RESUMEN
BACKGROUND: Microglial cells are the predominant immune cells in malignant brain tumors, but tumors may release some factors to reduce their defensive functions. Restoration of the anti-cancer function of microglia has been proposed as a treatment modality for glioblastoma. We examined the effect of intra-cranially administered recombinant adeno-associated virus encoding interleukin-12 (rAAV2/IL12) on transfection efficiency, local immune activity and survival in a rat model of glioblastoma multiforme. METHODS: F344 rats were injected with rAAV2/IL12 and implanted with syngeneic RG2 cells (glioblastoma cell line). Intracerebral interleukin-12 and interferon-γ concentrations were determined by ELISA. Activation of microglia was determined by expressions of ED1 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) which were evaluated by Western blotting and immunohistochemistry. The proliferation of cancer cells was evaluated with Ki67 immunohistochemistry and apoptosis of cancer cells with TUNEL. RESULTS: The brains treated with rAAV2/IL-12 maintained high expression of interleukin-12 and interferon-γ for at least two months. In syngeneic tumor model, brains treated with rAAV2/IL12 exhibited more infiltration of activated microglia cells as examined by ED1 and TRAIL stains in the tumor. In addition, the volume of tumor was markedly smaller in AAV2/IL12-treated group and the survival time was significantly longer in this group too. CONCLUSION: The intra-cerebrally administered rAAV2/IL-12 efficiently induces long lasting expression of IL-12, the greater infiltration of activated microglia cells in the tumor associated improved immune reactions, resulting in the inhibited growth of implanted glioblastoma and the increased survival time of these rats.
Asunto(s)
Dependovirus , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Terapia Genética , Glioblastoma/terapia , Interleucina-12/biosíntesis , Microglía/patología , Proteínas del Tejido Nervioso/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Transducción Genética , Animales , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Interleucina-12/genética , Masculino , Microglía/metabolismo , Trasplante de Neoplasias , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Endogámicas F344 , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Trasplante IsogénicoRESUMEN
Microglia have been found to infiltrate into malignant brain tumors. However, their function is usually reversed by cancerous cells thus contributing to cancer growth and metastasis. We propose that microglial immuno-activity can be modulated with interleukin-12, by which the anti-cancer ability might be restored. To this end, a strategy was designed using AAV2 carrying interleukin-12 to activate microglia to eliminate cancerous cells. Under this strategy, recombinant AAV2 encoding interleukin-12 was constructed and evaluated for its transduction efficacy on cancerous and CNS cells. The bioactivity of microglia modulated by interleukin-12 was examined and death receptors 4 and 5 were detected on cancerous cells. The effects of interleukin-12 on microglial cytotoxicity were evaluated by MTT assay. The human cell line DBTRG, surgical specimens of GBM and rat astrocytes expressed AAV2-mediated GFP quite strongly. Interleukin-12 secretion was detected in DBTRG, RG2 and astrocytes after the transduction of AAV2 encoding interleukin-12. TRAIL releasing and phagocytotic activity of microglia were significantly increased after interleukin-12 stimulation. MTT assay of microglial cytotoxicity elicited significant increase after the stimulation with interleukin-12 protein. In conclusion, AAV2 is an effective vector in transferring therapeutic genes such as interleukin-12 to enhance microglial anti-cancer activity and to eliminate cancerous cells.