Experimental Study Of Novel PMMA Bone Cement Loaded With TLR-7 Agonist Combined With Anti-CTLA-4 Antibody For Cancer Therapy

In recent years,percutaneous vertebroplasty(PVP)has gradually been used in the treatment of metastatic bone tumors.Polymethyl methacrylate(PMMA)bone cement is the most commonly used filling material in PVP procedure.PVP with PMMA can strengthen the affected vertebrae and relieve pain.Researchers generally believe that PMMA could kill the tumor cells by the hyperthermia during polymerization and the toxicity of MMA monomer,however,the antitumor effect of PMMA bone cement alone is weak and not lasting.Immunotherapy is the current research focus of tumor therapy.A large number of studies have confirmed that immunotherapy can effectively eradicate residual or metastatic tumor in the body.The recognition and presentation of tumor antigens by antigen-presenting cells(APCs)is the most important part of anti-tumor immune response.Imiquimod,also known as R837,is a common Toll-like receptor-7(TLR-7)agonist,whch could stimulate the body to produce anti-tumor immune response by promoting the maturation of dendritic cells(DCs).However,using R837 alone cannot elicit a potent anti-tumor immune response.Anti-cytotoxic T-lymphocyte-associated antigen 4 antibody(aCTLA4ab)is an immune checkpoint inhibitor(ICI)that can enhance the killing effect of the immune cells,and it has also been approved for clinical use.Herein,we propose to load R837 on PMMA to construct a novel bone cement(R837-PMMA)with immunomodulatory function,and hope to combine it with ICI to enhance the anti-tumor immune response produced by R837-PMMA.Thus,multiple tumor models were used to study the efficacy of novel bone cement combined with ICI in the treatment of tumors,and we made a preliminary exploration on its mechanism.The expected research results might offer some new perspectives and new ideas for the treatment of bone metastases.Part ? Experimental study of characterization and performance of novel PMMA bone cement loaded with TLR-7 agonistPurpose:To prepare a novel PMMA bone cement(R837-PMMA)loaded with R837,and to study its characterization characteristics and in vivo immunomodulatory function.Methods:R837 was mixed with methyl methacrylate(MMA)to form R837-MMA by ultrasonic dissolution method.PMMA powder and MMA liquid were mixed at a ratio of 3:2 to construct R837-PMMA.Infrared radiation(IR)thermal imaging camera was used to monitor the temperature of PMMA and R837-PMMA during polymerization.The release of R837-PMMA in the buffer solution was determined by HPLC analysis,and the microstructure of R837-PMMA was observed by scanning electron microscope(SEM).The inhibitory effect of R837-PMMA on the proliferation of different tumor cell lines was tested in vitro by the MMT.Subcutaneous CT26 murine tumor model was used to evaluate the effect of R837-PMMA on DCs maturation.Results:SEM scans showed small molecule R837 was attached to the surface of PMMA.IR thermal images showed both PMMA and R837-PMMA could reach a higher temperature during the polymerization process,and lasted for a long time.Significant differences were observed between the two bone cement regarding maximum temperature(t=11.352,P<0.001).It was shown that R837 could slowly released in the PMMA system,releasing about 15.24%within 3 hours.Both types of bone cement can inhibit the proliferation of tumor cells in vitro,and the cytotoxicity of bone cement was not enhanced after R837 loading.In vivo experiments showed the proportion of mature DCs in draining lymph nodes reached an average of 41.63%after intratumoral injection of R837-PMMA,which was higher than that of PMMA injection alone(p=0.015).Conclusions:R837-PMMA bone cement with immunomodulatory function was successfully prepared,and the heating effect was satisfactory.R837 in the bone cement can be slowly released,which could promote the maturation of DCs.Part ? Experimental study on inhibition of distant tumors by novel PMMA bone cement loaded with TLR-7 agonistPurpose:To evaluate the inhibitory effect of R837-PMMA on distant tumors after treating primary tumor,and to study the effect of R837-PMMA treatment on immune system.Methods:VX2 tumor suspension implantation was used to establish a model of paravertebral primary tumors and distant subcutaneous tumors in rabbits.Eighteen rabbits were randomly divided into untreated control group,PMMA group and R837-PMMA group(n=6 per group).Paravertebral primary tumors were treated with intratumoral PMMA injection and intratumoral R837-PMMA injection,respectively.Antitumor efficacy of R837-PMMA was determined by evaluating distant