RESUMO
Previously, by employing 3D organotypic tissue culture and patient-derived xenograft (PDX) model, oral myxoma response to a MAPK/MEK inhibitor was observed. Gross examination of the tumour fragments obtained after 55 days of PDX grafting revealed increased capsule vascularization. Microscopic analyses showed blood capillaries intermixed with myxoma cells, but the origin of these capillaries, from mice or humans, was not established. This study aimed to investigate whether the endothelial cells observed in the myxoma PDX model are derived from the mouse or from the primary human tumour. Immunohistochemistry was performed on five tumour fragments from the PDX of myxoma after 55 days of implantation in mice. Immunopositivity for antibodies against human (HLA-ABC) and mouse (H2 Db/H2-D1) major histocompatibility complex class I (MHCI) was assessed in the endothelial cells. The endothelial cells in the PDX fragments revealed a membrane staining for the human MHCI protein in the PDX tumour and adjacent connective tissue capsule, indicating that capillaries were derived from the human tumour fragment. Considering the probable human origin of the endothelial cells from capillary blood vessels in the myxoma PDX, we conclude that this PDX model is an interesting model to study myxoma angiogenesis.
Assuntos
Células Endoteliais , Mixoma , Animais , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Neovascularização Patológica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Central giant cell granulomas (CGCG) of the jaws are osteolytic lesions that may behave aggressively and respond poorly to surgery. Microscopically, in addition to giant cells, there is a mononuclear cell population composed of macrophage/monocytic cells and spindle-shaped cells of mesenchymal origin. Seventy two percent of these tumours harbour mutually exclusive TRPV4, KRAS and FGFR1 mutations. We aimed to assess the mutational status of mononuclear and giant cells and the osteogenic potential of stromal cells in vitro and in vivo. METHODS AND RESULTS: We screened CGCG for signature mutations and used laser-capture microdissection to demonstrate that the mutations are restricted to the mononuclear cells. Additionally, we established CGCG primary cell culture and observed that the cells retained the mutations throughout passages. By flow cytometry, we observed predominance of CD14- CD51- CD61- cells, consistent with the expected profile for stromal cells. Considering the mesenchymal origin of stromal cells, we assessed the osteogenic differentiation potential of CGCG cells in culture by cytochemistry (von Kossa and alizarin red staining), alkaline phosphatase (ALP) activity assay and gene expression of osteogenic markers. CGCG cells presented self-capacity to increase ALP levels in a time-dependent manner and under osteogenic induction presented increasing number of calcium deposits, and overall higher expression of osteocalcin, RUNX2, ALPL and osteopontin than cells without osteogenic induction. A patient-derived xenograft model for CGCG was established, and osteoid material deposition was observed. CONCLUSION: Collectively, the results confirm that the signature mutations are restricted to stromal cells in CGCG, and the in vitro and in vivo results support that these cells have the capacity to differentiate into osteoblasts, in line with the bone formation often observed in the stroma of these lesions.
Assuntos
Granuloma de Células Gigantes , Células-Tronco Mesenquimais , Fosfatase Alcalina , Diferenciação Celular , Células Cultivadas , Granuloma de Células Gigantes/genética , Humanos , Arcada Osseodentária , Mutação , Osteogênese/genética , Células EstromaisRESUMO
Loxoscelism is a recognized public health problem in Brazil, but the venom from Loxosceles similis, which is widespread in Brazil due to its adaptability to the urban environment, remains poorly characterized. Loxtox is a family of phospholipase D enzymes (PLDs), which are the major components of Loxosceles venom and are responsible for the clinical effects of loxoscelism. Loxtox toxins correspond to 15% of L. similis venom gland transcripts, but the Loxtox family of L. similis has yet to be fully described. In this study, we cloned and functionally characterized recLoxtox s1A and recLoxtox s11A. These recombinant toxins exhibited different in vitro activities depending on pH, and recLoxtox s1A had more intense effects on rabbit skin than did recLoxtox s11A in vivo. Both recombinant toxins were used in immunization protocols, and mapping of their epitopes revealed different immunological reactions for the produced immune serums. Additionally, polyclonal antibodies raised against recLoxtox s1A had greater capacity to significantly reduce the in vitro and in vivo effects of L. similis venom. In summary, we obtained and characterized two novel Loxtox isoforms from L. similis venom, which may be valuable biotechnological and immunological tools against loxoscelism.
