Journal: Advanced Science
Article Title: Osteosarcoma Cell‐Derived Migrasomes Promote Macrophage M2 Polarization to Aggravate Osteosarcoma Proliferation and Metastasis
doi: 10.1002/advs.202409870
Figure Lengend Snippet: MANPs promote macrophage phagocytosis and M2 polarization via Milk fat globule‐EGF factor 8 (MFGE8). A‐H) 4D proteome sequencing analysis of cell body, migrasome and MANP. (A‐D) Heatmaps and volcano plots of DEGs in protein‑seq analysis of cell body, migrasome and MANP ( n = 3). (E, F) Heatmaps of TSPAN proteins, integrin proteins, and histones in cell body, migrasome and MANP ( n = 3). (G) Venn diagram showing proteins co‐enriched by migrasomes and MANPs. (H) Relative expression of MFGE8 in cell body, migrasome and MANP ( n = 3). I) Immunostaining images of WGA (green) and MFGE8 (red) in OS cells ( n = 3). Magnified images of migrasomes and MANPs are shown on the right. Low magnification: scale bars = 10 µm; high magnification: scale bars = 1 µm. J,K) qRT‐PCR and WB analysis of MFGE8 knockdown efficiency in K7M2 wt cells ( n = 3). L,M) WB analysis of MFGE8 knockdown efficiency in migrasomes and MANPs ( n = 3). N‐Q) BMDMs treated with 10 µg mL −1 sh‐NC MANPs or sh‐MFGE8 MANPs for 24 h. (N, O) Representative immunostaining images and quantitative analysis of BMDMs (red) phagocytosis of microbeads (green) ( n = 3). Low magnification: scale bars = 100 µm; high magnification: scale bars = 50 µm. (P, Q) Flow cytometry analysis of the proportion of BMDMs (APC) that phagocytose apoptotic OS cells (CFSE) and quantitative analysis ( n = 3). R‐T) BMDMs treated with 10 µg mL −1 sh‐NC MANPs or sh‐MFGE8 MANPs for 24 h, followed by co‐incubation with apoptotic OS cells for 24 h. (R) qRT‐PCR analysis of the expression of M1 polarization and M2 polarization markers in BMDMs ( n = 3). (S, T) Flow cytometry analysis of the proportion of M2‐type macrophages and quantitative analysis ( n = 3). Results were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. One‐way ANOVA test or two‐way ANOVA test was used for multivariate analysis. Unpaired t‐tests were used for the comparison of two groups.
Article Snippet: The human OS cell lines 143b and MG63 and the mouse OS cell line K7M2 wt were obtained from the American Type Culture Collection (ATCC), and the OS cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, C11995500BT, Gibco, USA) supplemented with 1% penicillin/streptomycin (P/S, 15140122, Gibco, USA) and 10% fetal bovine serum (FBS, 10091148, Gibco, USA).
Techniques: Sequencing, Expressing, Immunostaining, Quantitative RT-PCR, Knockdown, Flow Cytometry, Incubation, Comparison
