Quantitative Changes in Tumor-Associated M2 Macrophages Characterize Cholangiocarcinoma and their Association with Metastasis

Abstract

The tumor microenvironment (TME) includes numerous non-neoplastic cells such as leukocytes and fibroblaststhat surround the neoplasm and influence its growth. Tumor-associated macrophages (TAMs) and cancerassociatedfibroblasts (CAFs) are documented as key players in facilitating cancer appearance and progression.Alteration of the macrophage (CD68, CD163) and fibroblast (α-SMA, FSP-1) cells in Opisthorchis viverrini (Ov)-induced cholangiocarcinoma (CCA) was here assessed using liver tissues from an established hamster modeland from 43 human cases using immunohistochemistry. We further investigated whether M2-activated TAMsinfluence CCA cell migration ability by wound healing assay and Western blot analysis. Macrophages andfibroblasts change their phenotypes to M2-TAMs (CD68+, CD163+) and CAFs (α-SMA+, FSP-1+), respectivelyin the early stages of carcinogenesis. Interestingly, a high density of the M2-TAMs CCA in patients is significantlyassociated with the presence of extrahepatic metastases (p=0.021). Similarly, CD163+ CCA cells are correlatedwith metastases (p=0.002), and they may be representative of an epithelial-to-mesenchymal transition (EMT)with increased metastatic activity. We further showed that M2-TAM conditioned medium can induce CCA cellmigration as well as increase N-cadherin expression (mesenchymal marker). The present work revealed thatsignificant TME changes occur at an early stage of Ov-induced carcinogenesis and that M2-TAMs are key factorscontributing to CCA metastasis, possibly via EMT processes.