The Levels And Clinicopathological Significance Of S100A9 And Mdscs In CRC Patients And Effects Of S100A9 On Migration And Activation Of MDSCs And Their Mechanisms

Backgrounds and objectivesColorectal carcinoma(CRC)is the third most common cancer and fourth leading cause of cancer death worldwide.At present,the treatment of CRC is surgery with radiotherapy,chemotherapy and targeted therapy.However,the 5-year survival rates of advanced CRC is only 10%which is still a serious threat to human health.Therefore,it is necessary to research into the CRC.S100A9 belongs to a family of more than 20 low molecular weight intracellular EF-hand motif calcium-binding protein found exclusively in vertebrates.S100A9 is constitutively expressed by myeloid cells,including granulocytes,monocytes,osteoclasts and early-differentiation cells of the myeloid lineage,but not by lymphocytes.And S100A9 participates in inflammatory response as a secretory inflammatory factor.Studies have demonstrated that the overexpressed S100A9 is correlated with the invasion and metastasis in varieties of cancers and directly enhanced tumor cell malignancy.Our previous studies have shown that S100A9 is highly expressed in CRC tissues and it has the direct effect of promoting the growth and migration of CRC cells in vitro.Furthermore,S100A9 is also considered as an important marker of myeloid-derived suppressor cells(MDSCs).Studies have shown that MDSCs is an obstacle to tumor immunotherapy.They represent a heterogeneous population of immature myeloid cells consisting of precursors for granulocytes,macrophage or dendritic cells(DCs)that are accumulated during chronic inflammation and tumor progression.The appearance of this tolerogenic population,MDSCs,represents a common trait of cancer and other noncancerous diseases.Being one of the most potent immunosuppressive cells,MDSCs promote tumor progression by inhibiting the anti-tumor functions of T and NK cells.MDSCs in peripheral blood and tumor tissue in patients with CRC,bladder cancer,and so on,are increased.Although the immunosuppressive function of MDSCs has been clarified,the molecule mechanisms related to its function have yet not to be clarified.Therefore,it is prospective to find the potential values of S100A9 and MDSCs in the diagnosis and treatment of CRC through intensively studying the levels of S100A9 and MDSCs in peripheral blood of CRC and their correlation,and the interactions among S100A9,MDSCs and CRC cells.Based on the above,we detect the level of serum S100A9 and the frequency of MDSCs in peripheral blood in patients with CRC and analyse the association between MDSCs and S100A9 in CRC samples and investigate the potential diagnostic value of the two indexes for CRC.Furthermore,we elaborate the effect of S100A9 on trafficking,expansion and activation of LoVo-MDSCs,which are induced with human colorectal cancer cell line LoVo in vitro,and as well as the molecular mechanisms underlying these effects.This study has accumulated experimental basis for elucidating the complex interactions among S100A9,cancer cells and MDSCs in CRC microenvironment and the effect on the progression and mechanism of CRC.Moreover,it also provides a new candidate marker and intervention target for the clinical diagnosis and treatment of CRC.Methods1.The detection of tissues and serum S100A9 levels and MDSCs frequency in CRC patients.1-1 Immunohistochemistry was used to determine S100A9 levels and the infiltrating of CD33~+cells in CRC tissues and paraneoplastic tissues.1-2 Enzyme-linked immunosorbent assay(ELISA)was used to detected S100A9 levels in CRC patients(n=52)and healthy controls(n=30).1-3 The peripheral blood mononuclear cells(PBMC)were separated from the whole blood of CRC patients and healthy controls.And the frequency of MDSCs was detected by flow cytometry.1-4 Spearman r analytical method was used to analyse the relation between S100A9 level and MDSCs frequency in CRC patients.1-5 The receiver operating characteristic(ROC)curve was used to analyse the diagnostic effect of S100A9 and MDSCs.2.Preparation and identification of recombinant protein GST-human S100A9(GST-S100A9):GST-S100A9 and GST were prepared by prokaryotic expression and identified by SDS-PAGE,coomassie blue staining and western blot.Then the recombinant protein was divided and stored at-80℃for use.3.Tumor-associated MDSCs induction and identification:PBMCs were extracted from peripheral blood samples of patients with CRC.CD33~+cells were screened out from PBMCs by magnetic beads and co-cultured with human colorectal cancer cell line LoVo for 