| Due to the lack of effective therapeutic targets for triple negative breast cancer(TNBC),surgery and conventional chemotherapy are the most prominent treatment modalities,but still exhibit high recurrence and mortality rates.Immunotherapy is a promising therapeutic modality,but has limited therapeutic effect when applied alone for TNBC.Chemoimmunotherapy is a promising therapeutic modality that combines the cytotoxic and immunomodulatory effects of chemotherapeutic agents with immunotherapeutic strategies to synergistically combat tumors.However,improving the efficacy of synergistic therapy and reducing the side effects of multiple drugs remain major challenges for chemoimmunotherapy.The emergence of drug delivery systems in the last decades is one of the most promising strategies to address this issue.The use of drug delivery systems as vehicles for chemoimmunotherapy can extend the half-life of drugs,increase drug accumulation,and target drug delivery to specific targets,thus minimizing the impact on non-target tissues and providing a more precise and targeted treatment modality for chemoimmunotherapy of TNBC.Living cells can be used as novel therapeutic and drug carriers with high specificity and persistence.Therapeutic drugs are usually loaded into cells or immobilized on the cell membrane surface using physical,chemical and material methods to confer new therapeutic functions to the cells.Previous studies have found that "dead cells" prepared by rapid freezing of live tumor cells in liquid nitrogen retain the primary structure and tropism of the source cells toward the lesion site,but lose their proliferative capacity and pathogenicity,making them ideal drug carriers.In addition,the fact that cell surface proteins and tumor associated antigens(TAAs)are preserved intact in the cells allows the cells treated with liquid nitrogen to act as tumor vaccines,activating and inducing dendritic cell(DC)maturation and promoting anti-tumor specific immune responses.When tumor cells die in response to external stimuli,the process of transformation from non-immunogenic to immunogenic to mediate a specific anti-tumor immune response is called immunogenic cell death(ICD).During the adaptive immune response and plasma membrane permeation triggered by ICD,dying tumor cells release multiple damage-associated molecular patterns(DAMPs),which bind to pattern recognition receptors on antigen-presenting cells such as DCs,activating DC maturation and phagocytosis of tumor antigens,which are subsequently presented to T lymphocytes,thus allowing the immunologically active host to trigger specific immune responses against dead cell-associated antigens in the absence of any adjuvant.Conventional chemotherapeutic drugs can not only induce apoptosis of tumor cells,but also exert immunomodulatory effects by inducing ICD to promote tumor specific immune response or eliminating immunosuppressive tumor microenvironment.In addition,the immunomodulatory effect of chemotherapy can be combined with immune checkpoint blockade therapy to promote DC chemotaxis and thus enhance the immunotherapeutic activity.Based on the above,we designed liquid nitrogen-treated(LNT)cells and loaded them with paclitaxel(PTX),a chemotherapeutic agent with ICD-inducing effect,as a carrier to prepare a cellular drug delivery system(PTX@LNT),and explored its potential application and mechanism of action in tumor treatment and prevention.In addition,we designed a combination therapy strategy of PTX@LNT with programmed cell death-ligand 1(PD-L1)inhibitor to investigate the therapeutic effect of the combination therapy on metastatic tumors and to realize the synergistic treatment of chemotherapy and immunotherapy.This study was divided into two parts.In the first part,we prepared breast cancer cells treated with liquid nitrogen freezing shock(LNT cells),and a cellular drug delivery system(PTX@LNT)based on LNT cells loaded with the chemotherapeutic drug PTX.The LNT cells,which were rapidly frozen in liquid nitrogen,retained their intact cell structure but lost their proliferative capacity and pathogenicity,making them ideal drug carriers.In addition,the endogenous danger signals and TAAs of the tumor cells were retained intact in the LNT cells,allowing the LNT cells to serve as an exogenous vaccine to stimulate DC maturation and elicit specific anti-tumor immune responses in the organism.PTX is one of the most commonly used chemotherapeutic agents for the treatment of TNBC,which not only directly kills tumor cells,but also induces ICD in tumor cells and increases the immunogenicity of tumor by releasing TAAs and DAMPs,promoting DC maturation and presenting antigen to T lymphocytes.We loaded PTX into LNT cells by mixed incubation,and the obtained PTX@LNT drug delivery system has the following advantages:(1)highly efficient drug delivery efficacy with a 50% drug delivery rate;(2)effective chemotherapy-based ICD induction,PTX@LNT has similar cytotoxic effects and ICD induction ability as free PTX,by increasing secretion of DAMPs,which improves the immunogenicity of tumors and thus induces DC maturation;(3)retains tumor antigens and endogenous danger signals,and is an immunostimulatory delivery system for inducing tumor antigen-specific immune responses;(4)has a simple preparation process and has a wide clinical application prospect.In the second part,the anti-tumor effects and immunomodulatory mechanisms of PTX@LNT were evaluated in a mouse TNBC model with a combination of a PD-L1 inhibitor for the treatment of lung metastases from breast cancer.LNT cells do not have proliferative capacity and pathogenic effect in animal models and can be used as an exogenous vaccine to effectively prevent tumor growth.PTX@LNT drug delivery system shows the strongest tumor suppressive effect in a tumor-bearing mouse model,PTX kills tumor cells while triggering ICD effect and enhances the immunogenicity of the tumor.Furthermore,LNT cells with immune activating effects induced antigen-specific immune cell responses,which synergistically increased the level of immune cell infiltration in tumor tissues with ICD effects and enhanced the antitumor therapeutic effect.PTX@LNT significantly promoted the infiltration of mature DCs and CD8+ cytotoxic T lymphocytes in tumor tissues,and improved the tumor microenvironment by reducing the proportion of regulatory T cells,and improved the tumor microenvironment of TNBC immunosuppression by reducing the proportion of regulatory T cells.However,the application of PTX led to enhanced PD-L1 expression in tumor tissues,so we combined PTX@LNT treatment with a PD-L1 inhibitor to effectively inhibit tumor lung metastasis and prolong survival time in mice by blocking the PD-1/PD-L1 pathway,reactivating the function of tolerant T cells and increasing their killing effect on tumors.In summary,this paper successfully constructed a therapeutic system for TNBC based on the LNT cellular drug delivery system by loading the chemotherapeutic drug PTX,which has shown great potential in the treatment of primary tumors and lung metastases.PTX@LNT has the immunostimulatory effect of LNT cells and the ICD-inducing ability of PTX,which can synergistically activate tumor-specific immune responses while killing tumor cells The combined therapeutic strategy with immune checkpoint inhibitors further enhances the therapeutic effect and has a wide clinical application prospect. |
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