Engineering Whole Mammalian Cells for Target‐Cell‐Specific Invasion/Fusion

Abstract Live mammalian cells are equipped with a synthetic cell invasion system that enables their target‐specific insertion into other live mammalian cells. By conjugating RhoA activator to a transmembrane protein that is segregated from cell–cell interface when specific cell contact occurs, polarization of RhoA activity is synthetically induced inside the cells in response to specific cell contact. This polarization is a sufficient condition for invader cells to selectively penetrate cells expressing a target antigen. Further, when an acid‐responsive fusogenic protein is expressed on invader cells, invader/receiver cell fusion occurs after invasion, and the invader's intracellular contents are released into the recipient's cytosol. It is shown that this system can be used for specific cell ablation. This synthetic‐biology‐inspired cell invasion/fusion system might open the door to using whole mammalian cells for cargo delivery purposes or for ablation of a specific cell type.


SEAP assay
The supernatant obtained from the transfected cells was incubated at 65°C for 30 min to inactivate endogenous alkaline phosphatase, and 80 µL of the heat-inactivated medium was placed in wells of a transparent 96-well plate. Then, 100 µL of 2 × SEAP buffer (20 mM homoarginine, 1 mM MgCl2, 21% (v/v) diethanolamine, pH 9.8) and 20 µL of p-nitrophenyl phosphate (pNPP) solution (120 mM) were added. The time course of absorbance at 405 nm was measured at 37 °C with an EnVision 2104 Multilabel Reader. SEAP production in U/L was quantified from the slope of the time-dependent increase S3 in absorbance by using an equation established with appropriate standards. All measurements were made in the region where the relationship was linear in order to avoid saturation effects.

Firefly luciferase viability assay
D-Luciferin (final 500 µM) was added to wells containing cells expressing firefly luciferase (in 96-well plates), and the plate was incubated for 10 min. The luminescence was measured with a Tecan M200 Infinite Pro plate reader. The ratio of luminescence intensity to the control was used as relative cell viability.

Poly-HEMA coating of the dishes
A solution of 6 mg/ml poly(2-hydroxyethyl methacrylate) (poly HEMA, Santa Cruz Biotechnology) in 95 % ethanol was prepared and used to coat the plates or slides (100 µL for a 24-well plate, 50 µL for an Ibidi µ-Slide 8 well), which were then dried under a cell culture hood overnight and used for experiments.

Observation of invasion and invasion/fusion
HEK-293 or HEK-293T cells were transfected with invasion or invasion/fusion components. These cells were mixed with HEK-HER2-iRFP and/or HEK-iRFP cells, and observed with a Nikon Confocal A1 or Leica SP8 microscope (usually equipped with a x63 oil lens).
Figure-specific protocols are as follows.      well. At 4 hrs after seeding, the cells were observed with a Leica SP8 microscope. The tile scan mode was used to obtain images, which is why the DIC image has a grid-like pattern.

Quantification of invasion using microscopy data.
HEK-293 cells were transfected as described in the legend of

Evaluation of invasion/fusion by reporter gene assay
Invader cells were transfected with invasion components, pMD2.G (PEF1-VSV-G-pA) and pDB24 (PhCMV-tTA-pA), while receiver cells were transfected with pMX9 (PTET-SEAP-pA), and the two were mixed. After 24 hours, SEAP activity in the cell culture supernatant was measured. Figure-specific protocols are as follows. Next, 500 µL of DMEM was added, and the cells were seeded on a 24-well plate. After 24 hr, SEAP activity in the cell-culture supernatant was assayed.    Then, 110 µL of invader cell suspension and 10 µL of receiver cell suspension were mixed (in Fig.S8, the following amounts were used; 60 µL each for 1:1 mixture, and 100 µL of invader cells and 20 µL of receiver cells for 5:1 mixture). The mixture was incubated at 37 °C in a 1.5 mL tube, 500 µL of DMEM was added, and the cells were seeded on a 24-well plate. After 1 day, the cells were trypsinized, spun down, and suspended in DMEM to give 4-fold-or 20-fold-diluted cell culture (compared to the 24-well plate culture after cell mixing). This cell suspension was seeded on a 96-well plate (110 µL/well), and cell viability was determined by firefly luminescence assay. (Four-fold dilution was used for measurement at 1 or 3 days after seeding, and 20-fold dilution was used for measurement at 5 days after seeding; luminescence intensity at 5 days after mixing was multiplied by 4 to compare it with the results at 1 and 3 days.)

Observation of the fate of fused cells.
See the following figure-specific protocols.    The invader cells and the receiver cells were mixed, and seeded on 24-well plates. SEAP activity in the supernatant was measured at 24 hours after seeding the cells. *9_26, G3: Anti-HER2 DARPin [4] . Error bars represent SEM of technical triplicates in a representative experiment. The red color is due to iRFP (from HEK-HER2-iRFP). The blue nucleus is from a receiver cell (from NlucBlue Live). The bright green nucleus is from an invader cell (from H2b-citrine). The error bar   3d)) (total cell number (invader + receiver) was fixed). Error bars represent SEM of three independent experiments (n=3). Statistical analysis was conducted at Day 5. * p<0.05, ** p<0.01 (against both mock and VSV-G only conditions), two-tailed Student's t-test. Cell killing efficiency appeared to increase as the proportion of invader cells increased. This was thought to be because receiver cells had a greater chance to encounter invader cells. Note that the invader cell population was transiently transfected and included cells that did not take up plasmids; this may account at least in part for the observation that some receiver cells were still growing even at the ratio of 11:1. Note S1: Note concerning the cell-suspension effect.
Entotic cell engulfment is induced by matrix detachment [5] , so the surface condition of cell culture dish was expected to be an important factor in our experiments. In our protocols, we first mixed the invader cells and receiver cells in a 1.5 mL tube. The mixture was incubated for 30 min, and then seeded on a plate or slide (either cell-adhesive condition (normal dish) or cell-suspension condition (dish coated with poly-HEMA). Since the invasion process was relatively fast, we could see cell-in-cell structures with a normal adherent dish. Cell fusion after invasion was also a relatively fast process, and there was no significant difference in the result of tTA transfer-based reporter gene assay between cell-adhesive and cell-suspension conditions (Fig.S7). So, we used a normal cell-adhesive dish for most of the experiments unless otherwise specified. There was a difference in the major cell fate after cell invasion (without VSV-G expression, Fig.S4a,b). In the cell-adhesive condition, escape of the engulfed cells from the receiver cells was more frequently observed, as compared with the suspension condition (in the suspension condition, cell death of the engulfed cell seemed to dominate). So, we show results using both the adhesion condition and the suspension condition in Fig.S4 and S7. Kojima et al [2] pRK21 Constitutive HER2-iRFP670 expression vector (PhCMV-HER2-iRFP-pAbGH).