Sage Journals: Discover world-class research

Abstract

Background

M2 macrophages and regulatory T cells (Tregs) can promote tumors and development by inhibiting the anti-tumor immune response. This study investigated the effect of CD163-positive M2 macrophages and Foxp3-positive Tregs in the progression of colorectal cancer and lymph node metastasis. It also investigated the correlation between M2 macrophages and Tregs.

Methods

Postoperative tissue specimens and clinical data were collected from 197 patients with colorectal cancer who underwent initial surgical treatment in The Second Ward of Colorectal Surgery of the First Affiliated Hospital of Jinzhou Medical University from March 2020 to December 2020. Immunohistochemical methods were used to detect the expression levels of CD163 protein-labeled M2 macrophages and Foxp3 protein-labeled Tregs in colorectal cancer tissues, matched paracancer tissues, and lymph node tissues. The correlation between CD163 and Foxp3 in cancer tissues and lymph node tissues were analyzed, as well as the relationship between clinicopathological characteristics and preoperative tumor markers.

Results

M2 macrophages and Tregs were importantly positively correlated in cancer and lymph node tissues, which significantly increased in cancer and metastatic lymph node tissues. Interestingly, M2 macrophages in non-metastatic lymph nodes also increased significantly in patients with metastatic lymph nodes. In addition, both CD163 and Foxp3 were upregulated with increasing tumor node metastasis stage, depth of infiltration, and lymphatic metastasis; and both were positively correlated with carcinoembryonic antigen.

Conclusion

CD163 may be a good predictor of pre-metastatic status of colorectal cancer lymph nodes. carcinoembryonic antigen affects the distribution of M2 macrophages and Tregs in colorectal cancer. There is a certain correlation between the two types of cells. It is possible that M2 macrophages, together with suppressor Tregs cells, promote an immunosuppressive environment.

Keywords

Colorectal cancer lymph node metastasis M2 macrophages regulatory T cells CD163 Foxp3

Introduction

Colorectal cancer (CRC) is one of the most common malignancies in the world and is the fourth leading cause of death among cancer patients.¹ It is a complex cancerous disease involving multiple cellular pathways starting from the initial process of tumor transformation to metastasis.² The tumor microenvironment consists mainly of immune cells and extracellular matrix, with specific immune cells playing an even more critical role in tumor progression.³

Tumor-associated macrophages (TAM) are one of the major tumor-infiltrating immune cell types and can be differentiated into “classically activated” M1 macrophages and “alternative activated” M2 macrophages.⁴ M1 macrophages and M2 macrophages play opposite roles in the tumor microenvironment. Increased M2/M1 ratio in CRC is closely associated with enhanced tumor cell invasion.^5,6 In solid tumors, including CRC, M2 macrophages are generally associated with poorer clinical outcomes.⁷ CD163 is a member of the cysteine-rich superfamily of scavenger receptors and is restricted to the monocyte/macrophage lineage. It is currently regarded as the most specific marker of M2 macrophages.⁸

Regulatory T cells (Tregs) are another type of immune cell. In malignancies, Tregs promote tumor progression by suppressing effective anti-tumor immunity.⁹ Tregs mostly have Foxp3 positive expression, and studies have shown that Foxp3 is the molecule that confers the suppressive activity of Tregs This transcription factor plays a key role in the immunosuppressive effects of Tregs and is closely related to the development and the performance of immunomodulatory functions of Tregs.^10,11 M2 macrophages and Tregs play an important immunomodulatory role in promoting CRC. M2 macrophages can induce the formation of Tregs.¹² Studies show a correlation between increased numbers of Tregs and M2 macrophages in several types of cancer^13–15; however, to the best of our knowledge, this has not been investigated in CRC progression, especially in the lymph nodes.

In this study, we analyzed the correlation and clinical significance of the number of M2 macrophages and Tregs in the paracancer, cancer, and lymph node tissues of 197 CRC patients by immunohistochemical methods, and further explored the mechanism of CRC infiltration and metastasis, thus providing new ideas for CRC diagnosis and treatment.