48h.The cells were collected for identification by FCM and used in subsequent experiments,that is called LoVo-induced MDSCs(LoVo-MDSCs).4.The effect of S100A9 on proliferation,migration and activation of LoVo-MDSCs4-1 CCK8 assay was used to detect the effect of GST-S100A9 on LoVo-MDSCs proliferation.4-2 Transwell system was used to analyse the effect of GST-S100A9 on migration of LoVo-MDSCs.4-3 To investigate the effect of S100A9 on LoVo-MDSCs activation,the Q-PCR was used to determine the mRNA expression of Arg-1,iNOS and IL-10 in LoVo-MDSCs after GST-S100A9 treated for 24h and the ROS level was detected by fluorescence immunoenzyme labeling apparatus.CD8~+T cells separated from healthy controls were co-cultured with LoVo-MDSCs and were treated with GST-S100A9 and GST for48h,then their proliferation was detected by FCM to reflect S100A9effect on the function of LoVo-MDSCs,which can inhibit CD8~+T cell proliferation.5.The mechanisms of S100A9-induced migration and activity of LoVo-MDSCs5-1 Western blot was used to detect the total and phosphorylated levels of some proteins in LoVo-MDSCs treated with GST-S100A9.They are RAGE,TLR4,p38/p-p38,p65/p-p65,ERK/p-ERK,JNK/p-JNK and AKT/p-AKT,which may be the key molecules in the related signal pathways.5-2 LoVo-MDSCs were treated with TAK-242(TLR4 inhibitor),FPS-ZM1(RAGE inhibitor),BAY11-7082(NF-κB inhibitor),SB203580(p38 inhibitor)and GST-S100A9 alone or combination for 24h.Transwell system and Q-PCR were used to detect MDSCs migration and activity,respectively.Results1.The level of S100A9 and the frequency of MDSCs in tissues and peripheral blood of patients with CRC.1-1 The level of S100A9 and the infiltration of CD33~+cells in CRC tissues were higher than those in paracancerous tissues.And the serum S100A9 level and the homologous frequency of MDSCs in PBMCs were increased compared with healthy control(P<0.001).1-2 There was a positive correlation between S100A9 level and MDSCs frequency in CRC patients(r=0.38,P=0.01).1-3 Compared with the patients at stage A/B or without lymph node metastasis,both S100A9 level and MDSC frequency were significantly increased in CRC patients at C/D phase(P<0.01and P<0.001)or with lymph node metastasis(P<0.01),but the two indexes had no significantly difference either between the colon cancer and rectal cancer or between the low and middle/high cell differentiation(P>0.05).2.Serum S100A9 and MDSCs had good diagnostic efficacy for CRC,especially for the staging of CRC and lymph node metastasis which suggests that both of them may be potential biomarkers of CRC.3.GST-S100A9 and GST were successfully prepared and the MDSCs were successfully induced to be LoVo-MDSCs in vitro.4.The effect of S100A9 on proliferation,migration and activation of LoVo-MDSCs.4-1 GST-S100A9 had no significant effect on the proliferation of LoVo-MDSCs(P>0.05).4-2 GST-S100A9 promoted the migration and activation of LoVo-MDSCs(P<0.05).5.The mechanisms of S100A9-induced migration and activity of LoVo-MDSCs5-1 The mRNA and protein levels of TLR4 and RAGE in LoVo-MDSCs were increased after treated with GST-S100A9(P<0.01).5-2 FPS-ZM1(RAGE inhibitor)reversed S100A9 effect on LoVo-MDSCs migration(P<0.05).TAK-242(TLR4 inhibitor)reversed the up-regulation of Arg-1,NOS,IL-10 caused by GST-S100A9 in LoVo-MDSCs(P<0.05)and the inhibition of T cell proliferation caused by GST-S100A9 in LoVo-MDSCs(P<0.05).These results suggested that S100A9 can promote migration and activation of LoVo-MDSCs via activating RAGE and TLR4,respectively.5-3 The phosphorylation levels of p38 and p65 were increased in LoVo-MDSCs treated with GST-S100A9.5-4 SB203580(p38 inhibitor)reversed the effect of GST-S100A9 on the migration of LoVo-MDSCs(P<0.05).And BAY11-7082(NF-κB inhibitor)reversed the up-regulation of LoVo-MDSCs activation molecules Arg-1,iNOS,IL-10 and the inhibition of T cell proliferation by GST-S100A9(P<0.05).These results suggested that S100A9 can promote the migration and activation of LoVo-MDSCs by activating RAGE/p38/MAPK and TLR4/p65/NF-κB signaling pathways,respectively.Conclusion1.S100A9 level and MDSCs frequency are increased in CRC patients,and there is a positive correlation between them.2.S100A9 level and MDSCs frequency in the blood are related to the stage of CRC and the lymph node metastasis.It is a potential marker for clinical diagnosis and treatment of CRC.3.S100A9 can promote LoVo-induced MDSCs migration,the mechanisms include activating RAGE/p38/MAPK signaling pathway4.S100A9 can promote LoVo-induced MDSCs activation,one of the mechanisms is activating TLR4/p65/NF-κB signaling pathway.