Cancer-associated Fibroblasts Promoted Salivary Adenoid Cystic Carcinoma Cell Invasion By IL6-induced Epithelial-to-mesenchymal Transition

Salivary gland adenoid cystic carcinoma(SACC)is a malignant tumor derived from the epithelium.Although growing slowly,it shows a great potential to invade surrounding tissues and tends to metastasize to distant organs.Our previous studies have confirmed that cancer associated fibroblasts(CAFs)existed in SACC tissues and promoted cancer invasion.It was known that epithelial-to-mesenchymal transition(EMT)is an important mechanism for cancer cells to enhance the ability of invasion.Objectives:This study aimed to elucidate whether CAFs promoted SACC invasion by inducing EMT and what is the molecular mechanism of EMT.Materials and methods: SACC cell lines,SACC-83 and SACC-LM,were used in this study.CAFs cells were two primary cells extracted from SACC tumor tissues,named CAF-A1 and CAF-A2.Conditional culture medium(CM)of CAFs was collected after culturing cells for one day without serum.Both SACC-83 and SACC-LM cells were treated with CAF-CM in vitro for different times.Then morphological changes of SACC cells were observed under an inversion microscope.The changes of SACC cell migration ability induced by CAF-A1-CM and CAF-A2-CM were examined by wound healing assay.A microfluidic chip was used to assess the invasion ability of cells.The invasion distance and area were recorded at 24,48 and 72 h.Then the invasion distance and area were calculated by Image-Pro Plus.The expression of EMT markers,including E-cadherin,N-cadherin and Vitmentin,was assessed by Western blot.Both SACC-83 and SACC-LM cells were treated with IL6 and EMT biomarkers were examined.Results: It was found that both SACC-83 and SACC-LM cells showed morphological changes induced by CAF-CM,such as decreased intercellular adhesion and appearing long processes.Cell wound healing assay confirmed that the migration ability of SACC-83 and SACC-LM was significantly increased after CAF-CM inducement,compared to the control.In the microfluidic invasion assay,both invasion distance and area of SACC cells were significantly increased in the CAF-CM group,compared to the control.Western blot results showed that E-cadherin down-regulated and N-cadherin and Vitmentin up-regulated significantly in SACC-83 and SACC-LM induced by CAF-CM.In addition,we found that IL6 showed high expression in CAFs by immunofluorecent staining and Western blot.Cytokine IL6 induced EMT in SACC-83 and SACC-LM cell in vitro,regarding by morphological changes and EMT biomarker expression.Conclusions: This study demonstrated that CAFs can induce EMT in SACC cells,thus promoted SACC invasion.IL6 might play an important role in CAF-induced EMT in SACC.