41 . To annotate the clusters, the FindAllMarkers function was used to determine the differentially expressed genes for the identified clusters. Finally, the FeaturePlot function was used to visualize the distribution of related genes. R software version 4.1.1 was used to conduct statistical analyses. The study schema is presented in Fig. 4 . Declarations Data Availability The GWAS data of cathepsins and Macrophage were derived from the (https://gwas.mrcieu.ac.uk.). The summary data of EAC/BE were collected from https://www.ebi.ac.uk/gwas/. The scRNA seq data of EAC/BE were downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), the accession number is GSE173950. Code Availability All packages for data analysis used in this study were open source in R software (version 4.1.1; R Development Core Team). Acknowledgments This study was funded by Jilin Province medical and health talents special (JLSWSRCZX2023-35), Jilin Provincial Science and Technology Development Plan Project (20220204115YY) and Natural Science Foundation of Jilin Province (YDZJ202301ZYTS007 and YDZJ202201ZYTS121). The funding body had no role in the design of the study and collection, analysis and interpretation of data and in writing the manuscript. We thank Sagesci (www.sagesci.cn) for its linguistic assistance during the preparation of this manuscript. The study schema figure was generated with the aid of Scidraw and Biorender. Authors' contributions S.T and W.L conceived and designed the experiment; J.L ran the analysis and verified the underlying data; J.L and S.T wrote the original manuscript. M.T and X.G involved in data interpretation. All authors have read and approved the final version of the manuscript. Competing interests All other authors declare no competing financial interests. References Smyth, E. C. et al. Oesophageal cancer. Nat Rev Dis Primers 3 , 17048, doi:10.1038/nrdp.2017.48 (2017). Cook, M. B., Chow, W. H. & Devesa, S. S. Oesophageal cancer incidence in the United States by race, sex, and histologic type, 1977-2005. Br J Cancer 101 , 855-859, doi:10.1038/sj.bjc.6605246 (2009). Thrift, A. P. & Whiteman, D. C. The incidence of esophageal adenocarcinoma continues to rise: analysis of period and birth cohort effects on recent trends. Ann Oncol 23 , 3155-3162, doi:10.1093/annonc/mds181 (2012). Salimian, K. J., Birkness-Gartman, J. & Waters, K. M. 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Genome-wide Association Study Identifies 27 Loci Influencing Concentrations of Circulating Cytokines and Growth Factors. Am J Hum Genet 100 , 40-50, doi:10.1016/j.ajhg.2016.11.007 (2017). Gharahkhani, P. et al. Genome-wide association studies in oesophageal adenocarcinoma and Barrett's oesophagus: a large-scale meta-analysis. Lancet Oncol 17 , 1363-1373, doi:10.1016/s1470-2045(16)30240-6 (2016). Burgess, S., Butterworth, A. & Thompson, S. G. Mendelian randomization analysis with multiple genetic variants using summarized data. Genet. Epidemiol. 37 , 658-665, doi:10.1002/gepi.21758 (2013). Hemani, G. et al. The MR-Base platform supports systematic causal inference across the human phenome. eLife 7 , doi:10.7554/eLife.34408 (2018). Yavorska, O. O. & Burgess, S. MendelianRandomization: an R package for performing Mendelian randomization analyses using summarized data. Int. J. Epidemiol. 46 , 1734-1739, doi:10.1093/ije/dyx034 (2017). Verbanck, M., Chen, C. Y., Neale, B. & Do, R. Detection of widespread horizontal pleiotropy in causal relationships inferred from Mendelian randomization between complex traits and diseases. Nat. Genet. 50 , 693-698, doi:10.1038/s41588-018-0099-7 (2018). Verbanck, M., Chen, C. Y., Neale, B. & Do, R. Detection of widespread horizontal pleiotropy in causal relationships inferred from Mendelian randomization between complex traits and diseases. Nat Genet 50 , 693-698, doi:10.1038/s41588-018-0099-7 (2018). Gribov, A. et al. SEURAT: visual analytics for the integrated analysis of microarray data. BMC Med Genomics 3 , 21, doi:10.1186/1755-8794-3-21 (2010). Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol , doi:10.1038/nbt.4314 (2018). Additional Declarations There is NO Competing Interest. Supplementary Files supplementtable1.xlsx Supplement table 1: Reverse MR analysis results between cathepsin family and EAC/BE supplementtable2.xlsx Supplement table 2: The detail two-Sample MR results between cathepsin B and various macrophage receptors. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3859370","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":269691657,"identity":"2fc5c012-0b3d-45d6-abf8-726b6ffa28d1","order_by":0,"name":"Suyan Tian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYLCCDzwWIMqAaA2MjTN4JEjU0szDQIoWg2tnzB/byEgkNrA3b5NgqLlDhJbbaYnNOTxALTzHyiQYjj0jrMXsdvJBiBaJHDMJxobDxGhJbGy2AGmRf0O0FqAtDGBbeIjUYg/0y8weHgnjNp60YouEY0RokZydY/DhZ4+NbD/74Y03PtQQoQUMGHsYGNhAjAQiNQDBD+KVjoJRMApGwQgEANVXNW+L0wfrAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5942-1542","institution":"The First Hospital of Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Suyan","middleName":"","lastName":"Tian","suffix":""},{"id":269691658,"identity":"3dc4e4da-d4de-4490-876d-8750e65cc13b","order_by":1,"name":"Jialin Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jialin","middleName":"","lastName":"Li","suffix":""},{"id":269691659,"identity":"7e32d227-14a1-470d-a916-660e14d48239","order_by":2,"name":"Mingbo Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mingbo","middleName":"","lastName":"Tang","suffix":""},{"id":269691660,"identity":"4aeabd83-30e1-466b-acdd-7f3042206970","order_by":3,"name":"Xinliang Gao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xinliang","middleName":"","lastName":"Gao","suffix":""},{"id":269691661,"identity":"28bbd2c9-c66a-472c-9ae5-56d319efe711","order_by":4,"name":"Wei Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-01-13 07:35:10","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3859370/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3859370/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50346669,"identity":"8a143897-8490-4198-ac9a-3b5440fee472","added_by":"auto","created_at":"2024-01-30 06:41:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":681024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eForest plot of the univariable Mendelian randomization analysis between nine cathepsins and esophageal adenocarcinoma/Barrett’s esophagus (EAC/BE) risk. \u003c/strong\u003eThe inverse variance-weighted method was used to determine the causal relationship between nine cathepsins types and EAC/BE and EAC and BE, independently. The red data indicate the statistically significant results and the error bars indicate the 95% confidence interval.