Feasibility study for the use of gene electrotransfer and cell electrofusion as a single-step technique for the generation of activated cancer cell vaccines

In the search for safe induction of cellular and humoral cancer immunity dendritic cell-based therapies hold great potential. This approach is based on manipulation of dendritic cells to activate immune system against specific cancer antigens. For the development of an effective cell vaccine platform, gene transfer, and cell fusion have been used for modification of dendritic or tumor cells to express immune (co)stimulatory signals and to load dendritic cells with tumor antigens. Both, gene transfer and cell fusion can be achieved by single technique, a cell membrane electroporation. The cell membrane exposed to external electric field becomes temporarily permeable, enabling introduction of genetic material, and also fusogenic, enabling the fusion of cells in the close contact. We tested the feasibility of combining gene electrotransfer and electrofusion into a single-step technique. We evaluated the effects of electroporation buffer, pulse parameters, and cell membrane fluidity on the efficacy of the combined method. We determined the percentage of fused cells expressing green fluorescence protein GFP in a murine cell model of melanoma B16F1, an often-used cell line in pre-clinical studies. Our results suggest that gene electrotransfer and cell electrofusion can be applied in a single procedure. The percentage of viable hybrid cells expressing GFP depends on electric pulse parameters and the composition of the electroporation buffer. Cell membrane fluidity cannot be related to the efficiency of this technique. Further optimization of electric pulse parameters and buffers has to be accomplished before this technique can be used for preparation of effective dendritic cell-based vaccines.


Introduction
Established cancer therapies frequently result in the development of drug resistance preventing successful treatment.To overcome such problems, an alternative strategy was proposed aiming to induce a potent antitumor response (Borghaei et al., 2009;Guo et al., 2013).The progress in the field of immunology provides new tools for development of prophylactic and therapeutic anti-cancer vaccines.DNA and whole cell vaccines have been designed and different clinical trials are underway.Among them, dendritic cells (DC) based vaccines are particularly promising.The unmatched ability of DC to initiate, control, and regulate cellular and humoral immunity make them optimal candidates for new vaccination strategies (Fajardo-Moser et al., 2008;Laureano et al., 2022;Patente et al., 2019;Verheye et al., 2022) with the main focus on cancer therapy (Keenan and Jaffee, 2012;Laureano et al., 2022;Lin et al., 2022;Patente et al., 2019;Perez and De Palma, 2019;Santos and Butterfield, 2018;Srivatsan et al., 2014;Verheye et al., 2022).Although strategies of targeting different DC subtypes in vivo are on the way, ex vivo preparation of monocyte-derived DC-based vaccines is still the most common approach.The basic vaccination strategy is quite straightforward (Patente et al., 2019); monocytes are obtained from peripheral blood and differentiated into dendritic cells which are further maturated and activated by different stimuli.Then DC are loaded with selected or total tumor antigens.Finally, prepared cells are injected into the patient where they can induce a tumor-specific immune response.Loading of DC with antigen can be accomplished by different approaches; by transfection with DNA/RNA encoding tumor associated antigens (TAAs) (Keenan and Jaffee, 2012), by loading them with tumor lysates, apoptotic bodies, exosomes or by cell fusion of DCs and tumor cells (de Gruijl et al., 2008).The later approach involving DC-tumor cell hybrids is an attractive alternative because it couples the antigen-processing and immune-stimulatory potential of DCs with the whole antigenic spectrum of the tumor cell (Barbuto et al., 2004;Koido, 2016;Rosenblatt et al., 2011).However, the promising DC vaccines approach have important limitations which may be attributed to several causes, such as tumor burden resulting in immune suppression (Saxena and Bhardwaj, 2018) and inadequate maturation status of DC used in vaccine preparation (Raaijmakers and Ansems, 2018).The effective vaccine has to be prepared from the mature DC.Mature immunogenic DC and their proinflammatory cytokines, so-called third signal (Lee, 2011) , are needed for T cell activation.The issues of DC maturation could be overcome by genetic manipulation.Namely, several studies have reported that the efficiency of DC-tumor cell fusion vaccines was higher if the vaccine was combined with exogenous administration of IL-12 (Hayashi et al., 2002;Vasir et al., 2008).Especially interesting is the approach where the B16 tumor model cell line (Tan et al., 2013), was genetically modified to secrete IL-12 (Shi et al., 2005;Suzuki et al., 2005;Tan et al., 2013) or to stimulate the IL-12 pathway (Xia et al., 2004).Based on this data some authors proposed that DC can be first genetically modified to promote DC maturation and then fused with tumor cells (Ogawa et al., 2004).The genetic manipulation resulting in overexpression of maturation stimuli in combination with antigen loading of DC have been shown as an effective alternative protocol for the production of DC vaccines (Saxena et al., 2018).Methods used for cell fusion and gene transfer are diverse; one of the possibilities for both, genetic manipulation and cell fusion is electroporation.Genetic manipulation is based on gene electrotransfer of plasmid DNA or different RNA molecules while antigen loading can be obtained by cell electrofusion of DC and tumor cells.
