1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and extrusion membranes were purchased from Avanti Polar lipids, Quant-iT™ PicoGreen^® from Invitrogen, luciferase (Luc) assay system from Promega, Pierce™ BCA Protein Assay Kit from Thermo Fisher Scientific, Resazurin sodium salt from Sigma-Aldrich, and D-luciferin potassium salt from Interchim Fluoprobes. CT26 murine colorectal carcinoma cell line [CT26. WT: American Type Culture Collection (ATCC)^® CRL-2638™] was purchased from ATCC (LGC Standards, Molsheim, France). Dulbecco’s modified Eagle’s medium (DMEM) completed with 10% fetal bovine serum (FBS), L-glutamine (29 mg/mL), penicillin (50 U/mL), and streptomycin (50 U/mL; Gibco, ThermoFisher).

3-[bis(3-aminopropyl)amino]propyl-[2-[[2-[di(tetradecyl)amino]-2-oxo-ethyl]amino]-2-oxoethyl] ammonium; hydrochloride (DMAPAP, also called RPR209120) was synthesized in our laboratory as described [24]. YUK1350 (also called RPR206252) was synthesized in our laboratory as described [25]. The chemical structures are presented in Figure 1.

Plasmid free of antibiotic resistance version 4 (pFAR4) gene vectors are antibiotic-free plasmids that are propagated using a specific Escherichia coli (E. coli) strain which is auxotrophic for thymidine [20]. The pFAR4-Luc plasmid encodes Luc expressed from the cytomegalovirus (CMV) promoter [20]. The pFAR4-IL-12 plasmid encodes the murine secreted IL-12 cytokine expressed from the CMV promoter. The IL-12 DNA sequence, synthesized by Geneart (Life Technologies SAS, Courtaboeuf, France), encodes the two P40 and P35 IL-12 subunits fused with an elastin linker (VPGVGVPGVG, pUN01-mIL12p40p35, Invivogen, Toulouse, France). Both resulting plasmids were entirely sequenced and purified using Endofree Plasmid purification kits (Macherey Nagel).

Cationic liposomes composed of DOPE, DMAPAP, YUK1350, or YUK1390 were obtained by the ethanolic injection method as described [26]. All the lipids were dissolved in ethanol (EtOH) at a concentration of 10 mmol/L and dropped in stirred water. After EtOH evaporation, liposomes were then stored at 4°C for further experiments. The formulation compositions tested are given in Table 1.

Typically, lipoplexes were obtained by dropwise addition of an equal volume of diluted pFAR4-Luc or pFAR4-IL-12 (in 10% glucose, 40 mmol/L NaCl) into a diluted solution of cationic liposomes to reach an amine/phosphate ratio = 6. The lipoplexes were left at room temperature for 30 min before being incubated with CT26 cells in DMEM + FBS for 24 h at 37°C using 1μg of plasmid per well. The transfection efficiency was evaluated by measuring IL-12 in the supernatant [DuoSet enzyme-linked immunosorbent assay (ELISA) mouse IL-12 p70]. Results were normalized according to the protein content quantifying with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Therefore, the amount of IL-12 was expressed as pg IL-12/μg of proteins.

A piezoelectric ultrasound transducer was used at 1.5 MHz with a spherical curvature of 20 mm and an active diameter of 25 mm (Imasonic, France) leading to a typical focal spot size of 1.2 mm × 1.2 mm × 6.2 mm. The transducer is coupled to the in vitro medium or the animals through a balloon filled with degassed water and closed by a latex membrane (Figure S2). Water was degassed prior to each experiment for 15–20 min. The transducer is also connected by a cable to a single-channel radiofrequency amplifier from Image Guided Therapy (France) with a programmable interface allowing to shoot custom sequences to measure in real-time the transmitted and reflected electrical powers (Figure S3).

Different HIFU sequences were tested in vitro on CT26 cells to choose the appropriate parameters for further studies. These parameters are detailed in Table 2. Briefly, on the day of experiment 5 × 10⁵ CT26 cells were loaded in a tube which was placed at the focus point of the transducer through a layer of ultrasound coupling gel with a reference tube near it. The temperature was also controlled during the experiment. The cell pellet was suspended in phosphate buffer saline (PBS) to perform an Annexin V viability test.

