Targeting surface nucleolin with a multivalent pseudopeptide delays development of spontaneous melanoma in RET transgenic mice.

BACKGROUND: The importance of cell-surface nucleolin in cancer biology was recently highlighted by studies showing that ligands of nucleolin play critical role in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, we recently reported that HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in the athymic nude mice without apparent toxicity. METHODS: The in vivo antitumoral action of HB-19 treatment was assessed on the spontaneous development of melanoma in the RET transgenic mouse model. Ten days old RET mice were treated with HB-19 in a prophylactic setting that extended 300 days. In parallel, the molecular basis for the action of HB-19 was investigated on a melanoma cell line (called TIII) derived from a cutaneous nodule of a RET mouse. RESULTS: HB-19 treatment of RET mice caused a significant delay in the onset of cutaneous tumors, several-months delay in the incidence of large tumors, a lower frequency of cutaneous nodules, and a reduction of visceral metastatic nodules while displaying no toxicity to normal tissue. Moreover, microvessel density was significantly reduced in tumors recovered from HB-19 treated mice compared to corresponding controls. Studies on the melanoma-derived tumor cells demonstrated that HB-19 treatment of TIII cells could restore contact inhibition, impair anchorage-independent growth, and reduce their tumorigenic potential in mice. Moreover, HB-19 treatment caused selective down regulation of transcripts coding matrix metalloproteinase 2 and 9, and tumor necrosis factor-alpha in the TIII cells and in melanoma tumors of RET mice. CONCLUSIONS: Although HB-19 treatment failed to prevent the development of spontaneous melanoma in the RET mice, it delayed for several months the onset and frequency of cutaneous tumors, and exerted a significant inhibitory effect on visceral metastasis. Consequently, HB-19 could provide a novel therapeutic agent by itself or as an adjuvant therapy in association with current therapeutic interventions on a virulent cancer like melanoma.

Spontaneous vitiligo in an animal model for human melanoma: role of tumor-specific CD8+ T cells.

Tumor antigen-reactive T cells can be detected in a large proportion of melanoma patients, but their efficacy on tumor control in vivo remains unclear. On the other hand, vitiligo, a skin disorder characterized by patchy depigmented macules, may occur spontaneously or after antitumor therapies. Moreover, vitiligo is significantly associated with positive clinical response, but the mechanism is not understood. Therefore, the establishment of a relevant animal model in which melanoma and vitiligo spontaneously develop stepwise may be useful for better understanding of the parameters involved in the destruction of both benign and malignant melanocytes. In a previous work, we established a mouse model for melanoma in which MT/ret transgenic mice express the ret oncogene fused to the metallothionein promoter. Here we report that melanoma leads to spontaneous vitiligo. We further investigate, for the first time in this model, the natural antitumor T-cell response and evaluate the role of cellular immunity in the development of the disease. Interestingly, the occurrence of spontaneous tumor nodules in MT/ret mice with melanoma-associated vitiligo is significantly delayed when compared in melanoma mice without vitiligo. Moreover, a significant proportion of mice with melanoma-associated vitiligo resisted a challenge with syngeneic melanoma cells in contrast to animals without vitiligo. Our results confirm that vitiligo is associated with clinical benefit and further demonstrate the crucial role of CD8+ T cells for tumor control in melanoma-associated vitiligo.

T cells contribute to tumor progression by favoring pro-tumoral properties of intra-tumoral myeloid cells in a mouse model for spontaneous melanoma.

Tumors affect myelopoeisis and induce the expansion of myeloid cells with immunosuppressive activity. In the MT/ret model of spontaneous metastatic melanoma, myeloid cells are the most abundant tumor infiltrating hematopoietic population and their proportion is highest in the most aggressive cutaneous metastasis. Our data suggest that the tumor microenvironment favors polarization of myeloid cells into type 2 cells characterized by F4/80 expression, a weak capacity to secrete IL-12 and a high production of arginase. Myeloid cells from tumor and spleen of MT/ret mice inhibit T cell proliferation and IFNγ secretion. Interestingly, T cells play a role in type 2 polarization of myeloid cells. Indeed, intra-tumoral myeloid cells from MT/ret mice lacking T cells are not only less suppressive towards T cells than corresponding cells from wild-type MT/ret mice, but they also inhibit more efficiently melanoma cell proliferation. Thus, our data support the existence of a vicious circle, in which T cells may favor cancer development by establishing an environment that is likely to skew myeloid cell immunity toward a tumor promoting response that, in turn, suppresses immune effector cell functions.

