The Biomechanical Mechanisms And Functions Of Cell-in-Cell Structure

Cell-in-cell structure refers to a unique eukaryotic cellular structure formed by the enclosure of one or more live cells inside of others.Based on the cell types involved,cell-in-cell structure can be classified as homotypic cell-in-cell which occurs between tumor cells commonly,and heterotypic cell-in-cell which occurs between different types of cells and is formed by the internalization of immune cells into tumor cells or other tissue cells.The outer cell of cell-in-cell structure is also known as target cell and the inner cell is known as effector cell.The cell-in-cell structure plays an important role in the occurrence and development of tumors,such as inducing tumor competition and selecting superior clones to promote cancer development.In addition,the cell-in-cell structure also participates in immune killing,for example,leading to immune escape of tumors by mediating effector NK cell death.In the process of cell-in-cell formation,the cell undergoes active large deformation,which is regulated by complex mechanical factors and biochemical signals.The researches about the formation mechanism of homotypic cell-in-cell(entosis)explore that the asymmetric distribution of E-cadherin and RhoA in the inner cell leads to the polarization distribution of the cytoskeleton protein,which in turn generates the driving force that pushes the inner cell into the outer cell.The key mechanical factor that determines a cell to become an inner cell or an outer cell is the rigidity of the cell,and the rigidity of the inner cell is significantly greater than that of the outer cell.However,there is no complete mechanical model for the homotypic cell-in-cell,and the mechanism of heterotypic cell-in-cell is still unclear.This paper has carried out a systematic study on the above scientific problems,and the content is divided into the following three parts:(1)I find that immune cell lines CCRF-CEM can be internalized into liver tumor cell PLC/PRF/5 to form heterotypic cell-in-cell structure.The expression level of CD44 is negatively correlated to the frequency of heterotypic cell-in-cell.Moreover,high expression of CD44 can increase the viscosity coefficient and characteristic relaxation time of PLC cells,and enhance the relative cellular rigidity by upregulating RhoA expression and downstream phosphorylated myosin light chain(pMLC),which suppress the formation of heterotypic cell-in-cell structure.(2)The time-lapse images of cell-in-cell process are taken by microscope,and a number of geometric parameters are used to describe the dynamic changes of cell morphology.The energy equation of cell deformation is established based on cell adhesion energy,cell deformation energy and myosin Ⅱ work.This mechanical model can quantitatively explain the dynamic process of cell-in-cell formation.In addition,I find that the different patterns of the contact area between the inner and outer cells over time can be explained by different energy dissipation mechanisms.(3)I quantitatively measure the death pH of inner cell based on fluorescent protein keima,and I also reveal that LC3 recruits to entotic vacuoles containing inner cell at a complex pattern associates with lysosome-dependent entotic death and caspase 3-dependent apoptotic death.Finally,I uncover that regulating the cellular pH by chemical compound and small interfering RNA can manipulate the inner cell fates and switch the ways whereby inner cells die.In conclusion,this thesis systematically studies the mechanical mechanism of homotypic and heterotypic cell-in-cell structure.By establishing a mechanical model,I discover the role of many mechanical factors on homotypic cell-in-cell formation and quantitatively explain the characteristics of cell morphological changes during this process.Furthermore,I clarify the mechanism of the gene CD44 regulating the mechanical properties of cell during heterotypic cell-in-cell process.Finally,I discover the role and dynamics of entotic vacuolar pH during cell-in-cell mediated death.