The increasing evidence that ;D T cells have potent antitumor activity suggests their value in immunotherapy, particularly in areas of unmet need such as metastatic carcinoma. To this end, we initiated a phase I clinical trial in metastatic hormonerefractory prostate cancer to examine the feasibility and consequences of using the ;D T-cell agonist zoledronate, either alone or in combination with low-dose interleukin 2 (IL-2), to activate peripheral blood ;D cells. Nine patients were enlisted to each arm. Neither treatment showed appreciable toxicity. Most patients were treated with zoledronate + IL-2, but conversely only two treated with zoledronate displayed a significant longterm shift of peripheral ;D cells toward an activated effectormemory-like state (T EM ), producing IFN-; and perforin. These patients also maintained serum levels of tumor necrosis factorrelated apoptosis inducing ligand (TRAIL), consistent with a parallel microarray analysis showing that TRAIL is produced by ;D cells activated via the T-cell receptor and IL-2. Moreover, the numbers of T EM ;D cells showed a statistically significant correlation with declining prostate-specific antigen levels and objective clinical outcomes that comprised three instances of partial remission and five of stable disease. By contrast, most patients treated only with zoledronate failed to sustain either ;D cell numbers or serum TRAIL, and showed progressive clinical deterioration. Thus, zoledronate + IL-2 represents a novel, safe, and feasible approach to induce immunologic and clinical responses in patients with metastatic carcinomas, potentially providing a substantially increased window for specific approaches to be administered. Moreover, ;D cell phenotypes and possibly serum TRAIL may constitute novel biomarkers of prognosis upon therapy with zoledronate + IL-2 in metastatic carcinoma. [Cancer Res 2007;67(15):7450-7]

BackgroundCD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.MethodsExosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.ResultsExosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.ConclusionsOur data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0426-x) contains supplementary material, which is available to authorized users.

SummaryThe potent anti-tumour activities of gd T cells have prompted the development of protocols in which gd-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vg9Vd2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vg9Vd2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vg9Vd2 T cell numbers emerged, as seven patients who failed to sustain Vg9Vd2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vg9Vd2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vg9Vd2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatmentrefractory patients with advanced breast cancer.

The identification of reciprocal interactions between tumor-infiltrating immune cells and the microenviroment may help us understand mechanisms of tumor growth inhibition or progression. We have assessed the frequencies of tumor-infiltrating and circulating γδ T cells and regulatory T cells (Treg) from 47 patients with squamous cell carcinoma (SCC), to determine if they correlated with progression or survival. Vδ1 T cells infiltrated SSC tissue to a greater extent than normal skin, but SCC patients and healthy subjects had similar amounts circulating. However, Vδ2 T cells were present at higher frequencies in circulation than in the tissue of either cancer patients or healthy donors. Tregs were decreased in the peripheral blood of SCC patients, but were significantly increased in the tumor compartment of these patients. Tumor-infiltrating γδ T cells preferentially showed an effector memory phenotype and made either IL17 or IFNγ depending on the tumor stage, whereas circulating γδ T cells of SCC patients preferentially made IFNγ. Different cell types in the tumor microenvironment produced chemokines that could recruit circulating γδ T cells to the tumor site and other cytokines that could reprogram γδ T cells to produce IL17. These findings suggest the possibility that γδ T cells in SCC are recruited from the periphery and their features are then affected by the tumor microenvironment. Elevated frequencies of infiltrating Vδ2 T cells and Tregs differently correlated with early and advanced tumor stages, respectively. Our results provide insights into the functions of tumor-infiltrating γδ T cells and define potential tools for tumor immunotherapy. .

