Matteo Bellone

Modulation of Microenvironment Acidity Reverses Anergy in Human and Murine Tumor-Infiltrating T Lymphocytes

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra-extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6-6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8(+) T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal-regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA-bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell-based cancer treatments.

Peripheral T-Cell Tolerance Associated with Prostate Cancer Is Independent from CD4+CD25+ Regulatory T Cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are thought to suppress the natural and vaccine-induced immune response against tumor-associated antigens (TAA). Here, we show that Treg accumulate in tumors and tumor-draining lymph nodes of aging transgenic adenocarcinoma of the mouse prostate (TRAMP) male mice, which spontaneously develop prostate cancer. TAA overexpression and disease progression associate also with induction of TAA-specific tolerance. TAA-specific T cells were found in the lymphoid organs of tumor-bearing mice. However, they had lost the ability to release IFN-gamma and kill relevant targets. Neither in vivo depletion of Treg by PC61 monoclonal antibody followed by repeated vaccinations with antigen-pulsed dendritic cells nor the combined treatment with 1-methyl-L-tryptophan inhibitor of the enzyme indoleamine 2,3-dyoxigenase, PC61 antibody, and dendritic cell vaccination restored the TAA-specific immune response. Treg did not seem to control the early phases of tolerance induction, as well. Indeed, depletion of Treg, starting at week 6, the age at which TRAMP mice are not yet tolerant, and prolonged up to week 12, did not avoid tolerance induction. A similar accumulation of Treg was found in the lymph nodes draining the site of dendritic cell vaccination both in TRAMP and wild-type animals. Hence, we conclude that Treg accrual is a phenomenon common to the sites of an ongoing immune response, and in TRAMP mice in particular, Treg are dispensable for induction of tumor-specific tolerance.

Crucial Role for Interferon γ in the Synergism between Tumor Vasculature-Targeted Tumor Necrosis Factor α (NGR-TNF) and Doxorubicin

NGR-TNF is a derivative of TNF-α, consisting of TNF fused to CNGRCG, a tumor vasculature-targeting peptide. Previous studies showed that NGR-TNF can exert synergistic antitumor effects with doxorubicin and with other chemotherapeutic drugs in murine models. In this study, we have investigated the role of endogenous IFN-γ on the antitumor activity of NGR-TNF in combination with doxorubicin. The study was carried out using murine B16F1 melanoma and TS/A mammary adenocarcinoma implanted subcutaneously in (a) immunocompetent mice, (b) athymic nude mice, and (c) IFN-γ–knockout mice. Synergism between NGR-TNF and doxorubicin was observed in immunocompetent mice but not in nude or IFN-γ–knockout mice. Preadministration of a neutralizing anti-IFN-γ antibody to immunocompetent mice inhibited the NGR-TNF/doxorubicin synergism, whereas administration of IFN-γ to nude and to IFN-γ–knockout mice restored the synergistic activity. The synergism in nude mice was restored also by transfecting tumor cells with the IFN-γ cDNA. Administration of NGR-TNF in combination with IFN-γ to nude mice, but not of NGR-TNF alone, doubled the penetration of doxorubicin in TS/A tumors. These findings point to a crucial role for locally produced IFN-γ in tumor vascular targeting with NGR-TNF and doxorubicin. Finally, addition of IFN-γ to the treatment of immunocompetent mice with NGR-TNF/doxorubicin induced only modest improvement in response, suggesting that exogenous IFN-γ can improve the therapeutic activity of these drugs only in case of suboptimal production of endogenous IFN-γ.

iNKT Cells Control Mouse Spontaneous Carcinoma Independently of Tumor-Specific Cytotoxic T Cells

Background CD1d-restricted invariant NKT (iNKT) cells are a subset of T lymphocytes endowed with innate effector functions that aid in the establishment of adaptive T and B cell immune responses. iNKT cells have been shown to play a spontaneous protective role against experimental tumors. Yet, the interplay between iNKT and tumor-specific T cells in cancer immune surveillance/editing has never been addressed. The transgenic adenocarcinoma of the mouse prostate (TRAMP) is a realistic model of spontaneous oncogenesis, in which the tumor-specific cytotoxic T cell (CTL) response undergoes full tolerance upon disease progression. Principal Findings We report here that lack of iNKT cells in TRAMP mice resulted in the appearance of more precocious and aggressive tumors that significantly reduced animal survival. TRAMP mice bearing or lacking iNKT cells responded similarly to a tumor-specific vaccination and developed tolerance to a tumor-associated antigen at comparable rate. Conclusions Hence, our data argue for a critical role of iNKT cells in the immune surveillance of carcinoma that is independent of tumor-specific CTL.

