RESUMO
The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective direct IQ-cytotoxic effect on transformed (H5V) vs. normal (1G11) endothelial cells (EC). In the present study, we investigated the mechanism underlying this selective cytotoxicity in terms of TLR7/8 receptor expression, NF-κB signalling and time-dependent modifications of oxidative stress parameters (ROS: reactive oxygen species, catalase and superoxide dismutase activities, GSH/GSSG and lipid peroxidation). TLR7/8 level was extremely low in both cell lines, and IQ did not upregulate TLR7/8 expression or activate NF-κB signalling. IQ significantly induced ROS in H5V after 2 h and strongly affected antioxidant defenses. After 12 h, enzyme activities were restored to baseline levels but a robust drop in GSH/GSSG persisted together with increased lipid peroxidation levels and a marked mitochondrial dysfunction. Although in normal IQ-treated EC some oxidative stress parameters were affected after 4 h, mitochondrial health and GSH/GSSG ratio remained notably unaffected after 12 h. Therefore, the early alterations (0-2 h) in transformed EC breached redox homeostasis as strongly as to enhance their susceptibility to IQ. This interesting facet of IQ as redox disruptor could broaden its therapeutic potential for other skin malignancies, alone or in adjuvant schemes.
Assuntos
Glutationa , NF-kappa B , Antioxidantes/metabolismo , Células Endoteliais/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Dissulfeto de Glutationa/farmacologia , Homeostase , Imiquimode/farmacologia , NF-kappa B/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Receptor 7 Toll-LikeRESUMO
This study aimed to investigate the acute effects of chlorpyrifos on biomarkers related to neurotoxicity and immunotoxicity in two allopatric freshwater gastropod species belonging to the family Planorbidae. For this purpose, Planorbarius corneus and Biomphalaria glabrata were exposed to chlorpyrifos (active ingredient or commercial formulation) for 48 h at environmentally realistic concentrations (1 and 7.5 µg L-1). Basal acetylcholinesterase activity in soft tissues and hemolymph was almost one order of magnitude higher in P. corneus than in B. glabrata. However, upon chlorpyrifos exposure, statistically significant inhibition of enzymatic activity was registered in both species. Acetylcholinesterase was more sensitive to inhibition in soft tissues than in hemolymph. The highest inhibition was observed in the B. glabrata soft tissues exposed to the commercial formulation (88 % at 1 µg L-1 and 93 % at 7.5 µg L-1). Hemocyte number and lysosomal membrane stability did not show significant changes with respect to controls in any of the exposed groups. Superoxide anion generation was diminished (21-46 %) in P. corneus hemocytes exposed to the active ingredient and in B. glabrata hemocytes exposed to the active ingredient or the formulation. In contrast, hemocyte phagocytic activity increased in all exposed groups. Phagocytosis was most stimulated (89 %) in hemocytes sampled from B. glabrata treated with 7.5 µg L-1 chlorpyrifos. Altogether the results suggest that the freshwater gastropods P. corneus and B. glabrata are suitable model animals for environmental monitoring studies in the Northern Hemisphere and Latin America, respectively. Furthermore, these results add information on the relevance of testing pesticide formulations and on the usefulness of acetylcholinesterase inhibition and immunological parameters as biomarkers of the acute effects of chlorpyrifos in these species.
Assuntos
Biomphalaria/fisiologia , Clorpirifos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Biomphalaria/efeitos dos fármacos , Inibidores da Colinesterase , Monitoramento Ambiental , Água Doce , Gastrópodes/fisiologia , Hemócitos/efeitos dos fármacos , Hemolinfa/efeitos dos fármacos , PraguicidasRESUMO
Infantile hemangiomas are the most common benign tumors of infancy, characterized by unregulated angiogenesis and endothelial cells with high mitotic rate. Although spontaneous regression occurs, sometimes treatment is required and alternatives to corticosteroids should be considered to reduce side effects. Imiquimod is an imidazoquinoline, approved for some skin pathologies other than hemangioma. It is proposed that the effectiveness of imiquimod comes from the activation of immune cells at tumor microenvironment. However, the possibility to selectively kill different cell types and to directly impede angiogenesis has been scarcely explored in vitro for endothelial cells. In this work we showed a dramatic cytotoxicity on hemangioma cell, with a significant lower IC50 value in hemangioma compared to normal endothelial cells and melanoma (employed as a non-endothelial tumor cell line). Nuclear morphometric and flow-cytometry assays revealed imiquimod-induced apoptosis on hemangioma and melanoma cells but a small percentage of senescence on normal endothelial cells. At sub-lethal conditions, cell migration, a key step in angiogenesis turned out to be inhibited in a tumor-selective manner along with actin cytoskeleton disorganization on hemangioma cells. Altogether, these findings pointed out the selective cytotoxic effects of imiquimod on transformed endothelial cells, evidencing the potential for imiquimod to be a therapeutic alternative to reduce extensive superficial hemangioma lesions.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Hemangioma/patologia , Neoplasias Cutâneas/patologia , Aminoquinolinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Endoteliais/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Humanos , Imiquimode , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestruturaRESUMO
Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.
RESUMO
Immune evasion strategies are important for the onset and the maintenance of viral infections. Many viruses have evolved mechanisms to counteract or suppress the host immune response. We have previously characterized two syncytial (syn) variants of Herpes simplex 1 (HSV-1) strain F, syn14-1 and syn17-2, obtained by selective pressure with a natural carrageenan. These variants showed a differential pathology in vaginal and respiratory mucosa infection in comparison with parental strain. In this paper, we evaluated the modulation of immune response in respiratory mucosa by these HSV-1 variants. We observed altered levels of Tumor Necrosis Factor-α and Interleukin-6 in lungs of animals infected with the syn14-1 and syn17-2 variants compared with the parental strain. Also, we detected differences in the recruitment of immune cells to the lung in syn variants infected mice. Both variants exhibit one point mutation in the sequence of the gene of glycoprotein D detected in the ectodomain of syn14-1 and the cytoplasmic tail of syn17-2. Results obtained in the present study contribute to the characterization of HSV-1 syn variants and the participation of the cellular inflammatory response in viral pathogenesis.
Assuntos
Citocinas/metabolismo , Herpesvirus Humano 1/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Animais , Feminino , Herpesvirus Humano 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Mucosa/patologia , Mutação Puntual , Infecções Respiratórias/virologia , Proteínas do Envelope Viral/genéticaRESUMO
Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high-cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.
Assuntos
Aterosclerose/prevenção & controle , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Hipercolesterolemia/fisiopatologia , Placa Aterosclerótica/prevenção & controle , Animais , Aorta/citologia , Aorta/metabolismo , Aterosclerose/fisiopatologia , Colesterol/sangue , Proteoglicanas de Sulfatos de Condroitina/química , Dermatan Sulfato/química , Dieta Aterogênica/efeitos adversos , Modelos Animais de Doenças , Glicosaminoglicanos/química , Cabras , Humanos , Hipercolesterolemia/metabolismo , Lipídeos/sangue , Masculino , Coelhos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
De novo ectopic lymphoid tissue formation is known to occur in certain disease and inflammatory settings. After an effective vaccination with dendritic cells (DC) charged with melanoma apoptotic/necrotic cells (Apo/Nec), a subcutaneous tertiary lymphoid structure was organized, where retained vaccine cells interacted with recruited inflammatory and T cells. In this work we report for the first time the recruitment of two morphologically different CD207(+) cells to vaccination site. The time-course behavior of CD207(+) cells was reciprocal between vaccination site and draining lymph nodes (DLNs). After 6-10 days, CD207(+) cells localized at the paracortical region of DLNs, in close contact with T cell population. DLNs were enriched in a peculiar MHCII(+) CD11c((-)) CD207(+) population, whose role remains to be determined. Whether CD207(+) cells migration to the vaccination site can be associated with a differential anti-tumoral response remains as an open and exciting question.
Assuntos
Antígenos de Superfície/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Linfonodos/imunologia , Tecido Linfoide/fisiologia , Lectinas de Ligação a Manose/imunologia , Animais , Movimento Celular , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo.
Assuntos
Apresentação de Antígeno/imunologia , Células da Medula Óssea/imunologia , Antígeno CD11c/imunologia , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Animais , Apresentação de Antígeno/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Células da Medula Óssea/metabolismo , Antígeno CD11c/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
Decorin and biglycan proteoglycans play important roles in the organization of the extracellular matrix, and in the regulation of cell adhesion and migration. Given morphological and functional endothelial heterogeneity, information is needed regarding whether endothelial cells (ECs) from different vascular beds possess different profiles of proteoglycan constituents of the basement membranes. Here, we report that endothelia from different murine organs and EC lines derived thereof produce and secrete different patterns of proteoglycans. A faint colocalization between decorin and PECAM/CD31 was found on tissue sections from mouse heart, lung and kidney by immunofluorescence. Three EC lines derived from these organs produced decorin (100-kDa) and its core protein (45-kDa). Extracellular decorin recognition in culture supernatant was only possible after chondroitin lyase digestion suggesting that the core protein of secreted proteoglycan is more encrypted by glycosaminoglycans than the intracellular one. Heart and lung ECs were able to produce and release decorin. Kidney ECs synthesized the proteoglycan and its core protein but no secretion was detected in culture supernatants. Although biglycan production was recorded in all EC lines, secretion was almost undetectable, consistent with immunofluorescence results. In addition, no biglycan secretion was detected after EC growth supplement treatment, indicating that biglycan is synthesized, secreted and quickly degraded extracellularly by metalloproteinase-2. Low molecular-mass dermatan sulfate was the predominant glycosaminoglycan identified bound to the core protein. ECs from different vascular beds, with differences in morphology, physiology and cell biology show differences in the proteoglycan profile, extending their heterogeneity to potential differences in cell migration capacities.
