Xenotransplantation Of Human Hepatocarcinoma Cells In Newborn Mice And The Resulting Cellular Immune Rejection Mechanism Study

Background and objectiveHepatocellular carcinoma(HCC),the third most common cause of cancer-related death worldwide,is the most frequent hepatic tumor with poor prognosis.Tumor xenograft models are widely used for human tumor biology and chemotherapy analysis, but they may be insufficient for studying tumor immunotherapy because most of them are established in immunodeficient hosts.Studies to elucidate the immune mechanisms of tumor-host interactions in immunocompetent tumor xenograft models will benefit our understanding of tumor immunotherapy.Previous studies have demonstrated that tumor xenografts can grow in some immunologically privileged sites,such as the anterior chamber of the eye,the hamster cheek pouch,or under the kidney capsule of mice or rats with limited cyclosporine A treatment.The potential barriers to successful xenograft survival are believed to be T cell mediated xenorejection and tissue microenvironments.The importance of T cells in xenorejection can be manifested by the growth of xenografts in mutant nude mice.However,the interaction between tumor xenografts and local microenvironments where the xenografts grow have received little extent attentions.As a common site for human HCC transplants,the liver is accepted as a complex immune or tolerance organ in recent years.It contains abundant innate immune cells, such as natural killer(NK),natural killer T(NKT) cells,kupffer cells and dendritic cells(DCs).Besides,classic CD4~+ T and CD8~+ T cells in the liver are present at a reversed ratio other than they are in the peripheral blood.All these innate and adaptive immune cells residing in liver dominate the local tumor surveillance,but their role in tumor xenograft survival has been inadequately explored.In this study we attempt to establish a human-mouse HCC model by transplanting human HCC cells to the liver of immunocompetent newborn mice.We based on three reasons:first,the elevated expression of alpha-fetaprotein(AFP) in the liver of newborn mice(at day 0～15 after birth) can suppress the anti-tumor immunity;second, newborn mice can generate weak immune rejection because of their incomplete thymus and peripheral immune organs;the third,as a immune privilege organ,the liver is apt to induce immune tolerance.In our previous experiments,we transplanted human HepG2 cells into the liver of immunocompetent newborn KM mice for the first time.We demonstrated that human HepG2 cells survived longer than those in adults.However,following the immune systems of newborn mice maturing,the immune xenorejection enhanced,and tumor cells began necrosis or apoptosis.The tumor cells encountered immune rejection in the liver of newborn mice,which immune cell participated in that process? Transplanted tumor cells were cleared from the liver,how the immune microenvironment of liver changed? In particular the aboundent NKT cells,how their activation and function changed? Whether the fate of different cells in the liver of newborn mice is same or not? No lymphocyte infiltration was detected in tumor tissues of CsA treated newborn mice,how the effect of CsA on cell-mediated xenorejection?Trying to solve these problems,we transplanted different human HCC lines (HepG2 and HCCLM3) to immunocompetent newborn mice.We intended to examine the fate of transplanted human tumor cells,the cell-mediated immune rejection,and the changes of activation and function of NKT cells.For better understanding this,we adopted low-dose CsA treated newborn and adult mice as controls.This study provides new information on tumor xenograft models in immature animals and throws new insight into NKT-mediated tumor xenorejection.Moreover,it adds to our understanding of the immunosuppressive effects of CsA.Methods1.In vitro cell imaging and Cytotoxicity analysis by DMAHAS1) HepG2 cells labeled with DMAHAS and cell imaging;2) Fluorescence release analysis of DMAHAS labeled cell;3) Cell viability was determined using MTT and neutral red assays;4) Total protein content of cells was measured by Coomassie brilliant blue assay;5) Cell proliferation and apoptosis were analyzed using the propidium iodide(PI) flow cytometric assay; 2.Orthotopic implantation of human