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/e861923666dc6c2ea1ebc412.jpg"},{"id":50346421,"identity":"5b7cf301-7b5b-49d7-b375-974fc7330f3e","added_by":"auto","created_at":"2024-01-30 06:33:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":719779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eForest plot of the multivariable Mendelian randomization analysis between cathepsins and esophageal adenocarcinoma/Barrett’s esophagus (EAC/BE) risk. \u003c/strong\u003eThe inverse variance-weighted method was used to determine the causal association between nine cathepsins (cathepsin B, E, F, G, H, L2, O, S, and Z) and EAC/BE and EAC and BE, independently. The red data indicate the statistically significant results, and the error bars indicate the 95% confidence interval.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/fe0c9225e87844a3a666a1c7.jpg"},{"id":50345676,"identity":"3f760063-bc45-452d-81d9-87719f10aa6c","added_by":"auto","created_at":"2024-01-30 06:25:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1939834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of the single-cell RNA sequencing data for esophageal adenocarcinoma (EAC) tissues. \u003c/strong\u003e(a) UMAP plot of 28,112 single cells in EAC tissues color-coded based on the eight major lineages. (b) Dot plot of the mean expression of the canonical marker genes for the eight major EAC lineages. (c) UMAP visualization of cathepsin B (CTSB) primary distribution in myeloid cells of esophageal adenocarcinoma patients. (d) UMAP plot for the myeloid cells in patients with EAC color-coded based on the three major lineages. (e) Dot plot of the mean expression of the canonical marker genes for macrophages, monocytes, and dendritic cells. (f) UMAP visualization of cathepsin B (CTSB) primary distribution in the macrophages of patients with EAC.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/9d7ba8e357e42f4a4d473309.jpg"},{"id":50345678,"identity":"775ec3be-8e17-4ef9-920b-3a5c10c7ee29","added_by":"auto","created_at":"2024-01-30 06:25:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2323857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy schema.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/5fcc1b66562d2521760dac71.jpg"},{"id":50347341,"identity":"21d2d385-46dc-4423-aeed-c808997b453c","added_by":"auto","created_at":"2024-01-30 06:49:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":821557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/69d13ac7-240e-4e6a-b0e2-49941a707583.pdf"},{"id":50345675,"identity":"fe4d1bb0-72b8-429d-be6b-d680ab7a135a","added_by":"auto","created_at":"2024-01-30 06:25:09","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13825,"visible":true,"origin":"","legend":"\u003cp\u003eSupplement table 1: Reverse MR analysis results between cathepsin family and EAC/BE\u003c/p\u003e","description":"","filename":"supplementtable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/c079b889730600405709fccc.xlsx"},{"id":50346423,"identity":"b759fb69-0c7c-45ae-ac2c-fdb7eb6361b1","added_by":"auto","created_at":"2024-01-30 06:33:09","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13298,"visible":true,"origin":"","legend":"\u003cp\u003eSupplement table 2: The detail two-Sample MR results between cathepsin B and various macrophage receptors.\u003c/p\u003e","description":"","filename":"supplementtable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3859370/v1/3e2a8d214185a9320ca2134f.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Causal relationship between cathepsins and esophageal adenocarcinoma: Mendelian randomization and single-cell RNA sequencing analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer is considered a health concern worldwide and is the sixth most common reason for cancer-related deaths\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Pathologically esophageal cancer is divided into two major subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma (EAC)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Over the past decades, the incidence of EAC has rapidly increased compared with any other cancer, particularly in developed countries\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Owing to the absence of an effective and noninvasive early screening option, most patients present with advanced-stage disease at diagnosis; such patients have a 5-year survival rate of \u0026lt;\u0026thinsp;20%\u003csup\u003e4\u003c/sup\u003e. EAC primarily originates from the Barrett\u0026rsquo;s mucosa in the lower esophagus, with characteristically occurring intestinal metaplasia forming Barrett\u0026rsquo;s esophagus (BE), an EAC precursor\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although BE is the predominant pathological type of esophageal cancer in Western countries, explaining the process from reflux esophagitis to BE to dysplasia and finally to adenocarcinoma using the Western model of EAC formation is difficult\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCathepsins are a group of lysosomal proteolytic enzymes that are involved in the autophagy\u0026ndash;lysosome pathway and ubiquitin-conjugating pathway, driving proteolytic degradation in the lysosomes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Dysregulation of the expression or activity of cathepsins plays a role in the development of cancer, neurodegeneration, and