Study On Enhancing Anti-tumor Immunity Of Hepatocellular Carcinoma With ¹²⁵I Seed Radiotherapy

Chapter I Study on Immune-related Biological Effects of ¹²⁵I seedRadiotherapy of Hepatocellular CarcinomaObjective:To explore the effects of iodine-125(¹²⁵I)seed irradiation on immune-related biological effects of liver cancer in mice by using an in vitro ¹²⁵I seed irradiation device and subcutaneous and orthotopic tumor models of liver cancer.Methods:Mouse hepatoma cell lines H22 and Hepa 1-6 were irradiated with ¹²⁵I seeds at different doses,and cell apoptosis,expression of immune-related phenotype molecules（MHC-I,PD-L1,GAL-9 and PVR）and immunogenic death（ICD）marker（CRT）was detected by flow cytometry（FCM）.Meanwhile,the expression of ATP and HMGB1,were detected by chemiluminescence assay and ELISA respectively,and the expression of CRT and HMGB1 were further verified by immunofluorescence confocal microscopy.The apoptosis of tumor cells was verified by TUNEL assay in vivo.The lymph nodes and tumor tissues were isolated and the DC mature and tumor-infiltrating T lymphocytes（TILs）in the lymph nodes were detected by FCM to further verify the effect of ¹²⁵I seed radiotherapy on T lymphocyte infiltration in orthotopic tumor of liver cancer in mice.Results:FCM showed that different doses of ¹²⁵I seed irradiation could increase the apoptosis rate of H22 and Hepa 1-6 of mouse hepatoma cells gradually,and reached the peak at 4 Gy.¹²⁵I seed irradiation enhanced the expression of MHC-I,decreased the expression of GAL-9 and PVR,and reached the peak at 4 Gy.FCM and immunofluorescence test results showed that ¹²⁵I seed irradiation could promote the expression of CRT and HMGB1 in HCC cells,and reached the peak at 4 Gy.Chemiluminescence experiments showed that ¹²⁵I seed irradiation could promote the release of ATP by tumor cells,which increased by 3 times at 4 Gy compared with the control group.Radiotherapy with ¹²⁵I seed implantation promoted DC maturation in subcutaneous tumor drainage lymph nodes.At 4 Gy,CD80 and CD86 increased by 1.47,1.41 and 1.73 times,respectively,compared with MHC-II of control group.Radiotherapy with ¹²⁵I seed implantation promoted CD8⁺T cell infiltration of subcutaneous tumor,which increased 2.16 times at 4 Gy,compared with the control group,and Treg decreased 4.5times.Radiotherapy with ¹²⁵I seed implantation promoted CD8⁺T cell infiltration in orthotopic carcinoma of mice,which increased by 1.6 times and decreased Treg by 1.29times compared with the control group.Conclusion:¹²⁵I seed irradiation promoted apoptosis,ICD,the expression of immune-related phenotypic molecule MHC-II,and decreased the expression of PD-L1,GAL-9 and PVR.¹²⁵I seed irradiation promoted DC maturation in lymph nodes,up-regulated CD8⁺T cell and down-regulated Treg cells infiltrated in tumor.Chapter II Study on ¹²⁵I Seed Radiotherapy Combined with DC to Enhance Anti-tumor Immunity of Hepatocellular CarcinomaObjective: To construct a subcutaneous tumor model of hepatocellular carcinoma and explore the effect of ¹²⁵I seed radiotherapy combined with DC on enhancing anti-tumor immunity of hepatocellular carcinoma.Methods: Bone marrow derived dendritic cells（BM-DC）were prepared and a mouse subcutaneous tumor model was established.Intratumoral ¹²⁵I seed irradiation was implanted,DC was injected into the tumor,and anti-PD-1 was injected intraperitoneally.Tumor growth and survival of mice were observed.The tumor draining lymph nodes were isolated and the maturation of DC was detected by FCM and immunofluorescence.Spleen cells were isolated,CD4+T and CD8+T cells were separated by magnetic beads,and the proliferation of CD4+T and CD8+T cells and the proportion of central memory T cells（TCM）and effector memory T cells（TEM）were detected by FCM.Tumor cells were isolated from mice and T cell infiltration was detected by FCM and immunofluorescence.Tumor tissue supernatant was collected and the release of cytokines（IFN-γ and IL12-p70）was detected by ELISA.Results: The results of tumor growth in mice showed that 125I+DC group could significantly inhibit tumor growth compared with ¹²⁵I group.When further combined with anti-PD-1,the inhibition effect on tumor growth was even more significant.Similarly,125I+DC treatment can significantly prolong the survival time of mice,up to 58 days,and the survival time of mice can be extended to 78 days after combined with anti-PD-1.In addition,DC mature experiment showed that 125I+DC could increase MHC-II,CD80 and CD86 by 58%,15% and 14.4%,respectively,compared with ¹²⁵I group.T cell proliferation results showed that 125I+DC could promote the proliferation of CD4+T and CD8+T cells by 8.6% and 1.2%,respectively,compared with ¹²⁵I group.T