CTSS is associated with immunity and affects prognosis in pan-cancer

CTSS is associated with immunity and affects prognosis in pan-cancer | 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 Research Article CTSS is associated with immunity and affects prognosis in pan-cancer De-yun Zhang, Yu Liu, Zhongjun Chen, Jinyu Zheng, Zongren Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2876916/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 Background: CTSS(Cathepsisn S) is a lysosomal cysteine protease which is found in many tissues and plays multifarious roles. However, the role of CTSS in tumorigenesis is poorly defined. We investigated the association between CTSS levels and prognosis of cancer patients to determine prognostic value in this study. Methods: In this study, the RNA-sequencing (RNA-Seq) gene expression profile and clinical data were downloaded from the TCGA database and the UCSC database. Immunohistochemical images of CTSS and its expression in normal tissues were obtained from HPA database.Statistical analysis and data visualization were performed on the relationship between CTSS expression and prognosis, TMB,MSI, immune score, immune checkpoint and enrichment pathway through R language version 4.1.1 and its additional package. Results: Overall, CTSS is differentially expressed in most cancers and adjacent normal tissues.In addition, multiple survival analyses showed that CTSS significantly affected patient prognoses.CTSS was strongly associated with TMB in 8 cancers and MSI in 12 cancers. In addition, CTSS expression level is positively correlated with immune score in many cancers. Finally, GSEA analysis showed that CTSS was closely related to cell cycle, immune function, JAK-STAT and other biological functions and signaling pathways Conclusion: In some tumor patients, the high expression of CTSS can be used as a biomarker with better prognosis.In contrast, high CTSS expression indicated poor prognosis in some tumors, especially in LGG. The relationship between CTSS and immune checkpoint and GSEA analysis of CTSS may provide some ideas for potential immunotherapy and related pathways of some tumors. pan-cancer CTSS biomarker prognosis immune infifiltration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Cancer is still a huge obstacle to human health, even though medical treatment is advancing rapidly with the progress of the times [ 1 ] .Immunotherapy is booming in the new generation of anti-tumor therapy.A variety of tumor suppressor genes have been shown to inhibit tumor development, while a variety of oncogenic genes have also been revealed to play a driving role in tumor progression [ 2 – 4 ] . Treating tumors by regulating the corresponding genes may be a good way to treat them [ 5 ] .The development and exploration of immune checkpoint inhibitors have achieved satisfactory results in the treatment of some tumors, such as melanoma and pancreatic cancer [ 6 , 7 ] .Despite these improvements, there are still many patients who have little response to current immunotherapies. Therefore, it is necessary to explore new and reliable immune checkpoints to achieve better antitumor efficacy [ 8 ] . Cathepsin S, a member of the cysteine protease family, is a group of intracellular peptide-bond hydrolases mainly present in lysosomes.Compared with normal tissues, CTSS expression levels have been demonstrated to be up-regulated in glioblastoma, gastric cancer,non-Hodgkin lymphoma and some other tumor tissues, suggesting a poor prognosis of cancer, which may be a potential therapeutic target for some cancer treatments [ 9 – 11 ] .CTSS is a key factor in the proliferation, invasion and metastasis of some tumor tissues, and its overexpression is also related to the role of angiogenesis in tumors previously confirmed [ 12 ] . While most studies to date have described the biological functions of CTSS in certain tumor types and in limited samples, a comprehensive understanding of CTSS in a variety of cancers is needed. In order to explain the biological function of CTSS in cancer, we conducted a pan-cancer study on CTSS in this study.The expression level of CTSS in tumor and normal tissues and its relationship with patient prognosis were studied using different databases. In addition, we also discussed the relationship between CTSS and immune invasion, TMB, MSI and immune checkpoint in various cancers.Gene set enrichment analysis (GSEA) was used to explore the possible Gene Ontology (GO) function and pathway enriched in 33 cancer types.This study provides a new perspective on the functional role of CTSS in different types of cancer, as well as its potential association with immune checkpoints. 2. Materials And Methods 2.1.Data Source and Processing Using UCSC Xena ( https://xenabrowser.net/ ), we collected the data required from various cancer samples in the TCGA database,including RNA-seq data,phenotype and survival data [ 13 ] .HPA( https://www.proteinatlas.org/)i s a program that uses a variety of techniques to integrate human proteins into cells, tissues and organs [ 14 ] .We obtained the CTSS gene expression data in diferent human normal tissues from this web site.Immunohistochemical images of CTSS protein were obtained in histologic map and pathological map panels. 2.2.Survival Analysis In order to explore the relationship between CTSS expression level and patient prognosis, including overall survival(OS), disease-specific survival(DSS), and progression-free interval(PFI), 33 cancers were detected by "surival" ,"survminer"and "forestplot" in R package. Hazard ratios (HRs) and 95% confidence intervals were calculated using single-factor survival analysis. 2.3.Correlation of PBK Expression With Tumor Mutation Burden and Tumor Microsatellite Instability Tumor mutation burden(TMB) is a quantitative assessment of the number of somatic mutations within a tumor genome and can be used as a biomarker of susceptibility to immunocheckpoint inhibitor therapy in some cancers [ 15 ] .High microsatellite instability (MSI-H), which is the gain or loss of nucleotides in repetitive DNA caused by genomic overvariation, is a powerful diagnostic phenotype for predicting phenotypes, which may also influence immune checkpoint therapy [ 16 ] .We calculated TMB scores and MSI scores and their correlation with CTSS expression.Whereafter,"fmsb" in R package is used to draw radar map for data visualization of the results. 