Long-Term Survival and Immune Response Dynamics in Melanoma Patients Undergoing TAPCells-Based Vaccination Therapy.

Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.

Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

BACKGROUND: Gallbladder cancer (GBC) is a prevalent and deadly biliary tract carcinoma, often diagnosed at advanced stages with limited treatment options. The 5-year survival rate varies widely from 4 to 60%, mainly due to differences in disease stage detection. With only a small fraction of patients having resectable tumors and a high incidence of metastasis, advanced GBC stages are characterized by significant chemoresistance. Identification of new therapeutic targets is crucial, and recent studies have shown that the Endothelin-1 (ET-1) signaling pathway, involving ETAR and/or ETBR receptors (ETRs), plays a crucial role in promoting tumor aggressiveness in various cancer models. Blocking one or both receptors has been reported to reduce invasiveness and chemoresistance in cancers like ovarian, prostate, and colon. Furthermore, transcriptomic studies have associated ET-1 levels with late stages of GBC; however, it remains unclear whether its signaling or its inhibition has implications for its aggressiveness. Although the role of ET-1 signaling in gallbladder physiology is minimally understood, its significance in other tumor models leads us to hypothesize its involvement in GBC malignancy. RESULTS: In this study, we investigated the expression of ET-1 pathway proteins in three GBC cell lines and a primary GBC culture. Our findings demonstrated that both ETAR and ETBR receptors are expressed in GBC cells and tumor samples. Moreover, we successfully down-regulated ET-1 signaling using a non-selective ETR antagonist, Macitentan, which resulted in reduced migratory and invasive capacities of GBC cells. Additionally, Macitentan treatment chemosensitized the cells to Gemcitabine, a commonly used therapy for GBC. CONCLUSION: For the first time, we reveal the role of the ET-1 pathway in GBC cells, providing insight into the potential therapeutic targeting of its receptors to mitigate invasion and chemoresistance in this cancer with limited treatment options. These findings pave the way for further exploration of Macitentan or other ETR antagonists as potential therapeutic strategies for GBC management. In summary, our study represents a groundbreaking contribution to the field by providing the first evidence of the ET 1 pathway's pivotal role in modulating the behavior and aggressiveness of GBC cells, shedding new light on potential therapeutic targets.

Co-Expression of Immunohistochemical Markers MRP2, CXCR4, and PD-L1 in Gallbladder Tumors Is Associated with Prolonged Patient Survival.

Gallbladder cancer (GBC) is a rare pathology in Western countries. However, it constitutes a relevant health problem in Asia and Latin America, with a high mortality in middle-aged Chilean women. The limited therapeutic options for GBC require the identification of targetable proteins with prognostic value for improving clinical management support. We evaluated the expression of targetable proteins, including three epithelial tumor markers, four proteins associated with multidrug and apoptosis resistance, and eleven immunological markers in 241 primary gallbladder adenocarcinomas. We investigated correlations between tumor marker expression, the primary tumor staging, and GBC patients' survival using automated immunohistochemistry, a semi-automatic method for image analysis, univariate and multivariate statistical analyses, and machine learning algorithms. Our data show a significant association between the expression of MRP2 (p = 0.0028), CXCR4 (p = 0.0423), and PD-L1 (p = 0.0264), and a better prognosis for patients with late-stage primary tumors. The expression of the MRP2/CXCR4/PD-L1 cluster of markers discriminates among short-, medium-, and long-term patient survival, with an ROC of significant prognostic value (AUC = 0.85, p = 0.0012). Moreover, a high MRP2/CXCR4/PD-L1 co-expression is associated with increased survival time (30 vs. 6 months, p = 0.0025) in GBC patients, regardless of tumor stage. Hence, our results suggest that the MRP2/CXCR4/PD-L1 cluster could potentially be a prognostic marker for GBC.

Whole tumour cell-based vaccines: tuning the instruments to orchestrate an optimal antitumour immune response.

