Expression of M2 Macrophages and Regulatory T Cells in Colorectal Cancer and Their Correlation with Lymph Node Metastasis

Background Colorectal cancer (CRC) is among the most common malignancies worldwide. M2 macrophages and regulatory T cells (Tregs) are immunosuppressive cells that can promote tumor progression via inhibiting anti-tumor immunities. However, the signicance and correlation of the two types of cells in colorectal cancer are still inconclusive. The purpose of this study is to detect the number of M2 macrophages and Tregs in colorectal cancer and lymph nodes and to explore the clinical and pathological signicance of their existence. Methods The pathologic specimens and clinical data of 197 patients with Colorectal cancer after radical resection were collected. Immunohistochemical methods were used to detect the expression of M2 macrophages and Tregs in colorectal cancer tissues, adjacent tissues, and lymph node tissues in each group. Results was


Introduction
Colorectal cancer is one of the most common diagnosed malignancies in the world, which is the fourth leading cause of cancer-related death [1]. In the pathogenesis of CRC, immune factors are important factors involved in the occurrence and development of tumors, including the body's tumor immunity and immune surveillance, immune escape effects, etc. Among the many factors that affect the prognosis of CRC, the microenvironment of colorectal cancer and various immune cells in the lymph nodes play a vital role.
Studies have shown that immunosuppressive cells, especially M2 macrophages and Tregs, can promote tumor development[2-6] M2 macrophages and Tregs play an important immunomodulatory role in the promotion of CRC. Studies have shown that M2 macrophages have the ability to induce the formation of Tregs [7], but whether the relationship between the two affects the progression of CRC and lymph node metastasis is still unknown.
We used immunohistochemical methods to detect the expression of M2 macrophages and Tregs in the tissues and lymph nodes of 197 colorectal cancer patients, and then analyzed the correlation between the two, and further explored the mechanism of colorectal cancer invasion and metastasis, so as to better monitor the progress of CRC And prognosis.

Clinical data
The clinical data and postoperative para n specimens of patients undergoing radical resection of colorectal cancer in the Department of Colorectal Surgery of the First A liated Hospital of Jinzhou Medical University from March 2020 to December 2020 were collected. After inclusion and exclusion, a total of 197 cases were collected. The detailed clinical data of all patients are shown in Table 1. Divide the colorectal cancer patients' specimens into two groups of cancer tissues and adjacent tissues (at least 2cm from the edge of the tumor); The lymph node tissue is divided into three groups according to the presence or absence of metastatic lymph nodes: In group A, one lymph node from each patient was randomly selected from the I and II cases, a total of 108 cases; In group B, one lymph node metastasized from each patient was randomly selected from the and cases, a total of 89 cases; In group C, one nonmetastatic lymph node in each patient was randomly selected from the and cases, a total of 89 cases.

Immunohistochemical analysis
Using immunohistochemistry streptomyces antibiotic protein-peroxidase link (SP) method: All specimens were xed in formalin, embedded in para n, and cut into 4 um thick sections. Place the section on a microscope slide, depara nize with xylene and hydrate with a series of graded ethanol, and perform antigen retrieval with ethylenediaminetetraacetic acid buffer (pH 9.0) at sub-boiling temperature for 20 minutes. Tissue sections were incubated with endogenous peroxidase blocker for 10 minutes at room temperature, and blocked with goat serum (

Statistical analysis
Use SPSS26.0 version software program and GraphPad Prism 8 for data analysis, The independent sample t test was used to evaluate the correlation between the expression number of M2 macrophages and Tregs and the pathological characteristics of the tumor, the Mann-Whitney U test was used to analyze and compare the expression differences between groups, and the Spearman correlation analysis was used to evaluate the expression of M2 macrophages and The relationship between Tregs and the correlation between the two and tumor markers. For the above results, P < 0.05 is considered to be statistically signi cant.

The relationship between the number of M2 macrophages and Tregs expression and the clinical characteristics of tumors
In order to clarify the in ltration characteristics of M2 macrophages and Tregs cells in colorectal cancer, we performed statistical analysis on the number of M2 macrophages and Tregs and clinicopathological parameters in 197 cases of colorectal cancer tissues (see Table 2) .It was found that the increase in the expression of CD163 + M2 and Foxp3 + tregs was related to the TNM staging, depth of invasion, lymph node metastasis, vascular tumor thrombosis, nerve invasion and distant metastasis of colorectal cancer, and the difference was statistically signi cant (p < 0.05); It has nothing to do with the patient's age, gender, tumor diameter, tumor location and degree of differentiation, and the difference is not statistically signi cant (p > 0.05). The main feature of CD163 is that yellow or brown medium particles appear on the membrane of M2 macrophages, while the staining in the cell matrix is weak (Fig. 1A-1D) ;The main feature of Foxp3 is the appearance of brown-yellow particles on the nucleus of Treg cells, which are distributed in various tissues of CRC patients ( Fig. 1E-1H). Cancer tissue (Fig. 1B, 1F) stained signi cantly more than adjacent tissues (Fig. 1A, 1E), and metastatic lymph node tissue (

