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A Study of the Correlation Between M2 Macrophages and Lymph Node Metastasis of Colorectal Cancer

Background: Lymph node metastasis is a major prognostic factor of colorectal cancer and an important indicator for individualized treatment. M2 macrophages play a key role in carcinogenesis and tumor development, not only enhancing invasiveness, but also promoting lymph node metastasis. The purpose of this study was to investigate the effect of CD163-positive M2 macrophages on lymph node metastasis in colorectal cancer. Methods: Postoperative lymph node tissues were obtained from 120 patients with colorectal cancer who underwent radical surgery in the First Aliated Hospital of Jinzhou Medical University between December 2019 and May 2020. We detected the expression of the CD163 protein in lymph nodes by immunohistochemistry. Furthermore, the relationship between M2 macrophages identied by this marker and lymph node metastasis were analyzed using the independent sample T-test and Chi-square test. Results: M2 macrophages were increased not only in metastatic lymph nodes, but also in non-metastatic lymph nodes adjacent to the cancer. The M2 macrophage count was higher in patients with macro-metastases than in those with micro-metastases. Conclusions: M2 macrophages represent an important factor for the promotion of lymph node metastasis in colorectal cancer, and may be a potential marker for its prediction. This may offer a new target for the comprehensive treatment of colorectal cancer.

Introduction Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, ranking third globally, with an annual incidence of about 1.2 million people. It results in more than 600,000 deaths annually, with a mortality rate increasing year by year [1,2]. Whether or not local lymph node metastasis (LNM) provides important information on tumor stage, clinical treatment and patient prognosis is unclear. However, the survival rate of colorectal cancer patients with LNM is signi cantly worse than in patients without LNM [3]. Nonetheless, the mechanisms associated with the origination of LNM remain to be fully elucidated because LNM is an extremely complex process. This involves many immune cells and changes in the expression of many different proteins to enable tumor cells to migrate away from the primary lesion, transit and adhere to, and implant in, the new environment. Compared to normal tissue, lymphatic drainage is increased in tumors. Regional lymph node immune tolerance is a necessary condition for the formation of LNM [4]. Studies have shown that M2 macrophages can promote tumor growth, angiogenesis and lymphangiogenesis, immune tolerance and anti-tumor immunity, and that CD163 is a speci c marker for these cells [5,6].
Many studies have found that the presence of large numbers of M2 macrophages in malignant tumor tissues such as gastric cancer, colorectal cancer, breast cancer and cervical cancer is signi cantly correlated with shortened overall survival [7][8][9][10]. However, few studies have reported the relationship between M2 macrophages and LNM. In the present study, the expression of CD163 in lymph node tissue was analyzed to explore the role of M2 macrophages in LNM in colorectal cancer, in order to provide more accurate prognostic information, help to identify new molecular therapeutic targets and understand the molecular mechanism of CRC progression.

Patients and specimens
We collected clinical data and postoperative lymph node specimens of 120 patients with colorectal Group B (one metastatic lymph node was randomly selected from stage and patients, with a total of 51 cases). Group C (one non-metastatic lymph node was randomly selected from stage and patients, with a total of 51 cases). All lymph nodes in group B were divided into two groups according to the tumor size in the lymph node: group D (n = 32) with macro-metastasis (> 2 mm) and group E (n = 19) with micro-

Statistical analysis
Page 4/13 SPSS 24.0 software program and GraphPad Prism 8 were used to analyze the data. Measurement data were expressed as mean ± standard deviation. The independent sample T-test was used to compare the differences in M2 macrophages in lymph node tissues with different clinicopathological parameters, as well as the differences in M2 macrophages between groups. The Chi-square test was used to analyze the association between lymph node metastasis and clinicopathological parameters. Spearman correlation analysis was used for assessing correlations between M2 macrophages and tumor markers. P < 0.05 was considered to indicate a statistically signi cant difference.

