Abstract

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies worldwide. More than 50% of patients are diagnosed with late-stage disease. Exosomes are a group of extracellular vesicles released by different types of cells, containing proteins, nucleic acids and lipids, mediating intercellular communication, and thus affecting physiological and pathological conditions. Tumor- derived exosomes have been shown to induce a pre-metastatic niche in the target organ to promote metastasis. Methods: We isolated exosomes from cell culture supernatants of a highly metastatic pancreatic cell line L3.6pl and a PDAC patient derived primary cell line TBO368 by ultracentrifugation. Exosomes were characterized by Western blotting, nanoparticle tracking analysis and transmission electron microscopy. The protein content of exosomes was analyzed by mass spectrometry. The potential effects of pancreatic cancer-derived exosomes on NK cells were investigated by immunofluorescence and flow cytometry. The exosomal TGF-β1 levels in serum of patients with PDAC were quantified by ELISA. Results: We found that adhesion receptors, especially integrins such as integrin αv and integrin β5, which are associated with liver-specific metastases, were enriched in pancreatic cancer-derived exosomes. These exosomes also displayed a variety of immune regulatory factors, such as TGF-β1, Nectin-2 and PVR. Then we co-cultured NK cells with exosomes derived from pancreatic cancer cells. After co-culture, the expression of NKG2D, CD107a, TNF-α and INF-γ in NK cells was significantly downregulated. NK cells also exhibited the decreased level of CD71 and CD98, as well as impaired glucose uptake ability. In addition, NK cell cytotoxicity against pancreatic cancer stem cells was attenuated. Moreover, pancreatic cancer-derived exosomes induced the phosphorylation of Smad2/3 in NK cells. Compared to healthy donors, serum exosomal TGF-β1 was significantly increased in patients with PDAC. Conclusion: In this study, we show that tumor-derived exosomes are responsible for pre-metastatic niche formation in the liver of PDAC. The inhibitory effects of pancreatic cancer-derived exosomes on NK cells represent a mechanism allowing metastatic tumor cells to escape from NK cell immune surveillance in the pre-metastatic niche. We also demonstrate that serum exosomal TGF-β1 was significantly increased in patients with PDAC. In conclusion, these findings emphasize the immunosuppressive role of pancreatic cancer-derived exosomes and provide new insights into our understanding of NK cell dysfunction in the pre-metastatic niche formation of PDAC.