Figure 2. DOX treatment induces upregulation of IDO1 and IDO1 impairs the therapeutic efficacy of DOX. (A) Slight inhibition of tumor growth by DOX in 4T1 tumor-bearing BALB/c mice (n = 6). DOX was i.v. injected at the dosage of 5.0 mg/kg for 5 times after every 3 days. (B) The mRNA expressions of CTLA-4, PD-1, PD-L1, IDO1, CSF-1, and CCL2 examined by real time polymerase chain reaction. The mRNA levels of these genes were normalized against the expression level of the housekeeping gene GAPDH. The IDO1 protein expression in tumor tissues from mice treated with phosphate buffer saline solution and DOX were examined by western blotting (C) and immunohistochemistry (D). (E) Protein levels of IDO1 in 4T1-shNC and 4T1-shIDO1 cells detected by western blot. β-actin was used as loading control. (F) Tumor growth in 4T1-shNC and 4T1-shIDO1 tumor bearing BALB/c mice. 4T1-shIDO1 tumor bearing BALB/c mice were injected with DOX at the dosage of 5.0 mg/kg.

Data represent means ± standard deviation.

DOX = doxorubicin; mRNA = messenger RNA; CTLA-4 = cytotoxic T-lymphocyte-associated protein 4; PD-1 = programmed cell death 1; PD-L1 = programmed cell death 1; CSF-1 = cerebrospinal fluid 1; CCL2 = chemokine (C-C motif) ligand 2; IDO1 = indoleamine 2,3-dioxygenase 1; shNC = short hairpin RNA non-target control; shIDO1 = short hairpin RNAs targeting indoleamine 2,3-dioxygenase 1.

^*p < 0.05; ^†p < 0.01; ^‡p < 0.001.