Characterization And Mechanisms Of Cell-in-Cell Death Induced By Interaction Of Immunocytes And Tumor Cells

Cell-in-cell refers to one or more living cells into another cell's cytoplasm with formationof overlapping structures. Cell-in-cell can occur both in homogeneous cells andheterogeneous cells. The representations of this phenomenon can almost be traced back to onehundred years ago. Most of those reports only focused on the phenomenon description, untilintracellular death of tumor cells after cell-in-cell was renamed for entosis and also consideredas a new kind of cell death in2007. Now cell-in-cell phenomenon was attracted muchattention and quickly became a research hotspot.Thirty years ago, our research group found that NK cells can go into tumor cells and killtarget cell from the inside. But main fate of inside NK cells is death in the tumor cells withtypical characteristics of apoptosis. Recently we further demonstrated that NK cell deathinside tumor cell was caspase-dependent death way. This kind of new immune cell death waywas named for emtosis (emperipolesis+entosis), different from entosis which iscaspase-independent and lysosome death way,also other than cannibalism phenomenonwhich was defined as tumor cells―eating‖immune cells.On the basis of above research, we further expanded on the different types of cell-in-cellphenomenon. The homogeneous and heterogeneous cell-in-cell death, especially cell-in-celldeath between immune cells and tumor cells' were compared. We focus on analysis ofmechanism of emtosis formation. In addition, we also try to explore biological significance ofcell-in-cell phenomenon between immune cells and tumor cells and establish a newcell-in-cell research system.Results were as follows: cell intracellular death was the dominant fate of cell-in-cell.Intracellular death of tumor cell in homogeneous or heterogeneous tumor cells was mainlylysosomal cell death (entosis), while immune cells, especially the cytotoxic immune cells'death inside tumor cells or normal cells, was mainly caspase-dependent apoptosis(emtosis).But intracellular death of non-cytotoxic immune cells or leukemia cells such as B cells,CCRF, Raji inside the tumor cell was mediated by lysosome (entosis). It was thereforeconcluded that intracellular cell death was not confined to homogeneous or heterogeneous cellinteractions and related to biological properties of cells. Previous research revealed that MCF7used in entosis was a caspase3-deficient cell line,intracellular cell death, which was characterized by caspase independency and lysosomemediation (entosis), was questioned due to caspase3deficiency. Our research also confirmedthat MCF7was a caspase3deficient cell in both DNA and mRNA level and can not expresscaspase3protein. Thus, cell death of MCF7induced by staurosporine did not show the typicalfeatures of apoptosis. In contrast, A431cells can be induced to typical apoptosis because ofcaspase3expression. However, both intracellular death of A431and MCF7after cell-in-cellshowed the same features-entosis. The results indicated that introcellular cell death dependedon the characteristics of cells and was not affected by caspase3Expression.Mechanism of entosis and emtosis was compared. There was significant aggregation ofROS and cytochrome C release in cytoplasm in dead cell with entosis; and also autophagyprotein marker LC3was observed to locate with vacuole structures in cytoplasm. Entosis canbe converted into emtosis when lysosomal function was inhibited, and the cell death rateincreased significantly and cell came into death more quickly than entosis. The aboveevidences suggested that entosis was actually a autophagy process by starting the autophagypathway to resist against apoptosis caused by internal environment for survival. However,continuous expansion of autophagy eventually led to cell degradation. The preliminary resultwas consistent with conclusion by Overholtzer's laboratory (NCB2011.10).However, mechanism of emtosis is completely different from entosis. Our study showedthat different immune cells died inside tumor cells in different ways and ishighly related tocell toxicity. Only death of cytotoxic immune cellsin tumor cells showed typicalcharacteristics of emtosis. Immunofluorescence, Granzyme B activity inhibitor and granzymeB deficient mice were used for further research on emtosis mechanism. The results were asfollows:(1) Immune cells expressing granzyme B or cytotoxic immune cells can die in tumorcells as emtosis way；other immune cells such as B cells and monocytes die in tumor cells asentosis way.(2) The granzyme B inhibitor and granzymeB gene deficiency has significantinhibitory effect on emtosis.(3) Through the microscope observation and dynamic statistics,it was also found that cytotoxic immune cells can release active granzyme B in tumor cells,but most of the released granzymeB gatheredto surround itself,it is quite different from,granzyme B spreading to the target cells' cytoplasm from outer cytotoxic immune cells. Therefore we hypothesize that emtosis is likely to be caused by granzyme B from itself. Thismay be a―manslaughter‖while immune cells attack tumor cells for keeping a balance andstability mechanism of immune cells.Moreover, our experiments also showed that cell fate and consequences after cell-in-cellare diverse: immune cells going into tumor cells and normal cells can obviously make targetcells form multi-nucleated aneuploid cell. Cell-in-cell phenomenon in vitro happened quitefrequently than expected: in nearly100kinds of cell lines that we studied, more than70%celllines can occur cell-in-cell phenomenon; and it also appeared widely in vivo including tumor,tumor adjacent tissues and other inflammation tissues. All the founding provided fundamentaldata to further study on biological significance of cell-in-cell.