The Mechanism Of γ-interferon-induced Tumor Dormancy And Its Related Applications

The interaction of tumor cells with the immune system is summarized as 3E theory,that is,Eradication,Equilibrium,Evasion,and that is what will come about when tumor cells meet with immune system.That is tumor cells to be eliminated by the immune system,under the supervision of the immune system to achieve its balance of power,or escape the immune system control,and then infringed the body.Immune-induced tumor dormancy,refers to the tumor cells under the surveillance of immune system,will depict cell cycle arrest,metabolic slowdown,proliferation-related gene expression down and so on.This phenomenon is the tumor and the body’s immune system interaction in the process of entering a balanced and stable state of the concentrated expression.Consistent with the hypothesis of immune surveillance,but due to the lack of effective recognition of in vivo and in vitro models,immune-induced tumor dormancy lacks an effective means of studying its in-depth mechanisms.The current research results only determine the presence of this phenomenon by the use of neutralizing antibodies and transgenic animal models,and to determine the relevance of certain molecules in the immune system,but its further mechanisms are not elucidated.Interferony(gamma interferon,IFNγ)as an effector cytokine in immune system,plays an important role in both innate and adoptive immunity.In tumor immunology,IFNγ is mainly secreted by cytotoxic T cells(CTLs)in tumor microenvironment which plays an important role in the control of tumor growth.In previously published literature,IFNγ has been shown to lead to tumor dormancy,but limited to the lack of a more appropriate model,and its intrinsic molecular mechanism is not well elucidated.Tumor tissue has heterogeneity,different subgroups,with different properties.Among them,cancer stem cells,or tumorigenic cells(TRCs),are a group of cells with strong tumorigenic ability and proliferating ability,and have strong resistance characteristics towards drug,which is related to the recurrence and metastasis of caner,But in the phenomenon of tumor dormancy,there is no conclusion whether tumor stem cells play a part and the internal mechanism has not been digging.The purpose of this study is to use the previously established soft three-dimensional in vitro expansion of tumorigenic cells culture technology to explain the above questions,that whether IFNγ can induce tumor dormancy,which group of tumor cells tends to be dormant,how to induce its dormancy.This study is divided into three parts:1.Can IFN-γ induce the dormancy of tumor cells in vitro;2.The mechanism of IFN-γ in the induction of tumor cell dormancy;3.Can tumor cell’s dormancy be broken to provide new ideas for tumor therapy.The first part:IFNγ induces tumor cell dormancy in vitroObjective:To explore whether IFNγ can establish an in vitro dormant model of tumor cells.Methods:In order to establish the immune model induced by IFNγ in vitro,we used the 3D culture method of fibrinogen originating from salmon and the mouse melanoma cell line B16 to establish the in vitro model.1.Treatment of traditional 2D culture dishes and 3D cultured B16 cells with 100 ng/ml IFNγ to see the effect on proliferating rates,apoptotic rates and metabolic rates;2.Treatment of traditional 2D dishes and other cell lines of 3D culture with 100 ng/ml IFNγ,and to test the rates of apoptosis and metabolism;3.To treat the IFNγ-treated 2D-cultured and 3D-cultured B16 cells with conventional chemotherapeutic agents to see their resistance to chemotherapeutic drugs.Results:1.The apoptosis of 2D cultured tumor cells was obvious after 4 days of treatment with traditional 2D culture dishes and 3D cultured B16 cells with different concentrations of IFNγ,and no obvious apoptosis was observed in 3D cells.3D cells were mainly depicting reduced clone size,cell cycle slowed down,decreased glucose consumption,and proliferation related genes’ expression going down;2.The clone size and cell cycle were also detected by treatment with 100 ng/ml IFNγ in 3D cultured mouse hepatocarcinoma cell H22 and mouse colon cancer cell CT26 for 4 days.It was found that the two cell lines could also be obtained with the previous B16 cells with the same conclusion;3.We use the commonly used chemotherapy drugs methotrexate(Methotrexate),paclitaxel(Paclitaxel)to treat IFNγ-induced dormant tumor cells and normal cultured 2D cells and 3D cells,to find that dormant tumor cells have stronger Drug resistance.Part Ⅱ:The mechanism of IFNy in inducing tumor cell dormancyObjective:To investigate the intrinsic mechanism of IFNy inducing tumor dormancy model in vitroMethods:In the model of IFN[gamma]induced by IFNy in vitro,we used PCR,Western-blot,plasmid construction,viral packaging,CRISPR-Cas9,immunofluorescence,flow analysis,sorting and other molecular biology,cell biology means to identify and verify the relevant signal pathway and mechanism.Results:1.According to the literature and PCR,we determined that indoleamine 2,3-dioxygenase(IDO)has