Figure 2.

Pep-20 inhibits tumor growth and activates antitumor T-cell immune response in MC38 tumor-bearing mice. C57BL/6 mice were transplanted with 1×10⁶ MC38 cells on the right flank, until the tumor volume reached 50 mm³. (A) Mice were treated s.c. with 2 mg/kg of pep-20 or normal saline as the negative control at the peritumoral site every day for 2 weeks (n=8). (B) Mice were treated with 2 mg/kg of pep-20 for 4 weeks and overall survival was monitored (n=12). (C) Tumors were detected for the percentage of tumor-infiltrating CD8⁺ T cells in total CD45⁺ cells (n=6). (D, E) Cells from draining lymph nodes (D) or spleen (E) were obtained and stimulated with 20 ng/mL of PMA and 1 µM ionomycin-containing protein transport inhibitor cocktail for 4 hours. Frequencies of IFN-γ expressing CD8⁺ T cells were detected by flow cytometry (n=6). (F, G) Draining lymph nodes (F) and spleen (G) were obtained and stimulated with 0.5 µg/mL of anti-CD3 and 0.5 µg/mL of anti-CD28 antibodies for 3 days. Cellular supernatant from draining lymph nodes or spleen for IFN-γ secretion was measured with ELISA assay (n=4 to 5). Data are represented as means±SEM. Statistical significance was determined by unpaired Student’s t-test. *p<0.05; **p<0.01. Kaplan-Meier survival curves were evaluated by log-rank analysis. IFN, interferon; PMA, phorbol 12-myristate 13-acetate.