Fig. 4: Sensitivity of ferroptosis of 4T1 cells relative to macrophages, and the effect of HMPB-H@M1EV on polarizing macrophages and stimulating DCs.

a Ferritin expression in 4T1 and macrophages are analyzed Western blotting. b Flow cytometric analysis of LPO levels in HMPB-H and laser treated 4T1 macrophages. c Flow cytometric analysis of iNOS expression in 4T1 macrophages. d Calcein/PI staining assay in HMPB-H and laser treated-4T1 macrophages. Scale bar: 50 µm. e, f Quantification of CD206⁺ and CD86⁺ cells in M2 macrophages treated with PBS, M0EV, M1EV, and HMPB-H@M1EV. g, h ELISA assay of TNF-α and IL-6 in M2 macrophages post-treatment with PBS, M0EV, M1EV, and HMPB-H@M1EV. ND stands for not detected. i Representative CLSM images of M2-like macrophages phagocytosing 4T1 cells. Macrophages and 4T1 cells were labeled with RhB (red) and CFSE (green), respectively. Scale bar: 50 µm. j 4T1 phagocytosis by macrophages. k Schematic diagram of the in vitro co-culture process of immature DCs and treated 4T1 cells. Created with MedPeer (www.medpeer.cn). l, m CD80⁺ CD86⁺ DCs by flow cytometric analysis. For (a–d, i), experiment was repeated three times independently with similar results. The data in (e–h, m) are presented as the mean ± SD (n = 3 independent experiments). Statistical differences were analyzed by one-way ANOVA with Tukey’s multiple comparisons test.