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Updated: Jun 29, 2026
Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo
Published on: May 9, 2016
Distribution and bioactivity of the Ret-specific D4 aptamer in three-dimensional collagen gel cultures
Maria Teresa Vento1, Marco Iuorio, Paolo A Netti
1Interdisciplinary Research Centre on Biomedical Materials and Department of Materials and Production Engineering, University of Naples Federico II, Naples, Italy.
Abstract:
The success of tyrosine kinase inhibitors in cancer therapy prompted intensive research efforts addressed to the development of new specific diagnostics and therapeutics. Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacologic challenge. The D4 RNA-aptamer, isolated applying the Systematic Evolution of Ligand by Exponential Enrichment procedure on living cells, has been proven a specific inhibitor of the human receptor tyrosine kinase Ret. In our attempts to generate new powerful probes for in vivo applications, in the present study, we addressed the ability of D4 to preserve its biological activity in cells embedded in three-dimensional collagen gels. These matrices provide a microenvironment mimicking the cell organization as seen in vivo, thus representing a suitable tool to approach the use of the aptamer in vivo. By taking advantage of transformed fibroblasts expressing Ret as a model system, we showed that the cells maintain normal phenotype and growth patterns when cultured in three-dimensional matrices and that the D4 aptamer preserves its ability to inhibit Ret on the surface of the cells embedded in collagen. Because the biological activity of RNA aptamers is largely dictated by their folded structure, the results indicate that a folded conformation of D4 responsible of its inhibiting function is preserved in the three-dimensional constructs, thus supporting its use in tumors in vivo.
Insights
The D4 RNA-aptamer retains its cancer-targeting activity within 3D collagen gels, mimicking in vivo conditions. This finding supports the potential use of this Ret inhibitor as a diagnostic or therapeutic agent in three-dimensional tumor environments.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Tyrosine kinase inhibitors are crucial in cancer therapy, but targeting large molecules like receptor tyrosine kinases remains challenging.
- The D4 RNA-aptamer specifically inhibits the human receptor tyrosine kinase Ret.
- Developing in vivo diagnostic and therapeutic probes is a key goal in cancer research.
Purpose of the Study:
- To evaluate the biological activity of the D4 RNA-aptamer in cells within a three-dimensional (3D) collagen gel environment.
- To determine if the D4 aptamer maintains its Ret-inhibiting function in a microenvironment that mimics in vivo conditions.
- To assess the potential of D4 for in vivo applications in cancer therapy.
Main Methods:
- Utilized transformed fibroblasts expressing Ret as a model system.
- Cultured cells within 3D collagen gels to simulate an in vivo-like microenvironment.
- Assessed the phenotype, growth patterns, and Ret-inhibiting activity of the D4 aptamer on cells embedded in the 3D matrix.
Main Results:
- Cells embedded in 3D collagen gels maintained normal phenotype and growth.
- The D4 RNA-aptamer preserved its ability to inhibit Ret on the surface of cells within the collagen matrix.
- The folded structure of D4, essential for its inhibitory function, remained intact in the 3D constructs.
Conclusions:
- The D4 RNA-aptamer maintains its biological activity and Ret-inhibiting function in a 3D collagen gel environment.
- The structural integrity required for D4's function is preserved in these 3D constructs.
- These findings support the potential use of the D4 aptamer for in vivo applications, particularly in targeting tumors.
