RIPK3 Mediates Myeloid-Derived Suppressor Cells-Potentiated Colorectal Carcinogenesis

Background:Colorectal cancer(CRC)is the most common digestive system malignancy and the third leading cause of cancer-associated mortality in the world^[1].The 5-year survival rate is only?10%for the patients with advanced CRC^[2].Exploring the pathogenesis and effective therapeutic targets of CRC is of great clinical significance.The CRC-infiltrating immune cells,including myeloid-derived suppressor cells(MDSC)are one of the most important causes for tumor progression and therapeutic failure^[3-5].Therefore,addressing mechanisms regulating MDSC will provide new ideas for the immunotherapy of CRC.Inflammation initiates necroptosis which parallels with caspases-mediated apoptosis and nuclear factor kappa B(NF-?B)-mediated proliferation and plays an essential role in carcinogenesis^[6].Receptor-interacting protein kinase 3(RIPK3)is a central regulatory molecule for necroptosis^[7],whereas its role in tumor immunity remains unknown.It has been reported that RIPK3 promotes the mucosal repair in IBD^[8].More importantly,RIPK3also inhibits the tumorigenesis of CRC and the expression of proinflammatory factors including interleukin(IL)-1?,IL-6,S100A8,chemokine(C-X-C motif)ligand 1(CXCL1),and tumor necrosis factor alpha(TNF?)^[9].Since these proinflammatory factors correlate with the accumulation and maintenance of MDSC^[10],RIPK3 may regulate the tumor-infiltrating MDSC.Methods:1.RIPK3 expression in MDSC from CRC tissues1.1 The AOM plus DSS-induced mouse colorectal cancer(CRC)model was employed to detect MDSC,CD45⁺cells,T cells,DC,M?which infiltrated in the tumor microenvironment(TME)by flow cytometry(FCM).1.2 RIPK3 expression in MDSC,CD45⁺cells,T cells,DC,M?were detected by FCM and confocal microscopy.1.3 RIPK3 expression in MDSC from clinical colorectal cancer tissue was detected by FCM1.4.RIPK3 expression in MDSC was detected by FCM when bone marrow cells(BMC)was stimulated by tumor supernatant.2.Effects of RIPK3 deficiency on MDSC infiltration and CRC tumorigenesis2.1 WT and RIPK3 knockout(KO)mice CRC model were employed;2.2 Body weight changes,blood in the stool,survival rate were detected;2.3 Colorectal length,spleen weight and tumor number were measured;2.4 MDSCs in spleen,colorectal and tumor tissues were examined by FCM and confocal microscopy.2.5 Immune cells in the TME were examined by FCM.3.Effects of RIPK3 absence in MDSC on the immunosuppressive activity in vitro3.1 WT and KO-BMC were isolated and stimulated by tumor supernatants for 48h,MDSC were detected by FCM.3.2 WT and KO-MDSC were sorted by the magnetic beads and were stimulated by GM-CSF for 48h,the proliferation rate was detected by CCK8 kit.3.3 WT and KO-MDSC were stimulated by GM-CSF for 48h,the cell death was detected by Annexin V Apoptosis Detection Kit with 7-AAD kit.3.4 WT and KO-MDSC were stimulated by GM-CSF for 48h,the differentiation and Arg-1,NOS2,ROS expression of MDSC were detected by FCM.3.5 WT and KO-MDSC were co-cultured with CD8⁺T cells,Gzm B and IFN-?expression of CD8⁺T cells were detected by FCM.3.6 WT and KO-MDSC were co-cultured with CFSE labeled CD8⁺T cells,CFSE expression was detected by FCM.3.7 WT and KO-MDSC were treated by NHNL for 48h,and then co-cultured with CD8⁺T cells,Gzm B and IFN-?expression of CD8⁺T cells were detected by FCM.3.8 WT and KO-MDSC were stimulated by GM-CSF for 48h,and then the supernatant of MDSC was added to CT-26 cell culture at a volume ratio of 1:1(medium:supernatant)for 48h,the proliferation rate of CT-26 was detected by CCK8 kit.4.Role of RIPK3 in myeloid derived cells in colorectal tumorigenesis.4.1 BMC chimerism model in CRC mice4.1.1 Body weight changes,mortality were detected during WT?WT group,WT?KO group,KO?WT group.4.1.2 Colorectal length,spleen weight and tumorigenecity were measured when mice were sacrificed during three groups.4.1.3 MDSC in the colorectal tissues and spleens were examined by FCM.4.2 MDSC depletion in KO CRC mice model was employed:4.2.1 Body weight changes,mortality were detected;4.2.2 Colorectal length and tumor number were measured;4.2.3 MDSC in colorectal tissue and spleen were examined by FCM.4.3 Inhibition of MDSC chemotaxis of KO CRC mice model were employed:4.3.1 Body weight changes,mortality were detected;4.3.2 Colorectal length and tumor number were measured;4.3.3 MDSC in the colorectal tissue and spleen were examined by FCM.5.The mechanism by which RIPK3 deficient-driven MDSC activation5.1 The expression of COX-2 in colorectal and tumor tissues were detected by IHC;5.2 The expression of COX-2 on MDSC in tumor tissues were detected by FCM;5.3 The expression of PGE₂ in MDSC was detected by UPLC-MS/MS;5.4 The gene expression of EP_1-4on MDSC were detected by RT-PCR.5.5 NF-?B/COX-2/PGE₂ signal pathway were detected by Western-blot5.6 Stat3/6 signal pathway were detected by Western-blot.5.7 The expression of COX-2 on CD45^-cells in colorectal and tumor tissues were detected by FCM.6.Roles of inhibitors targeting PGE2 biosynthesis or bioaction on MDSC and carcinogenesis6.1 In vitro6.1.1 WT and KO-MDSC were stimulated with PGE₂,aspirin(ASA),AH6809 for 48h,the differentiation and Arg-1 expression of MDSC were detected by FCM.6.1.2 WT and KO-MDSC were stimulated with PGE₂,ASA,AH6809 for 48h and then were co-cultured with CD8⁺T cells,Gzm B and IFN-?expression of CD8⁺T cells were detected by FCM.6.1.3 WT and KO-MDSC were stimulated with ASAor CAPE for 48h,the expression of PGE₂ in MDSC were detected by ELISA.6.1.4 CT-26 cells were stimulated with GSK872,ASA and CAPE for 48h,the expression of PGE₂ in CT-26 cells were detected by ELISA.6.2 In vivo:6.2.1 KO CRC model were treated with ASA,AH6809,ONO-AE3-204;Body weight changes were detected;6.2.2 Colorectal length,spleen weight and tumor number were measured;6.2.3 MDSC were examined in the colorectal tissues and spleens by FCM.7.RIPK3-PGE₂ circuit in MDSC and CRC cells.7.1 WT-MDSC were stimulated with PGE₂ for 48h,the expression of RIPK3,p65,COX-2 were detected by Western-blot.7.2 WT-MDSC were stimulated with ASA for 48h,the expression of RIPK3 were detected by FCM.7.3 WT-MDSC were stimulated with PGE₂,H89,or AH6809 for 48h,the expression of RIPK3 were detected by RT-PCR;the expression of RIPK3,PKA,CREB,and p-CREB were detected by Western-blot.7.4 CT-26 cells were stimulated with GSK872,PGE₂,H89,AH6809 for 48h,the expression of RIPK3,p65,COX-2,PKA,CREB,and p-CREB were detected by Western-blot.7.5 Clinical colorectal cancer tissues including polyps and different st ages tissues(stage I-IV)were collected to detect the fluorescence intensity of RIPK3 on CD33 positive cells by confocal microscopy.7.6 The correlation between RIPK3 and gene transcripts(CD33,S100A8,PTGS2)were examined in 148 patients with CRC from GSE21510.The correlation between the expression levels of RIPK3/PTGS2 and survival of CRC patients were examined from GSE17536.Results:1.RIPK3 is down-regulated in MDSC of CRC tissues1.1 AOM plus DSS-induced WT mouse CRC model were successfully established;1.2 Both leukocytes(CD45⁺)and MDSCs(CD11b⁺Gr-1⁺)were significantly higher in tumor than in colorectal tissues.1.3 CD8⁺Tcells and DC were significantly reduced while M?were higher in tumor than in colorectal tissues.1.4 The tumor-infiltrating MDSC showed lower RIPK3 expression.1.5 MDSC infiltrated in the human colorectal cancer showed lower RIPK3 expression.1.6 RIPK3 expression on MDSC increased in IBD,then significantly decreased in CRC.1.7 RIPK3 expression on CD45⁺cells,M?,DC,Treg,CD4⁺T cells,CD8⁺T cells were not different between tumor and colorectal tissue.1.8 The percentage of MDSC increased significantly whereas the expression of RIPK3was down-regulated in WT-BMC by CT-26 tumor supernatants.2.RIPK3 deficiency promotes MDSC infiltration and CRC tumorigenesis.2.1 AOM plus DSS-induced WT and KO mice CRC model were successfully established;2.2 KO mice exhibited decreased body weight,higher diarrhea score,reduced survival rate.2.3 KO mice exhibited shorter colorectal length,heavier spleen,higher tumor number in colorectal.2.4 In KO mice,accumulation of MDSC in tumor,colorectal and spleen increased.2.5 In KO mice,accumulation of CD45⁺cells,g-MDSC but not M?,DC,Treg,CD4⁺T cells,CD8⁺T cells in tumor increased.3.RIPK3 absence in MDSC enhances the immunosuppressive activity in vitro3.1 The percentage of MDSC was higher after BMC were stimulated by GM-CSF in KO group.3.2 RIPK3 absence in MDSC resulted in a modest higher proliferation.3.3 RIPK3 absence in MDSC did not show significant change in cell death.3.4 The percentage of M?s,especially M2 type M?s(F4/80⁺CD206⁺)were significantly higher in KO group,while DCs were lower in WT group.3.5 Arg-1 but not NOS2 or ROS