Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

Morgan, Rhys G; Chambers, Adam C; Legge, Danny N; Coles, S. J.; Greenhough, Alexander; Williams, Ann C

Abstract

© 2018 The Author(s). 3D tissue culture provides a physiologically relevant and genetically tractable system for studying normal and malignant human tissues. Despite this, gene-silencing studies using siRNA has proved difficult. In this study, we have identified a cause for why traditional siRNA transfection techniques are ineffective in eliciting gene silencing in situ within 3D cultures and proposed a simple method for significantly enhancing siRNA entry into spheroids/organoids. In 2D cell culture, the efficiency of gene silencing is significantly reduced when siRNA complexes are prepared in the presence of serum. Surprisingly, in both 3D tumour spheroids and primary murine organoids, the presence of serum during siRNA preparation rapidly promotes entry and internalization of Cy3-labelled siRNA in under 2 hours. Conversely, siRNA prepared in traditional low-serum transfection media fails to gain matrigel or spheroid/organoid entry. Direct measurement of CTNNB1 mRNA (encoding β-catenin) from transfected tumour spheroids confirmed a transient but significant knockdown of β-catenin when siRNA:liposome complexes were formed with serum, but not when prepared in the presence of reduced-serum media (Opti-MEM). Our studies suggest a simple modification to standard lipid-based transfection protocols facilitates rapid siRNA entry and transient gene repression, providing a platform for researchers to improve siRNA efficiency in established 3D cultures.

• Journal Article Type: Article
• Acceptance Date: May 4, 2018
• Online Publication Date: May 21, 2018
• Publication Date: May 21, 2018
• Deposit Date: Feb 12, 2019
• Publicly Available Date: Feb 14, 2019
• Journal: Scientific Reports
• Electronic ISSN: 2045-2322
• Publisher: Nature Research (part of Springer Nature)
• Peer Reviewed: Peer Reviewed
• Volume: 8
• Article Number: 7952
• DOI: https://doi.org/10.1038/s41598-018-26253-3
• Public URL: https://uwe-repository.worktribe.com/output/867955
• Publisher URL: https://doi.org/10.1038/s41598-018-26253-3
• Related Public URLs: https://doi.org/10.1038/s41598-018-26253-3
• Additional Information: Additional Information : Corresponding authors: [e-mail]; [e-mail]
• Contract Date: Feb 12, 2019