To determine the differential expression of CLDN10 in both gastric cancers and various other cancer types, we used the TIMER 2.0 database to investigate the CLDN10 expression in human cancers. To more accurately assess CLDN10 expression in gastric cancer, we used the Oncomine and UALCAN database to verify CLDN10 expression levels. As shown in Fig.1A, CLDN10 expression was significantly down-regulated in bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), glioblastoma multiforme (GBM), head and neck cancer (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), prostate adenocarcinoma (PRAD), stomach adenocarcinoma (STAD) and uterine corpus endometrial carcinoma (UCEC) compared to adjacent normal tissues. However, CLDN10 expression was higher in cholangiocarcinoma (CHOL), adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and thyroid carcinoma (THCA) than in the adjacent normal tissues. Furthermore, data from Oncomine (Fig.1B) and UALCAN (Fig.1C) confirmed that CLDN10 was down-expressed in gastric cancer.
Prognostic and clinicopathological significance of CLDN10 expression in gastric cancer
In order to better understand potential correlations of CLDN10 expression in gastric cancer, we used Kaplan-Meier Plotter and GEPIA2.0 database to investigate the prognosis and clinicopathological significance of CLDN10. From the Kaplan-Meier Plotter, the results showed that overexpression of CLDN10 corresponded with an unfavourable prognosis for gastric cancer patients (Fig.2A, OS HR=1.62, 95% CI=1.36–1.92, p=3.7e-8; Fig.2C, PFS HR= 1.48, 95% CI=1.21–1.81, p= 0.00011). Moreover, we used GEPIA2.0 to verify this association and got similar results (Fig.2B, OS HR=1.3, p=0.064; Fig.2D, DFS HR= 1.7, p= 0.0055).
To further understand the in-depth significance and underlying mechanism of the differential expression of CLDN10 in gastric cancer tissues and adjacent normal tissues, we explored the interrelation between CLDN10 expression levels and the clinicopathological characteristics of gastric cancer using the Kaplan-Meier Plotter database. High CLDN10 expression was associated with poor OS and PFS in both male and female gastric cancer patients (Table 1; p<0.05). TNM staging is a widely accepted standard for cancer classification, which can predict the prognosis of gastric cancer. It means the invasive depth of primary tumor (T), regional lymph node (N), and distant metastasis (M) [25, 26]. Regardless of the clinical stage, high CLDN10 expression was related to poorer OS, but only stage 2 was related to PFS. The expression of CLDN10 in T2 gastric cancer patients was associated with OS and PFS (OS p=0.03; PFS p=0.027), and the value of HR has a decreasing trend with increasing tumour development. A statistically significant correlation between CLDN10 expression and gastric cancer prognosis was observed in stage N0\N1\N3\N1+2+3 patients but not N2. The expression of CLDN10 was statistically correlated with OS and PFS in stage M0 gastric cancer, while only OS in stage M1. According to the Lauren classification, it was observed that the expression of CLDN10 in patients with intestinal-type gastric cancer was related to OS and PFS. In mixed-type group, it was associated with OS but not with PFS, while in diffuse-type gastric cancer, no statistically significant association was observed. In terms of treatment, the expression of CLDN10 in gastric cancer patients who have only undergone surgery was related to OS and PFS (OS p=0.017; PFS p=0.044). However, it was not significantly related to the prognosis of patients who had received 5-FU-based adjuvant chemotherapy. In addition, CLDN10 expression was associated with OS and PFS in HER2-negative gastric cancer patients (OS p=2.1e-07; PFS p=0.00033), but not with HER2- positive patients.
CLDN10 expression and somatic CNV is correlated with immune cell infiltration in gastric cancer
The gene module in the TIMER 2.0 database was used to investigate the relationship between CLDN10 expression and immune cell infiltration in STAD (Fig. 3). It was seen that CLDN10 expression level was negatively related to the infiltrating levels of T cell CD8+ central memory, T cell CD8+ effector memory, T cell CD4+ memory, T cell CD4+ Th1, T cell CD4+ Th2, T cell regulatory (Tregs), B cell plasma, macrophage M1, macrophage M2, plasmacytoid dendritic cell, and common lymphoid progenitor in STAD, while positively correlated with cancer associated fibroblast (CAF), endothelial cell, eosinophil, granulocyte−monocyte progenitor and hematopoietic stem cell (P<0.05). Most of the immune cells mentioned above were negatively associated with CLDN10 level, which meant that there were more infiltrating immune cells in the tumor microenvironment in gastric cancer patients with low expression of CLDN10. This could partly explain why a decline in CLDN10 was associated with better outcomes. Moreover, we analyzed and compared the immune infiltration levels among gastric cancer patients with the presence of different somatic copy number alterations (SCNA) for CLDN10 (Fig. 4). It was observed that high amplification in STAD was negatively associated with the infiltration of CD8+ T cells, CD4+ T cells, B cells, neutrophil, macrophages M1, neutrophils, myeloid dendritic cells, NK cells and T cell follicular helper (P<0.05). Furthermore, arm-level gain was also inversely correlated with the above immune cell infiltration (P<0.05), except for macrophage M1 and T cell follicular helper. However, only the level of NK cell infiltration was significantly correlated with the arm-level deletion (P<0.05). These results showed us that SCNA of CLDN10 may affect the level of immune cell infiltration.
Correlation analyses between immune marker genes and CLDN10 expression
The TIMER 2.0 database interrogation concentrated on the correlations between CLDN10 in STAD and related marker genes of infiltrating immune cells (Table 2). After purity-related adjustments, it was shown that CLDN10 levels were notably related to some marker genes in a variety of immune cells, including T cells (general), M1 macrophages, neutrophils, natural killer cells, dendritic cells (DCs) and different functional T cells, such as Tregs, Th1, Tfh, Th17 and exhausted T cells. It was of note that the levels of the main markers of B cell (CD19, CD79A), Tfh (BCL6, IL21) and exhausted T cells (PD1, CTLA4, LAG3, TIM3, GZMB) were all significantly correlated with CLDN10 levels in STAD. Tfh could promote the differentiation of B cells into memory B cells and the survival of plasma cells by providing signals to B cells, which played an important role in humoral immunity [27, 28]. In exhausted T cells, these gene markers (PD1, CTLA4, LAG3, TIM3 and GZMB) were consistently significantly correlated with the expression of CLDN10, which further suggested that the expression level of CLDN10 in gastric cancer is related to immune infiltration.