Article Figures & Data
Figures
- Fig. 1.
Characterization of human glioma-associated cancer-initiating cells from glioblastoma multiforme specimens. A, a representative image of neurospheres from one glioma-associated cancer-initiating cells. B, after 7 days of culture in differentiating medium, the glioma-associated cancer-initiating cells differentiated into glial fibrillary acidic protein+ astroglial lineage cells, microtubule-associated protein 2+ neuronal lineage cells, and galactosylceramidase+ oligodendroglial lineage cells (magnification, ×40), indicating that the glioma-associated cancer-initiating cells have multipotent differentiation potential. C, a representative image of a glioma-associated cancer-initiating cells xenografted into the frontal lobe of a nude mouse. The tumor that developed from the glioma-associated cancer-initiating cells caused enlargement of the brain and was diffusely infiltrative (arrow), including into white matter tracts such as the corpus callosum (arrow).
- Fig. 2.
Glioma-associated cancer-initiating cells mediate immunosuppression on human T cells. A, immune surface phenotype of representative glioma-associated cancer-initiating cells. The glioma-associated cancer-initiating cells were surface stained with antibodies to MHC-I, MHC-II, CD40, CD80, CD86, and B7-H1. Representative FACS histogram plots for one glioma-associated cancer-initiating cell are shown for target staining (solid line) with associated isotype controls (dotted line). Percentages of the positive populations are shown. B, glioma-associated cancer-initiating cells supernatants inhibit T-cell proliferation regardless of activating stimulus. When healthy donor PBMCs were cultured in the presence of the glioma-associated cancer-initiating cells supernatants, T-cell proliferation was inhibited as shown by FACS analysis of T-cell CFSE labeling. Representative FACS histogram plots with the percentages of the indicated cells are shown. C, glioma-associated cancer-initiating cells suppress T-cell proliferation through cell-to-cell contact. When autologous PBMCs were cultured in the presence of glioma-associated cancer-initiating cells from the same glioblastoma multiforme patient, proliferation of T cells was inhibited as shown by FACS analysis with CFSE labeling. A representative FACS histogram plot is shown and similar results were obtained with glioma-associated cancer-initiating cells and matched PBMCs from two other patients. Percentages of the indicated populations of proliferated CSFE-labeled T cells are shown. D, B7-H1 expressed on glioma-associated cancer-initiating cells mediates suppression of T-cell proliferation through cell-to-cell contact. When autologous PBMCs were cultured with cancer-initiating cells of the same glioblastoma multiforme patient in the presence of B7-H1 neutralizing antibody, the inhibition of T-cell proliferation was partially reversed by B7-H1 blockade. The addition of isotype control IgG failed to suppress the inhibition of T-cell proliferation, similar to autologous coculture without IgG as shown in Fig. 2C. E, glioma-associated cancer-initiating cells inhibit T-cell function by downregulating effector cytokine production through cell-to-cell contact. After coculturing with autologous glioma-associated cancer-initiating cells for 3 days, glioblastoma multiforme patients’ PBMCs were stimulated with anti-CD3/anti-CD28, surface-stained with anti-CD3, and then stained to detect intracellular IFN-γ and IL-2. Data were collected via FACScan. Compared with the medium (control), the glioma-associated cancer-initiating cells inhibited both IFN-γ and IL-2 production in gated CD3+ T cells. One representative FACS plot is shown with percentage in the top right quadrant indicating percentage of positive cells. Similar results were obtained for glioma-associated cancer-initiating cells and matched PBMCs from two other patients.
- Fig. 3.
Glioma-associated cancer-initiating cells induce functional Tregs. A, supernatants from the glioma-associated cancer-initiating cells induce an increase of the number of FoxP3+ Tregs in the gated CD4+ T cells as shown by the representative FACS analysis. B, FoxP3+ Tregs induced by the supernatants of cancer-initiating cells suppress T-cell proliferation. T cells that were treated with cancer-initiating cell supernatants were harvested, cocultured for 3 days with autologous PBMCs (labeled with CFSE, responder cells) at a 1:1 ratio in the presence of soluble anti-CD3, and subsequently analyzed via FACScan. The number above the line in each histogram represents proliferating responder cells. C, glioma-associated cancer-initiating cells also induce FoxP3+ Tregs through cell-to-cell contact. FACS analysis shows that the glioma-associated cancer-initiating cells increased the percentage of FoxP3+ Tregs in the gated CD4+ T cells. D, induction of FoxP3+ Tregs mediated by cell-to-cell contact is partially reduced after the addition of B7-H1 neutralizing antibody. One representative FACS plot is shown with percentage in the top right quadrant indicating percentage of positive cells. Similar results were obtained for glioma-associated cancer-initiating cells and autologous PBMCs from two other patients.
- Fig. 4.
Glioma-associated cancer-initiating cells trigger T-cell apoptosis. A, after culturing with the glioma-associated cancer-initiating cells supernatants, T cells were stimulated with anti-CD3/CD28 and stained with 7-amino-actinomycin D and Annexin V. Compared with medium alone (control), the glioma-associated cancer-initiating cells enhanced T-cell apoptosis. B, glioma-associated cancer-initiating cells induce T-cell apoptosis through cell-to-cell contact. The glioma-associated cancer-initiating cells were cocultured with autologous PBMCs at a 1:10 ratio, and an apoptotic assay was done after 3 days of culture. C, B7-H1 blockade in cell-to-cell contacting context reduces T-cell apoptosis. Both apoptotic and preapoptotic T-cell percentages decreased when addition of B7-H1 neutralizing antibody was added to culture conditions compared with the isotype control. One representative FACS plot is shown. Similar results were obtained for glioma-associated cancer-initiating cells and autologous PBMCs from two other patients. D, Galectin-3 can induce T-cell apoptosis in a dose-dependent manner within physiologic ranges produced by cancer-initiating cells supernatants.