Electroporation is a technique that uses high-voltage electric pulses to temporarily increase membrane permeability enabling the exchange of the molecules between the cell interior and its surroundings (Canatella et al., 2001;Kotnik et al., 2019).Additionally, the permeable cell membrane has been proven to be fusogenic enabling the fusion of neighboring cells in the close contact (Zimmermann, 1982).For effective application, one should choose the electric pulses to enable cell membrane permeabilization and at the same time to preserve cell viability.The effects of electric pulse parameters involved in the process have been studied extensively (Canatella et al., 2001;Rols and Teissié, 1998;Zimmermann, 1982) while biophysical characteristics of the treated cells, did not receive enough attention.Molecular dynamics studies reveal that electroporation affects the fluid-disordered membrane phases in local membrane regions of the lipid bilayer (Reigada, 2014) while in more complex biological membranes the role of cell membrane fluidity in gene electrotransfer or cell electrofusion was not established unambiguously (Golzio et al., 2001;Kanduser et al., 2008Kanduser et al., , 2006;;Rols et al., 1990).Control of the membrane fluidity is important for cell homeostasis (Levental et al., 2020) while the modulation of membrane fluidity enables cancer progression (Koundouros and Poulogiannis, 2020).In context with the latter we have indeed observed that higher electrofusion yield can be obtained with cancer cells compared to nonmalignant ones (Ušaj et al., 2010).Electroporation is a cell membrane-based phenomenon, however, cell electrofusion and gene electrotransfer are complex processes both involving several steps; some of them are cell membrane related, while others depend on cell signaling involved in post-pulse processes related to cell membrane resealing.Cell membrane-related processes in gene electrotransfer are: electropermeabilization, the interaction of the DNA with the cell membrane, complex formation, and translocation of the DNA across the membrane.The transport to the nucleus and gene expression are membrane-independent (Golzio et al., 2002(Golzio et al., , 2002;;Pavlin and Kanduser, 2015) and relay on post-pulse cell responses.The main phases in cell electrofusion are cell-cell contact, membrane permeabilization, fusion pore formation, membrane merging, cytoplasm mixing, and finally hybrid cell formation (Kozlovsky and Kozlov, 2002).One of the crucial parameters for efficient electrotransfer/electrofusion is the induced transmembrane voltage (ITV) which was shown to present the limiting condition for the efficiency of both applications (Kotnik et al., 2019;Marjanovič et al., 2010;Pavlin et al., 2005;Usaj et al., 2010).The present study aims to combine gene electrotransfer and cell electrofusion in a single-step procedure.We tested different electroporation buffers and pulse parameters to obtain a protocol that enables electrofusion and gene electrotransfer in one step.Additionally, we determined the effect of electroporation buffers on cell membrane fluidity as membrane fluidity was related to the efficacy of gene delivery and cell fusion.We evaluated the effect of cell membrane fluidity on the generation of hybrids expressing the delivered reporter gene.The application of one-step procedure for cell electrofusion and gene transfer has the potential application in DC-based cancer vaccines where fused cells could be simultaneously genetically modified to additionally secrete factors regulating DC maturation and/or to secrete other proinflammatory cytokines.As a proof of concept, we combined conditions that enable transient cell membrane permeabilization required for gene electrotransfer and cell electrofusion of B16-F1 cells that have been used as a simple cell model.The obtained results indicate the feasibility of the proposed methodology.They also show that further studies are needed to optimize the process for possible applications in the platforms used for effective dendritic cell vaccines.