Materials and methods

Clinical data

Clinical data and postoperative paraffin specimens of patients undergoing radical CRC surgery at the Second Ward of the First Affiliated Hospital of Jinzhou Medical University were collected between March 2020 and December 2020. After inclusion and exclusion, 197 cases were included. The clinical data of the patients are shown in Table 1. The tissue samples of patients with CRC were divided into cancer tissue, matched paracancer tissues, and lymph node tissue. In addition, they were divided into three groups according to the presence or absence of metastatic lymph nodes. Group A included 108 cases in which one normal lymph node was randomly selected from stage I and stage II patients. Group B included 89 cases in which one pathologically positive lymph node was randomly selected from stage III and IV patients. Group C included 89 cases in which one pathologically negative node was randomly selected from stage III and IV patients. Inclusion criteria were: (a) the patient received a diagnosis of primary colorectal carcinoma; (b) they had their first surgical treatment after diagnosis at our hospital; and (c) they consented to participate in this study. The exclusion criteria were: (a) patients older than 86 years; (b) young patients with a confirmed genetic background of the disease (familial adenomatous polyposis and hereditary non-polyposis CRC); (c) the patient had received chemotherapy or radiotherapy after the diagnosis; (d) the patient received targeted immune therapy after the diagnosis; and (e) the patient had intestinal malignant tumors with multiple sites or combined with other systemic malignancies. Clinical staging was according to the American Joint Committee on Cancer (AJCC) staging criteria (8th edition). This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University (trial number: KYLL202090) All patients signed an informed consent form.

Table 1.

Patient clinical data characteristics.

Clinicopathological parameters	Group A (I + II)	Group B or C (III + IV)
Clinicopathological parameters	(n = 108) (%)	(n = (89) (%)
Age
≥65	65 (60)	48 (54)
<65	43 (40)	41 (46)
Gender
Male	70 (65)	48 (54)
Female	38 (35)	41 (46)
Tumor site
Colon	58 (54)	42 (47)
Rectum	50 (46)	47 (53)
TNM staging
I	25 (23)
II	83 (77)
III		64 (82)
IV		25 (18)
N stage
N0	108 (100)
N1		60 (67)
N2		29 (33)
Differentiation
High/moderate	93 (86)	61 (69)
Low	15 (14)	28 (31)
Tumor diameter (cm)
≥5	63 (58)	40 (45)
<5	45 (42)	49 (55)
Distant metastasis
M0	108 (100)	64 (72)
M1		25 (28)

TNM: tumor node metastasis.

Immunohistochemical analysis

Using immunohistochemistry (IHC) streptomyces antibiotic protein-peroxidase linkage (SP) method: All specimens were formalin-fixed, paraffin-embedded, and cut into 4 um thick sections. Sections were placed on microscope slides, dewaxed in xylene, hydrated in a graded ethanol series, and subjected to antigen repair using ethylenediaminetetraacetic acid buffer (pH 8.0) at a sub-boiling temperature for 2.5 min. Tissue sections were incubated with endogenous peroxidase blocker for 10 min at room temperature, then closed with goat serum (KIT-9710; Maixin-Bio, Fuzhou, China) for 30 min. Sections were washed three times with phosphate buffered solution (PBS). Subsequently, anti-CD163 (1:500, ab182422, Abcam, Cambridge, UK), anti-Foxp3 (1:500, ab20034, Abcam) were added and incubated in a refrigerator at 4°C for 16 h. Biotin-labeled IgG polymer and Streptomyces anti-biotin protein-peroxidase (KIT-9710; Maixin-Bio) were added sequentially. Finally, sections were developed using the diaminobenzidine (DAB) kit (DAB-0031, Maixin-Bio) and restained with hematoxylin (G1140, Solarbio, Beijing, China).

Judgment of staining results

CD163 + staining was expressed as the presence of tan or yellow particles in the cell membrane or cytoplasm of M2 macrophages. The results were determined by two pathologists using a semi-quantitative scoring method under a double-blind method of independent film review. (Semi-quantitative score = cell membrane staining intensity score × cell membrane staining ratio score.) The staining intensity was divided into four levels: 0 (negative), 1 (weak), 2 (moderate), 3 (strong). The percentage of positive cells was 0 (0%–10%), 1 (11%–25%), 2 (26%–40%), 3 (41%–75%), and 4 (76%–100%). Semi-quantitative scores > 4 are positive cells, and < 4 are negative cells. Five randomly selected high magnification fields (400×) from stained pathology sections were counted and the mean calculated.

