| Objective:The objective of this experiment is to investigate the implications of DC vaccine pulsed with lysates of brain tumor stem cells for the treatment of animal brain glioma in vivo.1. To create a mouse orthotopic GL261brain glioma model, and observe all stages of GL261brain glioma progression and immune pattern in tumor-bearing mice.2. To explore the immunogenicity of GL261cells and biological characteristics of A2B5+GL261cells.3. To treat GL261brain glioma in vivo mediated by DC vaccine pulsed with lysates of A2B5+GL261cells, and investigate its possible action mechanisms.Methods:GL261cells were cultured in DMEM supplemented with10%FBS. To Create a mouse orthotopic GL261brain glioma model,1×10GL261cells were intracranially implanted in the right frontal lobe of syngeneic C57BL/6mice, symptoms and survival times were closely monitored. Mice were euthanized when moribund, and brains were harvested and prepared histologically for HE staining and immunohistochemical staining of GFAP、Ki-67and CD3, respectively.Three C57BL/6mice were sacrificed respectively at4、8、12、16、20and24days after intracranial implantation of1×105GL261cells, and brains and spleens were taken out for research. The progression of GL261brain glioma was observed by light microscope. The changes of spleen index、spleen cell counts、spleen T lymphocytes subgroups and infiltration of T lymphocytes in GL261brain glioma were calculated respectively.C57BL/6mice and Balb/c-nu mice were subcutaneously implanted with1X106GL261cells respectively, and take-rate of subcutaneous tumor was compared. GL261cells used in vaccination study were irradiated with20Gy X-ray. In prophylactic vaccination study. C57BL/6mice were subcutaneously inoculated with1X106 irradiated GL261cells, respectively12and5days before1×105GL261cells intracranial implantation; In therapeutic vaccination study, C57BL/6mice were subcutaneously inoculated with1×106irradiated GL261cells, respectively7、14and21days after1×105GL261cells intracranial implantation. A comparison in survival time and take-rate of GL261brain glioma was conducted. H-2Kb and H-2I-Ab molecules expression in GL261cells were detected by flow cytometry.GL261cells were cultured in serum-free DMEM/F12supplemented with20ng/mL EGF、20ng/mL bFGF and2%B27. A2B5+and A2B5-GL261cells were isolated by FACS, and single cell clone formation assay was conducted respectively. Different amounts of GL261cells(1×104、1×103、1×102A2B5+GL261cells and1×105、1×104、1×103A2B5-GL261cells) were intracranially implanted in C57BL/6mice, and take-rate of GL261brain glioma was compared.Dendritic cells were obtained from bone marrow of normal C57BL/6mice and cultured in RPMI1640supplemented with15%FBS、1Ong/mL mGM-CSF and lOng/mL mIL-4. DCs were pulsed with lysates of freeze-thawed A2B5+and A2B5-GL261cells respectively. Subsequently primed DCs were cocultured with spleen T lymphocytes to introduce CTL, and cytotoxicity of CTL was assayed through CFSE/PI flow cytometry. C57BL/6mice were intracranially implanted with1×105GL261cells (cultured in DMEM supplemented with10%FBS) on day0:in DC vaccination study before tumor formation, mice were treated with subcutaneous inoculation of1×106DCs (pulsed with lysates of A2B5+or A2B5-GL261cells) on day2、9and16; in DC vaccination study after tumor formation, mice were treated with subcutaneous inoculation of1×106DCs (pulsed with lysates of A2B5+or A2B5-GL261cells) on day12、19and26. A comparison in survival time and take-rate of GL261brain glioma was conducted. The mRNA expression of gp100and TRP-2in A2B5+and A2B5-GL261cells was detected by RT-PCR.Results:After intracranial implantation of1×105GL261cells, C57BL/6mice had a hundred percent take-rate of GL261brain glioma and a median survival of3to4weeks, which is highly reproducible. The GL261brain glioma showed an invasive behavior similar to human GBM. After intracranial implantation of1×105GL261cells, no tumor progression was observed in early stage. The early GL261brain glioma formation was observed on12days after the implantation; subsequently GL261brain glioma presented a radial growth pattern. In early stage, C57BL/6mice were in a state of immune activation, manifested as increased spleen index and spleen cell counts; after tumor formation, C57BL/6mice were in a state of immune tolerance, manifested as recovered spleen index and spleen cell counts; in end stage, C57BL/6mice were in a state of immune suppression, manifested as decreased spleen index and spleen cell counts and increased spleen Treg cells. There was an obvious infiltration of T lymphocytes in early GL261brain glioma, but sporadic in end stage.After1×106GL261cells were subcutaneously implanted in C57BL/6mice, GL261cells were circumscribed or rejected. In prophylactic vaccination study, six of eight C57BL/6mice remained tumor free; but in therapeutic vaccination study, no surviving mice could be found. GL261cells expressed low levels of H-2Kb and H-2I-Ab molecules.In serum free medium, the ratio of A2B5+GL261cells was10.36%. In single cell clone formation assay,11.5%A2B5+GL261cells formed tumor spheres whereas A2B5-did not. And we found that as few as1×103A2B5+GL261cells could initiate GL261brain glioma, whereas1×105A2B5-GL261cells could not.Murine bone marrow-derived DCs could be primed with lysates of A2B5+or A2B5-GL261cells. Cytotoxicity assays suggested that, DCs pulsed with lysates of A2B5+GL261cells could induce specific CTL against both A2B5-and A2B5+GL261cells; DCs pulsed with lysates of A2B5GL261cells could also induce specific CTL against A2B5-GL261cells, but poorly against A2B5+GL261cells. In DC vaccination study before tumor formation, three of eight C57BL/6mice treated with DCs pulsed with lysates of A2B5+GL261cells remained tumor free, and mice treated with DCs pulsed with lysates of A2B5+or A2B5-GL261cells had a prolonged survival. In DC vaccination study after tumor formation, mice treated with DCs pulsed with lysates of A2B5+or A2B5GL261cells all expired, no prolonged survival was found. The expression levels of TRP-2in A2B5+GL261cells were higher than in A2B5-GL261cells. Conclusions:The mouse orthotopic GL261brain glioma model is highly reproducible, and it is an attractive preclinical model for human GBM, but the moderate immunogenicity of GL261cells should be considered.Immune pattern in tumor-bearing mice experiences three stages:immune activation in early stage、immune tolerance after tumor formation and immune suppression in end stage.A2B5+GL261cells have brain tumor stem cell properties. DC vaccine pulsed with lysates of A2B5+GL261cells is superior to pulsed with lysates of A2B5-GL261cells in vitro and in vivo. |
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