Regulating The Tumor Microenvironment Of Pancreatic Cancer By Nanoimmunosystem And Its Synergistically Mechanism In Promoted Chemoradiotherapy

The absence of tumor-specific biomarkers limits the early detection of Pancreatic ductal adenocarcinoma(PDAC),which leads to bad preoperative conditions.Clinically,the anatomical obstacle further increases the surgerical difficulities and the easy postoperative relapse shortens survival time.Therefore,chemoradiotherapy and comprehensive therapy are preferred methods for treating advanced pancreatic cancers.However,the unique property of the tumor microenvironment(TME)strongly lowers the efficiency of comprehensive treatments,which is characterized by a desmoplastic stroma containing large amount of hyaluronan(HA)and abnormal vessels.Such dense stroma is the main obstacle that suppresses the intratumor drugs accumulation and infiltration of immune cells(T cell and NK cell).On the other hand,in the TME,pancreatic stellate cells can improve the radiation-toleration of pancreatic cancer cells.Additionally,the high expression of PD-L1 and immunosupressive factors in TME further inhibit the effect of cytotoxic T cells.Therefore,in clinics,re-construction of the TME through degradation of the hyaluronan,inhibition of pancreatic stellate cells and activation of the effective T cells would be an ideal strategy for improving the therapeutic index of PDAC.The key issue for this idea is how to design a system that can cover above mentioned targets.Nanomedicine offers new hope for solving above problems due to its unique enhanced permeability and retention effect(EPR),environmental responsive and tunable structure.In this thesis,the disulfide bond was use to crosslink the branched polyethylenimine(PEI)to form a nanogel,which can be degraded by the intratumoral GSH.The drug LDE225 was encapped in the nanogel by absorption.The anti-PD-L1 antibodies(Avelumab)and hyaluronidase(HAase)was then mass arrayed on the nanogel.Such smart novel nanoimmunosystem(NINS)was finely designed and fabricated with the ability to degrade the hyaluronan,responsive to GSH,inhibit pancreatic stellate cells,and active the effective T cell.Then the physical chemistry properties of the NINS such as the topological structure and the drug loading content were systemically analyzed by advanced dynamic and static light scattering(DLLS/SLLS),HPLC,TEM and Zeta potential tester.The moleculuar weight,total amount of Avelumab and HAase(~130)was further finely characterized.ELISA and enzyme activity experiments confirmed the activation of antibodies and HAase after its linking.But as the DTT was added to the system,the size and molecular weight of NINS decreased obviously indicating the breaking of nanogel.Moreover,the immunofluorescence testing showed that the NINS had a potential to penetrate the extracellular matrix.All the successful minic structure and biological experiments indicated that the fine-designed function of NINS could well work in vitro/vivo.Subsequently,the cellular level evaluation of NINS was systemically investigated.The results of CCk8 assay and flow cytometer showed that the biocompatibility of NINS was high,while the affinity of Avelumab was good.The 3D co-culture clone formation assay indicated the better penetrating of the NINS due to degradation of HA in mimic extracellular matrix.The pancreatic stellate cells were obviously inhibited by NINS.It was found that the PD-L1 expression of PANC-1 and SW1990 was not obvious,but BxPC-3 and radiation-resistant PANC-1 highly expressed the PD-L1.So the combination of drug LDE225 and radiotherapy significantly enhanced the PANC-1 killing effect.The cellular killing effect was enhanced when the NINS was added to the culture medium.In addition,as compared with the parental antibodies such as Avelumab,the antibody-dependent cellular cytotoxicity(ADCC)of NINS was significantly enhanced as treated by the human peripheral blood mononuclear cells(PBMC).The unexpected enhanced ADCC effect might attribute to the mass arrayed of antibodies on the cellular surface,which resulted in large amount of Fc fragments expose to the NK cell following the release of perforin and granzyme.Finally,the relation between HA matrix penetration ability and the pancreatic stellate cell killing,the immune effect with and without radiation was systemically evaluated.It was found that the chemoradiotherapeutic effects was enhanced which indicated that the all-in-one design of NINS worked well in vitro.The in vivo evaluation of NINS using the nude balb/c mice bearing subcutaneous pancreatic cancers was then conducted.Comparing with single chemotherapy,the antitumor effect was improved by drug loading NINS.Moreover,the tumor recession was futher improved by drug loading NINS as combined with radiotherapy,which indicated the high potential of synergistic therapy.Overall,the NINS constructed in this study can effectively albate the hyaluronan,suppress pancreatic stellate cells and blocking the PD-L1 receptor on pancreatic cancer,which synergically enhance the chemoradiotherapeutic efficiency.The results obtained in this work show us a new concept for comprehensive treatment of pancreatic cancer,which may promote the design,transformation of novel nano immuno system for clinical cancer treatment.