autoimmune diseases\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Previous study has revealed that the autophagy\u0026ndash;lysosome proteolytic system and the enzymatic activity of these systems are modulated in patients with esophageal cancer\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Therefore, some researchers have begun to elucidate the relationship between cathepsins and esophageal cancer. High cathepsin D levels have been observed in EAC cell lines\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, cathepsin E mRNA expression was found to be higher in EAC tissues; however, this was associated with a reduced risk of death\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Meanwhile, previous studies have revealed the unregular activities of cathepsin B (CTSB), C, and S in EAC/BE tissues \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, prospective observational studies and clinical trials on the causal association between various cathepsin types and EAC are limited.\u003c/p\u003e \u003cp\u003eMendelian randomization (MR) is an efficient method to elucidate the causal effects of exposure on an outcome using the genetic variants from genome-wide association studies (GWAS), particularly at the phenotype level. Furthermore, single-cell RNA sequencing (scRNA-seq) technology is a powerful tool to determine the microscopic pathogenic mechanisms of risk factors at the individual cell level. In the present study, we integrated MR and scRNA-seq analyses to comprehensively determine the causal effects of different cathepsin types on EAC risk and explored the possible mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eDefining the causal relationship between various cathepsins and EAC/BE\u003c/h2\u003e\n\u003cp\u003eThe causal relationship between nine cathepsins (CTSB and cathepsin E, F, G, H, L2, O, S, and Z) and EAC/BE was determined using two-sample MR analysis. The IVW method was used as a primary method to determine the effect of various cathepsins on EAC/BE risk. CTSB levels affected EAC and BE risk (p\u0026thinsp;=\u0026thinsp;0.001, odds ratio [OR]\u0026thinsp;=\u0026thinsp;0.890, 95% confidence interval [CI]\u0026thinsp;=\u0026thinsp;0.833\u0026ndash;0.951); the p-value of the MR-Egger intercept test was 0.02 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, the MR-PRESSO test was performed, and the p-value of the MR-PRESSO global test was 0.243 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eUnivariable MR analysis results of the sensitivity analyses between cathepsins and EAC/BE, EAC, and BE\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eexposure\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eoutcome\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMR-Egger intercept\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMR-PRESSO global test\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMR-IVW\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003eMR-Egger\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEgger intercept\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ep_value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ep_value\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQ_df\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQ_pval\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQ_df\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eQ_pval\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn B\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.051\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.243\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.613\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.411\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.859\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.829\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.580\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.954\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.655\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.991\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.335\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.989\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.064\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.067\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24.472\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.080\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.459\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.352\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.991\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.499\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.980\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.439\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.980\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.344\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.021\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.499\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.481\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.225\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.417\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.680\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.370\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.476\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.661\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.049\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.632\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.490\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.593\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn