| Background: Tumor-associated macrophages(TAMs)are the most abundant immune cells in solid tumors,and their proportion is even as high as 50% in ovarian cancer.The role of TAMs is similar to that of M2 macrophages,which is involved in chemotherapy resistance,invasion,metastasis,and immunotherapy invalidation in ovarian cancer.Nanodrug can change the pharmacokinetics and biodistribution of drugs by precisely regulating the particle size and surface properties of nanoparticles,promote drug targeted delivery to tumor,and show the advantages of more efficient tumor treatment.Therefore,in this study,PLGA nanocarrier was used to encapsulate the immune adjuvant Cp G-ODN,and coated with ovarian cancer cell membrane,thus the tumor vaccine of PLGA-Cp G@ID8-M NPs was constructed.In addition,the polarization of TAMs in the tumor microenvironment as well as the antitumor effects by nano-tumor vaccines were also explored,and the mechanism of nano-tumor vaccines regulating macrophages M1 polarization was further explored,so as to provide a new strategy for the treatment of ovarian cancer.Methods:1.Construction of nano-tumor vaccine: PLGA-Cp G nanoparticles were constructed by ultrasound double emulsion method(W-O-W),and mouse ovarian epithelial-derived ID8 tumor cell membrane were coated on PLGA-Cp G nanoparticles by liposome extrudator to form nano-tumor vaccines(PLGA-Cp G@ID8-M).2.Characterization of nano-tumor vaccines: The particle size,charge,morphology and stability of the nanovaccines were characterized by transmission electron microscopy(TEM)and dynamic light scattering(DLS).The loading capacity and loading efficiency of the nanoparticles were measured by ultraviolet and visible light spectrophotometer.The antigenic properties of the cell membrane on the surface of the nano-tumor vaccines were detected by coomassie blue staining.3.Evaluation of targeting and safety of nano-tumor vaccines: The Di I fluorescent dye was used to label the nano-tumor vaccines to detect the phagocytosis of macrophages.The cell cytotoxicity of nano-tumor vaccines was detected by CCK8 assay.The ID8 subcutaneous tumor model was constructed,and the tumor targeting ability of the nano-tumor vaccines was detected by IVIS imaging system.HE staining and biochemical test kits were used to detect the toxic and side effects of nano-tumor vaccines on major organs of mice.4.Detection of M1 macrophages regulated by nano-tumor vaccine: In vitro experiments,bone marrow-derived macrophages(BMDMs)and RAW264.7 cells were stimulated with nano-tumor vaccines to detect the immune stimulation and differentiation effects.The M1 and M2 differentiation markers of macrophages were detected by RT-PCT and flow cytometry,and the secretion of inflammatory factors was detected by ELISA kit.Western blot and immunofluorescence were used to verify the differentiation markers of M1 and M2 macrophages.5.Evaluation of antitumor effect of nano-tumor vaccine: The intraperitoneal metastasis model of ID8-Luci tumor was established,and the antitumor effect of the nano-tumor vaccines was detected by IVIS imaging system.Subcutaneous ID8 tumor model was constructed,and the tumor growth curve was measured to evaluate the antitumor effect of the nanovaccine.In addition,part of the tumor tissues were lysed into single cells,and the proportion of M1 and M2 macrophages in the tumor tissues were detected by flow cytometry.Furthermore,the proportion of M1 and M2 macrophages in tumor tissues of each group was further verified by multiple immunohistochemistry.6.Discussion on the mechanism of macrophages M1 polarization regulated by nano-tumor vaccine:After the intervention of BMDMs with nano-tumor vaccines,transcriptome sequencing analysis was performed,and single cell sequencing data of high-grade ovarian cancer tissues before and after chemotherapy were obtained through the analysis of database GSE165897,and to screen and verify whether upregulation of Gbp2 protein was involved in the promotion of macrophage M1 polarization.To screen and verify whether Pin1 was interacted with Gbp2 by Co-IP technology combined with protein spectrum and pull-down technology,and to verify whether Pin1 was involved in the regulation of macrophage M1 polarization.By transcriptome sequencing and GSEA analysis as well as the use of Gbp2 overexpression and knockdown lentiviru s,si RNA,Pin1 