Interferon Gamma Promotes The Responsiveness Of Anti-PD-1 Immunotherapy In Microsatellite Stability Colorectal Cancer

Colorectal cancer(CRC)is one of the most common digestive cancers worldwide.Although the treatments such as surgery,radiotherapy,and chemotherapy have been greatly improved,most patients are in the advanced stage when they are diagnosed.They have lost the opportunity to receive standard treatments so as to have a poor prognosis and face the plight of no medicine.The immune checkpoints targeted by programmed cell death-1(PD-1),which have appeared in the past decade,achieved exciting results in patients displayed with microsatellite instability-high(MSI-H)advanced colorectal cancer.However,the vast majority of patients with microsatellite instability(MSS)colorectal cancer may lose the opportunity for immune checkpoint inhibitor treatment due to their resistance to PD-1 antibody.IFNy is a cytokine with antitumor effect synthesized and secreted by activated immune cells in the tumor microenvironment.Recent studies involved PD-1 antibody treatment in patients with non-small cell lung cancer ord malignant melanoma have observed that the antitumor effect of PD-1 inhibitors is closely related to the activation level of programmed cell death ligand-1(PD-L1)in the tumor microenvironment before treatment.IFNy affects the therapeutic response of PD-1 inhibitors,which may be related to IFNy?-induced PD-L1 expression in tumor microenvironment cells,which promote the activation of PD-1/PD-L1 signaling pathway.In summary,the purpose of this study is to explore the involved mechanism of that IFNy promoted the responsiveness of anti-PD-1 immonotherapy in microsatellite stability colorectal cancerPart ?:IFN?-induced a different intensity of PD-L1 expression in MSI and MSS cell lines[Objectives]To evaluate whether-IFNy induced a different intensity of PD-L1 expression in in MSI and MSS cell lines.[Methods]We detected the expression of PD-L1 in MSI and MSS cell lines induced by IFNy via western blot analysis and flow cytometry.[Results]1.The flow cytometry result of PD-L1 expression:Extremely low PD-L1 expression was observed in all CRC cell lines under normal culture conditions.PD-L1 expression was respectively increased in MSI and MSS cell lines 12 h after treatment with 100 IU/mL IFN?,especially in MSI cell lines.2.The western blot analysis of PD-L1 expression:Treatment with 100 IU/mL IFN? significantly increased PD-L1 expression in MSI and MSS cell lines in vitro.The PD-L1 expression observed in MSI cells induced by 100 IU/mL IFNy was all significantly higher than that induced by the same treatment in MSS cells.[Conclusions]IFN? exposure induce the extensive expression of PD-L1 on CRC cells.Hyperresponsiveness to low doses of IFNy exposure existe in the MSI cell line.Part ?:Signaling mechanism of IFNy-induced differential expression of PD-L1 in MSI and MSS cells[Objectives]To observe the signal pathway of IFN-y-induced PD-L1 expression in colorectal cancer cells.We also explore the distinct molecular mechanisms of IFN? have been proposed to induce PD-L1 expression in MSI and MSS CRC cell lines.[Methods]In this experiment,we detected the expression of PD-L1 in HCT116 and SW480 cell induced by IFNy via RT-PCR and western blot analysis.To explore the signaling mechanism of PD-L1 expression induced by IFNy in HCT116 and SW480 cells,we evaluated the expression of PD-L1 in human CRC cells pretreated with PD98059(ERK1/2 inhibitor),wortmannin(AKT inhibitor),or Fludara(STAT1 inhibitor)by FCM.We further used a RT2 Profiler PCR Array and measured the mRNA levels of 84 genes associated with the JAK-STAT signaling pathway.Western blot was used to further observe the expression of signal transducer and activator of transcription(STAT1),p-STAT1,STAT3,and p-STAT3 in MSI and MSS cells.