Sample preparation
Mouse melanoma cells (B16-F1) were cultured in Dulbecco's minimal essential medium (DMEM), supplemented with 10% fetal bovine serum (FBS), L-glutamine, and antibiotics crystacillin, gentamicin and maintained in a humidified atmosphere at 37 ºC and 5 % CO 2 .Cells were grown in a 25 cm 2 culture flask to obtain 70-80 % confluence.For dual color microscopy cells were incubated with blue 7 µM CMAC in isotonic KPB and red CMRA in Krebs-Hepes buffer for 45 minutes at 37°C in two separate 25 cm 2 culture flasks.The staining solution was removed and cells were washed with culture media and maintained for an hour at 37°C in the DMEM medium.In other experiments, we used unstained cells.In both cases, we prepared homogenous cell suspension by 0.25 % trypsin/EDTA solution.Cells were centrifuged for 5 minutes at 1000 rpm (180 x g) and resuspended in iso-tonic potassium phosphate buffer -KPB (10 mM K 2 HPO 4 /KH 2 PO 4 , 1 mM MgCl 2 ) with 250 mM sucrose and osmolarity 262 mOsmol/kg.For dual-color electrofusion detection, we mixed blue and red cells in proportion 1:1, while for electrofusion detection via nuclear staining we have just resupended the cells.Close cell-cell contacts were established by a modified adherence method (Ušaj and Kandušer, 2015).A monolayer of spherical cells was obtained by placing 40 μl drop of cells in concentration 2×10 6 cells/ml in each well of the 24-well plate (TPP, Switzerland).Cells were then incubated in 5 % CO 2 at 37°C for twenty minutes to allow them to slightly attach to the surface of the culture dish, while maintaining a round shape.Before electroporation, cells were washed with iso-tonic buffer and then 350 μl of iso or hypotonic electroporation buffer containing 40 µg/ml of plasmid coding for green fluorescent protein pEGFP-N1 was added.Plasmid was purified with Qiagen HiSpeed Plasmid Mega kit (Qiagen).Expression of encoded reporter gene (GFP) was used to analyze efficiency of gene electrotransfer.Optimal parameters for gene electrotransfer and cell electrofusion were determined in our previous studies (Kandušer and Ušaj, 2014;Marjanovič et al., 2010;Pavlin and Kanduser, 2015;Ušaj et al., 2010;Usaj and Kanduser, 2012).Timing and changes in the cell radius due to the hypotonic cell swelling were recorded previously (Ušaj et al., 2009).For each treatment we calculated induced transmembrane potential, taking into account different cell sizes of cells exposed to isotonic or hypotonic buffer.The cells changed their size from radius r = 8.1 ± 1.1 µm in isotonic buffer to r = 9.3 ± 1.8 µm in hypotonic buffer (Ušaj et al., 2010).The induced transmembrane voltage (ITV) on a spherical cell can be obtained from Schwann equation (Pavlin et al., 2005;Zimmermann, 1982).
where r is the radius of the cell, E is strength of the external electric field and  is the angle between the direction of the external electric field and the normal from the center of the cell to the point of interest on the cell surface.
From this the maximal induced transmembrane voltage ITVmax can be calculated as described earlier (Usaj and Kanduser, 2012)

Electroporation and cell electrofusion
Cells were electroporated in different iso or hypotonic buffers (Table 1).In the protocols where we used hypotonic treatment cells were maintained in the hypotonic buffer for two minutes to reach their maximal volume, and then electric pulses were delivered.Electric pulse parameters were applied as a train of 8×100 μs electric pulses or 4×200 μs at repetition frequency 1 Hz and amplitude 1.4 kV/cm and 1.6 kV/cm.Besides we tested the combination of high voltage (HV) and low voltage (LV) electric pulses HV+LV with the HV 4×200 µs, 1.4 kV/cm, 1Hz and LV 1×100 ms, 0.014 kV/cm.The composition, and osmolarity of electroporation buffers used for gene electrotransfer and cell fusion and electric pulse parameters are presented in Table 1.As electroporation buffers, we used isotonic KPB (iso KPB), isotonic KPB with Ca 2+ ions (0.1 mM calcium acetate), or hypotonic KPB containing 75 mM sucrose with osmolarity 93 mOsmol/kg with conductivity 1.67 mS/cm and pH 7.2.Additionally, we tested, the commercially available Eppendorf® hypo-osmolar buffer (Epphypo; cat.number 36205-60) and the iso-osmolar buffer (Eppiso; cat.number 36205-62).

HV: 4×200µs
For electroporation we used two parallel wire electrodes (Pt/Ir = 90/10) with five mm gap.No pulses were applied in the control treatment.After pulse delivery, the cells were left undisturbed for ten minutes for cell fusion to take place.Fusion yield was determined 30 minutes and 24 hours after electroporation by dual-color fluorescence microscopy or by determination of polynucleated cells.Complete culture media was added 10 minutes after electroporation and images were acquired by inverted fluorescence microscope Zeiss Axiovert 200 (Zeiss, Germany) at 20× objective magnification.Brightfield and fluorescence images of blue CMAC (excitation/emission 353nm⁄466nm, Chroma, USA), red CMRA (excitation/emission 548 nm/576 nm, Chroma, USA) and green GFP positive cells (excitation/emission 488 nm/ 507 nm, Chroma, USA) were acquired.Alternatively, cell nuclei were stained with nucleic acid stain Hoechst 33342 (excitation/emission 361 nm/497 nm) for 15 minutes at 37°C and washed with cell culture media.

