Effectively Remodeling Tumor-associated Macrophages And Killing Cancer Stem Cells By Drug-loaded Extracellular Vesicles

Tumor tissues are composed of tumor cells（including cancer stem cells,CSCs）and tumor microenvironment.The immune cells especially immunosuppressive cells infiltrated in the tumor microenvironment affect the therapeutic efficacy of cancer treatment.Meanwhile,CSCs are considered to be the main population leading to tumor recurrence,metastasis and drug resistance.Therefore,how to efficiently remodel tumor immunosuppressive cells and kill CSCs is a major challenge for tumor therapy.It has been proved that the objective response rate of HCC patients to anti-PD-1therapy is only 15%～20%in clinical trials.The major factors attributed to the resistance to anti-PD1 therapy include the presence of tumor immunosuppressive microenvironment,the lack of infiltrating T lymphocytes in tumor tissues and the composition of CD8⁺T cells.Tumor-associated macrophages（TAMs）mainly pro-tumor M2-like TAMs are abundant in solid tumors.Targeting M2-like TAMs to repolarize them to anti-tumor M1-type,subsequently present tumor antigens as antigen-presenting cells to enhance antitumor immune response is a promising strategy to improve the therapeutic effect of anti-PD-1 antibody.In addition,the development of nanodrugs which can target different CSCs remains a big challenge.Extracellular vesicles as drug carriers have attracted extensive attention.In this study,M2pep-modified microparticles derived fromα-fetoprotein（AFP）-overexpressing macrophages(M2pep-MPs _AFP)are used for targeted delivery of Toll-like receptor 7/8agonists R848(denoted as R848@M2pep-MPs _AFP).R848@M2pep-MPs _AFPare used to targeted deliver R848 to M2-like TAMs and effectively reprogramm M2-like TAMs.Importantly,R848@M2pep-MPs _AFPsignificantly ameliorate tumor immunosuppressive microenvironment,and enhance CD8⁺T cells and stem-like CD8⁺T cells in tumor tissue.This strategy of reprogramming TAMs is promising to enhance the therapeutic efficacy of anti-PD-1 antibody in hepatocellular carcinoma.In addition,tumor cell-exocytosed exosome-biomimetic porous silicon nanoparticles（E-PSi NPs）loaded with doxorubicin（DOX@E-PSi NPs）are developed for targeted CSCs chemotherapy,which efficiently solve the problem that targeting different CSCs requires coupling of different target molecules.The main research contents and results of this thesis are as follows:（1）R848@M2pep-MPs _AFPwas preparaed and M2-like TAM reprogramming capacity was evaluated.RAW264.7_AFPcells were constructed by infecting murine RAW264.7macrophages with lentivirus expressing murine AFP gene.RAW264.7_AFPcells were treated with R848 to prepare R848@MPs _AFPand DSPE-PEG-M2pep was connected to R848@MPs _AFPsurface by membrane lipid exchange to prepare R848@M2pep-MPs _AFP.R848@M2pep-MPs _AFPefficiently targeted to and repolarized M2-like macrophages to M1-like phenotype in vitro and in vivo.R848@M2pep-MPs _AFP-educated M2-like macrophages,as antigen-presenting cells,efficiently processed and presented AFP to promote the proliferation and activation of CD8⁺T cells,ultimately specifically killing Hepa1-6 tumor cells which highly expressed AFP antigen.（2）The antitumor activity of R848@M2pep-MPs _AFPwas evaluated.R848@M2pep-MPs _AFPexhibited strong anticancer activity in subcutaneous or orthotopic Hepa1-6 tumor-bearing mice.In vivo immune microenvironment analysis showed that R848@M2pep-MPs _AFPeffectively repolarized M2-like TAMs to M1-like phenotype,ameliorated the tumor immunosuppressive microenvironment,promoted the proliferation and activation of CD8⁺T cells as well as altered the composition of CD8⁺T cells with enhanced number of stem-like CD8⁺T cells,terminally differentiated CD8⁺T cells and terminally differentiated CD8⁺T cells secreting Granzyme B.（3）The antitumor activity of R848@M2pep-MPs _AFPcombined with anti-PD-1antibody was determined in C57BL/6 mice bearing subcutaneous and orthotopic Hepa1-6tumors,as well as the diethylnitrosamine-induced autochthonous hepatocellular carcinoma.Tumor immune microenvironment analysis showed that R848@M2pep-MPs _AFPand anti-PD-1 antibody co-treatment further ameliorated the tumor immunosuppressive microenvironment.Tumor re-inoculation and in vitro antigen re-stimulation experiments of splenocytes showed that R848@M2pep-MPs _AFPand anti-PD-1 antibody co-treatment could induce antigen-specific immune memory response and prevent tumor recurrence.（4）Universal anti-tumor effects of R848@M2pep-MPs _X（X represents the specific antigen）combined with anti-PD-1 antibody were performed.R848@M2pep-MPs _OVAcarrying the model antigen ovalbumin（OVA）was constructed to efficiently repolarize M2-like macrophages to M1-like phenotype.The R848@M2pep-MPs _OVA-educated M2-like macrophages could process and present OVA peptide on the cells surface,and promoted the activated of CD8⁺T cells specifically.Combination of R848@M2pep-MPs _OVAand anti-PD-1 antibody significantly reduced the tumor growth and improved the immune microenvironment of B16-OVA tumor-bearing mice.（5）The non-homologous targeting mechanisms and anticancer activity of DOX@E-PSi NPs were evaluated.DOX@E-PSi NPs could efficiently be uptaken by both heterologous tumor cells and CSCs.Meanwhile,DOX@E-Psi NPs efficently increased cell membrane roughness,reduced cell membrane fluidity to inhibit P-gp expression,resulting in the enhanced accumulation of DOX in CSCs to kill tumor cells and CSCs.Besides,the CD54 adhesion molecule expressed on DOX@E-PSi NPs derived from tumor cells might be involved in regulating the non-homologous targeting effect,tumor accumulation and deep penetration of DOX@E-PSi NPs.In conclusion,this thesis studied the reprogramming effect of R848@M2pep-MPs _AFPon M2-like TAMs and the regulation tumor microenvironment.R848@M2pep-MPs _AFPefficiently relieves multiple inhibitory factors of anti-PD-1 antibody therapy at the same time,which provides a new idea for targeting TAMs to enhance the sensitivity of anti-PD-1 antibody against hepatocellular carcinoma.Meanwhile,the combination of the R848@M2pep-MPs _Xplatform and anti-PD-1 antibody is promising to realize personalized treatment in tumors expressing specific antigens,providing a new strategy for precise tumor treatment.In addition,this thesis explore the non-homologous targeting effect,deep penetration,intracellular transport process and non-homologous targeting mechanism of DOX@E-PSi NPs,which provides a new idea for application of extracellular vesicles as drug carriers in the treatment of CSCs.