undergone great changes in IFN-y-treated B16 tumor cells and IDO upregulation by TRCs treated with IFNy was more obvious than that of normal 2D cells,indicating that IDO plays an important role in the dormancy of TRCs.2.We analyzed the TRCs which are overexpressing IDO and found that the cell clone size and Cell cycle have showed a great reduction;3.IDO is a catalytic enzyme,the main catalysis of tryptophan(Tryptophan,Trp)metabolism,Trp can be degraded into Kynurenine(Kynurenine,Kyn),we used Kyn with 200 uM to 500 uM concentration to obtain the results of clonal size reduction and cell cycle slowing down,indicating that Kyn plays an important role in the dormancy of TRCs.4.We obtained from the literature that Kyn is a ligand of Aryl hydrocarbon receptor(Ahr),which can lead to the expression of Ahr-induced downstream genes,which may include protein related to cell cycle inhibition.We used immunofluorescence;western-blot and luciferase-based reporter experiments to confirm that Ahr was activated in TRCs.5.At the same time,we used western-blot to screen the relevant cyclin and found that Cdknlb(cyclin-dependent kinase inhibitor 1B,P27kip)showed a significant change after IFNy treatment,and other peripheral proteins did not show a significant change,indicating that P27 may play an important role in dormancy;6.We treated TRCs with Kyn for 72 hours and detected the expression level of P27 by western-blot and found that it was significantly up-regulated,the promoter of the P27 gene was constructed and the reporter cell was constructed.At the same time,the transfection of the AHR gene was significantly enhanced after treatment with Kyn and after treatment with Kyn,and the enhancement of the fluorescein signal after AHR,Kyn and IFNγtreatment showed that IFNγ P27 regulation is mainly through the IDO-Kyn-AHR signal pathway;7.The classic IFNγ signal mainly through STAT1(signal transducer and activator of transcription 1),and mainly on tumor cells caused by apoptosis,we used Western-blot and immunofluorescence and flow cytometry to confirm that this signaling pathway exists in the normally cultured B16,but in dormant cells,This pathway was significantly inhibited,and we found that through the Ip-western IFNγ-Statl signaling pathway and IFNy-IDO-AHR-P27 this signal pathway interaction,that P27 binding p-STAT1 protein to prevent its entry into the nucleus;8.We also later validated this conclusion by using IDO,AHR,and P27 knockdown cell lines that were later established.The third part:1.In vitro experiments using the relevant molecular inhibitors to see whether tumor dormancy can be brokenObjective:To improve the dormancy of tumor cells by using inhibitors of related molecules in combination with IFNy and to propose new ideas for tumor therapyMethods:The proliferation and apoptosis of tumor cells were detected by 3D culture,western-blot and flow cytometry.Results:1.We used the IDO inhibitor 1-MT(1-Methyl-L-tryptophan)and IFNγto treat 3D cultured B16 cells,and the number of clones decreased and clone size became smaller.and the emergence of the overall increase in cell apoptosis level,that is to say,IFN gamma and IDO inhibitor can break dormancy,effectively killing tumor;2.We treat with the combination of AHR inhibitor DMF(3’,4’-Dimethoxyflavone)and the IFN gamma in 3D cultured B16 cells,obtained the same results with the IDO inhibitor,namely the dormancy breaking apoptosis.These two inhibitors may have a good prospect in future immunotherapy.Summary:tumor dormancy in the tumor occurrence,development,metastasis and treatment of prognosis are important significance,immune-induced dormancy,it is a very good manifestation of this phenomenon,we use the 3D culture of tumor regeneration cells This method,which simulates the process of tumor dormancy in vitro,that is,treatment of 3D cultured cells with IFNy for 72 hours in vitro will induce its entry into dormancy,and in further studies we found that in the induction of tumor in vitro In the process of dormancy,indoleamine 2,3-dioxygenase(IDO),kynurenine(kyn),aromatic hydrocarbon receptor(AhR),IDO-Kyn-AhR,a metabolic regulatory transcription factor signaling pathway,plays an important role.IFN-y in the tumor-regenerating cells,it can induce its entry into dormancy,specifically manifested as IFNy-induced expression of IDO and AHR in tumor regenerated cells,upregulation of IDO expression and then produce a large number of endogenous Kyn,Kyn combined with AhR AhR,AhR into the nucleus and bind to the promoter of the corresponding gene,leading to the expression of the relevant cycle arrest gene,resulting in cell cycle arrest.We through further screening,found in this phenomenon dormancy,the cell cycle arrest protein P27 there was a very significant change,and by the AhR gene transcription regulation.The signal pathway of IFNy-IDO-AhR-P27 has a great effect on the resuscitation of tumor regenerated cells.We can also break down by using the related inhibitor and IFNy,and we can achieve good therapeutic effect.The future of clinical treatment of cancer put forward new ideas.