Determination of electrofusion yield and efficacy of gene electrotransfer
The fusion yield and gene transfer efficacy were determined in multi-channel images.The channels were brightfield, with fluorescence channels of blue CMAC, red CMRA, and green (GFP).Multiple images (bright field, blue, red, and green fluorescence) were acquired from five randomly chosen fields for each sample using cooled CCD video camera VisiCam 1280 (Visitron, Germany) and PC software MetaMorph 5.0.(Molecular Devices, USA).Images were merged into multichannel image in the image processing software ImageJ (NIH Image, USA).Alternatively, bright field and Hoechst fluorescence images were used to determine multinucleated cells resulting from cell fusion.The cells were manually counted and the electrofusion yield was calculated as a percentage of dual color cells (i.e. cells with red and blue fluorescence) (Ušaj and Kandušer, 2015) or multinucleated cells (i.e.presence of two or more nuclei in an individual cell) (Trontelj et al., 2008;Usaj et al., 2013).Hoechst is a membrane-permeable dye suitable for live cell imaging.With the combination of brightfield image and Hoechst fluorescence, we can readily determine the cells with more than one nucleus per cytoplasm.Since a certain amount of polynucleated cells is always present in the unsynchronized cell culture we also determined the number of the polynucleated cells in the control sample and subtracted them from the number of polynucleated cells in the treated sample (equation 3).
where Npc is the number of polynucleated cells in the control, Npt is the number of polynucleated cells in the treated sample and Nall cells represent the number of all cells per given sample.Besides cell fusion yield we determined the efficacy of gene electrotransfer as the percentage of GFP-positive cells.Uniquely for this study, the percentage of transfected fused cells expressing GFP (GFP-hybrids) was determined by detecting green staining in polynucleated cells (equation 4).
where NGFP-hybrids represent the number of GFP-positive fused cells and Nall cells represent the number of all cells per given sample.

Measurements of cell membrane fluidity
Membrane fluidity was determined in two cell lines, B16F1 and CHO-K1, used in our previous studies of gene electrotransfer and cell electrofusion.Cells were seeded at appropriate cell density.On the day of the experiment, we prepared aliquots of cells in suspension and centrifuged them for 5 min at 4°C at 1000 rpm (180 x g).We kept pellets at 4⁰ C. Cell pellet was resuspended to obtain a concentration of 10 5 cells/ml, which was stained for 5 min at 37°C with 1 µM TMA-DPH in iso-tonic KPB buffer.Stained cells were then centrifuged again and cell pellets were resuspended in iso-or hypo-tonic KPB buffer.Two minutes later cell membrane anisotropy was determined as described in (Rols et al., 1990) by polarized fluorescence intensity measurements using FP-6300 (Jasco, USA) equipped with a manual UV/VIS polarizer (FDP-223).The fluorescence anisotropy r was calculated as: where g is the correction factor of the instrument, lvv is the fluorescence emission intensity with the polarizer parallel, IvH is the fluorescence emission intensity with the polarizer perpendicular to the direction of the polarized excitation light.The fluorescence anisotropy r is inversely proportional to cell membrane fluidity.

Chemicals and Reagents
The chemicals were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Germany).Antibiotics (crystacillin and gentamicin) from Pliva (Pliva d.o.o, Croatia).Cell trackers blue CMAC and red CMRA as well as TMA-DPH were from Molecular probes (Invitrogen, USA) and pEGFP from Clontech.Hoechst 33341 dye was from Sigma (Sigma-Aldrich).

