Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression

Thomas S van Solinge; Erik R Abels; Lieke L van de Haar; Killian S Hanlon; Sybren L N Maas; Rosalie Schnoor; Jeroen de Vrij; Xandra O Breakefield; Marike L D Broekman

doi:10.3389/fmolb.2020.554649

Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression

Thomas S van Solinge^*
, Erik R Abels
, Lieke L van de Haar
, Killian S Hanlon
, Sybren L N Maas
, Rosalie Schnoor
, Jeroen de Vrij
, Xandra O Breakefield
, Marike L D Broekman

^*Corresponding author for this work

Neurosurgery

Harvard Pilgrim Health Care Institute
Utrecht University
University Medical Centre Utrecht

Research output: Contribution to journal › Article › Academic › peer-review

13 Citations (Scopus)

40 Downloads (Pure)

Abstract

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.

Original language	English
Article number	554649
Pages (from-to)	554649
Journal	Frontiers in Molecular Biosciences
Volume	7
DOIs	https://doi.org/10.3389/fmolb.2020.554649
Publication status	Published - 12 Nov 2020

Bibliographical note

Copyright © 2020 van Solinge, Abels, van de Haar, Hanlon, Maas, Schnoor, de Vrij, Breakefield and Broekman.

FUNDING:

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.

Research programs

EMC OR-01

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10.3389/fmolb.2020.554649Licence: CC BY

Versatile Role of Rab27a in GliomaFinal published version, 2.77 MBLicence: CC BY

http://hdl.handle.net/1765/132105

Cite this

van Solinge, T. S., Abels, E. R., van de Haar, L. L., Hanlon, K. S., Maas, S. L. N., Schnoor, R., de Vrij, J., Breakefield, X. O., & Broekman, M. L. D. (2020). Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression. Frontiers in Molecular Biosciences, 7, 554649. Article 554649. https://doi.org/10.3389/fmolb.2020.554649

@article{8e55a069c112427abcbf0dfa2079a4fc,

title = "Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression",

abstract = "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.",

author = "\{van Solinge\}, \{Thomas S\} and Abels, \{Erik R\} and \{van de Haar\}, \{Lieke L\} and Hanlon, \{Killian S\} and Maas, \{Sybren L N\} and Rosalie Schnoor and \{de Vrij\}, Jeroen and Breakefield, \{Xandra O\} and Broekman, \{Marike L D\}",

note = "Copyright {\textcopyright} 2020 van Solinge, Abels, van de Haar, Hanlon, Maas, Schnoor, de Vrij, Breakefield and Broekman. FUNDING: 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{\textquoteright}s Vaderlandsch Foundation, kfHein Foundation, and Foundation Vrijvrouwe van Renswoude. ",

year = "2020",

month = nov,

day = "12",

doi = "10.3389/fmolb.2020.554649",

language = "English",

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pages = "554649",

journal = "Frontiers in Molecular Biosciences",

issn = "2296-889X",

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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 - Copyright © 2020 van Solinge, Abels, van de Haar, Hanlon, Maas, Schnoor, de Vrij, Breakefield and Broekman. FUNDING: 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.

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.

U2 - 10.3389/fmolb.2020.554649

DO - 10.3389/fmolb.2020.554649

M3 - Article

C2 - 33282910

SN - 2296-889X

VL - 7

SP - 554649

JO - Frontiers in Molecular Biosciences

JF - Frontiers in Molecular Biosciences

M1 - 554649

ER -

Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression

Abstract

Bibliographical note

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