increased in the KO-MDSC.3.6 The co-cultivation of KO-MDSC dampened the proliferation,expression of Gzm B and IFN-?of CD8⁺T cells.3.7 NHNL significantly dampened MDSC activity.3.8 The supernatant from RIPK3-KO MDSC enhanced the proliferation of CT26 cells.4.RIPK3 in myeloid derived cells is essential for inhibiting colorectal tumorigenesis.4.1 BMC chimerism model were successfully employed:4.1.1 KO?WT group mice exhibited more severe weight loss,higher mortality;4.1.2 KO?WT group mice exhibited higher tumor formation ratio,shorter colorectal length,and splenomegaly.4.1.3 KO?WT group mice exhibited more MDSC and g-MDSC in colorectal and spleen.4.1.4 KO?WT group mice did not show significant change of M?s and DCs in colorectal and spleen.4.2 MDSC in KO CRC model were depleted:4.2.1 Anti-Gr-1 treatment reversed weight loss and reduced the mortality and exhibited longer colorectal length and less tumor number.4.2.2 Anti-Gr-1 treatment exhibited less MDSC in colorectal and spleen.4.3 Inhibition of MDSC chemotaxis in KO CRC model:4.3.1 CXCR2-a treatment reversed weight loss and reduced the mortality and exhibited longer colorectal length and lower tumor number.4.3.2 CXCR2-a treatment exhibited less MDSC infiltration in colorectal and spleen.5.NF-?B/COX-2/PGE₂ signaling is enhanced in RIPK3 deficient MDSC5.1 The expression of COX-2 in colorectal and tumor tissues were higher in KO mice CRC;5.2 The expression of COX-2 on MDSC in tumor tissues were higher in KO mice CRC;5.3 KO-MDSC produced more PGE₂;5.4 KO-MDSC showed higher gene expression of EP2 and EP4.5.5 The expression of NF-?B p65 and COX-2 were significantly upregulated in KO-MDSC.5.6 The expression of of stat3 and stat6 were not significantly altered in KO-MDSC.5.7 The expression of COX-2 on CD45^-cells in colorectal and tumor tissues were higher in KO mice CRC.6.Inhibitors targeting COX-2 and EP2 blunt the immunosuppressive activity of MDSC and carcinogenesis6.1 In vitro6.1.1 PGE₂ significantly enhanced Arg-1 expression in MDSC;6.1.2 Arg-1 expression in KO-MDSC were inhibited by ASA and AH6809;6.1.3 PGE₂ significantly enhanced MDSC differentiation towards M2 macrophages which were inhibited by ASA and AH6809;6.1.4 Antagonists of NF-?B/COX-2/PGE₂/EP2 signaling pathway rescued the KO-MDSC-dampened CD8⁺T cell activation6.1.5 ASA and CAPE inhibited PGE₂ production from KO-MDSC.6.1.6 GSK872 promoted PGE₂ production from CT-26 cells,but ASA and CAPE did not reverse PGE₂ production from GSK872-treated CT-26 cells.6.2 In vivo:6.2.1 KO CRC model were treated with ASA,AH6809 and ONO-AE3-204;ASA and AH6809 but not ONO-AE3-204 treatment reversed weight loss.6.2.2 ASA and AH6809 treatment exhibited longer colorectal length and less tumor number.6.2.3 ASA and AH6809 treatment exhibited less MDSC,g-MDSC and lower COX-2MFI in tumor,colorectum and spleen.7.PGE2 negatively regulated RIPK3 via the PKA-CREB signaling pathway,forming a RIPK3-PGE2 circuit to potentiate malignancy.7.1 PGE₂ significantly suppressed RIPK3 while enhanced the expression of p65 and COX-2 in MDSC.7.2 ASA could upregulate RIPK3 expression in MDSC.7.3 The gene expression of PGE₂-downregulated RIPK3 could be rescued by both H89and AH6809 in MDSC.7.4 The PGE₂-downregulated RIPK3 in MDSC could be rescued by both H89 and AH6809 which inhibited CREB expression upregulated by PGE₂.7.5 PGE₂ decreased RIPK3 via PKA-CREB pathway while both PGE₂ and GSK872promoted the expression of p65 and COX-2 in CT26 cells.7.6 With the development of CRC clinical stage,the expression of RIPK3 in tumor-infiltrating MDSC decreased.7.7 The RIPK3 expression negatively correlated with CD33 and S100A8,and PTGS2(COX-2)(GSE21510).7.8 The RIPK3^highPTGS2^low patients showed longest survival while low RIPK3 and high PTGS2 was associated with poor survival(GSE17536).Conclusion:1.RIPK3 is down-regulated in MDSC of CRC tissues2.Enhanced MDSC accumulation and tumorigenesis in RIPK3 deficient mice3.Deficiency of RIPK3 promotes the proliferation and immunosuppressive activi ty of MDSC in vitro4.Carcinogenesis is accelerated after RIPK3-deficient BMC chimerism5.NF-?B/COX-2/PGE₂ axis is upregulated in RIPK3-deficient MDSC.6.Inhibitors targeting COX-2 and EP2 blunt the immunosuppressive activity of MDSC and carcinogenesis7.PGE2 negatively regulated RIPK3 via the PKA-CREB signaling pathway,forming a RIPK3-PGE2 circuit to potentiate malignancy.