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

Abstract

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.

LAT1 and ASCT2 Related microRNAs as Potential New Therapeutic Agents against Colorectal Cancer Progression

“…In another study, Zoetemelk and colleagues established a robust, low-cost and reproducible short-term 3D colorectal cancer spheroids model to be used as a platform for screening the effect of combination therapies in CRC [119]. These enhanced research models are very useful for the study of miRNAs dynamics and for the development of the delivery systems for miRNA-based therapeutics [120,121]. Therefore, the next step should be focused on the delivery of the miRNA profile to CRC 3D culture models in order to see if it is sufficient to reverse the increased AA uptake caused by the increase of LAT1 and ASCT2 and inhibit cell proliferation.…”

5-Aminosalicylic acid inhibits stem cell function in human adenoma-derived cells: implications for chemoprophylaxis in colorectal tumorigenesis

“… 45 Cells were resuspended in Matrigel (Corning, NY, USA) as a single-cell suspension and seeded into 24-well plates (Corning, NY, USA) as described previously. 46 The Matrigel hemispheres were allowed to polymerise before being submerged in advanced DMEM:F12 (Gibco, Thermo Fisher Scientific, MA, USA) supplemented with 0.1% bovine serum albumin (Sigma-Aldrich, Merck, MO, USA), 2 mM glutamine (Gibco, Thermo Fisher Scientific, MA, USA), 10 mM HEPES (Sigma-Aldrich, Merck, MO, USA), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco; Thermo Fisher Scientific, MA, USA), 1% N2 (Thermo Fisher Scientific, MA, USA), 2% B27 (Thermo Fisher Scientific, MA, USA) and 0.2% N -acetylcysteine (Sigma-Aldrich, Merck, MO, USA). For spheroid culture of PC/AA/C1 adenoma-derived cells, the spheroid medium was further supplemented with human epidermal growth factor (Peprotech, London, UK), 50 ng/mL.…”

Current concepts in tumour-derived organoids

“… 48 In addition, the efficiency of gene silencing using small-interfering (si)RNA is significantly reduced in the presence of serum in 2D cultures, whereas serum improves knockdown efficacy in organoid cultures by promoting the internalisation of siRNA. 49 …”