Fig. 5
NK1.1Ab treatment partially reverses the CCP-evoked elimination of CD133(+) GBM stem cells and CD68high GBM cells. The GBM tumor in each mouse was cut into two parts and processed for IHC and Flow Cytometry, respectively. a-b IHC: the Vehicle-treated mouse GBM sections harbored a significant number of CD133(+) GBM stem cells (red) (a first row). Compared to the Vehicle-treated, the CD133(+) fluorescence showed a 72% decrease in the CCP-treated (*p = 6.5 × 10− 3 CCP versus Vehicle) (a, second row and b) and a 41% decrease in the CCP + NK1.1-treated mice (a third row and b) (Δ p < 0.02, CCP + NK1.1 versus CCP; **p = 0.03, CCP + NK1.1 versus Vehicle). (Scale bar: 47.62 μm). c-f Flow Cytometry: compared to the Vehicle-treated, the CD133(+) cells (c, top, red circle) were suppressed by 81% in the CCP-treated (c middle and CD133 IF shown in d) (*p < 0.05, CCP versus Vehicle) and by 32% in the CCP + NK1.1 mice (c, bottom, and d) (Δ p < 0.03, CCP + NK1.1 versus CCP; **p = 3.0 × 10− 3, CCP + NK1.1 versus Vehicle). e The fluorescence profiles of the CD133(+) cells in the Vehicle, CCP, and CCP + NK1.1 samples. f-h The GBM cells were also probed for CD68, and active-caspase-3 (Act. Cspse 3). f i In the Vehicle-treated, two segregated populations of CD68(+) but Cspse3(−) cells were identified (Lower Right, LR). The larger population (shown within the blue circle) was CD68high (presumably GBM cells) whereas a smaller population (shown within the green circle) was CD68low (expected to be the TAM) [8, 10, 18]. A very small population of CD68(+), Act. Cspse3(+) (double positive) cells was identified in the upper right (UR) quadrant (within the red ellipse). f ii In the CCP-treated, CD68high cells showed an 1187% increase in Act. Cspse 3 IF (red ellipse in the UR quadrant) with respect to Vehicle, along with the virtual disappearance of the CD68high but Cspse3(−) cells in the blue circle in the LR quadrant (*p = 0.036, CCP versus Vehicle) (f ii and g). The Act. Cspse 3 IF increased by 343% in the CCP + NK1.1 samples, along with the reappearance of some CD68hgh but Act. Cspse 3(−) cells in the blue circle in the LR quadrant (f iii and g) (Δ p < 0.04, CCP versus CCP + NK1.1; **p = 3.1 × 10− 3, CCP + NK1.1 versus Vehicle). (f iv) Fluorescence profiles for Act.Cspse 3. The CD68 fluorescence in CD68low cells (TAM) was not significantly different in the three groups (h). The graphs represent mean ± S.D. obtained from mice treated with Vehicle (n = 4), CCP (n = 4), and CCP + NK1.1 (n = 3)