Angiotensin II subtype 1a receptor signaling in resident hepatic macrophages induces liver metastasis formation

Abstract

Liver metastases from colorectal cancer (CRC) are a clinically significant problem. The renin-angiotensin system is involved in tumor growth and metastases. This study was designed to evaluate the role of angiotensin II subtype receptor 1a (AT1a) in the formation of liver metastasis in CRC. A model of liver metastasis was developed by intrasplenic injection of mouse colon cancer (CMT-93) into AT1a knockout mice (AT1aKO) and wild-type (C57BL/6) mice (WT). Compared with WT mice, the liver weight and liver metastatic rate were significantly lower in AT1aKO. The mRNA levels of CD31, transforming growth factor- β1 (TGF-β1), and F4/80 were suppressed in AT1aKO compared with WT. Double immunofluorescence analysis showed that the number of accumulated F4/80⁺ cells expressing TGF-β1 in metastatic areas was higher in WT than in AT1aKO. The AT1aKO bone marrow (BM) (AT1aKO-BM)→WT showed suppressed formation of liver metastasis compared with WT-BM→WT. However, the formation of metastasis was further suppressed in WT-BM→AT1aKO compared with AT1aKO-BM→WT. In addition, accumulated F4/80⁺ cells in the liver metastasis were not BM-derived F4/80⁺ cells, but mainly resident hepatic F4/80⁺ cells, and these resident hepatic F4/80⁺ cells were positive for TGF-β1. Angiotensin II enhanced TGF-β1 expression in Kupffer cells. Treatment of WT with clodronate liposomes suppressed liver metastasis by diminishing TGF-β1⁺ F4/80⁺ cells accumulation. The formation of liver metastasis correlated with collagen deposition in the metastatic area, which was dependent on AT1a signaling. These results suggested that resident hepatic macrophages induced liver metastasis formation by induction of TGF-β1 through AT1a signaling.

Keywords: AT1a; Angiotensin; Kupffer cell; colorectal cancer; liver metastasis.

Figure 1

Effect of AT1a signaling on liver metastasis formation. (a) Expressions of AT1a, AT1b, and AT2 receptor in metastatic livers 14 days after injection of CMT‐93 mouse colon cancer cells. Data are expressed as the means ± SD of six mice per group. *P < 0.05 versus PBS‐injected mice. (b,c). Effect of AT1a signaling on liver metastasis formation 14 days after injection of CMT‐93 cells. The weight of liver metastasis (b) and the metastasis rate (c). Data are presented as the means ± SD of six mice per group. P < 0.05 versus WT. (d) Typical gross appearance of liver metastasis in WT and AT1aKO injected intrasplenically with CMT‐93 cells. Arrows indicate metastatic colonization. Scale bar = 1 cm. (e) Typical appearance of H&E staining of metastatic livers from WT and AT1aKO after injection of CMT‐93 cells. Metastatic area is delineated with the black dashed line. T, tumor. Scale bar = 100 μm. (f,g) Effects of AT1a signaling on colony formation in liver in macro (f) and micro (g) after injection of CMT‐93 cells. Data are represented as the means ± SD of six mice per group. *P < 0.05 versus WT. (h) Mortality after injection of CMT‐93 cells. The survival rate in WT was lower than that in AT1aKO. n = 10 per group. *P < 0.05 versus WT mice, Kaplan–Meier analysis.

Figure 2

AT1a signaling enhances the expression of angiogenic factor and macrophage marker in metastatic liver. (a,b) CD31 expression in the liver 14 days after injection of CMT‐93 mouse colon cancer cells. Data are expressed as the means ± SD of six mice per group. *P < 0.05 versus WT. (a) mRNA level of CD31 was suppressed in AT1aKO mice. (b) Immunohistochemical staining of CD31 in metastatic areas from WT and AT1aKO mice. Scale bar = 100 μm. Arrows indicate CD31‐positive cells. (c–e) Vascular endothelial growth factor A (VEGF‐A) (c), stromal cell‐derived factor‐1 (SDF‐1) (d), and transforming growth factor‐β1 (TGF‐β1) (e) mRNA expression in the liver 14 days after CMT‐93 injection. Data are expressed as the means ± SD of six mice per group. *P < 0.05 versus WT. (f,g) F4/80 (f) and monocyte chemoattractant protein‐1 (MCP‐1) (g) mRNA expression in the liver 14 days after CMT‐93 injection. Data are expressed as the means ± SD of six mice per group. *P < 0.05 versus WT.

Figure 3

Angiotensin II (Ang II)/AT1a axis enhances transforming growth factor‐β1 (TGF‐β1) expression in Kupffer cells. (a) Accumulation of F4/80⁺ cells, TGF‐β1⁺ cells, and F4/80⁺/TGF‐β1⁺ cells in metastatic areas from WT and AT1aKO mice on day 14. Double‐staining of liver sections with antibodies against F4/80 (red) and TGF‐β1 (green) in WT and AT1aKO mice. Expression of TGF‐β1 was colocalized with F4/80⁺ cells. Metastatic area is delineated with the white dashed line. T, tumor. Scale bar = 100 μm. (b–d) Numbers of F4/80⁺ cells (b), TGF‐β1⁺ cells (c), and F4/80⁺ TGF‐β1⁺ cells (d) in metastatic areas from WT and AT1aKO mice. Data are expressed as the means ± SD of six mice per group. *P < 0.05. (e) Expression of AT1a and AT1b in KUP5 Kupffer cells. Data are expressed as the means ± SD of six mice per group. (f) Expression of TGF‐β1 on KUP5 cells under stimulation with Ang II. Expression of TGF‐β1 was enhanced 6 h after stimulation with Ang II compared with control. There was no significant difference at 12 h. Data are expressed as the means ± SD of six mice per group. *P < 0.05 versus control.