cell infiltration results showed that 125I+DC increased CD8+T cells by 27.7% and down-regulated Treg by 11.1% compared with ¹²⁵I group.Combined with anti-PD-1 therapy,T cell infiltration was further promoted.Memory immune cell results showed that 125I+DC increased TCM and TEM by 12% and 5.8%,respectively,compared with ¹²⁵I group.When combined with anti-PD-1 treatment,TCM and TEM increased by 20.4% and 35.8%.Cytokine results showed that 125I+DC increased IFN-γ and IL-12-p70 by 50% and 33.3%,respectively,compared with ¹²⁵I group.Conclusion: ¹²⁵I seed radiotherapy combined with DC significantly inhibited tumor growth,prolonged the survival of mice,promoted DC maturation and proliferation of CD4+T and CD8+T cells.In addition,¹²⁵I seed radiotherapy combined with DC improved the proportion of TCM and TEM of memory immune cells,CD8+T lymphocyte infiltration and reduced the proportion of Treg cells in tumor tissue;125I seed radiotherapy combined with DC also promoted the release of IFN-γ and IL-12-p70.Combining with anti-PD-1,they further promoted T cell proliferation,T cell infiltration,memory immune subsets ratio,and promote the release of cytokines.Chapter III Study on ¹²⁵I Seed Radiotherapy Combined with Flt-3L⁺pIC⁺DC to Enhance Anti-tumor Immunity of Hepatocellular CarcinomaObjective: To further enhance the anti-tumor immunity of ¹²⁵I seed radiotherapy combined with DC,DC（Flt-3L⁺pIC⁺DC）induced by DC amplifying agent（Flt-3L）and DC activator（p IC）was introduced.To explore the effect of ¹²⁵I seed radiotherapy combined with Flt-3L⁺pIC⁺DC on enhancing anti-tumor immunity of hepatocellular carcinoma in mice,primary and distant tumor models were constructed.Methods: FlT-3L⁺pIC⁺DC and primary tumor and distant tumor models of liver cancer in mice were constructed.The primary tumor was irradiated with ¹²⁵I seeds,Flt-3L⁺pIC⁺DCs were injected into the tumor,and anti-PD-1 was intraperitoneally injected to observe the tumor growth and survival of mice.The DC maturation was verified in vivo by FCM and immunofluorescence.The spleen,tumor draining lymph nodes and tumor tissues were isolated,and lymphocytes were stained with CFSE.The proliferation of CD4+ and CD8+T cells was detected by FCM,and the proportion of NK,CD4,CD8 and Treg lymphocyte subsets in spleen,tumor draining lymph nodes and tumor tissues were detected by FCM.The proportion of TCM and TEM subsets were determined by FCM.Cytometric Bead Array（CBA）was used to detect the release of IFN-γ,IL-2,TNF-ɑ,IL-12p70,IL-6,and IL-10 cytokines.Results: Tumor growth in mice showed that 125I+Flt-3L⁺pIC⁺DC group significantly inhibited tumor growth compared with ¹²⁵I +DC group.Among distant tumors,the 125I+Flt-3L⁺pIC⁺DC group showed more significant inhibition on distant tumor growth than 125I+DC group,and the combination of anti-PD-1 therapy showed more significant inhibition on tumor growth.The results of DC maturation showed that the expression of CD80 and CD86 in 125I+Flt-3L⁺pIC⁺DC group were 13%,6.3% and 6.1% higher than that in 125I+DC group,respectively.Combined treatment with anti-PD-1 further increased by 2.5%,3.3%,and 7.1%,respectively.Spleen T cell proliferation results showed that the 125I+Flt-3L⁺pIC⁺DC group promoted the proliferation of CD4+T and CD8+T cells by 18.7% and 0.6%,respectively,compared with 125I+DC group.Tumor lymphocyte subgroup infiltration results showed that NK and CD8+T cells were significantly increased and Treg proportion was down-regulated in 125I+Flt-3L⁺pIC⁺DC group,respectively,compared with 125I+DC group,in which,NK and CD8+T increased by 16.4% and 2.6%,respectively,and Treg decreased by 2.9%.After combined with anti-PD-1 treatment,NK and CD8+T cells could be further promoted and Treg ratio down-regulated.Compared with 125I+DC,TCM and TEM in 125I+Flt-3L⁺pIC⁺DC group increased by 0.6% and 0.95%,respectively.After further combined with anti-PD-1 treatment,TCM and TEM increased by 15.5% and 22.3%.Cytokine results showed that compared with 125I+DC group,IFN-γ,IL-2,TNFα and IL-12-p70 increased by 20%,8.7%,40% and 14.2% in 125I+Flt-3L⁺pIC⁺DC group,while IL-6 and IL-10 decreased by 4.2% and 20%,respectively.Conclusion: ¹²⁵I seed radiotherapy combined with Flt-3L⁺pIC⁺DC inhibited tumor growth,activated distant effect,induced DC maturation in tumor draining lymph nodes,promoted T cell proliferation and the proportion of TCM and TEM of immune memory cell subsets,increased tumor tissue lymphocyte infiltration,promoted the release of immunestimulating cytokines and reduced the secretion of immunosuppressive cytokines.Further combination with anti-PD-1 therapy can further promote T cell proliferation,lymphocyte infiltration,proportion of memory immune cell subsets and cytokine release.