M2-like tumor-associated macrophage-secreted CCL2 facilitates gallbladder cancer stemness and metastasis

Abstract

Background: The predominant immune cells in solid tumors are M2-like tumor-associated macrophages (M2-like TAMs), which significantly impact the promotion of epithelial-mesenchymal transition (EMT) in tumors, enhancing stemness and facilitating tumor invasion and metastasis. However, the contribution of M2-like TAMs to tumor progression in gallbladder cancer (GBC) is partially known.

Methods: Immunohistochemistry was used to evaluate the expression of M2-like TAMs and cancer stem cell (CSC) markers in 24 pairs of GBC and adjacent noncancerous tissues from patients with GBC. Subsequently, GBC cells and M2-like TAMs were co-cultured to examine the expression of CSC markers, EMT markers, and migratory behavior. Proteomics was performed on the culture supernatant of M2-like TAMs. The mechanisms underlying the induction of EMT, stemness, and metastasis in GBC by M2-like TAMs were elucidated using proteomics and transcriptomics. GBC cells were co-cultured with undifferentiated macrophages (M0) and analyzed. The therapeutic effect of gemcitabine combined with a chemokine (C-C motif) receptor 2 (CCR2) antagonist on GBC was observed in vivo.

Results: The expression levels of CD68 and CD163 in M2-like TAMs and CD44 and CD133 in gallbladder cancer stem cells (GBCSCs) were increased and positively correlated in GBC tissues compared with those in neighboring noncancerous tissues. M2-like TAMs secreted a significant amount of chemotactic cytokine ligand 2 (CCL2), which activated the MEK/extracellular regulated protein kinase (ERK) pathway and enhanced SNAIL expression after binding to the receptor CCR2 on GBC cells. Activation of the ERK pathway caused nuclear translocation of ELK1, which subsequently led to increased SNAIL expression. GBCSCs mediated the recruitment and polarization of M0 into M2-like TAMs within the GBC microenvironment via CCL2 secretion. In the murine models, the combination of a CCR2 antagonist and gemcitabine efficiently inhibited the growth of subcutaneous tumors in GBC.

Conclusions: The interaction between M2-like TAMs and GBC cells is mediated by the chemokine CCL2, which activates the MEK/ERK/ELK1/SNAIL pathway in GBC cells, promoting EMT, stemness, and metastasis. A combination of a CCR2 inhibitor and gemcitabine effectively suppressed the growth of subcutaneous tumors. Consequently, our study identified promising therapeutic targets and strategies for treating GBC.

Keywords: CCL2; Cancer stem cells; Epithelial-mesenchymal transformation; Gallbladder cancer; M2-like tumor-associated macrophage.

Fig. 1

Increased expression of CSCs and M2-like TAM markers in GBC is associated with metastasis (a) Representative immunohistochemical images of CD163, CD68, CD44, and CD133 in GBC and adjacent non-cancerous tissues in 24 patients with GBC (n = 24). (b) Expression of CD163, CD68, CD44, and CD133 in GBC and adjacent non-cancerous tissues from 24 patients with GBC (n = 24). (c) Expression of CD163, CD68, CD44, and CD133 and patient survival rates (n = 24). (d) SFE of NOZ and GBC-SD cells co-cultured with CD14⁺ TAMs obtained from fresh tissues of patients with GBC (n = 8). (e) Flow cytometry of the proportion of CD133⁺ CD44⁺ in NOZ and GBC-SD cells co-cultured with macrophages obtained from fresh tissues of patients with GBC (n = 8). (f) SFE in GBC cells co-cultured with M2-like TAMs (n = 3). (g) Western blotting of ALDH1, NANOG, SOX2, and OCT4 expression in GBC cells (n = 3). (h) Detection of the proportion of CD133⁺ CD44⁺ in GBC cells co-cultured with M2-like TAMs (n = 3). Data are presented as mean ± standard deviation (SD). GBC: gallbladder cancer, M2:M2-like tumor-associated macrophage, SFE: Sphere Formation Efficiency. Statistical significance was assessed using the Student’s t-test (b, d, e, f, g, h) and log-rank test (c). *P < 0.05, **P < 0.01

Fig. 2

M2-like TAMs drive GBC cell EMT, stemness, and migration by secreting CCL2. (a) Cytokine array analysis was used to detect cytokines in the supernatants of M0 and M2-like TAMs (n = 3). (b) Proteomics was used to identify cytokines in the supernatants of M0 and M2-like TAMs (n = 3). (c) ELISA was used to determine CCL2 expression in M0 and M2-like TAMs (n = 3). (d) SFE in GBC cells was determined using tumor sphere formation assay (n = 3). (e) Flow cytometry was used to measure the proportion of CD133⁺ CD44⁺ GBC cells (n = 3). (f) Western blotting was performed to determine the expression of ALDH1, NANOG, SOX2, and OCT4 in GBC cells (n = 3). Data are presented as mean ± SD. ELISA: Enzyme-linked immunosorbent assay, GBC: gallbladder cancer, M2:M2-like tumor-associated macrophage, R: RS504393, SFE: Sphere Formation Efficiency. Statistical significance was assessed using the Student’s t-test (a–f). *P < 0.05, **P < 0.01

