| Objective:This study analyzed and compared the effects of the novel third-generation Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs),Aumolertinib,and Pemetrexed,as well as their combination therapy on EGFR gene mutation with high expression and normal expression in lung adenocarcinoma xenograft nude mice.Methods:1.Real-time quantitative PCR(RT-q PCR)technique will be used to detect the expression of the EGFR gene in A549(wild type EGFR),HCC827(EGFR exon 19deletion mutation,19del),and PC9(EGFR exon 19 deletion mutation,19del)cells.HCC827 cells with EGFR mutation and high expression and PC9 cells with EGFR mutation and normal expression were selected for further experiments.2.Nude mouse models with HCC827 and PC9 cell xenografts will be established.HCC827 and PC9 cells will be prepared as a suspension of 1×10~7cells/m L and subcutaneously injected into the right axillary region of nude mice.Twelve nude mice will be inoculated with each cell line,with a volume of 0.2 m L per mouse.When the tumors reach an average size of 1.5-2.0 cm~3,the mice will be randomly divided into four groups of three mice each:combination group:oral administration of Aumolertinib(1.2 mg/kg/day,days 1-14)+intraperitoneal injection of Pemetrexed(50 mg/kg,day 1).Pemetrexed group:oral administration of an equal volume of saline(days 1-14)+intraperitoneal injection of Pemetrexed(50 mg/kg,day1).Aumolertinib group:oral administration of Aumolertinib(1.2 mg/kg/day,days1-14)+intraperitoneal injection of an equal volume of saline(day 1).Blank control group:oral administration of an equal volume of saline(days 1-14)+intraperitoneal injection of an equal volume of saline(day 1).Tumor volume and mouse weight will be measured every 3 days during the administration period.The mice will be euthanized on day 15 after drug administration,and tumor volume growth curves and body weight change curves will be plotted.3.Hematoxylin-eosin staining(HE)and CD34 fluorescence staining will be performed to observe the microscopic pathological changes and changes in microvessel density,respectively.Protein expression of EGFR and downstream ERK1 and AKT will be detected using Western blot analysis.Statistical analysis was conducted using Graph Pad Prism 8.0.1 software.Results:1.RT-q PCR experiment:Among the three cell lines,the m RNA expression level of EGFR was highest in HCC827 cells(9.00±0.832),followed by PC9 cells(0.986±0.023),and lowest in A549 cells(0.550±0.010).The difference in EGFR expression between HCC827 and PC9 cells/A549 cells was statistically significant(P<0.001),while there was no significant difference in EGFR m RNA expression between PC9 cells and A549 cells(P>0.05).2.Tumor volume:In the HCC827 and PC9 xenograft models,the combination group had the smallest tumor volume on the 15th day after drug treatment,measuring314.05±33.98 mm~3and 399.98±223.23 mm~3,respectively.The tumor volumes in the Aumolertinib group(366.8±111.96 mm~3,585.68±374.56 mm~3)were smaller than those in the Pemetrexed group(466.94±75.64 mm~3,635.51±172.62 mm~3),and the tumor volumes in the blank control group(542.45±288.24 mm~3,715.45±198.81 mm~3)were the largest.3.Nude mouse weight:In the HCC827 xenograft model,the weight of mice in the blank control group and Aumolertinib group showed stable growth,while the weight of mice in the combination group remained relatively unchanged,and mice in the Pemetrexed group showed a continuous decrease in weight.In the PC9 xenograft model,the weight of mice in the Aumolertinib group showed stable growth,the weight of mice in the blank control group remained relatively unchanged,and mice in the Pemetrexed group and combination group showed a decreasing trend in weight.4.Tumor inhibition rate:In the HCC827 and PC9 xenograft models,the combination group(42.11%,44.09%)showed higher tumor inhibition rates on the15th day compared to the Pemetrexed group(13.92%,11.17%)and the Aumolertinib group(32.74%,18.14%).The tumor inhibition rate in the Aumolertinib group was higher than that in the Pemetrexed group.5.HE staining:On the 15th day after drug administration,the tumor tissues of euthanized nude mice were dissected and processed into