Related Experiment Video
Updated: May 2, 2026
Three-Dimensional 3D Tumor Spheroid Invasion Assay
Published on: May 1, 2015
Multicellular tumor spheroids as a model for assessing delivery of oligonucleotides in three dimensions
Kyle Carver1, Xin Ming1, Rudolph L Juliano1
1Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, USA.
Abstract:
Oligonucleotides have shown promise in selectively manipulating gene expression in vitro, but that success has not translated to the clinic for cancer therapy. A potential reason for this is that cells behave differently in monolayer than in the three-dimensional tumor, resulting in limited penetration and distribution of oligonucleotides in the tumor. This may be especially true when oligonucleotides are associated with nanocarriers such as lipoplexes and polyplexes, commonly used delivery vehicles for oligonucleotides. The multicellular tumor spheroid (MCTS), a three-dimensional model that closely resembles small avascular tumors and micrometastases, has been utilized as an intermediate between monolayer culture and in vivo studies for the screening of small-molecule drugs. However, spheroids have been little used for the study of various oligonucleotide delivery formulations. Here, we have evaluated the uptake and efficacy of splice-switching antisense oligonucleotides using various delivery modalities in two- and three-dimensional culture models. We find that the size of the delivery agent dramatically influences penetration into the spheroid and thus the biological effect of the oligonucleotides. We hypothesize that the MCTS model will prove to be a useful tool in the future development of oligonucleotide delivery formulations.Molecular Therapy-Nucleic Acids (2014) 3, e153; doi:10.1038/mtna.2014.5; published online 11 March 2014.
Insights
Delivery agent size impacts how splice-switching antisense oligonucleotides penetrate multicellular tumor spheroids. This finding is crucial for developing effective oligonucleotide cancer therapies using 3D models.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Delivery
Background:
- Oligonucleotides show promise for gene expression manipulation in vitro but face challenges in clinical cancer therapy.
- Limited penetration and distribution in 3D tumors, especially with nanocarriers like lipoplexes and polyplexes, hinder clinical translation.
- Multicellular tumor spheroids (MCTS) offer a 3D model mimicking tumors, but their use in studying oligonucleotide delivery is underexplored.
Purpose of the Study:
- To evaluate the uptake and efficacy of splice-switching antisense oligonucleotides in 2D and 3D culture models.
- To investigate the influence of delivery agent size on oligonucleotide penetration and biological effect within MCTS.
- To assess the utility of the MCTS model for developing oligonucleotide delivery formulations.
Main Methods:
- Utilized two-dimensional (monolayer) and three-dimensional (MCTS) cell culture models.
- Tested various delivery modalities for splice-switching antisense oligonucleotides.
- Analyzed oligonucleotide uptake and biological efficacy in both culture systems.
Main Results:
- Delivery agent size significantly affects oligonucleotide penetration into MCTS.
- Penetration into the spheroid directly correlates with the biological effect of the oligonucleotides.
- Differences in cell behavior between 2D and 3D cultures impact oligonucleotide delivery and efficacy.
Conclusions:
- The size of the delivery agent is a critical factor for effective oligonucleotide delivery in 3D tumor models.
- Multicellular tumor spheroids are a valuable tool for optimizing oligonucleotide delivery formulations for cancer therapy.
- Further development of oligonucleotide delivery systems should consider the physical properties of the delivery agent for improved tumor penetration.
