Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer
Abstract
:1. Introduction
2. Results
2.1. Extracellular Vesicles Secreted by Non-Tumorigenic Human Mammary Cells and Triple Negative Breast Cancer Cells Demonstrate Classical Morphological and Molecular Features
2.2. MDA-MB-231 EVs Are Enriched in NDPK-B Expression Compared to Non-Tumorigenic Mammary EVs
2.3. MDA-MB-231 EVs Are Enriched in NDPK Phosphotransferase Activity
2.4. NDPK Inhibition and P2Y1 Receptor Antagonism Reverse the Pro-Migratory Effect of MDA-MB-231 EVs on Vascular Endothelial Cells
2.5. NDPK Inhibition Rescues MDA-MB-231 EV-Mediated Permeabilization of Vascular Endothelial Cell Monolayers
2.6. MDA-MB-231 EVs Enhance Vascular Leakage in the Lung and NDPK Inhibition or P2Y1 Receptor Antagonism Ameliorates This Effect
2.7. Attenuation of eNDPK Activity or P2Y1 Receptor Activation Blunts the Development of Lung Metastases in an Experimental Metastasis Model
2.8. Proteomic Analysis of the Lung Following MDA-MB-231 EV Treatment Identifies Purinergic Events Known to Support Pre-Metastatic Niche Formation
3. Discussion
4. Materials and Methods
4.1. Drug Reagents
4.2. Cell Culture of Human Cell Lines
4.3. Neonatal Pulmonary Endothelial Cell Isolation
4.4. Extracellular Vesicle Isolation
4.5. Transmission Electron Microscopy (TEM)
4.6. EV Analysis with Flow Cytometry
4.7. Western Blot Analysis
4.8. Wes Protein Assay
4.9. NDPK Transphosphorylation Activity Assay
4.10. Transwell Migration Assay
4.11. Transwell Permeability Assay
4.12. Imaging of Endothelial Barrier
4.13. Confocal Laser Scanning Microscopy (CLSM)
4.14. Animal Studies
4.15. TMT-Labeled Mass Spectrometry
4.16. Evans Blue Dye (EBD) Extravasation Study
4.17. Experimental Metastasis Study
4.18. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Duan, S.; Nordmeier, S.; Byrnes, A.E.; Buxton, I.L.O. Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer. Int. J. Mol. Sci. 2021, 22, 597. https://doi.org/10.3390/ijms22020597
Duan S, Nordmeier S, Byrnes AE, Buxton ILO. Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer. International Journal of Molecular Sciences. 2021; 22(2):597. https://doi.org/10.3390/ijms22020597
Chicago/Turabian StyleDuan, Suzann, Senny Nordmeier, Aidan E. Byrnes, and Iain L. O. Buxton. 2021. "Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer" International Journal of Molecular Sciences 22, no. 2: 597. https://doi.org/10.3390/ijms22020597