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Exosomes released from breast cancer carcinomas stimulate cell movement.

Harris DA, Patel SH, Gucek M, Hendrix A, Westbroek W, Taraska JW - PLoS ONE (2015)

Bottom Line: Exosomes have been proposed to act as regulators of cancer progression.Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance.We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

No MeSH data available.


Related in: MedlinePlus

Tumor-derived exosomes increase breast cell motility.(A) Time course of cell migration wound-healing assays using MCF-7 breast cancer cells as the “recipient” cell line with the three “donor” exosome preparations. Serum-free media was used as a control. Each point on the assay represents three independent experiments at 21 hours. (B) Representative images from the wound healing experiments. Quantification (C) and images (D) of wound-healing assay for MCF7/Rab27b cells in the presence of the three exosome preparations and serum-free media after 19 hours. Quantification (C) and images (D) of wound-healing assay for MDA-MB-231 cells in the presence of the three exosome preparations and serum-free media after 15 hours. Errors were calculated from wound closure at each time point and normalized to the wound closure at the initial time point (0 hour). Experiments were repeated two additional times to verify results.
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pone.0117495.g003: Tumor-derived exosomes increase breast cell motility.(A) Time course of cell migration wound-healing assays using MCF-7 breast cancer cells as the “recipient” cell line with the three “donor” exosome preparations. Serum-free media was used as a control. Each point on the assay represents three independent experiments at 21 hours. (B) Representative images from the wound healing experiments. Quantification (C) and images (D) of wound-healing assay for MCF7/Rab27b cells in the presence of the three exosome preparations and serum-free media after 19 hours. Quantification (C) and images (D) of wound-healing assay for MDA-MB-231 cells in the presence of the three exosome preparations and serum-free media after 15 hours. Errors were calculated from wound closure at each time point and normalized to the wound closure at the initial time point (0 hour). Experiments were repeated two additional times to verify results.

Mentions: Exosomes purified from these three cells by serial ultra-centrifugation [38] were observed by transmission electron microscopy to be small (50–120nm) spherical vesicles (Fig. 2A-B). To ensure that we isolated exosomes from our preparations, we conducted Western blotting to confirm the presence of several common exosome/vesicle markers, including TSG101, ALIX, HSP70, HSP90 (Fig. 2C). The presence of these markers is consistent with purified exosome samples. Additionally, we further analyzed (in triplicate) our exosome preparations using nanoparticle tracking analysis (NTA) which measures particles (eg. microvesicles). Based on these measurements and the shoulder associated with the major distribution peak (Figure A-C in S1 Fig.), it is likely that our exosome preparations isolated from MDA-MB-231 (and to a lesser extent also for MCF-7/Rab27b) contain a heterogeneous mixture of exosome and microvesicles consistent with other high speed ultracentrifugation protocols [39]. This finding is consistent with the work which suggested that several diverse population of vesicles (including exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles) are present in many exosome preparations obtained by differential ultracentrifugation [39,40]. To test the effect of these vesicles on cell motility, we added the same concentration of exosomes isolated from the three different “donor” breast cancer cells types (MCF-7, Rab27b, MDA-MB-231) to “recipient” cells of the same or different identity from which the exosomes were originally derived. Migration was measured in a standard wound healing assay. In this assay, cells were plated into two zones with a space separating each population. The decrease in the wound caused by the migration of cells over time was then quantified (Fig. 3). We observe a substantial and reproducible wound closure (or increase in cell motility) in cells incubated with exosomes isolated from all three donor cancer cell types compared to cells incubated in control media (Serum-free media (SFM), with PBS only). Exosomes isolated from the moderate and highly metastatic cells (Rab27b and MDA-MB-231) induced increases in cell migration in all three recipient cell lines (Fig. 3B, D, E). Interestingly, exosomes isolated from the cells with the highest metastatic potential (MDA-MB-231) induced the largest increase in motility, followed by Rab27b cell line (Fig. 3C-F). The effect on cell migration induced by the exosomes purified from the non-metastatic MCF-7 cells was modest compared to controls (Fig. 3A-B). These data show that the effect of exosomes on migration is linked to the underlying metastatic potential of the donor cells. Interestingly, exosomes from highly metastatic cells induced migration to a degree that depended on the metastatic potential of the donor cancer cell type. In short, exosomes derived from the donor metastatic lines (Rab27b and MDA-MB-231) promoted cell mobility faster and/or to a further extent than exosomes isolated from the non-metastatic cell line (MCF-7) or control media. The migration phenotype in the moderate metastatic line (Rab27b) occurred faster than the non-metastatic line (MCF-7) and the MDA-MB-231 cells healed the fastest overall.


Exosomes released from breast cancer carcinomas stimulate cell movement.

