Fig. 1.

Liraglutide enhances antitumor immune activities both in vivo and ex vivo. (A & B) 5 × 10⁶ Hepa1-6 cells were subcutaneous injected to 6–8-week C57BL/6 mice in the right flank. Then HCC tumor-bearing mice received an intratumoral injection of PBS (control, n = 5) or liraglutide (300 μg/kg per mouse, once a day for 7 days, n = 5). After 7 days, we killed the mice, and the tumor masses were isolated.

(A) IFN-γ-producing immune cells were identified by a mouse IFN-γ ELISpot assay kit. (B) qRT-PCR was conducted to analyze the mouse IFN-γ mRNA level which was extracted from tumor tissues. (C) Isolated PBMCs were cultured ex vivo as mentioned in the Materials and Methods. After 2 weeks, cells were detected via flow cytometry with anti-CD3, anti-CD4, anti-CD8 and anti-NKG2D antibodies. (D) HCC-LM3 cells were treated in the presence or absence of liraglutide (10 or 20 μM) for 12 h, and the cells were then further co-incubated with NKT cells. After 12 h, the supernatants were collected and IFN-γ secreted by NKT cells was quantified by ELISA. (E) IFN-γ mRNA levels were determined by qRT-PCR. HCC-LM3 cells were treated as described above in (D). Then, mRNA was obtained and analyzed by qRT-PCR. The data are shown as the means ± SD. *p < 0.05 and ****p < 0.0001.