Figure 5.

Osimertinib ameliorates tumor angiogenesis and triggers antitumor-macrophages infiltration. (A) Representative images of CD31 (green) and α-SMA (red) immunostaining, and DAPI nuclear staining (blue) of MC38 tumors treated with vehicle and osimertinib (1 mg/kg) daily for 14 days by oral gavage. (B) Relative CD31⁺ area in tumors of two indicated groups. Each dot indicates one tumor per mouse and represents the average of 5 images. n = 8. (C) Total number of CD31⁺ cells from tumor in vehicle group and osimertinib group determined by flow cytometry. n = 6. (D) Relative proportion of α-SMA⁺ pericyte-covered blood vessels in tumors of two indicated groups. Each dot indicates one tumor and represents the average of 5 images. n = 8. (E) Representative images of endothelial tubular network in HUVECs treated with osimertinib (1 μmol/L) or DMSO for 24 h after overexpression with GAPDH vector for 24 h. (F) Quantification of tubes in experiments as in (E). (G) Representative photographs of the tail area of zebrafish embryos treated with osimertinib (1 μmol/L) or DMSO for 12 h. (H, I) Quantification of filopodia length (H) and number of ISV (I) in experiments as in G. (J) Quantification of CD8⁺ T cells from MC38 tumors in vehicle group and osimertinib group. (K) Ratio of CD8⁺ T cells to FOXP3⁺CD25⁺ regulatory T cells in MC38 tumors treated with vehicle or osimertinib. (L) Left, PhenoGraph of cellular distribution and clustering, as defined by tSNE1 and tSNE2, colored by cellular phenotype of MC38 tumors in vehicle group and osimertinib group. Data show all normalized viable single cells, subjected to the PhenoGraph algorithm. Right, quantification of tumor-infiltrating immune (CD45⁺) cells in Vehicle group and osimertinib group, assessed by flow cytometry. Cell populations were identified as APCs (CD45⁺CD11b⁺CD11c⁺MHCII⁺), MΦ (CD45⁺CD11b⁺Gr-1⁻F4/80⁺), M1-like macrophages (CD45⁺CD11b⁺Gr-1⁻F4/80⁺CD11c⁺CD206⁻), M2-like macrophages (CD45⁺CD11b⁺Gr-1⁻F4/80⁺CD11c⁻CD206⁺), MDSCs (CD45⁺CD11b⁺Gr-1⁺F4/80^–), and neutrophils (CD45⁺CD11b⁺CD11c^–Gr-1⁺). (M) Left, viSNE analysis of immune cells from vehicle group and osimertinib group tumors colored by relative expression of MHCII in MΦ. Right, flow cytometry analysis of the major histocompatibility complex class II (MHCII) on MΦ from MC38 tumors. (N) HUVECs were treated with osimertinib (1 μmol/L) or DMSO in serum-free medium for 12 h. Left, statistical analyses of migrated LPS-activated M1-like or IL-4-activated M2-like macrophages attracted by conditioned medium (100 °C, 5 min) from either HUVEC-CM^DMSO or HUVEC-CM^OS and treated with osimertinib (1 μmol/L) or DMSO, additionally. Right, representative images of Transwell migration assays are shown. (E–N) n = 5. Statistical analysis by unpaired Student's t-test or one-way ANOVA. Data are presented as mean ± SEM. Scale bar = 100 μm.