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: OCDMs promote macrophage phagocytosis and M2 polarization. A‐D) Flow cytometry analysis of the proportion of M2‐type macrophages in tumor tissues and quantitative analysis ( n = 6). E‐H) Representative IHC images and quantitative analysis of CD86 in tumor sections ( n = 6). Scale bars = 100 µm. I‐L) Representative IHC images and quantitative analysis of CD206 in tumor sections ( n = 6). Scale bars = 100 µm. M,N) Heatmaps and volcano plots of DEGs in mRNA‑seq analysis of bone marrow‐derived macrophages (BMDMs) treated with migrasomes or PBS ( n = 3). O) KEGG enrichment analysis of up‐regulated genes in BMDMs treated with migrasomes. P‐S) BMDMs treated with PBS or 10 µg mL −1 migrasomes for 24 h. (P, Q) Representative immunostaining images and quantitative analysis of BMDMs (red) phagocytosis of microbeads (green) ( n = 3). Scale bars = 100 µm. (R, S) Flow cytometry analysis of the proportion of BMDMs (APC) that phagocytose apoptotic OS cells (CFSE) and quantitative analysis ( n = 3). T‐V) BMDMs treated with PBS or 10 µg mL −1 migrasomes for 24 h, followed by co‐incubation with apoptotic OS cells for 24 h. (T) Quantitative real‐time (qRT)‐PCR analysis of the expression of M1 polarization (TNFα, iNOS and CD86) and M2 polarization markers (CD206, CD163 and IL‐4) in BMDMs ( n = 3). (U, V) Flow cytometry analysis of the proportion of M2‐type macrophages and quantitative analysis ( n = 3). Results were shown as mean ± SD. ns 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: All the data were analyzed using FLowJo‐v10.8.1 flow cytometry analysis software (TreeStar, USA).

Techniques: Flow Cytometry, Derivative Assay, Immunostaining, Incubation, Quantitative RT-PCR, Expressing, Comparison

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: Migrasome‐associated nanoparticles (MANPs) promote macrophage phagocytosis and M2 polarization. A) TEM image of OS tissue in BALB/c mice ( n = 3). Red arrow points to MANPs. Low magnification: scale bars = 4 µm; high magnification: scale bars = 250 nm. B) Immunostaining images of RAB8a (red), TSPAN4‐EGFP + OS cells (green) and DAPI (blue) in OS tissue of BALB/c mice ( n = 3). White arrows point to MANPs. Scale bars = 3 µm. C) Immunostaining images of WGA labelled OS cells ( n = 3). Low magnification: scale bars = 10 µm; high magnification: scale bars = 1 µm. D) SEM images of OS cells ( n = 3). Low magnification: scale bars = 50 µm; high magnification: scale bars = 1 µm. E) Representative TEM images of purified migrasome by negative staining ( n = 3). Low magnification: scale bars = 500 nm; high magnification: scale bars = 200 nm. F) Representative TEM images of purified migrasomes by ultra‐thin section ( n = 3). G) Confocal time series images of WGA (green) labelled OS cells ( n = 3). White arrows point to the migrasome releasing the MANPs. Low magnification: scale bars = 10 µm; high magnification: scale bars = 2 µm. H) WB analysis of isolated MANPs with the indicated antibodies ( n = 3). I) Representative TEM images of purified MANPs by negative staining ( n = 3). J) Nanoparticle tracking analysis (NTA) of purified MANPs ( n = 3). K) Representative immunostaining images of BMDMs phagocytosis of MANPs (red) ( n = 3). Low magnification: scale bars = 20 µm; high magnification: scale bars = 10 µm. L‐O) BMDMs treated with PBS or 10 µg mL −1 MANPs for 24 h. (L, M) Representative immunostaining images and quantitative analysis of BMDMs phagocytosis of microbeads ( n = 3). (N, O) Flow cytometry analysis of the proportion of BMDMs that phagocytose apoptotic OS cells and quantitative analysis ( n = 3). P‐R) BMDMs treated with PBS or 10 µg mL −1 MANPs for 24 h, followed by co‐incubation with apoptotic OS cells for 24 h. (P) qRT‐PCR analysis of the expression of M1 and M2 polarization markers in BMDMs ( n = 3). (Q, R) Flow cytometry analysis of the proportion of M2‐type macrophages and quantitative analysis ( n = 3). Results were shown as mean ± SD. ** p < 0.01, *** p < 0.001. Unpaired t‐tests were used for the comparison of two groups.

