Supplementary Figures S1-S4; Supplementary Table S1.

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production and activation of the A3 Adenosine Receptor (A3AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A3AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A3AR on cell migration/invasion was evaluated using the A3AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A3AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAP-dependent activation of A3AR under hypoxia.

Creutzfeldt-Jakob disease has a higher incidence in Chile than in other countries. The postmortem pathological characterization of brain tissue is necessary to reach a definitive diagnosis. We report a 73 years old man with a history compatible with of a rapidly progressive dementia, in which the first electroencephalographic study showed a pattern consistent with non-convulsive status epilepticus. Besides discarding this diagnosis, it was necessary to rule out other causes of rapidly progressive dementia such as Hashimoto encephalopathy. Finally, the sustained clinical deterioration with no response to anticonvulsants and corticosteroids, the imaging studies, a serial electroencephalographic monitoring study and the detection of 14-3-3 protein in cerebrospinal fluid were the keys to achieve the diagnosis of the disease.

Creutzfeldt-Jakob disease has a higher incidence in Chile than in other countries. The post mortem pathological characterization of brain tissue is necessary to reach a definitive diagnosis. We report a 73 years old man with a history compatible with of a rapidly progressive dementia, in which the first electroencephalographic study showed a pattern consistent with non-convulsive status epilepticus. Besides discarding this diagnosis, it was necessary to rule out other causes of rapidly progressive dementia such as Hashimoto encephalopathy. Finally, the sustained clinical deterioration with no response to anticonvulsants and corticosteroids, the imaging studies, a serial electroencephalographic monitoring study and the detection of 14-3-3 protein in cerebrospinal fluid were the keys to achieve the diagnosis of the disease.

Background: Melanocortin 1 Receptor (MC1R) is expressed in a majority of melanoma biopsies and cell lines. We previously demonstrated that three hydrophobic low-affinity HLA-A2-restricted MC1R-derived peptides: MC1R(291-298), MC1 R244-252 and Mc1 R283-291 can elicit cytotoxic T-lymphocytes (CTL) responses from normal donor peripheral blood lymphocytes (PBL). Moreover, peptide-specific CTL recognized a panel of MHC-matched melanomas, demonstrating that human melanoma cell lines naturally present MC1R epitopes. However, the natural presence of MC1R-specific T cells in melanoma patient's tumour and blood remains unknown.Methods: The presence of anti-MC1R specific CD8(+) T cells was established in a population of melanoma-specific T cells derived from peripheral blood mononuclear cells (PBMC) and tumour-infiltrating lymphocytes (TIL) from HLA-A2(+) melanoma patients.Results: CTLs specific for the three MC1R-derived peptides that lysed allogeneic HLA-A2(+)MC1R(+) melanomas were elicited from PBMC, demonstrating the existence of an anti-MC1R T cell repertoire in melanoma patients. Moreover, TILs also recognized MC1R epitopes and HLA-A2(+) melanoma cell lines. Finally, HLA-A2/MC1R(244)-specific CD8(+) T cell clones derived from TILs and a subset of MCI R-291 specific TILs were identified using HLA-A2/MC1R tetramers.Conclusion: Our results demonstrate that MC1R-derived peptides are common immunogenic epitopes for melanoma-specific CTLs and TILs, and may thus be useful for the development of anti-melanoma immunotherapy. (C) 2013 Elsevier GmbH. All rights reserved.

Abstract Purpose: This study characterizes, biologically and clinically, a novel type of dendritic cells (DC) produced in the short term and called tumor antigen–presenting cells (TAPCells). In particular, we identified factors present in a lysate derived from heat-shocked allogeneic melanoma cells (TRIMEL) that are associated with TAPCells' enhanced capability to induce CD8+ T-cell responses in vitro and in vaccinated melanoma patients. Experimental Design: First, extensive phenotypic and functional characterization of TAPCells was performed, followed by vaccination of 45 melanoma patients with four doses of TAPCells over a period of 2 months. Specific delayed-type hypersensitivity (DTH) reaction was analyzed posttreatment and correlated with overall survival rates. Furthermore, heat-shock (HS)-induced factors present in TRIMEL and their effects on DC activation were identified and studied. Results: TRIMEL induced a committed, mature, DC-like phenotype in TAPCells and effectively activated melanoma-specific CD4+ and CD8+ T cells. Clinically, 64% of vaccinated patients showed positive DTH reaction against TRIMEL, and this was associated with improved overall survival. HS treatment of tumor cells increased calreticulin (CRT) plasma membrane translocation and induced the release of high-mobility group box 1 proteins (HMGB1). Both CRT and HMGB1 mobilization were associated with enhanced TAPCells' maturation and antigen (Ag) cross-presentation, respectively. DTH infiltration analysis revealed the presence of CD8+/CD45RO+ T cells, thus confirming TAPCells' ability to cross-present Ags in vivo. Conclusions: Our results indicate that lysates derived from heat-shocked tumor cells are an optimal source of tumor-associated Ags, which are crucial for the generation of DCs with improved Ag cross-presentation capacity and clinically effective immunogenicity. Clin Cancer Res; 17(8); 2474–83. ©2011 AACR.