Correlation analysis between M2 macrophages and Tregs in CRC and lymph nodes
Through comparative analysis (Fig. 1), we found that M2 type macrophages and Tregs are abundantly distributed in CRC and lymph nodes. In order to verify whether there is a certain correlation between the two in the development of colorectal cancer and lymph node metastasis, we used Spearman correlation analysis to nd: (Fig. 2a and 2b

Differences in the expression of M2 macrophages and Tregs in various tissues
We observed (Figs. 3A and 3B) that the medians of M2 macrophages and Tregs in colorectal cancer tissues were 19/HP and 23/HP, respectively; signi cantly higher than 7/HP and 10/HP in adjacent tissues HP, (P < 0.05). It is worth noting that in the adjacent tissues (Fig. 3C), the median of M2 macrophages stage + was 5/HP lower than 9/HP in stage + , (P < 0.05); however, ( Fig. 3D) The median of Tregs in stage + was 10/HP higher than 9/HP in stage + , (P < 0.05). In order to further analyze the prognosis of M2 macrophages and Tregs in colorectal cancer patients, we counted their lymph nodes, It was found (Figs. 3E and 3F) that the medians of M2 macrophages and Tregs in group A were 9/HP and 10/HP, respectively, signi cantly lower than 16/HP and 18/HP in group B, (P < 0.05); however; It is unclear whether the number of M2 macrophages and Tregs in the lymph nodes of patients with stage I + II is different from the number of M2 macrophages and Tregs in the non-metastatic lymph nodes of patients with stage III + IV. Therefore, we counted the M2 macrophages in group A and C, and found ( Fig. 3G) that the median of M2 macrophages in group A was 9/HP lower than 10/HP in group C, (P < 0.05). (Fig. 3H) The median of Tregs in group A and group C were both 10/HP, (P > 0.05).
The Mann-Whitney U test was used to analyze the statistical differences between the two groups.
( Figures A and B) M2 macrophages and Tregs expressed in colorectal cancer tissues were signi cantly higher than those in adjacent tissues, and the differences were statistically signi cant (***P < 0.001;***P < 0.001); In paracancerous tissues ( Figure C) the number of M2 macrophages expressed in stage + was lower than that in stage + , ( Figure D) the number of expressions of Tregs in stage + was higher than that in stage + . The difference was both There is statistical signi cance (***P < 0.001; **P < 0.01).
( Figures E and F) The expression of M2 macrophages and Tregs in group A was signi cantly lower than that in group B, and the differences were statistically signi cant (***P < 0.001; ***P < 0.001); in nonmetastatic lymph nodes ( Figure G) The expression of M2 macrophages in group A was lower than that in group C, and the difference was statistically signi cant (*P < 0.05). ( Figure H) There was no signi cant difference in Tregs between group A and group C. No statistical signi cance (P > 0.05).

