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

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.

Keywords: 3D cell cultures; Gene Electro-Transfer (GET); breast cancer; electroporation; scaffold.

Figure 1

Cell morphology in 3D and 2D cultures. HCC1954 (A) and MDA-MB231 (B) 3D and 2D cell culture images acquired in a bright field. White arrows indicate cells, whereas white stars indicate the extracellular matrix deposed by cells. Data are from triplicate wells for each condition in two independent experiments. Scale bar 100 µm. Magnification 20×.

Figure 2

Histological images of HCC1954 (A) and MDA-MB231 (B) cells cultured in the 3D scaffold for seven days. Representative images acquired by an inverted microscope after staining with Hematoxylin and Eosin (H&E), Masson Trichrome (MT), and Wiegert Van Gieson (WVG) methods. The blue arrows indicate cells, the white stars the extracellular matrix, and the white arrows indicate collagen. Data are from triplicate wells for each condition in two independent experiments. Scale bar 100 µm. Magnification 20× and 40×.

Figure 3

Efficiency of different Gene Electro-Transfer (GET) protocols in HCC1954 and MDA-MB231 cells according to the modality of cell culture (3D versus 2D). Cells were transfected with Electroporation (EP) employing linear electrodes or JetPrime^® transfection agent. Representative fluorescence images of HCC1954 (A,B) and MDA-MB231 (C,D) 3D and 2D cell culture transfected with green fluorescent protein (GFP) by EP with linear electrodes (A,C) and by JetPrime^® transfection agent (B,D). Representative micrographs of cells in bright field and using 4′,6-Diamidino-2-Phenylindole (DAPI) and GFP filters. Micrographs are representative of two separate blind experiments with similar outcomes. Not transfected cells are the Negative Control (Neg Ctrl). Observation at three days post-transfection. Magnification 20×. Scale bars, 100 µm.

Figure 3

Efficiency of different Gene Electro-Transfer (GET) protocols in HCC1954 and MDA-MB231 cells according to the modality of cell culture (3D versus 2D). Cells were transfected with Electroporation (EP) employing linear electrodes or JetPrime^® transfection agent. Representative fluorescence images of HCC1954 (A,B) and MDA-MB231 (C,D) 3D and 2D cell culture transfected with green fluorescent protein (GFP) by EP with linear electrodes (A,C) and by JetPrime^® transfection agent (B,D). Representative micrographs of cells in bright field and using 4′,6-Diamidino-2-Phenylindole (DAPI) and GFP filters. Micrographs are representative of two separate blind experiments with similar outcomes. Not transfected cells are the Negative Control (Neg Ctrl). Observation at three days post-transfection. Magnification 20×. Scale bars, 100 µm.

Figure 4

Percentage of cell transfection of HCC1954 (A) and MDA-MB231 (B) cell lines in 2D and 3D cultures measured three days after EP (with linear or plate electrode) or JetPrime^®. Data are from three replicates per experimental condition, in two independent blind experiments (Mean ± SD). Statistical analysis was performed using the Student’s t-test (* p < 0.01, ** p < 0.001, *** p < 0.0001).

Figure 5

The efficiency of different GET protocols in HCC1954 and MDA-MB231 3D and 2D cell cultures transfected by EP with plate electrodes. Representative fluorescence images of (A) HCC1954 and (B) MDA-MB231 3D and 2D cell cultures transfected with GFP by EP with plate electrodes. Observation at three days post-transfection. Representative micrographs of cells in bright field and using DAPI and GFP filters. Micrographs are representative of two separate blind experiments with similar outcomes. Not transfected cells are the Negative Control (Neg Ctrl). Magnification 20×. Scale bars, 100 µm.

Figure 6

Viability of HCC1954 and MDA-MB231 2D and 3D cell culture after three days post-GET using linear or plate electrodes. Percentage (%) viability obtained using PrestoBlue^TM Cell Viability assay. Control: untreated cells. Graphs represent quadruplicate biological repeats analyzed blindly and are displayed as Mean ± SD. Statistical analysis was performed using the Student’s t-test. * p < 0.01, ** p < 0.001, *** p < 0.0001.