Results
In Table 2, we present the efficiencies of cell electrofusion, gene electrotransfer, and feasibility of generation of fused cells expressing GFP (GFP-hybrids) in single-step procedure.Experiments were performed in commercially available iso and hypo-osmolar Eppendorf buffer (Epp iso, Epp hypo).We used two sets of electroporation parameters, 4×200 µs electric pulses (previously determined to be optimal for electro gene transfer in this cell line) and 8×100 µs electric pulses previously used for electrofusion of B16-F1 cells.The cells exposed to hypotonic buffer swell and increase their size, therefore electric pulse amplitudes have to be adjusted to obtain the comparable induced transmembrane potential for all treatments.The percentage of fused cells increases in hypotonic conditions.The percentage of gene electrotransfer (% GFP positive cells) and the electrofusion yield (% ECF) was higher in the hypotonic buffer and lower in the isotonic buffer when we compared commercially available iso and hypo-osmolar Eppendorf buffer.In these buffers, we did not obtain any GFP-hybrids.For further experiments, we used our own KPB buffer alone or with the addition of calcium ions for improved gene electrotransfer (Haberl et al., 2013(Haberl et al., , 2010)).In Figure 1, we present the percentages of cell electrofusion (hybrids), gene electrotransfer (transfection) and GFP-hybrids in different KPB buffers: isotonic KPB (iso KPB), hypotonic KPB (hypo KPB) and isotonic KPB with addition of [Ca 2+ ] =0.1 mM (iso KPB + Ca).As expected the highest fusion yield was obtained in hypotonic buffer.The percentage of cells expressing GFP was slightly higher in isotonic buffer containing Ca 2++ ions.The percentage of GFP-hybrids was higher in isotonic buffers (2%) compared to hypotonic buffers (0.4%).
Figure 1: Effect of different KPB electroporation media on the percentage of cell electrofusionfusion, GFP gene electrotransfer (transfection) and electrofused cells expressing GFP (GFP-hybrids) assessed 24h after electric pulses.Electric pulse parameters were 4×200 µs delivered at 1 Hz.Electric field amplitude [E] in isotonic buffer was 1.4 kV/cm and 1.2 kV/cm in hypotonic buffer preserving ITVmax at the same value of 1.7 V. Cell fusion was estimated by the presence of multinucleated cells detected by Hoechst staining 10 min after application of electric pulses.Bars are means of two independent experiments ± standard error.Differences among treatments were analyzed by 2-way ANOVA and compared by Bonferroni's multiple comparison test.The differences among the treatments were not statistically significant.
In Figure 2 we present the time course of cell electrofusion in hypotonic and isotonic buffer with respect to control treatment (first row -no electric pulse and hypotonic treatment).Panels on the left represent cells 30 minutes after while the panel on the right shows results 24h after the electric pulse application.The electric pulse amplitude was adapted to electroporation media (i.e.isotonic in the middle or hypotonic at the bottom of the figure) to preserve constant induced transmembrane voltage.
Figure 2: Images of B16F1 cells after electrofusion in isotonic (iso KPB) and hypotonic (hypo KPB) buffer were recorded at 30 min (separate channels and merged images) and 24h (merged image) after electric pulse application (4×200 µs delivered at 1 Hz).Electric pulse amplitudes were 1.2 kV/cm for hypotonic, 1.4 kV/cm for isotonic buffer, and 0 kV/cm for control.Cells were stained with red (CMRA) and blue (CMAC) cell tracker.Successfully fused cells are violet in color (the merge contains red and blue cell tracer in their cytoplasm) and some of them are marked with white arrows in merged images.
To increase the percentage of GFP-hybrid cells we have tested the combination of high voltage (HV) and low voltage (LV) pulses in iso KPB, because the percentage of GFP-hybrids was higher in isotonic buffers.In Figure 3 we present the percentages of cell electrofusion (fusion), GFP gene electrotransfer (transfection), and GFPhybrid cells for different electric pulse parameters.The highest percentage of GFP-hybrid cells was obtained when we used the combination of high voltage and low voltage pulses reaching 9 % of total cells used in experiment (Fig. 3 A).In tree channel micrographs we present treated cells.The combination of high voltage and low voltage pulses resulted in large polynucleated cells with dim fluorescence of GFP in GFP-hybrids (Fig. 3B).Polynucleated cells are the result of random unspecific cell pairing which is a current drawback of existing cell fusion methods.Dim fluorescence of GFP in GFP hybrids is most likely the consequence of large cytoplasmic volume and high number of nuclei in a single cytoplasm which are interrupting normal cell function required for proper expression of GFP.The situation might be specific for artificialy obtained polynucleated cells as such situation was not observed in naturally polynucleated muscle cells (Velayuthan et al., 2023).
In supplementary Figure S1 we present also the fraction of fused cells expressing GFP where we noted that 24% of all hybrids have expressed GFP when we used a combination of high voltage and low voltage pulses.
