Light (A and B) and scanning electron (C to F) microscopic images of intact (A, C, and E) and C-attacked (B, D, and F) T47D breast adenocarcinoma cell spheroids. Spheroids were selected for the experiments after 10 to 14 days of culture and exposed to YTH53.1B and S2 and NHS at 37°C for 24 hours (B, D, and F) as in Figure 1B ▶ . The spheroid shown in A was photographed before the NHS exposure. In C and E, the spheroids were exposed to NHS, S0, and normal rat IgG for 24 hours. The surface of a spheroid exposed to C-activating (S2) and CD59-neutralizing (YTH53.1B) antibodies and NHS (B) was less coherent than that of the control (A). Surface cells became swollen and detached. Scanning electron microscopy also showed damaged, swollen, and detaching cells on the spheroid surface (D). At higher magnification, remnants of cells with destroyed, porous membranes could be seen with released vesicles (F, arrow). Bar, 100 μm (A). Magnification, ×500 (C and D) and ×3000 in E and F.

Immunofluorescence microscopy analysis of antibody and C1q penetration into T47D spheroids. Multicellular spheroids grown for 10 days were exposed to S2 (50 μg/ml), YTH53.1 (anti-CD59; 25 μg/ml), and human serum (diluted 1:2) for 2 hours (A, C, and E) or 24 hours (B, D, and F; pulsed doses) at 37°C. In the controls (G, H, and I), S0 and normal rat IgG were used instead of S2 and YTH53.1 during a 24-hour incubation. Cryostat sections (5 μm) were fixed with 4% paraformaldehyde and incubated with antibodies against rat IgG (A, B, and G), human C1q (C, D, and H), and rabbit IgG (E, F, and I). Within 2 hours, YTH53.1 (A) and C1q (C) had penetrated four to five or two to three cell layers deep, respectively, into the spheroids. S2 IgG remained on the outermost surface (E). After a 24-hour exposure the YTH53.1 mAb (B) and C1q (D) had penetrated through the spheroids, whereas the polyclonal S2 IgG (F) did not penetrate completely to the core. In controls, no staining for rat IgG (G), C1q (H), or rabbit IgG (I) was detected. Bar, 100 μm (B, D, and E to I) and 200 μm (A and C).

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).

Visualization of C-mediated killing of PA-1 spheroids by the incorporation of PI into cellular DNA. A: PA-1 spheroids grown for 10 days were incubated with YTH53.1B (25 μg/ml) and S2 (50 μg/ml) antibodies and human serum for 4 hours at 37°C and with PI (50 μg/ml) for 1 hour. In the control (B), normal rat IgG (25 μg/ml) and S0 (50 μg/ml) were used instead of YTH53.1 and S2. After incubation, the spheroids were frozen in liquid nitrogen. Examination of cryostat sections (10 μm) by fluorescence microscopy shows PI staining of the nuclei of killed cells. In A, rims of dead cells can be seen on the surfaces of the spheroids. In the control (B), occasional necrotic cells in the center of a spheroid are stained. Bar, 200 μm (A).

Comparison between C (C3c and C5b-9) penetration and cell death as assessed by PI staining of T47D spheroids. Spheroids treated with S2, YTH53.1B, and human serum for 2 (A, C, and E) or 24 hours (B, D, and F) at 37°C as in Figure 4 ▶ . In the controls (G, H, and I), S0 and normal rat IgG were used instead of S2 and YTH53.1B. PI was added to the mixture 1 hour before termination of the incubation. Cryostat sections (10 μm) of the spheroids were fixed with 4% paraformaldehyde and incubated with antibodies to human C3c (A, B, and G) and the C5b-9 neoepitope (Wu; C, D, and H). Bound antibodies were detected with FITC-conjugated secondary antibodies. Deposition of C3c and C5b-9 after 2 hours of exposure to C can be seen as green fluorescence on the surface of the spheroid sections (A and C, respectively). Cell death after a 2-hour C exposure appears as yellow fluorescence in E. Some necrotic cells in the microtumor cores were also stained with PI. The deposition of C3c and C5b-9 remained peripheral after the 24-hour C exposure (B and D, respectively), and a large pool of dead and detaching cells was observed around the microtumors (F). No deposition of C3c (G) or C5b-9 (H) was detected in the control spheroids. In the control spheroids, only the necrotic cells in the cores of the microtumors were stained with PI (I). Bar, 100 μm.