Fig 1: Circulating sEVs from DIO mice deliver more ECM1 protein to the cancer cells, which in turn secret more ECM1 that acts on the cancer cells to increase invasion and migration.a Uptake of PKH67-labeled sEVs by E0771 cells. Expression of ECM1 protein in sEVs-treated E0771 cells shown by (b) immunofluorescence staining and (c) Western blot analysis. d Expression of ECM1 mRNA levels in the sEVs-treated E0771 cells. e Invasion and migration of sEVs-treated E0771 cells after C-sEVs or D-sEVs treatments. f ECM1 protein levels in the extracellular matrix derived from the C-sEV-treated or D-sEV-treated E-0771 cells, and ECM1 protein level in the extracellular matrix after ECM1 was depleted by immunoprecipitation (D-sEVs ECM1-depleted). g Invasion and migration of E0771 cells following treatments with extracellular matrix derived from E-0771 cells treated with C-sEVs, D-sEVs, or ECM1-depleted ECM. Shown is the mean ± SD; two-sided unpaired t-test for (c to e) and one-way ANOVA with Tukey’s multiple comparison test for (f to g); n = 3 independent experiments; p values are indicated in graphs. C-sEVs, circulating sEVs in CD mice; D-sEVs, circulating sEVs in high fat diet-induced obesity mice; ECM1 extracellular matrix protein 1; MMP3 matrix metallopeptidase 3. Source data are provided as a Source Data file.
Fig 2: Loading of ECM1 protein into sEVs is associated with integrin-β2.a Integrin-β2 protein expressions in the circulating sEVs of high-fat diet-induced obesity mice (D-sEVs) and control diet mice (C- sEVs). b Single cell specific transcriptome profiles showing ECM1 expressions in different cell types. c Single cell sequencing analysis showing ECM1 expressions in peripheral blood mononuclear cells (PBMC) including macrophages. d Single cell type specificity showing integrin-β2 expressions in PBMC including macrophages. e Integrin-β2 protein levels in the integrin β2-knockdown and control RAW264.7 cells. f ECM1 and (g) ICAM1 and GPX3 protein levels in the sEVs released by integrin-β2-knockdown and control RAW264.7 cells. Integrin-β2 protein levels (h) in integrin-β2-overexpressed and control RAW264.7 cells and (i) in the sEVs released by these cells. Shown is the mean ± SD; two-sided unpaired t-test for (a, e to i); n = 3 independent experiments; p values are indicated in graphs. si-β2 integrin-β2-knockdown cells, NC control cells, ICAM-1 intercellular cell adhesion molecule-1, GPX3 glutathione peroxidase 3, ECM1 extracellular matrix protein 1, CD control diet, DIO high-fat diet-induced obesity. Source data are provided in Source Data file.
Fig 3: ECM1 in the sEVs plays a role in enhancing BC growth and metastasis.a A schematic diagram showing the sEVs treatment protocol. b Protein expressions of ECM1, MMP3 and S100A/B in the tumors of E0771-bearing B6/J-Rab27a-Cas9-KO mice after C-sEVs, D-sEVs or ECM-sEVs treatments. c Tumors, d tumor size, e tumor weight and f lung metastasis of the E0771-bearing B6/J-Rab27a-Cas9-KO mice after sEVs treatments. Mouse tumor sizes are presented as the mean ± SEM, other data are presented as mean ± SD; one-way ANOVA with Tukey’s multiple comparison test for (b, d, e); n = 7 mice in each group, p values are indicated in graphs. Rab27aKO, B6/J-Rab27a-Cas9-KO mice; C-sEVs, circulating sEVs in CD-fed C57BL/6 mice; D-sEVs, circulating sEVs in high-fat-diet induced obesity C57BL/6 mice; ECM-sEVs, ECM1 construct-loaded C-sEVs; ECM1, extracellular matrix protein 1; MMP3, matrix metallopeptidase 3. Source data are provided as a Source Data file.
Fig 4: Injection of H-sEVs increases tumor ECM1 levels, cancer growth and metastasis in CD-fed 4T1-bearing mouse model.a Location of the injected 4T1 cells in the mammary fat pad of the female BALB/c mice (upper panel indicated by an arrow) and accumulation of the tail vein injected DIR-labeled sEVs in the mammary pad of the mice (lower panel). b A schematic diagram showing the sEVs treatment protocol. c Protein expressions of ECM1, MMP3 and S100A/B in tumors of CD-fed 4T1-bearing BALB/c mice after C-sEVs or H-sEVs treatments. d Tumors, e tumor size, f tumor weight and g lung metastasis of the CD-fed 4T1-bearing BALB/c mice after the sEVs treatments. Mouse tumor sizes are presented as the mean ± SEM, other data are presented as mean ± SD; two-sided unpaired t-test for (c, f, g); n = 7 mice in each group; p values are indicated in graphs. C-sEVs, circulating sEVs in CD-fed BALB/c mice; H-sEVs, circulating sEVs in HFD-fed BALB/c mice; ECM1 extracellular matrix protein 1; MMP3 matrix metallopeptidase 3. Source data are provided as a Source Data file.
Fig 5: Obesity increases ECM1 protein levels in the circulating sEVs of the human subjects with obesity or overweight and high-fat diet-induced obesity (DIO) mouse models.a Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) of the circulating sEVs purified from the plasma of normal weight healthy human subjects (NC) and human subjects with obesity or overweight (OB). b Volcano plot, c heatmap showing the differential expressed proteins (DEPs) in the sEVs. d Pathway enrichments analysis of the DEPs in the sEVs. e TEM and NTA of the circulating sEVs purified from plasma of the mouse models. f GO analysis of the DEPs in the sEVs of the mouse models. g Overlapping DEPs in the human and mouse sEVs. h ECM1 protein levels in the circulating sEVs in CD mice (C-sEVs) and DIO mice (D-sEVs). i ECM1 protein levels in the plasma and sEVs-depleted plasma of DIO mice. Shown is the mean ± SD; two-sided unpaired t-test for (b, d, h, i); n = 3 independent experiments for (a, e); n = 6 mice in each group; p values were indicated in graphs. C-sEVs, circulating sEVs in control diet mice; D-sEVs, circulating sEVs in high-fat diet-induced obesity mice; sEV-depleted, sEVs-depleted plasma in high-fat diet-induced obesity mice; ECM1 extracellular matrix protein 1. Source data are provided in Source Data file.
Supplier Page from Sino Biological, Inc. for Mouse ECM1 Gene ORF cDNA clone expression plasmid, C-GFPSpark tag