Foxp3 + staining was expressed as the presence of tan or yellow particles in the nucleus of Tregs. Five high magnification fields (400×) were randomly selected from the stained pathological sections. Image J was applied to determine the positive cells, which were then confirmed by two pathologists under double-blind methodological conditions. For pathology sections stained with the Anti-Foxp3 monoclonal antibody, the mean number of positive cells was calculated for the five high magnification fields, and this mean was the final score for this section.

Statistical analysis

The SPSS version 26.0 software program and GraphPad Prism 8 were used to analyze the data. Measurement data were expressed as means ± SD. The independent sample t-test was used to compare differences in M2 macrophages and Tregs in cancer tissues with different clinicopathological parameters, as well as differences within the M2 macrophage and Tregs groups. Spearman correlation analysis and linear regression analysis were used to analyze the correlation between the two variables. A value of P < 0.05 was considered significant.

Results

Immunohistochemical results of CD163 and Foxp3 in different tissues

CD163 is mainly characterized by the presence of yellow or brownish-yellow medium granules on the membrane of M2 macrophages and weak staining in the cell matrix (Figure 1(a) and (b)). Foxp3 is mainly characterized by the presence of brownish-yellow granules on the nuclei of Treg cells, distributed throughout the tissues of CRC patients (Figure 1(c) and (d)). Cancer tissue stained significantly more than paracancerous tissue (P < 0.05) (Figure 1(a) and (c)), and metastatic lymph node tissue stained significantly more than non-metastatic tissue (P < 0.05), (Figure 1(b) and (d)).

Figure 1.

Immunohistochemical results of CD163 and Foxp3 in different tissues. As shown in the figure: representative pictures of different tissues by IHC experiments (400× field of view). (a) CD163 staining and number in paraneoplastic and cancerous tissues; (b) CD163 staining and number in metastatic and non-metastatic lymph nodes; (c) Foxp3 staining and number in paraneoplastic and cancerous tissues; and (d) Foxp3 staining and number in metastatic and non-metastatic lymph nodes.

The relationship between the expression of M2 macrophages and Tregs and the clinical characteristics of tumors

To clarify the infiltration characteristics of M2 macrophages and Tregs cells within CRC, we performed a statistical analysis of the mean number of M2 macrophages and Tregs in 197 CRC tissues with clinicopathological parameters (Table 2). The mean number of M2 macrophages and Tregs increased with increasing tumor stage and depth of infiltration (P < 0.05). In addition, in the case of lymph node metastasis, vascular tumor thrombus, nerve invasion, and distant metastasis, the more significant the increase in the mean number of M2 macrophages and Tregs expression (P < 0.001). However, in patient age, gender, tumor diameter, tumor site, and degree of differentiation, there were no significant differences between the two types of cells (P > 0.05).

Table 2.

Clinicopathological characteristics of 197 patients with nodal cancer.

Clinicopathological parameters	Cases	CD163 + M2		Foxp3 + Tregs
Clinicopathological parameters	Cases	cell/HPF(mean)	P	cell/HPF(mean)	P
Gender			0.166		0.760
Male	118	18.45 ± 5.85		23.04 ± 6.89
Female	79	19.67 ± 6.33		22.72 ± 7.67
Age			0.156		0.544
≥65	118	19.44 ± 6.15		23.17 ± 7.09
<65	79	18.19 ± 5.89		22.53 ± 7.38
Tumor diameter (cm)			0.623		0.691
≥5	103	19.15 ± 5.17		22.72 ± 7.30
<5	94	18.71 ± 6.93		23.13 ± 7.13
Tumor site			0.541		0.927
Colon	100	19.20 ± 5.88		22.96 ± 7.46
Rectum	97	18.67 ± 6.26		22.87 ± 6.65
Lymph node metastasis			<0.001		<0.001
Have	89	22.57 ± 4.76		26.73 ± 6.06
No	108	15.94 ± 5.36		19.77 ± 6.53
TNM staging			<0.001		<0.001
I–II	108	15.94 ± 5.36		19.77 ± 6.53
III–IV	89	22.57 ± 4.76		26.73 ± 6.06
Differentiation			0.540		0.254
Medium + well differentiated	154	18.80 ± 5.98		22.60 ± 7.19
Poorly differentiated	43	19.44 ± 6.39		24.02 ± 7.19
Infiltration depth			<0.001		0.021
T1 + T2	26	10.27 ± 4.70		19.88 ± 5.83
T3 + T4	171	20.26 ± 5.07		23.37 ± 7.29
Vascular tumor thrombus			<0.001		<0.001
Have	85	22.48 ± 4.86		26.00 ± 6.47
No	112	16.25 ± 5.49		20.57 ± 6.86
Nerve invasion			<0.001		<0.001
Have	79	22.05 ± 5.37		26.06 ± 6.67
No	118	16.86 ± 5.61		20.81 ± 6.79
Distant metastasis			<0.001		<0.001
M0	172	18.02 ± 5.70		21.76 ± 6.55
M1	25	25.28 ± 4.61		30.84 ± 6.56

