BackgroundNecroptosis is a form of programmed cell death that is accompanied by release of intracellular contents, and reportedly contributes to various diseases. Here, we investigate the significance of necroptosis in pancreatic cancer. MethodsWe used immunohistochemistry and western blot analysis to evaluate expression of the key mediators of necroptosis-receptor-interacting serine/threonine protein kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL)-in human pancreatic cancer. We also tested the effects of conditioned media (CM) from necroptotic cells on pancreatic cancer cells in Transwell migration and Matrigel invasion assays. Protein array analysis was used to investigate possible mediators derived from necroptotic cells. ResultsRIP3 and MLKL are highly expressed in human pancreatic cancer tissues compared with normal pancreas. MLKL expression was particularly intense at the tumor invasion front. CM derived from necroptotic cells promoted cancer cell migration and invasion, but not CM derived from apoptotic cells. C-X-C motif chemokine 5 (CXCL5) was upregulated in CM derived from necroptotic cells compared with CM derived from control or apoptotic cells. Moreover, expression of the receptor for CXCL5, C-X-C-motif chemokine receptor-2 (CXCR2), was upregulated in pancreatic cancer cells. Inhibition of CXCR2 suppressed cancer cell migratory and invasive behavior enhanced by necroptosis.

Cancer-associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor-stromal 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.

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Pancreatic cancer is characterized by a desmoplastic reaction, which provokes treatment resistance. Recently, it has been reported that CAFs have heterogeneity, tumor-promoting or tumor-suppressive CAFs. The origin of CAFs on tumor progression and its mechanism remains unclear. In the pancreatic tumor, there are macrophages, but its origin is also unclear. Our previous data showed bone marrow-derived macrophages accumulated in the pancreatic tumor. Therefore, we aimed to investigate the involvement of peripheral blood (PB)-derived macrophages with CAF in pancreatic cancer microenvironment. Methods: Human pancreatic cancer cells (PCCs) were coinjected with PB-derived macrophages into immunodeficient mice to evaluate tumor development. Invaded or migrated PCCs were counted to investigate the involvement of PB-derived macrophages untreated or treated with PCCs-conditioned medium (CM) in the invasive and migratory capability of PCCs. We examined changes in phenotype and function of PB-derived macrophages treated with PCCs-CM. Results: PCCs co-injected with PB-derived macrophages grew invasively in xenotransplantation models. Invasive and migratory capability of PCCs increased significantly when they were co-cultured with PB-derived macrophages untreated or treated by PCCs-CM. Some PB-derived macrophages treated by PCCs-CM expressed CAF marker. PB macrophages-derived CAF-like cells produced tumor-promoting cytokines, increased their own migratory activity, and led the invasion of PCCs. Conclusion: These data revealed that PB-derived macrophages were interacted with PCCs and transformed into CAF-like cells and induced the invasion of pancreatic cancer. Therefore, it was indicated that there is a subset of CAFs derived from macrophages although the origins of CAFs is thought to be pancreatic stellate cells or MSCs.