The Efficiency of Gene Electrotransfer in Breast-Cancer Cell Lines Cultured on a Novel Collagen-Free 3D Scaffold.

Elisabetta Sieni,Annj Zamuner,Luca Giovanni Campana,Bianca Bazzolo,Monica Dettin,Mariangela De Robertis,Flavio Keller,Maria Teresa Conconi,Ramona Marino,Emanuela Signori

doi:10.3390/cancers12041043

Abstract

Gene Electro-Transfer (GET) is a powerful method of DNA delivery with great potential for medical applications. Although GET has been extensively studied in vitro and in vivo, the optimal parameters remain controversial. 2D cell cultures have been widely used to investigate GET protocols, but have intrinsic limitations, whereas 3D cultures may represent a more reliable model thanks to the capacity of reproducing the tumor architecture. Here we applied two GET protocols, using a plate or linear electrode, on 3D-cultured HCC1954 and MDA-MB231 breast cancer cell lines grown on a novel collagen-free 3D scaffold and compared results with conventional 2D cultures. To evaluate the electrotransfer efficiency, we used the plasmid pEGFP-C3 encoding the enhanced green fluorescent protein (EGFP) reporter gene. The novel 3D scaffold promoted extracellular matrix deposition, which particularly influences cell behavior in both in vitro cell cultures and in vivo tumor tissue. While the transfection efficiency was similar in the 2D-cultures, we observed significant differences in the 3D-model. The transfection efficiency in the 3D vs 2D model was 44% versus 15% (p < 0.01) and 24% versus 17% (p < 0.01) in HCC1954 and MDA-MB231 cell cultures, respectively. These findings suggest that the novel 3D scaffold allows reproducing, at least partially, the peculiar morphology of the original tumor tissues, thus allowing us to detect meaningful differences between the two cell lines. Following GET with plate electrodes, cell viability was higher in 3D-cultured HCC1954 (66%) and MDA-MB231 (96%) cell lines compared to their 2D counterpart (53% and 63%, respectively, p < 0.001). Based on these results, we propose the novel 3D scaffold as a reliable support for the preparation of cell cultures in GET studies. It may increase the reliability of in vitro assays and allow the optimization of GET parameters of in vivo protocols.

Highlights

Gene Electro-Transfer (GET) is a versatile and efficient method that involves the use of Electroporation (EP) after the injection of nucleic acids into target tissues
Several clinical trials based on GET have been started [8] and initial promising results have been obtained in a first phase I clinical trial, where 10% of the patients with metastatic melanoma were successfully treated by transfecting a plasmid encoding interleukin-12 (IL-12) [9]
3D in in vitro vitro models) models) directly directly influence influence membrane membrane permeabilization permeabilization and and the the cell viability, we evaluated the viability of MDA-MB231 cells cultured as a monolayer cell viability, we evaluated the viability of HCC1954 and MDA-MB231 cells cultured as a monolayer and in in the the new new 3D

Summary

Introduction

Gene Electro-Transfer (GET) is a versatile and efficient method that involves the use of Electroporation (EP) after the injection of nucleic acids (plasmid DNA, RNA, or oligonucleotides) into target tissues. This approach relies on the application of controlled electric pulses on a target tissue that induces the transient permeabilization of the cell membrane and the consequent uptake of therapeutic nucleotide sequences [1,2]. For instance, that EP parameters should be customized when applied to different tissues to produce the most effective electric field intensity [13,14]

Methods

Discussion

Conclusion