Role And Mechanism Of Chemotherapeutic Tumor Microparticles Combining Low Dose Irradiation In Tumor Therapy

Part Ⅰ Macrophages are required for the antitumor T cell immunity by Cis-MPs and LDIObjective:Tumor cell-derived microparticles(T-MPs)are capable of delivering chemotherapeutic drugs,enhancing their ability to kill tumor cells,and reducing their side effects.However,whether such drug-packaging MPs-based approach can be strengthened by other means is not clear.Radiotherapy is a conventional method for cancer treatment with typical side effects,low dose irradiation(LDI)is even more effective in cancer therapy than the conventional daily doses of 1-2 Gy.This study will be carried out by combining the cisplatin-packaging T-MPs(Cis-MPs)with LDI to explore the effect of combined treatment on tumors and the type of anti-tumor immunity mediated by combined treatment,as well as the role of macrophages in combined treatment.Methods:(1)To determine whether Cis-MPs combining LDI generates a better antitumor consequence,H22 hepatocarcinoma cells,CT-26 colon and Lewis lung cancer cells were s.c.injected into mice and then the growth of tumor was observed.(2)To investigate the type of anti-tumor immunity mediated by combined treatment,T cell-deficient nude mice,the mice pre-treated with CD3,CD4 and CD8 antibody that depleted T cells as well as clodronate liposomes that depleted macrophages was used.(3)Leukocytes in spleen and tumor tissues that pre-treated with clodronate liposomes were isolated,and the number of CD3+ and IFN-γ+ CD8 T cells was analyzed by flow cytometry.Treated by LDI and then the macrophage phenotype in tumor microenvironment was analyzed by flow cytometry.(4)To understand the influence of irradiation dose on mice,the weight and survival was monitored.(5)To understand the influence of combined treatment on mice,the survival was monitored and serum level of creatinine and alanine aminotransferase were measured by creatinine and alanine aminotransferase dectection kit.Results:(1)Combination of Cis-MPs and LDI inhibited tumor growth to generate better tumor treatment outcomes compared to single treatment.(2)T cell deficiency caused the complete loss of the combined treatment efficacy,which was also confirmed in the mice pre-treated with CD3,CD4,CD8 antibody that depleted T cells and clodronate liposomes.(3)Macrophage depletion resulted in less CD3+ T cell infiltration into tumor mass and the expression of IFN-y was downregulated in tumor-infiltrating T cells as well as splenic T cells.LDI had no effect on macrophage phenotype in tumor microenvironment.(4)Neither the weight nor the survival of the mice was influenced by the LDI.(5)Combining Cis-MPs and LDI significantly prolonged the survival of tumor-bearing mice,and had no effect on serum creatinine and alanine aminotransferase levels.Conclusions:Combination of Cis-MPs and LDI generates better tumor treatment outcomes,T cells are required to mediate the antitumor effect of Cis-MPs and LDI,and macrophages are required for the antitumor T cell immunity by Cis-MPs and LDI.In our settings,we do not find the effect of LDI on macrophage phenotype in tumor microenvironment,suggesting that the combination of drug-packaging MPs and LDI probably uses indirect means to act on macrophages.The weight and the survival of the mice are not influenced by the LDI,moreover,combining Cis-MPs and LDI significantly prolonged the survival of those tumor-bearing mice.Notably,this combination neither induced the hair and weight changes nor altered liver and kidney functions.Part Ⅱ TRCs as a major player in the polarization of tumor-promoting macrophagesObjective:Stem cell-like tumor-repopulating cells(TRCs)have a critical role in establishing a tumor immunosuppressive microenvironment.However,means to enhance antitumor immunity by disrupting TRCs is absent.Our previous studies have shown that tumor cell-derived microparticles preferentially abrogate TRCs by delivering anti-tumor drugs into nuclei of TRCs.Here,we will explore whether LDI can enhance the effect of Cis-MPs on TRCs and further study the effect of TRCs on the macrophages which are required for the antitumor T cell immunity by Cis-MPs and LDI.Methods:(1)H22,CT26 and Lewis tumoe cells were treated with cisplatin.H22 tumor cells were treated with Cis-MPs and LDI.The inhibition and survival rate of tumor cells were detected by CCK-8 method.(2)H22,CT26 and Lewis tumoe cells were treated with Cis-MPs.H22 tumor cells were treated with different concentrations of Cis-MPs,different radiation doses combined with Cis-MPs,as well as Cis,MPs and Cis-MPs alone or in combination with LDI.Apoptosis of tumor cells was analyzed by flow cytometry.(3)Primary H22 tumor cells or H22,CT-26,Lewis cell lines were treated with Cis-MPs or LDI alone or in combination.TRCs were separated by 3D soft fibrin gels culture.The growth of TRCs clones was observed.(4)H22,CT-26 and Lewis TRCs were treated with Doxo,Doxo-MPs or Doxo-MPs combined with LDI.The uptake of TRCs and outflow of H22 TRCs was analyzed by flow cytometry.(5)Macrophages were incubated with the supernatants from TRCs or control tumor cells and were analyzed for cell-surface expression of the indicated molecules by flow cytometry and then iNOS,CD86 and Argl mRNAs were assessed by Real-time PCR.