Immunotherapy, particularly those based on immune checkpoint inhibitors (ICIs), has become a useful approach for many neoplastic diseases. Despite the improvements of ICIs in supporting tumour regression and prolonging survival, many patients do not respond or develop resistance to treatment. Thus, therapies that enhance antitumour immunity, such as anticancer vaccines, constitute a feasible and promising therapeutic strategy. Whole tumour cell (WTC) vaccines have been extensively tested in clinical studies as intact or genetically modified cells or tumour lysates, injected directly or loaded on DCs with distinct adjuvants. The essential requirements of WTC vaccines include the optimal delivery of a broad battery of tumour-associated antigens, the presence of tumour cell-derived molecular danger signals, and adequate adjuvants. These factors trigger an early and robust local innate inflammatory response that orchestrates an antigen-specific and proinflammatory adaptive antitumour response capable of controlling tumour growth by several mechanisms. In this review, the strengths and weaknesses of our own and others' experiences in studying WTC vaccines are revised to discuss the essential elements required to increase anticancer vaccine effectiveness.

Preclinical evaluation of chimeric antigen receptor T cells targeting the carcinoembryonic antigen as a potential immunotherapy for gallbladder cancer.

Gallbladder cancer (GBC) is commonly diagnosed at late stages when conventional treatments achieve only modest clinical benefit. Therefore, effective treatments for advanced GBC are needed. In this context, the administration of T cells genetically engineered with chimeric antigen receptors (CAR) has shown remarkable results in hematological cancers and is being extensively studied for solid tumors. Interestingly, GBC tumors express canonical tumor-associated antigens, including the carcinoembryonic antigen (CEA). However, the potential of CEA as a relevant antigen in GBC to be targeted by CAR-T cell-based immunotherapy has not been addressed. Here we show that CEA was expressed in 88% of GBC tumors, with higher levels associated with advanced disease stages. CAR-T cells specifically recognized plate-bound CEA as evidenced by up-regulation of 4-1BB, CD69 and PD-1, and production of effector cytokines IFN-γ and TNF-α. In addition, CD8+ CAR-T cells up-regulated the cytotoxic molecules granzyme B and perforin. Interestingly, CAR-T cell activation occurred even in the presence of PD-L1. Consistent with these results, CAR-T cells efficiently recognized GBC cell lines expressing CEA and PD-L1, but not a CEA-negative cell line. Furthermore, CAR-T cells exhibited in vitro cytotoxicity and reduced in vivo tumor growth of GB-d1 cells. In summary, we demonstrate that CEA represents a relevant antigen for GBC that can be targeted by CAR-T cells at the preclinical level. This study warrants further development of the adoptive transfer of CEA-specific CAR-T cells as a potential immunotherapy for GBC.

A2B Adenosine Receptor Enhances Chemoresistance of Glioblastoma Stem-Like Cells under Hypoxia: New Insights into MRP3 Transporter Function.

Glioblastoma is the most common and aggressive primary brain tumor, characterized by its high chemoresistance and the presence of a cell subpopulation that persists under hypoxic niches, called glioblastoma stem-like cells (GSCs). The chemoresistance of GSCs is mediated in part by adenosine signaling and ABC transporters, which extrude drugs outside the cell, such as the multidrug resistance-associated proteins (MRPs) subfamily. Adenosine promotes MRP1-dependent chemoresistance under normoxia. However, adenosine/MRPs-dependent chemoresistance under hypoxia has not been studied until now. Transcript and protein levels were determined by RT-qPCR and Western blot, respectively. MRP extrusion capacity was determined by intracellular 5 (6)-Carboxyfluorescein diacetate (CFDA) accumulation. Cell viability was measured by MTS assays. Cell cycle and apoptosis were determined by flow cytometry. Here, we show for the first time that MRP3 expression is induced under hypoxia through the A2B adenosine receptor. Hypoxia enhances MRP-dependent extrusion capacity and the chemoresistance of GSCs. Meanwhile, MRP3 knockdown decreases GSC viability under hypoxia. Downregulation of the A2B receptor decreases MRP3 expression and chemosensibilizes GSCs treated with teniposide under hypoxia. These data suggest that hypoxia-dependent activation of A2B adenosine receptor promotes survival of GSCs through MRP3 induction.

Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells.