Figure 3: The effect of pulse parameters on the percentage of cell electrofusion (hybrids), gene expression (GFP), and fused cells expressing GFP (GFP-hybrids) in isotonic KPB buffer.We compared 4×200 µs and 8×100 µs (HV) pulses and a combination of HV (4×200 µs) and one LV55 pulse (100 ms) (HV + LV).Electric field amplitude E for HV was 1.4 kV/cm and 0.11 kV/cm for LV55.In the panel (A), we present the percentages of cell electrofusion, gene electrotransfer (GFP) and GFP-hybrids.Bars are means of 3 independent experiments ± standard deviation.The statistical differences among electric pulse treatments were tested by one-way ANOVA and Tukey's multiple comparisons test.The significantly higher number of GFP hybrids is obtained (P= 0.029) when we used HV + LV (4×200 µs + 1×100 ms) pulses compared with HV (4×200 µs) pulses alone, while all the other differences are statistically insignificant.In the panel (B) we present tree channel micrographs of the treated cells (brightfield, cell nuclei stained with Hoechst-blue, cells expressing GFP-green and merged images.Cell electrofusion and GFP expression were detected 24h after electric pulse application.Images were acquired at 20× objective magnification.Multinucleated cells expressing GFP (GFP-hybrids) are indicated with arrows.
To determine the role of biophysical properties of the cell membrane on the proposed one-step procedure we investigated the effect of cell membrane fluidity on gene electrotransfer and cell electrofusion.We performed polarization studies that measured the steady-state anisotropy (r) using fluorescent probe 1,6-diphenyl-1,3,5hexatriene; TMA-DPH that mimics the molecular movements in the membranes of the analyzed sample.Since r refers to the rigidity and fluidity refers to the viscosity of lipid layers, the fluidity index is the inverse value of r-anisotropy (i.e., 1/r).
To analyze the effect of cell membrane fluidity on gene electrotransfer, cell electrofusion and on one-step procedure we measured r-anisotropy of the cell line B16-F1 and compared it with CHO-K1 cell line that differs from B16F1 in gene electrotransfer and cell electrofusion efficiency.In Figure 4 we present the results of ranisotropy for the two cell lines and the results for gene electrotransfer (Marjanovič et al., 2010) and cell electrofusion (Usaj and Kanduser, 2012) obtained in our previous studies.Summarized results (Fig. 4) show no relation between the membrane fluidity and electrogene transfer or cell electrofusion.Even if a comparison in the percentage of cells expressing GFP between CHO (23 %) and B16F1 (58%) could be potentially related to the observed differences in r-anisotropy (0.171 vs 0.182; P=0.03, CHO and B16F1, respectively) it is interesting to note that we did not obtain any cell electrofusion of CHO cells compared to the 16% fused B16F1 cells already in isotonic conditions.Hypotonic buffer significantly increased electrofusion of both cell lines, but it does not affect r-anisotropy (supplementary Table 1).Interestingly, however, when we related r-anisotropy with the efficiency of generation of GFP-hybrids for B16F1 in hypotonic and isotonic buffers we observed a 2.7 % of GFP hybrids of GFP hybrids in isotonic buffer and only 0.4 % in hypotonic buffer (Fig. 1).
Figure 4: Comparison of r-anisotropy in two cell lines characterized in our previous work that significantly differ in their GFP gene electrotransfer (transfection) (Marjanovič et al., 2010) and cell electrofusion (fusion) (Usaj and Kanduser, 2012).In the panel (A) we present membrane r-anisotropy -fluidity is the inverse value of r (i.e., 1/ranisotropy) for both cell lines.In the panel (B) we present gene electrotransfer and cell electrofusion at ITVmax 1.7 V for the same cell lines in KPB isotonic buffer.
All the r-anisotropy measurements were performed at room temperature (22⁰C), while as additional control ranisotropy samples were also measured in isotonic buffer at 4⁰C (supplementary Table 1) where cell membrane fluidity was decreased as demonstrated also in our previous study (Kanduser et al., 2008).The measurements effectively validated the method used in this study for membrane fluidity measurements.

Discussion
The progress in immunology in recent years has led to breakthroughs in the development of prophylactic and therapeutic anti-cancer vaccines (Borghaei et al., 2009;Guo et al., 2013).For the development of effective vaccine platforms, different approaches have been used (Fajardo-Moser et al., 2008;Keenan and Jaffee, 2012;Laureano et al., 2022).The gene electrotransfer and cell electrofusion are versatile techniques that enable genetic manipulation and cell fusion (Lin et al., 2022;Patente et al., 2019;Perez and De Palma, 2019;Saxena et al., 2018) holding great potential for different vaccine platforms.We developed a one-step electroporation protocol enabling simultaneous cell fusion and delivery of genetic material.To understand the effect of membrane biophysical parameters on the protocol we also evaluated the role of cell membrane fluidity.