Harris DA, Patel SH, Gucek M, Hendrix A, Westbroek W, Taraska JW - PLoS ONE (2015)

Tumor-derived exosomes increase breast cell motility.(A) Time course of cell migration wound-healing assays using MCF-7 breast cancer cells as the “recipient” cell line with the three “donor” exosome preparations. Serum-free media was used as a control. Each point on the assay represents three independent experiments at 21 hours. (B) Representative images from the wound healing experiments. Quantification (C) and images (D) of wound-healing assay for MCF7/Rab27b cells in the presence of the three exosome preparations and serum-free media after 19 hours. Quantification (C) and images (D) of wound-healing assay for MDA-MB-231 cells in the presence of the three exosome preparations and serum-free media after 15 hours. Errors were calculated from wound closure at each time point and normalized to the wound closure at the initial time point (0 hour). Experiments were repeated two additional times to verify results.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4370373&req=5

pone.0117495.g003: Tumor-derived exosomes increase breast cell motility.(A) Time course of cell migration wound-healing assays using MCF-7 breast cancer cells as the “recipient” cell line with the three “donor” exosome preparations. Serum-free media was used as a control. Each point on the assay represents three independent experiments at 21 hours. (B) Representative images from the wound healing experiments. Quantification (C) and images (D) of wound-healing assay for MCF7/Rab27b cells in the presence of the three exosome preparations and serum-free media after 19 hours. Quantification (C) and images (D) of wound-healing assay for MDA-MB-231 cells in the presence of the three exosome preparations and serum-free media after 15 hours. Errors were calculated from wound closure at each time point and normalized to the wound closure at the initial time point (0 hour). Experiments were repeated two additional times to verify results.
Mentions: Exosomes purified from these three cells by serial ultra-centrifugation [38] were observed by transmission electron microscopy to be small (50–120nm) spherical vesicles (Fig. 2A-B). To ensure that we isolated exosomes from our preparations, we conducted Western blotting to confirm the presence of several common exosome/vesicle markers, including TSG101, ALIX, HSP70, HSP90 (Fig. 2C). The presence of these markers is consistent with purified exosome samples. Additionally, we further analyzed (in triplicate) our exosome preparations using nanoparticle tracking analysis (NTA) which measures particles (eg. microvesicles). Based on these measurements and the shoulder associated with the major distribution peak (Figure A-C in S1 Fig.), it is likely that our exosome preparations isolated from MDA-MB-231 (and to a lesser extent also for MCF-7/Rab27b) contain a heterogeneous mixture of exosome and microvesicles consistent with other high speed ultracentrifugation protocols [39]. This finding is consistent with the work which suggested that several diverse population of vesicles (including exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles, and apoptotic vesicles) are present in many exosome preparations obtained by differential ultracentrifugation [39,40]. To test the effect of these vesicles on cell motility, we added the same concentration of exosomes isolated from the three different “donor” breast cancer cells types (MCF-7, Rab27b, MDA-MB-231) to “recipient” cells of the same or different identity from which the exosomes were originally derived. Migration was measured in a standard wound healing assay. In this assay, cells were plated into two zones with a space separating each population. The decrease in the wound caused by the migration of cells over time was then quantified (Fig. 3). We observe a substantial and reproducible wound closure (or increase in cell motility) in cells incubated with exosomes isolated from all three donor cancer cell types compared to cells incubated in control media (Serum-free media (SFM), with PBS only). Exosomes isolated from the moderate and highly metastatic cells (Rab27b and MDA-MB-231) induced increases in cell migration in all three recipient cell lines (Fig. 3B, D, E). Interestingly, exosomes isolated from the cells with the highest metastatic potential (MDA-MB-231) induced the largest increase in motility, followed by Rab27b cell line (Fig. 3C-F). The effect on cell migration induced by the exosomes purified from the non-metastatic MCF-7 cells was modest compared to controls (Fig. 3A-B). These data show that the effect of exosomes on migration is linked to the underlying metastatic potential of the donor cells. Interestingly, exosomes from highly metastatic cells induced migration to a degree that depended on the metastatic potential of the donor cancer cell type. In short, exosomes derived from the donor metastatic lines (Rab27b and MDA-MB-231) promoted cell mobility faster and/or to a further extent than exosomes isolated from the non-metastatic cell line (MCF-7) or control media. The migration phenotype in the moderate metastatic line (Rab27b) occurred faster than the non-metastatic line (MCF-7) and the MDA-MB-231 cells healed the fastest overall.

Bottom Line: Exosomes have been proposed to act as regulators of cancer progression.Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance.We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
For metastasis to occur cells must communicate with to their local environment to initiate growth and invasion. Exosomes have emerged as an important mediator of cell-to-cell signalling through the transfer of molecules such as mRNAs, microRNAs, and proteins between cells. Exosomes have been proposed to act as regulators of cancer progression. Here, we study the effect of exosomes on cell migration, an important step in metastasis. We performed cell migration assays, endocytosis assays, and exosome proteomic profiling on exosomes released from three breast cancer cell lines that model progressive stages of metastasis. Results from these experiments suggest: (1) exosomes promote cell migration and (2) the signal is stronger from exosomes isolated from cells with higher metastatic potentials; (3) exosomes are endocytosed at the same rate regardless of the cell type; (4) exosomes released from cells show differential enrichment of proteins with unique protein signatures of both identity and abundance. We conclude that breast cancer cells of increasing metastatic potential secrete exosomes with distinct protein signatures that proportionally increase cell movement and suggest that released exosomes could play an active role in metastasis.

No MeSH data available.


Related in: MedlinePlus