Dacarbazine promotes stromal remodeling and lymphocyte infiltration in cutaneous melanoma lesions.

Dacarbazine (DTIC) is the standard first-line drug for advanced stage melanoma, but it induces objective clinical responses in only 15% of patients. This study was designed to identify molecular changes specifically induced by treatment in chemo-sensitive lesions. Using global transcriptome analysis and immunohistochemistry, we analyzed cutaneous metastases resected from patients with melanoma before and after DTIC treatment. The treatment induced similar functional changes in different lesions from the same patient. Stromal and immune response-related genes were the most frequently upregulated, particularly in lesions that responded to treatment by stabilizing or regressing. T-cell infiltration and enhanced major histocompatibility complex class II expression were observed in a subset of patients. Stable, chemo-sensitive lesions exhibited activation of genetic programs related to extracellular matrix remodeling, including increased expression of secreted protein acidic and rich in cysteine (SPARC) by tumor cells. These events were associated with local response to treatment and with superior survival in our group of patients. In contrast, SPARC expression was downregulated in lesions resistant to DTIC. Thus, chemotherapy drugs originally selected for their direct cytotoxicity to tumor cells may also influence disease progression by inducing changes in the tumor microenvironment.

Progressive upregulation of PD-1 in primary and metastatic melanomas associated with blunted TCR signaling in infiltrating T lymphocytes.

Programmed death-1 (PD-1) is involved in T-cell tolerance to self-antigens. For some cancers, it has been suggested that the expression of a ligand of PD-1, namely PD-L1, could contribute to tumor escape from immune destruction. Nevertheless, the relationship between PD-1 expression on tumor-infiltrating T lymphocytes (TILs), disease stage, and TIL responsiveness is still poorly documented. In this study, we show that freshly isolated CD4(+) and CD8(+) TILs express substantial levels of PD-1 in primary melanomas. The expression of PD-1 was further increased at later stages in distant cutaneous metastases, especially on CD8(+) TILs. The expression of PD-1 ligands was frequent only in metastases, on both tumor cells and tumor-derived myeloid cells. TILs isolated from these cutaneous tumors are poorly reactive ex vivo, with blunted calcium response and IFN-γ production after TCR stimulation. Surprisingly, in distinct parts of a primary melanoma, either invasive or regressing, we show that TILs similarly express PD-1 and remain dysfunctional. The expressions of PD-1 and PD-L1 in metastatic melanoma lesions could be considered as witnesses of an unsuccessful anti-tumoral immune response, but the direct involvement of PD-1 in the severity of the disease, and the importance of TILs in tumor regression, remain to be established.

Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors.

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.

Distinct role for CD8 T cells toward cutaneous tumors and visceral metastases.

The growth of immunogenic tumors in immunocompetent individuals is one of the oldest conundrums in tumor immunology. Although the ability of mouse CD8+ T cells to control transplanted tumors is well documented, little is known about their impact on autochthonous tumors. To gain insight into the role of CD8+ T cells during the course of cancer development, we produced a novel model of spontaneous melanoma. The metallothionein (MT)-ret/AAD mouse is transgenic for the RET oncogene and the chimeric MHC molecule AAD (alpha1-alpha2 domains of HLA-A2 linked to alpha3 domain of H2-Dd). This model recapitulates the natural history of human melanoma, and expression of the AAD molecule makes it suitable for analyzing CD8+ T cell responses directed against peptide Ags that have been previously identified in HLA-A2+ melanoma patients. We found that, as tumors grow, mice develop a broad melanoma-specific CD8+ T cell response. Occurrence of cutaneous nodules is not affected by CD8+ T cell depletion, showing that although CD8+ T cells are functional, they have no effect on established cutaneous tumors. However, depleted mice die from visceral disease much earlier than controls, showing that CD8+ T cells control metastasis spreading and disease progression. Antigenic modulation is observed in visceral metastases, suggesting that visceral nodules may be subject to immunoediting. Our data demonstrate that growth of melanoma in the MT-ret/AAD model involves several tolerance mechanisms sequentially. They also reveal a different role for CD8+ T cells toward early stage of cutaneous tumors and late visceral metastatic stage of the disease.