Cell viability after HIFU treatment was determined using the fluorescein isothyocyanate (FITC)-Annexin V Apoptosis Detection Kit II (BD Pharmingen). Cells were stained with FITC-Annexin V and propidium iodine (PI) following the manufacturer’s instructions. After that, 10,000 events were recorded per sample on Guava^® easyCyte™ Flow Cytometer. The CT26 cells were gated in the forward scatter (FSC)-side scatter (SSC) dot plot, then the viable/early apoptotic/dead cells were determined in the green and red channels using the Guavasoft 3.1.1 software (Figure S4).

CT26 tumor-bearing mice were used for the evaluation of transfection efficacy while CT26-Luc tumor-bearing mice were used for the anti-tumor effect of the HIFU. The CT26-Luc cell line was cultured in complete DMEM supplied with 0.4 mg/mL of Geneticin (G418 sulfate, Gibco Life Technologies at 37°C) in a 5% CO₂-humidified atmosphere. A subcutaneous CT26-Luc tumor was resected, placed into sterile phosphate buffer (Dulbecco’s phosphate buffer, Sigma), cut into fragments of 30 mm³, and inserted subcutaneously using a 12-gauge trocar (38 mm) into the mouse flank or in the back of the mouse. The tumor growth was monitored with a caliper for 8–11 days before further experiments. The volume was calculated according to the formula: Volume=Width×Width×Length2

In this approach, pFAR4-IL-12 was injected alone or complexed with lipids intratumorally 24 h before HIFU ablation to transfect viable cells and obtain IL-12 secretion before HIFU application. pFAR4-IL-12 (50 μg in 25 μL 5% glucose) was injected intratumorally. Six hours after injection of the plasmid, 25 μL of liposome DOPE:DMAPAP:YUK1350 (50:25:25 mol%) containing 0.16 μmol of cationic lipid YUK1350 in PBS were injected at the same site day 0 (D0). On the next day, the tumor was treated with HIFU 80T for 60 s [1 day after treatment (D+1)]. At D0 and D+1, mouse sera were collected to determine the IL-12 and IFN-γ production in the circulation. Tumor growth and body weight of the mice were monitored regularly until the end of the experiment or when the tumor reached more than 10 mm in one dimension. At the end point, mice were sacrificed and spleens were collected to analyze spleen lymphocytes activation. To do that, splenocytes were collected and 2 × 10⁵ cells were cocultured with 10⁴ CT26-Luc cells for 48 h.

Murine IFN-γ, TNF-α, and IL-12 levels were quantified in the supernatant of cell cultures or the mouse sera using the ELISA kit from the R&D system (DuoSet ELISA mouse IL-12 p70, TNF-α, or IFN-γ). The amount of each cytokine was calculated based on a calibration curve prepared at the same time with the samples.

Luc level in the tumor was monitored by optical imaging 20 min after injection intraperitoneally (i.p.) of 2 mg luciferin substrate. Bioluminescence was recorded for 10 min with the Optima camera (Biospace Lab). The quantification was performed over a region of interest (ROI) applied to the tumors using M3 vision software and the results were presented as the total bioluminescence signal in the chosen ROI normalized with the background signal in the same area [bioluminescence imaging (BLI)/bg].

Extracted mice spleen were washed with sterile PBS and sterile Roswell Park Memorial Institute (RPMI) medium, then cut into small pieces and grinded with a syringe piston rod. The mixture was passed through a 70 μm strainer and the red blood cells were then lysed using lysis buffer (2 mmol/L Tris, 0.84% NH₄Cl, pH 7.2) for 5 min. Splenocytes were collected after centrifugation at 1,600 rpm for 5 min at 20°C and resuspended in RPMI medium completed with 10% of FBS. At this step, about 2–3 × 10⁸ cells could be collected. The splenocytes were cultured in a 10 mL complete RMPI medium on 100 mm × 15 mm Petri dishes (P5606-400EA, Sigma Aldrich, France) at about 2 × 10⁶ cells/mL with 5% CO₂ at 37°C.

GraphPad Prism software was used to analyze the data and determine statistical significance between groups. Data are presented as mean ± standard error of the mean (SEM). One-way or two-way ANOVA test was used. Dunnett’s multiple comparisons test was also applied to compare each treatment to the control group. The meaning of the symbols used in the graphs was as follows: ns: not significant, P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

All plasmids used in this study were derivatives of the pFAR4 backbone which is an antibiotic-free gene vector. The pFAR4 plasmids present multiple advantages. They are devoid of dispensable and potentially inflammatory prokaryotic DNA sequences. They have a small size which is favorable for long-term transfection, and they allow the insertion of a multiple cloning site (MCS) for easy insertion of any eukaryotic expression cassette [27]. In this study, two pFAR4 derivatives were used. The pFAR4-Luc encoding the Luc gene allows monitoring the assessment of cell transfection efficacy in vitro or in mice using bioluminescence [28]. The pFAR4-IL-12 was designed to provide DCs activation and maturation in tumor-bearing mice. IL-12 (referred to as “P70”) is a heterodimeric cytokine composed of the P35 and P40 subunits. For this study, IL-12 was expressed as a P40:P35 fusion protein (Figure 1A).

