RESUMO
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
Assuntos
Armadilhas Extracelulares/metabolismo , Neoplasias Experimentais/terapia , Receptores de Quimiocinas/agonistas , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Células HT29 , Humanos , Microscopia Intravital/métodos , Células Matadoras Naturais/imunologia , Ligantes , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.
Assuntos
Quimiotaxia/imunologia , Armadilhas Extracelulares/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Acetilcisteína/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Transforming Growth Factor beta (TGF-ß) acts as a tumor suppressor early in carcinogenesis but turns into tumor promoter in later disease stages. In fact, TGF-ß is a known inducer of integrin expression by tumor cells which contributes to cancer metastatic spread and TGF-ß inhibition has been shown to attenuate metastasis in mouse models. However, carcinoma cells often become refractory to TGF-ß-mediated growth inhibition. Therefore identifying patients that may benefit from anti-TGF-ß therapy requires careful selection. METHODS: We performed in vitro analysis of the effects of exposure to TGF-ß in NSCLC cell chemotaxis and adhesion to lymphatic endothelial cells. We also studied in an orthotopic model of NSCLC the incidence of metastases to the lymph nodes after inhibition of TGF-ß signaling, ß3 integrin expression or both. RESULTS: We offer evidences of increased ß3-integrin dependent NSCLC adhesion to lymphatic endothelium after TGF-ß exposure. In vivo experiments show that targeting of TGF-ß and ß3 integrin significantly reduces the incidence of lymph node metastasis. Even more, blockade of ß3 integrin expression in tumors that did not respond to TGF-ß inhibition severely impaired the ability of the tumor to metastasize towards the lymph nodes. CONCLUSION: These findings suggest that lung cancer tumors refractory to TGF-ß monotherapy can be effectively treated using dual therapy that combines the inhibition of tumor cell adhesion to lymphatic vessels with stromal TGF-ß inhibition.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Regulação Neoplásica da Expressão Gênica , Integrina beta3/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Fator de Crescimento Transformador beta1/genética , Animais , Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Integrina beta3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Linfática , Camundongos , Terapia de Alvo Molecular , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.
Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Complemento C5a/fisiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Microambiente Tumoral/imunologia , Adenocarcinoma/sangue , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Carcinoma Pulmonar de Lewis/prevenção & controle , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Complemento C5a/biossíntese , Complemento C5a/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Tolerância Imunológica/genética , Neoplasias Pulmonares/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Microambiente Tumoral/genéticaRESUMO
Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/sangue , Carcinoma Adenoescamoso/metabolismo , Carcinoma de Células Grandes/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Apoptose , Western Blotting , Carcinoma Adenoescamoso/mortalidade , Carcinoma Adenoescamoso/patologia , Carcinoma de Células Grandes/mortalidade , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Ciclo Celular , Proliferação de Células , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Mutação/genética , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Fosforilação , Prognóstico , RNA Interferente Pequeno/genética , Fuso Acromático , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
CD137/TNFR9/41BB was originally described as a surface molecule present on activated T and NK cells. However, its expression is broader among leukocytes, and it is also detected on hypoxic endothelial cells and inflamed blood vessels, as well as in atherosclerotic lesions. Here, we demonstrate that lymphatic endothelial cells (LECs) up-regulate CD137 expression from undetectable baseline levels on stimulation with TNF-α, LPS, and IL-1ß. CD137 cross-linking with an agonistic mAb results in NF-κB nuclear translocation, followed by up-regulation of VCAM and a 3-fold increase in the production of the chemokine CCL21. Accordingly, there is a 50% increase in CCR7-dependent migration toward conditioned medium from activated LECs on CD137 cross-linking with the agonistic mAb or the natural ligand (CD137L). Such an enhancement of cell migration is also observed with monocyte-derived dendritic cells transmigrating across CD137-activated LEC monolayers. Using explanted human dermal tissue, we found that inflamed skin contains abundant CD137(+) lymphatic vessels and that ex vivo incubation of explanted human dermis with TNF-α induces CD137 expression in lymphatic capillaries. More interestingly, treatment with CD137 agonistic antibody induces CCL21 expression and DC accumulation close to lymphatic vessels. Collectively, our results demonstrate that the inflammatory function of lymphatic vessels can be regulated by CD137.