The feasibility of the single-step technique was evaluated in B16-F1 melanoma cancer cell line which was characterized in our numerous past studies (Kandušer et al., 2009;Marjanovič et al., 2010;Pavlin et al., 2010;Ušaj et al., 2010;Usaj and Kanduser, 2012) and it is frequently used in preclinical studies of DC-based cancer vaccines (Edele et al., 2014;Gordy et al., 2016;Mac Keon et al., 2015;Wang et al., 1998) .We successfully developed a protocol that combines electrofusion and gene electrotransfer in one step.We tested different electroporation media and electric pulse parameters previously optimized separately for electropermabilization, gene electrotransfer (Marjanovič et al., 2010;Kandušer et al., 2009;Pavlin et al., 2010;Haberl et al., 2013), and cell electrofusion (Trontelj et al., 2008;Usaj et al., 2010;Usaj and Kanduser, 2012) and evaluated the effectiveness of selected parameters for the one-step protocol.The efficiency of the protocol was evaluated as the yield of GFP transfected hybrids (GFP-hybrids), which depended on the electroporation buffer (Table 1, Fig. 1, Fig. 2) and on electric pulse parameters (Fig. 3).Our results demonstrate that the hypotonic treatment which is the best choice for cell electrofusion (Fig. 2) was not the best option for the one-step procedure (Fig 1), therefore we tested additional electric pulse parameters using isotonic buffer.The highest percentage of GFP-hybrids (9 %) was obtained with the combination of high voltage (HV) and low voltage (LV) electric pulses (Fig. 3).This is in agreement with our previous work where we have already shown that the combination of HV and LV pulses increased the efficiency of gene electrotransfer in CHO cells at suboptimal DNA concentrations (Kanduser et al., 2009;Pavlin et al., 2010).The results indicate that for the generation of transfected hybrids, it is also important to provide electrophoretic low voltage pulses to enable efficient contact between DNA and the cell membrane which is crucial for efficient gene electrotransfer (Golzio et al., 2002;Haberl et al., 2013;Kandušer et al., 2009;Pavlin et al., 2010).In the present study, we used optimal plasmid concentration for gene electrotransfer, however dense cell suspension and tight cell contacts required for cell fusion might hinder plasmid availability at the cell membrane level.The highest yield of GFP-hybrids (Fig. 3, Fig. S1) can be therefore explained with the improved DNA-membrane contact and higher degree of GFP expression (Fig. S1) like in conditions of suboptimal plasmid concentration.Interestingly, we observed that the efficiency of electrofusion increased on average by 15% when we used fewer pulses with longer duration (4×200 µs compared to 8×100 µs), with a total duration of pulses being the same (i.e., 800µs), however, more hybrids expressed GFP when we used 8×100 µs pulse application.Even if the difference was not statistically significant (P=0,136) the increase in electrofusion yield may pave a new way into optimization of cell electrofusion itself, especially for low fusogenic cell lines (Salomskaitė-Davalgienė et al., 2009).We would like to stress that we had to find a compromise to combine two protocols in one; we have to omit fetal bovine serum that is typically added after electroporation in gene electrotransfer protocols (Golzio et al., 2002;Kandušer et al., 2009;Pavlin et al., 2010) because such intervention has negative impact for electrofusion where cells have to be left undisturbed.The selected pulsing protocols (Fig .1, Fig. 3) otherwise enabled efficient electrotransfection and high fusion yield as shown in our previous studies (Kandušer et al., 2009;Marjanovič et al., 2010;Pavlin et al., 2010;Pavlin and Kanduser, 2015;Usaj and Kanduser, 2012).The optimal experimental conditions for these two separate processes are not trivial to achieve while combining them presents additional challenges.In the majority of common cell fusion protocols (bulk cell fusion), we face the problem of unspecific cell pairing and formation of large polynucleated hybrids.
We show here that such hybrids have a limited capacity to express GFP (Fig. 3 B), however, this challenge can be solved by specific cell fusion in microfluidic devices as discussed later.Our results indicate that one-step technique is possible with further optimization of electric pulse parameters, electroporation buffer, specific cell pairing and understanding of other experimental and biophysical parameters affecting both processes.
