SHH/GLI2-TGF-1 feedback loop between cancer cells and tumor-associated macrophages maintains epithelial-mesenchymal transition and endoplasmic reticulum homeostasis in cholangiocarcinoma

Background: Tumor-associated macrophages (TAMs) play a dual role in tumors. However, the factors which drive the function of TAMs in cholangiocarcinoma remain largely undefined. Methods: SHH signaling pathway and endoplasmic reticulum stress (ERS) indicators were detected in clinical tissues and cholangiocarcinoma cell lines. TAMs were co-cultured with cholangiocarcinoma cells under conditions of hypoxia/normoxia. Polarized TAMs were counted by flow cytometry, and TGF-beta 1 levels in cell supernatants were detected by ELISA. The effects of glioma-associated oncogene GLI2 on TAMs themselves and cholangiocarcinoma cells were examined by conducting interference and overexpression assays. Results: The SHH signaling pathway and ERS were both activated in tumor tissues or tumor cell lines under conditions of hypoxia. In co-culture experiments, the presence of cholangiocarcinoma cells increased the proportion of M2-polarized TAMs and the secretion of TGF-beta 1 by TAMs, while knockdown of SHH expression reversed those increases. Overexpression of GLI2 in TAMS or stimulation of TAMS with Hh-Ag1.5 increased their levels of TGF-beta 1 expression. Furthermore, under co-culture conditions, interference with GLI2 expression in TAMs reduced the tumor cell migration, invasion, and ER homeostasis induced by Hh-Ag1.5-pretreated TAMs. Under conditions of hypoxia, the presence of cholangiocarcinoma cells promoted the expression of GLI2 and TGF-beta 1 in Tams, and in turn, TAMs inhibited the apoptosis and promoted the migration and invasion of cholangiocarcinoma cells. In vivo, an injection of cholangiocarcinoma cells plus TAMs contributed to the growth, EMT, and ER homeostasis of tumor tissue, while an injection of TAMs with GLI2 knockdown had the opposite effects. Conclusion: Cholangiocarcinoma cells regulated TAM polarization and TGF-beta 1 secretion via a paracrine SHH signaling pathway, and in turn, TAMs promoted the growth, EMT, and ER homeostasis of cholangiocarcinoma cells via TGF-beta 1.