Fig. 3

Sequence-dependent synergistic effect on tumor growth and metastasis inhibition in EGFR-mutant NSCLC bearing mice. A A small-scale in vivo experiment (n = 3) was designed to compare the anti-tumor effects of different combination sequences. B Tumor volumes were measured every other day and tumor growth curves were plotted for mice bearing H1975 or HCC827 cell-derived tumor xenografts (n = 3). C At the endpoint of drug administration, mice in each group were sacrificed, and the tumors were dissected and photographed (n = 3). D Another systemic in vivo experiment (n = 8) was proposed to determine the advantage of P-A sequence over single drug administration. Schematic presentation of different administration strategies in H1975 and HCC827 tumor-bearing mice was shown. E Volumes of H1975 cell-derived tumor xenografts were measured every other day and tumor growth curves were plotted against time (n = 8). F H1975 tumor-bearing mice were sacrificed post 6-cycle drug administration, and the tumors were dissected and photographed (n = 8). G The weight of H1975 cell-derived tumor xenografts at the endpoint were analyzed and compared (n = 8). H Intratumoral expression of EMT-related protein, β-catenin, vimentin and snail, were analyzed by western blot. Quantification of the western blot band intensity was performed using ImageJ and GAPDH was used as loading controls (n = 6). I Metastatic nodule detection in liver was performed. J Similar to the experimental operation on H1975 tumor bearing mice, P-A sequence treatment and single drug administration were also applied on HCC827 tumor bearing mice. Tumor volumes were measured every other day and tumor growth curves were plotted (n = 8). K HCC87 tumor-bearing mice were sacrificed post 5 cycles drug administration, and the tumors were dissected and photographed (n = 8). L The weight of HCC827 cell-derived tumor xenografts at the endpoint were analyzed and compared (n = 8). Con represents control; P represents pemetrexed; A represents aumolertinib; Osi represents osimertinib; P + A represents concomitant treatment with pemetrexed and aumolertinib; P-A represents pemetrexed treatment followed by aumolertinib treatment; A-P represents aumolertinib treatment followed by pemetrexed treatment. All of the data were expressed as the mean ± SEM, * p < 0.05, ** p < 0.01 and *** p < 0.001