Angiotensin‑converting enzyme 2 as an immune and prognostic biomarker in colorectal cancer

Fig. 1. Expression of ACE2 in various tumour tissues. (A) ACE2 expression levels across different cancer types, as provided by GEPIA. (B) ACE2 mRNA expression levels in multiple types of tumors were analyzed using Xiantao Academic form the TCGA dataset. *p < 0.05; *** p < 0.001.

Fig. 2. Correlation between the expression of the ACE2 protein and the staging of tumours. (A) Correlation between the expression of the ACE2 protein and tumour staging in patients with colon adenocarcinoma (COAD). (B) Correlation between the expression of the ACE2 protein and tumour staging in patients with pancreatic cancer (PAAD). (C) Correlation between the expression of the ACE2 protein and tumour staging in patients with renal cell carcinoma (RCC). (D) Correlation between the expression of the ACE2 protein and tumour staging in patients with Gastric cancer (GC). *p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 3. Diagnostic and prognostic value of ACE2 in multiple types of tumors. (A) Diagnostic performance of ACE2 in COAD, READ, PAAD, KIRP, and STAD cancers. (B) Survival curves showing the prognostic relevance of the ACE2 expression in CRC, KIRP, PAAD, and STAD.

Fig. 4. ACE2 expression in CRC. (A) ACE2 expression in patients with CRC based on TCGA data. (B) ACE2 mRNA expression in CRC and corresponding normal tissues from the GSE31737 data set. (C) Relative mRNA expression levels of ACE2 in normal colon cells (NCM460) and colon cancer cell lines (SW480, RKO, and Caco-2) was quantified by qRT-PCR, NCM460 acts as the statistical comparison.(D) Evaluation of ACE2 protein expression in CRC tissues and adjacent normal tissues. (E) Immunohistochemical analysis of ACE2 in 119 CRC tissues and their adjacent normal tissues. *p < 0.05; ** p < 0.01.

Fig. 5. Correlation between the ACE2 expression and pathological characteristics/prognosis in patients with CRC. (A) ACE2 expression across different pathological stages. (B) Expression level of ACE2 in CRC patients stratified by different treatment responses, including the Complete Response (CR), Partial Response (PR), Stable Disease (SD) and Progressive Disease (PD) groups. (C) Correlation between the ACE2 expression and overall survival (OS) in the GSE106584 dataset. (D) Correlation between the ACE2 expression and OS in the GSE28722 dataset. (E) Correlation between the ACE2 expression and recurrence-free survival (RFS) in the GSE106584 dataset. (F) Correlation between the ACE2 expression and RFS in the GSE28722 dataset.

Fig. 6. ACE2 co-expression network analysis. (A) Co-expression network of ACE2 and associated genes, constructed using the STRING database, Metascape, and Cytoscape software. (B) Biological processes, molecular functions, and cellular components analyzed via Xiantao Academic. (C) KEGG pathway enrichment of the co-expressed genes, as analyzed using Xiantao Academic.

Fig. 7. ACE2 expression and its association with biological processes in cancer. (A) ACE2 expression is linked to various biological processes in multiple cancer types, including angiogenesis, apoptosis, cell cycle regulation, differentiation, DNA damage and repair, epithelial-mesenchymal transition (EMT), hypoxia, inflammation, invasion, metastasis, proliferation, quiescence, and stemness. (B) Investigation of the association between the ACE2 expression and biological processes in CRC. (C) Heatmap showing the ACE2 expression across different biological processes. (D) t-SNE plot of the ACE2 expression distribution in CRC.

Fig. 8. Effects of ACE2 knockdown on CRC RKO and SW480 cells proliferation and invasion. After transfection of RKO cells with either si-NC or si-ACE2, (A) qRT-PCR analysis were used to detect the transfection efficiency, (B) CCK-8 assay were used to detect cell proliferative ability, (C-D) transwell matrigel assays were used to detect cell metastatic ability, (E) qRT-PCR assay were used to detect the mRNA expression of CCNB1, CCND1, CCNE1, Snail1, β-Catenin, and MMP9, (F) Western blotting assay were used to detect the protein expression of CCND1, CCNE1, Snail1 and β-Catenin. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 9. Mutations and methylation of ACE2 in CRC. (A) ACE2 mutation levels in patients with CRC. (B) Assessment of the ACE2 methylation levels in patients with CRC. (C) After treating RKO and SW480 cells with the DNA methyltransferase inhibitor 5‑Aza, the expression of ACE2 was detected. (D) Analysis of drug sensitivity based on ACE2 expression. ** p < 0.01; *** p < 0.001.

Fig. 10. Association between the ACE2 expression and immune-related genes in CRC. (A) The ACE2 expression was associated with immune-stimulatory gene expression. (B) Two immune-stimulant factors most negatively correlated with ACE2 in CRC. (C) The ACE2 expression was associated with immunosuppressive factors. (D) Two immunosuppressive factors most negatively correlated with ACE2 in CRC. (E) The ACE2 expression was correlated with the major histocompatibility complex (MHC). (F) Two MHC factors most inversely correlated with ACE2 expression in CRC.

Fig. 11. Relationship between ACE2 and immune Cells in CRC. (A) Correlation between the ACE2 expression and various immune cell types in CRC. (B) Heatmap showing the ACE2 expression and its association with immune cell populations in CRC. (C) Correlation between the ACE2 expression and NK cell markers. (D) ACE2 expression levels in 25 CRC tissues cases and adjacent tissues. (E) CD94 expression levels in the same CRC samples. (F) Scatter plot depicting the correlation between ACE2 and CD94 expression in the 25 CRC samples.

Fig. 12. ACE2 and its association with the tumour mutational burden, microsatellite instability, neoantigen load, and immune checkpoint therapy response. (A) Correlation between the ACE2 expression and tumour mutational burden in various cancers. (B) Correlation between the ACE2 expression and microsatellite instability. (C) Relationship between the ACE2 expression and neoantigen load. (D) Radar chart illustrating the association between ACE2 expression and ESTIMATE scores across the cancer types. (E) Correlation between ACE2 expression and immunotherapeutic response in Melanoma. (F) Correlation between ACE2 expression and immunotherapeutic response in urothelial cancer.