2.4.Correlation of CTSS Expression With Immune Infifiltration and Immune Checkpoint Expression The immune score, interstitial score, estimated score, and tumor purity were calculated using an estimation algorithm based on transcriptome expression profiles in various cancers, which is a common method to assess the degree of invasion of stromal or immune cells [ 17 ] .We calculated CTSS expression in different tumors and its correlation with immune score through "estimate" and "corrplot" in R package, and plotted scatter plots to visualize the data.In addition, we analyzed the relationship between CTSS expression and some reported immune checkpoints.The result is shown in a heatmap which was visualized by “reshape2” and “RColorBrewer” in R package. 2.5.Enrichment Analysis To determine the biological function of CTSS in cancers,we downloaded and analyzed the gene sets “c2.cp.kegg.v7.1.symbols” and “c5.all.v7.1.symbols” from the offificial GSEA website ( https://www.gsea-msigdb.org/gsea/downloads.jsp),includin g gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.Enrichment results were visualized by “limma” ,“org.Hs.eg.db” ,“clusterProfifiler” and “enrichplot” in R package. 2.6.Statistical Analysis All statistical analyses in this study were calculated by R version 4.1.1 and its additional packages. The Wilcoxon test was used to calculate the difference in CTSS expression between tumor tissue and normal tissue.Log-rank test, Kaplan-Meier method and Cox regression model were used to analyze survival.Spearman method was used to test the correlation. p < 0.05 was considered statistically significant.In all figures:* p < 0.05,** p < 0.01,*** p < 0.001. 3. Results 3.1.CTSS expression profifiles in human normal and cancer tissues. We first analyzed the expression levels of CTSS in normal and tumor tissues, and compared the differences between them.The Human Protein Atlas (HPA) database was used to analyze the expression level of CTSS in Human normal tissues, and the results showed that the expression level of CTSS mRNA was high in spleen, bone marrow, lung, appendix and colon(Fig. 1 A).Compared to normal tissue, the expression of CTSS in COAD, LUAD, LUSC, PAAD, PRAD and READ was lower. However, the expression of CTSS was higher in BESC, GBM,KIRC,KIRP,STAD,THCA and UCEC than in normal tissues(Fig. 1 B).Immunohistochemistry (IHC) revealed CTSS protein was mainly distributed in cytoplasm and membrane, and was high expressed in glandular cells in normal gastric tissues and gastric adenocarcinoma tissues (Figs. 1 C, D). However, CTSS was moderate expressed in neuronal cells in normal cerebral cortex tissues and glioma tissues (Figs. 1 E, F). Specific IHC results were showed in Table 1 . Table 1 Clinical information and relative scores of immunohistochemistry results (IHC, immunohistochemistry). Protein Tissue Histological type Age Gender Location Quantity Intensity CTSS Stomach Normal tissue 71 Male Cytoplasmic/membranous > 75% Strong CTSS Gastric cancer Gastric adenocarcinoma 65 Female Cytoplasmic/membranous > 75% Strong CTSS Cerebral cortex Normal tissue 45 Female Cytoplasmic/membranous 75%-25% Moderate CTSS Cerebral cortex Glioma 77 Male Cytoplasmic/membranous 75%-25% Moderate 3.2.Correlation between CTSS expression level and OS. To explore the relationship between the expression level of CTSS and OS times, we analyzed the expression level of CTSS and clinical data of 33 types of tumors.Results revealed that high CTSS level was signifificantly linked to worse OS in patients with LGG(p < 0.001),PAAD(p = 0.042),THYM(p = 0.007),and UVM(p = 0.004)(Fig. 2 A).Besides, Kaplan–Meier curves revealed high levels of CTSS expression were associated with shorter survival times in patients with LGG(p = 0.006) or UVM(p = 0.002). On the contrary, CTSS expression in BLCA (p = 0.048),OV(p = 0.011),SARC(p = 0.007),and SKCM(p < 0.001) were positively associated with OS times(Fig. 2 B-G). 3.3.Correlation between CTSS expression level and DSS. High CTSS level was signifificantly linked to worse DSS in patients with LGG(p < 0.001) or UVM(p = 0.017).On the contrary,high CTSS level was signifificantly linked to better DSS in patients with BLCA(p < 0.001),CESC(p = 0.023),SKCM(p < 0.001),and THCA(p = 0.001).And what we reveal by analyzing Kaplan-Meier curves is that ,much like that of the OS analysis,higher CTSS expression to be signifificantly related to a poorer DSS in LGG(p = 0.003),UVM(p = 0.008),and KICH(p = 0.041).Conversely,higher CTSS expression to be signifificantly related to a better DSS in BLCA(p = 0.026),OV(p = 0.005),SARC(p = 0.034),SKCM(p < 0.001),and THCA(p = 0.013). 3.4.Correlation between CTSS expression level and PFI. High CTSS level was signifificantly linked to worse PFI in patients with GBM(p = 0.007),LGG(p < 0.001) or THYM(p = 0.027).On the contrary,high CTSS level was signifificantly linked to better PFI in patients with BLCA(p = 0.003),COAD(p = 0.045),SKCM(p = 0.014),and UCEC(p = 0.042).Kaplan-Meier curves showed that higher CTSS expression to be signifificantly related to a poorer PFI in GBM(p = 0.007) and LGG(p = 0.009).Conversely,higher CTSS expression to be signifificantly related to a better PFI in COAD(p = 0.039). 3.5.The correlations of CTSS expression and TMB, MSI in cancers. Mismatch repair pathways play a crucial role in repairing DNA replication errors in normal and cancer cells [ 18 ] . DNA mismatch repair defects and secondary microsatellite instability can increase the burden of oncogene mutations [ 19 ] .CTSS expression was positively correlated with TMB in LGG,ESCA and BRCA.Whereas,it was negatively correlated with TMB in LUSC,LUAD,LIHC and HNSC(Fig. 5 A).In TGCT,SKCM,PCPG,PAAD,OV,LUSC,LUAD,LIHC,LGG,KIRP,HNSC and DLBC,CTSS expression was negatively correlated with MSI(Fig. 5 B)(*p < 0.05,**p < 0.005,***p < 0.001). 3.6.High CTSS Expression Correlates With Immune Infifiltration in Cancers. By exploring the relationship between the degree of immune invasion and the expression level of CTSS, we explored the effect of CTSS on immune function, so as to further explore the potential role of CTSS in the occurrence and development of cancers. In the following tumors, the expression level of CTSS was positively correlated with immune score(Fig. 6 ). 