F\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.006\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26.575\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.736\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.294\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.026\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.042\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.556\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.774\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.237\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.032\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.427\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.123\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.267\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.609\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.178\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.966\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.345\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.613\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.282\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.019\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.509\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.801\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.742\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.837\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.756\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.669\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.245\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.010\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.264\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.734\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.204\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn H\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.669\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.646\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.601\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.283\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.321\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.216\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.591\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.354\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.686\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.080\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.040\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.061\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.968\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.629\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.230\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.313\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.227\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.222\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn L2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.169\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.739\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.646\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.989\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.545\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.002\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.955\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.971\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.699\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.911\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.696\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.846\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.581\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.861\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.481\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.723\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.141\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.658\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn O\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.227\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.693\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.079\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.718\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.988\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.638\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.042\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.197\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.750\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.617\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.666\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.675\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.772\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.613\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.370\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.514\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.913\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.445\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn S\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.021\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.080\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.588\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17.997\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.523\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.553\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.692\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.030\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.077\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.578\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e16.966\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.655\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.482\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.813\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.354\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.294\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24.222\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.188\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cp\u003ecathepisn Z\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma/\u003c/p\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.013\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.336\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.970\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.559\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.965\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.528\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.980\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eesophageal adenocarcinoma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.347\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.347\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.508\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.104\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.522\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.119\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.522\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBarrett\u0026rsquo;s oesophagus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e-0.009\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.562\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.576\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.496\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.581\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.132\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.521\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eAfter the combined EAC and BE cases were analyzed as a whole, subgroup analyses were performed for EAC and BE, respectively. For patients with EAC, univariable MR analysis revealed that both CTSB (p\u0026thinsp;=\u0026thinsp;0.004, OR\u0026thinsp;=\u0026thinsp;0.866, 95% CI\u0026thinsp;=\u0026thinsp;0.786\u0026ndash;0.954) and cathepsin S (p\u0026thinsp;=\u0026thinsp;0.007, OR\u0026thinsp;=\u0026thinsp;0.897, 95% CI\u0026thinsp;=\u0026thinsp;0.829\u0026ndash;0.971) decreased EAC risk. The causal association between CTSB and cathepsin S and OCA risk was tested using the MR-Egger intercept and MR-PRESSO global tests; neither horizontal pleiotropy nor outliers were observed (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the results. Furthermore, univariable MR analysis of cathepsins and BE revealed a causal relationship between CTSB and BE (p\u0026thinsp;=\u0026thinsp;0.017, OR\u0026thinsp;=\u0026thinsp;0.910, 95% CI\u0026thinsp;=\u0026thinsp;0.843\u0026ndash;0.983) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e); the p-values of the MR-Egger intercept and MR-PRESSO global tests were 0.016 and 0.67, respectively (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). However, cathepsin Z was associated with high BE risk (p\u0026thinsp;=\u0026thinsp;0.030, OR\u0026thinsp;=\u0026thinsp;1.086, 95% CI\u0026thinsp;=\u0026thinsp;1.008\u0026ndash;1.170). Subsequently, reverse MR analysis (supplement Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) was performed. No reverse causality was observed between all cathepsin types and the entire EAC/BE cohort or EAC and BE subgroups.\u003c/p\u003e\n\u003cp\u003eNext, multivariable MR analysis was performed to analyze the genetic predisposition for multiple cathepsin types in relation to EAC/BE risk. After controlling for other cathepsin types, CTSB levels were still inversely associated with EAC/BE risk (p\u0026thinsp;=\u0026thinsp;0.004, OR\u0026thinsp;=\u0026thinsp;0.896, 95% CI\u0026thinsp;=\u0026thinsp;0.831\u0026ndash;0.966). For the separate EAC and BE analyses, similar results were obtained (EAC: p\u0026thinsp;=\u0026thinsp;0.034, OR\u0026thinsp;=\u0026thinsp;0.899, 95% CI\u0026thinsp;=\u0026thinsp;0.814\u0026ndash;0.992; BE: p\u0026thinsp;=\u0026thinsp;0.019, OR\u0026thinsp;=\u0026thinsp;0.899, 95% CI\u0026thinsp;=\u0026thinsp;0.822\u0026ndash;0.982) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, after adjusting for other cathepsin types, no statistically significant causal associations were observed between cathepsin S and EAC and cathepsin Z and BE (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). There was no directional pleiotropy and heterogeneity in these causal associations. Overall, the results of univariable MR, multivariable MR, and sensitivity analyses suggest that high CTSB level decreases EAC risk.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eDefining the CTSB-related cell subpopulation\u003c/h2\u003e\n\u003cp\u003eTo understand the pathways or molecular mechanisms affecting EAC onset and progression at the single-cell level, we analyzed the scRNA-seq data of patients with EAC to determine the types and proportion of different cell types. After filtering low-quality cells, 28,112 cells were retained. Then, UMAP plots were constructed to visualize the cells in the two-dimensional space. We annotated the cells based on canonical markers and identified eight distinct cell subtypes: T lymphocytes, B lymphocytes, epithelial cells, fibroblasts, myeloid cells, endothelial cells, mast cells, and proliferative cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb presents the signature genes and canonical markers used to annotate the clusters. UMAP analysis revealed epithelial cells and lymphocytes as the major cell subpopulations of EAC. Overall, higher immune cell infiltration was observed in EAC tissues (48%). To determine CTSB-related target cells, the distribution of CTSB genes was visualized on the UMAP figure. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec demonstrates that CTSB was highly expressed in the myeloid subpopulation.\u003c/p\u003e\n\u003cp\u003eTo accurately identify the cell subpopulation associated with CTSB, the myeloid subpopulation was reclustered. Finally, three distinct cell subtypes were identified, namely, macrophages, monocytes, and dendritic cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed). Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee presents the signature genes and canonical markers used to annotate the clusters; macrophage was the main subpopulation, followed by monocytes. Thereafter, we again visualized CTSB distribution on the UMAP of the myeloid subpopulation; macrophages were identified as the CTSB-related target cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef). Because the remaining cells were few and the difference in marker gene expression between macrophage subtypes was not significant, futher division on the selected macrophages was not performed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eMR analyses exploring the causal relationship between cathepsin B and macrophages\u003c/h2\u003e\n\u003cp\u003eAs mentioned above, in patients with EAC, CTSB was highly expressed in macrophages. As a bridge between innate and adaptive immunities, the phenotypes and functions of macrophages are considerably complex\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. To elucidate the effect of CTSB on the macrophages in EAC, we investigated the causal association between CTSB and macrophage receptor types, which is needed for various macrophage functions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Supplementary table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e presents the results of two-sample MR analysis between cathepsin B and macrophage receptors. MR analysis revealed that CTSB affected the expression of macrophage scavenger receptor types I and II, which belong to class A receptors that play a role in antigen presentation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (known as MSR-A)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e (IVW method: p\u0026thinsp;=\u0026thinsp;0.004, OR\u0026thinsp;=\u0026thinsp;1.122, 95% CI\u0026thinsp;=\u0026thinsp;1.038\u0026ndash;1.213). The respective p-values of the MR-Egger intercept and MR-PRESSO global tests were 0.083 and 0.451, indicating no directional horizontal pleiotropy. Furthermore, reverse MR analysis revealed that the expression of macrophage scavenger receptor types I and II affected CTSB levels (IVW method: p\u0026thinsp;=\u0026thinsp;5.845 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, OR\u0026thinsp;=\u0026thinsp;1.285, 95% CI\u0026thinsp;=\u0026thinsp;1.137\u0026ndash;1.452); and no directional pleiotropy was observed. The p-values of the MR-Egger intercept and MR-PRESSO global tests were 0.334 and 0.316, respectively. Nevertheless, no causal association was observed between CTSB and another important receptor, macrophage mannose receptor, also called CD206, the cell expression marker for M2 macrophages\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. (IVW method: p\u0026thinsp;=\u0026thinsp;0.205, OR\u0026thinsp;=\u0026thinsp;1.052, 95% CI\u0026thinsp;=\u0026thinsp;0.973\u0026ndash;1.137; inverse results: OR\u0026thinsp;=\u0026thinsp;0.984, p\u0026thinsp;=\u0026thinsp;0.692, 95% CI\u0026thinsp;=\u0026thinsp;0.911\u0026ndash;1.064). Overall, CTSB affects the expression of macrophage scavenger receptor types I and II, and changes in the macrophage phenotype secondary to MSR-A upregulation may induce the upregulation of CTSB in patients with EAC. This possible positive feedback regulation between CTSB and macrophage scavenger receptor types I and II may accelerate the development of EAC.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe incidence of EAC has drastically increased, particularly in Western countries\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Except for gastroesophageal reflux and obesity, which are common risk factors, studies suggest that genetic susceptibility to EAC/BE and related gene variants play a role in inflammation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, DNA damage and repair\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and metabolism\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the present study, both univariable and multivariable MR analysis revealed that higher CTSB levels are associated with a decreased risk of EAC/BE. Furthermore, scRNA-seq analysis revealed that CTSB is significantly overexpressed in the macrophages of EAC tissues. Further two-sample MR analysis revealed that CTSB levels in patients with EAC are strongly associated with the expression of macrophage scavenger receptor types I and II.\u003c/p\u003e \u003cp\u003eThe human cysteine cathepsin protease family is mechanistically associated with the progression and metastasis of carcinomas. Particularly for CTSB, experimental and epidemiological studies suggest that it exerts different cancer-promoting or cancer-inhibiting effects in different tumors\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A recent study focused on the proteolytic activity of CTSB in the extracellular matrix and suggested that it plays an important role in tumor invasion and metastasis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, some other studies have revealed that CTSB induces lysosomal membrane permeabilization and subsequently leads to cathepsin-mediated cancer cell death, which is an important tumor-suppressor mechanism\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Specifically for EAC, a study by Ali et al. has revealed that the risk variant at chr8p23.1 in EAC cell lines implicates multiple gene targets, including CTSB\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Furthermore, the findings of this study clarified the dominant anticancer role of CTSB in EAC/BE, providing some evidence that the more effective function of CTSB in the EAC/BE course is cathepsin-mediated cancer cell death rather than its proteolytic activity in the extracellular matrix. Moreover, various stimuli can induce the CTSB present in the lysosome, leading to inflammation via the ATG7-dependent mechanism\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e; this finding also partially explains the relevance of EAC/BE and inflammation, the third risk factor for EAC/BE.