Figure 4

Effects of bone marrow (BM) transplantation on liver metastasis. (a) Typical appearance of H&E staining of metastatic livers from chimeric mice 14 days after injection of CMT‐93 mouse colon cancer cells. Metastatic area is delineated with the black dashed line. T, tumor. Scale bar = 100 μm. (b) Effect of BM transplantation on tumor area 14 days after CMT‐93 injection. Metastatic tumor areas in AT1aKO‐BM→WT and AT1aKO‐BM→AT1aKO mice were suppressed compared with those in WT‐BM→WT and WT‐BM→AT1aKO mice, respectively. Data are expressed as the means ± SD of six mice per group. *P < 0.05 versus control. (c) Accumulation of GFP ⁺F4/80⁺ cells in the metastatic area (delineated with white dashed line) on day 14. T, tumor. Scale bar = 100 μm.

Figure 5

Contribution of resident F4/80⁺ Kupffer cells to liver metastasis formation. (a–d) Numbers of GFP ⁺ (a), F4/80⁺ (b), GFP ⁺F4/80⁺ cells (c), and GFP ⁻F4/80⁺ cells (d) in metastatic areas. Data are expressed as the means ± SD of six mice per group. *P < 0.05. (e) Percentages of GFP ⁺F4/80⁺ cells and GFP ⁻F4/80⁺ cells in each group. BM, bone marrow.

Figure 6

Effect of AT1a signaling in bone marrow (BM) cells on accumulation of F4/80⁺ cells expressing transforming growth factor‐β1 (TGF‐β1). (a) Accumulation of TGF‐β1 ⁺F4/80⁺ Kupffer cells in the metastatic areas (delineated with the white dashed line) on day 14. T, tumor. Scale bar = 100 μm. (b,c) Total numbers of TGF‐β1⁺ (b) and TGF‐β1⁺F4/80⁺ Kupffer cells (c) in metastatic areas. Data are expressed as the means ± SD of six mice per group. *P < 0.05.

Figure 7

Effect of AT1a signaling on type I collagen expression in metastatic areas. Immunohistochemical analysis of type I collagen in metastatic livers from WT and AT1aKO mice. Metastatic areas are delineated with the black dashed line. T, tumor. Scale bar = 100 μm. (a) Type I collagen‐positive areas in metastatic livers from WT and AT1aKO mice. Data are expressed as the means ± SD of six mice per group. *P < 0.05. (b) Expression of collagen type I α1 (Col1a1) in metastatic livers from WT and AT1aKO mice. Data are expressed as the means ± SD of six mice per group. *P < 0.05. (c) Concentrations of type I collagen in metastatic livers from WT and AT1aKO mice at 0 and 14 days after CMT‐93 injection. Data are expressed as the means ± SD of six mice per group. *P < 0.05. (d) Immunohistochemical staining of type I collagen in metastatic areas (delineated with black dashed line) after bone marrow (BM) transplantation. T, tumor. Scale bar = 100 μm. (e) Type I collagen‐positive areas in metastatic livers from chimeric mice given transplants of BM from WT and AT1aKO. Data are expressed as the means ± SD of six mice per group. *P < 0.05.

Figure 8

Effects of macrophage deletion on liver metastasis formation. (a) Metastasis area in WT with control liposomes and clodronate liposomes 14 days after injection CMT‐93 mouse colon cancer cells. Data are expressed as the means ± SD of four mice per group. *P < 0.05 versus control liposomes. (b–e). F4/80 (b), transforming growth factor‐β1 (TGF‐β1) (c), collagen type I α1 (Col1a1) (d), and CD31 (e) mRNA expression in metastatic liver 14 days after CMT‐93 injection. Data are expressed as the means ± SD of four mice per group. *P < 0.05 versus control liposomes. (f) Accumulation of TGF‐β1⁺ cells, F4/80⁺ cells, and TGF‐β1⁺/F4/80⁺ cells in metastatic areas on day 14. Double‐staining of liver sections with antibodies against TGF‐β1 (green) and F4/80 (red). T, tumor. Scale bar = 100 μm. (g–i) Numbers of TGF‐β1⁺ cells (g), F4/80⁺ cells (h), and TGF‐β1⁺ F4/80⁺ cells (i) in metastatic areas from WT mice with control liposomes and clodronate liposomes. Data are expressed as the means ± SD of four mice per group. *P < 0.05. (j) Representative images showing immunofluorescence staining for type I collagen in metastatic livers of WT mice with control liposomes and clodronate liposomes 14 days after CMT‐93 injection. Metastatic area is delineated with the white dashed line. T, tumor. Scale bar = 100 μm. (k) Type I collagen‐positive area in metastatic livers of WT mice with control liposomes and clodronate liposomes. Data are expressed as the means ± SD of four mice per group. *P < 0.05.