Figure 3.

Macrophage phenotype in lung adenocarcinoma cells with osimertinib resistance. (a). Schematic diagram of immunophenotype analysis in xenograft tumors with osimertinib resistance. The BALB/c mice with M109 tumors or BALB/c-nude mice with PC-9 L792F^cis tumors were treated with control (5% dimethyl sulfoxide, 40% PEG300, 5% Tween 80, and 50% water) or osimertinib (10 mg/kg, 5 days a week) for 4 weeks. The resected tumors were further analyzed with immunophenotype by flow cytometry on the markers, including CD45e, CD326, 7AAD, CD118, Ly6C, CD206, F4-80, CD3e, CD49B, CD86, and CD27, and immunohistochemistry on the staining of CD206, SMA, F4-80, iNOS, and DAPI. (b). t-distributed stochastic neighbor embedding plot of blood and formed tumor from the mice injected with M109 cells by multiplex fluorescence flow cytometry. (c). F4-80, CD45, CD11b, and CD206 staining on tumors of M109 control and M109 osimertinib resistance (OR) by multiplex fluorescence flow cytometry analysis. (d). Histograms show CD3e for T cell and Ly6C/CD49B for NK cell analysis in the tumors of M109 control and M109 OR. (e). CD326, 7AAD, CD3e, CD45, CD11b, Ly6C, F4-80, CD86, and CD206 staining (indicated by colors) on tumors of H1975 control, H1975 L792F^cis and PC-9 control, and PC-9 L792F^cis by multiplex fluorescence flow cytometry analysis for tumor-associated macrophages. (f). Multiplex immunohistochemistry staining, including CD206, SMA, F4-80, iNOS, and DAPI, with the multiple colors as indicated on tumors of H1975 control, H1975 L792F^cis, PC-9 control, and PC-9 L792F^cis. The scale bar was 200 um. (g). Graphs show the F4-80+/CD206+ influence ratio in the tumors of H1975 control, H1975 L792F^cis, PC-9 control, and PC-9 L792F^cis by multiplex immunohistochemistry analysis (n=25, both p<0.001. Statistical differences were evaluated using t test).