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation. The Journal of Immunology, 2011, 187: 3121-3132.

BACKGROUND Gastrointestinal stromal tumors (GIST) are the most common mesenchymatous tumors of the digestive tract. The pathological diagnosis is based on microscopy and immunohistochemistiy. AIM To review the experience of our surgical unit in patients with GIST MATERIAL AND METHODS: Review of medical records of 15 patients (aged 66+/-13 years, 11 women), with a pathological diagnosis of GIST, treated between 1999 and 2005. RESULTS The main presenting symptoms were melena in 40%, hematemesis in 20%, abdominal pain in 60% and anemia in 13%. In only one patient, the tumor appeared as an incidentaloma. All patients underwent upper gastrointestinal endoscopy A CAT scan was done in 87%, a barium swallow in 60% and a digestive endosonography in 20%. Thirteen tumors were located in the stomach and two in the small bowel. Mean tumor diameter was 5.3+/-1.7 cm. Surgical management was a tumor resection in 40%, a partial gastrectomy in 27%, a total gastrectomy in 20% and an intestinal excision in the rest. Mean hospital stay was 6.9+/-4.2 days. No postoperative complications were recorded. CONCLUSIONS The main clinical presentation of GIST in this retrospective series was an upper gastrointestinal bleeding. Surgical treatment was devoid of complications.

PURPOSE:Uveal melanoma is the most common primary malignant ocular cancer in adults. This tumor has a distinct expression pattern of markers compared with cutaneous melanoma. MC1R is under study as a potential target for antitumor immunity. Because of the potential immunogenicity of MC1R, it is important to evaluate its expression on uveal melanomas.METHODS:Two novel monoclonal antibodies (MP1.1C11 and MP1.1B7) were used to examine the expression of MC1R in uveal melanomas. Tissue samples obtained from 17 patients were analyzed for expression of MC1R by immunohistochemistry. Additionally, uveal melanoma cell lines were treated with proinflammatory cytokines, after which MC1R cell surface expression was analyzed by flow cytometry.RESULTS:Results demonstrated that MC1R is expressed by uveal melanoma to a significantly greater extent than other melanoma markers. With the use of MP1.1C11 or MP1.1B7, MC1R was detected in 95% of the tested melanoma tissues, including one liver metastasis. In contrast, MART-1, S100-specific protein, and gp-100 were only expressed by 66%, 33%, and 67% of the analyzed samples, respectively. Results also demonstrated that even though MC1R is mainly located intracellularly, its cell surface expression can be promoted by cytokines such as IFN-gamma, TNF-alpha, IL-4, and IL-10.CONCLUSIONS:These observations support the inclusion of MC1R in the panel of markers for the diagnosis of uveal melanoma. Therapeutic use of MC1R-specific antibodies targeting cytokine-induced MC1R potentially requires expression of the target molecule on the surfaces of tumor cells. Data presented here support MC1R as a new marker and a putative therapeutic target for uveal melanoma.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-α (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymatous tumors of the digestive tract. The pathological diagnosis is based on microscopy and immunohistochemistiy.To review the experience of our surgical unit in patients with GIST MATERIAL AND METHODS: Review of medical records of 15 patients (aged 66+/-13 years, 11 women), with a pathological diagnosis of GIST, treated between 1999 and 2005.The main presenting symptoms were melena in 40%, hematemesis in 20%, abdominal pain in 60% and anemia in 13%. In only one patient, the tumor appeared as an incidentaloma. All patients underwent upper gastrointestinal endoscopy A CAT scan was done in 87%, a barium swallow in 60% and a digestive endosonography in 20%. Thirteen tumors were located in the stomach and two in the small bowel. Mean tumor diameter was 5.3+/-1.7 cm. Surgical management was a tumor resection in 40%, a partial gastrectomy in 27%, a total gastrectomy in 20% and an intestinal excision in the rest. Mean hospital stay was 6.9+/-4.2 days. No postoperative complications were recorded.The main clinical presentation of GIST in this retrospective series was an upper gastrointestinal bleeding. Surgical treatment was devoid of complications.

