Engineered anti-EGFRvIII targeted exosomes induce apoptosis in glioblastoma multiforme

Abstract

Background

The drug delivery for treatment of glioblastoma multiforme (GBM) has been unsatisfactory mainly due to the drug resistance and low targeting efficiency. The selective targeting of GBM cells and using a cocktail of therapeutic agents to synergistically induce apoptosis may help enhance the drug delivery.

Methods

In this study, mesenchymal stem cells (MSCs) were engineered to produce exosomes, i.e. nanosized natural vesicles presenting anti-EGFRvIII (ab139) antibody on their surface while encapsulating two apoptosis-inducing gene therapy agents, i.e. cytosine deaminase (CDA) and miR-34a. Exosomes were characterised for their size, morphology, protein content and markers using dynamic light scattering and nanoparticle tracking analysis, cryo-TEM, Western blotting, respectively. miR-34a overexpression and Lamp2-ab139 protein expression were analysed using real-time PCR and flow cytometry, respectively. The armed exosomes were delivered to EGFRvIII positive GBM cells (U87EGFRvIII) as well as wild type cell line (U87), which was EGFRvIII negative. Apoptosis was quantified using flow cytometry in both EGFRvIII negative and positive U87 cells, receiving one gene therapy agent (either CDA or miR-34a) or a combination of them (CDAmiR).

Results

Spherical shape exosomes with an average diameter of 110 nm and a membrane thickness of 6.5 nm were isolated from MSCs. Lamp2-ab139 was successfully expressed on the surface of transfected cells and their secreted exosomes. Induced apoptosis rates was significantly higher in U87EGFRvIII cells than for U87 cells, indicating selectivity. The cell death rate was 6%, 9% and 12% in U87, 13%, 21% and 40% in U87EGFRvIII cells for CDA, miR-34a and CDAmiR treatment respectively, showing a higher apoptosis rate in the cells receiving both drugs compared to when single therapy was applied.

Conclusion

The experimental findings clearly show the improved apoptosis rate of GBM cells when treated by engineered exosomes armed with two gene therapy agents and targeted towards EGFRvIII antigen.

Keywords:

• Exosomes
• MSC-derived exosomes
• glioblastoma multiforme
• EGFRvIII
• gene therapy
• microRNA
• apoptosis

Acknowledgements

The authors thank Jacinta White from CSIRO for her help in TEM imaging. They also are grateful to Sandy Fung for her help in operating the FACS analysis. The authors also acknowledge the support received from Prof. Suresh Mathivanan and Dr. Pamali Fonseka in Western Blot analysis and providing exosome-free FBS. The authors appreciate Dr. Fatemeh Gheidari for gifting U87EGFRvIII cells. The authors acknowledge the Iran National Science Foundation(INSF) for their support (Project ID: 96013284). The authors also thank Dr. Mojtaba Hedayati Marzbali for his advice on manuscript improvement and professional English editing.

Consent for publication

Not applicable.

Availability of data and materials

The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Disclosure statement

The authors declare that they have no competing interests.

Authors’ contributions

Rana Rahmani: Conceptualisation, Methodology, Experimentation, Characterisation and Analysis, Visualisation, Writing original draft and editing. Jafar Kiani: Genetic constructs designing. Wing Yin Tong: Advisor, Methodology. Masoud Soleimani: Resources. Nicolas H. Voelcker: Advisor, Resources, Manuscript reviewing. Ehsan Arefian: Conceptualisation, Methodology, Supervision, Resources, Manuscript editing.

Additional information

Funding

Not applicable.