Immune cells play a crucial role in the development of tumors in the tumor microenvironment . In this study, immunohistochemical staining was performed on paracancerous tissue, cancerous tissue and its surrounding lymph node tissue of 197 patients with colorectal cancer. The mean number of M2 macrophages and Tregs was found to be significantly higher in cancer tissue than in paracancerous tissue. The mean number of M2 macrophages was higher in stage III+IV than in stage I+II in paraneoplastic tissue, yet the mean number in stage III+IV Tregs was lower than in stage I+II. In lymph node tissue, the mean number of M2 macrophages and Tregs was significantly higher in metastatic lymph nodes. In addition, M2 macrophages also significantly increase in the non-metastatic lymph node tissue of patients with metastatic lymph nodes. Notably, our study found a positive correlation between M2 macrophages and Tregs in cancer tissues and lymph node tissues.
M2 macrophages are a kind of activated macrophages induced by Th2 cytokines interleukin (IL-4), interleukin-10 (IL-10) and interleukin 13 (IL-13). It promotes tumor angiogenesis and leads to tumor progression by suppressing T-cell-mediated anti-tumor immune responses [18, 19]. Our study found that the mean number of M2 macrophages was significantly higher in cancer tissue than in paracancerous tissue. The mean number of M2 macrophages was significantly greater in stage III+IV than in stage I+II in paraneoplastic tissue. With the rapid growth of tumor tissue leads to increased metabolism and uneven vascularization around it. Therefore, the lack of blood vessels in the tumor area causes tumor hypoxia. Hypoxia causes tumor cells to release factors such as HIF-1α and HIF-2α, which can increase the expression of miRNAs through the PI3K/AKT/mTOR pathway and promote the polarization of M2 cells [20-22]. Lian et al also noted that colon cancer cells secrete EGF, which can bind to EGFR on monocytes, activate the smad-PI3K-Akt-MTOR pathway and promote the differentiation of monocytes into M2 macrophages. We also found higher mean numbers of M2 macrophages in tumor tissue in the presence of more severe vascular cancer thrombosis, nerve invasion, and distant metastases. Meanwhile, the increase in the mean number of M2 macrophages was closely associated with the TMN stage, lymph node metastasis and depth of infiltration of the tumor. It indicates that M2 macrophages are involved in the formation of the immunosuppressive microenvironment in cancer tissues, which may be one of the important factors leading to the occurrence of colorectal cancer. The epithelial growth factor (EGF) secreted by M2 macrophages activates the factor receptor (EGFR) on tumor cells, which in turn up-regulates the VEGF/VEGFR signals in surrounding tumor cells to support tumor cell proliferation and migration . Our study found that in lymph node tissue, the mean number of M2 macrophages not only increased significantly in metastatic lymph node tissue, but also in non-metastatic lymph nodes in patients with metastatic lymph nodes. It suggests that in patients with metastatic lymph nodes, there is more M2 macrophage infiltration in the part where tumor metastasis has not occurred. It shows that the microenvironment of the lymph node has changed before metastasis, and M2 macrophages are involved in it. This is the same result as our previous study . Therefore, we speculate that M2 macrophages play an important role in the process of lymph node metastasis in colorectal cancer, which indicates a poor prognosis. Tacconi C et al  stated that VEGF-C promotes proliferation and expansion of lymphatic vessels, thereby increasing the pathway of tumor metastasis to lymph nodes. Therefore, M2 macrophages may change the tumor microenvironment and promote the lymph node metastasis of colorectal cancer [16, 26].
Tregs are a subset of T lymphocytes with immunosuppressive effects, which can induce tumor immune escape by inhibiting effector T cells, and induce tumor growth and immune tolerance. To investigate the changes in Tregs during CRC progression, we examined the changes in Tregs in paracancerous, cancerous and lymph node tissues. CRC was divided into early (stage I+II) and late (stage III+IV) stages for analysis. It was found that Tregs in cancer tissues were significantly higher than those in paracancerous tissues, but in the late phases, the paracancerous tissues were lower than those in the early stage. We analyze tumor cells and microenvironmental macrophages to produce chemokine CCL22, which can bind to the CCR4 receptor highly expressed on the surface of FOXP3 + Tregs. This helps recruit Treg cells to tumor tissues[28, 29]. In addition, the dysregulation of colon inflammation is an important cause for the development of CRC. The increased inflammation of paracancerous tissues in the early stage of CRC promotes the continuous accumulation of Treg cells to inhibit inflammation [30, 31]. Märkl et al analyzed 136 specimens of patients with early stage (stage I and II) colon cancer. It was found that Tregs infiltrated in cancerous tissues higher than in paracancerous tissues, and high Tregs in paracancerous tissues indicated a better prognosis, which just confirms our research. In addition, we analyzed the correlation between Tregs and clinicopathological characteristics. It is found that Tregs in CRC are closely related to TNM staging, lymph node metastasis and distant metastasis. TNM stage and lymph node metastasis are important indicators of prognosis and survival time of tumor patients. Thus, our results suggest that Tregs infiltration is associated with CRC metastasis and poor prognosis.
Our research found that there is a certain correlation between M2 macrophages and Tregs in cancer tissues and lymph node tissues. M2 type macrophages can produce immunosuppressive cytokines and chemokines. These cytokines and chemokines are involved in recruiting lymphocytes and stimulating their development into Tregs [28, 33]. In addition, Tregs produce high levels of IL-10, IL-32 and TGF-β, which further inhibit the anti-tumor inflammatory response and stimulate M2 macrophages to increase the production of cytokines and chemokines, thereby being able to recruit additional Tregs. Studies have pointed out  M2 macrophages and Tregs have synergistic effects in promoting proliferation, tumor angiogenesis and metastasis in ovarian cancer. Sun et al  studied 65 patients with laryngeal squamous cell carcinoma (LSCC) and found that Tregs and M2 type macrophages were positively correlated with each other in LSCC, and demonstrated that the two formed a positive feedback loop. In addition, we also preliminarily confirmed that M2 macrophages may induce the production of regulatory T cells by activating the TGF-β/Smad signaling pathway. Therefore, we speculate that M2 macrophages in CRC may have the ability to induce the formation of Tregs and increase the expression of Tregs in tumor tissues. The interaction between the two may change the tumor microenvironment and promote the development of CRC and lymph node metastasis.
The specific mechanism of action of M2 macrophages and Tregs in CRC, especially the molecular mechanism of interaction in local lymph nodes, will be the next focus of our group's research. In addition, studies have pointed out [37, 38] that CD163 can be used as a potential prognostic biomarker for CRC patients, and Foxp3 can directly affect the prognosis of CRC patients. To further verify the impact of both on the monitoring and prognosis of CRC patients, our study analyzed the preoperative CEA, CA199 and CA724 levels in correlation with the number of M2 macrophages and Tregs. It was found that the level of CEA was significantly positively correlated with the mean number of M2 macrophages and Tregs. This is consistent with our previous findings . We know that CEA plays an important role in monitoring the recurrence and metastasis of colorectal cancer. Therefore, we speculate that CEA has an important influence on the distribution of M2 macrophages and Tregs in colon cancer.
In summary, the presence of M2 macrophages and Tregs in colorectal cancer patients is an important indicator of colorectal cancer progression and lymph node metastasis. Our results provide some insights for M2 macrophages and Tregs in the progression of colorectal cancer and its lymph node metastasis. There is a certain correlation between the two. The relationship between M2 macrophages and Tregs may affect the progression of colorectal cancer and lymph node metastasis. This could be a potential future target for immunotherapy and provide important clinical value for colorectal cancer diagnosis, treatment, and prognosis.