The cationic lipids used are linear polyamine-based lipids (Figure 1B) which can interact very efficiently with DNA [13]. Cationic liposomes composed of DOPE and DMAPAP (50:50 mol%) were shown to be as efficient as lipofectamine for transfection in vitro [13]. As plasmid DNA does not efficiently transfect cells without a carrier, cationic liposomes made with DMAPAP were used to evaluate the validity of IL-12 expression in vitro from the pFAR4-IL-12. As the cationic lipid YUK1350 was previously shown to interact with TLR2 [12, 15], we selected it to be used as an adjuvant in vivo. To ensure that YUK1350 was able to properly deliver the plasmid into the cells, we compared the transfection efficiency of pFAR4-IL-12 delivered with the formulation DMAPAP/YUK1350 with the transfection mediated by our standard DMAPAP liposomes by quantifying the amount of secreted IL-12 protein. The transfection efficacy expressed as pg of IL-12 per μg intracellular proteins is presented in Figure 1C. The level of IL-12 (< 0.1 pg/mL) was similar for untreated cells or cells incubated with naked pFAR4-IL-12. An amount of 4.9 pg and 7.0 pg IL-12/μg intracellular proteins was measured when adherent or suspended CT26 cells, respectively, were transfected with DOPE/DMAPAP/pFAR4-IL-12 lipoplexes. A similar amount of IL-12 was produced from cells transfected with lipoplexes containing YUK1350 (25 mol%). A minor enhancement of IL-12 production was measured in suspended cells as regard to adherent cells with both DOPE:DMAPAP:pFAR4-IL-12 and DOPE/DMAPAP/YUK1350/pFAR4-IL-12 lipoplexes (8.0 pg/μg and 7.2 pg/μg cell protein, respectively) thanks to a probable enhanced interaction between the cells and the lipoplexes. As there was no statistical difference between the two formulations, we concluded that the incorporation of YUK1350 within the formulation did not alter the transfection efficiency and could be further used for in vivo experiments (Figure 1C).

To determine the optimal parameters for HIFU treatment (Figures S2–S3), CT26 cells were suspended in a medium containing either the free plasmid or the lipoplexes. After applying HIFU, the cells were transferred to a plate and cultured for 24 h to determine the proportion of viable, apoptotic, and dead cells. First, an amplitude of 30% of the maximum amplitude of our ultrasound amplifier (called “HIFU 30T”) corresponding to about 1.7 MPa peak negative pressure (PNP) as measured with a calibrated hydrophone in a water tank was applied during 10 s and showed a slight decreased in cell viability to 76%, compared to 80% in the control. By increasing the amplitude to 40% (“HIFU 40T” i.e. about 2.1 MPa PNP) the proportion of viable cells decreased to 66%. The HIFU conditions with 30% and 40% amplitude could be interesting to perform in vitro combination experiments on viable cells but would not result in tumor ablation. When HIFU amplitude was further raised to 60% or 80% (3.0 MPa and 3.9 MPa PNP in water, respectively) for 10 s, about 90% of the cells were killed due to hyperthermia. These two last conditions were then considered later for thermal ablation in vivo (Figure 2; Figure S4).

Prior to combining lipoplexes administration and HIFU application in vivo, we first assessed the transfection efficacy of our formulations in vivo, and we secondly, assessed the HIFU conditions to reach immunogenic death.

