First explanations for differences in electrotransfection efficiency in vitro and in vivo using spheroid model

Area of Science:

• Biomedical Engineering
• Cancer Research
• Molecular Biology

Background:

• Electro-gene-therapy is a promising cancer treatment, but in vivo gene transfer efficiency is limited.
• The mechanisms of gene transfer via electric fields in tumors remain poorly understood.
• In vitro studies show efficient electrotransfection in cell suspensions, contrasting with weak gene expression in vivo.

Purpose of the Study:

• To investigate the reasons for reduced gene expression in vivo compared to in vitro electrotransfection.
• To elucidate gene transfer mechanisms using a multicellular tumor spheroid model ex vivo.
• To compare gene transfer efficiency between cells in suspension and within a 3D tumor spheroid structure.

Main Methods:

• Utilized multicellular tumor spheroids as a 3D ex vivo model for electrotransfection studies.
• Compared transfection rates under classical electrical conditions (10 pulses, 500V/cm, 5ms, 1Hz) in cell suspensions and spheroids.
• Quantified plasmid DNA interaction with permeabilized cells and transgene expression (GFP).

Main Results:

• Suspension cells showed a transfection rate of 23.75%±2.450 SEM under tested conditions.
• In spheroids, less than 1% of cells expressed the transgene, despite plasmid DNA interacting with half of permeabilized cells.
• Identified cell mortality due to the electric field and the physical barrier of the 3D spheroid structure as key factors limiting gene transfer.

Conclusions:

• Multicellular tumor spheroids accurately replicate in vivo electrotransfection challenges.
• Cellular mortality and the physical constraints of the tumor microenvironment significantly impede gene delivery.
• The validated spheroid model enables ex vivo optimization of electro-gene-therapy strategies before in vivo application.