| Background:Breast cancer is one of the most prevalent malignancies in the world.In 2020,breast cancer surpassed lung cancer(2.21 million cases)for the first time and became the largest cancer in the world,with 680,000 deaths from breast cancer worldwide.Triple-negative breast cancer(TNBC)is the most severe subtype of breast cancer,mainly treated with surgery and chemoradiotherapy,but there is a lack of effective targeted therapy.Tumor cell-derived microparticles(TMPs)are a class of microparticles secreted by tumor cells,which have lipid bilayer membrane structure and carry a large number of DNA,RNA and proteins derived from parental cells,and play an important role in information exchange between tumor cells and between tumor cells and other cells.Recent studies have confirmed that the integrity and stability of tumor cell-derived microparticle structure has good biocompatibility with tumor cells,and can carry large-sized biological agents including chemotherapeutic drugs,targeted therapy drugs and oncolytic viruses,which is one of the most promising anti-tumor drug delivery carriers.In addition,TMPs carry a large number of antigens derived from tumor cells and can be applied in the development of tumor vaccines.Radiotherapy(RT)is one of the conventional methods used to treat cancer and promotes tumor cell apoptosis mainly by inducing DNA damage.Recent studies have shown that it can also facilitate exposure of tumor antigens to host immune cells.However,radiotherapy alone is often difficult to activate an effective antitumor immune response due to the low immunogenicity of tumor cells and the formation of a strong immunosuppressive microenvironment in tumors.Combination therapy is a potential solution to reverse this unfavorable condition.The application of TMPs as delivery carriers of anti-tumor drugs,combined with a novel therapeutic strategy of radiotherapy,may not only enhance the killing effect of the two on tumor cells,but also facilitate the stimulated immune response,which can enhance the anti-tumor effect through a variety of modes of action and is expected to provide a new therapeutic regimen for refractory tumors such as triple-negative breast cancer.Objective:We will investigate the feasibility of combining tumor cell-derived microparticle-loaded paclitaxel(MP-PTX)with radiotherapy as a novel treatment modality for TNBC treatment.Methods:1.Characterization of MP-PTX:The morphology,particle size,zeta potential and in vitro stability of MP-PTX were investigated by transmission electron microscopy(TEM)and dynamic light scattering(DLS);the characteristics of its protein components were investigated by whole protein mapping technique;and the drug loading was detected by high performance liquid chromatography(HPLC).2.Detection of tumor killing ability of MP-PTX in vitro:In order to clarify the feasibility of TMPs as drug carriers combined with radiotherapy,we first detected the uptake efficiency of tumor cells into drug-loaded tumor cell-derived microparticles;MTT assay was used to evaluate the killing effect of different treatment groups on 4T1 or MAD-MB-231 cells;scratch assay and plate cloning assay were used to evaluate the effect of different treatment groups on the proliferation of 4T1 or MAD-MB-231 cells;flow cytometry and hoechst33342 staining assay were used to detect the apoptosis induction of 4T1 or MAD-MB-231 cells in different treatment groups;3.Establish a subcutaneous xenograft model of triple-negative breast cancer,administer it by tail vein injection,investigate the metabolism of mouse tumors after treatment with each treatment system using PET/CT imaging technique,and then investigate the distribution of drug-loaded microparticles in mice using mouse in vivo imaging technique;Lung tissues were dissected and lung metastasis was evaluated in each treatment system;HE staining was performed on important organs to evaluate the toxic and side effects of different treatment systems;TUNEL staining and HE staining were performed on tumor tissues to evaluate the induction of tumor cell apoptosis by different treatment systems;immunohistochemical analysis was performed on tumor tissues to investigate the anti-tumor effect and anti-tumor mechanism of different treatment systems.Results:1.TEM images show that both MP and MP-PTX have vesicular morphology with diameters ranging from 100 nm to 500 nm.The particle size of MP measured by dynamic laser scattering was consistent with the TEM results.The zeta potential of MP and MP-PTX were similar,indicating that PTX loading did not cause significant effects on the surface potential of MP.MP-PTX was stable at different temperatures,and these results suggest that MP-PTX has high stability;MP-PTX and MP have similar protein profiles in tumor cells;the corresponding protein profiles could be detected in MP-PTX;paclitaxel loading in MP-PTX was positively correlated with the drug dose added to the medium;2.Cellular uptake experiments demonstrated that MP loading group could improve the drug uptake efficiency of tumor cells.In vitro cytotoxicity experiments showed that the killing power or efficiency of the drug could be enhanced after MP encapsulation.In addition,the cytotoxicity of MP-PTX combined with radiotherapy was significantly higher than that of MP-PTX,indicating the enhanced inhibitory effect of MP-PTX combined with radiotherapy on cell proliferation.3.Small animal live imaging showed that almost no fluorescence signal was detected in the tumor-bearing mice in the saline group,while fluorescence signal in the lungs could be detected in the group injected with Di R.However,in the group injected with Di R-MP-PTX,in addition to fluorescent signals detected in the lung,obvious fluorescent signals could also be detected in the tumor site,indicating that Di R-MP-PTX had a good targeting effects on tumor tissues;In vivo antitumor experiments showed that MP-PTX combined with radiotherapy significantly inhibited the proliferation of tumor cells,thereby reducing the tumor weight and volume,reducing lung metastases,and prolonging the survival time of tumor-bearing mice.Further studies showed that CD4~+,CD8~+and CD86~+cells were significantly increased in the tumors of the MP-PTX combined with radiotherapy group,while the number of CD206~+cells was significantly reduced,indicating that this combined treatment regimen can effectively stimulate the anti-tumor immune response and reverse the immunosuppressive tumor microenvironment.Conclusion:In this study,we successfully prepared breast cancer cell-derived MP-PTX,which has the advantages of good stability and high tumor targeting.The results of in vitro and in vivo studies showed that the combination of MP-PTX and radiotherapy significantly inhibited tumor cell proliferation and enhanced the killing effect on tumor cells.More importantly,this combination therapy could effectively alleviate the immunosuppressive tumor microenvironment and enhance the antitumor immune killing effect.Our study provides a novel therapeutic strategy for refractory tumors,including TNBC,with promising clinical applications. |
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