Besides external factors, we investigated also an intrinsic biophysical characteristic of the cell membrane which have not been clarified yet and might affect the one-step protocol for the generation of GFP-hybrids.Cell membrane fluidity could be involved in complex or contact formation required for gene electrotransfer or electrofusion respectively (Golzio et al., 2002;Kandušer and Ušaj, 2014;Kozlovsky and Kozlov, 2002;Pavlin and Kanduser, 2015).Besides, electroporation and cell fusion depend on the lipid composition of the cell membrane (Gabriel and Teissie, 1994;Kanduser et al., 2019;Kotnik et al., 2019;Kozlovsky and Kozlov, 2002;Maccarrone et al., 1995;Markelc et al., 2012).We observed higher electrofusion yield in cancer cell lines (Usaj and Kanduser, 2012), which could be related to changes in membrane fluidity characteristic for malignant transformation (Koundouros and Poulogiannis, 2020).Besides, some authors reported that membrane fluidity was implicated in cell fusion (Grobner et al., 1996;2014;Velizarov et al., 1998) and molecular dynamics simulations have shown that hydrophobic pores are formed mainly in small disordered membrane domains in the lipid bilayer (Reigada, 2014;Shigematsu et al., 2014), while on the other hand the experimental evidence on the role of cell membrane fluidity in gene electrotransfer is not conclusive yet (Golzio et al., 2001;Kanduser et al., 2008Kanduser et al., , 2006;;Rols et al., 1990).To clarify the role of cell membrane fluidity we summarized the results in conditions that result in different gene electrotransfer (Marjanovič et al., 2010) and electrofusion yields (Usaj and Kanduser, 2012) and related them to membrane fluidity (Fig. 4, Supplementary Table S1).Our results demonstrate that membrane fluidity (Fig. 4 A) could not be related to the differences in gene electrotransfer or electrofusion obtained with different cell lines or electroporation protocols (Fig. 3; Fig. 4 B).This observation is in line with previous results (Golzio et al., 2002;Kanduser et al., 2008Kanduser et al., , 2006) ) and demonstrates that cell membrane fluidity as defined in simplified lipid models is not relevant neither for gene electrotransfer nor for electrofusion.This observation is further supported by our previous results (Pavlin et al., 2007;Pavlin and Miklavčič, 2008) demonstrating that the stability of long-lived pores and their time-constant of resealing do not behave as pores in purely lipid systems that have very short-resealing time.Therefore, new models (Kotnik et al., 2019;Pavlin and Miklavčič, 2008;Sözer et al., 2018) that take into account membrane complexity are still needed to match experimental observations.Taken together, the one-step protocol presented here is a proof of a concept; we can combine electrofusion and gene electrotransfer in a single step.We show that the optimization of gene electrotransfer and cell electrofusion is pre-detrimental for the preparation of therapeutic plasmid DNA or RNA based dendritic cancer cell vaccines.Our methodology could be upgraded for platforms of genetic (Chattergoon et al., 1997;Flingai et al., 2013;Lin et al., 2022;Perez and De Palma, 2019) and cell (Keenan and Jaffee, 2012;Laureano et al., 2022;Palena and Schlom, 2010;Patente et al., 2019;Santos and Butterfield, 2018;Verheye et al., 2022) vaccines to produce genetically modified hybrids expressing genes encoding adjuvants or cytokines required to improve vaccine efficacy.Technological advances in microfluidics have been addressing the current limitation of random cell pairing present in bulk cell fusion protocols and will enable the formation of specific binucleated hybrids (He et al., 2019;Tang et al., 2022) resulting in viable cells efficiently expressing the transferred genes.From this prospective our one-step protocol presents an advantage as it can be implemented in microfluidic devices as shown schematically in Figure 5.In conclusion, we demonstrated that gene electrotransfer and cell electrofusion can be applied as a one-step protocol.The percentage of viable fused cells expressing GFP mainly depends on electric pulse parameters and electroporation buffers.Selected electroporation buffers that improve gene electrotransfer, cell electrofusion and/or formation of GFP-hybrids do not affect cell membrane fluidity; therefore, in this aspect cell membrane fluidity cannot be related to the efficiency of this technique.Additionally, we provide further evidence that more complex molecular and biophysical cell membrane models are needed considering the high concentration of membrane proteins and the interactions of the cell membrane with the cell cortex and cytoskeleton to bridge the gap between current models and experimental results.The effect of pulse parameters on a fraction of obtained hybrids that were efficiently transfected with a plasmid encoding GFP in isotonic KPB buffer.

Figure 5 :
Figure 5: The schematic of the implementation of the single-step protocol (demonstrated here in our study) with proposed microfluidic design for the one-step electrofusion-electrotransfection device.
Figure S1:The effect of pulse parameters on a fraction of obtained hybrids that were efficiently transfected with a plasmid encoding GFP in isotonic KPB buffer.The fraction was calculated as   ℎ =

Table 1 :
Different electroporation buffers and electric pulse parameters used in gene electrotransfer/ electrofusion.
*at selected pulse amplitude ITVmax was always 1.7 V