[Results]1.Different concentrations of IFN? treatment influenced the PD-L1 expression in vitro.1.1 PD-L1 mRNA relative expression:Treatment with different concentrations of IFNy significantly increased PD-L1 expression in HCT116 and SW480 cells in vitro,especially with 100 IU/mL IFNy.The PD-L1 expression observed in HCT116 cells induced by IFNy was significantly higher than that induced by the same treatment in SW480 cells.1.2 The western blot result of PD-L1 expression:Treatment with 100 IU/mL IFN? significantly increased PD-L1 expression in HCT116 and SW480 cells in vitro,especially with 100 IU/mL IFNy.The PD-L1 expression observed in HCT116 cells induced by 100 IU/mL IFNy or 500 IU/mL IFNy were all significantly higher than that induced by the same treatment in SW480 cells.2.Signal pathways involved in IFNy-induced PD-L1 expression in colorectal cancer cells.PD-L1 expression was significantly downregulated in different MSI status cell lines pretreated with Fludara.By contrast,there was no significant alteration in PD-L1 expression after treatment with PD98059 or wortmannin,which inhibited the MAPK and PI3K-AKT pathways,respectively.3.Different JAK-STAT signaling molecules participated in low-dose IFNy-induced PD-L1 expression in MSI and MSS colorectal cancer cell lines in vitro3.1 The different JAK-STAT signaling molecules under normal culture conditions:Compared with the gene fold changes in mRNA expression of JAK-STAT signaling molecules in cultures SW480 cell,there are 19 genes upregulated with greater than two-fold changes.There are 2 genes down-regulated with greater than two-fold changes.3.2 The different JAK-STAT signaling molecules after IFNy treatment.After treatment with 100 IU/mL IFNy for 12 h,there are 15 genes changed with greater than two-fold in SW480 cell line,and 24 genes with greater than two-fold in HCT116.The most striking result was the strong upregulation of chemokine(C-X-C motif)ligand 9(CXCL9)in HCT116(68.53-fold)and SW480 cells(100.19-fold).However,JAK2(2.11-fold),as well as SATA2/3(2.06-fold and 2.04-fold,respectively),was upregulated in SW480 cells only,and downregulation of JAK3(2.87-fold)and STAT5A/5B(2.14-fold and 2.06-fold,respectively)was observed in HCT116 cells only after stimulation with a low level of IFN?.4.The analysis of STAT1,p-STAT1,STAT3 and p-STAT3 in MSI and MSS cell linesUnder normal culture conditions,CRC cell lines displayed expression of STAT1 and STAT3 to varying degrees.With the treatment of 100IU/ml IFNy,both MSI and MSS cell lines showed significant phosphorylation of STAT1.After 12 hours,the expression of STAT1 in both types of CRC cell lines significantly increased;under the effect of IFNy,STAT3 was only obviously expressed in MSS cell lines(SW480 and SW620).It showed significantly increased phosphorylation and protein levels,while MSI cell line STAT3 phosphorylation level was low,and no significant change in protein expression was observed after 12 hours.[Conclusions]Hyperresponsiveness to low doses of IFN? exposure existed in the MSI cell line.The upregulation of PD-L1 induced by IFN? was mainly associated with activation of the JAK-STAT1 in CRC cell lines in vitro.The upregulation of STAT3 might contribute to hypo-responsiveness to IFN? in MSS cell lines.Part ?:IFN-?/PD-L1 signal promote the responsiveness to anti-PD-1 therapy in microsatellite stable colorectal cancer animal model[Objectives)To investigate whether the expression of IFN-y and the downstream target molecular PD-L1 influence the responsiveness to anti-PD-1 therapy in the Balb/c mice with a subcutaneous microsatellite stable tumor challenge as well as the involved mechanism.[Methods]Lentiviruses were produced by transient transfection of 293T cells using the constructed PD-L1 overexpressed plasmid.Transduction of CT26 cells was performed with the harvested virus.When CRC animal models subcutaneously injected with CT26 cells or PD-L1-transduced CT26 cells were successfully established,they were divided into six groups treated with PD-1 blockade therapy with IFNy plus PD-1 blockade or saline.The expression of PD-L1 and CXCL9 induced by IFNy exposure in tumor-infiltrated tissues was performed.PD-L1 expression was localized to tumor cells or tumor-infiltrating CD8+ cells were also determined by means of immunofluorescence costaining.PD-1 antibody was used to treat MSS CRC animal models,and umor weights as well as volumes in each group were also observed.The effects of IFNy injection and PD-L1 overexpression intervention on the tumor weight/volume changes induced by PD-1 immunotherapy were fuether evaluated.CD8 and E-cadherin immunofluorescence staining were used to analyze the cell percentage of CT26 cells and CD8+lymphocytes in tumors harvested from animal models.The involved apoptotic mechanism was also explored by TUNEL analysis.Additionally,we performed RT-PCR and western blot to assess the effect of PD-L1 overexpression on the average extent of adaptive immunity factors in response to anti-PD-1 immunotherapy.