Assuntos
Movimento Celular/efeitos dos fármacos , Células Dendríticas/citologia , Células Endoteliais/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Anticorpos Monoclonais/farmacologia , Quimiocina CCL21/fisiologia , Dermatite/patologia , Dermatite/fisiopatologia , Humanos , Inflamação/imunologia , Vasos Linfáticos/metabolismo , NF-kappa B/farmacologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
The study of lymphatic tumor vasculature has been gaining interest in the context of cancer immunotherapy. These vessels constitute conduits for immune cells' transit toward the lymph nodes, and they endow tumors with routes to metastasize to the lymph nodes and, from them, toward distant sites. In addition, this vasculature participates in the modulation of the immune response directly through the interaction with tumor-infiltrating leukocytes and indirectly through the secretion of cytokines and chemokines that attract leukocytes and tumor cells. Radiotherapy constitutes the therapeutic option for more than 50% of solid tumors. Besides impacting transformed cells, RT affects stromal cells such as endothelial and immune cells. Mature lymphatic endothelial cells are resistant to RT, but we do not know to what extent RT may affect tumor-aberrant lymphatics. RT compromises lymphatic integrity and functionality, and it is a risk factor to the onset of lymphedema, a condition characterized by deficient lymphatic drainage and compromised tissue homeostasis. This review aims to provide evidence of RT's effects on tumor vessels, particularly on lymphatic endothelial cell physiology and immune properties. We will also explore the therapeutic options available so far to modulate signaling through lymphatic endothelial cell receptors and their repercussions on tumor immune cells in the context of cancer. There is a need for careful consideration of the RT dosage to come to terms with the participation of the lymphatic vasculature in anti-tumor response. Here, we provide new approaches to enhance the contribution of the lymphatic endothelium to radioimmuno-oncology.
RESUMO
Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.
Assuntos
Anticorpos Biespecíficos , Antígeno Carcinoembrionário , Neoplasias do Colo , Linfócitos T , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Complexo CD3/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Humanos , Ativação Linfocitária , Organoides/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor CTLs. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin-mediated cDC1 depletion prior to immunotherapy treatment with anti-PD-1 and/or anti-CD137 immunostimulatory mAbs completely ablated antitumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti-PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors. SIGNIFICANCE: These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.
Assuntos
Toxina Diftérica , Neoplasias Hepáticas , Camundongos , Animais , Células Dendríticas , Imunoterapia/métodos , Linfócitos T CD8-Positivos , Anticorpos Monoclonais , Camundongos Transgênicos , Neoplasias Hepáticas/tratamento farmacológicoRESUMO
CD137 artificial costimulation results in complete tumor rejection in several mouse models. Type I interferons (IFN) exert antitumor effects through an array of molecular functions on malignant cells, tumor stroma and immune system cells. The fact that agonist anti-CD137 mAb induce tumor regressions in mice deficient in the unique receptor for Type I IFNs (IFNAR(-/-) ) indicated potential for treatment combinations. Indeed, combination of intratumor injections of mouse IFN-α and intraperitoneal injections of anti-CD137 mAb synergized as seen on subcutaneous lesions derived from the MC38 colon carcinoma, which is resistant to each treatment if given separately. Therapeutic activity was achieved both against lesions directly injected with IFN-α and against distant concomitant tumors. Experiments in bone marrow chimeras prepared with IFNAR(-/-) and WT mice concluded that expression of the receptor for Type I interferons is mainly required on cells of the hematopoietic compartment. Synergistic effects correlated with a remarkable cellular hyperplasia of the tumor draining lymph nodes (TDLNs). Enlarged TDLNs contained more plasmacytoid and conventional dendritic cells (DC) that more readily cross-presented. Importantly, numbers of both DC subtypes inversely correlated with the tumor size. Numbers of CD8 T cells specific for a dominant tumor antigen were increased at TDLNs by each separate treatment but only with slight augments due to the combination. Combined antitumor effects of the therapeutic strategy were also seen on subcutaneous TC-1 tumors established for 24 days before treatment onset. The described strategy is realistic because (i) agents of each kind are clinically available and (ii) equivalent procedures in humans are feasible.