Results
Relationships between clinicopathological parameters and lymph node metastasis in colorectal cancer patients As shown in Table 1, LNM in colorectal cancer patients was correlated with the degree of tumor differentiation, depth of invasion, preoperative CEA, CA199 and CA724 levels (P < 0.05), but not with gender, age or tumor diameter (P > 0.05). Expression of CD163 protein in lymph node tissues Immunohistochemistry showed that the expression of CD163 was characterized by the appearance of yellow or brown granules on the cell membrane and in the cytoplasm of M2 macrophages, whereas nuclear staining was slightly weaker. In groups A and C (non-metastatic lymph node tissues), M2 macrophages mainly in ltrated into the medullary sinus ( Fig. 1A-C). In group B (metastatic lymph node tissues), M2 macrophages mainly in ltrated into the peritumoral region and intra-tumoral area (Fig. 1D,E).

Mean numbers of M2 macrophages are different in different patient groups
We found that the mean number of M2 macrophages in group B (26.8 ± 7.4) was signi cantly higher than in group A (14.0 ± 3.4) ( Figure. 2A). However, no clear differences were seen in M2 macrophages between stage I and II patients and the non-metastatic lymph nodes in stage III and IV.Therefore, we counted the M2 macrophages in group A and group C, and found that the mean number in group C (17.4 ± 3.4) was signi cantly higher than in group A (14.0 ± 3.4) ( Fig. 2A). In addition, we also found that the mean number of M2 macrophages in group D (30.0 ± 7.0) was higher than in group E (21.2 ± 3.9) (Figure. Correlations between the mean number of M2 macrophages in lymph node tissues and tumor markers As shown in Table 2, Spearman analysis was used to seek correlations between the mean number of M2 macrophages and preoperative CEA, CA199, and CA724 levels. These were all found to be positively correlated with the mean number of M2 macrophages (P < 0.05).