Damage-associated molecular patterns (DAMPs) play a critical role in dendritic cells (DCs) ability to trigger a specific and efficient adaptive immune response for different physiological and pathological scenarios. We have previously identified constitutive DAMPs (HMGB1 and Calreticulin) as well as new putative inducible DAMPs such as Haptoglobin (HP), from a therapeutically used heat shock-conditioned melanoma cell lysate (called TRIMEL). Remarkably, HP was shown to be the most abundant protein in the proteomic profile of heat shock-conditioned TRIMEL samples. However, its relative contribution to the observed DCs phenotype has not been fully elucidated. Human DCs were generated from monocytes isolated from PBMC of melanoma patients and healthy donors. DC lineage was induced with rhIL-4 and rhGM-CSF. After additional stimulation with HP, the proteome of these HP-stimulated cells was characterized. In addition, DCs were phenotypically characterized by flow cytometry for canonical maturation markers and cytokine production. Finally, in vitro transmigration capacity was assessed using Transwell plates. Our results showed that the stimulation with HP was associated with the presence of exclusive and higher relative abundance of specific immune-; energy production-; lipid biosynthesis-; and DAMPs-related proteins. Importantly, HP stimulation enhanced the expression of specific DC maturation markers and pro-inflammatory and Th1-associated cytokines, and an in vitro transmigration of primary human DCs. Taken together, these data suggest that HP can be considered as a new inducible DAMP with an important role in in vitro DC activation for cancer immunotherapy.

Dysregulated Immune Responses in COVID-19 Patients Correlating With Disease Severity and Invasive Oxygen Requirements.

The prognosis of severe COVID-19 patients has motivated research communities to uncover mechanisms of SARS-CoV-2 pathogenesis also on a regional level. In this work, we aimed to understand the immunological dynamics of severe COVID-19 patients with different degrees of illness, and upon long-term recovery. We analyzed immune cellular subsets and SARS-CoV-2-specific antibody isotypes of 66 COVID-19 patients admitted to the Hospital Clínico Universidad de Chile, which were categorized according to the WHO ten-point clinical progression score. These included 29 moderate patients (score 4-5) and 37 severe patients under either high flow oxygen nasal cannula (18 patients, score 6), or invasive mechanical ventilation (19 patients, score 7-9), plus 28 convalescent patients and 28 healthy controls. Furthermore, six severe patients that recovered from the disease were longitudinally followed over 300 days. Our data indicate that severe COVID-19 patients display increased frequencies of plasmablasts, activated T cells and SARS-CoV-2-specific antibodies compared to moderate and convalescent patients. Remarkably, within the severe COVID-19 group, patients rapidly progressing into invasive mechanical ventilation show higher frequencies of plasmablasts, monocytes, eosinophils, Th1 cells and SARS-CoV-2-specific IgG than patients under high flow oxygen nasal cannula. These findings demonstrate that severe COVID-19 patients progressing into invasive mechanical ventilation show a distinctive type of immunity. In addition, patients that recover from severe COVID-19 begin to regain normal proportions of immune cells 100 days after hospital discharge and maintain high levels of SARS-CoV-2-specific IgG throughout the study, which is an indicative sign of immunological memory. Thus, this work can provide useful information to better understand the diverse outcomes of severe COVID-19 pathogenesis.

C-type lectin receptors MR and DC-SIGN are involved in recognition of hemocyanins, shaping their immunostimulatory effects on human dendritic cells.

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1-biased cell-mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose-rich structures. It remains unclear whether these structures influence hemocyanin-induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C-type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose-binding CLRs on monocyte-derived human DCs: MR (mannose receptor) and DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose-sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC-SIGN, and were partly internalized through clathrin-mediated endocytosis. The hemocyanin-mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC-SIGN in a carbohydrate-dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose-sensitive CLRs, initiating an immune response and promoting a Th1 cell-driving potential.

CD73 Ectonucleotidase Restrains CD8+ T Cell Metabolic Fitness and Anti-tumoral Activity.

