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Luc2-EVs expressing CD44 form small aggregates via CD44 and HA (hyaluronan). ( A ) Analysis of CD44 on Luc2-EVs by transmission electron microscopy (TEM) for homophilic interaction between CD44 on Luc2-EVs. The top row shows EVs incubated in PBS (CD44 + -EVs) for 0 and 3 days (black arrows indicate EV aggregates), and the bottom row shows EVs in PBS after treatment with a neutral anti-CD44 antibody (CD44 − -EVs). ( B ) The bottom graph shows statistical analysis between CD44 + -EVs and CD44 − -EVs for forming EV aggregates. The average aggregate area was measured per pixel and displayed as pixels. * p < 0.01. ** p < 0.001. ( C ) TEM image of Luc2-EVs incubated in 0.05% HA solution for 7 days. Single or aggregated EVs (black arrowheads) were on the HA (high electron-density materials: blue asterisks). ( D ) TEM of hyaluronan-binding protein (HABP) in Luc2-EVs. Twenty nm gold (blue arrowheads) on the EVs demonstrated HA conjugated on the Luc2-EVs. ( E ) Analysis of the effect of HA on the aggregate, EVs were treated with or without HA (HA + , HA − ) and anti-CD44 neutral antibody (CD44 + , CD44 − ). The transmission electron micrograph depicts a clump of extracellular vesicles (blue arrows) observed in a single field of view at a magnification of 5000×. The area of the clump of extracellular vesicles observed in the field of view was subsequently measured using the ImageJ program 1.54j, a software program designed for image analysis. The surface area of clumps comprising two or more extracellular vesicles was quantified. ( F ) Statistical analysis of results are shown in ( E ). * p < 0.01. ** p < 0.001.

Journal: International Journal of Molecular Sciences

Article Title: Role of CD44-Positive Extracellular Vesicles Derived from Highly Metastatic Mouse Mammary Carcinoma Cells in Pre-Metastatic Niche Formation

doi: 10.3390/ijms25179742

Figure Lengend Snippet: Luc2-EVs expressing CD44 form small aggregates via CD44 and HA (hyaluronan). ( A ) Analysis of CD44 on Luc2-EVs by transmission electron microscopy (TEM) for homophilic interaction between CD44 on Luc2-EVs. The top row shows EVs incubated in PBS (CD44 + -EVs) for 0 and 3 days (black arrows indicate EV aggregates), and the bottom row shows EVs in PBS after treatment with a neutral anti-CD44 antibody (CD44 − -EVs). ( B ) The bottom graph shows statistical analysis between CD44 + -EVs and CD44 − -EVs for forming EV aggregates. The average aggregate area was measured per pixel and displayed as pixels. * p < 0.01. ** p < 0.001. ( C ) TEM image of Luc2-EVs incubated in 0.05% HA solution for 7 days. Single or aggregated EVs (black arrowheads) were on the HA (high electron-density materials: blue asterisks). ( D ) TEM of hyaluronan-binding protein (HABP) in Luc2-EVs. Twenty nm gold (blue arrowheads) on the EVs demonstrated HA conjugated on the Luc2-EVs. ( E ) Analysis of the effect of HA on the aggregate, EVs were treated with or without HA (HA + , HA − ) and anti-CD44 neutral antibody (CD44 + , CD44 − ). The transmission electron micrograph depicts a clump of extracellular vesicles (blue arrows) observed in a single field of view at a magnification of 5000×. The area of the clump of extracellular vesicles observed in the field of view was subsequently measured using the ImageJ program 1.54j, a software program designed for image analysis. The surface area of clumps comprising two or more extracellular vesicles was quantified. ( F ) Statistical analysis of results are shown in ( E ). * p < 0.01. ** p < 0.001.

Article Snippet: For visualization of hyaluronan localization, avidin conjugated hyaluronan-binding proteins (Merck SA, 1:50) and streptavidin Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific Inc.) were used.

Techniques: Expressing, Transmission Assay, Electron Microscopy, Incubation, Binding Assay, Software

Hypothesis of pre-metastatic EVs’ metastasis. (1) EV secretion in the early stage of tumorigenesis. (2) EVs contained VEGF-A, VEGF-C, and VEGFR-1, -2, and -3. EVs expressed CD44, which is an HA receptor. (3) EVs aggregated by CD44–CD44 interaction. (4) The aggregates of EVs are attached to HA, and EVs enter the vasculature with hyaluronan, the beginning of pre-metastatic EVs’ metastasis. (5) In this organ, EVs are trapped by endothelial cells that express HA receptors. (6) Endothelial proliferation induced by VEGF-A, VEGF-C, and their receptors: the final stage of premetastatic niche formation.

Journal: International Journal of Molecular Sciences

Article Title: Role of CD44-Positive Extracellular Vesicles Derived from Highly Metastatic Mouse Mammary Carcinoma Cells in Pre-Metastatic Niche Formation

doi: 10.3390/ijms25179742

Figure Lengend Snippet: Hypothesis of pre-metastatic EVs’ metastasis. (1) EV secretion in the early stage of tumorigenesis. (2) EVs contained VEGF-A, VEGF-C, and VEGFR-1, -2, and -3. EVs expressed CD44, which is an HA receptor. (3) EVs aggregated by CD44–CD44 interaction. (4) The aggregates of EVs are attached to HA, and EVs enter the vasculature with hyaluronan, the beginning of pre-metastatic EVs’ metastasis. (5) In this organ, EVs are trapped by endothelial cells that express HA receptors. (6) Endothelial proliferation induced by VEGF-A, VEGF-C, and their receptors: the final stage of premetastatic niche formation.

Article Snippet: For visualization of hyaluronan localization, avidin conjugated hyaluronan-binding proteins (Merck SA, 1:50) and streptavidin Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific Inc.) were used.

Techniques: