Invader cells as delivery vesicles for various cargo molecules

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.

Schematic illustration and characterization of the target-cell-specific invasion system

a) Engineered cells are equipped with a chimeric protein consisting  of  extracellular  and  transmembrane  domains  of  CD43  and  constitutively  active  RhoA  (CD43EX-RhoACA),  as  well  as  a  chimeric  protein  of  ML39 (HER2 recognition moiety)-CD28 transmembrane domain-dominant negative RhoA (ML39-CD28TM-RhoADN). In the absence of target antigen, RhoA polarization does not occur, so the engineered cells do not invade nontarget cells. Upon binding to HER2 on target cells, the desired polarization of RhoA occurs and the engineered cells invade the target HER2-positive cells. b) Image of cell-in-cell structure (invader cells: yellow; receiver cells: red). Invader cells were transfected with the above-mentioned invasion components (pRK47: PhCMV-ML39-CD28TM-RhoADN-pA, pRK48: PhCMV-CD43EX-RhoACA-pA) and pEYFP-C1 (PhCMV-EYFP-pA), and mixed with HEK-HER2-iRFP or HEK-iRFP cells. After 6 h, the cells were observed under a confocal fluorescence  microscope.  (Bottom  right  image  is  with  HEK-iRFP,  and  the  other  three  images  are  with  HEK-HER2-iRFP  cells.)  Scale  bars  indicate  10 μm.  c)  Quantification  of  cell-in-cell  structure.  Cells  were  transfected  with  the  following  plasmid  together  with  pEYFP-C1.  Correct  polarization:  pRK47 and pRK48. Opposite polarization: pRK66 (PhCMV-ML39-CD28TM-RhoACA-pA) and pRK67 (PhCMV-CD43EX-RhoADN-pA). No polarization: pRK64 (PhCMV-RhoADN  (full  length)-pA)  and  pRK65  (PhCMV-RhoACA  (full  length)-pA).  Mock:  pcDNA3.1(+).  The  proportion  of  the  cells  that  invaded  receiver  cells among YFP-positive cells is shown. The ratio of the number of invader cells (including cells that did not take up plasmids) and receiver cells was 1:  1.  Error  bars  represent  SEM  (n=  3;  ≈200  cells  were  observed  per  experiment,  and  the  average  of  three  independent  experiment  was  calculated).  **p< 0.01, two-tailed Student’s t-test.