Modification Of Cell Surface By Synthetic Glycopolymers And Its Influence On Cell Behavior In Tumor Microenvironments

The metastasis and development of tumors are closely related to the migration of cancer cells,and thus tumor metastasis can be slowed down by inhibiting the migration of cancer cells.In addition,in cancer treatment,besides the inhibition of metastasis,cancer cells need to be destroyed at the same time,when the immune cells in the tumor microenvironment play an important role.T cells are the most critical functional cells in the immune response of tumors.Since dendritic cells(DCs)can induce T cell activation,vaccines prepared based on DCs are currently one of the most promising tumor immunotherapy approaches.Promoting the interaction between DCs and T cells directly is an effective way to improve the efficiency of DC vaccines.Cell membrane engineering can alter the interaction between cells and their microenvironments,providing an effective way to regulate cancer cell migration and DC-T cell interaction.Glycans are an important functional molecule on cell membranes.Its interaction with Lectins can affect the recognition and adhesion between cells,making it an ideal material for modifying cell membranes.However,the complex molecular structure of natural oligose is not conducive to studying the effect of a single sugar unit on cell behavior.Therefore,we can use synthetic glycopolymers with uniform structures to modify the cell surface.Then studying the effect of glycopolymers on the cell behavior in the tumor microenvironments,which plays an important role in the research of cancer treatment.Based on the above ideas,our research focused on the topic of "Modification of cell surface by synthetic glycopolymers and its influence on cell behavior in tumor microenvironments".We first obtained two glycopolymers by reversible additionfragmentation chain transfer polymerization(RAFT)and grafted the glycopolymers onto mouse melanoma(B16)cells by genetic engineering combined with chemical modification.Subsequently,we investigated the morphology,adhesion to the substrate,and single cell migration behavior of glycopolymer-modified cancer cells.Based on these,we further studied the interaction between glycopolymer-modified cancer cells and the surrounding cells as well as the effects on their collective migration behaviors.This provides a new perspective for studying the treatment of cancer metastasis.In addition,we modified DCs in the same way and studied the promoting effect of glycopolymer-modified DCs on T cell activation and the mechanism of glycopolymermodified DCs in promoting DC-T cell interaction.This facilitates the design and optimization of cellular vaccines.The specific research contents are as follows:(Ⅰ)Effect of surface modification of cancer cells by glycopolymers on the migration behavior of single cells.Glycopolymers(pMAG/pMAM)were obtained by RAFT polymerization using N-methacrylamide glucosamine(MAG)and Nmethacrylamide mannosamine(MAM)as monomers,and they were bonded to B16 cell membranes,which stably expressed HaloTag protein(HTP).The glycopolymers could stay on the cell surface for at least 5 days.The cytotoxicity test showed that the glycopolymers had good biocompatibility with B16 cells.Microscopic observations showed that the morphology of B16 cells modified by HTP changed from spindle to circle.However,after the cells were further modified with the glycopolymers,the cell morphology did not change again.To understand these obseravtions,the expression level of integrin αvβ6 by different modified cells is determined.It is found that the HTP increased the expression of integrin and hence the adhesion between the cell and the substrate while the glycopolymers did not change the expression of integrin.In addition,for cancer cells with up-regulated integrin expression,the cell morphology tended to be round,while for cells with less integrin expression,the morphology tended to be spindle-shaped.These results demonstrated that after the surface modifications,the morphological changes of cells were mainly caused by the HTP and the glycopolymers did not change the cell morphology since they had little effect on the adhesion between cells and the substrate.Finally,via the time-lapse imaging of living cells,it was found the single-cell migration behavior of glycopolymer-modified cancer cells was changed.The migration direction of the modified cancer cells was constantly changed,and hence the directional motion was reduced.(Ⅱ)The effect of glycopolymer-modified cancer cells on collective migration of unmodified cancer cells.pMAG/pMAM promoted the expression of E-cadherin by different types of cancer cells,and the degree of promotion of E-cadherin expression varied depending on the content of the glycopolymers.In addition,glycopolymers bound to the cell surface interacted consistently and stably with the cells compared to glycopolymers in solution.The addition of glycopolymer-modified B16 cells to B16 cell cohorts resulted in a vortex-like motion around the modified cells in B16 cell cohorts and inhibition of collective migration of B16 cells.In contrast,stimulation of B16 cells with free glycopolymers resulted in overexpression of E-cadherin on B16 cells,and did not effectively inhibit the collective migration of B16 cells.It is proposed that the glycopolymers on the cell surface can promote the cells to express E-cadherin which enhances the intercellular junctions.Thus via such enhanced intercellular junctions,the modified cancer cells will affect the migration of the surrounding unmodified cells,resulting in vortex-like movement and reduction of the speed of collective migration.These results show the potentiality of glycopolymer-modified cancer cells in inhibiting the spread and metastasis of cancer.(Ⅲ)Effect of interaction between glycopolymer-modified DCs and T cells on T cell immune activity.DCs was modified with glycopolymers via HTP gene transfection combined with chemical modification.The fluorescence labeling showed that the glycopolymers was successfully bound to the DC surface,and remained on the DC surface for 7 days.The toxicity test revealed that the glycopolymers were not cytotoxic to DCs.Glycopolymer-modified DCs induced T cells to secrete higher cytokines,and T cells after stimulation with glycopolymer-modified DCs showed greater proliferation capacity.These results indicated that glycopolymer-modified DCs promoted the activation of T cells.Through microscopic observation and LDH release detection,it was found that glycopolymer-modified DCs had a higher antigen presentation efficiency to T cells,indicating the T cells stimulated with glycopolymer-modified DCs had a stronger killing ability to tumor cells.In addition,stimulation of T cells with glycopolymer-modified DCs did not change the specificity of activated T cells for specific tumors.Through time-lapse imaging analysis of living cells,it was found that the glycopolymers modified on DCs increased the connection between DCs and T cells by binding to the mannose receptor on T cells,thus promoting the interaction between DCs and T cells.In addition,pMAG might also interact with other proteins on the T cell surface.This work demonstrates that the addition of suitable synthetic glycopolymers to the cell surface is a valuable approach to the design and optimization of cellular vaccines.