University of Worcester Worcester Research and Publications

Optimized Delivery of siRNA Into 3D Tumor Spheroid Cultures in Situ

Morgan, R.G., Chambers, A., Legge, D.N., Coles, Steven ORCID:, Greenhough, A. and Williams, A.C. (2018) Optimized Delivery of siRNA Into 3D Tumor Spheroid Cultures in Situ. Scientific Reports, 8 (7952). ISSN 2045-2322

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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.

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Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
Divisions: College of Health, Life and Environmental Sciences > School of Science and the Environment
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Depositing User: Steven Coles
Date Deposited: 08 May 2018 14:46
Last Modified: 19 Mar 2020 13:14

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