Correlation analysis between the number of M2 macrophages and Tregs in CRC and tumor markers
In order to analyze the in uence of M2 macrophages and Tregs on the condition and prognosis of CRC patients, we used Spearman correlation analysis to evaluate the correlation between the number of M2 macrophages and Tregs and the preoperative CEA, CA199, and CA724 concentrations. as shown in Table  3, It was found that the number of M2 macrophages was positively correlated with the concentration of CEA and CA199 before surgery (P < 0.001; P < 0.05); it has nothing to do with the concentration of CA724 before surgery (P > 0.05). The number of Tregs is positively correlated with the preoperative CEA concentration (P < 0.001); it has nothing to do with the preoperative CA199 and CA724 concentrations (P > 0.05). surrounding lymph nodes. Mainly study the expression of M2 macrophages and Tregs in the microenvironment of colorectal cancer and the expression of each group of lymph nodes. It was found that the number of expressions of M2 macrophages and Tregs was signi cantly reduced compared with cancer tissues. The number of M2 macrophages of stage + in the adjacent tissues was signi cantly more than that of stage + , but the number of stage + in Tregs was less than that of stage + . In the lymph nodes, we found that compared with the metastatic lymph node tissue, the number of the two types of cells in the non-metastatic nodes was signi cantly reduced. In non-metastatic lymph nodes, the number of M2 macrophages in stage + was signi cantly higher than that in stage + , but there was no signi cant difference in the number of Tregs. In addition, our research also found that the number of expressions of these two types of cells in cancer tissues and lymph node tissues is signi cantly positively correlated.
Macrophages have functional and phenotypic plasticity that can be categorized into M1 and M2 macrophages [9]. M1 macrophages and M2 macrophages play opposite roles in the tumor microenvironment. The increase in the ratio of M1 to M2 in colorectal cancer is closely related to the enhancement of tumor cell invasion [10,11]. Tumor-derived cells secrete granulocyte macrophage colonystimulating factor, monocyte chemotactic protein, and so on. These factors are related to the nonclassical activation pathway of macrophages, which promotes the differentiation of monocytes into M2 macrophages, which increases their expression in tumor tissues [12]. In our study, the expression level of M2 macrophages in tumor tissues is signi cantly up-regulated, and it is closely related to tumor TMN staging, lymph node metastasis and depth of invasion. These results indicated that M2 macrophages are involved in the formation of immunosuppressive tumor microenvironment,which is one of the important factors in tumor microenvironments that cause the poor prognosis of patients. Lian et al. [13] 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. The number of M2 macrophages of stage + in the adjacent tissues is signi cantly more than that of stage + . In our analysis, with the proliferation of the tumour leads to increased metabolism and uneven vascularization around it, so the lack of blood vessels in the tumor area causes tumor hypoxia. The main cellular response to hypoxia is mediated by HIF-1α and HIF-2α. The above factors can increase the expression of miRNAs through the PI3K/AKT/mTOR pathway and promote the polarization of M2 cells [14][15][16]. In the lymph node tissue, it merits our attention. The number of M2 macrophages in non-metastatic lymph nodes of stage + was signi cantly higher than that of stage + . In the lymph node tissue deserves our attention that the number of M2 macrophages in the non-metastatic lymph nodes of stage + is signi cantly higher than that in stage + . It was suggested that in patients with metastatic lymph nodes, there are morein ltration of M2 macrophages in the part where tumor metastasis has not occurred, indicating that the lymph node microenvironment has changed before metastasis, and M2 macrophages are involved, which is the same as our previous research results [17]. Therefore, we speculate that M2 macrophages play an important role in metastasis of lymph node colorectal cancer, and it indicated that poor prognosis. Tacconi C et al.
[18] pointed out that VEGF-C can promote the proliferation and expansion of lymphatic vessels, thereby increasing the way for the metastatic spread of tumor cells to lymph nodes. Thus, M2 macrophages may change the tumor microenvironment and promote colorectal cancer lymph node metastasis [17,19].
So far, the role of Treg cells in CRC is controversial. The main reason for our analysis is that Tregs have duality in CRC. This duality may be affected by many factors, including the role of Tregs in the occurrence and development of CRC is not clear, such as the function and distribution in the development of the disease. And changes in the number, including external factors (such as treatment) interference. In order to study the potential functions of Tregs in CRC. We analyzed the correlation between Tregs and clinicopathological characteristics, and divide CRC into early stage ( + stage) and late stage ( + stage) for discussion. We found that Tregs in CRC are closely related to TNM staging, lymph node metastasis, and distant metastasis. TNM staging and lymph node metastasis are important indicators for judging the prognosis and survival time of tumor patients. Thus, our results indicate that Tregs in ltration is related to metastasis and poor prognosis of colorectal cancer Macrophage-derived chemokine CCL22, which can bind to the CCR4 receptor highly expressed on the surface of FOXP3 + Tregs, thereby helping to recruit Treg cells to tumor tissues [2,20]. It is noteworthy that we found that the number of Tregs in the late stage (stage + ) adjacent to the cancer is less than that in the early stage (stage + ). We analyzed that the dysregulation of colonic in ammatory response is an important inducement for the development of CRC. The increased in ammation in adjacent tissues in the early stage of CRC promotes the continuous accumulation of Treg cells to inhibit in ammation [21,22]. Märkl et al. [23] analyzed the specimens of 136 patients with early-stage (stage I, II) colon cancer and found that Tregs in ltrated more cancerous tissues than adjacent tissues, and high Tregs values in adjacent tissues suggested a better prognosis.
In our study, we found that there is a certain correlation between M2 macrophages and Tregs in CRC and lymph nodes. The possible explanation is that M2 macrophages secrete immune suppressive cytokines and chemokines. These cytokines and chemokines participate in the recruitment of lymphocytes and stimulate them to develop into Tregs [2,24]. In addition, Tregs produce high levels of IL-10, IL-32 and TGFβ, which further inhibit the anti-tumor in ammatory response and stimulate M2 macrophages to increase the production of cytokines and chemokines, thereby being able to recruit additional Tregs [25]. Studies have pointed out [26] that M2 macrophages and Tregs have a synergistic effect in promoting the proliferation, tumor angiogenesis ,and metastasis of ovarian cancer. Sun et al. [27] studied 65 patients with laryngeal squamous cell carcinoma (LSCC) and found that Tregs and M2 macrophages in LSCC were positively correlated with each other, and proved that the two formed a positive feedback loop. Therefore, we speculate that M2 macrophages in CRC may have the ability to induce the formation of Tregs, which will 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. Because our experiment is relatively limited, the correlation between M2 macrophages and Tregs and its speci c mechanism need to be further studied. In addition, studies have pointed out [28,29] that CD163 can be used as a potential prognostic biomarker for CRC patients, and Foxp3 can directly affect the prognosis of CRC patients. In order to further verify the impact of the two on the monitoring and prognosis of CRC patients, our study analyzed the correlation between the levels of CEA, CA199 and CA724 before surgery and the number of M2 macrophages and Tregs. CEA level is closely related to the number of M2 macrophages and Tregs, and the correlation coe cient is higher than CA199 and CA724 levels. Therefore, we speculate that M2 macrophages and Tregs have a close relationship with CEA, which is expected to become an important observation index for monitoring the condition of colorectal cancer and judging the prognosis.
In summary, M2 macrophages participate in the formation of lymph node immunosuppressive environment and may promote the development of CRC and lymph node metastasis together with Tregs. Our results provide some insights into the role of M2 macrophages and Tregs in colorectal cancer and its lymph node metastasis. M2 macrophages and Tregs are up-regulated in CRC, which is expected to be an effective indicator for monitoring the condition of CRC and judging the prognosis.