pathological wax blocks for HE staining.In the blank control group of the HCC827 and PC9 xenograft models,diffuse patches of tumor cells were observed under the microscope,with crowded and densely packed tumor cells.The cells showed significant pleomorphism,irregular polygonal,spindle-shaped,and oval shapes,forming solid patches without glandular luminal structures.The nuclei exhibited irregular deep staining,increased nuclear-cytoplasmic ratio,eosinophilic cytoplasm,and scattered infiltration of inflammatory cells in the surrounding area.The tumor cell density was highest in the blank control group,followed by the Pemetrexed group,and the combination group had the sparsest tumor cell density.The tumor cells in the combination group showed similar morphology,with necrotic foci and infiltration of acute and chronic inflammatory cells.6.CD34 fluorescence staining:Pathological wax blocks were sectioned and stained with CD34.Under fluorescence microscopy,the nuclei of tumor cells exhibited blue fluorescence,while the labeled blood vessels appeared as green fluorescence.In the tumor tissues of the HCC827 and PC9 xenograft models,the combined group showed significantly lower microvessel density compared to the blank control group,the Aumolertinib group,and the Pemetrexed group,with all differences being statistically significant(P<0.0001).The Pemetrexed group had slightly lower microvessel density than the blank control group,but the difference was not statistically significant(P>0.05).In the tumor tissues of the HCC827xenograft model,the Aumolertinib group had significantly decreased microvessel density compared to the blank control group and the Pemetrexed group,with both differences being statistically significant(P<0.0001).In the tumor tissues of the PC9xenograft model,the Aumolertinib group showed minimal change in microvessel density compared to the blank control group and the Pemetrexed group,and the differences were not statistically significant(P>0.05).In the comparison of microvessel density within the same group,there were statistically significant differences among the blank control group,Aumolertinib group,Pemetrexed group,and combined group(P<0.001,P<0.01,P<0.0001,P<0.001).7.Western blot experiment:On the 15th day after administration,in the tumor tissues of both HCC827 and PC9 xenograft models,compared to the blank control group,the protein expression levels of EGFR,ERK1,and AKT showed no significant change in the Pemetrexed group(P>0.05),while the Aumolertinib and combination groups exhibited a significant decrease in protein expression compared to the blank control group(P<0.01).The combination group showed a significant reduction in protein expression compared to the Pemetrexed group(P<0.05).In the HCC827xenograft tumor with nude mice,the combination group displayed a lower expression of EGFR,ERK1,and AKT proteins compared to the Aumolertinib group,but the difference was not statistically significant(P>0.05).In the PC9 xenograft tumor,the combination group showed an increase in the expression of EGFR,ERK1,and AKT proteins compared to the Aumolertinib group,but the difference was not statistically significant(P>0.05).No statistically significant differences were observed between the control and Pemetrexed groups in terms of EGFR,ERK1,and AKT protein expression(P>0.05).Statistically significant differences were observed between the Aumolertinib and combination groups in terms of EGFR,ERK1,and AKT protein expression(P<0.05,P<0.01,P<0.05).Conclusions:1.RT-q PCR confirmed that PC9 cells exhibited normal expression of EGFR,while HCC827 cells showed high expression of EGFR.2.The combination of Aumolertinib and Pemetrexed demonstrated a certain degree of tumor growth inhibition in EGFR high expressing HCC827 and EGFR normal expressing PC9 xenograft mouse models of lung adenocarcinoma compared to Aumolertinib or Pemetrexed.3.The combination of Aumolertinib and Pemetrexed may inhibit tumor growth by reducing the expression of EGFR and downstream proteins ERK1 and AKT.The reduction of these proteins in HCC827 xenograft mice was more significant compared to PC9. |
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