inhibitor and NFk B inhibitor,together with tumor models by co-transplantation with RAW264.7 cells and ID8-Luci cells or 4T1 cells,we explored whether PLGA-Cp G@ID8-M NPs promote the M1 polarization of macrophages and exert antitumor effects by up-regulating Gbp2/Pin1 and activating NFk B signaling pathway.Results:1.The PLGA-Cp G@ID8-M NPs with a particle size of 144 nm was successfully constructed by ultrasonic double emulsion method and liposome extrusion.The surface δpotential was-29.4m V,and the drug loading efficiency was up to 92%.In addition,PLGA-Cp G@ID8-M NPs have good stability.2.In vitro and in vivo experiments showed that the nano-tumor vaccines could be phagocytosed by macrophages,and the nanovaccines modified by tumor cell membrane could be significantly enriched in tumor tissues of ovarian cancer.CCK8 assay confirmed that the nano-tumor vaccines had no significant effect on cell survival within the effective concentration range as well as no obvious toxic and side effects in vivo confirmed by H&E staining and serum biochemical test kit assay.3.The results of RT-PCR showed that M1 macrophages biomarkers(IFN-γ,TNF-α,i NOS)were significantly increased and M2 macrophages biomarkers(CD206,CD163,Arg1)were significantly decreased in BMDMs treated with nano-tumor vaccines for 12 hours.Flow cytometry analysis showed that the expression of CD80,a surface M1 biomarker of BMDMs,was increased,while CD206 was decreased.ELISA showed that the secretion of IFN-γ,TNF-α and IL-6 was significantly increased.In addition,immunofluorescence and Western blot results showed that the expression of i NOS was increased and Arg1 was decreased in RAW264.7 cells treated with nano-tumor vaccines for 12 hours.4.Nano-tumor vaccine can significantly inhibit the growth of ovarian cancer in intraperitoneal injection tumor model and subcutaneous tumor model.In addition,nano-tumor vaccine can significantly inhibit the growth of ovarian cancer in combination with chemotherapy.Flow cytometry and multiple immunohistochemistry analysis of tumor tissues showed that the nano-tumor vaccines could promote the transformation of TAMs into M1 macrophages.5.Transcriptome sequencing combined with single cell sequencing of ovarian cancer showed that nano-tumor vaccine may be involved in macrophage M1 polarization through regulation of Gbp2,and KEGG and GSEA analysis showed that NFk B signaling pathway may play an important role in this process.TCGA database analysis showed that Gbp2 expression was higher in normal ovarian tissues than that of ovarian cancer tissues.In ovarian cancer patients,the patients with higher expression of Gbp2 in ovarian cancer tissues have better prognosis.Finally,GEPIA database analysis showed that G bp2 was significantly expressed in M1 macrophages compared with M2 macrophages in ovarian cancer tissues.6.After RAW264.7 cells were infected with Flag-Gbp2 lentivirus,the Gbp2 interacting protein Pin1 was screened by Co-IP technology combined with protein spectrum.The GST-Gbp2 and His-Pin1 plasmids were constructed,and the fusion protein was purified,and it was confirmed that Gbp2 and Pin1 had direct protein interaction involved in the regulation of macrophage M1 polarization.7.After Gbp2 overexpression in RAW264.7 cells,the protein expression of Pin1 and p-NFk B was increased,while the protein expression of IL-1β(M1 marker)was increased and the protein expression of Arg1(M2 marker)was decreased.The protein expression of Pin1 and p-NFk B decreased after Gbp2 knockdown.After Gbp2 overexpression,inhibition of Pin1 expression or inhibition of NFk B expression can antagonize Gbp2-induced M1 polarization of macrophages.Finally,co-transplantation of RAW264.7cells overexpressing Gbp2 with ID8-Luci cells or 4T1 cells significantly inhibited tumor growth and prolonged the survival of mice.Conclusions:In this study,we constructed a novel nanotumor vaccine(PLGA-Cp G@ID8-M)and demonstrated its anti-ovarian cancer effect by promoting macrophage M1 polarization.At the same time,it is revealed that PLGA-Cp G@ID8-M can promote the polarization of macrophage M1 to play an anti-ovarian cancer effect by up-regulating Gbp2/Pin1 and activation of NFk B signaling pathway.This study provides a new strategy and a new target for the treatment of ovarian cancer... |
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