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.

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.

The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8 + T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to ''feed'' in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of ''feeding'' activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. (Cancer Res 2006; 66(7): 3629-38)

contributed equally to this work CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Here we show that human T cells that are susceptible to CD95-mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4 + T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides speci®cally protected from the CD95-mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These ®ndings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis. Keywords: apoptosis/CD95 Fas/cytoskeleton/ezrin/ polarization IntroductionBetween the major programmed cell death (PCD) pathways, CD95 (APO-1/Fas)-mediated apoptosis seems one of the most involved in both the physiological control of cell proliferation and in the pathogenesis of viral, autoimmune and neoplastic diseases (Linch et al., 1995;Giordano et al., 1997; Apoptosis, special section, 1998;Peter and Krammer, 1998). Particularly, the CD95 interaction with its ligand (FasL) plays a crucial role in homeostasis and self-tolerance of lymphocytes in both humans and mice (Nagata and Suda, 1995). However, despite abundant surface expression, cells may be either susceptible or refractory to CD95-mediated PCD (Klas et al., 1993;Suda et al., 1997). In fact, susceptibility to the CD95-mediated apoptosis may not be merely due to the surface expression of the CD95 antigen, in that lymphocytes equally expressing CD95 on the membrane are differently triggerable to PCD (Klas et al., 1993;Alderson et al., 1995;Brunner et al., 1995). Intracellular mechanisms involved in the positive or negative regulation of the CD95-signaling pathway have been described (Tschopp et al., 1998). However, cellular susceptibility to CD95-mediated PCD remains an unresolved issue and the search for novel mechanisms is necessary. Asymmetric organization of the plasma membrane and cytosolic organelles is fundamental for a variety of cells, including pro-and eukaryotic cells (Nelson, 1992). The degree to which cells polarize is characterized by their ability to create and maintain morphologically and biochemically distinct plasma membrane domains. The prototype of stable polarized membrane domains are the apical and basolateral surfaces of simple epithelial cells. However, T lymphocytes continuously change their shape and polariza...

P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of Pglycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ionpump-related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies. (Blood. 2002;99:641-648)

SUMMARY:Features of phagocytosis have been observed in human tumors, but the phagocytic apparatus of tumor cells and the mechanism(s) underlying this phenomenon have yet to be defined. To address the phenomenon of phagocytosis, its underlying mechanism(s), and its possible role in tumor biology, we used human melanoma cells as a prototypic model. Our results showed that a process of phagocytosis of apoptotic cells occurs in vivo in human melanoma. This finding was consistent with evidence that human melanoma cells in vitro express all of the known lysosomal and phagocytic markers on their cytoplasmic vesicles and that a process of phagocytosis occurs in these vesicles. However, exclusively human melanoma cells deriving from metastatic lesions possess an efficient phagocytic machinery responsible for a macrophage-like activity against latex beads, yeast, and apoptotic cells of different origins, which was comparable to that of human primary macrophages. Moreover, the actin-binding protein ezrin was expressed on phagocytic vacuoles of melanoma cells and of cells deriving from a human adenocarcinoma; both treatment with cytochalasin B and specific inhibition of ezrin synthesis strongly affected the phagocytic activity of melanoma cells. This suggests that the association with the actin cytoskeleton is a crucial requirement for the development of this phenomenon. Hence our data provide evidence for a potent phagocytic activity exerted by metastatic melanoma cells possibly involved in determining the level of aggressiveness of human melanoma. This suggests that the assessment of phagocytic activity may be exploited as a new tool to evaluate the malignancy of human melanoma. Moreover, our data suggest that gene therapy or drug treatments aimed at inhibiting actin assembly to the phagosomal membranes may be proposed as a new strategy for the control of tumor aggressiveness. (Lab Invest 2003, 83:1555-1567.

The CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential requirement for susceptibility to the CD95-mediated apoptosis in CD4؉ T cells. We have previously shown that moesin was not involved in the binding to CD95. Here we further support the specificity of the ezrin/CD95 binding, showing that radixin did not bind CD95. The ezrin region specifically and directly involved in the binding to CD95 was located in the middle lobe of the ezrin FERM domain, between amino acids 149 and 168. In this region, ezrin, radixin, and moesin show 60 -65% identity, as compared with the 86% identity in the whole FERM domain. Transfection of two different human cell lines with a green fluorescent protein-tagged ezrin mutated in the CD95-binding epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells, the mutated ezrin did not co-localize or co-immunoprecipitate with CD95. Further analysis showed that the mutated ezrin, while unable to bind CD95, was fully able to bind actin, thus preventing the actin linkage to CD95. Altogether, our results support the specificity of ezrin in the association to CD95 and the importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover, this study suggests that a major role of ezrin is to connect CD95 to actin, thus allowing the CD95 polarization on the cells and the occurrence of the following multiple cascades of the CD95 pathway.A major requirement for the susceptibility to CD95 (APO-1/ Fas)-mediated apoptosis in CD4ϩ T cells is the CD95 polarization on cell uropods, as a result of CD95 linkage to the actin cytoskeleton through ezrin (1). Ezrin, radixin, and moesin (ERM) 1 are closely related proteins involved in cellular polarization and in various cellular functions (2-4). ERM are found in microvilli, filopodia, membrane ruffles, and cell-to-cell contact sites, where they co-localize and associate with F-actin (5-10). ERM are members of the erythrocyte protein 4.1 superfamily, characterized by a ϳ300-residue globular N-terminal domain highly conserved in the ERM family (FERM domain) (3,(11)(12)(13)(14). ERM interact directly with various membrane proteins, such as CD43, CD44, and intercellular adhesion molecule-1, -2, and 3, through their FERM amino-terminal domain (reviewed in Ref.3). These proteins can also indirectly bind to H ϩ /Na ϩ exchanger 3 via the cytoplasmic protein EBP50 (15, 16). ERM are present both in an inactive/closed and in an active/opened form (9, 14), and a direct consequence of ERM activation is their recruitment to the plasma membrane, allowing the ERM linkage to the membrane molecules (17, 18). The ERM/membrane protein interaction is stabilized at the plasma membrane by the ERM association to phosphatidylinositol 4, 5-bisphosphate (19, 20) and regulated by tyrosine phosphorylation in Tyr 145 and Tyr 353 (21-26). However, whereas the ERM domains involved in actin binding are known (27-30), the specific sites accounting for the binding to membrane proteins are much less defined. Particularly, the specific a...

These data support the possibility to exploit NK cells and V␦1 ␥␦ T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.