| Background:Neuroblastoma(NB)is the most common extracranial solid tumor in children,accounting for about 8%of all pediatric tumors.It is an embryonal tumor of the autonomic nervous system,and its median age at diagnosis is about 18 months.Genome-wide association studies(GWAS)have shown that neuroblastoma is a complex genetic disease associated with common polymorphic alleles that can affect tumorigenesis.At present,12 groups of susceptibility loci which are highly related to the occurrence of neuroblastoma have been identified,which provide potential drug screening targets for molecular targeted therapy of NB.However,the current drug treatment of NB is still dominated by traditional chemotherapy,although its tissue selectivity is poor,and long-term administration can cause many adverse reactions such as liver and kidney toxicity,bone marrow suppression,etc.,which has severely limited the clinical application of this drug.In recent years,with the rise of immunotherapy,tumor-targeted immunotherapy is considered to be the most potential drug treatment strategy,bringing new hope to tumor patients.However,although immunotherapy has made great breakthroughs in adult tumors,its application in NB is still in its infancy,and the clinical benefit of the existing representative treatment drug dartuximab β is also limited,due to the inhibitory factors in immune microenvironment,including immunogenicity of tumor cells,infiltration and activity of immune cells,and inhibition of immune cells by tumor cells.The current problems in the clinical treatment of NB are as follows:the exploration of the genetic and molecular biological characteristics of neuroblastoma is not thorough enough,and there are few potential drug screening targets;immunotherapy research is still in its infancy,and the choice of immunotherapy drugs is extremely limited.less;the overall efficacy of high-risk neuroblastoma is poor and so on.This experiment was carried out in order to further explore the molecular biological targets of NB and develop immune preparations against the tumor immune microenvironment.Aim:1.Taking YTHDC1 gene as the research object,from the perspective of epigenetics,carry out genotype analysis based on a large number of clinical samples and explore the correlation between YTHDC1 gene single nucleotide polymorphisms and the occurrence and development of NB.2.Explore the efficacy and mechanism of nano-immune adjuvant loaded with cGASSTING pathway agonist ADU-S100 on neuroblastoma both in vitro and in vivo,provide experimental support for the development of such nano-preparations,and offer new targeted immunotherapy to "Cold tumor" with immunogenicity and low tumor mutation burden.Methods:Based on the above facts,this thesis mainly reports two parts of the work:In the first part of the study,a multicenter case-control study was carried out in eight hospitals in China with the YTHDC1 gene as the research object.A total of 898 NB patients and 1734 healthy volunteers were recruited to extract fresh tumor tissue or tumor paraffin from NB patients.DNA from slices and blood samples from healthy children were genotyped,and 3 polymorphic loci(rs2293596 T>C,rs2293595 T>C,rs3813832 T>C)of the YTHDC1 gene were selected for analysis.siRNA corresponding to the gene was constructed for verification the role of this gene on NB cell tumor biology.The second part of the work used a "one-pot method" to construct a metal polyphenol nanoparticle TA-Mn/S100 NPs co-loaded with ADU-S100 immune agonist for comprehensive regulation of the NB tumor immune microenvironment to achieve NB targeting immunity therapy.The nano-formulation is formed by the coordination of polyphenolic material tannic acid(TA)and Mn2+,and encapsulates the cGAS-STING pathway agonist ADU-S100.The tumor immunological effect fully activates the anti-tumor immune pathway.Firstly,the basic pharmaceutical properties such as the hydrated particle size,morphology,and spectroscopic characteristics of the nanoparticles were systematically characterized,and the nanoparticles were explored using inductively coupled plasma optical emission spectrometer(ICP-OES)and high-performance liquid chromatography(HPLC).In vitro release behavior.On the basis of clear formulation characteristics,it was verified at the cellular level that the nanoformulation could be effectively delivered to tumor cells and induced immunogenic cell death.The activation of the cGAS-STING pathway by nano-formulation at protein and gene levels was verified,and Activation of antigen presenting cells.Finally,a mouse subcutaneous tumor transplantation model was constructed,and the anti-tumor effect of the nano-formulation was evaluated by systemic administration.Results:1.We found that at the polymorphic site rs3813832 T>C,TC genotype can significantly reduce the susceptibility of NB;in addition,the results of combined genotype analysis showed that individuals with three protective genotypes had significantly lower risk of NB than other carriers Individuals with 1-2 protective genotypes.At the same time,we constructed the corresponding siRNA to interfere with the YTHDC1 gene in two NB cell lines,and used a series of molecular biological methods(CCK-8,Western Blot,scratch assay and migration assay)to verify the promotion of this gene on tumor cells.effect.2.The constructed immunomodulatory nano-formulation has an amorphous structure of about 180 nm,which has the typical properties of metal coordination polymers.The particle size of the nanoparticles is uniform and the colloidal stability is good.Both UV-Vis and IR absorption spectra proved the effective encapsulation of the drug,and the encapsulation rate was 54%.In vitro release studies have found that the drug can not only be stably encapsulated in a physiological environment,but also release the drug in response to intracellular slightly acidic organelles(such as endosomes/lysosomes)and high glutathione levels,with intelligent drug release characteristics.3.The preparation can be efficiently transported into cells,induce apoptosis,and activate the cGAS-STING pathway to stimulate immunological effects.In vivo pharmacodynamic studies have shown that the nano-formulations have good tumor suppressive activity,and immunopathological studies have confirmed that the anti-tumor activity mainly comes from the regulation of the tumor immune microenvironment,including the activation of DC cells,the activation of T cells,and the activation of macrophages.At the same time,the in vivo application safety of the nano-formulation was evaluated,and it was found that there was no significant change in the body weight of the mice during the treatment process,and no changes in the major organs of the mice were found in the histopathological characterization after treatment,suggesting the excellent biocompatibility of the nano-formulation.Conclusion:1.A significant correlation was found between genetic variants of the m6A-modifying gene YTHDC1 and the risk of NB,and this finding has important implications for the pathogenesis,epidemiological features,epigenetic properties of NB.In addition,it has important reference significance for the design of new targets for anti-tumor drugs.2.Aiming at the molecular biological mechanism of YTHDC1 gene promoting tumorigenesis and development and the tumor immunology characteristics of NB,we used metal polyphenol nanoparticles to co-load the immune agonist ADU-S100 for the first time to synergistically activate the cGAS-STING pathway and induce tumor cell apoptosis.At the same time,it activates antigen-presenting cells,increases the infiltration of lymphocytes in the tumor microenvironment,and regulates the tumor immunosuppressive microenvironment of NB.3.This study provides an experimental basis for elucidating the correlation between the YTHDC1 gene and susceptibility to NB,and also offers new strategies of tumor targeted immunotherapy for "cold tumor" targeted immunotherapy lots of "cold tumors" which has low immunogenicity and low TMB,such as NB. |
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