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imunoterapia/métodos , Neoplasias Experimentais/terapia , Receptor de Interferon alfa e beta/deficiência , Animais , Anticorpos Monoclonais/administração & dosagem , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Injeções Intralesionais , Injeções Intraperitoneais , Interferon-alfa/administração & dosagem , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Receptor de Interferon alfa e beta/genética , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Migration of DC into lymphatic vessels ferries antigenic cargo and pro-inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured lymphatic endothelium monolayers pretreated with TNF-α were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-α2b or IFN-α5 showed enhanced adhesion to cultured lymphatic endothelial cells. These pro-adhesive effects were mediated by DC, not the lymphatic endothelium, and correlated with increased DC transmigration across lymphatic endothelial cell monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA-1. Furthermore, incubation of DC with IFN-α led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN-α also increased DC migration from inflammed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Endotélio Linfático/imunologia , Interferon-alfa/imunologia , Animais , Antígeno CD11a/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Interferon-alfa/farmacologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Collapsin response mediator protein 2 (CRMP2) is an adaptor protein that adds tubulin dimers to the growing tip of a microtubule. First described in neurons, it is now considered a ubiquitous protein that intervenes in processes such as cytoskeletal remodeling, synaptic connection and trafficking of voltage channels. Mounting evidence supports that CRMP2 plays an essential role in neuropathology and, more recently, in cancer. We have previously described a positive correlation between nuclear phosphorylation of CRMP2 and poor prognosis in lung adenocarcinoma patients. In this work, we studied whether this cytoskeleton molding protein is involved in cancer cell migration. To this aim, we evaluated CRMP2 phosphorylation and localization in the extending lamella of lung adenocarcinoma migrating cells using in vitro assays and in vivo confocal microscopy. We demonstrated that constitutive phosphorylation of CRMP2 impaired lamella formation, cell adhesion and oriented migration. In search of a mechanistic explanation of this phenomenon, we discovered that CRMP2 Ser522 phospho-mimetic mutants display unstable tubulin polymers, unable to bind EB1 plus-Tip protein and the cortical actin adaptor IQGAP1. In addition, integrin recycling is defective and invasive structures are less evident in these mutants. Significantly, mouse xenograft tumors of NSCLC expressing CRMP2 phosphorylation mimetic mutants grew significantly less than wild-type tumors. Given the recent development of small molecule inhibitors of CRMP2 phosphorylation to treat neurodegenerative diseases, our results open the door for their use in cancer treatment.
Assuntos
Adenocarcinoma de Pulmão/genética , Movimento Celular/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Proteínas Ativadoras de ras GTPase/genética , Adenocarcinoma de Pulmão/patologia , Animais , Proliferação de Células/genética , Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Microtúbulos/genética , Fosforilação/genética , Tubulina (Proteína)/genéticaRESUMO
BACKGROUND: Lung cancer is one of the most prevalent neoplasias in developed countries. Advances in patient survival have been limited and the identification of prognostic molecules is needed. Resistance to treatment is strongly related to tumor cell adhesion to the extracellular matrix and alterations in the quantity and nature of molecules constituting the tumor cell niche. Recently, transforming growth factor beta-induced protein (TGFBI), an extracellular matrix adaptor protein, has been reported to be differentially expressed in transformed tissues. Loss of TGFBI expression has been described in several cancers including lung carcinoma, and it has been suggested to act as a tumor suppressor gene. RESULTS: To address the importance of TGFBI expression in cancer progression, we determined its expression in NSCLC clinical samples using immunohistochemistry. We identified a strong association between elevated TGFBI expression and the response to chemotherapy. Furthermore, we transiently over-expressed and silenced TGFBI in human NSCLC cell lines. Cells over-expressing TGFBI displayed increased sensitivity to etoposide, paclitaxel, cisplatin and gemcitabine. We observed that TGFBI-mediated induction of apoptosis occurred through its binding to alphavbeta3 integrin. We also determined that full-length TGFBI did not induce caspase 3/7 activation but its proteolytic fragments that were < 3 kDa in size, were able to activate caspase 3, 7 and 8. This pro-apoptotic effect was blocked by anti-alphavbeta3 integrin antibodies. CONCLUSIONS: The results shown here indicate that TGFBI is a predictive factor of the response to chemotherapy, and suggest the use of TGFBI-derived peptides as possible therapeutic adjuvants for the enhancement of responses to chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspases/metabolismo , Ativação Enzimática , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , PrognósticoRESUMO
Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat-mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Cápsulas/administração & dosagem , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Células Dendríticas/imunologia , Hibridomas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Materiais Biocompatíveis , Antígenos CD40 , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/fisiologia , Imunização , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Taxa de Sobrevida , Carga Tumoral , Células Tumorais Cultivadas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.
