A Strong Anti-tumor Immune Effects Mediated By Dendritic Cells Pulsed With Exosomes From Endoplasmic Reticulum Stress Hepatocellular Carcinoma Cells

BackgroundHepatocellular carcinoma（HCC）is a malignant tumor with high degree of malignancy,rapid progression and refractory.Patients are often diagnosed in the advanced stage,the treatment is very limited,traditional chemotherapy,radiotherapy and other methods are not ideal.So finding a new treatment strategy for HCC is an urgent problem.At present,immunotherapy is a hot filed in the cancer research and is considered to be a promising treatment strategy.DCs-based immunotherapy has received much attention and has been extensively studied in many human malignancies and has shown a certain therapeutic effect.Researchers have also extensively explored DC-stimulating antigens,such as the use of lysates of tumor cells as broad-spectrum antigens for DCs-based immunotherapy.However,response rate of clinical is limited.Therefore,DCs-based immunotherapy requires optimization of antigens and attaching meaningful clinical effects.Malignant tumor cells are often accompanied by endoplasmic reticulum stress（ERs）during the process of rapid progress.ERs often promote tumor growth and inhibit the response of anti-tumor immune.However,studies have found that excessive ERs occurred in tumor may lead to cell apoptosis and release a large number of immunogenic molecules to activate anti-tumor immune response.Studies have also shown that tumor immunogenic apoptosis caused by ERs in vivo is not sufficient to reverse the immunosuppressive state of tumor microenvironment.The reason may be:tumor microenvironment exist a large number of immunosuppressive factors and cells.Therefore,how to promote the immunogenicity of tumors cells and improve the tumor microenvironment is worth exploring.The discovery of exosomes has aroused great interest among researchers.Exosomes are membrane-like vesicles secreted by living cells.Tumor exosomes contain a large number of antigenic substances from parental tumor cells and represent a new class of non-cellular tumor antigens.Studies have found that anti-tumor immune response mediated by DCs pulsed with tumor exosomes were stronger than tumor lysates.The results of our study have also showed that DCs pulsed with HCC exosomes mediated anti-tumor immune responses in vitro and in vivo.Importantly,the anti-tumor immune effect mediated by DCs pulsed with ERs HCC exosomes are stronger than non-ERs HCC exosomes,but the cause and mechanism are still unclear.Therefore,exploring the antigenic components packaged by ERs HCC exosomes and the mechanisms of anti-tumor immune is very important for the development of new antigens for DCs-based immunotherapy.ObjectiveTo observe the effect of ERs HCC exosomes on DCs and the anti-tumor reponse mediated by DCs in vitro and in vivo,further to search for differentially protein expressed in ERs and non-ERs HCC exosomes by protein profiling,analyze and explore the role of this protein in the effects of ERs HCC exosomes on DCs,and provide a theoretical basis for the discovery of new antigens for DCs-based immunotherapy.Methods（1）HepG2 were treated with Tunicamycin,the expression of ERs-related protein were detected by Western-blot.（2）Exosomes were extracted from the culture supernatants of ERs and non-ERs HepG2.The morphology of exosomes were observed by electron transmission microscopy,and the expressions of CD63 and TSG101 were detected by Western-blot.ERs and non-ERs HCC exosomes are named:"EXO-TM"and"EXO-CON".（3）Exosomes was labeled with PKH67（membrane fluorescent dye）,and PKH67-exosomes was incubated with DCs for 12 hours.The uptake of exosomes by DCs was observed under a laser confocal microscope.（4）The DCs were pulsed with ERs or non-ERs HepG2 exosomes.The morphology of DCs were observed under inverted microscope.The surface molecules of DCs were tested by flow cytometry.The inflammatory factors were detected by CBA kit.Immature DCs are named:“iDC”,The DCs that pulsed with ERs and non-ERs HCC exosomes are named:“DC_EXO-TM”and“DC_EXO-CON”.（5）Fluorescent dye CFSE prestained lymphocytes were co-cultured with DCs at a ratio of 5:1,and the cell proliferation of lymphocytes was detected by flow cytometry,and lymphocyte factor was detected by CBA kit.（6）RTCA（real-time cell analyzer）DP system monitored the tumor killing effect of lymphocytes.Effector cells:The target（CD8+T cells:Tumor cells）was at 25:1.