\u003c/p\u003e \u003cp\u003eCathepsins are secreted from tumor and immune cells, including tumor-associated macrophages\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In the present study, scRNA-seq analysis revealed that CTSB in patients with EAC/BE is associated with infiltrating macrophages. Furthermore, MR analysis of CTSB and various macrophage receptors revealed the mutual relationship between CTSB levels and macrophage scavenger receptor types I and II; this indicates that the effect of CTSB on macrophages is related to MSR-A upregulation. As a pattern recognition receptor, MSR plays a vital role in phagocytosis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and proinflammatory cytokine release of macrophages\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It is essential for promoting the immune responses of patients with EAC. However, no causal effect was observed between CTSB and the macrophage mannose receptor, a marker receptor of M2 macrophages, which is related to immunosuppression. Therefore, it is reasonable to assume the presence of a possible positive feedback regulation between CTSB and the proinflammatory phenotype of macrophages rather than the immunosuppressive phenotype of macrophages, which possibly exerts an important inhibitory effect on EAC/BE occurrence and development. This study has its own limitations., first, all results of this study were from European individuals, which may lead to inevitable selection bias. Besides, considering the partial overlap between macrophage-related GWAS data and cathepsin-related GWAS data, further mechanistical studies on cathepsin B and macrophage phenotypes are warranted.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is the first study in which MR analysis and scRNA-seq data mining were integrated to determine the causal relationship between cathepsins and EAC and the potential mechanism underlying the identified relationship. Our findings may provide avenues for the diagnosis and treatment of EAC in the future. In summary, we revealed that CTSB levels are associated with EAC risk and that infiltrating macrophages in EAC may be involved in this process. These insights imply CTSB as a potential target for EAC intervention and treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eData collection\u003c/h2\u003e\n\u003cp\u003eThe GWAS data of cathepsins (\u0026micro;g/L) were acquired from the INTERVAL study, which included 3,301 European individuals\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The summary data for macrophages were acquired from the INTERVAL study and FINRISK surveys, which included 8,293 European individuals\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (accession: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk\u003c/span\u003e\u003c/span\u003e). Furthermore, the summary data for EAC/BE (confirmed by pathological diagnosis) were collected from the GWAS Catalog (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/gwas/\u003c/span\u003e\u003c/span\u003e), which included 6,167 patients with BE, 4,112 individuals with EAC, and 17,159 controls\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The scRNA-seq data for EAC were retrieved from the Gene Expression Omnibus (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003c/span\u003e) (accession number: GSE173950). All donors were asked to complete the trial consent, related studies were reviewed and approved by institutional ethics review committees at the involved institutions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eSelection of instrumental variables\u003c/h2\u003e\n\u003cp\u003eThe following criteria were used to select cathepsin-related genetic variants: (a) r\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e measure of LD among instruments\u0026thinsp;\u0026lt;\u0026thinsp;0\u0026middot;001 within a 10,000 kb window, and (b) p-value\u0026thinsp;\u0026lt;\u0026thinsp;the genome-wide significance level identified in the corresponding study, i.e., 5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eMR analyses\u003c/h2\u003e\n\u003cp\u003eA genetic variant was justified as a valid instrument if it satisfied the following three core assumptions: (i) highly correlated with the exposure, (ii) was independent of any confounders between the exposure and the outcome, and (iii) was not directly associated with the outcome. The inverse variance-weighted (IVW) method was used as the primary method to determine the overall effect size of an exposure on the outcome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In IVW, the effect was elucidated using the Wald ratio method for each SNP. Subsequently, by using a random-effect inverse variance meta-analysis, these individual MR estimates were combined to achieve an overall summary value. The R TwoSampleMR package was used to perform MR analyses\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo evaluate the validity of the core instrumental variables assumptions, various sensitivity analyses and statistical tests were employed. Cochran\u0026rsquo;s Q test was performed to determine the heterogeneity of the SNPs. A p-value of \u0026lt;\u0026thinsp;0\u0026middot;05 suggested the presence of heterogeneity. When significant heterogeneity was observed among the SNPs, the random-effect model was used. Otherwise, a fixed-effect model\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e was used. MR-PRESSO global test and MR-Egger intercept were used to determine outliers and identify horizontal pleiotropy \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The MR-Egger intercept assessed the presence of a directional pleiotropic effect (intercept p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On the other hand, the MR-PRESSO outlier test was performed to correct for horizontal pleiotropy by removing or down-weighting the outliers when horizontal pleiotropy was significant (p-value of the MR-PRESSO global test\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, the MR-PRESSO distortion test was performed to identify significant distortion in the causal estimates before and after removing the outliers. The R MR-PRESSO package was used to perform the MR-PRESSO global, outlier, and distortion tests\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In reverse MRs, the GWAS datasets mentioned above were used, i.e., EAC/BE was the exposure, whereas the levels of cathepsin types were the outcomes.