RESUMEN
Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.
Asunto(s)
Exosomas/metabolismo , Melanoma/metabolismo , Melanoma/patología , Proteínas rab27 de Unión a GTP/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Medios de Cultivo Condicionados , Exosomas/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanosomas/genética , Melanosomas/metabolismo , Ratones , Invasividad Neoplásica , Nevo/genética , Nevo/metabolismo , Proteómica , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Esferoides Celulares , Proteínas rab27 de Unión a GTP/biosíntesis , Proteínas rab27 de Unión a GTP/genéticaRESUMEN
Immunotherapies based on the autologous adoptive transfer of ex vivo-manipulated T cells are rapidly evolving for the treatment of both metastatic and primary malignancies. However, extended ex vivo culturing reduces the functionality of isolated T cells. Cryopreservation of rapidly expanded T cells for subsequent use throughout an immunotherapeutic regimen is a highly desirable recourse, thus far encumbered by a lack of studies investigating its effects on effector T-cell functionality. Here we directly compare murine tumour-reactive CD8(+) T cells cryopreserved during ex vivo expansion to freshly isolated populations. We show that cryopreservation fully conserves the differentiation potential of effector T cells, secretion of pro-inflammatory cytokines, cytotoxic function and does not impair the three-dimensional scanning motility of T cells or their capacity to infiltrate and reject tumours.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Criopreservación , Inmunoterapia Adoptiva , Neoplasias/terapia , Animales , Antígenos de Neoplasias/inmunología , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Estudios de Factibilidad , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunologíaRESUMEN
Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.
Asunto(s)
Progresión de la Enfermedad , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/farmacología , Melanoma Experimental/patología , Imitación Molecular , Metástasis de la Neoplasia , Animales , Línea Celular Tumoral , Citometría de Flujo , Melanoma Experimental/irrigación sanguínea , Ratones , Resonancia por Plasmón de SuperficieRESUMEN
Recruitment of leukocytes to glomeruli is fundamental to the pathogenesis of many forms of glomerulonephritis. In a model of glomerulonephritis induced by in situ immune complex deposition, we previously observed that, in addition to leukocytes, platelets accumulate in glomerular capillaries, where they contribute to leukocyte recruitment. However, the mechanisms of platelet recruitment and the role of platelet-expressed P-selectin in leukocyte recruitment require further investigation. We used intravital microscopy to examine the mechanisms of platelet and leukocyte recruitment to glomeruli of mice following administration of an antibody against the glomerular basement membrane (anti-GBM antibody). Platelet recruitment was initiated within five minutes of administration of anti-GBM antibody. This was unaltered by inhibition of platelet GPIbalpha but was prevented by the absence of platelet GPVI. Fibrinogen was deposited in glomerular capillaries via a partially intercellular adhesion molecule 1 (ICAM-1)-dependent mechanism, and inhibition of alpha(IIb)beta(3), fibrinogen and ICAM-1 inhibited platelet recruitment. Notably, neutrophil depletion also reduced platelet accumulation, indicating a cooperative interaction underlying recruitment of platelets and neutrophils. Finally, using bone marrow chimeras to restrict expression of P-selectin to platelets or endothelial cells, platelet but not endothelial P-selectin was required for glomerular leukocyte recruitment. Together these data indicate that platelet recruitment in this model is dependent on the combined actions of GPVI and the alpha(IIb)beta(3)/fibrinogen/ICAM-1 pathway and that platelet P-selectin is crucial for subsequent leukocyte recruitment.
