Anti-tumor And Immune Mechanism Of IL-33 Modified Melanoma Stem Cells Vaccine

Background:With the in-depth study of tumor immune mechanism,tumor immunotherapy has become another breakthrough therapy strategy after surgery,chemotherapy and radiotherapy.Immunotherapy for melanoma is a research hotspot in recent years,especially the immune checkpoint inhibitors(ICIs)targeting cytotoxic T-lymphocyte antigen-4(CTLA-4)and programmed cell death protein-1(PD-1),which have achieved good clinical efficacy.As an important branch of immunotherapy,vaccines are rarely studied in melanoma.The gp100 peptide vaccine can produce high level circulating T cells in vitro,and can recognize and kill melanoma cells,but the clinical efficacy is still not ideal.Therefore,the effective tumor vaccine can be used as an important supplement in the field of melanoma immunotherapy.The concept of cancer stem cells(CSCs)broadens our understanding of melanoma,targeting melanoma stem cells(MSCs)can inhibit the growth of melanoma and reduce the risk of tumor recurrence and metastasis.Therefore,tumor vaccines targeting MSCs will improve the efficacy of current immunotherapy regimens.However,it is still a great challenge to stimulate the body to produce anti-tumor immune response by using vaccine.The simple MSCs vaccine is not enough to induce strong anti-tumor immune effect.At present,the research of MSCs vaccine in melanoma is mostly to induce protective anti-tumor immunity by pulsing DCs with MSCs.In addition,immune adjuvant can enhance the anti-tumor immune response of MSCs vaccine,and immune adjuvant IL-21 gene modified MSCs can enhance complement dependent cytotoxic activity and NK cytotoxic activity.As a pleiotropic immune effector,IL-33 plays an important role in activating anti-tumor immunity,including stimulating the maturation of DCs,enhancing the tumor killing effect of CD8~+T cells and NK cells,and enhancing the immune response of antigen-specific effector T cells and memory T cells in vivo.It can significantly inhibit tumor growth and lung metastasis in mice.Similarly,IL-33 can be used as an effective immune adjuvant.The effect of IL-33 modified MSCs on melanoma has not been reported,and its anti-tumor immune mechanism needs to be further explained.Objectives:In this study,IL-33 modified melanoma stem cell vaccine is prepared to study its inhibitory effect on the growth and metastasis of melanoma,and to explore its antitumor immune mechanism,so as to lay a theoretical and experimental foundation for the application of melanoma stem cell vaccine in the immunotherapy of melanoma.Methods:1.Preparation of melanoma stem cells vaccine(1)Immunomagnetic beads were used to separate cells: B16F10-CD44~+CD133~+ cells(i.e.melanoma stem cells)and B16F10-CD44~-CD133~-cells were isolated from B16F10 cells by immunomagnetic beads.(2)Identification of having been sorted melanoma stem cells: The expressions of CD44,CD133 and MHC I were detected by flow cytometry;The ability of cell colony forming is measured: The colony forming ability of B16F10 and B16F10-CD44~+CD133~+ cells in two dimension(2D)and three dimension(3D)culture before and after sorting were detected;The total RNA of cells before and after sorting was extracted.The expression of SOX-2,OCT-4 and KLF-4 was detected by RT-q PCR.(3)B16F10-CD44~+CD133~+ was infected by HBAD-IL33-EGFP: B16F10-CD44~+CD133~+ was inoculated into the 6-well plate.On the second day,a suitable volume of virus(HBAD-EGFP overexpression control or HBAD-IL33-EGFP)was added.The infection time is 8 h.(4)Tumor cells were inactivated and identified after inactivation: Mitomycin C(MMC)50 μg/ml was used to inactivate tumor cells for 4 h.MTT method was used to determine the cell proliferation rate at different time points;The expression of IL-33 was detected by enzyme linked immunosorbent assay(ELISA).2.Evaluation of anti-tumor effect induced by melanoma stem cells vaccine(1)Establishment of preventive animal model: The tumor bearing mice model was established: The inactivated tumor cells(including equal volume PBS)of different experimental groups were inoculated subcutaneously into the groin of C57BL/6 mice for 3 times,with an interval of 7 days.After the last immunization for 7 days,5 × 10~5 B16F10 cells were injected subcutaneously into the back of all mice,and the tumor size was measured regularly;Lung metastasis model: the immune mode was the same as above.After the last immunization for 7 days,all mice were injected with 5 × 105 B16F10 cells via tail vein.The lung metastasis and survival time were observed and recorded.(2)Histopathological examination of tumor: Three weeks after the last immunization,tumor tissues of different groups of mice were taken for histopathological examination,including Hematoxylin-eosin(HE)staining,CD8,Ki67 and Caspase 3 immunohistochemical staining.3.The mechanism of anti-tumor immunity induced by melanoma stem cells vaccine(1)The effect of vaccine on the maturation of DCs: DCs from mouse bone marrow were co-cultured indirectly with inactivated tumor cells(including equal volume PBS)of different experimental groups by suspension cell culture chamber.After co-culture for 4 days,the morphological changes of DCs were observed,and the expressions of CD11 c,CD80,CD86 and MHC II were determined by flow cytometry.And then DCs were collected and co-cultured directly with CFSE labeled splenic lymphocytes at a ratio of 1:10 for 6 days.The expression of CFSE in CD8~+T cells was determined by flow cytometry.The secretion of IFN-γ and TNF-α in the supernatant of each group was determined by ELISA.