Declarations
Ethics approval and consent to participate The study was conducted in accordance with the Ethics Committee of the First A liated Hospital of Jinzhou Medical University and the 1964 Helsinki Declaration. Informed consent was obtained from all participants included in the study.

Consent for publication
Not applicable The main feature of CD163 is that yellow or brown medium particles appear on the membrane of M2 macrophages, while the staining in the cell matrix is weak ( Figure 1A-1D) ;The main feature of Foxp3 is the appearance of brown-yellow particles on the nucleus of Treg cells, which are distributed in various tissues of CRC patients ( Figure 1E-1H). Cancer tissue ( Figure 1B, 1F) stained signi cantly more than adjacent tissues ( Figure 1A, 1E), and metastatic lymph node tissue ( Figure 1C, 1G) stained signi cantly more than non-metastatic tissue ( Figure 1D, 1H)

Figure 2
In order to verify whether there is a certain correlation between the two in the development of colorectal cancer and lymph node metastasis, we used Spearman correlation analysis to nd: (Figure 2a and 2b) M2 type macrophages and Tregs are both present in CRC and lymph nodes. Positive correlation (r=0.269, P<0.001; r=0.541, p<0.001).

Figure 3
We observed ( Figures 3A and 3B) that the medians of M2 macrophages and Tregs in colorectal cancer tissues were 19/HP and 23/HP, respectively; signi cantly higher than 7/HP and 10/HP in adjacent tissues HP, (P<0.05). It is worth noting that in the adjacent tissues ( Figure 3C), the median of M2 macrophages stage + was 5/HP lower than 9/HP in stage + , (P<0.05); however, ( Figure 3D) The median of Tregs in stage + was 10/HP higher than 9/HP in stage + , (P<0.05). In order to further analyze the prognosis of M2 macrophages and Tregs in colorectal cancer patients, we counted their lymph nodes, It was found ( Figures 3E and 3F) that the medians of M2 macrophages and Tregs in group A were 9/HP and 10/HP, respectively, signi cantly lower than 16/HP and 18/HP in group B, (P<0.05); however; It is unclear whether the number of M2 macrophages and Tregs in the lymph nodes of patients with stage I+II is different from the number of M2 macrophages and Tregs in the non-metastatic lymph nodes of patients with stage III+IV. Therefore, we counted the M2 macrophages in group A and C, and found ( Figure 3G) that the median of M2 macrophages in group A was 9/HP lower than 10/HP in group C, (P< 0.05). ( Figure 3H) The median of Tregs in group A and group C were both 10/HP, (P>0.05).