Exosome RNA Exosome RNA Research & Industry News
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.
