The Establishment Of IFN-γ-induced Tumor Regenerative Cell Dormancy Model And Its Mechanism

Malignancy is one of the most common causes of death in modern society,and it is important to study the biology of cancer.2011 Weinberg suggested malignant tumor 10 big features,on the basis of the original,added to avoid immune to destroy,promoting tumor related to inflammation,cell energy metabolism abnormality and,genomic instability and mutations.Avoiding immune destruction is the result of a vicious tumor and an immune system that is at the heart of cancer therapy.Relationship between tumor and immune system is complex,Burnet in 1970 proposed the "tumor immune surveillance" hypothesis,in 2002 Schreiber put forward the "theory of tumor immune editor".Tumor immune editing theory is more comprehensive and accurate expounds the interaction of tumor and the immune system,mainly divided into three stages:elimination,equilibrium and escape.The equilibrium is the key stage in these three,has direct relationship with subsequent tumor recurrence,in this phase,the immune system may limit the tumor cells in a dormant state.Tumor dormancy is a clinical concept,mainly refers to the cancer patients after surgical treatment,the residual tumor cells in the body of the long-standing and no significant growth of a state.Tumor dormancy is an important part in the process of cancer development,also is the root cause of tumor recurrence,so will dormant tumor cells to become "the seeds of evil".Although this concept has been put forward for a long time,but its mechanism is not clear,the reason may be that the model is scarce,and so far there is no ideal of tumor dormancy models to simulate the clinical behavior;And because of the heterogeneity of the tumor,the study had to go deep into the various subgroups of the tumor cell population;Moreover,the immune cells and cytokines that play a role in the immune system are more complex,making it much more difficult to study.This research adopts the co-culture tumor specific CTL and tumor cells in vitro and to observe the effects of CTL cells of different subsets,discovered the specific CTL can induce TRCs into dormancy,on the basis of screening the IFN-gaimma the cytokines,IFN-gamma as the breakthrough point,the establishment of a tumor dormancy in vitro model,in a new level,to prove the existence of tumor immune surveillance,has the important value of immunology and biology.The research content of this topic mainly consists of three parts:(1)IFN-y induced tumor regeneration cell dormancy;(2)IFN-γ induced tumor regeneration cell dormancy;(3)IFN-γ induced tumor regeneration mechanism was first explored.Part I:IFN-γ induces tumor repopulating cells dormancy in vitroObjective:To investigate whether IFN-γ can induce dormancy of tumor repopulating cells in vitro.Methods:Tumor-specific OT-I cytotoxic T lymphocytes(CTL)were incubated with OVA-B16 melanoma cells(1 × 105),then adjust the ratio of tumor-specific OT-I CTL to OVA-B16 melanoma cells 1:1,25:1,50:1,100:1,200:1,respectively.The cells were collected after 12 hours,and the tumor specificity was analyzed by flow cytometry(FCM);(1× 104)OVA-B16 melanoma cells or the same number(1 × 104)of live OVA-B16 melanoma cells,respectively,from each group,were seeded into 3D fibrin gels,and the number of cloney were detected after 5 days;The tumor-specific OT-1 CTLs were coclutured with OVA-B16 melanoma cells(10:1)or OVA-B16 melanoma TRCs(10:1),After 12 hours,the killing effect was analyzed by FCM;Co-culture supernatant(CM)or PBS were used to culture TRCs,The number of cloney and the size of TRCs were compared.The effects of CM on TRCs cell cycle were analyzed by FCM;The CM was analysised for the expression of panel factors by ELISA.And then neutralizing antibodies were used to screen the cytokines that affected the proliferation of TRCs;TRCs were treated with different concentration IFN-y(50ng/mL,100ng/ML,200ng/mL),then the expression of PCNA was detected by Real-time PCR,FCM was used to analyze the cell cycled and the anti-apoptotic ability,glucose content test was used to detect the glucose consumption,β-galactosidase assay kit to detect senescence.Results:1.The killing efficiency was increased from 13.76%to 14.20%,45.62%,61.45%,72.10%,74.86%,76.18%,79.87%in different groups.The killing efficiency was positively related with the CTLs number(in a certain range,from 1:1 to 50:1),the correlation coefficient was 0.90.However,when the ratio increases to 50:1,there was no significant difference in the killing efficiency between the two groups(P>0.05).19.68%of OVA-B16 melanoma cells survived once the CTL ratio exceeded the critical point(50:1),even at the increased ratio of CTL(100:1，200:1).2.5 days later,the number of cloney in the same number group co-culture system was no difference than that in the control group(P>0.05).While the number of cloney in the same alive number co-culture system was significantly higher than that in the control group(P<0.01);3.FCM analysis showed that the killing efficiencies of OVA-B16:32.15%,OVA-B16 TRCs:8.07%,the killing efficiency between the two groups was statistically significant(P<0.01);4.There was no significant difference in the volume or number of TRCs between 2 days and 2 days after treated with CM(P>0.05).However,the cloney volume of TRCs cultured in CM group was much smaller than that in PBS group.There was significant difference between the two groups(P<0.01),Cell cycle analysis showed that the percentage of TRCs in G0/G1 phase in CM group increased to 77.98%compared with PBS group(G0/G1:47.21%).There was significant difference between the two groups(P<0.01);5.IFN-γ(680.25 