Initial Research On The Inhibition Of Postoperative Recurrence And Metastasis Of Triple Negative Breast Cancer With Durvalumab And PLGA-GEM Gel

At present,the treatment of triple-negative breast cancer（TNBC）is a hot and difficult point of concern for breast cancer.Studies have reported that immunotherapy has a good therapeutic effect on many solid tumors.Therefore,we designed a hydrogel material（a PD-L1+PLGA-GEM@Gel）that can release gemcitabine（GEM）and anti-programmed death ligand receptor 1（a PD-L1）locally in the wound.We established a mouse tumor model of 4T1 cell transplantation and performed incomplete tumor resection（TRS）,using flow cytometry,TUNEL,Ki67,and CD31immunohistochemical methods to evaluate the effects of a PD-L1+PLGA-GEM@Gel combined with radiotherapy（RT）on the recurrence and metastasis of TNBC after TRS.The effects of a PD-L1+PLGA-GEM@Gel+RT on liver and kidney function and organ toxicity of mice were evaluated by blood routine,blood biochemistry and H&E test results.The results showed that a PD-L1+PLGA-GEM@Gel+RT further reduced the size,proliferation,angiogenesis and lung metastasis of breast cancer without significant side effects on the liver and kidney of mice.The above results indicate that in the process of breast cancer postoperative treatment,a PD-L1+PLGA-GEM@Gel+RT promoted immune-mediated tumor regression in tumor-bearing mice and has a good effect of inhibiting tumor recurrence and metastasis.It is suggested that immunotherapeutics combined with GEM nano-formulations may have a good application prospect in the treatment of TNBC.PurposeEstablish a TRS model of TNBC and implant the gel a PD-L1+PLGA-GEM@Gel in the wound after the operation to observe whether the gel can improve the therapeutic effect of TNBC and whether it can inhibit the recurrence and metastasis of TNBC after surgery.It is intended for clinical application Provide experimental basis.Method（1）Characterize the particle size and morphology of PLGA-GEM nanoparticles by particle size analyzer,scanning electron microscope（SEM）and transmission electron microscope（TEM）.High performance liquid chromatography（HPLC）was used to determine the drug loading and encapsulation efficiency of PLGA-GEM,as well as the drug release behavior.Murine 4T1 cells were cultured in vitro,and different concentration gradients of GEM and PLGA-GEM were given to detect the cytotoxicity of PLGA-GEM to 4T1 cells.We established TRS model of TNBC,and used TUNEL,Ki67 and CD31 immunohistochemical methods to explore the mechanism of PLGA-GEM in inhibiting recurrence and metastasis after TNBC.（2）Observe the state of a PD-L1+PLGA-GEM@Gel at 25°C and37°C,and use Cryo-SEM and infrared spectroscopy to further analyze the characterize of a PD-L1+PLGA-GEM@Gel and use the BCA protein quantification kit to determine the drug release rate of a PD-L1+PLGA-GEM@Gel in vitro.We established TRS model of TNBC and detected the expression of CD3,CD4 and CD8 in mouse spleen by flow cytometry.Use immunohistochemical methods to detect the expression of tumor tissue-related proteins,includingγH2AX,TUNEL,Ki67,CD31,and analyze the pathological characteristics of mouse organs（heart,liver,spleen,lung and kidney）by H&E staining to explore a PD-L1+PLGA-GEM@Gel inhibits the immune molecular mechanism of TNBC recurrence and metastasis.At the same time,we tested blood routines（including red blood cells,white blood cells and platelets）and blood biochemistry（AST,ALT,CR,BUN）,and evaluate the biological toxicity of a PD-L1+PLGA-GEM@Gel.Result1 The effect of nanoparticle PLGA-GEM on the recurrence and metastasis of TNBC1.1 Characterization of PLGA-GEMThe particle size of the PLGA-GEM nanoparticles is 223.50 nm,and the zeta potential is-25.40 m V.The SEM image shows that the PLGA-GEM particles have a smooth morphology,and the TEM image further