The Role And Mechanism Of Mesenchymal Stem Cell Subsets And Its Derived Extracellular Vesicles On Ovarian Cancer And Breast Cancer

Objective:This study is the first attempt to apply our newly found subsets of anti-tumor mesenchymal stem cells（MSCs）and its derived extracellular vesicles（EVs）to the treatment of female malignant tumors（ovarian cancer and breast cancer）.Further exploration of the role of CD90^low MSC subsets in the treatment of tumors provides a theoretical basis and therapeutic targets.This study will not only provide a basis for EVs as a new and effective treatment or drug delivery medium,but also provide new ideas and insighs for the treatment of other types of tumors.Methods:（1）Bone marrow-derived MSCs（BM-MSCs）,Compact bone-derived MSCs（CB-MSCs）and Adipose-derived mesenchymal stem cells（ADSCs）were isolated and cultured from different tissues by enzyme digestion.（2）MSCs was identified by morphological characteristics of cells,flow cytometry analysis of cells surface markers and detection of multi-directional differentiation ability.（3）The anti-tumor effects of CB-MSCs,BM-MSCs,CB-MSCs and VIC-008 was evaluated in mouse ovarian cancer models.（4）qRT-PCR was used to evaluate the cytokine changes of CB MSCs in 3D culture.（5）CD90^high ADSCs were induced into CD90^low ADSCs by LPS and the antitumor effects of CD90^high ADSCs and CD90^low ADSCs subsets were evaluated by mouse tumor models.（6）EVs(CD90^high ADSC-EVs and CD90^low ADSC-EVs)from CD90^high ADSCs and CD90^low ADSCs were separated by ultracentrifugation.（7）The morphological characteristics of EVs were detected by Transmission electron microscope（TEM）and Scanning electron microscope（SEM）;the protein markers of EVs were detected by Western blot;the concentration and size distribution of EVs were detected by nanoparticle tracking analysis（NTA）.（8）The effects of CD90^high ADSC-EVs and CD90^low ADSC-EVs on the proliferation of E0771 cells were evaluated by plate cloning assay.（9）Transwell and scratch assays to evaluate the effects of CD90^high ADSC-EVs and CD90^low ADSC-EVs on the migration ability of E0771 cells.（10）The antitumor effects of CD90^high ADSC-EVs and CD90^low ADSC-EVs were evaluated in mouse breast cancer models.（11）miR-16-5p mimics were loaded into CD90^low ADSC-EVs（EVs-miR-16-5p mimic）by liposome fusion.（12）The effects of CD90^low ADSC-EV-miR-16-5p on tumor cells in vitro and in vivo were evaluated by flow cytometry and mouse tumor models.Results:（1）The shape of cultured cells was fibroblast-like;the surface markers of cells were consistent with the general characteristics of MSCs;and these cells can differentiate into adipogenesis,osteogenesis and chondrogenesis.CD90 expression of CB-MSCs was lower than that of BM-MSCs and CB-MSCs had antitumor effects.Gene expressions of immune activating cytokines IL-12,IL-21,IFN-γand a pro-inflammatory cytokine CXCL10 in CB-MSCs were increased in 3D culture whereas gene expression of anti-inflammatory cytokine IL-10 and CCL5 was downregulated.Treatment that combines CB-MSCs and VIC-008 significantly decreased ovarian tumor growth and prolonged mouse survival.Increase of activated anti-tumoral CD4⁺and CD8⁺T cells while decrease of Treg cells in tumor microenvironment conferred the synergistic antitumor efficacy of combination therapy.（2）We identified CD90^high and CD90^low ADSC subsets and demonstrated that CD90^high ADSCs could be converted into CD90^low ADSCs by stimulation with LPS.CD90^low ADSCs and its derived EVs significantly inhibited tumor growth in breast tumor-bearing mice.Benefit of tumor control were associated with decreased tumor cell proliferation and migration,and enhanced tumor cell apoptosis mediated by ADSC-EVs.Antioncogenic miR-16-5p loaded CD90^low ADSC-EVs further significantly enhanced antitumor activities.Conclusion:In this study,we characterized the difference of CD90 expression in CB-MSCs and BM-MSCs and evaluated the anti-tumoral effects of these cells in a mouse model of ovarian cancer.Our data demonstrated the synergistic anti-tumoral efficacy by application of CB-MSCs combined with fusion protein VIC-008 and revealed mechanisms by which CB-MSCs modulates immunity alone and in combination with VIC-008.In addition,we evaluated antitumor activities of CD90^high and CD90^lowow ADSCs,and ADSC-derived primary and miR-16-5p-modified EVs in mouse models of breast cancer.Taken together,this study represents for the first time to apply our newly identified antitumor MSCs and its derived EVs in preclinical treatment of ovarian and breast cancer.This study also provides the evidence that EVs can serve as a novel and effective therapeutics or drug delivery vesicle.