subcutaneous tumor size,serum cytokine levels,and survival rate.Then a paravertebral tumor model(n=3 per group)was established to evaluate the T cell subsets in peripheral blood.Results:After treating the paravertebral primary tumors,the growth of subcutaneous tumor of rabbits treated with R837-PMMA was inhibited with longer survival time than that of control group or PMMA gourp.Serum IFN-y and TNF-? significantly increased compared to control group(both p<0.05).The results of flow cytometry showed both CD4+and CD8+T cells in peripheral blood also significantly increased.Conclusions:This study confirms that intratumoral injection of the R837-PMMA bone cement could slow the growth of distant tumor and cause an increase in peripheral blood CD4+T cells and CD8+T cells.Part ? Inhibitory effect of a novel PMMA bone cement loaded with TLR-7 agonist combined with anti-CTLA-4 antibody on distant tumor and metastasisPurpose:To evaluate the inhibitory effect on distant tumors and metastasis after treatment of primary tumor by R837-PMMA combined with aCTLA4ab.Methods:CT26 bilateral murine colorectal tumor model and Luc-4T1 orthotopic murine breast cancer model were established to evaluate the efficacy of R837-PMMA combined with aCTLA4ab.After various treatments,breast cancer metastasis was monitored by IVIS,and CT26 distance tumor size were measured.Serum cytokines such as IFN-y,TNF-?,IL-6,IL12p70 were detected by ELISA.The survival of mice were observed.Results:In murine CT26 bilateral tumor model,volume of distant tumor in R837-PMMA combined with aCTLA4ab group was significantly smaller than that of the other 4 groups(p<0.001).In Luc-4T1 orthotopic murine breast cancer model,IVIS images showed that combination therapy significantly inhibits and delays the onset of metastasis.In both murine tumor models,R837-PMMA combined with aCTLA-4ab could significantly prolong survival and increase serum TNF-?,IFN-y,IL-6,IL12p70 levels.Conclusions:This study confirms that intratumoral injection of the R837-PMMA could induce anti-tumor immune response,and combining R837-PMMA with aCTLA4ab could enhance the anti-tumor immune response and further inhibit the development of distant tumor growth and metastasis.Part ? Mechanism study of anti-tumor immune response produced by novel PMMA bone cement loaded with TLR-7 agonist combined with anti-CTLA-4 antibodyPurpose:To investigate whether R837-PMMA combined with aCTLA-4ab could produce immune memory effects to suppress rechallenged tumors,and to elucidate the mechanism of combined therapy on inhibiting distant,metastastic and recurrent tumors.Methods:Murine CT26 bilateral tumor model was established,and distant tumors were collected on the 7th day after the primary tumors eliminated.Fifteen mice were randomly divided into 5 groups:surgery,PMMA,R837-PMMA,PMMA+aCTLA4ab,and R837-PMMA+aCTLA4ab,with 3 in each group.Distant tumors were collected on day 7 after treatment of primary tumors in each groups,and the contents of CD8+cytotoxic T cells(CTL),CD4+effector T cells(Teff),and regulatory T cells(Treg)in distant tumors were studied by flow cytometry.Then a CT26 unilateral murine tumor model was established(n=9 per group)and eliminated by various treatments.On the 40th day after primary tumor treatment,3 mice were sacrificed before rechallenge in each group and the contents of central memory T cells(TCM)and effector memory T cells(TEM)in the spleen were studied by flow cytometry.The remained 6 mice in each group were rechallenged with CT26 tumor cells.Tumor size,survival time,and serum TNF-? and IFN-? levels on the 6th day after tumor rechallenge were observed to evaluate the immune memory effect.Results:After eliminated the primary tumors with R837-PMMA combined with aCTLA-4ab,the proportion of CD3+T cells in distant tumors was the highest.The proportions of CD8+CTL was 13.31%,accompanied by a significant increase in CTL/Treg and Teff/Treg ratios.In long-term immune memory studies,mice treated with R837-PMMA combined aCTLA-4ab had a significantly higher proportion of TEMs in the spleen before tumor rechallenge.Serum TNF-? and IFN-y levels were significantly increased on the 6th day after tumor rechallenge.The rechallenged tumor growth was significantly inhibited and the survival time was significantly prolonged.Conclusions:R837-PMMA combined with aCTLA-4ab therapy can enhance the antitumor immune response by promoting the infiltration of T cells,especially CD8+CTL into distant tumors,and blocking the inhibitory effect of Treg on T cells through aCTLA-4ab.The combined treatment could also generate strong immune memory effect to suppress recurrence of the tumor.