Article Snippet: All the data were analyzed using FLowJo‐v10.8.1 flow cytometry analysis software (TreeStar, USA).

Techniques: Immunostaining, Purification, Negative Staining, Isolation, Flow Cytometry, Incubation, Quantitative RT-PCR, Expressing, Comparison

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: OCDMs promote macrophage phagocytosis and M2 polarization via MANPs. A) Representative TEM images of purified migrasomes by ultra‐thin section ( n = 3). Scale bars = 500 nm. B) Immunostaining images of WGA labelled OS cells, with magnified images of migrasome and young migrasome on the right ( n = 3). White arrows point to the migrasome and the young migrasome. Low magnification: scale bars = 10 µm; high magnification: scale bars = 1 µm. C) SEM images of OS cells ( n = 3). Red arrows point to young migrasomes. Low magnification: scale bars = 10 µm; high magnification: scale bars = 500 nm. D) Schematic diagram of migrasomes, MANPs and young migrasomes purification (created with BioRender.com). E) Representative TEM images of purified young migrasomes by negative staining ( n = 3). Scale bars = 200 nm. F) Representative TEM images of purified young migrasomes by ultra‐thin section ( n = 3). Scale bars = 200 nm. G) Representative immunostaining images of BMDMs phagocytosis of young migrasomes (red) ( n = 3). Scale bars = 10 µm. H‐K) BMDMs treated with 10 µg mL −1 migrasomes or young migrasomes for 24 h. (H, I) 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. (J, K) Flow cytometry analysis of the proportion of BMDMs (APC) that phagocytose apoptotic OS cells (CFSE) and quantitative analysis ( n = 3). L‐N) BMDMs treated with 10 µg mL −1 migrasomes or young migrasomes for 24 h, followed by co‐incubation with apoptotic OS cells for 24 h. (L) qRT‐PCR analysis of the expression of M1 polarization and M2 polarization markers in BMDMs ( n = 3). (M, N) Flow cytometry analysis of the proportion of M2‐type macrophages and quantitative analysis ( n = 3). Results were shown as mean ± SD. ** p < 0.01, *** p < 0.001. Unpaired t‐tests were used for the comparison of two groups.

Article Snippet: All the data were analyzed using FLowJo‐v10.8.1 flow cytometry analysis software (TreeStar, USA).

Techniques: Purification, Immunostaining, Negative Staining, Flow Cytometry, Incubation, Quantitative RT-PCR, Expressing, Comparison

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: All the data were analyzed using FLowJo‐v10.8.1 flow cytometry analysis software (TreeStar, USA).

Techniques: Sequencing, Expressing, Immunostaining, Quantitative RT-PCR, Knockdown, Flow Cytometry, Incubation, Comparison

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: OCDMs promote OS progression through MFGE8. A‐D) BMDMs treated with 10 µg mL −1 sh‐NC migrasomes or sh‐MFGE8 migrasomes for 24 h. (A, B) 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. (C, D) Flow cytometry analysis of the proportion of BMDMs (APC) that phagocytose apoptotic OS cells (CFSE) and quantitative analysis ( n = 3). E‐G) BMDMs treated with 10 µg mL −1 sh‐NC migrasomes or sh‐MFGE8 migrasomes for 24 h, followed by co‐incubation with apoptotic OS cells for 24 h. (E) qRT‐PCR analysis of the expression of M1 polarization and M2 polarization markers in BMDMs ( n = 3). (F, G) Flow cytometry analysis of the proportion of M2‐type macrophages and quantitative analysis ( n = 3). H‐W) After tibia injection of OS cells, mice were treated with sh‐NC migrasomes or sh‐MFGE8 migrasomes. (H) Schematic diagram of animal experiment (created with BioRender.com). (I) Representative images of tumors ( n = 6). (J) Quantification of tumor volumes ( n = 6). (K) Quantification of tumor and bone weight ( n = 6). (L, M) Representative HE staining images and quantitative analysis of pulmonary metastatic nodules ( n = 6). Scale bars = 500 µm. (N‐W) Representative IHC images and quantitative analysis of Ki67, Vimentin and E‐cadherin in tumor sections ( n = 6). Scale bars = 100 µm. Results were shown as mean ± SD. ** 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: All the data were analyzed using FLowJo‐v10.8.1 flow cytometry analysis software (TreeStar, USA).

Techniques: Immunostaining, Flow Cytometry, Incubation, Quantitative RT-PCR, Expressing, Injection, Staining, Comparison