Assuntos
Diester Fosfórico Hidrolases/metabolismo , Venenos de Aranha/metabolismo , Aranhas/metabolismo , Animais , Clonagem Molecular , Epitopos/química , Feminino , Concentração de Íons de Hidrogênio , Soros Imunes/imunologia , Testes de Neutralização , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases/genética , Isoformas de Proteínas , Coelhos , Proteínas Recombinantes/metabolismo , Pele/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Venenos de Aranha/genéticaRESUMO
OBJECTIVE: The aim of this study was to describe the clinicopathologic features of a series of gnathic epithelioid osteoblastomas. As high levels of Proto-oncogene c-Fos proteins resulting from FOS-FOSB translocation were recently demonstrated in osteoblastomas, we also evaluated the immunoexpression of these proteins. STUDY DESIGN: Records of all cases of epithelioid osteoblastoma of the jaws were retrieved from oral pathology services, and their clinicopathologic and immunohistochemical data were collected. Immunohistochemistry was also performed by using anti-FOS and anti-FOSB antibodies. RESULTS: Six cases of epithelioid osteoblastomas were obtained, 4 in men and 2 in women, and they were mainly located in the posterior body of the mandible (nâ¯=â¯4). Radiographically, the tumors showed mixed radiolucent and radiopaque images, most with poorly defined margins. Microscopically, large epithelioid cells with eccentrically located nuclei predominated among osteoid and immature bone trabeculae. Sharp delineation from adjacent normal bone was observed in all cases. FOS immunostaining was diffuse and strong in the cytoplasm and nucleus of neoplastic cells in all cases, whereas FOSB was only focally positive, with few epithelioid osteoblasts showing nuclear staining. CONCLUSIONS: Although epithelioid osteoblastomas of the jaws are locally aggressive, widespread metastasis does not occur, and, as with conventional osteoblastomas, there is wide expression of the FOS protein.
Assuntos
Neoplasias Ósseas , Osteoblastoma , Feminino , Humanos , Imuno-Histoquímica , Masculino , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fosRESUMO
OBJECTIVE: Odontogenic myxoma (OM) occasionally responds poorly to surgical treatment. The MAPK pathway is constitutively activated in several neoplasms and we aimed to test if the MAPK pathway is activated in OM, in order to pave the way for an alternative therapy for aggressive and recurrent cases. MATERIALS AND METHODS: The immunoexpression of phosphorylated ERK1/2 (pERK1/2) was assessed in OM. We established a 3D organotypic culture model for the in vitro study and patient-derived xenografts (PDX) in mice for the in vivo study. The MEK inhibitor U0126 was used to inhibit phosphorylation of ERK1/2 in the in vitro and in vivo models. RESULTS: All OM showed strong pERK1/2 immunoexpression, consistent with MAPK pathway activation. Treatment of the 3D culture with U0126 resulted in a reduced pERK1/2/ERK1/2 ratio. Consistent with the in vitro results, all PDX of animals treated with U0126 showed a decreased volume fold change compared with controls. CONCLUSIONS: The MAPK pathway is activated in OM and its inhibition leads to tumor shrinkage in PDX and cell culture models. CLINICAL RELEVANCE: Our results offer a pre-clinical frame for OM-targeted therapy. Further work is needed to determine if this initial finding holds clinical promise.
