Exosomes in ascites from patients with human pancreatic cancer enhance remote metastasis partially through endothelial-mesenchymal transition.

BACKGROUND: Despite advances in multidisciplinary treatment, the prognosis of pancreatic cancer remains poor. Since distant metastasis defines prognosis, elucidation of the mechanism of metastasis is important for improving survival. Exosomes are extracellular secretory vesicles and are responsible for intercellular communication. In this study, we investigated whether exosomes secreted by human pancreatic cancer cells are involved in promoting distant metastasis of cancer and the mechanism that underlies the promotion of metastasis. METHODS: Exosomes were isolated from ascites of a patient with pancreatic cancer and a patient with liver cirrhosis as a control. Three days after the administration of exosomes to nude mice, GFP-labeled human pancreatic cancer cells were injected via the spleen or tail vein, and then the liver and lungs were histologically analyzed. To elucidate the mechanism, vascular permeability was estimated using FITC-dextran in place of pancreatic cancer cells in vivo and human umbilical vascular endothelial cells (HUVECs) were used to analyze vascular permeability and the induction of endothelial-mesenchymal transition (EndMT) in vitro. RESULTS: Distant metastasis and vascular permeability were significantly enhanced in mice treated with exosomes from pancreatic cancer patients in comparison to exosomes from a control patient in vivo. In addition, exosomes from pancreatic cancer patients significantly enhanced vascular permeability and the induction of EndMT in HUVECs in vitro. CONCLUSION: Exosomes derived from pancreatic cancer cells form a pre-metastatic niche and promote the extravasation and colonization of pancreatic cancer cells to remote organs, partially through endothelial-mesenchymal transition.

Pancreatic cancer cell-derived exosomes induce epithelial-mesenchymal transition in human pancreatic cancer cells themselves partially via transforming growth factor ß1.

Distant metastasis is a dismal prognostic factor of pancreatic cancer. Metastasis is established in several steps, but the mechanism underlying the very early stages remains unclear. Epithelial-mesenchymal transition (EMT) is involved in these stages. Although signaling molecules have been reported to induce EMT, the mechanism underlying their origin is unclear. In this study, we hypothesized that pancreatic cancer cell-derived exosomes induce EMT in cancer cells themselves, a notion we entertained because we found EMT in in vitro three-dimensional colonies of cancer cells, with vimentin-positive cells observed in some of the budding pancreatic cancer cells and in single cells outside the colony as well. First, we clarified that pancreatic cancer cell-derived exosomes induce EMT in cancer cells themselves. Next, we examined the involvement of transforming growth factor-ß1 (TGF-ß1), and TGF-ß1 knock-down in pancreatic cancer cells with TGF-ß1 siRNA significantly suppressed TGF-ß1 gene expression in cancer cells, and exosomal TGF-ß1 was significantly reduced in the secretory exosomes. Exosomes from TGF-ß1 knock-down cells suppressed EMT induction in cancer cells themselves and TGF-ß1 protein expression in target cells. Taken together, these findings suggest that TGF-ß1 is involved in EMT induction via exosomes, results that may support the production of effective metastasis inhibitors.

Novel HIT antibody detection method using Sonoclot® coagulation analyzer.

INTRODUCTION: Since heparin-induced thrombocytopenia (HIT), caused by the generation of antibodies against platelet factor 4 (PF4)/heparin complexes (HIT antibodies), may induce serious complications due to thrombosis, a prompt diagnosis is desirable. Functional tests with platelet activation to detect HIT antibodies are useful for diagnosis of HIT, in particular (14)C-selotonin release assay (SRA). However, they are complicated and so can be performed only in limited laboratories. We tested if a blood coagulation test using Sonoclot® analyzer can serve for the detection of HIT antibodies. MATERIALS AND METHODS: A murine monoclonal antibody (HIT-MoAb) against PF4/heparin complexes was used as an alternative to human HIT antibodies. To the mixture of HIT-MoAb and heparin (0.5 U/mL, final), whole blood obtained from a healthy volunteer was added, and then the activated clotting time (ACT), clot rate (CR), and area under the curve (AUC) were measured with Sonoclot® analyzer for 30minutes. RESULTS: The HIT-MoAb (30 to 100µg/mL, final) concentration dependently suppressed the anticoagulation activity (prolongation of ACT and decrease of CR and AUC) of heparin. CONCLUSIONS: The suppression of anticoagulation effect of heparin by HIT-MoAb was demonstrated by measurements using Sonoclot® analyzer. This method may provide a new tool for screening of HIT antibodies.

Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia.

Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.