（7）Mouse-derived DC2.4 was treated with ERs and non-ERs mouse HCC exosomes,and the expression of surface molecules of DCs was detected.DC2.4 of each treatment group was co-cultured with mouse lymphocytes to detect lymphocyte proliferation effect,the ratio CD8⁺/CD4⁺T cells,lymphocyte factor IFN-γsecretion,and T lymphocyte-mediated tumor killing effect.（8）Mouse-derived DC2.4 pulsed with ERs or non-ERs mouse HCC cells Hepa1-6exosomes were injected into ectopic HCC mouse model and systematic observation of its mediated antitumor effects.（9）Differential proteins of ERs and non-ERs HCC exosomes were identified by protein profiling and verified by Western blot.（10）Construct a stable HCC cell line with low expression of HSP90 by specific shRNA interference technology(named“HepG2_shHSP90”),further to exam the effect of ERs HepG2_shHSP90 exosomes on DCs and mediated antitumor effects.(ERs HepG2_shHSP90 exosomes named"EXO-TMshHSP90").Results（1）The expression of ERs-related protein in HepG2 was correlated with the concentration and time of TM treatment.When treated with 2.5μmol TM for 24 hours,the expression of ERs-related protein changed most significantly.（2）Transmission electron microscopy captured a set of cup-shaped circular small vesicles with a diameter of 30-120 nm in medium extract from HCC cells.Western blot confirmed that vesicles rich in CD63 and TSG101,lack of Calnexin,consistence with the feature of"exosomes"described in the literature.（3）Spotted green fluorescence was found in the cytoplasm of DCs captured by laser confocal microscopy,suggesting that exosomes can be taken up by DCs.（4）Under the inverted microscope,the dendrites of DC_EXO-TM were slightly longer than DC_EXO-CON and iDC;the surface molecule of DC_EXO-TM was higher than DC_EXO-CON and iDC;and the secretion of pro-inflammatory cytokines was also increased,such as IL-1β,IL-6,IL-8,IL-12 and TNFα（p<0.01）.（5）The induction effect of DC_EXO-TM on T lymphocyte proliferation was stronger than iDC（p<0.05）,but no significant difference with DC_EXO-CON（p>0.05）;Co-incubation of DC_EXO-TMXO-TM and T lymphocytes increased CD8⁺/CD4⁺cell ratio（p<0.05）and secretion of cytokines IL-2,IL-4,IL-6,IL-10,TNFαand INF-γ（p<0.05）.（6）Both DC_EXO-TM and DC_EXO-CON can trigger T cell-mediated specific and non-specific tumor killing,and the effect of DC_EXO-TM was higher than DC_EXO-CONXO-CON at24 hous（p<0.05）.（7）Treatment of murine DC2.4 with ERs and non-ERs murine HCC exosomes yielded results similar to humans DCs.The results showed that the surface molecules MHC-I,MHC-II,CD80,CD86 and CD83 of DC_EXO-TM were up-regulated more significant than iDC and DC_EXO-CON;The DC_EXO-TM induced the most obvious effect of lymphocyte proliferation（p<0.001）,increased CD8⁺/CD4⁺T cell ratio（p<0.05）;and promoted lymphocyte IFN-γsecretion（p<0.05）.The tumors killing effect of T lymphocytes was observed.The results showed that at 24 hours and 36 hours,the tumor killing effect of T lymphocytes was mediated by DC_EXO-TM was higher than iDC and DC_EXO-CON（p<0.05）.（8）Ectopic HCC mice were inoculated with mouse-derived DC_EXO-TM and DC_EXO-CON,and found that the group of DC_EXO-TM had slower tumor growth rate（p<0.01）and smaller tumor volume（p<0.01）than the group of DC_EXO-CON and iDC;The ratio of CD8⁺/CD4⁺cells increased significantly in tumor infiltration and spleen（p<0.05）;The level of INF-γin the serum increased（p<0.05）.（9）160 proteins were differentially expressed in EXO-TM and EXO-CON by protein profiling,and 71 proteins were higher（>1.25 times）in EXO-TM than in EXO-CON,of which HSP90 was up to 2.548 times;more chaperone proteins and immune function related proteins were discovered by GO analysis.（10）The surface molecules of DC_{EXO-TMshHSP90}（DCs pulsed with EXO-TMshHSP90）have a certain degree of down-regulation relative to DC_EXO-TM,and the tumor killing effect of T lymphocytes was also weakened.ConclusionERs HCC cells exosomes pulsed DCs mediated a stronger anti-tumor immune response than non-ERs HCC cells exosomes.Moreover,DCs pulsed with ERs HCC exosomes can mediate tumor killing effects of specific and non-specific of T lymphocytes.Furthermore,DCs pulsed with ERs HCC exosomes can help to improve tumor microenvironment in ectopic HCC mice.Suggesting that ERs HCC exosomes may be a potential and broad-spectrum antigen for DCs-based immunotherapy.