TNM: tumor node metastasis.

Different expressions of M2 macrophages and Tregs in different tissues

We observed (Figure 2(a) and (b)) the mean number of M2 macrophages and Tregs in cancer tissues (18.94 ± 6.06) and (22.91 ± 7.20), significantly higher than the paracancerous tissues (7.08 ± 2.97) and (9.55 ± 2.82), (P < 0.05); In cancer tissues, the mean number of M2 macrophages at stage III + IV (22.57 ± 4.76) was significantly higher than at stage I + II (15.94 ± 5.36) (Figure 2(c)), (P < 0.05). Meanwhile, the mean number of Tregs at stage III + IV (26.73 ± 6.07) was also significantly higher than at stage I + II (19.77 ± 6.53) (Figure 2(d)), (P < 0.05). To further investigate the progression of M2 macrophages and Tregs in CRC patients. We examined their lymph nodes and counted them. It was found that the mean number of M2 macrophages and Tregs in group A (8.82 ± 3.28) and (9.99 ± 3.70) were significantly lower than those in group B (16.65 ± 3.60) and (18.07 ± 4.33), (P < 0.05) (Figure 2(e) and (f)). However, it is not clear 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 2(g)) that the mean number of M2 macrophages in group A (8.82 ± 3.28) was lower than that in group C (9.89 ± 2.47), (P < 0.05). (Figure 2(h)) There was no difference in the mean number of Tregs (9.99 ± 3.70) and (10.42 ± 3.06) between group A and group C, respectively, (P > 0.05).

Figure 2.

Different expressions of M2 macrophages and Tregs in different tissues. (a) and (b) M2 macrophages and Tregs were significantly higher in cancer tissue than in paracancerous tissue, with statistically significant differences (both ***P < 0.001). In cancer tissues (c) and (d), M2 macrophages and Tregs in stage I + II were significantly lower than those in stage III + IV, and the difference was statistically significant (both ***P < 0.001). (e) and (f) M2 macrophages and Tregs in group A were significantly lower than those in group B, and the differences were statistically significant (both***P < 0.001). In the non-metastatic lymph node tissue (g), the mean number of M2 macrophages in group A was lower than that in group C, and the difference was statistically significant (**P < 0.05). (h) There was no significant difference in Tregs between group A and group C, and the difference was not statistically significant (*P > 0.05).

Correlation analysis of M2 macrophages and Tregs in cancer tissues and lymph nodes

By comparative analysis (Figure 3) we found that M2 macrophages and Tregs were abundantly distributed in cancerous tissues and lymph nodes. In order to verify whether there is a certain correlation between the two in the development of CRC and lymph node metastasis, we used Spearman correlation analysis to find that: Figure 3(a) and (b) M2 macrophages and Tregs are positively correlated in CRC and lymph nodes (r = 0.269, P < 0.001; r = 0.541, P < 0.001).

Figure 3.

(a) Correlation of M2 macrophages and Tregs in cancer tissue (r = 0.269, P < 0.001); (b) correlation of M2 macrophages and Tregs in lymph node tissue (r = 0.541, P < 0.001).

Correlation analysis of the number of M2 macrophages and Tregs with tumor markers in CRC

According to Supplementary Table 3, Spearman correlation analysis was used to determine the relationship between the mean number of M2 macrophages and Tregs and preoperative carcinoembryonic antigen (CEA), CA199, and CA724 levels. It was found that the mean number of M2 macrophages and Tregs were found to be significantly and positively correlated with preoperative CEA levels (both P < 0.001).

