Objective  This study was performed using preclinical transplanted animal experiments to analyce the effects and mechanisms of third-generation EGFR-TKIs combined with anti-angiogenic therapy, thereby providing theoretical basis for further clinical trials.

  Methods  Researchers constructed the transplant BALB/C nude mice models with H1975 lung adenocarcinoma cell line (EGFR T790M) and divided the mice into four groups and treated them with osimertinib (2.5 or 5 mg/kg/day, gavage) alone or plus bevacizumab (5 mg/kg/twice weekly, i.p.) when the tumors reached approximately 0.4-0.6 cm³ in volume. The tumor growth curve of tumor volume was drawn according to the time in every group. After 2 weeks of treatment, the mice were killed and the tumors were processed for immunohistochemical staining and Western blot analysis. Immunostaining was performed to detect: HIF-1α, VEGF, and microvessel density (MVD) by using SP method on paraffin sections. Western blot analysis was used to analyze the protein expression levels of EGFR, AKT, and ERK signal transduction pathways.

  Results  After 2 weeks of treatment in high-and low-dose osimertinib alone, tumor volume in the high-dose group was significantly less than in low-dose osimertinib-alone group (P < 0.05). VEGF, HIF-1α expression, and MVD were significantly low in the high-dose osimertinibalone group (P < 0.05). Increasing doses of osimertinib induced dose-dependent weakening of the p-EGFR, p-AKT, and p-ERK expression levels (P < 0.05). In the low-dose osimertinib-plus-bevacizumab group and low-dose osimertinib-alone group, no significant difference in tumor volume and the above factors was observed. In the low-dose osimertinib-plus-bevacizumab group and high-dose osimertinib-alone group, tumor volumes did not exhibit significant difference (P=0.178). Moreover, VEGF, HIF-1α expression, and MVD exhibited no significant difference. No significant difference in the p-EGFR, p-AKT, and p-ERK expression levels was found between high-dose osimertinib-alone group and low-dose osimertinib-plus-bevacizumab group (P > 0.05). In the high-dose osimertinib-plus-bevacizumab group, tumor growth was not significantly greater than that in the high-dose osimertinib-alone group (P=0.642). No significant difference was observed in the above factors. In the high-and low-dose osimertinib-plus-bevacizumab groups, tumor volume and the above factors did not exhibit significant differences (P > 0.05).

  Conclusion  Osimertinib has obvious antitumor effects in EGFR-mutant lung adenocarcinoma with T790M mutation cell xenografts. Bevacizumab has a synergetic inhibitory effect with osimertinib against EGFR-mutant lung adenocarcinoma with T790M mutation cell xenografts. Bevacizumab enhanced the antitumor effects of osimertinib by reducing VEGF expression and the microvascular density of the tumor, thereby improving the tumor microenvironment. Bevacizumab can enhance the effect of osimertinib by suppressing EGFR, ERK, and AKT phosphorylation, thereby synergistically inhibiting EGFR activation and downstream signaling.