TY - JOUR
T1 - Versatile Role of Rab27a in Glioma
T2 - Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression
AU - van Solinge, Thomas S.
AU - Abels, Erik R.
AU - van de Haar, Lieke L.
AU - Hanlon, Killian S.
AU - Maas, Sybren L.N.
AU - Schnoor, Rosalie
AU - de Vrij, Jeroen
AU - Breakefield, Xandra O.
AU - Broekman, Marike L.D.
N1 - Funding Information:
The Breakefield laboratory acknowledges grant support from NIH NCI P01 CA069246, NIH NCI R35 CA232103 and U19 CA179563 was supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director. TS was supported by grants from the Bontius Stichting, the Nijbakker-Morra Fund, Foundation Vrijvrouwe van Renswoude, and the Bekker-la Bastide Fund. LH was supported by grants from the Jo Kolk Foundation, Dr. Hendrick Muller’s Vaderlandsch Foundation, kfHein Foundation, and Foundation Vrijvrouwe van Renswoude.
Publisher Copyright:
© Copyright © 2020 van Solinge, Abels, van de Haar, Hanlon, Maas, Schnoor, de Vrij, Breakefield and Broekman.
PY - 2020/11/12
Y1 - 2020/11/12
N2 - Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo. Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment. Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed. Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth.
AB - Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo. Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment. Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed. Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth.
KW - exosomes
KW - extracellular vesicles
KW - glioblastoma
KW - glioma
KW - Rab27a
KW - tumor microenvironment
UR - http://www.scopus.com/inward/record.url?scp=85096772200&partnerID=8YFLogxK
U2 - 10.3389/fmolb.2020.554649
DO - 10.3389/fmolb.2020.554649
M3 - Article
C2 - 33282910
AN - SCOPUS:85096772200
VL - 7
JO - Frontiers in Molecular Biosciences
JF - Frontiers in Molecular Biosciences
M1 - 554649
ER -