Discussion

Immune cells play a crucial role in the development of tumors in the tumor microenvironment.¹⁶ In this study, IHC staining was performed on paracancerous tissue, cancerous tissue, and its surrounding lymph node tissue of 197 patients with CRC. The number of M2 macrophages and Tregs was found to be significantly higher in cancer tissue than in paracancerous tissue. In lymph node tissue, increased numbers of M2 macrophages and Tregs infiltrated into the metastatic lymph nodes compared to the non-metastatic lymph nodes. Also, M2 macrophages infiltrated into non-metastatic lymph nodes in patients who also exhibited metastatic lymph nodes. These observations indicate that M2 macrophages were closely associated with lymph node metastasis in colorectal carcinoma. 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, IL-10, and IL-13. It promotes tumor angiogenesis and leads to tumor progression by suppressing T-cell-mediated anti-tumor immune responses.^17,18 The rapid growth of tumor tissue leads to increased metabolism and uneven vascularization around it. 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 micro RNAs (miRNAs) through the PI3K/AKT/mTOR pathway, and promote the polarization of M2 cells.^19–21 Therefore, M2 macrophages are significantly increased in tumor tissue. Lian et al.²² also noted that colon cancer cells secrete epithelial growth factor (EGF), which can bind to the epithelial growth factor receptor (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. The increase in the mean number of M2 macrophages was closely associated with the tumor node metastasis (TNM) 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 CRC. The EGF secreted by M2 macrophages activates the EGFR on tumor cells, which in turn up-regulates the vascular (V)EGF/VEGFR signals in surrounding tumor cells to support tumor cell proliferation and migration.²³ Our study found that in lymph node tissue, the mean number of M2 macrophages increased significantly not only 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.²⁴ Therefore, we speculate that M2 macrophages play an important role in the process of lymph node metastasis in CRC, which indicates a poor prognosis. Tacconi et al.²⁵ 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 CRC.^24,26

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.²⁷ 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, and significantly higher in late stages than in early stages, which is consistent with previous studies.²⁸ We analyzed 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 helped to recruit Tregs to tumor tissues.^29,30 In addition, we analyzed the correlation between Tregs and clinicopathological characteristics. It was 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 the 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 M2 macrophages and Tregs are closely related in tumor tissue and lymph node tissue. M2 macrophages can produce immunosuppressive cytokines and chemokines. These cytokines and chemokines are involved in recruiting lymphocytes and stimulating their development into Tregs.^29,31 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.³² Studies have pointed out that M2 macrophages and Tregs have synergistic effects in promoting proliferation, tumor angiogenesis and metastasis in ovarian cancer.³³ Sun et al.¹⁵ studied 65 patients with laryngeal squamous cell carcinoma (LSCC) and found that Tregs and M2 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.³⁴ 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 that CD163 can be used as a potential prognostic biomarker for CRC patients, and Foxp3 can directly affect the prognosis of CRC patients.^35,36 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.²⁴ We know that CEA plays an important role in monitoring the recurrence and metastasis of CRC. In addition, studies have shown that IL-10 stimulated by CEA can activate the JAK1/STAT3 signaling pathway to differentiate macrophages into M2 macrophages.^37,38 At the same time, TGF-β combined with CEA in malignant tumors is a key regulatory signal of Tregs and one of the main functional cytokines secreted by Tregs.^39,40 It is evident that CEA has an important effect on the distribution of M2 macrophages and Tregs in colon cancer.

In summary, the mechanisms leading to CRC progression and lymph node metastasis are very complex. The presence of M2 macrophages and Tregs in CRC patients is an important indicator of CRC progression and lymph node metastasis. Our results provide some insights for M2 macrophages and Tregs in the progression of CRC and its lymph node metastasis. CD163 may be a good predictor of pre-metastatic status of CRC lymph nodes. CEA affects the distribution of M2 macrophages and Tregs in CRC. There is a certain correlation between the two. The relationship between M2 macrophages and Tregs may affect the progression of CRC and lymph node metastasis. This could be a potential future target for immunotherapy and could provide important clinical value for CRC diagnosis, treatment, and prognosis.