Assuntos
Doenças da Boca , Mixoma , Animais , Fosfatase 1 de Especificidade Dupla/efeitos dos fármacos , Humanos , Camundongos , Doenças da Boca/tratamento farmacológico , Mixoma/tratamento farmacológico , FosforilaçãoRESUMO
OBJECTIVES: To investigate the molecular pathogenesis of implant-associated peripheral giant cell granuloma (IA-PGCG). METHODS: A convenience sample of 15 IA-PGCG cases was selected. Hotspot mutations of KRAS, FGFR1, and TRPV4 genes, previously reported in conventional giant cell lesions of the jaws, were investigated by Sanger sequencing. As these mutations could activate MAPK/ERK pathway, the expression of phospho-ERK1/2 was also evaluated by immunohistochemistry. RESULTS: KRAS mutations were detected in 8/15 (53.4%) samples. Similar to conventional peripheral giant cell granuloma, the KRAS mutations most frequently occurred in codon 146 (p.A146V, n = 3), followed by codon 12 (p.G12A and p.G12D, n = 1 each) and codon 14 (p.V14L, n = 1). Variants of unknown significance (VUS) were also detected in two cases, affecting codons 37 (p.E37K) and 127 (p.T127I). All samples showed wild-type (WT) sequences for FGFR1 and TRPV4 genes. Consistent with MAPK/ERK pathway activation, all mononuclear cells of the lesion showed strong staining for phospho-ERK1/2 protein in the immunohistochemical analysis. CONCLUSIONS: KRAS mutations and activation of the MAPK-ERK signaling pathway occur in IA-PGCG. This is the first study to demonstrate cancer-associated gene mutations in a non-neoplastic reactive condition associated with dental implants.
Assuntos
Implantes Dentários/efeitos adversos , Granuloma de Células Gigantes/etiologia , Granuloma de Células Gigantes/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Transdução de SinaisRESUMO
BACKGROUND: Pyogenic granuloma (PG) is a benign nodular lesion with a prominent vascular component which may affect different sites. Recently, BRAF, KRAS, HRAS, NRAS, GNA11, and GNA14 mutations were reported on PGs of the skin. The present study assessed the role of the MAPK/ERK pathway in oral PG pathogenesis. METHODS: Mutations in hotspot regions of genes involved in the MAPK/ERK pathway activation were investigated by Sanger sequencing. The expression of phospho-ERK1/2 was evaluated by immunohistochemistry. RESULTS: Oral PGs did not show mutations in the sequenced regions of the genes BRAF, KRAS, HRAS, NRAS, GNA11, or GNA14. Our results also showed activation of the MAPK/ERK pathway demonstrated by phospho-ERK1/2 immunohistochemical positivity. CONCLUSIONS: Although oral PG shows MAPK/ERK pathway activation, the driver molecular event remains to be elucidated.
Assuntos
Granuloma Piogênico/metabolismo , Sistema de Sinalização das MAP Quinases , Mutação , Adolescente , Adulto , Idoso , Feminino , GTP Fosfo-Hidrolases/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Granuloma Piogênico/genética , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
Adenomatoid odontogenic tumor is a benign encapsulated epithelial odontogenic tumor that shows an indolent clinical behavior. We have reported in a few adenomatoid odontogenic tumors mutations in KRAS, which is a proto-oncogene frequently mutated in cancer such as lung, pancreas, and colorectal adenocarcinomas. We aimed to assess KRAS mutations in the hotspot codons 12, 13, and 61 in a large cohort of adenomatoid odontogenic tumors and to test the association of these mutations with clinical (age, site, tumor size, follicular/extrafollicular subtypes) and histopathological parameters. Thirty eight central cases were studied. KRAS codon 12 mutations were assessed by TaqMan allele-specific qPCR (p.G12V/R) and/or Sanger sequencing, and codon 13 and 61 mutations were screened by Sanger. Histological tumor capsule thickness was evaluated by morphometric analysis. Additionally, the phosphorylated form of the MAPK downstream effector ERK1/2 was investigated. Statistical analysis was carried out to test the association of KRAS mutations with clinicopathological parameters. KRAS c.35 G >T mutation, leading to p.G12V, was detected in 15 cases. A novel mutation in adenomatoid odontogenic tumor, c.34 G >C, leading to p.G12R, was detected in 12 cases and the other 11 were wild-type. Codon 12 mutations were not associated with the clinicopathological parameters tested. RAS mutations are known to activate the MAPK pathway, and we show that adenomatoid odontogenic tumors express phosphorylated ERK1/2. In conclusion, a high proportion of adenomatoid odontogenic tumors (27/38, 71%) have KRAS codon 12 mutations, which occur independently of the clinicopathological features evaluated. Collectively, these findings indicate that KRAS mutations and MAPK pathway activation are the common features of this tumor and some cancer types. Although it is unclear why different codon 12 alleles occur in different disease contexts and the complex interactions between tumor genotype and phenotype need clarification, on the basis of our results the presence of KRAS p.G12V/R favors the adenomatoid odontogenic tumor diagnosis in challenging oral neoplasm cases.