3.7.The correlations between CTSS and confifirmed immune checkpoints in cancers. Immune checkpoint is a signal pathway on the surface of T cells that inhibits its activation and participates in the Immune response [ 20 ] . Immune checkpoints, if activated, inhibit the action of immune cells [ 21 ] . Cancer cells, in order to evade the immune system, usually activate immune checkpoints to suppress the immune system from attacking them. Blocking cancer cells from activating immune checkpoints would allow the immune system to function normally, attacking and killing cancer cells [ 22 – 24 ] .In the following 33 types of cancer, the expression level of CTSS was positively correlated with recognized immune checkpoints expression levels including CTLA4,CD200R1,HAVCR2,CD86,and TNFRSF9.Although these immune checkpoints are not highly correlated with the expression level of CTSS in some tumors, they are positively correlated in most cases, which more or less gives us some inspiration for the elaboration of the mechanism of CTSS in the development of tumors, in order to be applied to clinical tumor treatment in future studies. 3.8.GO and KEGG analysis of CTSS-related signatures in cancers GO enrichment analysis showed that CTSS gene was closely related to cell cycle, gene silencing, negative regulation of myocardial tissue growth and negative regulation of cytoamide metabolism in BLCA.In GBM, CTSS gene is closely related to gene silencing, mRNA-binding, negative motor regulation, olfaction and other functions.In UVM, CTSS gene plays an important role in immune response regulation of cell surface receptor signals, leukocyte migration, immune process, cytokine production, immune effects and other processes.KEGG enrichment analysis showed that CTSS gene was closely involved in allograft rejection, antigen presentation and processing, leishmaniasis infection, primary immune deficiency and viral myocarditis in BLCA.In addition, CTSS and cell adhesion molecules, IgA produced by intestinal immune network, natural killer cell-mediated cytotoxicity, nod-like receptor signaling pathway are closely related to type I diabetes in GBM.In UVM, CTSS gene is strongly associated with cytokine receptor, hematopoietic cell lineage, JAK-STAT signaling pathway, natural killer cell-mediated cytotoxicity and T-cell receptor signaling pathway. 4. Discussion In this study, we evaluated CTSS expression levels in 33 cancers using a public database and identified 13 cancers with significant differences in CTSS expression levels compared to normal tissues.Compared to normal tissue, the expression of CTSS in COAD, LUAD, LUSC, PAAD, PRAD and READ was lower. Similar to previous studies, the expression of CTSS was higher in BESC, GBM,KIRC,KIRP,STAD,THCA and UCEC than in normal tissues [ 25 ] .In addition,we downloaded representative IHC images of CTSS expression in normal stomach tissues,gastric adenocarcinoma tissues,normal cerebral cortex tissues, and glioma tissues from HPA.CTSS is a cysteine protease that is thought to play a role in many physiological and pathological processes, including tumor growth, angiogenesis, and metastasis [ 26 , 27 ] . Previous studies have shown that CTSS is highly expressed in a variety of malignant tumor cells, and its expression level is positively correlated with tumor invasion and metastasis [ 28 – 30 ] .This was also confirmed to a certain extent by analyzing the survival time of tumor patients. Because we observed high CTSS expression consistent with poor prognosis in GBM,LGG,UVM and KICH .According to our results, CTSS dose have malignant biological characteristics and complex prognostic value in some cancers. Another key finding of this study is that the expression of CTSS is closely related to immunity, and there is also evidence from previous studies that CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation [ 31 ] .CTSS has been demonstrated to be involved in the activation of NKT cells in vivo and in vitro [ 32 ] .Therefore, we believe that CTSS has an immune-like function in cancer and may act as a costimulatory molecule mediating the activation of NKT cells. Of course, further work is needed to determine whether CTSS fulfils this function. Immune checkpoint inhibitors, particularly anti-CTLA4 and anti-PD-1 antibodies, have achieved unprecedented clinical results in the treatment of some cancers, such as melanoma and non-small cell lung cancer [ 33 – 35 ] .Therefore, we analyzed the correlation between CTSS and some immune checkpoints and found significant correlation between CTSS and most of the immune checkpoints in 33 cancers. This may provide some reference for us to regulate CTSS and influence related immune checkpoints, so as to achieve therapeutic effect on some tumors.Finally, GSEA enrichment analysis was performed on CTSS, and CTSS in 33 tumor tissues were analyzed by GO and KEGG. The results showed that the gene was involved in cell cycle, immunity and other functions, and the gene was also involved in JAK-STAT signaling pathway in UVM. In summary, CTSS may be partially effective by influencing immune processes through complex mechanisms and multiple tumor interactions, and its expression levels affect patient survival outcomes in different cancers. 5. Conclusions In conclusion, we used comprehensive bioinformatics methods to show that CTSS expression may mediate immune infiltration and influence the prognosis of patients with generalized cancer, and may serve as an important biomarker of poor prognosis in some tumors. CTSS provides a new direction for exploring the malignant pathogenesis of these cancers. We conclude that CTSS is closely associated with a variety of immune responses, and that immunotherapy combining targeted CTSS with existing checkpoint inhibitors may be a viable approach to suppress these unpleasant tumors. Declarations Data Availability The data used in this paper can be obtained in the relevant introduction of materials and methods. Specific data sets can be obtained by contacting the corresponding author. Authors ’ Contributions Deyun Zhang and Zongren Zhao designed the framework of this paper, Yu Liu, Deyun Zhang and Zhongjun Chen collected data and performed data analysis and data visualization, Deyun Zhang wrote the manuscript, and all authors contributed to this paper and approved the submitted version. Competing Interests The authors declare no competing financial interest. Acknowledgments This work was supported by a grant from Huai 'an Natural Science Research Program. （NO:HAB202118） References Tang J, Pearce L, O'Donnell-Tormey J, Hubbard-Lucey VM (2018) Nov;17(11):783–784 Trends in the global immuno-oncology landscape. Nat Rev Drug Discov. doi: 10.1038/nrd.2018.167 . Epub 2018 Oct 19. 