[Results]1.The tumorigenesis rate of subcutaneous injection with CT26 cells:The tumorigenesis rate of subcutaneous injection with CT26 cells or PD-L1 transduced CT26 cells on day 15 was 59.1%(26/44)and 87.5%(14/16)respectively.Compared the CRC animal models subcutaneously injected with CT26 cells,the tumorigenesis rate of tumor challenged mice injected with PD-L1 transduced CT26 cells was significantly higher(?2=4.26,p?0.05).2.The PD-L1 expression in cancer tissues after IFN y exposure2.1 The RT-PCR results of PD-L1 expression:the PD-L1 gene fold changes of mRNA abundance in tumor infiltrated tissues harvested from group 1 CRC mice was assigned as 1.The gene fold changes in group 2,group 5 and group 6 CRC mice were(0.19±0.23),(4.02±0.81)and(0.42±0.28)respectively.The expression of PD-L1 induced by IFNy exposure in tumor-infiltrated tissues was increased compare with tumors harvested from the CRC animal models subcutaneously injected with CT26 cells,however,significantly decreased when challenged with PD-1 blockade(p=0.000).Compared with the PD-L1 gene fold changes of mRNA abundance in tumor infiltrated tissues harvested from group 2 CRC mice,the gene fold changes of group 6 was significantly higher(p=0.006).2.2 The western blot results of PD-L1 expression:PD-L1 protein expression was observed in tumor tissue of microsatellite-stabilized tumor-bearing Balb/c mice.The expression of PD-L1 induced by IFNy exposure in tumor-infiltrated tissues was increased compare with tumors harvested from the CRC animal models subcutaneously injected with CT26 cells.2.3 The expression rate of PD-L1 in tumor tissue by immunofluorescence staining:The positive rate of PD-L1 in tumor tissue of microsatellite-stabilized tumor-bearing Balb/c mice was(42.83±6.05)%,and the positive rate of PD-L1 in tumor tissue after IFNy treatment is(58.33±3.50)%.Exogenous IFNy significantly increased the positive rate of PD-L1 expression in microsatellite stabilized colorectal cancer tumor tissues(p=0.002).2.4 PD-L1 is widely expressed on the surface of tumor infiltrating tumor cells(CT26 cells)and CD8 cells in each group.3.The western blot results of of STAT1/3 and CXCL9:Exogenous IFNy treatment caused increased expression of STAT1/3 and CXCL9 proteins in tumor tissues of microsatellite-stabilized tumor-bearing Balb/c mice..4.Observation of tumor mass of Balb/c tumor-bearing mice in each experimental group4.1 The observation of tumor weights:When CRC animal models subcutaneously injected with CT26 cells(group 1,2,5,6)or PD-L1-transduced CT26 cells(group 3,4)were successfully established,they were divided into six groups treated with PD-1 monoclonal antibody(PD-1 mAb),IFNy exposure combined with PD-1 mAb,or saline.Group 1 and group 3 were treated with saline,and group 5 was treated with IFNy and saline.Group 2 represented mice treated with CT26 cells and PD-1 mAb;group 4 included mice treated with PD-L1-overexpressing CT26 cells plus PD-1 mAb;group 6 included mice treated with CT26 cells plus IFNy and PD-1 mAb.PD-1 mAb was injected peritumorally after the successful establishment of animal models,and IFNy was administered before PD-1 immunotherapy.Those animal models were sacrificed,and tumors were harvested on day 30.The tumor weights in six groups was(10.39±0.84)g,(0.14±0.15)g,(17.06±0.59)g,(0.28±0.13)g,(14.82±0.69)g and(0.21±0.08)g respectively.IFNy exposure as well as PD-L1 overexpression on CT26 cells resulted in obviously enhanced subcutaneous tumor weights in CRC mice(p?0.001).Furthemore,immunotherapy with PD-1 blockade significantly inhibited tumor growth,especially in mice challenged with PD-L1-transduced CT26 cells.Mice treated with the combination of PD-1 blockade and IFN? exposure(or PD-L1 transduction)had a slightly larger tumor weight on day 30.4.2.The tumor weight changes induced by PD-1 blockade:PD-1 blockade in tumor challenged mice with IFNy exposure as well as PD-L1 overexpression induced obviously tumor weight