Immunotherapy effect of dendritic cells pulsed by lysate of tumor cell Ar-He cryoablation on rat models of intracraniai gliomas

Abstract

Objective To investigate the role of C6 glioma cells mediated by rapid freezing and thawing ofAr-He cryoablation in the maturation of marrow-derived dendritic cells (BM-DCs) in Wistar rats,and the anti-tumor effect of these DCs on rat models of intracranial gliomas. Methods C6 glioma cells were routinely cultured in vitro; rapid freezing and thawing of Ar-He cryoablation was employed in C6 glioma cells of the experimental group, and C6 glioma cells of the negative control group were only performed insertion of the probe; blank control group (using rapid freezing and thawing of Ar-He cryoablation on the same amount of PBS) was also employed.Bone marrow-derived mononuclear cells (MNCs) were first prepared from tibia and femur bones of Wistar rats. These cells were cultured with such cytokines as recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF),recombinant interleukin-4 (rmIL-4) and tumor necrosis factor-alpha (TNFα) to induce their maturation; BM-DCs were pulsed with or without tumor cell lysate obtained by rapid freezing and thawing of Ar-Hecryoablation at a ratio of (DC:tumor cells =1:3) 7 d after that.Morphological observation of BM-DCs was performed by light microscopy and the expression of DCs costimulatory molecules CD80 and CD86 were measured by flow cytometry 48 h after the addiction; the IL-12 level in the supematant of DCs was detected by ELISA. In order to determine whether or not vaccination with C6 TP DCs can induce the therapeutic potential in the established glioma-bearing models, the C6 cells cultured in vitro were stereotaxically implanted into the left caudate nucleus of Wistar rat brain; glioma-bearing rats were injected with vaccination with DCs,cells from the blank control group and negative control group on the 3rd and 10th d. Survival time was observed and determined using the method of Kaplan-Meier and Log-Rank analysis. Results DCs from rats' bone marrow cells cultured with cytokines and pulsed with tumor lysates showed the characters of typical mature DCs.Morphologically,these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, they expressed high levels of CD80 and CD86 antigens (71.8 1％± 1.10％ and 74.66％± 1.48％ in experimental group,49.49％±1.08％ and 51.20％±2.06％ in negative control group, and 48.47％±1.09％ and 49.53％±1.89％ in blank control group); significant difference was noted between each 2 groups (P＜0.05).Functionally,the IL-12level in the supernatant of DCs showed obvious increment in the experimental group (245.99 ±3.20pg/mL) as compared with that in the negative control group (138.68±3.20 pg/mL) and blank control group (135.16±2.88 pg/mL,P＜0.05).These cells gained the capacity of mediating immunotherapy against intracranial gliomas in rats:the median survival in the experimental group (33 d) was significantly higher than that in the negative control and blank control groups (22 and 24 d, respectively, P＜0.05).Conclusion C6 glioma cells mediated by rapid freezing and thawing of Ar-He cryoablation can induce maturation of BM-DCs in Wistar rats; these BM-DCs pulsed with tumor lysates, as new therapeutic vaccines,can mediate immunotherapy against intracranial gliomas in rats. Key words: Ar-he Cryoablation; Glioma; Dendritic cell; Vaccine; Immunotherapy