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Carlino MS, Larkin J, Long GV (2021) Immune checkpoint inhibitors in melanoma. Lancet. Sep 11;398(10304):1002–1014. doi: 10.1016/S0140-6736(21)01206-X . PMID: 34509219 Vansteenkiste J, Wauters E, Reymen B, Ackermann CJ, Peters S, De Ruysscher D (2019) Current status of immune checkpoint inhibition in early-stage NSCLC. Ann Oncol. Aug 1;30(8):1244–1253. doi: 10.1093/annonc/mdz175 . PMID: 31143921 Souquet PJ, Couraud S (2019) Oct;20(10):1334–1335 Immune checkpoint inhibitors: a game changer for metastatic non-small-cell lung cancer. Lancet Oncol. doi: 10.1016/S1470-2045(19)30508-X. Epub 2019 Aug 14. PMID: 31422027 Additional Declarations No competing interests reported. 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. 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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-2876916","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":196910185,"identity":"0ae66134-098a-4223-b3bf-373dfd117327","order_by":0,"name":"De-yun Zhang","email":"","orcid":"","institution":"Xuzhou Children's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"De-yun","middleName":"","lastName":"Zhang","suffix":""},{"id":196910190,"identity":"797ea6eb-56f3-48f1-b1e8-71ddb0f82967","order_by":1,"name":"Yu Liu","email":"","orcid":"","institution":"Affiliated Huaian Hospital of Xuzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liu","suffix":""},{"id":196910195,"identity":"9cff1431-7f7f-4ffa-a95d-ce49e8a7f6f0","order_by":2,"name":"Zhongjun Chen","email":"","orcid":"","institution":"Affiliated Huaian Hospital of Xuzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhongjun","middleName":"","lastName":"Chen","suffix":""},{"id":196910197,"identity":"f740b4b7-2ec2-409f-aeb7-e8db5f74311f","order_by":3,"name":"Jinyu Zheng","email":"","orcid":"","institution":"Affiliated Huaian Hospital of Xuzhou Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinyu","middleName":"","lastName":"Zheng","suffix":""},{"id":196910199,"identity":"1fdbc03f-0826-4e52-adf0-c4d2c90e0928","order_by":4,"name":"Zongren Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYDACCQSTmSGBgUGOjb39AGlajPl4ziSQoAUIEudJOBjg1SE/u/nhwx8Vd+y2t/ceNni4oza9TQJo2Y+KbTi1MM45ZmwgceZZ8pwz55ITEs8cz22TbjzA2HPmNk4tzBIJZhKGbYeTJSRyjA8kth3LbZM5kMDM2IZbC5tE+jeJRJAW+TdgLelsEgkGeLXwSOSYSRxsO2wnIcFjnJDYVpNAUAvQPcWGDWcOJ0jw5BgbJLYdMGwDBvJBfH6Rn5G+ERhih+0l2M8YS/5sq5OXb28/+OBHBW4tMJDYAKEPg8kDBNUDgT2UriNG8SgYBaNgFIwwAABKe1fIedKxRAAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Huaian Hospital of Xuzhou Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zongren","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2023-04-29 15:59:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2876916/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2876916/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36650332,"identity":"cbf88e8b-72e4-42cd-8273-f88d1bef617d","added_by":"auto","created_at":"2023-05-05 14:58:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1212649,"visible":true,"origin":"","legend":"\u003cp\u003eCTSS expression profifiles in human normal and cancer tissues.(A) CD96 expression profifiles in normal human tissues.(B)The expression level of HSF1 in difffferent cancers.(C-D)Representative IHC images of CTSS expression in normal stomach tissues,gastric adenocarcinoma tissues,normal cerebral cortex tissues, and glioma tissues.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/651d87eaaf420fe52d717efe.png"},{"id":36650331,"identity":"770d40ce-5b1d-4229-8002-915514f67acb","added_by":"auto","created_at":"2023-05-05 14:58:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209456,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between CTSS level and OS. (A) Forest plot showing OS after Cox analysis in pan-cancer. (B–G) Kaplan-Meier curves showing OS in pan-cancer. Only signifificant results were shown(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/a2a319a904c0eb09e6b74b5b.png"},{"id":36650336,"identity":"83b99d8c-a432-4776-b6bf-befe777d433d","added_by":"auto","created_at":"2023-05-05 14:58:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":270091,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between CTSS level and DSS.(A) Forest plot showing DSS after Cox analysis in pan-cancer. (B–I) Kaplan-Meier curves showing DSS in pan-cancer. Only signifificant results were shown(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/a492dceba59ab0ac89c1d29f.png"},{"id":36650640,"identity":"5cadf137-1c68-46ad-b2a2-e18684f0951a","added_by":"auto","created_at":"2023-05-05 15:06:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":214615,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between CTSS level and PFI.(A) Forest plot showing PFI after Cox analysis in pan-cancer. (B–D) Kaplan-Meier curves showing PFI in pan-cancer. Only signifificant results were shown(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/d5a2de5ef9973ce72e2387db.png"},{"id":36650641,"identity":"f96f107a-bb9b-4abd-81b1-2efc59104388","added_by":"auto","created_at":"2023-05-05 15:06:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":233943,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlations of CTSS expression and TMB, MSI in cancers(A,B).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/e7bd249006739be745bad2dc.png"},{"id":36650338,"identity":"586c2ba5-9f32-4baa-86a6-173e5930d251","added_by":"auto","created_at":"2023-05-05 14:58:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":571516,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plots of correlation between CTSS and immune score in cancers.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/7d3074e4370a14f7c1538c12.png"},{"id":36650642,"identity":"547c9276-455f-4a04-972f-99b8a67ee94d","added_by":"auto","created_at":"2023-05-05 15:06:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":873937,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlations between CTSS and confifirmed immune checkpoints in cancers (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/eac8f2545660d5ae9fb09740.png"},{"id":36650334,"identity":"02a028e1-1662-44fa-94ea-1cc56cd06320","added_by":"auto","created_at":"2023-05-05 14:58:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":341745,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG analysis of CTSS-related signatures in BLCA, GBM and UVM.