For in vivo transfection, mice-bearing CT26 tumors were injected intratumorally with either naked pFAR4-Luc (Figure 3A) or DMAPAP:DOPE:YUK1350:pFAR4-IL-12 complexes (Figure 3B) in the presence (right tumor) or absence (left tumor) of HIFU. The HIFU condition 80T for 30 s was chosen as the number of cells in the tumor was much higher than that in the in vitro experiment. According to a study from our group, a CT26 tumor fragment of 20–30 mm³ was evaluated to contain approximately 9 × 10⁵ tumor cells [29]. If we want to transpose the HIFU parameters in vivo, the acoustic absorption of skin and fat tissue leads to a high degree of attenuation and thus lowers the energy delivery, moreover, the center of the tumor should be reached to have an apoptotic effect on all tumor cells. Therefore, we chose the highest amplitude of the HIFU (80%) in a primary experiment. The expression of pFAR4-Luc was evidenced by the bioluminescence signal measured in the tumor 24 h and 72 h after intratumoral injection of free or complexed pFAR4-Luc (Figure 3A and B). In opposite to the in vitro transfection results, we found that the complexation of the pFAR4-Luc to cationic lipid led to undetectable transfection. The discrepancy between in vitro and in vivo results is not surprising and has previously been shown by Nomura et al. [30] using pCMV-Luc/3β-[N-(N’,N’-dimethylaminoethane) carbamoyl] cholesterol (DC-chol) lipoplexes or Coll et al. [31] who injected linear polyethyleneimine (L-PEI)/DNA complexes. Finally, from these results, we concluded that naked pFAR4-Luc was more efficient than lipoplexes for gene transfection in vivo in the tested conditions.

The application of HIFU at 80% of maximum amplitude (80T) for 30 s reduced by a factor of 2 the expression of Luc encoded by pFAR4-Luc (Figure 3C) meaning that viable cells were still present in the HIFU-treated tumor. The decreased transfection might be due to the fact that this HIFU condition which induces tumor cell necrosis in vitro (Figure 2), was not sufficient to induce complete necrosis in vivo. Two other thermal HIFU conditions at 80% of amplitude for 60 s and 120 s (named HIFU 80T 30 s/60 s/120 s) were tested to find the best one which could give a complete ablation of a 150–200 mm³ tumor (unshown). From this preliminary study, we could conclude that the thermal HIFU 80T could possibly be applied for tumor necrosis in vivo. Moreover, the HIFU 80T for 60 s or 120 s could be chosen for tumor ablation for a tumor volume of about 150 mm³.

The designed combination protocol resulted from the above study. Because DNA formulation by cationic lipids did not enhance transfection in vivo, we chose to inject separately lipids and DNA. Indeed, we found that pFAR4-IL-12 was able to induce IL-12 secretion (as shown through in vitro experiments) and transgene expression in vivo (as proven with the pFAR4-Luc experiments). This thus suggested that pFAR4-IL-12 administered naked in vivo should allow an improved production of Th1-derived cytokines, such as IFN-γ, and the activation of cytotoxic effector cells (Figure 4). As the cationic lipid YUK1350 was shown in a previous study to interact with TLR2 receptors, we assumed that its role as an adjuvant could be better obtained via a separate injection. A long application of 80T HIFU for 60 s once or repeated 3 times was chosen to insure in a first combination study thermal ablation of the tumor, resulting in antigen release and APC recruitment (Figure 4).

Naked pFAR4-IL-12 was injected intratumorally at a dose of 50 μg/mouse as published [32]. The cationic liposomes composed of DOPE/DMAPAP/YUK1350 (50/25/25 mol%) were used. Finally, the “HIFU 80T 60 s” protocol was applied for tumor ablation (Figure 5A). The treatment was well tolerated as referred the mouse weight monitored over 8 days (Figure 5B). The weight loss was below 10% in all groups after D0 of treatment and remained stable during the following days. In terms of tumor growth, the tumors in the control group grew from 94 ± 26 mm³ on the D0 to 347 ± 168 mm³ on D+8. Similarly, in pFAR4-IL-12 and lipid group, the tumors developed at the same rate as the control ones. After 8 days, they reached 411 ± 143 mm³ and 449 ± 188 mm³ in pFAR4-IL-12 and lipid group, respectively. In contrast, in the HIFU and the complete group, tumor growth was clearly inhibited. Volumes remained similar one week after the last HIFU session (Figure 5C). The effect of HIFU alone repeated three times seems really interesting to provide complete thermal ablation. In terms of survival, a significant difference was obtained between the groups with and without HIFU. Indeed, median survival of 9 versus > 20 for the HIFU groups was observed (Figure 5D).

