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Modulation of exosome release by cancer cells: a screening of potential inhibitors

Author
Organization
Abstract
Background: The molecular mechanisms of exosome biogenesis and secretion are poorly understood. An important role is dedicated to the small GTPase Rab27B, a regulator of vesicle exocytosis that delivers pro-invasive signals for increased tumor size, invasiveness and metastasis of various estrogen receptor positive breast cancer cell lines, both in vitro and in vivo. In vitro MCF-7 GFP-Rab27B cells are used to hunt down the influence of a series of compounds on exosome secretion. V-ATPase inhibitors, bafilomycin A1 and concanamycin A, because of demonstrated presence of V-ATPase subunits on GFP-Rab27B vesicles. And cytoskeleton inhibitors, cytochalasin D and nocodazole to undermine intracellular vesicle transport routes. Methods: MTT assays are performed to study the influence of the compounds on cell proliferation. To study the compounds their effect the design consists of two legs. In the quantitative leg nanoparticle tracking analysis is performed on isolated particles from conditioned medium of treated cells to study the difference in total particle secretion. In the morphological leg fluorescence microscopy is performed on treated cells to observe changes in intracellular localization of GFP-Rab27B vesicles containing exosomes. Results: All used compounds display reversible lowering in total particle concentration. Individual reversible inhibition of V-ATPase activity and cytoskeleton morphology demonstrates that the substrates control peripheral localization of Rab27B vesicles. This suggests the particle lowering observed in the quantitative leg is due to reduction in exosome secretion. Conclusion: Inhibiting V-ATPase activity and interfering cytoskeleton architecture by administration of drugs is potentially linked to a lowering of exosome secretion in ERα-positive breast cancer. The positive screening of these inhibitors reveals essential compounds in the exosome releasing pathway and proposes new perspectives for potential prevention of further metastasis in the future. We established a readily and cheap screening method for inhibitors of exosome release in cancer cells.
Keywords
Rab27B, Exosomes, Cytoskeleton, V-ATPase

Citation

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MLA
Rasschaert, Gertjan, An Hendrix, and Olivier De Wever. “Modulation of Exosome Release by Cancer Cells: a Screening of Potential Inhibitors.” Abstract Book : 9th Leiden International Medical Student Conference. Leiden, The Netherlands: Leiden University Medical Center, 2015. 87–87. Print.
APA
Rasschaert, Gertjan, Hendrix, A., & De Wever, O. (2015). Modulation of exosome release by cancer cells: a screening of potential inhibitors. Abstract book : 9th Leiden international medical student conference (pp. 87–87). Presented at the 9th Leiden International Medical Student conference (LIMSC): Key to the future, Leiden, The Netherlands: Leiden University Medical Center.
Chicago author-date
Rasschaert, Gertjan, An Hendrix, and Olivier De Wever. 2015. “Modulation of Exosome Release by Cancer Cells: a Screening of Potential Inhibitors.” In Abstract Book : 9th Leiden International Medical Student Conference, 87–87. Leiden, The Netherlands: Leiden University Medical Center.
Chicago author-date (all authors)
Rasschaert, Gertjan, An Hendrix, and Olivier De Wever. 2015. “Modulation of Exosome Release by Cancer Cells: a Screening of Potential Inhibitors.” In Abstract Book : 9th Leiden International Medical Student Conference, 87–87. Leiden, The Netherlands: Leiden University Medical Center.
Vancouver
1.
Rasschaert G, Hendrix A, De Wever O. Modulation of exosome release by cancer cells: a screening of potential inhibitors. Abstract book : 9th Leiden international medical student conference. Leiden, The Netherlands: Leiden University Medical Center; 2015. p. 87–87.
IEEE
[1]
G. Rasschaert, A. Hendrix, and O. De Wever, “Modulation of exosome release by cancer cells: a screening of potential inhibitors,” in Abstract book : 9th Leiden international medical student conference, Leiden, The Netherlands, 2015, pp. 87–87.
@inproceedings{5896105,
  abstract     = {Background: The molecular mechanisms of exosome biogenesis and secretion are poorly understood. An important role is dedicated to the small GTPase Rab27B, a regulator of vesicle exocytosis that delivers pro-invasive signals for increased tumor size, invasiveness and metastasis of various estrogen receptor positive breast cancer cell lines, both in vitro and in vivo. In vitro MCF-7 GFP-Rab27B cells are used to hunt down the influence of a series of compounds on exosome secretion. V-ATPase inhibitors, bafilomycin A1 and concanamycin A, because of demonstrated presence of V-ATPase subunits on GFP-Rab27B vesicles. And cytoskeleton inhibitors, cytochalasin D and nocodazole to undermine intracellular vesicle transport routes.

Methods: MTT assays are performed to study the influence of the compounds on cell proliferation. 
To study the compounds their effect the design consists of two legs. In the quantitative leg nanoparticle tracking analysis is performed on isolated particles from conditioned medium of treated cells to study the difference in total particle secretion. In the morphological leg fluorescence microscopy is performed on treated cells to observe changes in intracellular localization of GFP-Rab27B vesicles containing exosomes.

Results: All used compounds display reversible lowering in total particle concentration. Individual reversible inhibition of V-ATPase activity and cytoskeleton morphology demonstrates that the substrates control peripheral localization of Rab27B vesicles. This suggests the particle lowering observed in the quantitative leg is due to reduction in exosome secretion. 

Conclusion: Inhibiting V-ATPase activity and interfering cytoskeleton architecture by administration of drugs is potentially linked to a lowering of exosome secretion in ERα-positive breast cancer. The positive screening of these inhibitors reveals essential compounds in the exosome releasing pathway and proposes new perspectives for potential prevention of further metastasis in the future. We established a readily and cheap screening method for inhibitors of exosome release in cancer cells.},
  articleno    = {abstract 333},
  author       = {Rasschaert, Gertjan and Hendrix, An and De Wever, Olivier},
  booktitle    = {Abstract book : 9th Leiden international medical student conference},
  keywords     = {Rab27B,Exosomes,Cytoskeleton,V-ATPase},
  language     = {eng},
  location     = {Leiden, The Netherlands},
  pages        = {abstract 333:87--abstract 333:87},
  publisher    = {Leiden University Medical Center},
  title        = {Modulation of exosome release by cancer cells: a screening of potential inhibitors},
  year         = {2015},
}