CD39 and CD73 are ectoenzymes that dephosphorylate ATP into its metabolites; ADP, AMP, and adenosine, and thus are considered instrumental in the development of immunosuppressive microenvironments. We have previously shown that within the CD8+ T cell population, naïve and memory cells express the CD73 ectonucleotidase, while terminally differentiated effector cells are devoid of this enzyme. This evidence suggests that adenosine might exert an autocrine effect on CD8+ T cells during T cell differentiation. To study the possible role of CD73 and adenosine during this process, we compared the expression of the adenosinergic signaling components, the phenotype, and the functional properties between CD73-deficient and WT CD8+ T cells. Upon activation, we observed an upregulation of CD73 expression in CD8+ T cells along with an upregulation of the adenosine A2A receptor. Interestingly, when we differentiated CD8+ T cells to Tc1 cells in vitro, we observed that these cells produce adenosine and that CD73-deficient cells present a higher cytotoxic potential evidenced by an increase in IFN-γ, TNF-α, and granzyme B production. Moreover, CD73-deficient cells presented a increased glucose uptake and higher mitochondrial respiration, indicating that this ectonucleotidase restrict the mitochondrial capacity in CD8+ T cells. In agreement, when adoptively transferred, antigen-specific CD73-deficient CD8+ T cells were more effective in reducing the tumor burden in B16.OVA melanoma-bearing mice and presented lower levels of exhaustion markers than wild type cells. All these data suggest an autocrine effect of CD73-mediated adenosine production, limiting differentiation and cytotoxic T cells' metabolic fitness.

The Evaluation of 17 Gastrointestinal Tumor Markers Reveals Prognosis Value for MUC6, CK17, and CD10 in Gallbladder-Cancer Patients.

Gallbladder cancer (GBC) is an aggressive and highly lethal disease with relatively low global incidence, but one that constitutes a major health problem in Asian and Latin American countries, particularly in Chile. The identification of new tumor-associated markers with potential prognosis value is required for GBC clinical practice. Using immunohistochemistry/tumor tissue microarray, we evaluated the expression of 17 gastrointestinal tumor-associated protein markers (CK7, CK17, CK19, CK20, CKLMW, CKHMW, MUC1, MUC2, MUC5AC, MUC6, CA125, CD10, CEA, vimentin, villin, claudin-4, and CDX2) in primary gallbladder adenocarcinomas from 180 Chilean patients and analyzed potential associations with their pathological and clinical characteristics. Younger female patients with well- to moderately differentiated tumors had a better prognosis than that of older female or male patients with tumors with a similar tumor differentiation grade. Among all analyzed markers, MUC6 expression was associated with better prognosis in patients with well- to moderately differentiated tumors, whereas CK17 or CD10 was associated with worse prognosis in patients with poorly differentiated tumors. In addition, the MUC6+CK17- expression pattern was strongly associated with better prognosis in patients with well- to moderately differentiated tumors, whereas patients with poorly differentiated tumors and with the CK17+CD10+ expression pattern showed worse prognosis. Our results suggest that tumor MUC6, CK17, and CD10 can be considered as potential prognosis markers for GBC.

Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cells.

Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological processes, including in anti-tumor immune responses. Here, we described a simple flow cytometry method to test in vitro antigen-dependent GJ-mediated cell-to-cell coupling between cytotoxic T cells and target melanoma cells.

Hypoxic Melanoma Cells Deliver microRNAs to Dendritic Cells and Cytotoxic T Lymphocytes through Connexin-43 Channels.

Alterations in microRNA (miRNA) profiles, induced by tumor microenvironment stressors, like hypoxia, allow cancer cells to acquire immune-resistance phenotypes. Indeed, hypoxia-induced miRNAs have been implicated in cancer progression through numerous cancer cell non-autonomous mechanisms, including the direct transfer of hypoxia-responsive miRNA from cancer to immune cells via extracellular vesicles. Connexin-43 (Cx43)-constituted gap junctions (GJs) have also been involved in miRNA intercellular mobilization, in other biological processes. In this report, we aimed to evaluate the involvement of Cx43-GJs in the shift of miRNAs induced by hypoxia, from hypoxic melanoma cells to dendritic cells and melanoma-specific cytotoxic T lymphocytes (CTLs). Using qRT-PCR arrays, we identified that miR-192-5p was strongly induced in hypoxic melanoma cells. Immune cells acquired this miRNA after co-culture with hypoxic melanoma cells. The transfer of miR-192-5p was inhibited when hypoxic melanoma cells expressed a dominant negative Cx43 mutant or when Cx43 expression was silenced using specific short-hairpin RNAs. Interestingly, miR-192-5p levels on CTLs after co-culture with hypoxic melanoma cells were inversely correlated with the cytotoxic activity of T cells and with ZEB2 mRNA expression, a validated immune-related target of miR-192-5p, which is also observed in vivo. Altogether, our data suggest that hypoxic melanoma cells may suppress CTLs cytotoxic activity by transferring hypoxia-induced miR-192-5p through a Cx43-GJs driven mechanism, constituting a resistance strategy for immunological tumor escape.

