Background Plasma and other body fluids contain membranous extracellular vesicles (EVs), which are considered to derive from activated or apoptotic cells. EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. Knowledge on EVs is, however, limited, mainly due to their sub‐micrometer size and to intrinsic limitations in methods applied for their characterization. Objectives Our aim was to provide a comprehensive description of EVs from plasma of healthy subjects. Methods Cryo‐transmission electron microscopy combined with receptor‐specific gold labeling was used to reveal the morphology, size and phenotype of EVs. An original approach based on sedimentation on electron microscopy grids was developed for enumerating EVs. A correlation was performed between conventional flow cytometry and electron microscopy results. Results We show that platelet‐free plasma samples contain spherical EVs, 30 nm to 1 μm in diameter, tubular EVs, 1–5 μm long, and membrane fragments, 1–8 μm large. We show that only a minority of EVs expose the procoagulant lipid phosphatidylserine, in contrast to the classical theory of EV formation. In addition, the concentrations of the main EV sub‐populations are determined after sedimentation on EM grids. Finally, we show that conventional flow cytometry, the main method of EV characterization, detects only about 1% of them. Conclusion This study brings novel insights on EVs from normal plasma and provides a reference for further studies of EVs in disease situations.

ATF6α, a membrane-anchored transcription factor from the endoplasmic reticulum (ER) that modulates the cellular response to stress as an effector of the unfolded-protein response (UPR), is a key player in the development of tumors of different origin. ATF6α activation has been linked to oncogenic transformation and tumor maintenance; however, the mechanism(s) underlying this phenomenon remains elusive. Here, using a phenotypic small interfering RNA (siRNA) screening, we identified a novel role for ATF6α in chemoresistance and defined the protein disulfide isomerase A5 (PDIA5) as necessary for ATF6α activation upon ER stress. PDIA5 contributed to disulfide bond rearrangement in ATF6α under stress conditions, thereby leading to ATF6α export from the ER and activation of its target genes. Further analysis of the mechanism demonstrated that PDIA5 promotes ATF6α packaging into coat protein complex II (COPII) vesicles and that the PDIA5/ATF6α activation loop is essential to confer chemoresistance on cancer cells. Genetic and pharmacological inhibition of the PDIA5/ATF6α axis restored sensitivity to the drug treatment. This work defines the mechanisms underlying the role of ATF6α activation in carcinogenesis and chemoresistance; furthermore, it identifies PDIA5 as a key regulator ATF6α-mediated cellular functions in cancer.

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl–positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance. [Cancer Res 2008;68(23):9809–16]

We have related the release of procoagulant microparticles from platelets to calcium movement and the activation of the Ca²⁺‐dependent protease calpain. The effects of the Ca²⁺‐ATPase inhibitors thapsigargin, cyclopiazonic acid and 2,5‐di‐(t‐butyl)‐1,4‐benzohydroquinone were compared with those of the Ca²⁺ ionophore A23187. Whereas all three Ca²⁺‐ATPase inhibitors induced aminophospholipid exposure on platelets, only thapsigargin and cyclopiazonic acid promoted microparticle formation and only when strong Ca²⁺ influx, calpain activation and proteolysis of cytoskeletal proteins occurred concomitantly. Preincubation with dibutylbenzohydroquinone inhibited the responses to thapsigargin and cyclopiazonic acid but not to A23187. When platelets were suspended in a Ca²⁺‐free medium, calpain activation and microparticle formation were not observed, even with maximum mobilisation of internal Ca²⁺ stores by A23187. Incubation of fluo‐3–loaded platelets with A23187 in 0.1 mM EGTA followed by the sequential addition of 25 μM Ca²⁺ increments to the medium showed that calpain activation occurred when the intraplatelet [Ca²⁺] reached 3–8 μM. To assess the physiologic significance of these results, the subpopu‐lation of platelets that expressed procoagulant activity after stimulation by a thrombin/collagen mixture was isolated by means of annexin‐V–coupled magnetic beads. Subsequent western blotting experiments confirmed that this subpopulation contained activated calpain. Overall, our results provide evidence that microparticle formation and calpain activation require an elevated intraplatelet [Ca²⁺] that is brought about by influx across the plasma membrane.

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy.