Tumor-associated macrophages promote bladder cancer metastasis through the CCL20-CCR6 axis

Abstract

We investigated the mechanisms of interaction between bladder cancer (BC) cells and tumor-associated macrophages (TAMs). Coculturing BC cell lines (UMUC3 and T24) with macrophage-like cells differentiated from THP-1 into M2-like TAMs revealed a decrease in Cluster of Differentiation (CD) 68 expression and an increase in CD206 expression. This differentiation enhanced BC cell migration and invasion. Additionally, M2-like TAMs significantly increased the secretion of C-C motif chemokine ligand (CCL) 20, which promotes BC cell migration and invasion via the MEK/ERK signaling pathway through its paracrine effects. Coculturing with TAMs also elevated the expression of CC chemokine receptor (CCR) 6 in BC cells, indicating increased sensitivity to CCL20. Immunohistochemistry analysis of human BC tissues showed a significant correlation between CCR6 expression levels and BC prognosis. Inhibition of CCR6 reduced BC cell metastasis both in vitro and in vivo. Additionally, CXCL1 secretion from BC cells was found to contribute to the M2-like polarization of macrophages and to enhance BC cell migration and invasion through autocrine and indirect effects. In summary, CCL20 and CXCL1 play crucial roles in the interaction between BC cells and TAMs.

Keywords: Bladder cancer; CCL20; CCR6; Tumor assosociated Macrophage.

Fig. 1

. M2-like tumor-associated macrophage (TAM) as a prognostic marker and promoter of bladder cancer (BC) Progression. A. Examples of low and high expression of CD68 and CD206 in BC tissue. B. ROC curve analysis for 2-year metastasis-free survival (MFS). The cutoff value for the CD206/CD68 ratio was 0.6191. C. Kaplan–Meier curves for MFS in low and high CD206/CD68 ratio groups. D. Macrophages treated with PMA for THP-1 cells were defined as ST1, ST1 exposed to the conditioned media (CM) of BC cells as ST2, and ST1 cocultured with BC cells as ST3. Decreased expression of CD68 and increased expression of CD206 were observed in ST3. E. Proliferation assay of BC cells cocultured with each type of macrophage. Parental refers to untreated THP-1 cells. Macrophages were placed in the upper chamber, and BC cells were placed in the lower chamber. Coculture with macrophages did not alter the proliferation ability of BC cells. F. Migration assay of BC cells cocultured with each type of macrophage. Macrophages were spread in the lower chamber, and BC cells were placed in the upper chamber. Coculture with macrophages enhanced the migration of BC cells, particularly in the presence of ST3. G. Invasion assay of BC cells cocultured with each type of macrophage. Coculture with macrophages enhanced the invasion of BC cells, particularly in the presence of ST3. H. Western blot (WB) analysis showing alterations in epithelial–mesenchymal transition (EMT) markers of BC cells. Coculture with macrophages led to phosphorylation of the MEK/ERK pathway and elevated expression of EMT markers in BC cells.

Fig. 2

Alterations in chemokine expression through polarization to tumor-associated macrophages (TAM). A. Chemokine array analysis of various conditioned media. Coculture of macrophages and BC cells markedly increased the expression levels of CCL20, CCL2, and CXCL7. Items not detected in any of the conditioned media (CCL14, CCL15, CCL17, CCL18, CCL19, CCL21, CCL22, CCL26, CCL28, Chemerin, CX3CL1, CXCL4, CXCL9, CXCL11, CXCL12, CXCL17, IL-16, XCL1) were not listed in the figure. B. ELISA analysis of CCL20 secretion. High secretion of CCL20 from ST1 was further enhanced when cocultured with BC cells. C. RT-qPCR analysis of CCL20. mRNA expression levels of CCL20 were markedly elevated in ST3 compared to BC cells, indicating that CCL20 was mainly secreted from TAMs. D. Migration assay of BC cells treated with rhCCL20. Migration was activated in a CCL20 concentration-dependent manner. E. Invasion assay of BC cells treated with rhCCL20. Invasion was activated in a CCL20 concentration-dependent manner. F. Western blot (WB) analysis showing that rhCCL20 treatment enhanced phosphorylation of the MEK/ERK pathway and increased expression levels of epithelial–mesenchymal transition (EMT) markers in BC cells. G WB analysis of CCR6. Coculture with TAMs increased the expression level of CCR6 in BC cells. H. Comparison of MFS by immunohistochemistry (IHC) analysis and Kaplan-Meier curve of CCR6 in radical cystectomy specimens. Patients with strong CCR6 positivity had a significantly worse prognosis.

