The Biological Characterization And Effect Of Heterotypic Cell-in-cell Structure

Cell-in-cell, which means one cell or more can invade and wonder round about in thecytoplasm of other cells without damage to either cell, has been found for over a century(‘effector cell’ denotes the cell penetrating another cell and ‘target cell’ denotes the cell thathas been penetrated). Cell-in-cell can take place among the same type of cells (homotypiccell-in-cell) or different types of cells (heterotypic cell-in-cell). The types of effector andtarget cells vary extensively from terminally differentiated cells to stem cells, from immunecells to nonimmune cells, and from normal tissue cells to abnormal cells. Alternatively,cell-in-cell can be divided into cannibalism, entosis and emperipolesis based on biologicalacnhdar adcetesrcirsitbicesd anfodr simgnainfiyc andceec.a Adeltsh, oubguht ththe eo cmcuercrehnacnei somf (cse)l l-ains-cell has been recognizedfsriogmni faic vaanlcide suonludteiorlny.i nOgu rt hstius dpyh ceennotrmese nono nly amnpdh oitcsy tpeos sisnitbol etu mrooler wcienel lsillm tomas fu onitrmosl ohgbeyiote lraoortegy icpfaaicrlcell-in-cell structure, referred as emperipolesis.At first, we approached standardized methods to quantify heterotypic cell-in-cellfrequencies and sorting the group based on fluorescent labels and two-color flow cytometryby Fluorescence Activated Cell Sorting (FACS) instead of manual counting and identificationby Confocal Scanning Laser Microscopy (CSLM), which we consider to be the most efficientand objective method to study heterotypic cell-in-cell formed by emperipolesis as theaccuracy rate is about85%. Furthermore, the heterotypic cell-in-cell population is moredifficult to be internalized (the control group is6%and the experimental group is4.7%) orkilled (the control group is16.5%and the experimental group is9.6%) by the same effectorcells for the second time, we suggest heterotypic cell-in-cell has made some changes of targetcells in order to escape immune surveillance.Then we took the opportunity to gain some insights into the formation mechanism ofheterotypic cell-in-cell structure by emperipolesis. Using digitalized time-lapse microscopyand confocal scanning laser microscopy, we found speric-shaped resting lymphocytes become polarized, and develop a well defined cytoplasmic projection designated as cellularlampodium and uropod with polymerization/contraction of the actin/tubulin cytoskeletonduring penetrating. To gain further insights into the molecular process, we testedproinflammatory chemokine RANTES (regulated upon activation normal T cell expressed andsecreted) were able to induce NK cells polarity prior invading that CCR2, CCR5chemokinereceptors and LFA-1(lymphocyte function-associated antigen-1) rapidly redistributes toconcentrate in the lampodium, as well as cytoskeletal components involved in thedevelopment of contractile force, such as actin and MLC (myosin light chain). In contrast,different adhesion molecules, such as ICAM-1(intercellular adhesion molecule1), CD44areclustered into the other pole of the cell comprises a slender posterior appendage called theuropod, along with components of the cytoskeleton, such as the centre of microtubulin.Finally, we approached the role of MLCK (myosin light chain kinase) and ROCK(rho-associated protein kinase1), two predominant kinases in regulating lyphocytesinternalizing.Furthermore, diversity outcomes derived from different kinds of heterotypic cell-in-cellstructures. A very interesting event is the multinucleated or aneuploid target cells produced byheterotypic cell-in-cell process. On the one hand, internalized cells cause chromosomeinstability (CIN) of target cells probably by exchanging chromosomal components throughpenetrating directly into the nucleus of target cells or fusing with them. On the other hand,effector cell inside could interfere mitosis of target cell to induce aneuploidy. After coculturedeffector cells and target cells for24hrs, the aneuploidy of single cell was2.1%and the targetcell aneuploidy of cell-in-cell structure was28%. More importantly, we analyzed thecell-in-cell death pathway of cytotoxic effector cells. The study illustrated that activatedgranzyme B (GzmB) existing in intracellular cytotoxic cells cannot directly get into thecytoplasm of target cells due to the vacuole formation. Instead, active GzmB wasre-picking-up into the effector cells and induced the effect cell in-cell apoptosis afterwards,just as the suicide of killer cell inside tumor cells. We define this type of apoptotic cell-in-celldeath as emperitosis. In addition, we explored the associated heterotypic cell-in-cell with diseases such as liver cirrhosis, hepatocellular carcinoma.Concerning the biological significance and clinic value of heterotypic cell-in-cell, westudied the impact of monoclonal antibodies on heterotypic cell-in-cell. We found monoclonalantibody Trastuzumab could induce cell-in-cell formation as well as its universal role inantibody-dependent cell-mediated cytotoxicity (ADCC). More strikingly, the death of effectorcell could reserve to target cell by Trastuzumab. It is rational that Trastuzumab could bindinside efffector cell to vacuole, which is conducive for GzmB directly gets into the cytosol oftarget cells. According to this opinion, monoclonal antibodies involved in heterotypiccell-in-cell might serve as a new insight into the monoclonal antibody research and clinicindividualation.