pg/mL),TNF-α(170.40 pg/mL),IL-2(15 pg/mL)and IL-10(32 pg/mL)were all detected in the supernatant by ELISA.The cytotoxicity of OT-I CTL on OVA-B16 melanoma cells was significantly decreased from 30.62%to 12.88%after using IFN-γ neutralizing antibody.There was significant difference between the two groups(P<0.01).But there was no significant difference in the TRCs group(P>0.05).Cell cycle analysis showed that the percentage of TRCs in the G0/G1 phase decreased to 52.81 compared to the CM group(77.65%);Conclusions:Tumor-specific OT-I CTL can significantly kill OVA-B16 melanoma cells,but there is always a part of the cells alive,and this small number of cells was TRCs.Although CTL could not kill TRCs,CTL can secrete a large amount of IFN-γ,by which to induce TRCs into dormancy.IFN-γcould induce the size of TRCs remained unchanged,the cell cycle was arrested in G0/G1 phase,all we call domante stage.Part Ⅱ:IFN-γ induces tumor repopulating cells dormancy in vivoObjective:To explore whether IFN-γ can induce the dormancy of tumor regeneration cells in vivo.Methods:C57 mice were subcutaneously inoculated with 2× 104OVA-B16 TRCs.One group was injected with PBS and the other was injected with 1 × 107 CTL in vitro.10 days later,TRCs cells were analyzed by FCM;C57 mice were inoculated subcutaneously with 2× 105 OVA-B16 TRCs,3 d ays later,one group of injection of PBS,one of anti-IFN-γ(1 ug per mouse),one were injected with anti-IFN-γ(1 ug per mouse)by tail vein injection of CTL at the same time,and then injected again in 5 days,7 days and 9 days.FCM was used to analyze the cell cycle of OVA-B16 TRCs;C57 mice were subcutaneously inoculated with 5 × 13 B16 melanoma TRCs.One group was injected with PBS and the other one was injected with IFN-γ(1 ×105 U per mouse),and TRCs cell cycle was analyzed by FCM;C57 mice were inoculated subcutaneously with 5×103 B16 TRCs.One group was injected with PBS and the other one was injected with IFN-γ(1 ×105 U per mouse).Then the skins were stained with HE and IHC,and tumor volume was counted;C57 mice were inoculated subcutaneously with 5 ×103 B16 TRCs.One group was injected with PBS and the other one was injected with IFN-γ(1 ×105 U per mouse)/anti-IFN-γ(10 ug per mouse),then every other day,5d,10d,respectively,the corresponding position of skin was stained with HE and IHC,and tumor volume was counted;Results:1,Cell cycle analysis showed that G0/G1 phase of OVA-B16 TRCs increased to 76.53%in CTL group compared with PBS group(30.12%).There was significant difference between the two groups(P<0.01).2.Cell cycle analysis showed that G0/G1 phase of OVA-B16 TRCs decreased to 51.94%in the group treated with anti-IFN-γ/CTL compared with CTL group(75.98%).There was significant difference between the two groups(P<0.01).3.Cell analysis showed that G0/G1 phase of B16 TRCs increased to 66.80%in IFN-γ treated group compared with PBS group(17.20%).There was significant difference between the two groups(P<0.01).4.IHC and HE staining showed that the size of tumor in PBS group was significantly greater in day 20 than that in day 5.There was significant difference between the two groups(P<0.01).But there was no significant difference in the IFN-γ group(P>0.05).5.IHC and HE staining showed that there was no significant difference in the IFN-γ in day 10 compared to day 5(P>0.05).But the size of tumor in IFN-γ/anti-IFN-γ group was significantly greater in day 10 than that in day 5.There was significant difference between the two groups(P<0.01).Conclusion:Tumor-specific OT-I CTL could also induce OVA-B16 TRCs into dormancy in vivo.This effect could be blocked by anti-IFN-γ,which indicates that CTL is induced TRCs dormancy by IFN-γ,and further in vivo experiments confirmed that INF-γ could indeed induce TRCs into dormant state.Part Ⅲ:Mechanism of IFN-γ-induced dormancy in tumor repopulating cellsObjective:To investigate how IFN-γ induces TRCs into dormancy,combined with inhibitors of IFN-γand IDO signaling pathways that can break down tumor dormancy.Methods:B16 TRCs were cultured in vitro and injected into mice subcutaneously.When the tumor grew to 5×5mm,mice were injected with IFN-γ subcutaneously once every other day.The expression of IDO1 and AhR protein was analyzed.The changes of cell cycle were analyzed.Combined IFN-γeiht IDO inhibitors in the treatment of tumor-bearing mice,observe the tumor growth and survival changes in mice.Results:1.Confocal,Western Blot results showed that after treatment with IFN-γ,confocal,Western Blot detection of IDO1 levels were significantly increased,AhR significantly into the nucleus.2.Compared with the normal group,the number of TRCs clone formation in IFN-γ group was not significantly changed(P>0.05).The number of TRCs clone formation in combination with IFN-γ and IDO signal pathway inhibitor(1MT,DMF)(P<0.01).3.Compared with the normal group,most of the IFN-γ cells(34%)were in the G0/G1 phase,after treated with combination of IFN-γ and IDO related inhibitor(1MT,DMF),most TRCs are in sub-GO phase.There was significant difference between the two groups(P<0.01).4.Animal experiments showed that the combination of IFN-γ and IDO inhibitor in the treatment of tumor-bearing mice could significantly reduce the tumor volume and prolong the survival time.The data of each group were statistically significant differences(P<0.01).Conclusion:IFN-γ could significantly upregulate the IDO1 of TRCs.By raising the IDO and inducing AhR into the nucleus,TRCs enter into the dormant state.And the combination of IFN-γ and IDO realted inhibitors in tumor-bearing mice can significantly reduce the tumor volume,prolong the survival time.