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor currently used for the treatment of alcoholism. Here, we show that multiple myeloma (MM) cell lines and primary cells from newly diagnosed and relapsed/resistant patients affected by MM, acute myeloid and lymphoblastic leukemia are significantly sensitive to DSF alone and in combination with copper. These effects are present at doses lower than those achievable in vivo after DSF standard administration. The cytotoxic effect achieved by this treatment is comparable to that obtained by conventional chemotherapy and is absent in normal hematopoietic cells. In addition, we found that DSF plus copper induces loss of mitochondrial membrane potential, triggers reactive oxygen species (ROS) production and activates executioner caspases. DSF-copper-induced apoptosis and caspases activation are strongly reversed by antioxidant N-acetylcysteine, thus indicating a critical role of ROS. These results might suggest the use of the old drug DSF, alone or in combination with copper, in the treatment of hematological malignancies.The commonly used alcohol-abuse deterrent disulfiram (DSF) is a member of the dithiocarbamate family, a broad class of molecules with the ability to complex metals, such as copper, and react with sulfhydryl groups and glutathione (GSH). DSF has been used for decades in the treatment of alcoholism for its ability to inhibit irreversibly the aldehyde dehydrogenase.1,2 Several studies from 1970s (e.g., Lewison 1977) 3 have shown that DSF and its metabolites can enhance the effect of some chemotherapeutics, and it is considered a convincing anticancer drug both in vivo and in human patients. The case report described by Lewison was the first on DSF activity against breast cancer.3 Then the compound or its main metabolite was successfully used in a phase II clinical trial with 64 high-risk breast cancer patients and in hepatic metastases and in a patient with stage IV metastatic ocular melanoma. 4 More recently, there are ongoing clinical trials for DSF (Antabuse) as an adjuvant therapy (ClinicalTrials.gov Identifier NCT00312819) or combined with copper gluconate (ClinicalTrials.gov Identifier NCT00742911) against advanced solid cancers. However, a clear explanation for the antitumor effect of DSF is still missing. 2,4It has been shown that association of DSF with copper forms a complex with potent proteasome inhibiting and apoptosis-inducing activity on several tumors, both in vitro and in vivo. 1,2,4-6Moreover, DSF has also been found to reverse the resistance of human tumors to chemotherapeutic drugs by blocking maturation of the P-glycoprotein membrane pump; inhibit activation of nuclear factor-kB; decrease angiogenesis, tumor growth in vivo, matrix metalloproteinases activity and cancer cell invasiveness. 4,5 The proapoptotic activity of DSF has been attributed to redox-related mitochondrial membrane permeabilization, followed by complexation with zinc/copper and inhibition of Zn-dependent matrix metalloproteinases or Cu/Zn superoxide dismutase. The...

A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized with the aim of improving activity toward activation of human gammadelta T cells and in turn their in vivo antitumor activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen, appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater ability to activate gammadelta T cells expression (100 times more) and a proapoptotic effect that is better than zoledronic acid. The potent activation of gammadelta T cells, in addition to evidence of the in vivo antitumor activity of 6a, suggests it may be a new potential drug candidate for cancer treatment.

The influence of Th2 cytokines in tuberculosis has been a matter of dispute. Here we report that IL‐4 has a profound regulatory effect on the infection of BALB/c mice with Mycobacterium tuberculosis. Depletion of IL‐4 with a neutralizing mAb caused only evanescent reduction of lung infection, but when combined with i.n. inoculations of IgA anti‐mycobacterial α‐crystallin mAb and mouse rIFN‐γ, we observed a 40‐fold reduction of the bacterial counts in the lungs at 3 wks following i.n. infection (p<0.001). In genetically deficient IL‐4–/– BALB/c mice, infection in both lung and spleen was substantially reduced for up to 8 wks without further treatment. Reconstitution of IL‐4–/– mice with rIL‐4 increased bacterial counts to wild‐type levels and made the mice refractory to protection by IgA/IFN‐γ. Analysis of the lungs showed increased granulomatous infiltration and proinflammatory mediators in anti‐IL‐4/IgA/IFN‐γ‐treated and infected mice. We conclude that the action of IL‐4 in tuberculosis is targeted at macrophages and that it may include an antagonistic effect on their IgA/IFN‐γ‐induced activation and nitric oxide production. The described novel immunotherapy, combining treatments with anti‐IL‐4, IgA antibody and IFN‐γ, has potential for translation toward the passive immunoprophylaxis of tuberculosis.

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