Assuntos
Ameloblastoma/genética , Ameloblastoma/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Adolescente , Adulto , Criança , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Pessoa de Meia-Idade , Mutação , Proto-Oncogene Mas , Adulto JovemRESUMO
BACKGROUND: Benign neoplasms exhibit most of the cellular phenomena considered hallmarks of cancer, except the capacity to metastasize. Thus, the elucidation of the mechanisms associated with the progression of benign neoplasms may complement and clarify the mechanisms involved in carcinogenesis. Benign odontogenic tumours often result in facial deformities and morbidities, and have complex pathogenesis, mainly due to the diversity of interactions between the odontogenic epithelium and the ectomesenchyme. Primary cell culture of such tumours is not only difficult to be established and maintained, but also tumour cells lose characteristic cellular morphology. Considering gene expression, growth, migration, proliferation and cellular morphology are controlled by cell-cell interactions and cell-extracellular matrix interactions, cell culture in 3D substrates has gained space as a way to overcome some of the limitations of traditional monolayer cell culture systems. METHODS: In this study, fragments obtained from mesenchymal odontogenic tumours were cultured in type I collagen scaffolds. Invasion tests were performed in these models, as well as phenotypic characterization of the cultured tumours. RESULTS: The results obtained for the odontogenic myxoma and the cemento-ossifying fibroma demonstrate a good reproduction of the growth pattern of these tumours under ex vivo conditions. Microscopic evaluation showed maintenance of cell viability in the explants for more than 30 days, without the presence of necrosis. CONCLUSION: This is the first study involving long-term 3D primary cultures of benign odontogenic tumours, which is expected to support complex approaches to cell and molecular biology, and to serve as an experimental model for testing molecular therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Técnicas In Vitro , Tumores Odontogênicos/patologia , Carcinogênese , Comunicação Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Cementoma , Expressão Gênica , Humanos , Tumores Odontogênicos/genética , Células Tumorais CultivadasRESUMO
A crucial component of the integration between foreign implants and the host is angiogenesis. However, to date, none of the available techniques and/or endothelial markers employed to assess angiogenesis in the implant/host interface seems to be able to highlight vascular structures convincingly. In the present study we investigated and compared the expression of two endothelial cell markers: platelet endothelial cell adhesion molecule (PECAM-1) (CD31) and endoglin (CD105) using immunohistochemistry (IHC) and immunofluorescence (IF) to identify and quantify newly formed blood vessels in subcutaneous implants of polyether-polyurethane sponge of formalin-fixed paraffin-embedded tissue. At day 14 post implantation the discs of the synthetic matrix were removed and processed for histological and morphometric analysis. In IHC staining for CD31 antibody the number of vessels was 2.27±0.69 and 5.25±0.46 for CD105. In IF for CD31 the number of vessels was 15.36±1.295 and 10.54±0.8213 for CD105. The level of cross-reaction was lesser in IF images compared with IHC images. Co-localization of CD31/CD105 using confocal images showed positive correlation (Pearson's co-relation and Manders' equation). The double labeling for blood vessels using the IF technique for CD31/CD105 may be an important tool for evaluation of angiogenesis in biomaterial/host integration.