pFluc was obtained from Icosagen (Tartu, Estonia). This pDNA construct encodes firefly luciferase 2 under control of a CAG promoter, and contains a backbone comprising an ampicillin resistance gene and pUC origin of replication. pFluc production and purification was performed as described previously17, except that pFluc was eluted with sterile milliQ water instead of D-PBS.
Mouse tumour model
The MC38 murine colon cancer cell line was purchased from Kerafast (ENH204-FP, Boston, MA, USA) in March 2017 and was shown to be free of Mycoplasma. Cells were grown in Dulbecco’s Modified Eagle Medium, supplemented with 10% heat-inactivated foetal bovine serum, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES and 50 U/ml penicillin/streptomycin (Thermo Fischer Scientific, Waltham, MA, USA), in a humidified incubator at 37 °C and 5% CO2. Before tumour injections, cells were harvested with 0.05% trypsin–EDTA (25300-054, Thermo Fischer Scientific) and resuspended in D-PBS (14190-094, Thermo Fischer Scientific). 1 × 106 MC38 cells in 100 µl were injected subcutaneously in the right flank of 6- to 7-week-old female C57BL/6J mice. Tumour volumes were evaluated with an electronic calliper (500-712-20, Mitutoyo, Kawasaki, Japan), and calculated with the formula length x width2 × 0.5. Mice were sacrificed when tumour volume exceeded 1500 mm3, or when reporter expression was comparable to background. C57BL/6J mice were purchased from Charles River Laboratories (Saint Germain Nuelles, France) or bred at the KU Leuven Animal Research Center. All animal experiments were approved by the KU Leuven Animal Ethics Committee (P130/2017) and were performed in accordance with the regulations of the European Union and Belgium concerning the protection of laboratory animals.
Intratumoral DNA transfection
Intratumoral electroporation was performed according to a previously described protocol3,11. 20 µg pDNA in 50 µl sterile milliQ water was injected intratumorally, immediately followed by two series of four 5-ms square-wave pulses of 600 V/cm in perpendicular directions at a frequency of 1 Hz. Electrical pulses were delivered by the NEPA21 Electroporator (Sonidel Limited, Dublin, Ireland) with CUY650P5 tweezer electrodes (Sonidel Limited) at a fixed width of 5 mm and covered with Eco Ultrasound Transmission Gel (G0066, Fiab, Vicchio, Italy). Pulse current and total energy were verified with the NEPA21 readout.
Complexes of pDNA with in vivo-jetPEI (201-10G, Polyplus-transfection, Illkirch, France) were prepared according to the manufacturer’s instructions. 20 µg pDNA and 3.2 µl in vivo-jetPEI (N/P = 8) were each diluted in 25 µl of 5% sterile D-glucose. Both dilutions were mixed, and following a 15-min incubation at room temperature, 50 µl was injected intratumorally per mouse.
For electroporation- and jetPEI-driven transfection, pDNA injection was performed manually with a syringe without thorough control of the injection speed. On average, injection of 50 µl lasted 10–20 s.
Fluc expression was visualised and quantified by bioluminescence imaging (IVIS Spectrum, PerkinElmer, Waltham, MA, USA) at the Molecular Small Animal Imaging Center (MoSAIC) at KU Leuven. For in vivo imaging, mice were subcutaneously injected with 126 mg/kg D-luciferin substrate (E6551, Promega, Madison, WI, USA) at 15 mg/ml in D-PBS, after which bioluminescence intensity was measured every two minutes. Intratumoral fluc expression was defined as the maximal total radiance (in photons per second) measured in a specified region of interest covering the tumour area. Mice were anesthetised by isoflurane inhalation during the whole procedure. For ex vivo imaging, mice received a second subcutaneous injection with D-luciferin after in vivo bioluminescence imaging. Five minutes later, mice were sacrificed. Different organs were excised and analysed. Fluc expression in the individual organs was calculated as the total bioluminescent signal measured in a region of interest covering the organ.
At the start of experiments, mice were randomised based on tumour volume and weight. Data of different groups were compared at different time points with Mann–Whitney tests. Outliers were detected with the Grubb’s test. Statistical analyses were performed with Graphpad Prism 8.4.3 (Graphpad Software, San Diego, CA, USA), with two-sided P values below 0.05 considered as significant.