FIGURE 7.

Intrapulmonary delivery of immRNA using EGFR‐targeted RBCEVs suppresses breast cancer metastasis in the lung. (a) Schematic intrapulmonary treatments for mice bearing breast cancer metastasis. (b) Flow cytometry analysis of tumour burden in the lungs using anti‐human‐EGFR antibody after five treatments. (c) Average percentage of hEGFR‐positive tumour cells in the lungs as shown in (b) (n = 4 mice). (d) Average percentage of metastatic area in the lungs treated with 25 mg/kg uncoated, Ctrl‐VHH‐coated and EGFR‐VHH‐coated RBCEVs containing immRNA after five treatments (n = 4 mice). (e) Representative H & E stained lung sections from mice as in (d). Scale bar, 50 μm. (f) qPCR analysis of the RIG‐I pathway gene expression relative to Gapdh in the lungs of mice as in (a) (n = 4 mice). (g) Flow cytometry analysis of immune cells in the lungs of mice as in (a) (n = 4 mice). (h) Representative images of TUNEL staining (orange fluorescence) of lung sections from mice as in (a). Nuclei were stained with Hoechst (blue). Scale bar, 20 μm. (i) Average mean intensity per nucleus area of TUNEL staining signals (n = 4 mice). (j) Multiplex immunoassay of cytokines in the lung homogenates of mice with 4T1‐hEGFR metastatic tumours (n = 3 mice). All bar graphs represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 determined by Student's two‐tailed t‐test