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. 2015 Jan 22;4(3):e994391. doi: 10.4161/2162402X.2014.994391

Figure 5.

Figure 5.

The calreticulin exposure induced by Tras-Permut CrossMab is essential for induction of antitumor T cell immunity against breast cancer. (A), the inhibition of cell death by caspase inhibitor Z-VAD-fmk in the range from 50 μM to 0.4 μM were evaluated after 5 d (in the absence of ligands). Mean ± SD (n = 3). *p < 0.05. (B), the surface exposure of CRT was determined by immunofluorescence cytometry 48 h after treatment with HER2 antibodies. The antibody-untreated group stained with an anti-CRT antibody was used as the negative controls. *p < 0.05. (C), in vivo antitumor protection depends on CRT. 4T1-HER2 cells were transfected with the indicated siRNAs, then treated with rCRT and/or Tras-Permut CrossMab. The antitumor response was measured by challenging BALB/c mice simultaneously with Tras-Permut CrossMab-treated tumor cells in one flank and untreated, live tumor cells in the opposite flank (n = 20). The siRNA-transfected cells were measured by immunoblotting. (D), BALB/c mice were inoculated with Matrigel-mixed 4T1-HER2 cells and then treated with Tras-Permut CrossMab or control Ig (2 mg/kg). After the first inoculation, the CrossMab-treated mice were secondarily inoculated with Matrigel-mixed 4T1-HER2 cells in the opposite flank (n = 25). Some mice were treated with anti-CD4 mAb, anti-CD8 mAb, anti-CD4 and anti-CD8 mAbs or isotype Ig. Naive mice were inoculated with Matrigel-loaded 4T1-HER2 cells as the control. *p < 0.05, Mann–Whitney test. (E), after injection of siCRT-treated 4T1-HER2 cells (containing 0.1mL Matrigel) into the inguinal mammary fat pads of female BALB/c mice, the mice were treated with Tras-Permut CrossMab (2 mg/kg). After the first challenge, the CrossMab-treated mice were subsequently inoculated with Matrigel-mixed 4T1-HER2 cells in the opposite flank (n = 25). Some mice were treated with rCRT and/or anti-CD4 and anti-CD8 mAbs (n = 25). Naive mice were inoculated with Matrigel-loaded 4T1-HER2 cells as the control. The primary tumor material is examined through measurement of tumor size. *p < 0.05, Mann–Whitney test.