Cancer-associated fibroblasts promote M2 polarization of macrophages in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by remarkable desmoplasia with infiltration of distinct cellular components. Cancer-associated fibroblasts (CAFs) has been shown to be among the most prominent cells and played a significant role in shaping the tumor microenvironment by interacting with other type of cells. Here, we aimed to investigate the effect of CAFs in modulating phenotype of tumor-associated macrophages (TAM). Under treatment of CAFs conditioned medium (CM) or direct co-culture with CAFs, monocytes exhibited enhanced expression of CD206 and CD163 compared with control group (P < 0.01). The induction of M2 polarization was mediated by increased reactive oxygen species (ROS) production in monocytes as ROS elimination abolished the effect of CAFs (P < 0.05). The supernatant analysis showed that pancreatic CAFs produced increased macrophage colony-stimulating factor (M-CSF). Upon treatment of M-CSF neutralizing antibody, the ROS generation and M2 polarization of CAFs CM-stimulated monocytes were significantly inhibited (P < 0.05). In addition, the CAFs-induced M2 macrophages significantly enhanced pancreatic tumor cell growth, migration, and invasion. Collectively, our data revealed that pancreatic CAFs were able to induce a tumor-promoting TAM phenotype partly through secreted M-CSF and enhanced ROS production in monocytes, indicating possible treatment strategies by targeting stromal cell interaction within PDAC microenvironment.

Keywords: Cancer-associated fibroblasts; Macrophage colony-stimulating factor (M-CSF); pancreatic adenocarcinoma; reactive oxygen species; tumor-associated macrophages.

Figure 1

The characterization of cancer‐associated fibroblasts (CAFs) isolated from human pancreatic ductal adenocarcinoma (PDAC). Immunohistochemistry showed abundant α‐SMA ⁺ CAFs from a PDAC sample. The scale bar is 100 μm (A). The primary CAFs exhibited typical spindle‐like mesenchymal morphology. The scale bar is 20 μm (B). The expression for α‐SMA in CAFs was analyzed by western blot (C). The polarization status for human monocytes either treated by CAFs CM or directly co‐cultured with CAFs was detected by flow cytometry analysis for CD206 or CD163 expression (D–E). ** indicated P < 0.01.

Figure 2

The M2 phenotype induced by pancreatic CAFs was caused by increased reactive oxygen species (ROS) production. Monocytes were treated by CAFs CM with or without antioxidant BHA, and ROS level was measured through DCFH‐DA (A–B). M2 polarization was examined via flow cytometry analysis of CD206 expression (C–D). ** indicated P < 0.01, * indicated P < 0.05.

Figure 3

Secreted M‐CSF from pancreatic CAFs led to enhanced reactive oxygen species (ROS) production and M2 polarization. The secretion of soluble factors by CAFs was determined by ELISA (A). ROS level was measured when M‐CSF was blocked (B). The effect of blocking M‐CSF on the M2 polarization was evaluated by flow cytometry (C). * indicated P < 0.05, ** indicated P < 0.01.

Figure 4

The effect of CAFs‐induced M2 macrophages on pancreatic cancer cells. The effect of CAFs‐induced M2 phenotype on Panc1 and Miapaca2 cells growth (A–B), migration (C–D), and invasion (E–F). Western blot analysis for p‐Stat3 and p‐Akt (Fig. 4G). * indicated P < 0.05, ** indicated P < 0.01.