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/595a004eda40057e49140a07.png"},{"id":36651377,"identity":"2c03cc97-6fa2-43a0-8f67-fe0d873df7bb","added_by":"auto","created_at":"2023-05-05 15:15:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4713707,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2876916/v1/e55787b9-3e0d-4ef4-ba26-dc8f502c6020.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CTSS is associated with immunity and affects prognosis in pan-cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer is still a huge obstacle to human health, even though medical treatment is advancing rapidly with the progress of the times\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.Immunotherapy is booming in the new generation of anti-tumor therapy.A variety of tumor suppressor genes have been shown to inhibit tumor development, while a variety of oncogenic genes have also been revealed to play a driving role in tumor progression\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Treating tumors by regulating the corresponding genes may be a good way to treat them\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.The development and exploration of immune checkpoint inhibitors have achieved satisfactory results in the treatment of some tumors, such as melanoma and pancreatic cancer\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.Despite these improvements, there are still many patients who have little response to current immunotherapies. Therefore, it is necessary to explore new and reliable immune checkpoints to achieve better antitumor efficacy\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCathepsin S, a member of the cysteine protease family, is a group of intracellular peptide-bond hydrolases mainly present in lysosomes.Compared with normal tissues, CTSS expression levels have been demonstrated to be up-regulated in glioblastoma, gastric cancer,non-Hodgkin lymphoma and some other tumor tissues, suggesting a poor prognosis of cancer, which may be a potential therapeutic target for some cancer treatments\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.CTSS is a key factor in the proliferation, invasion and metastasis of some tumor tissues, and its overexpression is also related to the role of angiogenesis in tumors previously confirmed\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. While most studies to date have described the biological functions of CTSS in certain tumor types and in limited samples, a comprehensive understanding of CTSS in a variety of cancers is needed.\u003c/p\u003e \u003cp\u003eIn order to explain the biological function of CTSS in cancer, we conducted a pan-cancer study on CTSS in this study.The expression level of CTSS in tumor and normal tissues and its relationship with patient prognosis were studied using different databases. In addition, we also discussed the relationship between CTSS and immune invasion, TMB, MSI and immune checkpoint in various cancers.Gene set enrichment analysis (GSEA) was used to explore the possible Gene Ontology (GO) function and pathway enriched in 33 cancer types.This study provides a new perspective on the functional role of CTSS in different types of cancer, as well as its potential association with immune checkpoints.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Data Source and Processing\u003c/h2\u003e \u003cp\u003eUsing UCSC Xena (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://xenabrowser.net/\u003c/span\u003e\u003cspan address=\"https://xenabrowser.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we collected the data required from various cancer samples in the TCGA database,including RNA-seq data,phenotype and survival data\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.HPA(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.proteinatlas.org/)i\u003c/span\u003e\u003cspan address=\"https://www.proteinatlas.org/)i\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003es a program that uses a variety of techniques to integrate human proteins into cells, tissues and organs\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.We obtained the CTSS gene expression data in diferent human normal tissues from this web site.Immunohistochemical images of CTSS protein were obtained in histologic map and pathological map panels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2.Survival Analysis\u003c/h2\u003e \u003cp\u003eIn order to explore the relationship between CTSS expression level and patient prognosis, including overall survival(OS), disease-specific survival(DSS), and progression-free interval(PFI), 33 cancers were detected by \"surival\" ,\"survminer\"and \"forestplot\" in R package. Hazard ratios (HRs) and 95% confidence intervals were calculated using single-factor survival analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3.Correlation of PBK Expression With Tumor Mutation Burden and Tumor Microsatellite Instability\u003c/h2\u003e \u003cp\u003eTumor mutation burden(TMB) is a quantitative assessment of the number of somatic mutations within a tumor genome and can be used as a biomarker of susceptibility to immunocheckpoint inhibitor therapy in some cancers\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.High microsatellite instability (MSI-H), which is the gain or loss of nucleotides in repetitive DNA caused by genomic overvariation, is a powerful diagnostic phenotype for predicting phenotypes, which may also influence immune checkpoint therapy\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.We calculated TMB scores and MSI scores and their correlation with CTSS expression.Whereafter,\"fmsb\" in R package is used to draw radar map for data visualization of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4.Correlation of CTSS Expression With Immune Infifiltration and Immune Checkpoint Expression\u003c/h2\u003e \u003cp\u003eThe immune score, interstitial score, estimated score, and tumor purity were calculated using an estimation algorithm based on transcriptome expression profiles in various cancers, which is a common method to assess the degree of invasion of stromal or immune cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.We calculated CTSS expression