Preliminary Study Of Tumor Cell-derived Microparticles Mediating Intestinal Mucosal Anti-tumor Immunity

Objective:The intestinal mucosal immune system,as an important part of the systemic immune system,profoundly affects the occurrence and development of tumors.Remodeling intestinal mucosal immunity through oral tumor vaccines to induce systemic antitumor immune protection may be a new approach to tumor immunity.The tumor cell-derived microparticles(T-MPs)are a type of vesicles with a diameter ranging from 100 to 1000 nm and composed of cell membranes encapsulating cytoplasmic contents,which are released by tumor cells changing the cytoskeletal structure,when they are stimulated by the outside or undergo apoptosis.Our previous studies have confirmed that T-MPs carry both tumor antigens and innate signal molecules like DNA,and its unique lipid raft structure has excellent physicochemical stability.In addition,subcutaneous immunization with T-MPs as a vaccine achieves ideal outcomes.On this basis,this study will use T-MPs as an oral vaccine to thoroughly explore the changes of T-MP-mediated intestinal mucosal immune system,clarify the relevant mechanisms of intestinal anti-tumor immune responses,and provide possibilities for the development of novel oral tumor vaccines.Methods:(1)To prove that oral T-MPs can inhibit tumor growth in vivo,we extracted B16-MPs and CT-26-MPs to immunize mice orally followed by tumor cell injection subcutaneously,and drew tumor growth curves;to prove that the immune response caused by oral administration of T-MPs is antigen-specific,mice were immunized i.g.with OVAB16-MPs,B16-MPs and Ovalbumin followed by s.c.injection of OVAB16 cells;to prove that T-MPs have advantages over tumor cell lysates and apoptotic tumor cells,mice were immunized i.g.with B16-MPs,B16 cell lysates and apoptotic B16 cells followed by s.c.injection of B16 cells;we immunized i.g.nude mice with B16-MPs to study the type of T-MPs-mediated immune response.(2)To explore whether oral T-MPs can mediate intestinal local and systemic T cell immune responses,mice were immunized i.g.with B16-MPs,and the percentages of IFN-y+ cells in CD8+ and CD4+ T cells and the CD4+ T cell subsets in spleen and mesenteric lymph node(MLN)were analyzed by flow cytometry,while the expression of relevant cytokines was detected by qPCR;lymphocytes were separated for secondary stimulation in vitro after immunization with OVAB16-MPs orally,and the IFN-y production was analyzed by flow cytometry,demonstrating that T-MPs can induce specific T cells in vivo.(3)Establishing conditional clearance and reconstruction models of DCs in mice,the percentages of IFN-y+ cells in CD8+ and CD4+ T cells were analyzed by flow cytometry to determine whether T-MP-induced T cell immunity is initiated and investigate whether DCs participate in T-MP-mediated antitumor T cell immunity.(4)To study the uptake of T-MPs in the intestine,mice were given i.g.PKH26-T-MPs,and the uptake of T-MPs by different intestinal segments at multiple time points were analyzed by flow cytometry;the intestinal cells were divided into CD45+ immune cells and CD45-non-immune cells,and the uptake of PKH26-T-MPs by which cells was detected by flow cytometry and immunofluorescent staining;the cytoskeletal inhibitor colchicine was used to study the transcytosis of T-MPs by intestinal epithelial cells(IECs).(5)mice were given i.g.H22-MPs followed by detection of the percentages of CD11c+,CD 103+ and CD3+ CD8+ cells in intestine by flow cytometry and immunofluorescent staining,to investigate the mobilization of immune cells in intestine by T-MPs;at the same time,the expression of chemokines in IECs on gene and protein levels was analyzed by qPCR and ELISA to investigate whether the migration of intestinal immune cells is related to chemokines.(6)To explore the signal transduction of chemokines in IECs,Western blot and immunofluorescence were used to detect the activation of MAPK and NF-κB in IECs uptaking H22-MPs,and MAPK or NF-κB inhibitors were used to achieve reverse proof;to further study the upstream molecules of MAPK and NF-κB signaling,the expression of NOD receptor in IECs uptaking H22-MPs was analyzed by qPCR and Western blot,and additionally the MAPK and NF-κB activation,chemokine expression,recruitment of immune cells,and tumor growth inhibition mediated by oral T-MPs were detected again in knockout mice.Results:(1)Mice were orally immunized with B16-MPs and CT-26-MPs followed by tumor cell injection subcutaneously.As a result,B16-MPs and CT-26-MPs both inhibited tumor growth well in different strains of mice;oral immunization with OVAB16-MPs had more significant anti-OVAB16 tumor activity than B16-MPs and Ovalbumin;oral immunization with B16-MPs could inhibit the growth of B16 tumors more efficiently than the B16 cell lysates and apoptotic B16 cells;oral B16-MPs could not inhibit tumor growth in nude mice.(2)after oral immunization of mice with B16-MPs,the proportion of CD8+and CD4+ T cells secreting IFN-y in MLN and spleen was increased,whereas the percentages of other CD4+ T cell subsets remained unaltered,and the expression of Th1 related cytokines was upregulated;when lymphocytes from mice orally immunized with OVAB16-MPs,B16-MPs and Ovalbumin were separated for secondary stimulation by OVA peptides in vitro,lymphocytes from OVAB16-MPs group produced more IFN-y.(3)CD1 lc-DTR mice were injected with DT for DC depletion and adoptively transferred with additional DCs for reconstruction.The results showed that removal of DCs did not alter the proportion of IFN-y+ T cells,whereas reconstitution of DCs restored T-MP-induced increase in the proportion of IFN-y+ T cells.(4)It was found that the majority of T-MPs were taken up by the ileum at 1h when mice were given i.g.PKH26-T-MPs.The cells that took up T-MPs were mainly IECs,while the CD11c+ CD103+ DCs only took up a small portion;colchicine was used to inhibit the transcytosis of IECs and we found that T-MPs obtained by DCs were released from IECs.(5)The increased proportion of CDllc+,CD 103+,and CD3+ CD8+ cells in ileac cells was observed after oral administration of H22-MPs in mice;at the same time,the expression of chemokines CCL2,CCL20 and CX3CL1 was upregulated at both gene and protein levels in ileac IECs,among which the upregulation of CCL2 was the most significant and maintained for the longest time.(6)The increased levels of phosphorylated ERK and p38,and the translocation of NF-κB from cytoplasm to nucleus were observed in IECs that have taken up T-MPs.The upregulation of CCL2 could be blocked by the use of MAPK or NF-κB inhibitors;the expression of NOD2 receptor in IECs that have taken up T-MPs was upregulated at both gene and protein levels,and the MAPK and NF-κB activation,increased expression of CCL2,recruitment of CD 103+ DCs and CD8+ T cells,and suppression of tumor growth by oral T-MPs were all blocked in NOD2 knockout mice.Conclusion:Most of the T-MPs can be taken up by the ileac epithelial cells after reaching the intestinal tract through oral route.On one hand,T-MPs interact with NOD2 receptors of IECs to activate the downstream MAPK and NF-κB signals,resulting in the upregulation and release of CCL2 as the dominant chemokine,which subsequently recruits CD103+ DCs.On the other hand,T-MPs are transcytosed by IECs from tract surface to basolateral surface,which results in the capture and antigen presentation of T-MPs by migrated CD 103+ DCs,triggering mucosal and systemic antitumor T cell immune responses.T-MPs as oral tumor vaccines not only have efficacy and broad spectrum,but also have greater advantages than other tumor vaccine.