Fig. 3

Antibody dependent cytotoxicity and antitumor activity of CRA-induced antisera collected from CT26.CL25 tumor-bearing mice. Panel A. Cytotoxicity of CRA antisera. CT26.CL25 cells were treated with 2 µL antisera and incubated for 48 h. Cell viability was determined by MTT assay. To prepare sCRA antisera, the anti-CT26.CL25 antibodies from CRA antisera were pre-subtracted by CT26.CL25 cell binding. The data are reported as the proliferation index. N: normal sera; Ctrl: control antisera; sCRA antisera: CRA antisera with the subtraction of anti-CT26.CL25 antibodies. Panel B. CRA antisera-mediated ADCC. CSFE labeled CT26.CL25 cells serving as target cells were incubated with mouse splenic NK cells (effector cells) in an E:T ratio of 8:1 with 2 µL normal sera or antisera for 4 h. Panel C. CRA antisera-mediated ADCP. RAW264.7 macrophages treated with unopsonized or opsonized CT26.CL25 cells (incubated with 2 µL antisera) for 4 h. The percentage of phagocytosis for normal sera and antisera is shown. Panel D. CRA antisera induced CDC activity. CDC activity of CRA antisera used horse complements. Cell lysis was determined after the addition of antisera for 4 h. Panel E. Schematic representation of animal study with CRA antisera. Panel F. Tumor volumes in the CT26.CL25 tumor-bearing SCID mice (n = 5/group) after treatment with 100 μL of control or CRA antisera weekly. *: P < 0.05, compared to the untreated group (NC); #: P < 0.05, compared to the control antisera group. Panel G. Image of tumors from control or CRA antisera treated mice. Panel H. Schematic representation of animal study with in vivo B cell depletion and CRA treatment. Panel I. Effects of CRA on tumor growth in immunocompetent or B cell-depleted BALB/c mice. B cell-depleted BALB/c mice (n = 5/group) bearing CT26 tumors were treated with 50 mg/kg of CRA by oral injection every week. Tumor volumes were measured every 3 days after treatment. **: P < 0.01, compared to the control group