Figure 1.

Resistance of differentiated DPSCs and OSCCs but not stem-like OSCSCs or DPSCs to untreated, IL-2-treated, and IL-2 + anti-CD16-treated NK cell cytotoxicity; loss of NK cell cytotoxicity and gain in secretion of IFN-γ after NK cell receptor signaling. OSCCs or OSCSCs were seeded at 1 × 10⁵ cells/well in 24-well plate for 24 h prior to the addition of highly purified NK cells pre-treated with IL-2 (1000 units/ml) for 24 h. NK cells were added to tumor cells at 2:1 effector to target ratio. At time 0 when NK cells were added to the tumor culture, a final concentration of 10 μg/ml of propidium iodide (PI) was also added. The cells were then subsequently tracked for over 72 h using time-lapse microscopy with Nikon Eclipse Ti-E inverted microscope fitted with a culture chamber to provide cells with a stable temperature of 37°C with 5% CO₂. An image was taken every 15 min and a representation is shown at day 1, day 1½, and day 3. OSCSCs but not OSCCs which are lysed take up PI and appear orange in the time-lapse (A). The surface expression of CD26, CD44, CD166, and CD326 on OSCCs and OSCSCs were assessed with flow cytometric analysis after staining with the respective PE-conjugated antibodies. Isotype control antibodies were used as control. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (B). The surface expression of CD338 on OSCCs and OSCSCs was assessed by flow cytometric analysis after staining with PE-conjugated CD338 (right graphs in the histogram). Isotype control antibodies were used as control (left graphs in the histograms) (C). OSCCs and OSCSCs were left untreated or treated with 10–80 μg/ml of cisplatin for 18 h, after which the tumor cells were washed with 1× PBS, detached, and stained with propidium iodide (PI) and percent cell death was determined using flow cytometric analysis (D). NK cells were left untreated or treated with IL-2 (1000 units/ml), anti-CD16 mAb (3 μg/ml), or a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 18 h before they were added to ⁵¹Cr-labeled OSCSCs and OSCCs (E) or undifferentiated and differentiated DPSCs (G). NK cell-mediated cytotoxicity was determined using a standard 4 h ⁵¹Cr release assay and the lytic units 30/10⁶ cells were determined using inverse number of NK cells required to lyse 30% of the target cells × 100. NK cells were treated as described in (E) and each NK sample was either cultured in the absence or presence of OSCSCs and OSCCs (F) or undifferentiated and differentiated DPSCs (H) at an NK cell to target cell ratio of 0.5:1. After an overnight incubation, the supernatant was removed from the co-cultures and the levels of IFN-γ secretion were determined using specific ELISAs. One of minimum three representative experiments is shown in each of (B–H).