| Objective: Type I interferons(IFNs)are cytokines secreted by various types of cells(such as dendritic cells,lymphocytes,macrophages,epithelial cells,stromal cells,and tumor cells).They can up-regulate the expression of hundreds of IFN-stimulated genes(ISGs).These up-regulated ISGs can not only directly exert anti-tumor effects by affecting proliferation,apoptosis,angiogenesis,etc.,but also modulate innate immune and adaptive immune by promoting antigen presentation and activating CD8+ T cells,respectively.Therefore,the exogenous activation of type I interferon signaling through drugs may be very suitable for the treatment of immunosuppressive tumors,such as pancreatic cancer.Since human recombinant interferons(such as IFNα-2a and IFNα-2b)have a short half-life and exhibit great toxicity in most clinical trials related to pancreatic cancer,their clinical application is limited.In recent years,experts have begun to try to explore the role of endogenous IFNs in anti-tumor by targeting its upstream signaling.The production of IFNs is mainly mediated by pattern recognition receptors(PRRs).In tumor cells,cyclic GMP-AMP synthase/stimulator of IFN genes,c GAS/STING)pathway is the main source of IFNs production.In view of the important role of the STING-IFN signaling,a variety of STING agonists have shown significant anti-tumor effects in preclinical models and have been tested in clinical trials.However,since excessive activation of STING can trigger an inflammatory storm and produce great toxicity,there is an urgent need for methods that can be used to monitor STING-IFN signaling to help optimize the usage of STING agonists.Positron emission tomography(PET)may be a potential solution to this problem.PET is a non-invasive and highly sensitive imaging technology that can monitor the distribution and concentration of positron emitting radionuclide labeled imaging probes injected into the body.The current research and development of specific probes for human metabolism,cell proliferation,tumor antigens,etc.provide us with a lot of important information for preclinical or clinical diagnosis and treatment.Recently,it has been reported that IFNs also play an important role in the regulation of cell metabolism in immune cells and epithelial cells.Therefore,we wonder whether IFNs can cause specific metabolic changes in pancreatic cancer cells that can be identified by specific PET probes.In order to systematically explore the effect of IFNs on cell metabolism,we used IFNβ to treat a human pancreatic cancer cell line SUIT2 for 24 hours and then performed RNAseq analysis.The results showed that in addition to the classic ISGs upregulation,IFNβ also caused changes in the transcription of multiple genes involved in cell metabolism,such as SCL7A11,which is related to cysteine ??homeostasis,and PARP 9/10/14 and OAS1/ 2/2/L which are related to NAD/NADP metabolism,IDO1 which is related to tryptophan metabolism,and TYMP/SAMHD1/CMPK2/NT5C3 A which are related to purine and pyrimidine metabolism.It has been reported that both exogenous administration of thymidine phosphorylase(TYMP)or high intrinsic level of TYMP in the tumor can promote the accumulation of 3′-fluoro-3′-deoxythymidine(FLT)in tumor cells,so we propose our hypothesis: whether we can monitor the STING-IFN signaling through 3′-[18F] fluoro-3′-deoxythymidine([18F]FLT)PET.This study will explore the effects of STING activation on the proliferation of pancreatic cancer in vitro and in vivo,and evaluate whether[18F]FLT PET can be used to monitor IFN signaling induced by small molecule STING agonists in mouse model,so as to optimize the clinical application of small molecule STING agonists.Methods: In this study,we first analyzed the regulation of IFNβ on the transcription of pancreatic cancer metabolism-related genes through RNAseq and then put forward our experimental hypotheses based on the analysis results and literature review.Then the[18F]FLT uptake experiment was performed to verify the effect of IFNβ on the accumulation of [18F]FLT in pancreatic cancer cells in vitro.In order to verify the role of TYMP in the effect of IFNβ on [18F]FLT accumulation,we constructed a TYMP downregulation model by using sh RNA.Through immunohistochemical staining of 33 types of pancreatic cancer cell lines derived tumors and 138 tumors from pancreatic cancer patients,we verified whether the expression of TYMP in pancreatic cancer is universal.In 21 pancreatic cancer cell lines,immunoblot analysis was performed to verify the universality that TYMP can be upregulated by IFNβ.In order to study the effect of STING activation on IFN signaling production,cell proliferation,TYMP expression and[18F]FLT accumulation in pancreatic cancer,we engineered genetic STING activation model which can be switched by doxycycline(DOX).The Incu Cyte Zoom live cell imaging system was used to study the effect of STING activation on the proliferation of pancreatic cancer cells in 3D culture in vitro.The effect of STING activation on tumor growth was evaluated in a subcutaneous mouse xenograft model.Immunoblot analysis was used to explore the effect of STING activation on TYMP expression in vitro and in vivo and LC-MS/MS analysis was used to explore the effect of STING activation on nucleoside metabolism.In pancreatic cancer mouse subcutaneous xenograft models and orthotopic xenograft models,PET/CT scan was used to explore the effect of STING activation on [18F]FLT accumulation in tumors.[18F]FDG PET scan was performed in the same subcutaneous xenograft model to prove whether the [18F]FLT probe can be used to monitor STING-IFN signaling specifically.In order to verify whether these results obtained in the genetic STING activation model can be extended to small molecule STING agonists,a new small molecule STING agonist di ABZI was selected to be used in the following experiments.Firstly,RT-PCR and immunoblot analysis were used to verify whether di ABZI can activate the STING-IFN signaling pathway in vitro.By constructing luciferase linked interferon-stimulated response elements(ISRE)promoter reporter,we can use bioluminescence imaging system to dynamically track the influence of di ABZI on IFN signaling in vivo.Immunoblot analysis of tumors was used to detect the change of protein markers related to STING-IFN signaling caused by di ABZI.[18F]FLT uptake experiment was used to verify the effect of di ABZI on the accumulation of [18F]FLT in pancreatic cancer cell in vitro,and CRISPR/Cas9 was used to knock out IFNα receptor 1 to prove that whether the effect of di ABZI on the accumulation of[18F]FLT in pancreatic cancer cell is dependent on IFN signaling.Finally,in two pancreatic cancer mouse subcutaneous xenograft models,PET/CT scan was performed to explore the effect of di ABZI on the accumulation of [18F]FLT in tumors.Results: 1.IFNβ can cause transcriptional changes of many genes related to cell metabolism(such as TYMP)in pancreatic cancer cells.2.IFNβ can promote the accumulation of [18F]FLT in pancreatic cancer cells,and this effect depends on the upregulation of TYMP.3.TYMP is generally expressed in pancreatic cancer cell lines and human pancreatic cancer tumors,and IFNβ can generally up-regulate the expression of TYMP in pancreatic cell lines.4.STING is generally highly expressed in pancreatic cancer cell lines.5.STING activation can significantly inhibit the proliferation of pancreatic cancer in vitro and in vivo.6.STING activation can up-regulate the expression of TYMP in vitro and in vivo,and the up-regulation of TYMP expression can be blocked by a JAK inhibitor in vitro.7.STING activation can promote the accumulation of [18F]FLT in tumors in both pancreatic cancer mouse subcutaneous xenograft models and orthotopic xenograft models,but it has no effect on the accumulation of [18F]FDG in tumors.8.The small molecule STING agonist di ABZI can activate the STING-IFN signaling in pancreatic cancer cells both in vitro and in vivo.9.di ABZI can up-regulate the expression of TYMP both in vitro and in vivo,and both IFNAR1 knockout and JAK inhibitor can block the up-regulation of TYMP expression by di ABZI in vitro.10.di ABZI can promote the accumulation of [18F]FLT in pancreatic cancer cells both in vitro and in vivo,and IFNAR1 knockout can block this phenomenon in vitro.Conclusion: This research proved that STING activation can inhibit the proliferation of pancreatic cancer cells in vitro and in vivo and [18F]FLT PET can be used to monitor IFN signaling induced by small molecule STING agonist di ABZI in pancreatic cancer mouse xenograft models.Therefore,PET imaging may have utility as a pharmacodynamic readout in the translation of emerging interferon-inducing therapies,including STING agonists,for cancer treatment. |
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