(6)Splenic T cells purified from WT mice were incubated with TRCs or control-educated macrophages.T-cell proliferation was examined by CFSE dilution assay.(7)Mice,pre-inoculated with B16-F10 tumor cells,were injected the TRCs or control-educated macrophages into tumor mass.Tumor-infiltrating leukocytes were isolated from tumor tissues in each groups and analyzed by flow cytometry.(8)TRCs or control-configured macrophages were incubated with B16-F10 cells and then the tumor cells were subcutaneously or intravenously injected into the tumor cells into mice.The growth of subcutaneous melanoma and lung metastasis were observed.(9)PKH67(Green)-conjugated MPs were i.v.injected to H22 subcutaneous tumor-bearing mice alone or in combination with LDI treatment,frozen sections of tumor tissues were used for the analysis by fluorescence microscope and tumor cells were collected for flow cytometric analysis of PKH67-positive population.Results:(1)Cisplatin could inhibit the proliferation of tumor cells,combination of Cis-MPs and LDI reduced the survival ratio of H22 cell lines.(2)Cis-MPs promoted tumor cell apoptosis.With the increase of Cis-MPs concentration and irradiation dose,the apoptosis of tumor cells increased.2 Gy twice irradiation significantly enhanced the killing effect of Cis-MPs on tumor cells.(3)Combination of Cis-MPs and LDI could inhibit the growth of TRCs derived from in vivo and in vitro.(4)Treated by combination of Doxo-MPs and LDI increased the drugs uptake of H22,CT-26 and Lewis TRCs and decreased the outflow of H22 TRCs.(5)TRCs-educated macrophages expressed higher M2 phenotype markers such as CD206 and arginase 1.(6)TRCs-educated macrophages suppressed T cell activation and proliferation.(7)Immunosuppressive cell types MDSCs and Treg cells were significantly increased in tumor microenvironment.(8)TRCs-configured macrophages significantly promoted subcutaneous melanoma growth and lung metastasis.(9)LDI had no effect on the infiltration of drug-packaging MPs into tumor mass.Conclusions:Combination of Cis-MPs and LDI Cis-MPs can more effectively disrupt different types of TRCs derived from in vivo and in vitro as well as lead to increased drug uptake and reduced outflow of TRCs,suggesting that combined treatment of drug-packaging MPs and LDI may well curtail tumor-repopulating cells in tumor microenvironment.TRCs-educated macrophages expresse higher M2 phenotype markers and their significant antitumor effects,indicating that TRCs plays an important role to in the polarization of tumor-promoting macrophages.Part Ⅲ Remodeling macrophages by combined LDI and Cis-MPs is explained by their disrupting tumor-repopulating cellsObjective:Firstly,we tried to confirm that TRCs were the main factors of polarized macrophages by in vivo experiments.And to further study the effect of combined treatment on reprogramming macrophages by disrupting TRCs as well as the effect of reprogramming macrophage on tumor microenvironment.Methods:(1)Leukocytes in tumor tissues and spleen that treated with LDI and Cis-MPs on mice were isolated,and the number of CD3+ T cells,IFN-γ+ CD8 T cells,CD11b+F4/80+IL-1β+ and CD11b+F4/80+IL-10+ macrophages was analyzed by flow cytometry.The expression of IFN-y in TILs and splenocytes was detected by Elisa kit.(2)The growth of replenished tumor was observed.Leukocytes in tumor tissues and spleen that replenished TRCs into tumor mass were isolated,and the number of CD3+T cells,IFN-γ+ CD8 T cells,CD11b+F4/80+IL-1β+ and CD11b+F4/80+IL-10+macrophages was analyzed by flow cytometry.The expression of IFN-γ in TILs and splenocytes was detected by Elisa kit.(3)Mice were pre-treated with clodronate liposomes and then splenocytes and bone marrow cells were isolated,and the number of MDSCs and Treg was analyzed by flow cytometry.(4)The expression of VEGF,GM-CSF,RANTES and TARC in tumor tissues and the peripheral blood of the mice was detected by Elisa kit.Results:(1)Combination of Cis-MPs and LDI resulted in more CD11b+F4/80+IL-1β+ macrophages,CD3 T cells and IFN-γ+ CD8 T cells but less CD11b+F4/80+IL-10+ macrophages in TILs and splenocytes compared to single treatment.The expression of IFN-γ was upregulated in TILs as well as splenocytes.(2)The replenishment of TRCs promoted tumor growth,and the increase of TRCs resulted in more CD11b+F4/80+IL-10+ macrophages but less CD3 T cells,IFN-γ+ CD8 T cells and CD 11b+F4/80+IL-1β+ macrophages in TILs and splenocytes.The expression of IFN-y was downregulated in TILs as well as splenocytes.(3)Macrophage depletion resulted in a significant decrease of CD11b+Ly-6G+granulocytic and CD4+CD25+Foxp3+ Treg cells in splenocytes and bone marrow cells.(4)Macrophage depletion resulted in the upregulation of the expression of VEGF,GM-CSF and TARC but not RANTES in tumor tissues and the peripheral blood of the mice.Conclusions:TRCs is the main factor of polarized macrophages in vivo.Combination of Cis-MPs and LDI reprogram macrophages from tumor-promotion to tumor-inhibition by disrupting TRCs and curtailing their vicious education on macrophages and then remodel tumor microenvironment in a macrophage dependent manner,leading to improve anti-tumor immune responses.