Fig. 6: The combination of osimertinib and leelamine effectively inhibits EGFR C797S mutant tumor growth in vivo.
a Confirmation of epidermal growth factor receptor (EGFR) mutant (EGFR19del747_750+T790M (19DM)-blue fluorescent protein (BFP)) or EGFR19del747_750+T790M+C797S (19DMS)-green fluorescent protein (GFP) overexpression in LLC cells using qPCR analysis of EGFR mRNA expression. b Assessment of the cytotoxic effects of osimertinib on EGFR-mutant LLC cells (19DM-BFP or 19DMS-GFP) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay after treatment with the indicated concentrations of osimertinib for 72 h. c Fluorescence microscopy images showing a 1:1 mixture of EGFR-mutant 19DM-BFP and 19DMS-GFP cells treated with osimertinib (0, 1, 5 μM) for 72 h. The scale bar represents 50 μm. d Flow cytometry data confirming the ratio of BFP to GFP in LLC 19DM-BFP and LLC 19DMS-GFP cells mixed at a 1:1 ratio and treated with osimertinib (0, 1, 5 μM) for 72 h. e–k Allograft models were established by injecting a 1:1 mixture of 19DM-BFP and LLC 19DMS-GFP LLC cells into C57BL/6 mice. After one week, the mice were treated with corn oil (CON), 2 mg/kg osimertinib (O2), 2 mg/kg osimertinib + 1 mg/kg leelamine (O2L1), or 2 mg/kg osimertinib + 2 mg/kg leelamine (O2L2) for two weeks. Body weight (e) and tumor volume (f) were measured during the treatment period. The mice were euthanized, tumor samples were harvested, and the tumors were imaged (g). Tumor volume (h) and weight (i) were also analyzed. Imaging of the tumors was performed using a confocal microscope (j). The scale bar represents 20 μm. The fluorescence intensity ratio of 19DM-BFP and 19DMS-GFP LLC cells was quantified using ImageJ software, and the bar length indicates the relative tumor volume across different treatment groups (k). Data information: The data in b, f, h, and i are presented as the mean ± standard error of the mean (SEM) values. Statistical analysis in b was conducted using Student’s t-test, with comparison to the 19DM group. Statistical analyses in f, h, and i were performed using a one-way analysis of variance with Dunnett’s post hoc test, with comparison to the O2 group. *p < 0.05, **p < 0.01, and ***p < 0.001.