- Fig. 5.
Glioma-associated cancer-initiating cells lose immunosuppressive properties on altering their state of differentiation. Glioma-associated cancer-initiating cells were cultured in neural stem cell medium or exposed to differentiating medium for two to three passages (5-7 days per passage). A, CD133 expression was reduced on the glioma-associated cancer-initiating cells on exposure to differentiation medium. B, glioma-associated cancer-initiating cells exposed to differentiating medium were less suppressive of T-cell proliferation. Healthy donor PBMCs were cocultured with supernatants from undifferentiated glioma-associated cancer-initiating cells or altered differentiated glioma-associated cancer-initiating cells in the presence of anti-CD3/CD28 stimulation, and cell proliferation was measured by Cell Counting Kit-8 after 4 days. T-cell proliferation in medium alone (control) was used as a baseline (100%), and the relative change in T-cell proliferation was plotted relative to this baseline. C, glioma-associated cancer-initiating cells exposed to differentiating medium do not induce FoxP3+ Tregs. Cultured T cells on day 4 from A were stained for CD4 and FoxP3, and FACS data were converted into bar graphs showing fold change in the percentage of FoxP3+ Tregs versus medium alone (control; set at baseline of 1). The results are averages from three independent experiments, with error bars showing SD. D, glioma-associated cancer-initiating cells exposed to differentiating medium reduces T-cell apoptosis. Cultured T cells on day 4 were analyzed for apoptosis, and the increase in apoptotic T cells was calculated as follows: percentage of apoptotic T cells in the presence of supernatants from the glioma-associated cancer-initiating cells minus percentage of apoptotic T cells in medium alone (control) divided by percentage of apoptotic T cells in medium alone. B to D, gray bars represent the results from conditioned medium from glioma-associated cancer-initiating cells; black bars represent the results from conditioned medium from differentiated glioma-associated cancer-initiating cells. This entire data set was obtained with similar results from two other patients. **, P < 0.01, significant difference between undifferentiated and differentiated glioma-associated cancer-initiating cells.
Tables
- Table 1.
Characterization of immunosuppressive properties of cloned cancer-initiating cells
Neurosphere parental line Clone number CD133+ (%) CFSE-labeled dividing T cells (%) Change compared with medium (%) Apoptotic T cells (%) Change compared with medium (%) Foxp3+ Tregs (%) Change compared with medium (%) 6-15 1 25.9 6.6 ↓92 60.7 ↑163 40 ↑205 2 88.9 3.8 ↓95 48.9 ↑112 27.8 ↑112 3 84.5 3.2 ↓96 47.5 ↑106 31.7 ↑142 4 47 7.2 ↓91 55.8 ↑142 35.9 ↑174 5 34.6 46.7 ↓42 51.7 ↑124 36.1 ↑176 6 89.6 3.1 ↓96 53.4 ↑131 24.4 ↑86 7 86.3 3.5 ↓96 55.6 ↑141 35.7 ↑173 8 84.7 5.7 ↓93 61.1 ↑165 44 ↑236 9 64 3.7 ↓96 66.5 ↑188 29 ↑121 10 70.2 4 ↓95 67.3 ↑191 31.7 ↑142 11-28 1 97.5 4.1 ↓95 47.8 ↑107 24.2 ↑85 2 93.5 4.8 ↓94 64.1 ↑177 18.6 ↑42 3 76 5.6 ↓93 54.5 ↑136 28.7 ↑119 4 68.4 11.2 ↓86 51.5 ↑123 21.6 ↑65 5 93.6 5.6 ↓93 55.9 ↑142 21.6 ↑65 6 91.1 1.4 ↓98 70 ↑203 11.8 ↓10 7 81.5 5.2 ↓94 54.5 ↑136 29.8 ↑127 8 96.5 4.3 ↓95 52.2 ↑126 28.3 ↑116 9 96.2 6.1 ↓92 50.6 ↑119 29.6 ↑126 10 81.1 3.9 ↓95 57.6 ↑149 13.3 ↑1.5 Control (cancer-initiating) medium None None 81 — 23.1 — 13.1 — U-87 None None 55.2 ↓12 15.7 ↓10 8.9 ↓8 U-87 medium None None 63.3 — 17.4 — 9.7 — U-87 cells propagated in cancer-initiating medium None None 82.9 ↑2 21.8 ↓6 14.5 ↑11 Astrocyte None None 42.7 ↓8 10.9 ↓7 10.6 ↑36 Astrocyte medium None None 46.4 — 11.7 — 7.8 — U-87 medium* None None 34.1 — 29.3 — 11.5 — Glioblastoma multiforme cell suspension in U-87 medium* None — 14.8 ↓56 51.2 ↑78 22.7 ↑97 Glioblastoma multiforme cell suspension in U-87 differentiation medium* None — 28.7 ↓16 33.8 ↑15 10.9 ↓5 ↵*Contact-dependent coculture with autologous PBMCs.
Volume 16, Issue 2
