Stem-like protein expression and function modulated by T cell activity.
(A) Flow cytometric staining and analysis was performed on GL26 tumor section from brains of nude (GL26nu), C57BL6J (WT; not shown), and C57BL/6J mice vaccinated with 2×106 GL26 lysate-pulsed DC2.4 cells 3 and 7 days post-tumor implantation (“WT, vac”). Percentage of positive cells appears above gating bar. Markers shown had significantly different (P<0.05) positive cell percentages in 3 independent samples of GL26nu and GL26B6V cells by ANOVA and/or one-tailed T-test. (B) Immunofluorescence staining was performed on GL26 tumor section from brains of nude, and C57BL/6J mice vaccinated with 2×106 GL26 lysate-pulsed DC2.4 cells 3 and 7 days post-tumor implantation (“WT, vac”). Results are representative of ≥ samples/group, with the exception of CD133, which revealed strongly positive tumor staining in 2 of 4 brains. WT brains generally exhibited staining similar to nude, or intermediate staining between that of nude and WT, vac (not shown). Marker expression of WT, vac (Prominin+, GFAP-, Sox-2+, Nestin+) is characteristic of cancer stem cells. However, Sox-2 expression was not isolated to nuclei in WT, vac GL26 (right panel, top left inset), though Sox-2 staining of contralateral ventricular cells in the same brain was (right panel, top right inset). (C) Cell numbers in absence or presence of the indicated concentrations of Erlotinib (Tarceva) or Cyclopamine were determined for low-passage (≤5) GL26nu and GL26B6V by Coulter counter. Differences between GL26nu and GL26B6V for either drug, and between erlotinib and cyclopamine within the same recovered tumor lines were significant (P<0.03 by one-tailed T-Test and ANOVA), at concentrations above 4 uM. Distinct glioma lines exhibited opposite patterns of drug sensitivity: GL26nu cells were sensitive to erlotinib but not to cyclopamine, and GL26B6V cells were sensitive to cyclopamine but not to erlotinib, consistent with their reciprocal expression of EGFR and SHH target genes.