Supplemental Material

sj-docx-1-jbm-10.1177_03936155221132572 - Supplemental material for A study on the correlation between M2 macrophages and regulatory T cells in the progression of colorectal cancer

Supplemental material, sj-docx-1-jbm-10.1177_03936155221132572 for A study on the correlation between M2 macrophages and regulatory T cells in the progression of colorectal cancer by Yanlei Chen, Yu Gao, Xueqian Ma, Yanping Wang, Jinhao Liu, Chunyu Yang, Yue Wang, Cuifen Bao, Xiaoyu Song, Yang Feng, Yan Sun and Shifeng Qiao in The International Journal of Biological Markers

Footnotes

Acknowledgments

The authors would like to thank the laboratory of Jinzhou Medical University for providing technical guidance

Authors’ contributions

YLC, YG, XQM, CYY, and YW performed the experiments. YLC, YPW, JHL, and CFB contributed to the statistical analysis and drafted the manuscript. YLC, XYS, YF, and YS participated in collecting the data, and contributed to the statistical analysis and manuscript writing. SFQ conceived the current study and helped revise the manuscript. All authors read and approved the final manuscript.

Ethics approval

The study was conducted in accordance with the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University (KYLL202090).

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.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Yanlei Chen

Shifeng Qiao

Supplemental material

Supplemental material for this article is available online.

References

Bray

Ferlay

Soerjomataram

, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394–424.

Yahaya

MAF

Lila

MAM

Ismail

, et al. Tumour-associated macrophages (TAMs) in colon cancer and how to reeducate them. J Immunol Res 2019; 2019: 2368249.

Hinshaw

Shevde

. The tumor microenvironment innately modulates cancer progression. Cancer Res 2019; 79: 4557–4566.

Sica

Mantovani

. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest 2012; 122: 787–795.

Solinas

Germano

Mantovani

, et al. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol 2009; 86: 1065–1073.

Yang

Wei

Wang

, et al. Elevated CD163( + )/CD68( + ) ratio at tumor invasive front is closely associated with aggressive phenotype and poor prognosis in colorectal cancer. Int J Biol Sci 2019; 15: 984–998.

Yang

Zhang

Peng

, et al. The prognostic and clinicopathological value of tumor-associated macrophages in patients with colorectal cancer: a systematic review and meta-analysis. Int J Colorectal Dis 2020; 35: 1651–1661.

Komohara

Hirahara

Horikawa

, et al. AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype. J Histochem Cytochem 2006; 54: 763–771.

Takeuchi

Nishikawa

. Roles of regulatory T cells in cancer immunity. Int Immunol 2016; 28: 401–409.

10.

Fontenot

Gavin

Rudensky

. Foxp3 programs the development and function of CD4 + CD25 + regulatory T cells. Nat Immunol 2003; 4: 330–336.

11.

Sakaguchi

Asano

, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995; 155: 1151–1164.

12.

Savage

de Boer

Walburg

, et al. Human anti-inflammatory macrophages induce Foxp3 + GITR + CD25 + regulatory T cells, which suppress via membrane-bound TGFbeta-1. J Immunol 2008; 181: 2220–2226.

13.

Dannenmann

Thielicke

Stöckli

, et al. Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma. Oncoimmunology 2013; 2: e23562.

14.

Wartenberg

Zlobec

Perren

, et al. Accumulation of FOXP3 + T-cells in the tumor microenvironment is associated with an epithelial-mesenchymal-transition-type tumor budding phenotype and is an independent prognostic factor in surgically resected pancreatic ductal adenocarcinoma. Oncotarget 2015; 6: 4190–4201.

15.

Sun

Wei

, et al. A positive-feedback loop between tumour infiltrating activated Treg cells and type 2-skewed macrophages is essential for progression of laryngeal squamous cell carcinoma. Br J Cancer 2017; 117: 1631–1643.

16.

Joyce

Fearon

. T cell exclusion, immune privilege, and the tumor microenvironment. Science 2015; 348: 74–80.

17.

Mantovani

Sica

Locati

. New vistas on macrophage differentiation and activation. Eur J Immunol 2007; 37: 14–16.

18.

Ngambenjawong

Gustafson

Pun

. Progress in tumor-associated macrophage (TAM)-targeted therapeutics. Adv Drug Deliv Rev 2017; 114: 206–221.

19.

Sengupta

Pal

Nath

, et al. Anticancer efficacy of noble metal nanoparticles relies on reprogramming tumor-associated macrophages through redox pathways and pro-inflammatory cytokine cascades. Cell Mol Immunol 2018; 15: 1088–1090.