Discussion
The results of our studies showed that there was greater M2 macrophage in ltration into metastatic than into non-metastatic lymph nodes, and that more in ltration was seen in patients with macro-metastases than in patients with micro-metastases. It is therefore suggested that M2 macrophages are closely associated with LNM in colorectal cancer, and that metastasis is more likely to occur in non-metastatic lymph nodes due to the presence of M2 macrophages. In addition, in patients with metastatic lymph nodes, M2 macrophage in ltration into the non-metastatic lymph nodes was also seen, suggesting that the lymph node microenvironment had changed before metastasis, and that M2 macrophages were involved in that process. Therefore, we speculate that M2 macrophages play an important role in lymph node metastasis of colorectal cancer.
The occurrence and development of tumors is closely related to the tumor microenvironment (TME). Macrophages are generally the most abundant component of immune cells in the TME, making up to 50% of tumor stroma-in ltrating cells [11]. Because macrophages have the characteristics of plasticity and functional diversity, they can be polarized into two types, namely, M1 macrophages (classical activation) and M2 macrophages (alternative activation) depending on changes in the TME [12]. Agents such as lipopolysaccharide (LPS), and cytokines such as Interferon-γ (IFN-γ) or granulocyte colonystimulating factor (G-CSF) in the TME in uence macrophage differentiation along the M1 pathway. In contrast, macrophages exposed to anti-in ammatory cytokines such as IL-4, IL-10, IL-13 or TGF-β can be polarized into M2 macrophages. M1 macrophages produce a variety of different pro-in ammatory cytokines, which mainly kill pathogens and tumor cells, and are useful for immune monitoring. M2 macrophages produce less pro-in ammatory cytokines but a large number of anti-in ammatory cytokines, which mainly play an immunosuppressive role and contribute to immune tolerance [13]. Previous studies on colorectal cancer indicated that a high M2:M1 ratio was closely related to the enhancement of tumor cell invasion [14]. Our study found that the number of M2 macrophages in lymph nodes was signi cantly correlated with the depth of tumor invasion, degree of differentiation, degree of lymph node involvement and clinical TNM stage. This indicates that M2 macrophages are involved in the formation of an immunosuppressive environment in lymph nodes and are one of the important factors leading to lymph node metastasis, consistent with previous research results.
Tumor cell metastasis is a stage of deterioration in disease progression and is associated with poor prognosis. Lymphatic metastasis is the most common form of tumor metastasis in various types of malignancies. We speculate that M2 macrophages may facilitate LNM for the following reasons: On the one hand, several studies [15][16][17] have shown that the number of lymphatic vessels in tumor tissues or metastatic lymph node tissues is signi cantly higher than that in normal tissues and is related to the presence of M2 macrophages. It has been con rmed that M2 macrophages produce VEGF-C which induces lymphangiogenesis. Tacconi et al. [18]reported that VEGF-C binding to VEGFR3 on lymphatic vessels can inhibit the expression of vascular endothelial cadherin (VE-Cad), resulting in damage to the endothelial barrier of lymphatic vessels around the tumor. This is conducive to the entry of tumor cells into lymphatic vessels. VEGF-C also promotes the proliferation and expansion of lymphatic vessels, which can increase the routes for tumor metastasis to lymph nodes [19]. Therefore, M2 macrophages may reshape the lymphatic network to provide favorable conditions for tumor cell metastasis.
On the other hand, it may that the presence of a large number of cytokines such as IL-4, EGF, and IL-6 [20] in the TME is most important. When IL-4 binds to IL-4R, it leads to phosphorylation of JAK-1 and JAK-3, and activates the downstream STAT6 signaling pathway [21]. Choi et al. [22] con rmed that STAT6 phosphorylation increased mRNA expression of M2 macrophage activation markers (FIZZ-1, ARG-1 and CD163), and conversely, that its inhibition reduced the number of M2 macrophages. Yin et al. [23]found that the IL-6/JAK/STAT3 signaling pathway was inhibited during M1 macrophage polarization but activated during M2 macrophage polarization. Thus, a new mechanism of IL-6/JAK/STAT3 signaling pathway regulating macrophage polarization was revealed. In addition, Lian et al. [24] noted that colon cancer cells secrete EGF and bind to EGFR on monocytes, which activates the smad-PI3K-Akt-MTOR pathway and promotes monocyte differentiation into M2 macrophages. Therefore, when the above factors are present in normal lymph nodes, they can promote polarization into M2 macrophages.
Because these M2 macrophages recruit Tregs into the TME by releasing chemokines (such as CCL 22 and CCL 24) [25], high expression of arginase-1,2 (ARG1,2) and indoleamine-2,3-dioxygenase 1 (IDO1) on the surface of M2 macrophages can greatly deplete arginine and tryptophan from the TME, both of which are indispensable for the metabolism of immune cells, and their depletion leads to T cell and NK cell dysfunction [26][27][28]. Therefore, M2 macrophages enhance immunosuppression in lymph nodes to create conditions for tumor cell metastasis.
Our study found that high preoperative levels of CEA, CA199 and CA724 were closely correlated with LNM, and that the preoperative concentration of CEA was positively correlated with the number of M2 macrophages, with a correlation coe cient higher than that of CA199 and CA724. Therefore, we speculated that CEA is also closely related to the differentiation of M2 macrophages.
In this study, we comprehensively analyzed the presence of M2 macrophages in lymph node tissues of patients with different stages of colorectal cancer, and determined the relationships between them. We found that M2 macrophages were higher not only in the metastatic lymph nodes, but also in the remaining non-metastatic lymph nodes in patients with lymph node metastasis. Therefore, we speculated that M2 macrophages are important factors leading to lymph node metastasis in patients with colorectal cancer. Although the speci c molecular mechanism whereby M2 macrophages achieve this in colorectal cancer is not clear, the results of this study provide a foundation for further research. Due to the plasticity of macrophages, a variety of markers should be applied to label M2 macrophages, which may make their quanti cation more accurate.
In conclusion, M2 macrophages are involved in local lymph node immunosuppression and promotion of lymph node metastasis in colorectal cancer. Our ndings provide a reference for understanding lymph node metastasis of this cancer and suggest treatment targets, especially because M2 macrophages are a good predictor of status prior to the occurrence of lymph node metastasis in colorectal cancer.

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 Availability of data and materials The analyzed data sets generated during the study are available from the corresponding author on reasonable request. Inquiries for data access may be sent to the following e-mail address: shifengqiao2020@163.com.