Assuntos
Biglicano/metabolismo , Decorina/metabolismo , Células Endoteliais/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Dermatan Sulfato/metabolismo , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Rim/citologia , Rim/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/citologia , Miocárdio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
Antigen presentation by dendritic cells (DC) is of key importance for the initiation of the primary immune response. Mice vaccinated with DC charged with apoptotic/necrotic B16 cells (DC-Apo/Nec) are protected against B16 challenge. The aim of this study was to assess vaccine cell migration in our system and to find out if there is an immunological response taking place at the vaccination site. The formation of a pseudocapsule, peripheral node addresin expression in small venules, and the recruitment of a wide variety of cellular populations, including macrophages, polymorphonuclear lymphocytes, and CD8+ and CD4+ T lymphocytes found in association with DC, evidenced the formation of tertiary lymphoid tissue in the vaccination site in our experimental system.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Tecido Linfoide/fisiologia , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Vacinação/métodos , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Histocitoquímica , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tela Subcutânea/imunologia , Análise de SobrevidaRESUMO
Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.
Assuntos
Quimiocina CCL2/antagonistas & inibidores , Ácido Clodrônico/administração & dosagem , Indazóis/uso terapêutico , Macrófagos/fisiologia , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Propionatos/uso terapêutico , Animais , Linhagem Celular Tumoral , Quimiocina CCL2/fisiologia , Humanos , Lipossomos , Masculino , Melanoma Experimental/irrigação sanguínea , Camundongos , Transplante de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Transplante HeterólogoRESUMO
Se analizaron biopsias de melanoma metastásico humano para elucidar la relación entre la expresión de la quimioquina MCP-1/CCL2 (monocyte chemoattractant protein-1), la angiogénesis y la agrasividad del tumor. Se encontró que esta quimioquina se expresa en el 100% de los casos, con heterogeneidad en el porcentaje de células positivas dentro del tumor. Estos tumores presentaron gran cantidad de macrófagos infiltrantes, particularmente asociados a las áreas de mas activa angiogénesis. Se obtuvo correlación positiva entre el porcentaje de células que expresan MCP-1 y el grado de vascularización. Asimismo, se encontró asociación entre una mayor angiogénesis y la proliferación tumoral evaluada como índice mitótico. Estos resultados sugieren que el aumento en la vascularización podría ser predictivo de metástasis más agresivas, donde la expresión de MCP-1 estaría estrechamente vinculada al desarrollo de vasos a través del reclutamiento de macrófagos.
Assuntos
Humanos , Melanoma/patologia , Neovascularização Patológica/etiologia , Melanoma/fisiopatologia , Metástase Neoplásica , Neovascularização Patológica/fisiopatologiaRESUMO
Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle alpha-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13-20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.
Assuntos
Endotélio Vascular/citologia , Rim/irrigação sanguínea , Actinas/metabolismo , Animais , Linhagem Celular , Separação Celular/métodos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Queratinas/metabolismo , Lipopolissacarídeos/toxicidade , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Inoculações Seriadas/métodos , Toxina Shiga I/toxicidade , Células Vero , Vimentina/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Para estudiar la evolución de la angiogénesis a lo largo del desarrollo tumoral en tumores mamarios murinos con diferente tasa de crecimiento y capacidad metastásica se utilizó el Anticuerpo Monoclonal MEC 13.3, que reconoce la molécula CD31/PECAM murino en células endoteliales. Los vasos detectados por la técnica inmunohistoquímica se cuantificaron en campos de 200 x en las áreas de mayor densidad vascular. Todos los tumores presentaron un incremento de la densidad vascular a lo largo de su crecimiento. En los estadios iniciales del desarrollo tumoral (10 días post-trasplante) se observó una correlación positiva de la densidad vascular con la tasa de crecimiento y la capacidad metastásica. En los estadios tardíos la densidad vascular se correlacionó con la capacidad metastásica pero no con la tasa de crecimiento. Además, se identificaron células aisladas CD31+ en los primeros estadios de desarrollo tumoral que disminuyeron con la progresión de la neovascularización. Estos resultados muestran la contribución del anticuerpo MEC 13.3 en la identificación de la neovascularización y sugieren que la misma se correlaciona siempre con la capacidad metastizante y sólo en los estadios tempranos con la velocidad de crecimiento.
Assuntos
Animais , Camundongos , Anticorpos Monoclonais , Neoplasias Mamárias Experimentais , Neovascularização Patológica , Fatores de Crescimento Endotelial , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Estadiamento de NeoplasiasRESUMO
In order to clearly visualize blood vessels, the monoclonal antibody (mAb) MEC 13.3 was used for an immunohistochemical staining on frozen sections of different mice mammary tumors. MEC 13.3 mAb is specific for endothelial cells (ECs) of mouse blood vessels and recognizes a molecule related to the murine form of CD31/PECAM. This mAb with immunoenzymatic technique or immunofluorescent labelling, was found to be a useful tool to quantify tumor neovascularization. Specifically, membrane reinforcement could be observed in vessel ECs, indicating the expression of CD31/ PECAM in their surface. The staining of ECs from tumors and from normal tissues was also compared. In this work, the use of MEC13.3 mAb is reported to recognize mice mammary tumor ECs as a useful tool to identify neovascularization. It would also be helpful for research on the origin and function of vascular endothelium in murine tumor experimental models.