20.

Tan

Shi

Zhang

, et al. Inhibition of Rspo-Lgr4 facilitates checkpoint blockade therapy by switching macrophage polarization. Cancer Res 2018; 78: 4929–4942.

21.

Wang

Luo

Zhang

, et al. Hypoxic tumor-derived exosomal miR-301a mediates M2 macrophage polarization via PTEN/PI3Kγ to promote pancreatic cancer metastasis. Cancer Res 2018; 78: 4586–4598.

22.

Lian

Chen

Ouyang

, et al. Colon cancer cell secretes EGF to promote M2 polarization of TAM through EGFR/PI3K/AKT/mTOR pathway. Technol Cancer Res Treat 2019; 18: 1533033819849068.

23.

Yin

Tan

, et al. Tumor-associated macrophages drive spheroid formation during early transcoelomic metastasis of ovarian cancer. J Clin Invest 2016; 126: 4157–4173.

24.

Wang

Yang

, et al. A study of the correlation between M2 macrophages and lymph node metastasis of colorectal carcinoma. World J Surg Oncol 2021; 19: 91.

25.

Tacconi

Correale

Gandelli

, et al. Vascular endothelial growth factor C disrupts the endothelial lymphatic barrier to promote colorectal cancer invasion. Gastroenterology 2015; 148: 1438–1451.e1438.

26.

Breslin

Gaudreault

Watson

, et al. Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism. Am J Physiol Heart Circ Physiol 2007; 293: H709–H718.

27.

Chen

Lin

, et al. CD4 + CD25 + regulatory T cells in tumor immunity. Int Immunopharmacol 2016; 34: 244–249.

28.

Ling

Zhang

, et al. Immunohistochemical distribution of FOXP3 + regulatory T cells in colorectal cancer patients. Int J Clin Exp Pathol 2018; 11: 1841–1854.

29.

Curiel

Coukos

Zou

, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004; 10: 942–949.

30.

Wang

Schmoeckel

Kost

, et al. Higher CCL22 + cell infiltration is associated with poor prognosis in cervical cancer patients. Cancers (Basel) 2019; 11: 2004.

31.

Cao

Wang

Zheng

, et al. IL-10/TGF-beta-modified macrophages induce regulatory T cells and protect against Adriamycin nephrosis. J Am Soc Nephrol 2010; 21: 933–942.

32.

Tiemessen

Jagger

Evans

, et al. CD4 + CD25 + Foxp3 + regulatory T cells induce alternative activation of human monocytes/macrophages. Proc Natl Acad Sci U S A 2007; 104: 19446–19451.

33.

Zhu

Wang

. Differential distribution of tumor-associated macrophages and Treg/Th17 cells in the progression of malignant and benign epithelial ovarian tumors. Oncol Lett 2017; 13: 159–166.

34.

Gao

Chen

, et al. M2-type macrophages induce Tregs generation by activating the TGF-β/Smad signalling pathway to promote colorectal cancer development. Onco Targets Ther 2021; 14: 5391–5402.

35.

Krijgsman

De Vries

Andersen

, et al. CD163 as a biomarker in colorectal cancer: the expression on circulating monocytes and tumor-associated macrophages, and the soluble form in the blood. Int J Mol Sci 2020; 21: 5925.

36.

Saito

Nishikawa

Wada

, et al. Two FOXP3( + )CD4( + ) T cell subpopulations distinctly control the prognosis of colorectal cancers. Nat Med 2016; 22: 679–684.

37.

Jessup

Laguinge

Lin

, et al. Carcinoembryonic antigen induction of IL-10 and IL-6 inhibits hepatic ischemic/reperfusion injury to colorectal carcinoma cells. Int J Cancer 2004; 111: 332–337.

38.

Sanjurjo

Aran

Téllez

, et al. CD5L Promotes M2 macrophage polarization through autophagy-mediated upregulation of ID3. Front Immunol 2018; 9: 480.

39.

Zhao

Yin

, et al. CD25 And TGF-β blockade based on predictive integrated immune ratio inhibits tumor growth in pancreatic cancer. J Transl Med 2018; 16: 294.

40.

Beauchemin

Arabzadeh

. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev 2013; 32: 643–671.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.01 MB