A heat-shocked melanoma cell lysate vaccine enhances tumor infiltration by prototypic effector T cells inhibiting tumor growth.

BACKGROUND: Immune checkpoint blocker (ICB) therapy has shown survival benefits for some patients with cancer. Nevertheless, many individuals remain refractory or acquire resistance to treatment, motivating the exploration of complementary immunotherapies. Accordingly, cancer vaccines offer an attractive alternative. Optimal delivery of multiple tumor-associated antigens combined with potent adjuvants seems to be crucial for vaccine effectiveness. METHODS: Here, a prototype for a generic melanoma vaccine, named TRIMELVax, was tested using B16F10 mouse melanoma model. This vaccine is made of heat shock-treated tumor cell lysates combined with the Concholepas concholepas hemocyanin as adjuvant. RESULTS: While B16F10 lysate provides appropriate melanoma-associated antigens, both a generic human melanoma cell lysate and hemocyanin adjuvant contributes with danger signals promoting conventional dendritic type 1 cells (cDC1), activation, phagocytosis and effective antigen cross-presentation. TRIMELVax inhibited tumor growth and increased mice survival, inducing cellular and humoral immune responses. Furthermore, this vaccine generated an increased frequency of intratumor cDC1s but not conventional type 2 dendritic cells (cDC2s). Augmented infiltration of CD3+, CD4+ and CD8+ T cells was also observed, compared with anti-programmed cell death protein 1 (PD-1) monotherapy, while TRIMELVax/anti-PD-1 combination generated higher tumor infiltration of CD4+ T cells. Moreover, TRIMELVax promoted an augmented proportion of PD-1lo CD8+ T cells in tumors, a phenotype associated with prototypic effector cells required for tumor growth control, preventing dysfunctional T-cell accumulation. CONCLUSIONS: The therapeutic vaccine TRIMELVax efficiently controls the weakly immunogenic and aggressive B16F10 melanoma tumor growth, prolonging tumor-bearing mice survival even in the absence of ICB. The strong immunogenicity shown by TRIMELVax encourages clinical studies in patients with melanoma.

Connexin-Mediated Signaling at the Immunological Synapse.

The immunological synapse (IS) is an intercellular communication platform, organized at the contact site of two adjacent cells, where at least one is an immune cell. Functional IS formation is fundamental for the modulation of the most relevant immune system activities, such as T cell activation by antigen presenting cells and T cell/natural killer (NK) cell-mediated target cell (infected or cancer) killing. Extensive evidence suggests that connexins, in particular connexin-43 (Cx43) hemichannels and/or gap junctions, regulate signaling events in different types of IS. Although the underlying mechanisms are not fully understood, the current evidence suggests that Cx43 channels could act as facilitators for calcium ions, cyclic adenosine monophosphate, and/or adenosine triphosphate uptake and/or release at the interface of interacting cells. These second messengers have relevant roles in the IS signaling during dendritic cell-mediated T and NK cell activation, regulatory T cell-mediated immune suppression, and cytotoxic T lymphocyte or NK cell-mediated target tumor cell killing. Additionally, as the cytoplasmic C-terminus domain of Cx43 interacts with a plethora of proteins, Cx43 may act as scaffolds for integration of various regulatory proteins at the IS, as suggested by the high number of Cx43-interacting proteins that translocate at these cell-cell interface domains. In this review, we provide an updated overview and analysis on the role and possible underlying mechanisms of Cx43 in IS signaling.

Dendritic Cells Loaded with Heat Shock-Conditioned Ovarian Epithelial Carcinoma Cell Lysates Elicit T Cell-Dependent Antitumor Immune Responses In Vitro.

Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mortality in advanced stages where conventional therapies are not effective. Based on the role of the immune system in the progression of this disease, immunotherapy using checkpoint blockade has been considered as a therapeutic alternative. Nevertheless, its results do not match up to the positive results in entities like melanoma and other malignancies, suggesting the need to find other therapies to be used alone or in combination. Dendritic cell- (DC-) based vaccines have shown promising results in several types of cancer, such as melanoma, prostate, and lung cancers, due to the essential role played by DCs in the activation of specific T cells, thus using other ways of activating the immune response than immune checkpoint blockade. During the last decade, we have used DC-based vaccines loaded with an allogeneic heat shock-conditioned melanoma cell lysate in the treatment of advanced stage patients in a series of clinical trials. In these studies, 60% of treated patients showed immunological responses which correlated positively with improved survival. Considering the relevance of ovarian cancer and the promising results of our DC-based vaccine, we show here that heat shock-conditioned cell lysates derived from ovarian epithelial carcinoma cell lines have the potential to induce the phenotypic and functional maturation of human DC, which in turn, is able to induce an efficient CD4+ and CD8+ T cell-mediated immune responses against ovarian cancer cell lines in vitro. In summary, OEC heat shock-conditioned cell lysate-loaded DCs may be considered for future combined immunotherapy approaches against ovarian tumors.

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing.

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell-tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.

Interleukin-8 Secreted by Glioblastoma Cells Induces Microvascular Hyperpermeability Through NO Signaling Involving S-Nitrosylation of VE-Cadherin and p120 in Endothelial Cells.

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

Coagulation Factor Xa Promotes Solid Tumor Growth, Experimental Metastasis and Endothelial Cell Activation.

Hypercoagulable state is linked to cancer progression; however, the precise role of the coagulation cascade is poorly described. Herein, we examined the contribution of a hypercoagulative state through the administration of intravenous Coagulation Factor Xa (FXa), on the growth of solid human tumors and the experimental metastasis of the B16F10 melanoma in mouse models. FXa increased solid tumor volume and lung, liver, kidney and lymph node metastasis of tail-vein injected B16F10 cells. Concentrating on the metastasis model, upon coadministration of the anticoagulant Dalteparin, lung metastasis was significantly reduced, and no metastasis was observed in other organs. FXa did not directly alter proliferation, migration or invasion of cancer cells in vitro. Alternatively, FXa upon endothelial cells promoted cytoskeleton contraction, disrupted membrane VE-Cadherin pattern, heightened endothelial-hyperpermeability, increased inflammatory adhesion molecules and enhanced B16F10 adhesion under flow conditions. Microarray analysis of endothelial cells treated with FXa demonstrated elevated expression of inflammatory transcripts. Accordingly, FXa treatment increased immune cell infiltration in mouse lungs, an effect reduced by dalteparin. Taken together, our results suggest that FXa increases B16F10 metastasis via endothelial cell activation and enhanced cancer cell-endothelium adhesion advocating that the coagulation system is not merely a bystander in the process of cancer metastasis.

Dexamethasone turns tumor antigen-presenting cells into tolerogenic dendritic cells with T cell inhibitory functions.

BACKGROUND: Dendritic cells (DCs) are usually immunogenic, but they are also capable of inducing tolerance under anti-inflammatory conditions. Immunotherapy based on autologous DCs loaded with an allogeneic melanoma cell lysate (TRIMEL/DCs) induces immunological responses and increases melanoma patient survival. Glucocorticoids can suppress DC maturation and function, leading to a DC-mediated inhibition of T cell responses. METHODS: The effect of dexamethasone, a glucocorticoid extensively used in cancer therapies, on TRIMEL/DCs phenotype and immunogenicity was examined. RESULTS: Dexamethasone induced a semi-mature phenotype on TRIMEL/DC with low maturation surface marker expressions, decreased pro-inflammatory cytokine induction (IL-1ß and IL-12) and increased release of regulatory cytokines (IL-10 and TGF-ß). Dexamethasone-treated TRIMEL/DCs inhibited allogeneic CD4+ T cell proliferation and cytokine release (IFNγ, TNF-α and IL-17). Co-culturing melanoma-specific memory tumor-infiltrating lymphocytes with dexamethasone-treated TRIMEL/DC inhibited proliferation and effector T cell activities, including cytokine secretion and anti-melanoma cytotoxicity. CONCLUSIONS: These findings suggest that dexamethasone repressed melanoma cell lysate-mediated DC maturation, generating a potent tolerogenic-like DC phenotype that inhibited melanoma-specific effector T cell activities. These results suggest that dexamethasone-induced immunosuppression may interfere with the clinical efficacy of DC-based melanoma vaccines, and must be taken into account for optimal design of cellular therapy against cancer.