*3.10. Possible Role of the Expression of CPA4 in Various Infiltrating Immune Cells*

We used the TIMER2 and GEPIA2 databases to further identify the possible role of the expression of CPA4 in various infiltrating immune cells including T cells (general), M1/M2 macrophages, tumor-associated macrophages, B cells, neutrophils, monocytes, NK, CD8+ T cells, and functional DCs as well as T cells such as Th1, Th2, Th9, Th17, Th22, Tfh, exhausted T cells, and Treg. From the results, Th1, T cell exhaustion, and TAM sets marking were greatly connected with the expression of CPA4 in BLCA (Table 2).

**Table 2.** Correlation analysis between CPA4 and markers of immune cells in BLCA patients found in the TIMER2 and GEPIA2.



**Table 2.** *Cont.*

BLCA, Bladder Urothelial Carcinoma; Tfh, Follicular helper T cell; Th, T helper cell; Treg, Regulatory T cell; TAM, tumor-associated macrophage;.None, correlation without adjustment correlation; Purity, correlation adjusted by purity; Tumor, correlation analysis in the tumor tissue of TCGA; Normal, correlation analysis in normal tissue of TCGA; Cor, R value of Spearman's correlation \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

#### **4. Discussion**

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family and is a zinc-containing exopeptidase that catalyzes the release of carboxy-terminal amino acids [34]. In recent years, CPA4 has shown the potential to be a biomarker in the early diagnosis with clinical benefit for certain cancers. Some studies revealed that CPA4 is connected with various cancer cells in its differentiation and growth, including non-smallcell lung cancer and gastric cancer [35,36]. Furthermore, it is reported that CPA4 is located on chromosome 7q32 in a region linked to prostate cancer aggressiveness [11], and Sun suggested that CPA4 is closely associated with colorectal cancer liver metastasis [37]. Although CPA4 expression has been confirmed to have potential significance in multiple types of cancer, no studies have shown the expression level and clinical significance of CPA4 in BLCA. In this study, based on a pan-cancer analysis, we demonstrated that human CPA4 expression levels were highly expressed in 11 types of cancer with corresponding normal tissues (Figure 1), which are consistent with the findings in the previous study reported by Sun and Handa et al. [17,35,38]. We also confirmed that CPA4 is significantly upregulated in BLCA (Figure 1b). Moreover, a previous study has shown that CPA4 expression was detected specifically in the cytoplasm of cancer tissue cells, and in the CPA4-suppressed triple-negative breast cancer (TNBC), viability, and migration were decreased [38]. It can act as a potential biomarker of poor prognosis in TNBC. It is reported that CPA4 might be used as an independent poor prognostic factor in esophageal squamous cell carcinoma [39]. In our study, the results in BLCA are consistent. However, one trial showed that CPA4 is a protective factor in muscle-invasive bladder cancer, contrary to the role of CPA4 in most cancers [40]. A potential reason for the difference is due to updates in the TCGA database and different objects. We studied BLCA, while that study investigated muscle-invasive bladder cancer. We compared the different expression levels of BLCA with age; gender; T, N, and M stage; pathologic stage; subtype; and OS. Surprisingly, we found that higher dead events, higher pathologic stages, and the subtype non-papillary were associated with higher expressions of CPA4 in BLCA, with statistical differences (Figure 2). These findings suggest that CPA4 may be a potential biomarker of poor prognosis in identifying BLCA with poor clinical outcome.

Currently, the function of CPA4 in tumors had not been fully reported. Previous trials suggested that the inhibition of CPA4 could reduce the number of breast cancer cells with stemness properties and may be a potential target for TNBC therapy [41]. The CircCPA4 sponge let-7 regulates the expression of CPA4 and glioma progression [42]. All of these results suggest that CPA4 could be regarded as an emerging target or promising biomarker for cancer therapy. Since the mRNA expression of CPA4 in BLCA was significantly higher than that in normal bladder tissue, we speculated that CPA4 can be regarded as a biomarker to detect BLCA from normal controls. To verify the clinical value of CPA4, an ROC curve analysis was performed to verify the clinical value of CPA4 in the diagnosis of BLCA; our results showed that CPA4 may be a potential diagnostic biomarker between bladder cancer and normal tissues, with an AUC of 0.798 (Figure 2d).