Nitric Oxide Confers Therapeutic Activity to Dendritic Cells in a Mouse Model of Melanoma

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

Critical role of indoleamine 2,3-dioxygenase in tumor resistance to repeated treatments with targeted IFNγ

Targeted delivery of IFNγ to tumors has been achieved by fusing this cytokine with GCNGRC, a tumor neovasculature homing peptide. Although the therapeutic efficacy of this protein (called IFNγ-NGR) in animal models is greater than that of IFNγ, frequent administrations of IFNγ-NGR may result in lower efficacy and tumor resistance. We investigated the role of indoleamine 2,3-dioxygenase (IDO), an IFNγ-inducible enzyme that may down-regulate T cells by affecting local tryptophan catabolism in tumor resistance to repeated treatments with IFNγ-NGR. The study was carried out in immunocompetent mice and in nu/nu mice bearing RMA lymphoma, B16F melanoma, or WEHI-164 fibrosarcoma and in vitro using cultured tumor cells. IDO activity was increased in lymphoma homogenates after multiple treatments with IFNγ-NGR but not after a single treatment. Coadministration of 1-methyl-tryptophan, an inhibitor of IDO, increased tumor responses to multiple treatments in the lymphoma, melanoma, and fibrosarcoma models. No synergism between IFNγ-NGR and 1-methyl-tryptophan was observed in vitro in tumor cell proliferation assays or in nu/nu tumor-bearing mice, suggesting that the antitumor effect was host mediated. We conclude that IDO is critically involved in tumor resistance to repeated treatments with IFNγ-NGR, likely causing excessive stimulation of tryptophan catabolism and inhibiting antitumor immune mechanisms. Coadministration of IFNγ-NGR with IDO inhibitors could represent a new strategy for increasing its antitumor activity. [Mol Cancer Ther 2008;7(12):3859–66]

Antisense transcription at the TRPM2 locus as a novel prognostic marker and therapeutic target in prostate cancer

Overwhelming evidence indicates that cancer is a genetic disease caused by the accumulation of mutations in oncogenes and tumor suppressor genes. It is also increasingly apparent, however, that cancer depends not only on mutations in these coding genes but also on alterations in the large class of non-coding RNAs. Here, we report that one such long non-coding RNA, TRPM2-AS, an antisense transcript of TRPM2, which encodes an oxidative stress-activated ion channel, is overexpressed in prostate cancer (PCa). The high expression of TRPM2-AS and its related gene signature were found to be linked to poor clinical outcome, with the related gene signature working also independently of the patients Gleason score. Mechanistically, TRPM2-AS knockdown led to PCa cell apoptosis, with a transcriptional profile that indicated an unbearable increase in cellular stress in the dying cells, which was coupled to cell cycle arrest, an increase in intracellular hydrogen peroxide and activation of the sense TRPM2 gene. Moreover, targets of existing drugs and treatments were found to be consistently associated with high TRPM2-AS levels in both targeted cells and patients, ultimately suggesting that the measurement of the expression levels of TRPM2-AS allows not only for the early identification of aggressive PCa tumors, but also identifies a subset of at-risk patients who would benefit from currently available, but mostly differently purposed, therapeutic agents.

Long non-coding RNAs as novel therapeutic targets in cancer

Thanks to impressive technology advancements, pervasive expression of non-coding RNAs (ncRNAs) has been recently identified in the genome of numerous cancers. Long ncRNAs (lncRNAs) belong to a new class of ncRNAs including tens of thousands different species. A fraction of these molecules shows a striking cancer-enriched expression pattern, suggesting an essential role in tumor cells and, possibly, a utility in therapeutic terms. This review aims at summarizing current knowledge for the identification and validation of lncRNAs as therapeutics targets in tumors. Both in-silico and wet-biology resources are presented in relation to the many challenges that the scientific community still needs to address in terms of lncRNA identification, stratification, patient personalization, drug delivery and toxicity.

Modulators of arginine metabolism do not impact on peripheral T-cell tolerance and disease progression in a model of spontaneous prostate cancer

Purpose: Chronic inflammation, recruitment of myeloid-derived cells, and perturbation of the arginine metabolism have been all proposed as mechanisms favoring prostate carcinogenesis and tumor immunoescape. Objective of this study was to evaluate whether accumulation of CD11b+Gr1+ cells, also defined myeloid-derived suppressor cells, occur in mice affected by transplantable or spontaneous prostate cancer (PC). We also investigated whether N(G) nitro-l-arginine methyl ester (l-NAME) and sildenafil, both modulators of the arginine metabolism, restrain tumor growth and restore tumor-specific immunity. Experimental Design: Wild-type C57BL/6 mice bearing TRAMP-C1 PC and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice were treated with vehicle, l-NAME or sildenafil, and evaluated for CD11b+ cells accumulation in the blood, several organs, and the tumor mass and for disease progression. Results: CD11b+Gr1high, CD11b+Gr1int, and CD11b+Gr1− cells differently accumulated in different organs and especially in the tumor of the two mouse models. l-NAME and sildenafil impaired the immunosuppressive function of CD11b+ cells in both models and restrained TRAMP-C1 growth, but they neither break tumor-specific immune tolerance nor limit tumor progression in TRAMP mice. Conclusions: Collectively, our results emphasize substantial differences in tumor-induced alteration of myelopoiesis and sensitivity to modulators of the arginine metabolism between a transplantable and a spontaneous model of PC. They also suggest that perturbation of the arginine metabolism is dispensable for PC progression and the associated T-cell tolerance. Clin Cancer Res; 17(5); 1012–23. ©2011 AACR.

Thymoma associated with systemic lupus erythematosus and immunologic abnormalities

The association between Systemic Lupus Erythematosus (SLE) and thymoma occurs with a greater frequency than dictated by coincidence alone. The immunologic effects of thymectomy on the appearance and/or the course of SLE are still to be elucidated. We report one case of SLE diagnosed at the same time as thymoma, and two cases of thymoma associated with immunologic disorders in the absence of clinical signs and symptoms diagnostic of SLE.