Distribution and bioactivity of the Ret-specific D4 aptamer in three-dimensional collagen gel cultures.
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.
Interdisciplinary Research Centre on Biomedical Materials and Department of Materials and Production Engineering, University of Naples Federico II, Naples, Italy.
Maria Teresa
MT
Vento
Marco
M
Iuorio
Paolo A
PA
Netti
Frederic
F
Ducongè
Bertrand
B
Tavitian
Vittoriode
V
Franciscis
Laura
L
Cerchia
D000818:Animals; D000918:Antibody Specificity / Q000187:drug effects; D052157:Aptamers, Nucleotide / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D004058:Diffusion / Q000187:drug effects; D005453:Fluorescence; D005782:Gels; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034443:RNA Transport / Q000187:drug effects; D051381:Rats; D013057:Spectrum Analysis
D052157:Aptamers, Nucleotide; D005782:Gels; D050505:Mutant Proteins; D003094:Collagen; D051096:Proto-Oncogene Proteins c-ret