rejection,especially in mice challenged with PD-L1-transduced CT26 cells.5.E-cadherin and CD8 immunofluorescence staining5.1 E-cadherin immunofluorescence stainingThe percentage of E-cadherin+ CT26 cell was(66.33±9.61)%in group 1(CT26),(13.67±3.79)%in group 2(CT26+PD-1),(75.67±4.93)%in group 5(CT26+IFNy),and(15.33±2.51)%in group 6(CT26+IFNy+PD-1).PD-1 blockade immunotherapy significantly induced a decreased E-cadherin+ CT26 cell percentage in CT26-injected(p=0.012),or CT26-injected plus IFNy exposure(p=0.048)CRC animal models.5.2.CD8 immunofluorescence stainingIFNy treatment induced higher PD-L1 expression in the tumor tissues of Balb/c mice challenged with CT26 cells subcutaneously.The percentage of CD8+cell was(33.67±3.79)%in group 1(CT26),(77.67±9.61)%in group 2(CT26+PD-1),(12.00±3.00)%in group 5(CT26+IFN?),and(83.33±7.09)%in group 6(CT26+IFN?+PD-1).The CD8+ cell percentage of group 5 was significantly decreased compared with that of group 1(p=0.004).PD-1 blockade immunotherapy significantly induced an increased CD8+ cell percentage in CT26-injected(p=0.021),or CT26-injected plus IFN? exposure(p=0.005)CRC animal models.The CD8+ cell percentage enhance in CT26-injected plus IFNy exposure animal models induced by PD-1 blockade was significantly higher than that in the CT26 cell intervention group(p=0.023).6.TUNEL analysis:The rate of cell apoptosis was(5.23±0.91)%in group 1(CT26),(3.47±0.35)%in group 3(PD-L1 transduced CT26),(4.13±0.91)%in group 5(CT26+IFNy),(30.67±5.13)%in group 2 CT26+PD-1),(29.33±4.04)%in group 4(PD-L1-transduced CT26+PD-1),and(24.33±5.03)%in group 6(CT26+IFNy+PD-1).The apoptosis rate of group 3 was significantly decreased compared with that of group 1(p=0.042).PD-1 blockade significantly induced an increased apoptosis rate in CT26-injected(p=0.018),PD-L1-transduced CT26 cell-challenged(p=0.007)or IFNy-exposed(p=0.026)CRC animal models.No significant difference was observed among the three PD-1 blockade groups.Apoptotic neoplastic cells were observed in many groups,especially in CRC mice treated with PD-1 blockade.7.PD-L1 overexpression on CT26 cells influenced the adaptive immune microenvironment after PD-1 blockade7.1 In response to immunotherapy with PD-1 mAb,many adaptive immune factors were prominently enhanced in tumors harvested from the PD-L1 transduced CT26 group.The expression of CD8 was prominently increased in response to PD-1 mAb when challenged with PD-L1 overexpression on CT26 cells.After the PD-1 blockade,there are adaptive immune factors(CD4,IL-17A and LAG3)displayed with decreased mRNA expression induced by PD-L1 overexpression on CT26 cells,however,no significant changes were observed in PRF1 and FOXP3.The expression of CD8a was clearly decreased(the gene fold change is 0.37±0.25)when challenged with PD-L1 overexpression,whereas it was significantly increased(the gene fold change is 16.49±2.19)in response to PD-1 mAb.7.2 The percentage of CD8+ cell was(12.67±2.08)%in group 1(CT26),(71.33±8.08)%in group 2(CT26+PD-1),(5.00±1.00)%in group 3(PD-L1 transduced CT26),and(73.00±5.57)%in group 4(PD-L1-transduced CT26+PD-1).The CD8+cell percentage of group 3 was significantly decreased compared with that of group 1(p=0.007).PD-1 blockade immunotherapy significantly induced an increased CD8+cell percentage in CT26-injected(p=0.004),or PD-L1-transduced CT26 cell-challenged(p=0.002)CRC animal models.No significant difference was observed among the two PD-1 blockade groups.PD-1 blockade in tumor challenged mice with PD-L1 overexpression on CT26 cells induced obviously infiltrated CD8+cells,especially in mice challenged with PD-L1-transduced CT26 cells(p=0.009).[Conclusions]Colorectal cancer is a kind of tumor involved with PD-1/PD-L1 signal.IFN? exposure induced the expression of PD-L1 on tumor cells.PD-1 blockade therapy plays a remarkable role in tumor rejection,possibly by reinvigoration CD8+ T lymphocytes to maintain their effector function and enhancement of cell apoptosis in tumor-infiltrated tissues.IFN y possibly promotes the responsiveness of anti-PD-1 immonotherapy in microsatellite stability colorectal cancer.The mechanism may be related to the expression of PD-L1 in tumor cells mediated by STAT1/3 and the recruitment of CD8+T lymphocytes associated with chemokine CXCL9.