Fig. 1

Osimertinib-resistant NSCLC cell lines were established, and the IC50 values of osimertinib and anlotinib were determined. An MTT assay was used to explore cell viability in (A) H1975 and H1975-OR cells and (B) PC9 and PC9-OR cells treated with different concentrations of osimertinib. A CCK-8 assay was conducted to explore the proliferative capacities of (C) H1975 and H1975-OR cells and (D) PC9 and PC9-OR cells. An MTT assay was conducted to measure cell viability in (E) H1975 and H1975-OR cells and (F) PC9 and PC9-OR cells treated with different concentrations of anlotinib. P values were calculated using two-way ANOVA analysis; ***, P<0.001. OD, optical density; NSCLC, non-small cell lung cancer; IC50, half-maximal inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide; CCK-8, Cell Counting Kit-8; ANOVA, analysis of variance.

Fig. 2

Effects of combination therapy with anlotinib and osimertinib on NSCLC cells in vitro. A CCK-8 assay was conducted to explore the proliferative capacities of (A) H1975, (B) PC9, (C) H1975-OR, and (D) PC9-OR cells, pre-treated with the NC, osimertinib monotherapy (1 µM), anlotinib monotherapy (3 µM), and a combination of anlotinib (3 µM) and osimertinib (1 µM) for 72 h. An MTT test was conducted to determine cell viability in (E) H1975-OR and (F) PC9-OR cells treated with different concentrations of osimertinib monotherapy or the combination of anlotinib (3 µM) and osimertinib. P values were calculated by two-way ANOVA analysis; ***, P<0.001; NS, not significant. OD, optical density; NC, negative control; Osi, osimertinib; Anl, anlotinib; Osi + Anl, osimertinib combined with anlotinib; NSCLC, non-small cell lung cancer; CCK-8, Cell Counting Kit-8; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide; ANOVA, analysis of variance.

Fig. 3

Determination of the MTD of anlotinib in a zebrafish model. (A) Dose-survival rate curves of zebrafish embryos soaked in different concentrations of anlotinib. (B) Summary of the survival number of the zebrafish embryos at each anlotinib concentration after 3 days of anlotinib soaking. dpi, day post-injection; MTD, maximum tolerated dose.

Fig. 4

The inhibitory effect of anlotinib combined with osimertinib on NSCLC cell proliferation was investigated in vivo in zebrafish xenograft models. (A) Representative images and quantitative analysis of cell proliferation in the H1975 and PC9 zebrafish xenograft models (scale bar, 500 µm). (B) Representative images and quantitative analysis of cell proliferation in the H1975-OR and PC9-OR zebrafish xenograft models (scale bar, 500 µm). The number of xenografts analyzed is indicated in the corresponding images. P values were calculated by unpaired Student’s t-tests; *, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant. Each data point represents an independent biological replicate. NC, negative control; Osi, osimertinib; Anl, anlotinib; Osi + Anl, osimertinib combined with anlotinib; NSCLC, non-small cell lung cancer.

Fig. 5

The inhibitory effect of anlotinib combined with osimertinib on NSCLC cell migration was investigated in vivo in zebrafish xenograft models. (A) Representative images and quantitative analysis of cell migration in the H1975 and PC9 zebrafish xenograft models (scale bar, 200 µm). (B) Representative images and quantitative analysis of cell migration in the H1975-OR and PC9-OR zebrafish xenograft models (scale bar, 200 µm). The number of xenografts analyzed is indicated in the corresponding images. P values were calculated by unpaired Student’s t-tests; *, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant. Each data point represents an independent biological replicate. NC, negative control; Osi, osimertinib; Anl, anlotinib; Osi + Anl, osimertinib combined with anlotinib; NSCLC, non-small cell lung cancer.

Fig. 6

Representative images of cell proliferation in a zPDX model derived from patients who are clinically sensitive to osimertinib (scale bar, 500 µm). The number of xenografts analyzed is indicated in the corresponding images. NC, negative control; Osi, osimertinib; Anl, anlotinib; Osi + Anl, osimertinib combined with anlotinib; zPDX, zebrafish patient-derived xenograft.

Fig. 7

Representative images of cell proliferation in a zPDX model derived from patients who are clinically resistant to osimertinib (scale bar, 500 µm). The number of xenografts analyzed is indicated in the corresponding images. NC, negative control; Osi, osimertinib; Anl, anlotinib; Osi + Anl, osimertinib combined with anlotinib; zPDX, zebrafish patient-derived xenograft.

Acknowledgments
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