Asunto(s)
Plaquetas/inmunología , Glomérulos Renales/inmunología , Activación Plaquetaria/fisiología , Transducción de Señal/fisiología , Análisis de Varianza , Animales , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Plaquetas/metabolismo , Inmunohistoquímica , Glomérulos Renales/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Activación Neutrófila/fisiología , Selectina-P/inmunología , Selectina-P/metabolismo , Glicoproteínas de Membrana Plaquetaria/inmunología , Glicoproteínas de Membrana Plaquetaria/metabolismoRESUMEN
Patients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 microg anti-MPO induced LFA-1-dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO(-/-) mice. In addition, a higher dose of anti-MPO (200 microg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was beta2-integrin independent, instead requiring the alpha4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Adhesión Celular/inmunología , Integrina alfa4/fisiología , Glomérulos Renales/irrigación sanguínea , Antígeno-1 Asociado a Función de Linfocito/fisiología , Monocitos/inmunología , Neutrófilos/inmunología , Peroxidasa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Reacciones Antígeno-Anticuerpo , Antígenos CD18/inmunología , Antígenos CD18/fisiología , Endotoxemia/inmunología , Endotoxinas/farmacología , Endotoxinas/toxicidad , Hidronefrosis/inmunología , Hidronefrosis/patología , Inmunización , Integrina alfa4/metabolismo , Glomérulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/enzimología , Neutrófilos/enzimología , Selectina-P/inmunología , Peroxidasa/deficiencia , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Interferon (IFN) gamma's ability to localise in the nucleus and function in gene activation has been known for some time, although the role of the conventional nuclear transporting importin molecules is unclear. Here, we demonstrate for the first time the direct recognition of IFNgamma and an IFNgamma mimetic peptide by IMPalpha and the IMPalpha/beta heterodimer, where the IFNgamma mimetic shows higher affinity. Significantly, this correlates well both with in vivo ability to target green fluorescent protein to the nucleus in transfected cells as determined by quantitative confocal laser scanning microscopy, as well as GAS promoter activity of a luciferase reporter. This has important implications for IFNgamma's anti-viral action, and the potential use of the IFNgamma mimetic in antiviral therapies.
Asunto(s)
Núcleo Celular/metabolismo , Interferón gamma/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Secuencia de Aminoácidos , Animales , Materiales Biomiméticos/metabolismo , Células COS , Chlorocebus aethiops , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interferón gamma/genética , Datos de Secuencia Molecular , Señales de Localización Nuclear/metabolismo , Péptidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Activación TranscripcionalRESUMEN
Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens alpha-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (theta-toxin) is less well understood. Similarly, Clostridium septicum is a highly virulent causative agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion, consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the alpha-toxin or perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise correlating with the level of the C. septicum alpha-toxin. Together, these data indicate that as a result of its ability to produce alpha-toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function is central to the pathogenesis of clostridial myonecrosis, irrespective of the causative bacterium.
Asunto(s)
Toxinas Bacterianas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Clostridium perfringens/patogenicidad , Clostridium septicum/patogenicidad , Gangrena Gaseosa/microbiología , Proteínas Hemolisinas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Toxinas Bacterianas/genética , Proteínas de Unión al Calcio/genética , Muerte Celular/efectos de los fármacos , Clostridium perfringens/fisiología , Clostridium septicum/fisiología , Gangrena Gaseosa/fisiopatología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas Hemolisinas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Microcirculación/efectos de los fármacos , Microscopía por Video , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Mutagénesis Insercional , Perfusión , Flujo Sanguíneo Regional/efectos de los fármacos , Fosfolipasas de Tipo C/genéticaRESUMEN
Most humans with microscopic polyarteritis and anti-myeloperoxidase (anti-MPO), anti-neutrophil cytoplasmic antibodies (ANCA) develop "pauci-immune" crescentic glomerulonephritis. For dissection of the roles of ANCA and cell-mediated effectors in microscopic polyarteritis, experimental autoimmune anti-MPO glomerulonephritis was induced by immunizing C57BL/6 mice with human MPO. Autoimmunity to mouse MPO (ANCA and CD4+ cell reactivity) was induced. Challenge with anti-glomerular basement membrane globulin resulted in accumulation of neutrophils, CD4+ cells and macrophages, and significant numbers of crescentic glomeruli compared with similarly challenged control-immunized mice. MPO-deficient (Mpo(-/-)) mice immunized with MPO developed similar immune responses to MPO but failed to recruit effector cells to glomeruli or develop significant crescent formation, suggesting that MPO is acting as a planted glomerular autoantigen. Effector CD4+ cell depletion in this model attenuated crescentic glomerulonephritis and effector cell influx without altering ANCA titers. However, B cell-deficient mice, with no ANCA, still developed severe crescentic glomerulonephritis with accumulation of effector cells. Intravital microscopy studies demonstrated that passive transfer of sera from MPO-immunized Mpo(-/-) mice to LPS-primed mice rapidly induced glomerular neutrophil accumulation and release of MPO. These studies provide in vivo evidence in a relevant vascular bed for both humoral and cellular anti-MPO responses as key inducers of injury. ANCA induces glomerular neutrophil infiltration and MPO deposition. Subsequently, anti-MPO CD4+ cells recognize MPO as a planted glomerular antigen and act with macrophages to amplify severe glomerular injury.