Role of tumor-associated macrophages at the invasive front in human colorectal cancer progression

Abstract

Macrophages are an essential component of antitumor activity; however, the role of tumor-associated macrophages (TAMs) in colorectal cancer (CRC) remains controversial. Here, we elucidated the role of TAMs in CRC progression, especially at the early stage. We assessed the TAM number, phenotype, and distribution in 53 patients with colorectal neoplasia, including intramucosal neoplasia, submucosal invasive colorectal cancer (SM-CRC), and advanced cancer, using double immunofluorescence for CD68 and CD163. Next, we focused on the invasive front in SM-CRC and association between TAMs and clinicopathological features including lymph node metastasis, which were evaluated in 87 SM-CRC clinical specimens. The number of M2 macrophages increased with tumor progression and dynamic changes were observed with respect to the number and phenotype of TAMs at the invasive front, especially at the stage of submucosal invasion. A high M2 macrophage count at the invasive front was correlated with lymphovascular invasion, low histological differentiation, and lymph node metastasis; a low M1 macrophage count at the invasive front was correlated with lymph node metastasis. Furthermore, receiver operating characteristic curve analysis revealed that the M2/M1 ratio was a better predictor of the risk of lymph node metastasis than the pan-, M1, or M2 macrophage counts at the invasive front. These results suggested that TAMs at the invasive front might play a role in CRC progression, especially at the early stages. Therefore, evaluating the TAM phenotype, number, and distribution may be a potential predictor of metastasis, including lymph node metastasis, and TAMs may be a potential CRC therapeutic target.

Keywords: colorectal neoplasms; epithelial-mesenchymal transition; lymphatic metastasis polarization; neoplasm metastasis; tumor-associated macrophages.

FIGURE 1

Phenotype and distribution of tumor‐associated macrophages. A, Double immunofluorescence for CD68 (red) and CD163 (green). Nuclei were stained with DAPI (blue), and the overlay depicts fluorescence detected by all channels. a: CD68⁺CD163⁻ cells (entirely red). M1 macrophages were defined as CD68⁺CD163⁻ cells. b: CD68⁺CD163⁺ cells (the center is red and edge is yellow). M2 macrophages were defined as CD68⁺CD163⁺ cells. Scale bars, 50 µm. B, Schema of the tumor. The number of macrophages was counted in 4 different regions: (a) the invasive front of the tumor, (b) the center of the tumor, (c) the lateral periphery of the tumor, and (d) the non‐neoplastic mucosa adjacent to the tumor

FIGURE 2

Immunofluorescence in specimens from patients with colorectal neoplasia. A, Representative images of immunofluorescence in the IM group (20 patients with IM). A low number of macrophages homogenously infiltrated into the tumor stroma, with most of them being M1 macrophages. Images are of the sigmoid colon with high‐grade dysplasia [type 0‐IIa (12 × 10 mm)]. B, Representative images of immunofluorescence in the SM group (20 patients with SM‐CRC). In the SM group, the number of pan‐macrophages was higher compared with that in the IM group, and M2 macrophages were present in each region. Pan‐macrophages were abundant, especially at the invasive front. Images are of the ascending colon with moderately differentiated adenocarcinoma [type 0‐IIa+IIc, pT1 (submucosal invasion depth: 2000 μm), 15 × 10 mm]. C, Representative images of immunofluorescence in the AD group (13 patients with AD). In the AD group, the number of M2 macrophages was higher compared with that in the SM group, with a dense accumulation of M2 macrophages detected at the invasive front. Images are of the ascending colon with moderately differentiated adenocarcinoma [type 2, pT2 (20 × 15 mm)]. Scale bars, 50 μm. AD, advanced cancer; IM, intramucosal neoplasia; SM‐CRC, submucosal invasive colorectal cancer

FIGURE 3

Comparison of the number of tumor‐associated macrophages in the IM, SM, and AD groups. A, The numbers of pan‐macrophages and M2 macrophages, and the M2/M1 ratio in each tumor region were higher in the SM group relative to those in the IM group and in the AD group relative to those in the SM group, with these differences most significant at the invasive front. *P < .05, **P < .01, ***P < .001. Significant predictive variables: P < .0167. B, Mean counts of M2 macrophages at the invasive front were measured in each group. In the IM group, the number of M2 macrophages at the invasive front was low in most cases. In the SM group, there was a mix of cases with high and low numbers of M2 macrophages at the invasive front. In the AD group, the number was high in most cases. AD, advanced cancer; IM, intramucosal neoplasia; SM‐CRC, submucosal invasive colorectal cancer

FIGURE 4

Areas with high and low numbers of TAMs at the invasive front. A‐D, An invasive front in AD; representative images of the area with a low TAM number (M‐low area). A, Hematoxylin and eosin (H&E) staining (low‐power field). B, H&E staining (high‐power field). C, Double immunofluorescence for CD68 (red) and CD163 (green) (high‐power field). D, Immunohistochemistry (IHC) for E‐cadherin (high‐power field). In the M‐low area, the glandular structure of the tumor was preserved, cell differentiation was observed, and E‐cadherin expression was not decreased. E‐H. An invasive front in SM‐CRC; representative images of the area with a high M2 macrophage number (M2‐high area). E, H&E staining (low‐power field). F, H&E staining (high‐power field). G, Double immunofluorescence for CD68 (red) and CD163 (green) (high‐power field). H, IHC for E‐cadherin (high‐power field). In the M2‐high area, cancer cells were poorly differentiated, morphology of cancer cells changed from epithelioid to fibroid, and E‐cadherin expression in cancer cells was decreased. I‐K, An invasive front in SM‐CRC; representative images of the area with a high M1 macrophage number (M1‐high area). I, H&E staining (low‐power field). J, H&E staining (high‐power field). K, Double immunofluorescence for CD68 (red) and CD163 (green) (high‐power field). In the M1‐high area, TAMs infiltrated destructively and the glandular structure of the tumor collapsed and was destroyed. Scale bars, 50 µm. AD, advanced cancer; IM, intramucosal neoplasia; SM‐CRC, submucosal invasive colorectal cancer; TAMs, tumor‐associated macrophages

FIGURE 5

Association of TAMs at the invasive front with lymph node metastasis. A, Association between the number and phenotype of TAMs at the invasive front and the presence of lymph node metastasis. There was no significant difference in the number of pan‐macrophages between the lymph node metastasis‐positive (LN+) and metastasis‐negative (LN−) groups, but the number of M1 macrophages was significantly lower and the number of M2 macrophages and M2/M1 ratio were significantly higher in the LN+ group compared with those in the LN− group. Significant predictive variables: P < .05. B, Lymph node metastasis prediction based on TAMs at the invasive front. We compared the receiver operating characteristic curves for predicting lymph node metastasis based on the pan‐macrophage, M1 macrophage, and M2 macrophage numbers, and M2/M1 ratio at the invasive front. The AUCs of the pan‐macrophage, M1 macrophage, and M2 macrophage numbers, and M2/M1 ratio were 0.64, 0.78, 0.87, and 0.90, respectively. The AUCs of the M2 macrophage number and M2/M1 ratio were significantly higher compared with those of the pan‐macrophage number; the AUC of the M2/M1 ratio was the highest among the 4 parameters. Significant predictive variables: P < .0083. AUC, area under the curve; CI, confidence interval; TAMs, tumor‐associated macrophages