Assuntos
Primers do DNA/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/metabolismo , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Moldes Genéticos , Sinergismo Farmacológico , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Humanos , Taninos Hidrolisáveis/uso terapêutico , Inibidores da Síntese de Ácido Nucleico , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/uso terapêutico , Terminalia/química , Zidovudina/metabolismo , Zidovudina/uso terapêuticoRESUMO
The quantity of T-lymphocytes reaching the draining lymph nodes from tumors is likely important to mount effective distant responses and for the establishment of long term systemic memory. Looking into mechanisms behind lymphocyte egress, we directed our attention to leukocyte adhesion mechanisms inside tumors. Here we demonstrate that activated T-cells form intra-tumor aggregates in a LFA-1-ICAM-1-dependent fashion in mouse models of melanoma and breast cancer. We also provide evidence of the presence of T-cell clusters in primary human melanoma. Disruption of LFA-1-ICAM-1 interactions, and thereby T-cell clustering, enhances the arrival of activated CD8+ T-cells to tumor draining lymph nodes in both transplanted and spontaneous cancer models. Interestingly, upon ICAM-1 blockade, the expression of the chemotactic receptor CCR7 augments in tumor infiltrating lymphocytes and in in-vitro de-clustered T cells, as well as their ability to transmigrate across lymphatic endothelial cells. We propose that ICAM-1-mediated homotypic T-lymphocyte aggregation may serve as a tumor-mediated immune retention mechanism entrapping activated CD8+ T cells in the tumor microenvironment. Modulation of T-cell adhesion may be of use to improve the transit of activated lymphocytes toward the lymph nodes and their subsequent recirculation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Linfonodos/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Proteínas de Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Feminino , Molécula 1 de Adesão Intercelular/genética , Linfonodos/patologia , Antígeno-1 Associado à Função Linfocitária/genética , Linfócitos do Interstício Tumoral/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genéticaRESUMO
T and NK lymphocytes express CD137 (4-1BB), a costimulatory receptor of the TNFR family whose function is exploitable for cancer immunotherapy. Mitochondria regulate the function and survival of T lymphocytes. Herein, we show that CD137 costimulation provided by agonist mAb and CD137L (4-1BBL) induced mitochondria enlargement that resulted in enhanced mitochondrial mass and transmembrane potential in human and mouse CD8+ T cells. Such mitochondrial changes increased T-cell respiratory capacities and were critically dependent on mitochondrial fusion protein OPA-1 expression. Mass and function of mitochondria in tumor-reactive CD8+ T cells from cancer-bearing mice were invigorated by agonist mAb to CD137, whereas mitochondrial baseline mass and function were depressed in CD137-deficient tumor reactive T cells. Tumor rejection induced by the synergistic combination of adoptive T-cell therapy and agonistic anti-CD137 was critically dependent on OPA-1 expression in transferred CD8+ T cells. Moreover, stimulation of CD137 with CD137 mAb in short-term cultures of human tumor-infiltrating lymphocytes led to mitochondria enlargement and increased transmembrane potential. Collectively, these data point to a critical link between mitochondrial morphology and function and enhanced antitumor effector activity upon CD137 costimulation of T cells. Cancer Immunol Res; 6(7); 798-811. ©2018 AACR.
Assuntos
Ligante 4-1BB/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Linfócitos T/metabolismo , Ligante 4-1BB/genética , Animais , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Inativação Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , RNA Interferente Pequeno/genética , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
Neurons are highly polarized cells composed of two structurally and functionally distinct parts, the axon and the dendrite. The establishment of this asymmetric structure is a tightly regulated process. In fact, alterations in the proteins involved in the configuration of the microtubule lattice are frequent in neuro-oncologic diseases. One of these cytoplasmic mediators is the protein known as collapsin response mediator protein-2, which interacts with and promotes tubulin polymerization. In this study, we investigated collapsin response mediator protein-2 transcriptional regulation during all-trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells. All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro. Therefore, we first demonstrated that collapsin response mediator protein-2 mRNA levels are downregulated during the differentiation process. After completion of deletion construct analysis and mutagenesis and mobility shift assays, we concluded that collapsin response mediator protein-2 basal promoter activity is regulated by the transcription factors AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate in its regulation. Furthermore, we finally established that reduced expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus sequences in the collapsin response mediator protein-2 promoter. Decreased attachment of AP-2 is a consequence of its accumulation in the cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-retinoic acid-mediated transcriptional repression. Unraveling the molecular mechanisms behind the action of all-trans-retinoic acid on neuroblastoma cells may well offer new perspectives for its clinical application.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Transcrição Gênica , Tretinoína/metabolismo , Antineoplásicos/farmacologia , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Neuroblastoma/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/metabolismo , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição AP-2/metabolismoRESUMO
Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether ß3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-ß induction of ß3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-ß exposure. Invadopodia formation and degradation activity is dependent on ß3 integrin expression since ß3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in ß3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for ß3 integrin in invadopodia. Our results suggest that ß3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. ß3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Matriz Extracelular/metabolismo , Integrina beta3/metabolismo , Neoplasias Pulmonares/metabolismo , Podossomos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Células A549 , Adesão Celular , Linhagem Celular Tumoral , Humanos , Metástase Neoplásica , Podossomos/efeitos dos fármacos , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process.