Fig. 3

Alteration of migration and invasion of bladder cancer (BC) cells by CCR6 inhibition. A. Migration assay of BC cells with CCR6 inhibitor treatment. CCR6 inhibition reduced the migration of BC cells cocultured with tumor-associated macrophages (TAMs). B. Invasion assay of BC cells with CCR6 inhibitor treatment. CCR6 Inhibition reduced the invasion of BC cells cocultured with TAMs. C. Western Blot (WB) analysis showing that CCR6 inhibition suppressed MEK/ERK phosphorylation and decreased the expression levels of EMT markers in BC cells. D. Mouse subcutaneous transplantation model. CCR6 inhibitor was administered intraperitoneally twice a week starting 7 days after UMUC3 transplantation. Tumors were removed 25 days after transplantation. No significant difference in tumor size and body weight was observed between the control and CCR6 inhibitor groups. IHC analysis of Vimentin. The weakly positive rate was significantly higher in the CCR6 inhibitor group. E. Pulmonary metastasis model in mice. UMUC3 cells were transplanted via the tail vein of nude mice. Intraperitoneal administration of CCR6 inhibitor was performed twice a week starting from the day after tumor transplantation. Lung metastases were assessed by CT and IHC 42 days after transplantation. While three out of four mice in the control group had lung metastases, none of the mice in the CCR6 inhibitor group showed metastases (p = 0.0285).

Fig. 4

The effect of CXCL1 on bladder cancer (BC) cells. A. ELISA analysis of CXCL1. There was no significant difference in CXCL1 concentration between conditioned media (CM) from BC cells alone and CM from ST3 generation. B. RT-qPCR analysis of CXCL1. CXCL1 mRNA expression was significantly upregulated in BC cells, indicating that CXCL1 is mainly secreted by BC cells. C. Treatment of ST1 with rhCXCL1 decreased CD68 expression and increased CD206 expression in a concentration-dependent manner. D ELISA analysis of CCL20. Treatment with rhCXCL1 enhanced CCL20 secretion from ST1 cells. E. Migration assay of BC cells. Migration was enhanced by rhCXCL1 in a concentration-dependent manner. F. Invasion assay of BC cells. Invasion was enhanced by rhCXCL1 in a concentration-dependent manner. G. Western blot (WB) analysis showed that rhCXCL1 treatment increased MEK/ERK phosphorylation and expression levels of epithelial–mesenchymal transition (EMT) markers. H. Migration assay of BC cells. ST1 cells treated with CXCL1 showed significantly enhanced migration compared to untreated ST1 cells. I. Invasion assay of BC cells. ST1 cells treated with CXCL1 showed significantly enhanced invasion compared to untreated ST1 cells. J. Coculture with ST1 cells treated with CXCL1 further enhanced MEK/ERK phosphorylation and the expression of epithelial–mesenchymal transition (EMT) markers. K. CCL20 administration significantly enhanced BC cell migration more than CXCL1 administration. The combined administration of CXCL1 and CCL20 did not result in significantly different migration enhancement compared to CCL20 alone.

Fig. 5

Schematic illustration of chemokine mechanisms of interaction between bladder cancer (BC) cells and macrophage. A. Macrophages polarize into M2-like tumor-associated macrophages (TAMs) via interaction with BCs, and CXCL1 secretion from BC cells contributes to their polarization. B. CXCL1 also contributes to the activation of BC cell migration and invasion through an autocrine effect. C. CCL20 induces BC cells to increase MEK/ERK phosphorylation and expression of epithelial–mesenchymal transition (EMT) markers, activating BC cell migration and invasion. D. Interaction with TAMs increases CCR6 expression in BC cells, enhancing their sensitivity to tumor-associated macrophage (TAM)-derived CCL20. E. BC cells activated by CCL20 secretion from TAMs have enhanced metastatic potential.