Many studies have shown that CPA4 is a significant biomarker of poor prognosis in lots of cancers and is associated with the upregulation of CPA4 with poor overall survival. In hepatocellular carcinoma, AC10364 inhibited cell proliferation and viability through the abnormal expression of genes including CPA4 associated with tumorigenesis or growth [43]. A paper from Yan suggested that the inhibition of CPA4 might be of great significance for improving early stage non-small cell lung cancer survival after ablation [43]. However, the prognostic value in BLCA of CPA4 has not been investigated. With the increased level of CPA4 related to a higher number of dead events and higher pathologic stages, we speculated that CPA4 is involved in the development of BLCA. In light of the Kaplan–Meier curves, we confirmed that the overexpression of CPA4 is associated with shorter overall survival (OS), disease-specific survival (DSF), and progress-free intervals (PFIs) (Figure 2). Moreover, by univariate and multivariate regression analysis, we found that high CPA4 expression; high pathologic stage; T, N, and M stage; and the subtype papillary were negative predictors for OS in BLCA patients and that CPA4 can be an independent factor correlated with OS (Figure 5). The nomogram more accurately predicted 1-, 3-, and 5-year OS in BLCA patients and could help to screen and determine those high-risk patients (Figure 6).

Through GSEA, CPA4 was found to be involved in epidermal cell differentiation, keratinocyte differentiation, keratinization, etc., indicating CPA4 potentially playing a role in cell metabolism and protein synthesis (Figure 4).

The PPI network of CPA4 protein, which were constructed by STRING, showed the relationship of CPA4 in the progression of BLCA such as LXN, CMA1, SGCE, TPSAB1, etc. (Figure 7). It has been reported that latexin (LXN) can inhibit human CPA4, in which the expression is induced in prostate cancer cells after treatment with histone deacetylase inhibitors [44]. The level of CMA1, a key gene, is significantly correlated with gastric cancer prognosis and infiltration level [45]. SGCE promotes breast cancer stem cell self-renewal, chemoresistance, and metastasis both in vitro and in vivo by stabilizing EGFR [45]. Thus, it is speculated that a high expression of CPA4 may increase the degree of malignancy of tumors through CPA4 interacting proteins, leading to the deterioration of patients' conditions.

Moreover, CPA4 plays a specific role in immune infiltration in bladder cancer. Compellingly, we unraveled that several infiltrating immune cells (Th1cell, Th2 cell, T cell exhaustion, and TAM) were correlated with the expression of CPA4 in bladder cancer using TIMER2 and GEPIA2. Type 1 T helper (Th1) cells produce interferon-gamma [46] (Figure 8, Table 2). The dual inhibition of STAT1 and STAT3 activation downregulates the expression of PD-L1 in cancer cells [47]. T-cell exhaustion is a state of T-cell dysfunction that occurs in many chronic infections and cancers [48]. Scholars have observed that CTLA4 was identified as a crucial negative regulator of the immune system, which transmits an inhibitory signal [49].

There are some limitations in our study. First, basic experiments are needed to verify the results, which were conducted with online public databases. Second, in vivo/vitro experiments are needed to further investigate the potential mechanism of the effect of CPA4 on immune invasion in BLCA.

#### **5. Conclusions**

In conclusion, our study first demonstrated that CPA4 expression increased in BLCA, and univariate and multivariate regression analyses and a nomogram were used to prove that increased CPA4 is correlated with shorter overall survival, which means high risk factors in BLCA patients. Moreover, we illustrated that a high level of CPA4 was positively related to a high pathologic grade; high T, N, and M stages; and the subtype papillary. The immune infiltration in the tumor microenvironment has also been shown to be associated with CPA4. Collectively, this study partially unveiled that CPA4 in BLCA could be regarded

as a potential biomarker for diagnosis and prognosis and may play a special role in immune infiltration.

**Author Contributions:** C.W. designed the original idea, organized the model of TCGA data with relative pictures, and wrote part of the article. Y.Z. was responded for the modification of manuscript and wrote part of the manuscript. Q.X. was responded for the statistics problems and grammar check. M.X. was responsible for the language, bioinformation problems and correcting them. Z.C. and X.Y. was a supporter of this manuscript. Y.H. edited the whole manuscript. All of the authors of this manuscript are responsible for the accuracy, appropriation, and completing any part of this research and agree to open this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All of the data in this manuscript are available and approved by the institution.

**Acknowledgments:** The authors would like to thank all who supported this manuscript as well as provided any useful suggestions and help.

**Conflicts of Interest:** All of the authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

