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

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.