HCC cells in young and adult mice1) In vitro culture of tumor cells and labeled by DMAHAS;2) Mice were treated with lose-CsA(10mg/kg) and sera CsA was measured;3) Orthotopic implantation of human HCC cells in the liver of mice;4) In vitro frozen section analysis and fluorescence imaging of tumor xenografts;5) Conventional HE staining and pathological examination of tumor xenografts:3.Different cellular rejection occurred in young and adult mice was analyzed1) Orthotopic implantation of human HCC cells in the liver of mice;2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;3) Isolation of hepatic or splenic lymphocytes and counting;4) Method based on DMAHAS fluorescence release for cytotoxicity analysis;5) Specific antibodies toward minor cells were measured in the serum from mice;6) Phenotypic analysis of hepatic and splenic lymphocytes from mice by FCM;4.Analysis of the activation and function of NKT from tumor-bearing mice1) Orthotopic implantation of human HCC cells in the liver of mice;2) Mice treated with lose-CsA(10mg/kg) through subcutaneously injection;3) Isolation of hepatic or splenic lymphocytes and counting;4) NKT activity analysis by LDH release assay;5) Analysis of NKT activation and CD1d expression of hepatic/splenic lymphocytes;6) Levels of IL-4 and IFN-γwere measured by ELISA using a quantitative kit;7) In vitro immune cells depleted by spcific antibodies;8) Cytotoxicity analysis by LDH release assay.ResultsThe first partCell imaging in vitro using DMAHASWe found that both live and fixed cells took up DMAHAS rapidly within 30min. The final recommended working concentration is 5μM.DMAHAS exhibited strong one photon induced blue fluorescence and stable cytosolic localization. Cytotoxicity of DMAHAS toward HepG2 cellsNo significant cytotoxicity was determined following 48h incubation of HepG2 cells with DMAHAS at a working concentration of 5μM.According to MTT,NR,CB and FCM assay.Tumor tracking in vivo using DMAHAS and fluorescence imagingDMAHAS labeled HepG2 cells could survive in recipient mice for more than 4 weeks and the same results were obtained with mice receiving HCCLM3 xenografts. Smaller tumor xenografts were observed as survival time increased,and tumor cells tended to aggregated.At six weeks after transplantation,no tumor cells or fluorescence were detected in the liver of recipient mice.At any given time,no significant decline in fluorescence intensity was observed.Pathological examination of tumor xenografts in newborn miceLymphocyte infiltration appeared in and around the xenografts at day 8 after implantation of human HCCLM3 cells in the liver of newborn mice,no macrophages or neutrophils was visible,and the recruitment of lymphocytes appeared in the normal liver tissue near the tumor xenografts.Some of them still survived for more than four weeks,and no evidence of distant metastasis was observed.Low-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks.The second partNumber changes of hepatic and splenic lymphocytes in mice bearing tumor cellsIn young mice,the absolute number of lymphocytes increased in the liver,but decreased in the spleen at days 10 and 16 postimplantation(P＜0.05).Similar results were found in adult mice at days 4,8 and 10 postimplantation.No significant apoptosis of splenic or hepatic lymphocytes was detected in young mice.Immune analysis of hepatic and splenic lymphocytes in mice bearing tumor cellsIn young mice,the absolute number and proportion of hepatic CD4~+ T cells was significantly increased at day 16 postimplantation(P＜0.01);the absolute number of NK cells and CD8~+ T cells increased in the liver,but decreased in the spleen(P＜0.05). In adult mice,the proportion of CD8~+ cells increased in the liver,but decreased in the spleen at day 2 after transplantation.CTL responses induced by HCCLM3 xenografts in young miceSplenic lymphocytes from tumor-bearing young mice showed higher cytotoxicity against HCCLM3 cells at the ratio of 10:1,20:1 and 50:1(P＜0.05).Effects of low-dose CsA on the survival of xenografts and immune responseLow-dose CsA treatments significantly reduced the lymphocytes infiltration and prolonged the survival of xenografts in young mice to five weeks posttransplantation. However,CsA had no obvious effect on the survival of xenografts in adults.Besides the inhibiting