RESUMEN
As miRNAs emerge as potential circulating biomarkers for the diagnosis or prognosis of a wide variety of diseases, the quantification of miRNA necessitates careful preanalytic considerations and sample quality control becomes crucial. This study comprehensively analyzed the profiles of 356 miRNAs by quantitative RT-PCR in various blood sample types, with various processing protocols. The comprehensive analysis investigated the correlations of individual miRNAs with certain confounding factors. On the basis of these profiles, a panel of 7 miRNAs was established for the quality control of samples corresponding to hemolysis and platelet contamination. The panel was used to investigate the confounding impacts based on the size of the blood collection tube, the centrifugation protocol, post-freeze-thaw spinning, and whole blood storage. A standard dual-spin workflow for the processing of blood had been established for optimal sample quality. The real-time stability of 356 miRNAs was also investigated with demonstration of the temperature and time-induced miRNA degradation profile. Stability-related miRNAs were identified from real-time stability study and further incorporated into the quality control panel. This quality control panel enables the assessment of sample quality for more robust and reliable detection of circulating miRNAs.
Asunto(s)
MicroARN Circulante , MicroARNs , Humanos , MicroARNs/genética , MicroARN Circulante/genética , MicroARN Circulante/metabolismo , Biomarcadores , Control de Calidad , Recolección de Muestras de Sangre/métodosRESUMEN
We have recently demonstrated that a 4-in-1 gene therapy strategy that contains two anti-angiogenic genes [endostatin and pigment epithelium-derived factor] and two cytokine genes [granulocyte macrophage colony-stimulating factor and interleukin 12] has a considerable antitumor effect on large tumors in a woodchuck hepatoma model. The current study further investigates the underlying mechanisms for the antitumor effect observed by using small rodent models. We found that immunotherapy alone increased immunosuppressive cells in large tumors over time, whereas the anti-angiogenic therapy contained in the 4-in-1 strategy alleviated immunosuppression and made tumors vulnerable to immunotherapy, thus resulting in a synergistic antitumor effect.
Asunto(s)
Endostatinas/genética , Endostatinas/inmunología , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Terapia Genética/métodos , Inmunoterapia/métodos , Neoplasias Hepáticas Experimentales/terapia , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/inmunología , Serpinas/genética , Serpinas/inmunología , Adenoviridae/genética , Animales , Apoptosis/genética , Apoptosis/inmunología , Línea Celular Tumoral , Terapia Combinada , Endostatinas/biosíntesis , Proteínas del Ojo/biosíntesis , Humanos , Neoplasias Hepáticas Experimentales/irrigación sanguínea , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/terapia , Factores de Crecimiento Nervioso/biosíntesis , Serpinas/biosíntesis , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
UNLABELLED: Immunotherapy is often effective only for small tumor burdens and, in many cases, is restricted to subcutaneous tumors. Here, we investigated the antitumor effects of combination therapy with GM-CSF and IL-12 on orthotopic liver tumors with intermediate or large tumor volumes, or on chemically-induced multifocal liver tumors in animals. Adenoviruses encoding GM-CSF or IL-12 were injected intratumorally to animals bearing transplanted tumors, or injected via intrahepatic artery in animals with primary multifocal liver tumors induced by diethylnitrosamine. Our results demonstrated that IL-12, but not GM-CSF, monotherapy displayed significant therapeutic effects, whereas combination therapy with both cytokines displayed synergistic antitumor effects not only on transplanted tumor models with intermediate or large tumor loads, but also on carcinogen-induced multifocal liver tumors. Effector cell analyses, revealed by in vivo cell subset depletion, flow cytometry analysis, and immunohistochemical staining of tumor infiltrates, indicated that NK cells were the prominent antitumor effectors for the IL-12-mediated antitumor activity, whereas CD8+ T cells, NKT cells, and macrophages were more important than NK cells in the combination therapy-mediated antitumor effects. Both IL-12 monotherapy and combination therapy could induce various types of effectors and high levels of IFN-gamma; however, the latter induced much higher levels than the former, which may explain why combination therapy is superior to IL-12 monotherapy. CONCLUSION: Combination therapy with GM-CSF and IL-12 represents a promising immunotherapy strategy for treating orthotopic, widespread liver tumors.