At present, the role of viruses in cancer has attracted enough attention. The colon and rectum itself has a very complex intestinal microbial system, and some scholars gradually put forward the view of the effect of the interaction between microorganisms and epithelium on the carcinogenesis of CRC (14).
In our study, EBV showed a higher infection rate in colorectal cancer than other carcinogenic viruses, consistent with previous studies(15). However, some studies have shown that other viruses have a higher expression rate in colorectal cancer such as HPV(16). These divergent results may be attributed to differences in geographical and sample sources. Previous studies have demonstrated the positive expression of CMV and JCV in colorectal cancer(17, 18), yet these viruses were not detected in the samples obtained from our region.
EBV belongs to the family of human herpesviruses(19, 20). The virus is spread through saliva and latently infects B cells in the body for a long time(21). EBV is involved in the development of several cancers, mainly lymphomas of B-cell origin, in addition to epithelial cancers including nasopharyngeal carcinoma (NPC) and gastric cancer. At present, there is clear evidence that EBV is involved in the occurrence and development of gastric cancer, mainly manifested by PI3K mutation, lack of p53 mutation, and high CpG methylation(22). According to previous reports, the infection rate of EBV in colorectal cancer ranges from 20–50%(23–25). However, some studies have failed to detect the presence of EBV in human CRC samples(26, 27). Given that colorectal and gastric tissues share similar histological structures, it is reasonable to question whether EBV is also involved in the development of colorectal cancer.
The analysis of the function and composition of different immune cells and immune components of EBVaCRC is of great value for further understanding the unique tumor immune microenvironment of EBVaCRC patients. Our study showed a significantly higher density of immune cells in the tumor immune microenvironment in EBVaCRC than in EBVnCRC. These results suggest that the immune response is more closely associated with EBVaCRC. This immune response may be triggered by EBV in tumor cells or lymphocytes, resulting in dense lymphocyte infiltration. These immune cells can recognize and attack tumor cells infected with the virus, causing the tumor cells to die. However, in some cases, tumor cells can evade the attack of immune cells through a variety of mechanisms, leading to the growth and spread of tumor cells. Thus, viral infection and immune cell infiltration play an important role in tumorigenesis and development. T cells are the most abundant and characteristic immune cells in TIME, which are divided into cytotoxic T cells, helper T cells, suppressor T cells and NK cells(28). In our study, CD3+ T cells and CD8+ cytotoxic T lymphocytes (CTL) have higher expression in EBVaCRC. T cells participate in tumorigenesis through different mechanisms, especially CD8+ CTL, which seems to play an anti-tumor role in EBV-related cancers(29). Conversely, the inhibitory function of regulatory T cells (Tregs) may prevent CTL from responding effectively to cancer cells, thereby promoting tumor development(30). FOXP3+ Tregs has a higher infiltration density in EBVaCRC. although it may be involved in immune escape of tumor cells, studies have confirmed that high-density FOXP3+ Tregs infiltration is associated with better survival(31), so we prefer the anti-tumor effect played by Tregs in EBVaCRC. B lymphocytes are involved in immune regulation mainly by producing immunoglobulins in response to antigenic stimulation(32). T cells and B cells can also interact to enhance local immune activation. Higher infiltration of CD20+ B cells in EBVaCRC suggests that EBV promotes a stronger anti-tumor immune environment. NK cells are the effector cells of the immune system. It has been shown that NK cell levels are lower in CRC tissues compared to NK cells in adjacent normal tissues, suggesting that less NK cell infiltration may be a mechanism for immune escape(33). In contrast, EBVaCRC expressed stronger CD56+ NK cell expression more strongly in our study suggesting that EBV may inhibit the immune escape mechanism of CRC through NK cells. In addition to lymphocytes, macrophages are also the main cells that make up TIME. Tumor-associated macrophages (TAM) are considered to be the most abundant immune cells in solid tumor tissues(34). In colorectal cancer, TAM is concentrated in the high incidence of epithelial mesenchymal transformation (EMT), TAM promotes the growth and invasion of colon cancer cells through EMT remodeling(35). In addition, a large amount of TAM is beneficial to the prognosis of colorectal cancer(36). In our study, the density of CD68+ TAM increased significantly in patients with EBVaCRC, which was consistent with previous studies on EBV-associated gastric cancer (EBVaGC)(37). However, there was no difference in M2-TAM in EBVaCRC. It is reported that EBV-associated gastric cancer has a higher ratio of M1/M2 macrophages(38). Another study reported that the decrease of M2-TAM ratio in EBVaGC was associated with poor prognosis(39). Therefore, further analysis of the phenotypic changes of macrophages in EBVaGC will help to clarify the mechanism of macrophage infiltration and obtain a better prognosis.
Anti-PD-(L)1 antibodies and Anti-CTLA-4 antibodies are the most representative immune checkpoint inhibitors (ICIs) in recent years(40). CRC patients, especially those with deficient mismatch repair (dMMR) / microsatellite instability-high (MSI-H) tumors, have a high sensitivity to ICIs(41). Our study found that PD-L1 and PD-1 immune cell infiltration occurred more frequently in EBVaCRC. Moreover, both PD-1 and PD-L1 immune cells were present in the matrix of PD-L1 positive cancer, while CTLA-4 immune cells did not show differential expression in EBVaCRC. This suggests that anti-PD-1/PD-L1 therapy may have a superior effect in treating EBVaCRC. In our study, there was no correlation between EBV infection and prognosis, which was also reflected in previous studies(42). However, many studies have concluded that the survival rate of EBV-positive gastric cancer is improved, but the mechanism is still uncertain, which may be due to the extensive infiltration of cytotoxic CD8+ lymphocytes into the tumor nest and promote the eradication of EBV-positive malignant cells(37). Although our study did not observe a better survival rate, EBVaCRC showed a lower clinical stage. After that, we will collect a larger sample size to evaluate the prognostic value in more aspects.
However, this study is retrospective and requires a larger sample size and a multicenter prospective study. TIME is a complex landscape, which is not only closely related to the growth and development of colorectal cancer, but also affects the treatment and prognosis of patients with colorectal cancer. Therefore, an in-depth study of TIME in order to find better treatments for CRC is currently a very important goal to explore.