Figure 1.

C-mediated lysis of T47D breast adenocarcinoma spheroids. A: The effect of pulsed versus single C treatment on microtumor lysis by antibodies and C. In one set of experiments, ⁵¹Cr-labeled spheroids were exposed to biotinylated anti-CD59 antibody (YTH53.1B; 25 μg/ml), polyclonal rabbit IgG against the tumor cells (S2; 50 μg/ml), and NHS (diluted 1:2) at the beginning of the experiment (□), and the released radioactivity was counted from the supernatants after 24 hours of incubation at 37°C. In another set of experiments, the consumed C was replaced by fresh NHS at 2, 4, and 8 hours during the 24-hour incubation. The pulsed addition (▪) of C increased the lysis of spheroids from 16 ± 4 to 33 ± 2.3% (mean ± standard deviation (SD)). In the controls, normal rat IgG (25 μg/ml) and preimmune rabbit IgG (S0; 50 μg/ml) were used instead of YTH53.1B and S2, or YTH53.1B and S2 were used separately. B: Time course of spheroid lysis. ⁵¹Cr-labeled T47D spheroids were exposed to S2 and YTH53.1 (•), S0 and Rat IgG (○), S2 (▵), YTH53.1 (▴), and NHS as above for the times indicated. During the incubation the microtumors were isolated and placed into new tubes containing fresh serum and the respective antibodies at 2, 4, and 8 hours. In C and D, the lysis of the spheroids was examined for 48 and 50 hours, and fresh Complement was added during the incubation 7 or 11 times, respectively. The radioactivity released was counted from the supernatants. A to D: Cell lysis was determined as the percentage of total radioactivity: ((Released cumulative radioactivity − spontaneous ⁵¹Cr release)/(Total radioactivity − spontaneous ⁵¹Cr release)) × 100%. The vertical bars indicate the SD of quadruplicate determinations. Two-tailed Student’s t-test; *P < 0.05; comparison between lysis values of spheroids exposed to a single dose and multiple doses of complement (A); ***, P < 0.001 lysis of spheroids exposed to S2, YTH53.1B (•) and NHS was significantly different from control spheroids (S0 + rat IgG; ○; B).