Cell culture. Human lung adenocarcinoma cell line HCC827 (STR identification, E746-A750 deletion, Wuhan Prose Life Technology Co., Ltd.), 10% fetal bovine serum RPMI1640 medium, were cultured and reproduced at 37 Celsius degrees in 5% CO2 incubator. 0.5ml-1ml of 0.25% trypsin solution was kept at 37°C for 1 min and centrifuged at 1000 r/min for 3 min to prepare cell suspension.
Grouping of tumor-bearing mice and establishment of EGFR-M + xenograft animal model. Sixty SPF BALB/c-nu nude mice [SPF (Beijing) Biotechnology Co. Ltd. SCXK (jing) 2019-0010], six weeks old, weight (21.49 ± 1.63)g, half male and half male, were randomly divided into control group (Group C) and Treatment Group (Group T), 30 rats in each. The cells in the logarithmic growth phase were digested with trypsin to prepare a cell suspension with a concentration of 1.0×107 cells/mL, and 0.2 mL/ was inoculated into the right hind limb of each of the nude mice subcutaneously to establish animal models of lung adenocarcinoma transplantation tumor. Observation of the growth of subcutaneous transplanted tumors was performed on a daily basis. When the long diameters of the transplanted tumors grew to 5 mm, 10 mm, 15 mm, and 20 mm in group C, and 20 mm in group T, the intervention of Gefitinib by gastric infusion was introduced. After the transplanted tumor retreated to 15 mm, 10 mm, 5 mm, and 0 mm in long diameter, the same method was used for sub-clinical lesion measurement on the nude mice with transplanted tumors in their hind limbs. The nude mice were fed in the SPF animal room of the Animal Experiment Center by the principles of animal experiment 3R (Reduction, Replacement, Refinement), observing the ethic requirements and sterilized conditions.
EGFR-TKI treatment. After sugar-coating removal, Gefitinib Tablets (AstraZeneca) was ground, and dissolved in sterilized ultrapure water to prepare a 7.5 mg/mL solution and stored in a constant temperature refrigerator at 4°C. Nude mice in group T were given the prepared solution at 0.5mL/100g by gastric perfusion, once a day, to observe the changes in the long diameter of the transplanted tumor in nude mice . When the long diameters of the transplanted tumors decreased to 1.5cm, 1.0cm, 0.5cm, At 0.0cm (complete response, CR), the hind limbs of nude mice were obtained to measure the subclinical lesion of the transplanted tumor.
Definition and detection methods of subclinical lesions. The subclinical lesion area is defined as the distance between the small lesion in the three-dimensional direction and the edge of the transplanted tumor by HE staining and 200x to 400x microscope examination [20–21]. In group C, when the longest diameters of the transplanted tumors grew to 5mm, 10mm, 15mm, and 20mm, the hind limbs with transplanted tumors were taken from three nude mice and fixed with formalin solution respectively, altogether 12 nude mice were sacrificed. The transplanted tumors were sectioned on the largest diameter plane, HE stained, and observed under a microscope. The extent of subclinical lesions is recorded. In group T, Gefitinib was injected into the stomach when the long diameter of the transplanted tumor grew to 20mm, and the changes in the long diameter of the transplanted tumor were observed daily. When the cancer was reduced to 15mm, 10mm, 5mm, and 0mm, the same method as group C was followed for pathological observation of clinical lesions.
Observation indicators. Observed under the microscope, the subclinical lesion range and change the rule of the transplanted tumor in group C under different long diameters, the longest diameter of the transplanted tumor was reduced with the treatment of gefitinib in group T, the subclinical lesion range and change rule.
Statistical analysis. SPSS26.0 data statistics software was used for analysis. T-testing was adopted for the inter-group comparison. There is a statistical difference between group C and T(p < 0.05). The range included in 95% of subclinical lesions was calculated as a cumulative percentage.