ABSTRACT
Cancerassociated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumorstromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.
Subject(s)
Cancer-Associated Fibroblasts/immunology , Carcinoma, Pancreatic Ductal/immunology , Liver Neoplasms/immunology , Neutrophils/immunology , Pancreatic Neoplasms/pathology , Aged , Animals , Carcinoma, Pancreatic Ductal/secondary , Carcinoma, Pancreatic Ductal/surgery , Cell Culture Techniques , Cell Line, Tumor/transplantation , Cell Movement/immunology , Cell Proliferation , Coculture Techniques , Deoxyribonuclease I/administration & dosage , Disease Models, Animal , Extracellular Traps/drug effects , Extracellular Traps/immunology , Extracellular Traps/metabolism , Hepatic Stellate Cells , Humans , Injections, Intraperitoneal , Liver Neoplasms/secondary , Male , Neoplasm Micrometastasis/immunology , Neoplasm Micrometastasis/prevention & control , Neutrophils/metabolism , Pancreas/immunology , Pancreas/pathology , Pancreas/surgery , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/surgery , Pancreaticoduodenectomy , Primary Cell CultureABSTRACT
Lymph node metastasis is an independent prognostic factor in pancreatic cancer. However, the mechanisms of lymph node colonization are unknown. As a mechanism of lymphatic metastasis, it has been reported for other types of cancer that spheroids from tumor cells cause circular chemorepellentinduced defects (CCIDs) in lymphatic endothelial monolayers. In pancreatic cancer, such mechanisms of metastasis have not been elucidated. The present study evaluated the involvement of this new mechanism of metastasis in pancreatic cancer and investigated the associated factors. In human pancreatic cancer tissue, it was observed that clusters of cancer cells penetrated the wall of lymphatic ducts around the primary tumor. An in vitro coculture system was then used to analyze the mechanisms of tumor cellmediated disruption of lymphatic vessels. Timelapse microscopic imaging revealed that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. CCID formation ability differed depending on the cell line. Neither aggregation of spheroids nor adhesion to lymphatic endothelial cells (LECs) exhibited a significant correlation with this phenomenon. The addition of supernatant from cultured cancer cells enhanced CCID formation. Microarray analysis revealed that the expression of S100 calcium binding protein P (S100P) was significantly increased when LECs were treated with supernatant from cultured cancer cells. Addition of a S100P antagonist significantly suppressed the migration of LECs and CCID formation. The present findings demonstrated that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. These CCIDs in pancreatic cancer were partly regulated by S100P, suggesting that S100P may be a promising target to inhibit lymph node metastasis.
