Figure 5. Immune‐dependent effect of DACT on WEHI 164 tumors growth and sensitization to immunotherapy.

• A–K
  3 × 10⁵ mouse fibrosarcoma WEHI 164 cells were injected subcutaneously (s.c.) into the flank of immunocompetent syngeneic Balb/c mice with n mice per group (n = 7 for DACT + anti‐PD‐1, n = 8 for DACT, and n = 9 for Ctr, anti‐PD‐1, anti‐CD4/anti‐CD8, and DACT + anti‐CD4/anti‐CD8). When tumors became palpable, the mice were injected intraperitoneally (i.p.) with solvent control (Ctr) or with 0.5 mg/kg dactinomycin (DACT). A second injection of chemotherapy was performed 4 days later. Anti‐CD4 and anti‐CD8 were administered i.p. at days −1, 0, and 7 days before/after chemotherapy and anti‐PD‐1 at days 8, 12, and 16 (A). Tumor size was assessed regularly, and individual tumor growth curves of DACT versus Ctr (B), DACT + anti‐CD4/CD8 versus anti‐CD‐4/anti‐CD‐8 (C), and DACT + anti‐PD‐1 versus anti‐PD‐1 (F) are depicted. Mean tumor area for each group was calculated, and significances were tested using a type II ANOVA test (D, G). Overall survival is depicted, and P‐values were calculated with a log‐rank test (E, H). Stars indicate the P‐values of each treatment versus Ctr (D, E, G, H), and hashes indicate the P‐values of the DACT + anti‐CD‐4/anti‐CD‐8 versus DACT alone (D, E) and of DACT + anti‐PD‐1 versus anti‐PD‐1 alone (G, H) (*^/# P < 0.05, **^/## P < 0.01, ***^/### P < 0.001). Five naïve mice and the eleven mice that were cured by treatment with DACT alone or in combination with PD‐1 blockade were (re)challenged with WEHI 164 cells, and individual tumor growths (I, J), as well as overall survival (***P < 0.001, log‐rank test) (K), were monitored.