Combination index of apatinib (A) and gemcitabine (B1), paclitaxel (B2), and pemetrexed (B3) in A549 cell line. There are four combination proportions of apatinib and cytotoxic agents (from 1/5 A + 4/5 B to 4/5 A + 1/5 B). The combination index (CI) was used to estimate the combination effects.

Toxicity and safety of apatinib and pemetrexed in the zebrafish model. (a) The number of animal situations observed after apatinib treatment. (b) Treatment with 2.5 μM apatinib for 48 h caused pericardial edema (red arrow) and heart failure in all zebrafish embryos. The complications were caused due to vascular defects in the SIV (white arrow). (c) The number of animal situations observed after pemetrexed treatment. Zebrafish Strain: Tg(fli1a:EGFP)y1. Route of Administration: Soaking in 0.1% DMSO (in fish water). Animal number: Total of 20 embryos for each condition.

Apatinib inhibits angiogenesis in zebrafish in a dose-dependent manner. (a) (a–o) Representative bright field and fluorescent images of zebrafish embryos at 49 hpf treated with 0.1% DMSO control, apatinib (1, 2.5, 5, and 10 μM), or 5 μM PTK787 (positive control) for 26 h were observed. (f–o) Compared with controls, embryos treated with apatinib presented fewer incomplete ISVs and only occasional sprouts (asterisk) of DA. The boxed regions are shown at higher magnification in the right panels. (b) Quantification of the number of complete ISVs showed a significant decrease in the apatinib-treated embryos. The antiangiogenic effect of apatinib in zebrafish embryos was dose-dependent. # ISVs: The number of complete ISVs (the number of ISVs that connect the DA to the DLAV). Error bars, SEM; ***P < 0.0001 (n = 10; ANOVA); ns, not significant. DLAV, dorsal longitudinal anastomotic vessels; ISV, intersegmental vessels; DA, dorsal aorta; PCV, posterior cardinal vein. Zebrafish strain: fli1a-EGFP; casper. Route of administration: soaking in 0.1% DMSO (in fish water). Animal number: 30 embryos for each condition.

Co-administration of apatinib and pemetrexed inhibited tumor growth in vivo. (a) DiI-stained human A549 cells were successfully grafted into the yolk sac of a zebrafish embryo 2 dpf without immunosuppressant treatment. Approximately 200 cells were injected into the yolk sac and assessed by fluorescence microscopy. (b) Tumor growth at 4 dpi was observed by fluorescence microscopy. (c) Quantitative analysis of inhibitory effects of Apatinib, Pemetrexed, or combination concerning tumor growth. Columns, mean; bars, SEM (n = 10; ANOVA; ***P < 0.0001, **P < 0.01, *P < 0.05, compared to vehicle control group; *P < 0.05, compared to combination group). RFI, relative fluorescence intensity; dpi, days post-injection; MNLC, maximum non-lethal concentration. Zebrafish Strain: fli1a-EGFP; casper. Route of Administration: Soaking in 0.1% DMSO (in fish water).

Effect of apatinib combined with pemetrexed on signaling pathways in zebrafish models. (a) Endogenous VEGFaa, VEGFR2, and VEGFR1 in control and lead compounds treated embryos were assessed by qRT-PCR (n = 10–15 individual embryos). APT, apatinib; PEM, pemetrexed; ns, not significant. (b) Endogenous Dll4, Notch1a, Hey2, and EFNB2A in control and lead compounds treated embryos were assessed by qRT-PCR (n = 10–15 individual embryos). (c) Endogenous SLIT2, SLIT3, ROBO1, ROBO2, and ROBO4 in control and lead compounds treated embryos were assessed by qRT-PCR (n = 10–15 individual embryos). (d) Endogenous FGFR11, FGFR 2, FGFR 3, and FGFR 4 in control and lead compounds treated embryos were assessed by qRT-PCR (n = 10–15 individual embryos). Zebrafish strain: Tg (fli1a:EGFP)y1. Animal number: 90 embryos for each condition.