shows that the PLGA-GEM particles are spherical.The drug loading and encapsulation efficiency were 9.28%and 63.7%,respectively.In 0.05%Tween80（v/v）PBS solution（0.02 M,p H 6.0）,0.05%Tween80（v/v）PBS solution（Under the mixed solution conditions of 0.02 M,p H 7.4）and 37°C（0.01 M PBS（p H 7.4）+1%SDS:methanol=60:40）,the GEM release rates after 72 h were0.22%,7.19%and 6.99%,respectively.1.2 The cytotoxicity of different concentrations of GEM and nanoparticle PLGA-GEMThe experimental results of MTT show that GEM and PLGA-GEM have a concentration-dependent effect on the cytotoxicity of 4T1.The IC 50 value of GEM is 0.1537,and the IC50 value of PLGA-GEM is 29.44.In comparison,GEM has a higher cell killing effect.1.3 PLGA-GEM inhibits recurrence and metastasis after TNBCWe established TRS model of TNBC.Through the growth curve and tumor size of the transplanted tumor,we found that the treatment of nanoparticle PLGA-GEM after the operation can inhibit the growth of TNBC in mice.The results of TUNEL immunofluorescence staining showed that the number of positive apoptotic cells in the TRS+PLGA-GEM group was significantly higher than that in the other experimental groups,suggesting that TRS+PLGA-GEM can promote the apoptosis of 4T1 breast cancer cells.The staining results of Ki67 and CD31 indicate that TRS+PLGA-GEM can inhibit the proliferation and angiogenesis of TNBC cells.In addition,compared with the GEM treatment group alone,PLGA-GEM further inhibited the occurrence of lung metastasis.2 The effect of a PD-L1+PLGA-GEM@Gel on the recurrence and metastasis of TNBC2.1 Characterization of a PD-L1+PLGA-GEM@Gela PD-L1+PLGA-GEM@Gel is liquid at 25°C,and solidified at37°C.Under Cryo-SEM,a PD-L1+PLGA-GEM@Gel is fluffy and loose porous structure.Comparing the infrared spectra of the blank hydrogel（Gel）and PLGA-GEM hydrogel（PLGA-GEM@Gel）,it can be found that the infrared characteristic peaks of the two are basically the same.a PD-L1 hydrogel（a PD-L1@Gel）and a PD-L1+PLGA-GEM hydrogel（a PD-L1+PLGA-GEM@Gel）showed-NH₂ stretching vibration peak at 3300cm^-1,N-H bending vibration peak at 1620cm^-1 and C-N bending vibration peak at990cm^-1 both indicate that a PD-L1 is loaded in the hydrogel（the antibody contains a lot of amino groups）.In addition,under the condition of 0.5%Tween 80 in 10 m L PBS（10 m M,p H 6.0）and 0.5%Tween 80 in 10 m L PBS（10 m M,p H 7.4）solution conditions,after72h,the release rates of a PD-L1 in a PD-L1+PLGA-GEM@Gel were50.08%and 43.37%,respectively.2.2 a PD-L1+PLGA-GEM@Gel inhibits recurrence and metastasis after TNBCPerforming TRS on tumor-bearing mice,we found that the a PD-L1+PLGA-GEM@Gel combined with RT can inhibit the growth of TNBC in mice.TUNEL immunofluorescence staining showed that a PD-L1+PLGA-GEM@Gel+RT can promote the apoptosis of 4T1cells.The results of Ki67 and CD31 staining indicate that a PD-L1+PLGA-GEM@Gel+RT can inhibit the proliferation and bloodvesselsof4T1cells.Inaddition,a PD-L1+PLGA-GEM@Gel+RT also produced a systemic anti-immune response that inhibits the growth of distant tumors（lung metastasis）,and it was found through blood routine and blood biochemical tests that the mice treated with a PD-L1+PLGA-GEM@Gel+RT did not show any obvious toxicity.ConclusionThe study revealed for the first time that the postoperative injection of a PD-L1+PLGA-GEM@Gel into the surgical lesion,and then postoperative radiotherapy,can focus on the disease site to break the local immune tolerance,so as not to cause serious side effects,generating systemic anti-tumor immunity,enhancing the sensitivity of chemotherapeutic drug GEM to tumor cells,thereby inducing apoptosis of breast cancer cells,suggesting that our diversified therapeutic strategy of immunization combined with radiochemical therapy may provide a platform for the treatment of recurrence and metastasis of TNBC.