Immunogenicity of mGC cells. (A) Anti-CEA antibodies were determined in the serum of C57BL/6 and CEA-transgenic mice using flow cytometry. All mice had progressively growing tumors without overt necrosis after transplantation and growth of the indicated cell lines for 35–40 days. mGC4CEA cells were incubated with the serum of the indicated mice at different dilutions and bound primary antibodies were detected with a PE-conjugated anti-mouse antibody. (B, C) Three C57BL/6 mice each were injected subcutaneously three times at weekly intervals with 1 × 106 mGC8 cells killed by two freeze-thaw cycles. Two weeks after the last vaccination, the mice were challenged by injection with 1 × 106 (B) and 3 × 106 (C) live mGC8 cells, respectively (filled-in circles). As a control, tumor cells were injected in non-immunized mice (open circles). Tumor volumes were calculated as described in Material and Methods section. Results are shown as mean values +/- SD.