Figure 1.

Selection of MC32-S2–1 and MC32-S4–1 cells showing tumor growth in mice immunized with CEA DNA vaccines. (A) Each group of mice (n = 6–8/group) was immunized by IM-EP with 50 μg of CEA DNA vaccines (pCEA) per mouse at 0 and 1 weeks. The animals were also injected intraperitoneally with 50 μg of anti-4–1BB Abs at 0 and 1 weeks. At 3 weeks, the mice were challenged s.c. with 1 × 10⁵ MC32 cells per mouse. Tumor sizes were measured and recorded at 41 d following tumor cell challenge. In the group immunized with pcDNA3+anti-4–1BB Abs, one mouse showing no tumor formation was not included in the tumor size calculation to reduce variation. The values and bars represent mean tumor sizes and the SD, respectively. The numbers in (/) denote the number of mice showing no tumor formation at 50 d post-tumor cell challenge/the number of mice tested. Of the 8 mice challenged with MC32 cells following immunization with pCEA+anti-4–1BB Abs, 4 tumor-forming mice were sacrificed. Tumor tissues were surgically removed and then expanded more than 5 times in cDMEM. The resultant 4 tumor cells were designated as MC32-S1, MC32-S2, MC32-S3 and MC32-S4. Of the 8 mice challenged with MC32 cells following immunization with pCEA+anti-4–1BB Abs, 4 tumor-free mice were re-immunized with pCEA at 60 d post-tumor cell challenge. At 7 d following final immunization, each of the mice (F–I) was re-challenged with 5 × 10⁵ MC32 cells per mouse on the right flank and with 5 × 10⁵ MC32-S1 (F), MC32-S2 (G), MC32-S3 (H), or MC32-S4 (I) cells per mouse on the left flank. Each of the control mice was also challenged with 5 × 10⁵ MC32 cells per mouse on the right flank and with 5 × 10⁵ MC32-S1 (B), MC32-S2 (C), MC32-S3 (D) or MC32-S4 (E) cells per mouse on the left flank. Tumor sizes were measured at each time point, and each tumor volume was recorded. *P < 0.05 using ANOVA compared with pcDNA3.