(2)The effect of the vaccine on spleen CD8~+T cells: Three weeks after the last immunization,splenic lymphocytes of mice in different groups were collected for flow cytometry.The expression of CD3,CD8 and CD69 on the surface of splenic lymphocytes was detected to determine the effect of the vaccine on the proportion and activity of splenic CD8~+T cells;The expression of PD-1,CTLA-4,Tim-3 and central memory phenotypes CD44,CD62 L and CD127 were detected to determine the effect of the vaccine on CD8~+T cell immune checkpoint and CD8~+ central memory T cell;The expression of IFN-γ and Gzm B in CD8~+T cells was detected to determine the effect of the vaccine on the expression of anti-tumor effector molecules.(3)The effect of the vaccine on the cytotoxicity ability of spleen lymphocytes in mice: Three weeks after the last immunization,splenic lymphocytes of mice in different groups were taken as effector cells,and B16F10 cells cultured in vitro were taken as target cells;similarly,spleen lymphocytes of mice in vaccine group were taken as effector cells,and B16F10,sorted B16F10-CD44~-CD133~-and B16F10-CD44~+CD133~+ cells were taken as target cells.The cytotoxicity effect of spleen lymphocytes on tumor cells was determined by Calcein AM release assay.(4)The expression of CD44,CD133 and MHC I in melanoma tissue cells: three weeks after the last immunization,the expression of CD44,CD133 and MHC I in melanoma tissue cells of different groups were determined by flow cytometry.(5)Detection of serum cytokine levels: three weeks after the last immunization,the serum of mice in different groups was taken,and the levels of IFN-α,IL-2,TNF-α and IFN-γ in different groups were detected by ELISA.Results:We successfully isolated B16F10-CD44~+CD133~+ cells through immunomagnetic beads,the purity of B16F10-CD44~+CD133~+ cells was 96.14%,and the clone forming ability of B16F10-CD44~+CD133~+ cells was stronger.The expression of SOX-2,OCT-4 and KLF-4 was significantly increased,and the expression of MHC I was reduced.B16F10-CD44~+CD133~+ was infected by HBAD-IL33-EGFP,following MMC was used to inactivate the cells,and the cell activity and IL-33 secretion were measured,At the same time,the inactivated cells were inoculated subcutaneously into the groin of mice,and no tumor growth was found.Finally,B16F10-IL-33 CD44~+CD133~+,whose cell proliferation was inhibited and can still secrete IL-33,was obtained,namely melanoma stem cell vaccine(hereinafter referred to as "vaccine").In the preventive mice model,the vaccine could significantly inhibit the growth of melanoma and lung metastasis,and induced apoptosis of melanoma cells.The median and total survival of mice in the tumor bearing mice model were significantly prolonged(40 days and 55 days respectively),while in the model of lung metastasis,the median and total survival time of mice in the vaccine group were prolonged more significantly(55 days and 70 days respectively).To further study the mechanism of anti-tumor effect of vaccine,we found that:(1)the vaccine could promote the maturation of mouse bone marrow-derived DCS and up-regulate the expression of DCs maturation markers,and DCs could activate CD8~+ T,including stimulating the proliferation of CD8~+T cells and promoting the secretion of IFN-γ and TNF-α by CD8~+T cells;(2)Three weeks after vaccination,the vaccine could increase the proportion and activity of CD8~+T cells in spleen and decrease the expression of immune checkpoint.In addition,the vaccine could induce the formation of CD8~+ central memory T cells and increase the expression of anti-tumor effector molecules of CD8~+ T cells.(3)Three weeks after immunization,the vaccine could significantly enhance the cytotoxicity effect of spleen lymphocytes on tumor cells,and the cytotoxicity effect was specific,and showed target killing effect on B16F10-CD44~+CD133~+ cells.(4)The expression of CD44 and CD133 in tumor cells of tumor bearing mice in vaccine group decreased,while MHC I expression increased,and CD8~+ T cells infiltrated in melanoma tissue increased.(5)The vaccine could increase the secretion of anti-tumor cytokines,including IL-2,IFN-α,TNF-α and IFN-γ.Conclusions:1.B16F10-CD44~+CD133~+ cells with high purity and stem cell properties are successfully isolated from B16F10 melanoma cells,and B16F10-IL-33 CD44~+CD133~+ whole cell vaccine with inhibited proliferation activity but still secreted IL-33 is prepared.2.The vaccine can inhibit the growth and lung metastasis of melanoma in mice,and induce apoptosis of melanoma cells,and prolong the median and total survival period of mice.3.The anti-tumor immune effect induced by the vaccine is to stimulate the maturation of DCs,promote the proliferation of CD8~+T cells,inhibit the differentiated depletion of CD8~+T cells in vivo,and induce the formation of memory T cells.4.The vaccine can activate the host’s cellular immunity.It can promote the infiltration of CD8~+T cells in melanoma tissue and activate the cytotoxic T lymphocyte immune response,and then target specifically to kill melanoma stem cells.