effects on CD4~+ and CD8~+ cells as seen in data from young and adult mice,low-dose CsA treatment efficiently suppressed the activation of hepatic NKT cells at day 2 after implantation in adult mice without the loss of NK1.1 expression on NKT cells.Moreover,low-dose CsA treatment efficiently suppressed the splenic CTL activity in tumor-bearing young mice.The third partHepatic NKT cell activation in mice induced by HCCLM3 xenograftsAfter HCCLM3 cells were transplanted into the livers of 3-day-old mice,the hepatic NKT cells lost expression of NK1.1 at days 5 and 10.NK1.1 molecules reappeared at day 16,and elevated NKT-like activity was detected.In adult mice,a loss of NK1.1 expression was detected in hepatic NK/NKT cells at days 2 and 4 posttransplantation.Then the NK1.1 marker was reexpressed and returned to normal levels at day 10 posttransplantation.There were no significant changes in NK1.1 expression on splenic NK/NKT cells after transplantation.CD1d expression on lymphocytes from the liver and spleen of tumor-bearing mice In young mice,the CD1d expression first increased on hepatic NK1.1~+ cells at day 10 after transplantation.Then up-regulation of CD1d expression was detected on hepatic CD3~+ T cells and NK1.1~+ cells at days 16 and 28 after transplantation.Similar results were detected in hepatic or splenic lymphocytes from adults at day 4 and 10 posttransplantation.Secretions of IL-4 and IFN-γin tumor-bearing young and adult miceSera were collected from the retro-orbital veins at days 10 and 16 in young mice and at days 4 and 10 in adult mice after transplanted with human HCCLM3 cells. Elevated secretion of IL-4 was detected at day 10 in young mice and at day 4 in adult mice(P＜0.05),and elevated secretion of IFN-γat any given time in young and adult mice(P＜0.05),compared with control groups.Effects of low-dose CsA treatment on the activation and function of NKT cellslow-dose CsA treatment efficiently suppressed the activity of NKT cells from tumor-bearing young mice,and the activation of hepatic NKT cells in adult mice at day 2 after implantation.Moreover,low-dose CsA treatment inhibited the secretions of IL-4 and IFN-γin recipient mice(P＜0.05).No down-regulation of CD1d expression on hepatic or splenic T cells and NK1.1~+ cells was detected in mice with low-dose CsA treatment.CTL analysis after in vitro deletion of specific lymphocytesIn vitro deletion of CD3 and CD8 cells efficiently reduced the cytotoxicity against HCCLM3 cells(P＜0.05),however,deletion of CD4 and NK1.1 cells showed no evident effect. Conclusion1.DMAHAS was used to label human HCC cells for fluorescence imaging for the first time.It was demonstrated that DMAHAS had no evident effect on the cell viability,synthesis of total protein or DNA,as well as cell apoptosis.In vivo study indicated that DMAHAS was a reliable probe for tumor cell tracking and imaging.2.It was demonstrated that the liver of newborn mice was more suitable for the survival of tumor xenografts than that of adults.3.Lymphocytes significantly increased in the liver,but decreased in the spleen after tumor implantation without enhanced cell apoptosis,suggesting that splenic lymphocytes may participate in the tumor xenorejection.4.cell-mediated xenorejection in the liver of young mice was different from that in adult liver.In young mice the delayed cellular rejection was mediated mainly by CD4~+T cells,CD8~+T cells and NK1.1~+cells.In contrast,in adult mice the acute cellular rejection was mediated mainly by CD8~+T cells,NK1.1~+cells,macrophages or neutrophils.5.The rapid activation of hepatic NKT cells with enhanced activity and function, suggested that NKT cells acted as an important role in the xenorejection.6.Increased expression of CD1d on the surface of hepatic or splenic lymphocytes, implying that CD1d molecules participate in the tumor xeno-antigens presenting and the subsequent tumor clearance.7.Low-dose CsA treatments significantly prolonged the survival of xenografts in young mice by inhibiting the cell-mediated rejection.However,it had no obvious effect on the survival of tumor xenografts in adults.8.Besides the inhibiting effects on CD4~+ and CD8~+ cells,low-dose CsA treatment efficiently suppressed the activation and function of hepatic NKT cells,maybe through a CD1d-independent manner.