Fig. 3

M2-like TAMs promote GBC EMT, stemness, and metastasis in vivo through the secretion of CCL2. (a) Images of subcutaneous tumors (n = 6). (b) Weight of subcutaneous tumors (n = 6). (c) Growth curve of subcutaneous tumors (n = 6). (d) Immunohistochemistry of CD44 and CD133 expression in subcutaneous tumors (n = 6). (e) Images of BLI in the lung metastasis model (n = 6). (f) Images of lung metastasis tumor (n = 6). (g) Total BLI intensity of lung metastases (n = 6). (h) Number of lung metastases tumor (n = 6). Data are presented as mean ± SD. BLI: bioluminescence, GBC: gallbladder cancer, M2:M2-like tumor-associated macrophage, R: RS504393, BLI: Bioluminescence image. Statistical significance was assessed using Student’s t-test (b, c, d, g, h). *P < 0.05, **P < 0.01

Fig. 4

M2-like TAMs promote GBC EMT, stemness, and migration via CCL2/MEK/ERK. (a) Western blotting was used to identify the expression of p-MEK and total-MEK in GBC cells (n = 3). (b) SFE of GBC cells (n = 3). (c) Flow cytometry was used to determine the proportion of CD133⁺ CD44⁺ in GBC cells (n = 3). (d) Western blotting was performed to examine the expression of ALDH1, NANOG, SOX2, and OCT4 in GBC cells (n = 3). Data are presented as mean ± SD. GBC: gallbladder cancer, M2:M2-like tumor-associated macrophage, R: RS504393, U: U0126, SFE: Sphere Formation Efficiency. Statistical significance was assessed using the Student’s t-test (a–d). *P < 0.05, **P < 0.01

Fig. 5

CCL2 activates the MEK/ERK/ELK1/SNAIL signaling pathway in GBC cells. (a) Western blotting for ELK1, p-ELK1, ERK, and p-ERK expression in GBC cells (n = 3). (b) Western blotting showed increased intranuclear expression of ELK1 and p-ELK1 in GBC cells (n = 3). (c) Immunofluorescence staining was used to determine the expression of ELK1 and p-ELK1 in GBC cells (n = 3). (d) Double luciferase reporter gene assay for the fluorescence intensity of truncated fragments (n = 3). (e) Double luciferase reporter gene assay was used to measure the fluorescence intensity of mutant fragments (n = 3). (f) CHIP validated the binding sites (n = 3). (g) The diagram of the ELK1 binding site on the SNAIL promoter. Data are presented as mean ± SD. CHIP: Chromatin immunoprecipitation, GBC: gallbladder cancer, U: U0126. Statistical significance was assessed using the Student’s t-test (a–f). *P < 0.05, **P < 0.01

Fig. 6

CCL2 triggers augmented stemness in GBC cells through the MEK/ERK/ELK1/SNAIL pathway (a) Western blotting was performed to determine the expression of ELK1, p-ELK1, ERK, p-ERK, SNAIL, and CSC markers in GBC cells (n = 3). (b) Western blotting was conducted to determine the expression of ELK1, p-ELK1, SNAIL, and CSC markers in GBC cells (n = 3). Data are presented as mean ± SD. GBC: gallbladder cancer. Statistical significance was assessed using the Student’s t-test (a, b). *P < 0.05, **P < 0.01

Fig. 7

Promotion of M2-like TAMs by GBCSCs. (a) qPCR was performed to evaluate CD163, CD206, ARG-1, IL-10, INOS, and IL-12 expression in M0 (n = 3). (b) Flow cytometry was used to measure the expression of CD68 and CD163 in M0 (n = 3). (c) Migration of M0 (n = 3). (d) qPCR was used to determine CCL2 expression in M0 co-cultured with GBCSCs (n = 3). (e) ELISA was used to determine CCL2 secretion from M0 co-cultured with GBCSCs (n = 3). (f) qPCR was performed to evaluate the expression of M2-like TAM markers in M0 (n = 3). (g) Flow cytometry was used to measure CD68 and CD163 expression in M0 (n = 3). (h) Migration of M0 (n = 3). (i) qPCR was performed to determine CCL2 expression in M0 (n = 3). Data are presented as mean ± SD. ELISA: Enzyme-linked immunosorbent assay, GBCSCs: gallbladder cancer stem cells. Statistical significance was assessed using the Student’s t-test (a–i). *P < 0.05, **P < 0.01

Fig. 8

Targeted inhibition of CCL2 to treat GBC. (a) Images of subcutaneous tumors (n = 4). (b) Growth curves of subcutaneous tumors (n = 4). (c) Weight of subcutaneous tumors (n = 4). (d) Survival of nude mice (n = 4). (e) BLI of subcutaneous tumors. Mice were anesthetized to death (n = 4). (f) Total BLI intensity of live imaging (n = 4). (g) Positive feedback between GBCSCs and TAMs-M2. Data are presented as mean ± SD. BLI: bioluminescence, D: Dissolution agent, GBC: gallbladder cancer, M2:M2-Like tumor-associated macrophage, R: RS504393, G: gemcitabine. Statistical significance was assessed using the Student’s t-test (b, c, and e) and log-rank (d). *P < 0.05, **P < 0.01