BackgroundMetastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up.Methodology/Principal FindingsWe designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group.Conclusions/SignificanceWe describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.

Type I interferons (IFNs) are cytokines exhibiting antiviral and antitumor effects, including multiple activities on immune cells. However, the importance of these cytokines in the early events leading to the generation of an immune response is still unclear. Here, we have investigated the effects of type I IFNs on freshly isolated granulocyte/macrophage colony-stimulating factor (GM-CSF)–treated human monocytes in terms of dendritic cell (DC) differentiation and activity in vitro and in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID) mice. Type I IFNs induced a surprisingly rapid maturation of monocytes into short-lived tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)–expressing DCs endowed with potent functional activities, superior with respect to the interleukin (IL)-4/GM-CSF treatment, as shown by FACS® analyses, mixed leukocyte reaction assays with allogeneic PBLs, and lymphocyte proliferation responses to HIV-1–pulsed autologous DCs. Type I IFN induced IL-15 production and strongly promoted a T helper cell type 1 response. Notably, injection of IFN-treated HIV-1–pulsed DCs in SCID mice reconstituted with autologous PBLs resulted in the generation of a potent primary immune response, as evaluated by the detection of human antibodies to various HIV-1 antigens. These results provide a rationale for using type I IFNs as vaccine adjuvants and support the concept that a natural alliance between these cytokines and monocytes/DCs represents an important early mechanism for connecting innate and adaptive immunity.

Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.

Proton pumps like the vacuolar-type H + ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose-and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI. [Cancer Res 2007;67(11):5408-17]

IntroductionDendritic cells (DCs) are the most potent antigen-presenting cells playing a pivotal role in the induction of the immune response. [1][2][3] DCs are located in peripheral tissues, in sites where they can optimally survey for incoming pathogens. The interaction of DCs with pathogens leads to migration to secondary lymphoid organs where they initiate a specific immune response. Notably, the migration capability of DCs is strictly regulated by their response to soluble factors, namely chemokines 4,5 that characterize maturation stage and shape functional activities of DCs.Chemokines represent a family of 8-to 10-kDa secreted proteins capable of regulating migration and activation not only of leukocytes, including DCs, but also of stromal cells. 6,7 It is well documented that migration of DCs is tightly regulated as a function of maturation. [8][9][10][11][12] In particular, immature DCs respond to many CC and CXC chemokines, such as MIP-1␣, MIP-1␤, RANTES, and MIP-3␣, whereas mature DCs have lost their responsiveness to most of these chemokines, as a result of down-regulation of receptor expression or activity. However, mature DCs have been reported to respond to MIP-3␤/ELC and 6Ckine/SLC as a consequence of an up-regulation of their receptor (CCR7). Of interest, studies in knock-out mice for CCR7 have shown the crucial importance of the CCR7/MIP-3␤ interaction for the generation of a primary immune response. 13 All this emphasizes the essential role of certain chemokines/chemokine receptors and DC migration properties in the generation of the immune response.DCs are derived from hematopoietic progenitor cells, 2,3 and distinct subtypes of human circulating DCs have been detected in the blood. 14,15 However, the mechanisms regulating generation, functions, and survival of blood-circulating DCs in response to infections are largely unknown. The rapid generation of active DCs endowed with potent migratory capabilities would be advantageous for a prompt immune response to incoming pathogens.Blood monocytes are highly versatile cells playing crucial roles in the maintenance of immune homeostasis. These cells circulate in the bloodstream, transmigrate through vascular endothelium, and localize in peripheral and mucosal tissues, where they differentiate into different cell types. 16,17 Monocyte-derived mature DCs are currently generated in vitro by 2 sequential treatments, 18 leading first to the so-called "immature DCs," after exposure for several days to both granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), and then to mature DCs, after a subsequent addition of stimuli such as lipopolysaccharide (LPS), CD40L, or virus infection. However, the in vivo relevance of monocyte differentiation into DCs remains unclear, especially because exposure of monocytes to IL-4 can hardly mimic the cytokine milieu likely to be present under in vivo conditions at the infection site.Although results indicate that DC maturation can occur directly from monocytes during transendothelial migration...