in different tumors and its correlation with immune score through \"estimate\" and \"corrplot\" in R package, and plotted scatter plots to visualize the data.In addition, we analyzed the relationship between CTSS expression and some reported immune checkpoints.The result is shown in a heatmap which was visualized by \u0026ldquo;reshape2\u0026rdquo; and \u0026ldquo;RColorBrewer\u0026rdquo; in R package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5.Enrichment Analysis\u003c/h2\u003e \u003cp\u003eTo determine the biological function of CTSS in cancers,we downloaded and analyzed the gene sets \u0026ldquo;c2.cp.kegg.v7.1.symbols\u0026rdquo; and \u0026ldquo;c5.all.v7.1.symbols\u0026rdquo; from the offificial GSEA website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gsea-msigdb.org/gsea/downloads.jsp),includin\u003c/span\u003e\u003cspan address=\"https://www.gsea-msigdb.org/gsea/downloads.jsp),includin\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eg gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.Enrichment results were visualized by \u0026ldquo;limma\u0026rdquo; ,\u0026ldquo;org.Hs.eg.db\u0026rdquo; ,\u0026ldquo;clusterProfifiler\u0026rdquo; and \u0026ldquo;enrichplot\u0026rdquo; in R package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6.Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses in this study were calculated by R version 4.1.1 and its additional packages. The Wilcoxon test was used to calculate the difference in CTSS expression between tumor tissue and normal tissue.Log-rank test, Kaplan-Meier method and Cox regression model were used to analyze survival.Spearman method was used to test the correlation.\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.In all figures:*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05,**\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01,***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1.CTSS expression profifiles in human normal and cancer tissues.\u003c/h2\u003e \u003cp\u003eWe first analyzed the expression levels of CTSS in normal and tumor tissues, and compared the differences between them.The Human Protein Atlas (HPA) database was used to analyze the expression level of CTSS in Human normal tissues, and the results showed that the expression level of CTSS mRNA was high in spleen, bone marrow, lung, appendix and colon(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).Compared to normal tissue, the expression of CTSS in COAD, LUAD, LUSC, PAAD, PRAD and READ was lower. However, the expression of CTSS was higher in BESC, GBM,KIRC,KIRP,STAD,THCA and UCEC than in normal tissues(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).Immunohistochemistry (IHC) revealed CTSS protein was mainly distributed in cytoplasm and membrane, and was high expressed in glandular cells in normal gastric tissues and gastric adenocarcinoma tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). However, CTSS was moderate expressed in neuronal cells in normal cerebral cortex tissues and glioma tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F). Specific IHC results were showed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical information and relative scores of immunohistochemistry results (IHC, immunohistochemistry).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHistological type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eQuantity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIntensity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStomach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic/membranous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eStrong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGastric cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGastric adenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic/membranous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eStrong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCerebral cortex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormal tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic/membranous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e75%-25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTSS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCerebral cortex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlioma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCytoplasmic/membranous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e75%-25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2.Correlation between CTSS expression level and OS.\u003c/h2\u003e \u003cp\u003eTo explore the relationship between the expression level of CTSS and OS times, we analyzed the expression level of CTSS and clinical data of 33 types of tumors.Results revealed that high CTSS level was signifificantly linked to worse OS in patients with LGG(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001),PAAD(p\u0026thinsp;=\u0026thinsp;0.042),THYM(p\u0026thinsp;=\u0026thinsp;0.007),and UVM(p\u0026thinsp;=\u0026thinsp;0.004)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).Besides, Kaplan\u0026ndash;Meier curves revealed high levels of CTSS expression were associated with shorter survival times in patients with LGG(p\u0026thinsp;=\u0026thinsp;0.006) or UVM(p\u0026thinsp;=\u0026thinsp;0.002). On the contrary, CTSS expression in BLCA (p\u0026thinsp;=\u0026thinsp;0.048),OV(p\u0026thinsp;=\u0026thinsp;0.011),SARC(p\u0026thinsp;=\u0026thinsp;0.007),and SKCM(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were positively associated\u003c/p\u003e \u003cp\u003ewith OS times(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3.Correlation between CTSS expression level and DSS.\u003c/h2\u003e \u003cp\u003eHigh CTSS level was signifificantly linked to worse DSS in patients with LGG(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) or UVM(p\u0026thinsp;=\u0026thinsp;0.017).On the contrary,high CTSS level was signifificantly linked to better DSS in patients with BLCA(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001),CESC(p\u0026thinsp;=\u0026thinsp;0.023),SKCM(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001),and THCA(p\u0026thinsp;=\u0026thinsp;0.001).And what we reveal by analyzing Kaplan-Meier curves is that ,much like that of the OS analysis,higher CTSS expression to be signifificantly related to a poorer DSS in LGG(p\u0026thinsp;=\u0026thinsp;0.003),UVM(p\u0026thinsp;=\u0026thinsp;0.008),and KICH(p\u0026thinsp;=\u0026thinsp;0.041).Conversely,higher CTSS expression to be signifificantly related to a better DSS in BLCA(p\u0026thinsp;=\u0026thinsp;0.026),OV(p\u0026thinsp;=\u0026thinsp;0.005),SARC(p\u0026thinsp;=\u0026thinsp;0.034),SKCM(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001),and THCA(p\u0026thinsp;=\u0026thinsp;0.013).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4.Correlation between CTSS expression level and PFI.\u003c/h2\u003e \u003cp\u003eHigh CTSS level was signifificantly linked to worse PFI in patients with GBM(p\u0026thinsp;=\u0026thinsp;0.007),LGG(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) or THYM(p\u0026thinsp;=\u0026thinsp;0.027).On the contrary,high CTSS level was signifificantly linked to better PFI in patients with BLCA(p\u0026thinsp;=\u0026thinsp;0.003),COAD(p\u0026thinsp;=\u0026thinsp;0.045),SKCM(p\u0026thinsp;=\u0026thinsp;0.014),and UCEC(p\u0026thinsp;=\u0026thinsp;0.042).Kaplan-Meier curves showed that higher CTSS expression to be signifificantly related to a poorer PFI in GBM(p\u0026thinsp;=\u0026thinsp;0.007) and LGG(p\u0026thinsp;=\u0026thinsp;0.009).Conversely,higher CTSS expression to be signifificantly related to a better PFI in COAD(p\u0026thinsp;=\u0026thinsp;0.039).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5.The correlations of CTSS expression and TMB, MSI in cancers.\u003c/h2\u003e \u003cp\u003eMismatch repair pathways play a crucial role in repairing DNA replication errors in normal and cancer cells\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. DNA mismatch repair defects and secondary microsatellite instability can increase the burden of oncogene mutations\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.CTSS expression was positively correlated with TMB in LGG,ESCA and BRCA.Whereas,it was negatively correlated with TMB in LUSC,LUAD,LIHC and HNSC(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).In TGCT,SKCM,PCPG,PAAD,OV,LUSC,LUAD,LIHC,LGG,KIRP,HNSC and DLBC,CTSS expression was negatively correlated with MSI(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB)(*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05,**p\u0026thinsp;\u0026lt;\u0026thinsp;0.005,***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6.High CTSS Expression Correlates With Immune Infifiltration in Cancers.\u003c/h2\u003e \u003cp\u003eBy exploring the relationship between the degree of immune invasion and the expression level of CTSS, we explored the effect of CTSS on immune function, so as to further explore the potential role of CTSS in the occurrence and development of cancers. In the following tumors, the expression level of CTSS was positively correlated with immune score(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.7.The correlations between CTSS and confifirmed immune checkpoints in cancers.\u003c/h2\u003e \u003cp\u003eImmune checkpoint is a signal pathway on the surface of T cells that inhibits its activation and participates in the Immune response\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Immune checkpoints, if activated, inhibit the action of immune cells\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Cancer cells, in order to evade the immune system, usually activate immune checkpoints to suppress the immune system from attacking them. Blocking cancer cells from activating immune checkpoints would allow the immune system to function normally, attacking and killing cancer cells\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.In the following 33 types of cancer, the expression level of CTSS was positively correlated with recognized immune checkpoints expression levels including CTLA4,CD200R1,HAVCR2,CD86,and TNFRSF9.Although these immune checkpoints are not highly correlated with the expression level of CTSS in some tumors, they are positively correlated in most cases, which more or less gives us some inspiration for the elaboration of the mechanism of CTSS in the development of tumors, in order to be applied to clinical tumor treatment in future studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.8.GO and KEGG analysis of CTSS-related signatures in cancers\u003c/h2\u003e \u003cp\u003eGO enrichment analysis showed that CTSS gene was closely related to cell cycle, gene silencing, negative regulation of myocardial tissue growth and negative regulation of cytoamide metabolism in BLCA.In GBM, CTSS gene is closely related to gene silencing, mRNA-binding, negative motor regulation, olfaction and other functions.In UVM, CTSS gene plays an important role in immune response regulation of cell surface receptor signals, leukocyte migration, immune process, cytokine production, immune effects and other processes.KEGG enrichment analysis showed that CTSS gene was closely involved in allograft rejection, antigen presentation and processing, leishmaniasis infection, primary immune deficiency and viral myocarditis in BLCA.In addition, CTSS and cell adhesion molecules, IgA produced by intestinal immune network, natural killer cell-mediated cytotoxicity, nod-like receptor signaling pathway are closely related to type I diabetes in GBM.In UVM, CTSS gene is strongly associated with cytokine receptor, hematopoietic cell lineage, JAK-STAT signaling pathway, natural killer cell-mediated cytotoxicity and T-cell receptor signaling pathway.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we evaluated CTSS expression levels in 33 cancers using a public database and identified 13 cancers with significant differences in CTSS expression levels compared to normal tissues.Compared to normal tissue, the expression of CTSS in COAD, LUAD, LUSC, PAAD, PRAD and READ was lower. Similar to previous studies, the expression of CTSS was higher in BESC, GBM,KIRC,KIRP,STAD,THCA and UCEC than in normal tissues\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.In addition,we downloaded representative IHC images of CTSS expression in normal stomach tissues,gastric adenocarcinoma tissues,normal cerebral cortex tissues, and glioma tissues from HPA.CTSS is a cysteine protease that is thought to play a role in many physiological and pathological processes, including tumor growth, angiogenesis, and metastasis\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that CTSS is highly expressed in a variety of malignant tumor cells, and its expression level is positively correlated with tumor invasion and metastasis\u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.This was also confirmed to a certain extent by analyzing the survival time of tumor patients. Because we observed high CTSS expression consistent with poor prognosis in GBM,LGG,UVM and KICH .According to our results, CTSS dose have malignant biological characteristics and complex prognostic value in some cancers.\u003c/p\u003e \u003cp\u003eAnother key finding of this study is that the expression of CTSS is closely related to immunity, and there is also evidence from previous studies that CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.CTSS has been demonstrated to be involved in the activation of NKT cells in vivo and in vitro\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.Therefore, we believe that CTSS has an immune-like function in cancer and may act as a costimulatory molecule mediating the activation of NKT cells. Of course, further work is needed to determine whether CTSS fulfils this function.\u003c/p\u003e \u003cp\u003eImmune checkpoint inhibitors, particularly anti-CTLA4 and anti-PD-1 antibodies, have achieved unprecedented clinical results in the treatment of some cancers, such as melanoma and non-small cell lung cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.Therefore, we analyzed the correlation between CTSS and some immune checkpoints and found significant correlation between CTSS and most of the immune checkpoints in 33 cancers. This may provide some reference for us to regulate CTSS and influence related immune checkpoints, so as to achieve therapeutic effect on some tumors.Finally, GSEA enrichment analysis was performed on CTSS, and CTSS in 33 tumor tissues were analyzed by GO and KEGG. The results showed that the gene was involved in cell cycle, immunity and other functions, and the gene was also involved in JAK-STAT signaling pathway in UVM. In summary, CTSS may be partially effective by influencing immune processes through complex mechanisms and multiple tumor interactions, and its expression levels affect patient survival outcomes in different cancers.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, we used comprehensive bioinformatics methods to show that CTSS expression may mediate immune infiltration and influence the prognosis of patients with generalized cancer, and may serve as an important biomarker of poor prognosis in some tumors. CTSS provides a new direction for exploring the malignant pathogenesis of these cancers. We conclude that CTSS is closely associated with a variety of immune responses, and that immunotherapy combining targeted CTSS with existing checkpoint inhibitors may be a viable approach to suppress these unpleasant tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this paper can be obtained in the relevant introduction of materials and methods. Specific data sets can be obtained by contacting the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo; \u003c/strong\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeyun Zhang and Zongren Zhao designed the framework of this paper, Yu Liu, Deyun Zhang and Zhongjun Chen collected data and performed data analysis and data visualization, Deyun Zhang wrote the manuscript, and all authors contributed to this paper and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from Huai 'an Natural Science Research Program. （NO:HAB202118）\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTang J, Pearce L, O'Donnell-Tormey J, Hubbard-Lucey VM (2018) Nov;17(11):783\u0026ndash;784 Trends in the global immuno-oncology landscape. 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PMID: 31422027\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"pan-cancer, CTSS, biomarker, prognosis, immune infifiltration","lastPublishedDoi":"10.21203/rs.3.rs-2876916/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2876916/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCTSS(Cathepsisn S) is a lysosomal cysteine protease which is found in many tissues and plays multifarious roles. However, the role of CTSS in tumorigenesis is poorly defined. We investigated the association between CTSS levels and prognosis of cancer patients to determine prognostic value in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn this study, the RNA-sequencing (RNA-Seq) gene expression profile and clinical data were downloaded from the TCGA database and the UCSC database. Immunohistochemical images of CTSS and its expression in normal tissues were obtained from HPA database.Statistical analysis and data visualization were performed on the relationship between CTSS expression and prognosis, TMB,MSI, immune score, immune checkpoint and enrichment pathway through R language version 4.1.1 and its additional package.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eOverall, CTSS is differentially expressed in most cancers and adjacent normal tissues.In addition, multiple survival analyses showed that CTSS significantly affected patient prognoses.CTSS was strongly associated with TMB in 8 cancers and MSI in 12 cancers. In addition, CTSS expression level is positively correlated with immune score in many cancers. Finally, GSEA analysis showed that CTSS was closely related to cell cycle, immune function, JAK-STAT and other biological functions and signaling pathways\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eIn some tumor patients, the high expression of CTSS can be used as a biomarker with better prognosis.In contrast, high CTSS expression indicated poor prognosis in some tumors, especially in LGG. The relationship between CTSS and immune checkpoint and GSEA analysis of CTSS may provide some ideas for potential immunotherapy and related pathways of some tumors.\u003c/p\u003e","manuscriptTitle":"CTSS is associated with immunity and affects prognosis in pan-cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-05 14:58:53","doi":"10.21203/rs.3.rs-2876916/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":"ad45cb3c-38c6-4e59-b34f-c6405c5648fe","owner":[],"postedDate":"May 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-05T14:58:55+00:00","versionOfRecord":[],"versionCreatedAt":"2023-05-05 14:58:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2876916","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2876916","identity":"rs-2876916","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}