Immunotherapy has become a novel therapeutic alternative for various kinds of tumours. Recently, we have finalized the first phase I clinical study in Chile for the treatment of advanced malignant melanoma, using dendritic cells (DCs) loaded with allogeneic melanoma cell lysate. This study included 20 patients and the obtained results, pioneer in Latin America, showed that DC-based immunotherapy is innocuous, even provided in combination with IL-2. In addition, immunological responses were detected in 50% of the treated patients, establishing a positive correlation between the delayed type hypersensitivity (DTH) reaction, which indicates induction of in vivo immunological memory, and patients surviving. Nevertheless, objective clinical responses in vaccinated patients are still insufficient. Only sporadic objective metastasis regressions have been registered and an important proportion of the treated patients did not respond, or their responses were weak. Several strategies have been described to be used by tumours to escape from the immune response. Actually, we have demonstrated that IL-10 inhibits antigen presentation in melanoma, reducing tumour sensitivity to melanoma-specific cytotoxic T lymphocytes (CTLs). Regulation of the immunological response by inhibitory cells could be another possible cause of clinical unresponsiveness. Lately, the existence of subpopulations of regulatory T lymphocytes (RTL) able to limit the immune response in a specific form has been established, specially inhibiting the proliferation and activity of CD4+ and CD8+ effector T lymphocytes. These cellular subpopulations, mostly CD4+/CD25+/Foxp3+ T lymphocytes (Treg) of thymic origin, or TR1 lymphocytes able to release IL-10, and tumour growth factor β (TGF-β) producing TH3 lymphocytes, would be accumulated in the body during tumour growth, inhibiting the immune response. In relation to RTL and cancer, evidence indicates that Treg cell numbers are increased in blood and other tissues in different types of cancer. Additionally, it has been demonstrated that in patients with refractory metastatic melanoma, the adoptive transference of anti-tumour CD8+ T lymphocytes after non-myeloablative chemotherapy was able to induce important tumour regressions that would be due to elimination of RTL populations. Additionally, chemotherapeutical drugs like decarbazine, besides their effect on tumour proliferation, also have an immunosuppressive effect on T lymphocyte populations, as well as on accumulated RTL. In this article, a novel strategy for the study of RTL is proposed, including potential therapeutic innovations, which is being pioneered in current clinical trials.

Dendritic cell (DC)-based therapy has proved to be effective in patients with a variety of malignancies. However, an optimal immunization protocol using DCs and the best means for delivering antigens has not yet been described. In this study, 20 patients with malignant melanoma in stages III or IV were vaccinated with autologous DCs pulsed with a melanoma cell lysate, alone (n = 13) or in combination with low doses of subcutaneous (s.c.)interleukin (IL)-2 injections (n = 7), to assess toxicity, immunological and clinical responses. Monocyte-derived DCs were morphological, phenotypic and functionally characterized in vitro. Peripheral blood mononuclear cells (PBMC), harvested from patients either prior to and after the treatment, were analysed using enzyme-linked immunosorbent spot (ELISPOT). After vaccination, 50% of the patients tested (seven of 13) from the first group and (three of seven) from the second, showed an increase in interferon (IFN)-gamma production in response to allogeneic melanoma cell lines but not to controls. Four of five tested human leucocyte antigen (HLA)-A2(+) patients with anti-melanoma activity also showed specific T cell responses against peptides derived from melanoma-associated antigens. Delayed type IV hypersensitivity reaction (DTH) against melanoma cell lysate was observed in six of 13 patients from the group treated with DC vaccines only and four of seven from the group treated with the combination of DCs and IL-2. Significant correlations were found between DTH-positive responses against tumour lysate and both disease stability and post-vaccination survival on the stage IV patients. There were no toxicities associated with the vaccines or evidence of autoimmunity including vitiligo. Furthermore, no significant enhancement was observed as a result of combining DC vaccination with IL-2. Our data suggest that autologous DCs pulsed with tumour lysate may provide a standardized and widely applicable source of melanoma specific antigens for clinical use. It is safe and causes no significant side effects and has been demonstrated to be partially efficient at triggering effective anti-melanoma immunity.