Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by “normal” cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

IntroductionOver the past years, it has become apparent that type-I interferons (IFNs) affect adaptive immunity through their effects on monocytes. In particular, IFN-␣ has been shown to act as a potent inducer of the rapid differentiation of human monocytes into highly activated and partially mature dendritic cells (DCs), known as IFN-DCs. 1 We demonstrated previously that human monocytes exposed to granulocyte macrophage colony-stimulating factor (GM-CSF) and IFN-␣ are rapidly induced to express a set of membrane molecules involved in antigen (Ag) presentation and T-cell costimulation, as well as to strongly promote T helper (Th)-1 response and CD8 ϩ T cell cross-priming. 2 Moreover, IFN-DCs were shown to cross-present very efficiently low amounts of nonstructural-3 protein (NS3) of hepatitis C virus (HCV) to a specific CD8 ϩ T cell clone, even in the absence of CD4 ϩ T-cell help. 2 The cross-presentation efficiency of DCs is not dictated solely by their Ag capture capability 3 ; it also is affected by critical factors such as (1) the route of Ag uptake, (2) acidification-sensitive Ag degradation in endosomal-lysosomal compartments, and (3) Ag entry into the major histocompatibility complex class-I (MHC-I) pathway. [4][5][6][7][8] At present, 4 nonmutually exclusive models have been proposed to explain cross-presentation. [8][9][10] In the canonical cytosolic pathway, endocytosed Ags are translocated into the cytosol, where they are degraded by the proteasome, and then the antigenic peptides are transported into the lumen of the endoplasmic reticulum (ER) by the transporters associated with Ag processing (TAPs), 9,10 or alternatively, reimported from the cytoplasm into the early endosomes and loaded onto endosomal 12 According to a less well-defined TAP-and proteasomeindependent endosomal pathway, Ags can be processed by endosomal proteases and loaded onto MHC-I molecules directly within early and late endosomes and lysosomes. [13][14][15] An additional model involves the delivery of components of the ER to endocytic organelles or the transport of incoming Ags to the ER. [16][17][18] Here, we have investigated the mechanisms underlying the superior efficiency of IFN-DCs in the cross-presentation of soluble proteins, by studying (1) the Ag uptake and trafficking to the class-I processing pathway, (2) the maturation kinetic of the organelles containing the internalized proteins, (3) the Ag stability within endosomes, and (4) the Ag processing and cross-presentation to specific CD8 ϩ T cells. The results reveal that IFN-DCs exhibit a delayed endosomal acidification associated with a prolonged Ag survival and retention in the early endosomal compartment, as well as with Ag trafficking to recycling pathways. In IFN-DCs, both early and recycling endosomal compartments serve as important stores of MHC-I molecules, allowing rapid presentation of exogenous Ags. These findings provide novel mechanistic insight into the cross-presentation efficiency of IFN-DCs and underscore the potential advantage of using these cellula...

IntroductionOverexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation.MethodsLocalization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab.ResultsPC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with HER2 in raft domains. PC-PLC inhibition resulted in enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 reexpression on the membrane and a decrease in the overall cellular contents of HER2, HER2-HER3, and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced antiproliferative effects, especially in trastuzumab-resistant cells.ConclusionsThe results pointed to PC-PLC inhibition as a potential means to counteract the tumorigenic effects of HER2 amplification and complement the effectiveness of current HER2-targeting therapies.

Introduction Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231. Methods PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography. Results PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/μg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 μg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, β-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells. Conclusions These results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20–50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods.