| BackgroundPD-L1 highly expressed on various tumor cells surface,binding to PD-1 expressed by tumor infiltrating T cells,inhibiting the function of T cells,and mediating tumor immune escape.Therefore,blocking the PD-1/PD-L1 signaling pathway is one of the most important ways to reverse the "dysfunction" of T cells and restore the anti-tumor immune response.At present,monoclonal antibodies such as PD-1 or PD-L1 are commonly used to block this signaling pathway,but the response rate of this therapy is extremely low.Only about 20%of patients can produce an effective response,and some patients develop resistance in subsequent treatment.The combination therapy based on immune checkpoint blockade may increase response rates,prolong response times,and even activate anti-tumor-related immune memory.Oxaliplatin is a third-generation platinum-based chemotherapeutic drug that exhibits a broad spectrum of in vitro cytotoxicity and in vivo antitumor activity in a variety of tumor models.Despite this,tumor patients receiving chemotherapy are highly recurrent due to secondary expansion of immunosuppressive cells lead to failure of immune function,and development tolerance to chemotherapeutic drugs.Under this premise,adding chemotherapy to the immunotherapy regimen is expected to increase the sensitivity of the tumor to the immunological checkpoint,enhance the anti-tumor effect,and reduce the probability of tumor recurrence.Recent studies have shown that certain cytotoxic chemotherapeutics(such as oxaliplatin and doxorubicin)can activate the immune system by inducing immunogenic cell death(ICD)in tumor cells,promote DC cell maturation to uptake of tumor-associated antigens and enhance anti-tumor immunity.In view of the above studies,we constructed a short hairpin RNA(shRNA)recombinant vector targeting PD-L1,interfering the expression of PD-L1 and block the PD-1/PD-L1 signaling pathway.At the same time,the chemotherapy drug oxaliplatin was administered to study the effect of the combination therapy on tumor treatment.Methods1.Design and synthesize a short hairpin RNA sequence targeting PD-L1 suitable for constructing pLKO.1 lentiviral vector.The pLKO.1 recombinant vector was constructed successfully.2.Using lentiviral packaging technology to obtain lentiviral particles containing the shRNA of interest,select mouse melanoma cell line B16 with high expression of PD--L1 as target cells,and use lentiviral particles for in vitro transduction.The expression of PD-L1 was detected by qPCR and flow cytometry.3.Select the appropriate treatment concentration by plotting the concentration-inhibition rate of oxaliplatin on tumor cells.The transduced tumor cells were ligated into 96-well plates at 5000 cells per well.After 4 hours adherence,oxaliplatin was added to the cells,and cell proliferation was detected by MTT assay.4.Setting control group,shPD-L1 group,oxaliplatin group and combination group.Apoptosis was detected by Annexin V/PI double staining after 24 h.5.The effect of silencing PD-L1 and oxaliplatin on cell cycle was detected by PI single staining.6.The effects of PD-L1 and oxaliplatin on cell migration were examined by Transwell chamber and scratch assay.7.Establish a subcutaneous tumor-bearing model of C57/BL6J mice,and set doses of three oxaliplatin at 1.5 mg/kg,3 mg/kg,and 6 mg/kg,once every three days,and three times.The tumor volume after treatment was compared,and immunological indicators such as DC activation and T cell typing of tumor sites and various immune organs were detected by flow cytometry,and the therapeutic dose was selected.8.Establish a subcutaneous tumor-bearing model of C57/BL6J mice,intraperitoneal injection of oxaliplatin,and transfection of shPD-L1 in vivo with transfection reagents.Comparing tumor volume and lymphocyte populations of tumor tissues.Result1.Sequencing results showed that a lentiviral vector targeting silencing PD-L1 was successfully constructed.2.After lentiviral transduction,PD-L1 in B16 and Hepal-6 cells was significantly down-regulated in mRNA and protein levels.3.Oxaliplatin and PD-L1 expression can inhibit the proliferation of B16 and Hepal-6 to a certain extent,and the inhibitory effect on proliferation after combination is enhanced.4.Low concentration of oxaliplatin does not cause tumor cell apoptosis,while silencing PD-L1 promotes tumor cell apoptosis.5.Low concentration of oxaliplatin can significantly block the cell cycle,but silencing PD-L1 has little effect on the development of the cycle,and the cycle block is enhanced after the combination.6.Low concentration of oxaliplatin and down-regulation of PD-L1 expression in tumor cells can inhibit Hepal-6 migration to a certain extent,and the inhibitory effect enhanced after combination.7.All three therapeutic doses inhibit tumor growth,activate DC,and up-regulate the proportion of CD4+ T cells and CD8+ T cells.Finally,the lowest dose of 1.5 mg/kg was selected as the combined dose.8.After combined treatment,further inhibit tumor growth,significantly up-regulate the proportion of CD8+ T cells,NK cells and memory T cells in the tumor infiltration site,and promote the expression of IFN-?.Conclusions1.In vitro experiments confirmed that interfering PD-L1 expression can promote apoptosis to inhibit cell proliferation;while low concentrations of oxaliplatin inhibit proliferation by blocking cell cycle progression.Therefore,cell proliferation is further inhibited by the combined action of apoptosis and cycle arrest after combination.In addition,we observed that the combined treatment was better than the single treatment in the cell migration experiment.The rationality of the combination therapy was verified in vitro.2.In vivo experiments,we demonstrated that lower doses of oxaliplatin have a significant inhibitory effect on tumor growth and activate anti-tumor immune responses.The therapeutic effect of the tumor is enhanced after combined with PD-L1 blockade,indicating that the combined strategy has certain feasibility.3.The research results of this subject expand the clinical application of immunological checkpoint blockade and traditional chemotherapy drugs,and also provide brand-new ideas for cancer treatment... |
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