| Aims:To culture and isolate BTSCs (brain tumor stem-like cells) from glioma first, then characterize BTSCs including morphology, clonogenicity, growth curve, tumorigenicity and differentiation. To compare the expression ratio of negative co-stimulatory molecular B7-H1 between BTSCs and non-BTSCs through multiple methods. To describe the immunological characteristics of BTSCs in vitro, and whether it can utilize B7-H1 to escape immune surveillance better than non-BTSCs.Materials and methods:Six primary GBM (glioblastomas multiform) samples obtained from surgical patients suffered from GBM and one glioma cell line (U87) were cultured in serum free medium (SFM, containing EGF, bFGF and B27) and in serum medium respectively. The growth characteristics of tumor cells in different culture conditions (SFM and SCM, serum containing medium) were observed. The expression ratio of A2B5 and CD133 were analyzed with flowcytometry. A2B5-positive and A2B5-negative U87 and primary glioma cells were sorted by fluorescence activated cell sorting. Cell morphology under SFM condition, proliferation capacity, expression of stem cell markers and differentiation markers, and tumorigenicity were compared between these two populations by monoclonal formation assay, XTT assay, immunofluorescence assay and nude mice subcutaneous xenografts, to identify BTSCs.B7-HI expression was compared between A2B5-positive and A2B5-negative glioma cells isolated from six primary GBM by immunofluorescence assay, western blot assay and flowcytometry assay. A2B5 and B7-H1 expression of U87 cells, SHG62 cells and SHG62 cells (cultered in long-term) was analyzed by double-labeling flowcytometry assay, and B7-H1 expression was compared between A2B5-positive and A2B5-negative cells isolated from glioma cell lines which were in long-term maintained in vitro.Antigens were prepared from A2B5-positive and A2B5-negative glioma cells isolated from primary GBM. Autologous peripheral blood mononuclear cells (PBMCs) were collected from patient’s peripheral blood. Mature DCs were obtained from adherent PBMCs after co-culture with GM-CSF and IL-4 first, and then loaded with tumor lysates later. Effector cells (cytotoxic T cells, CTLs) were obtained from non-adherent PBMCs after co-cultured with mature DCs. The immunological characteristics of A2B5-positive and A2B5-negative cells before and after intervention of B7-H1 were compared and analyzed by CSFE/PI cytotoxicity assay and XTT specific T cell proliferation assay. Unsorted U87 cells were cultured in the following three conditions:1) SCM; 2) SFM; 3) SFM under concurrent chemoradiotherapy intervention. CTLs cytotoxicity effects were compared between groups before and after intervention of B7-H1.Results:Primary GBM cells cultured in SFM were partially suspended to form tumorspheres, partially adherent to the dish; while U87 cells cultured in SFM were all suspended. Sorted A2B5-positive cells were suspended to form tumorspheres. but A2B5-negtive cells could hardly form tumorspheres. Colony efficiency of A2B5-positive cells, unsorted glioma cells and A2B5-negtive cells were 5.73%±0.64%,3.01%±0.50% and 0% respectively. A2B5-positive cells grew faster than the unsorted and A2B5-negtive cells in terms of their growth curves examined by XTT assay.106 A2B5-positive cells could generate subcutaneous xenografts, which exhibited typical biological features of glioma, in nude mice; while A2B5-negtive cells, up to 107 were not tumorigenic. A2B5-positive cells cultured in SFM expressed stem cell markers such as nestin and CD133; and expressed differentiated markers such as GFAP,β-tubulin and CNPase when culture in SCM, examined by immunofluorescencing assay. The result of double-labeling flowcytometry assay indicated:A2B5-positive cells contained CD133-positeve and CD133-negtive cells, A2B5-negtive cells contained CD133-negtive cells only, A2B5-CD133+ cells did not exist, A2B5-positive cells covered the whole population of CD133-positive cells.The expression level of negative co-stimulatory molecule B7-H1 was higher in A2B5-positive primary glioma cells than in A2B5-negtive cells as indicated by the results of immunofluorescencing assay, western blot assay and flowcytometry assay (39.50%±8.76% Vs 16.41%±5.41%, respectively). The expression level of B7-H1 in A2B5-positive and A2B5-negtive cell lines long-termly maintained in vitro was 31.22%±24.04% and 42.52%±25.20% respectively, the difference did not reach statistic significance.The results of CTLs cytotoxicity assay indicated:the cytotoxic efficiency of CTLs to tumor cells (BTSCs or non-BTSCs) was higher when activated by BTSCs antigens than by non-BTSCs antigens (19.09%±2.89% Vs 12.69%±3.47%; 30.05%±4.98% Vs 23.09%±1.04%, P<0.05, t test); the cytotoxic efficiency of CTLs activated by the same type of antigens (BTSCs or non-BTSCs antigens) was lower to BTSCs than to non-BTSCs (19.09%±2.89% Vs 30.05%±4.98%; 12.69%±3.47% Vs 23.09%±1.04%, P<0.01, t test); the cytotoxic efficiency of CTLs to BTSCs augmented about 55.95% after B7-H1 blockade (29.77%±3.05% Vs 19.09%±2.89%, P<0.01, t test), while the augmentation of cytotoxic efficiency to non-BTSCs was not obvious (33.71%±6.08% Vs 30.05%±4.98%, P>0.05, t test); the difference between cytotoxic efficiency of CTLs to BTSCs and non-BTSCs after B7-H1 blockade was not obvious (29.77%±3.05% Vs 33.71%±6.08%,P>0.05, t test).The results of specific T cell proliferation assay indicated:A2B5-positive BTSCs could inhibit autologous T cell proliferation, and this inhibition was partially reversed by the addition of B7-H1 blocking antibody; while the inhibition of autologous T cell proliferation induced by A2B5-negtive non-BTSCs was not obvious.The CTLs cytotoxic efficiency to U87 cell lines cultured in the three culture conditions mention above, and before and after B7-H1 blockade was 24.56%,25.70%, 17.00%,12.80%,24.30% and 23.18% respectively,. The mean cytotoxic efficiency was 21.95%±4.29% and 20.56%±6.84%, respectively before and after B7-H1 blockade, the difference did not reach statistic significance.Conclusion:A2B5-positive GBM glioma cells have BTSCs characteristics, and cover the whole population of CD133-positive cells, indicating that A2B5 can be an efficient biomarker for BTSCs isolation. A2B5-positive BTSCs can induce immunosuppression while A2B5-negtive non-BTSCs do not. A2B5-positive BTSCs is better to escape from immune surveillance, not only quantitatively but also functionally by utilizing negative co-stimulatory molecule B7-H1 pathway. Blocking of B7-H1 pathway can augment T cell cytotoxic efficiency to A2B5-positive BTSCs. B7-H1 molecule expressing on U87 cell lines, which is cultured in immune-deficient condition for innumerable passages, might not possess negative immune modulation function. |
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