\u003c/p\u003e\n\u003cp\u003eLastly, when analyzing the causal effects on EAC/BE and determining the direct causal effects of each exposure in univariable analysis, multivariable MR analysis was used to consider multiple cathepsins. The \u0026ldquo;MendelianRandomization\u0026rdquo; package was used\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Furthermore, reverse MRs where lung cancer was regarded as the exposure and cathepsins as the outcome were used to evaluate the reverse casualties and justify the presence of bidirectional causality.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003escRNA-seq analysis\u003c/h2\u003e\n\u003cp\u003eThe droplet-based sequencing (DropSeq) was used to process the scRNA-seq data. The Seurat package was used to analyze the downloaded digital gene\u0026ndash;cell matrix of the 17 EAC cohorts\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Cells from different samples were filtered based on the following criteria: (a) the detected genes per cell should be \u0026gt;\u0026thinsp;200, and (b) the percentage of mitochondrial genes should be \u0026lt;\u0026thinsp;20%. A global scaling method was used to normalize the gene expression matrices of the remaining cells, with a default scale factor, followed by natural log transformation using log(1\u0026thinsp;+\u0026thinsp;x). The NormalizeData function was used for this. Then, the FindVariableFeatures method was used to identify the top 2000 highly variable genes. The \u0026ldquo;RunPCA\u0026rdquo; function was used to process the scaled and normalized gene expression data via linear dimensional reduction. The inflection point of the ElbowPlot function was used to determine the final number of principal components. Based on the Euclidean distance, the \u0026ldquo;FindNeighbors\u0026rdquo; function was used to prepare a K-nearest neighbor graph of the selected principal components. Then, the Louvain algorithm was performed to optimize modularity using the \u0026ldquo;FindClusters\u0026rdquo; function. Using the \u0026ldquo;RunUMAP\u0026rdquo; function, the cluster results of EAC scRNA-seq data were visualized using uniform manifold approximation and projection (UMAP)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To annotate the clusters, the FindAllMarkers function was used to determine the differentially expressed genes for the identified clusters. Finally, the FeaturePlot function was used to visualize the distribution of related genes. R software version 4.1.1 was used to conduct statistical analyses. The study schema is presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GWAS data of cathepsins and Macrophage were derived from the (https://gwas.mrcieu.ac.uk.). The summary data of EAC/BE were collected from https://www.ebi.ac.uk/gwas/. The scRNA seq data of EAC/BE were downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), the accession number is GSE173950.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll packages for data analysis used in this study were open source in R software (version 4.1.1; R Development Core Team).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Jilin Province medical and health talents special (JLSWSRCZX2023-35), Jilin Provincial Science and Technology Development Plan Project (20220204115YY) and Natural Science Foundation of Jilin Province (YDZJ202301ZYTS007 and YDZJ202201ZYTS121). The funding body had no role in the design of the study and collection, analysis and interpretation of data and in writing the manuscript. We thank Sagesci (www.sagesci.cn) for its linguistic assistance during the preparation of this manuscript. The study schema figure was generated with the aid of Scidraw and Biorender.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.T and W.L conceived and designed the experiment; J.L ran the analysis and verified the underlying data; J.L and S.T wrote the original manuscript. M.T and X.G involved in data interpretation. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll other authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSmyth, E. 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Cathepsins are a group of lysosomal proteolytic enzymes; they are associated with the occurrence and progression of various tumors. However, the causal relationship between the cathepsin family and EAC remains unelucidated. To investigate this association, Mendelian randomization (MR) and bioinformatics analyses of single-cell RNA sequencing (scRNA-seq) data were performed. MR analyses revealed that high cathepsin B (CTSB) levels decreased EAC risk. Furthermore, scRNA-seq revealed that CTSB expression was primarily distributed in macrophages. In addition, MR analysis of CTSB and macrophage scavenger receptor types I and II verified their interrelationship; CTSB primarily affects the proinflammatory phenotype of macrophages. Our findings suggest that CTSB levels affect EAC progression by regulating the expression of macrophage scavenger receptor types I and II, which induce the proinflammatory phenotypes of macrophages. Therefore, targeting CTSB may provide avenues for EAC diagnosis and treatment.\u003c/p\u003e","manuscriptTitle":"Causal relationship between cathepsins and esophageal adenocarcinoma: Mendelian randomization and single-cell RNA sequencing analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 06:25:04","doi":"10.21203/rs.3.rs-3859370/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b592c96-cd29-4f7b-b611-f8c67bff539a","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28411147,"name":"Health sciences/Oncology/Cancer/Cancer epidemiology"},{"id":28411148,"name":"Health sciences/Oncology/Cancer/Cancer genomics"}],"tags":[],"updatedAt":"2024-01-30T06:25:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-30 06:25:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3859370","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3859370","identity":"rs-3859370","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}