As shown in the schedule diagram (Figure 5A), the level of immunocytokines IL-12 and IFN-γ were quantified 24 h after transfection (Figure 5E, F). We found that the IL-12 level in the serum of mice transfected with pFAR4-IL-12 was 3 times higher than in the control or the lipid group. These results point out that the pFAR4-IL-12 was successfully transfected and IL-12 was secreted into the systemic circulation. However, 48 h after transfection, the level of IL-12 in the serum decreased to the background level in all groups. Moreover, the HIFU conditions leading to a full tumor ablation resulted in the elimination of cells expressing IL-12. Therefore, a lower HIFU condition should be applied to maintain APC viability.

The effect of IL-12, cationic lipid, and HIFU on the production of IFN-γ and TNF-α was also studied. While the level of TNF-α was similar in all groups, indicating either no change or weak modifications under the assay detection limit at this time point, we found that 24 h after the transfection of pFAR4-IL-12, the level of IFN-γ was significantly higher in the serum (6.8 pg/mL and 7.2 pg/mL in pFAR4-IL-12, and complete combined treatment, respectively, versus 2.8 pg/mL in the control group). The concentration of IFN-γ did not increase with the cationic lipid or HIFU treatment. Moreover, the level of IFN-γ decreased rapidly to reach the baseline in the pFAR4-IL-12 group 24 h after HIFU application. Interestingly, the IFN-γ concentration remained stable in the complete group 24 h after HIFU. The production of IFN-γ in pFAR4-IL-12 group could be attributed to IL-12-induced enhancement of IFN-γ production by CD4 Th1 and CD8 cytotoxic T lymphocytes as well as NK cells. Meanwhile, 48 h after transfection, the higher IFN-γ level in the complete group as regard to pFAR4-IL-12, lipid, and HIFU group 48 h after transfection suggests that there might be a synergistic effect of these components in the production of IFN-γ.

To determine the impact of local tumor immunomodulation on the systemic immune response, we analyzed the ability of splenocytes to produce IFN-γ and TNF-α ex vivo. Splenocytes, taken at the end of the experiment, were co-cultured with CT26 cells to evaluate the specific response. Interestingly, the production of TNF-α from splenocytes in all treated groups was higher than that in the control group. In detail, the concentration of TNF-α secreted from splenocytes in the culture medium for pFAR4-IL-12 and lipid group was about 1.5 times higher than in the control group, and about 2 folds higher for the HIFU and complete treatment groups. The enhancement of TNF-α production suggests that the lymphocytes were activated in vivo, meaning that there was a probable immunomodulation effect of IL-12 and the cationic lipid YUK1350 even though, it was not strong enough to elicit an antitumor response. The fact that TNF-α also increased in the YUK1350 group suggests that an inflammatory response has been elicited since monocytes/macrophages are the main suppliers of this master inflammatory cytokine. The application of HIFU alone slightly increased the stimulation of T cells as regard to pFAR4-IL-12 and lipid group (Figure 5G). Even though an additional effect related to the combination of plasmid, lipid, and HIFU was not proven in that experiment, the activation of cytotoxic T cells suggests that the systemic immune response of these treatments would be interesting to evaluate.

In order to investigate in more detail, the synergistic effect of the HIFU combined with lipid and pFAR4-IL-12, we then chose, to apply HIFU with the same sequence (power and duration) but only once. We had indeed observed in the above study that a single HIFU application was insufficient to prevent tumor recurrence. We, therefore, chose to apply a single HIFU session with or without prior injection of both lipid and pFAR4-IL-12 one day before. We then observed the effect on the growth of the distant tumor implanted 2 days after the beginning of the treatment. The schedule is indicated in Figure 6A, the tolerance of the treatment in Figure 6B.

By reducing the amplitude of HIFU, we could see this time that HIFU was insufficient to remove the tumor and we could evidence an additional effect of the complete treatment. The HIFU significantly limited the primary tumor growth as regards the untreated control (P < 0.0001 at D+7) which is an important result for these fast-growing tumors, and the complete group showed similar efficacy. At D+10, CT26 tumors escape from this single HIFU application, which is not the case when injecting plasmid and lipid in addition to this single HIFU application. In this case, at D+17, the difference is still significant (Figure 6C).

To evaluate if an abscopal effect could be obtained thanks to this combined therapy, a secondary tumor was injected (~1–2 mm³) at D+2. Even if the bioluminescence signals of the Luc expressing tumors were quite dispersed, we could clearly evidence an absence of tumor growth for the secondary tumor injected for the complete treatment group (Figure 6D), showing a distal effect which was reflected by an improved survival rate for the complete treatment (Figure 6E). A significant increase in TNF-α is observed with HIFU and complete treatment (Figure 6).