Efficient gene transfer by pulse parameters for electrochemotherapy of cells in vitro and in muscle and melanoma tumors in mice

Abstract

Background: In recent years, various gene therapy strategies have been developed for cancer treatment. One of these strategies is electroporation-based delivery of therapeutic transgenes - gene electrotransfer (GET). Electrochemotherapy and GET have been combined in several contemporary preclinical and veterinary studies. In most cases, two different pulse protocols are used, each for a specific treatment. The aim of our current study was to test whether the standard pulse protocol used in daily clinical practice for electrochemotherapy can also be used for effective GET.

Materials and methods: Experiments were performed in vitro in a tumor (B16F10) and two normal tissue cell lines (C2C12 myoblasts and L929 fibroblasts). Four different GET protocols, three using monopolar electric pulses and one bipolar electric pulses, were tested for the GET of plasmid DNA, which codes for green fluorescent protein in vitro. In addition, two GET protocols were chosen for in vivo tumor and muscle transfection.

Results: Two GET protocols using monopolar electric pulses of different voltages delivered at 1 Hz transfected B16F10 tumor cells significantly better than normal cells. GET4 protocol, which uses monopolar electric pulses at 5 kHz, again transfected the B16F10 tumor cells significantly better, but the difference to the C2C12 myoblast cells was not significant. Compared with other GET protocols, GET3 using bipolar electric pulses at 1 Hz was significantly less effective. Both the GET2 (1 Hz) and GET4 (5 kHz) protocols resulted in similar tumor transfection efficiencies, whereas only the GET4 protocol was effective for muscle transfection in vivo.

Conclusions: Our study demonstrated the efficient transfection of tumors and muscles with the GET4 pulse protocol, which is used clinically for electrochemotherapy. The use of this protocol could enable simultaneous electrochemotherapy and GET of the therapeutic gene in one session, which will significantly shorten the procedure and thus will be more tolerable for patients.

Keywords: cells; electrochemotherapy; gene electrotransfer; muscle; tumor.

FIGURE 1.

The transfection efficiency timeline of different cell lines according to the gene electrotransfer (GET) protocol. Percent of transfected B16F10 (A), C2C12 (B) and L929 (C) cells and fluorescence intensity of B16F10 (D), C2C12 (E) and L929 (F) cells. *p < 0.05, statistically significant difference in GET1/GET2 compared with GET4 (A); *p < 0.05, statistically significant difference in GET1/GET4 compared with GET3 and GET2 (B); **p < 0.05, statistically significant difference in GET2 compared with GET3 (B); *p < 0.05, statistically significant difference in GET1/GET2/GET4 compared with GET4 (D and E)

FIGURE 2.

Transfection efficiency of different pulse parameter protocols in three different cell lines. Percent of transfected tumor (B16F10) and normal (C2C12, L929) cells after GET1 (A), GET2 (B), GET3 (C) and GET4 (D) treatment. *p < 0.05 indicates a statistically significant difference; GET = gene electrotransfer

FIGURE 3.

Cell survival after different pulse parameters and gene electrotransfer (GET) in B16F10 (A), C2C12 (B) and L929 (C) cells. * = p < 0.05 indicates a statistically significant difference; EP = electroporation only; pGFP = plasmid only, which encodes the green fluorescent protein

FIGURE 4.

Transfection of B16F10 tumors. Untreated control tumors (A), GET2 (B) and GET4 (C) tumors. Nuclei are stained blue with Hoechst. The transfected cells are presented in green, indicating transfection with the GFP plasmid. Scale bar = 1000 μm

FIGURE 5.

Transfection of muscle. Control (A), GET2 (B), GET4 (C) and 1HV-4LV (D). The nuclei are stained blue with Hoechst. The transfected cells are presented in green, indicating transfection with the GFP plasmid. Scale bar = 1000 μm.