Tumor Necrosis-induced CD61-positive M2-type Macrophage Recruits Neutrophils In HCC Through TLR4/CXCL5 Signaling

Background and aims:Tumor necrosis is a prominent feature of solid tumor including Hepatocellular Carcinoma.However,the effect of tumor necrosis on the progression of HCC is still unrevealed.We have found there is a kind of IL-1β-positive M2-type tumor-associated macrophages in HCC,which is induced by tumor necrosis.And it may play an important role in the progression of HCC by producing IL-1β.The purpose of this study is to further explain the phenotype and function of the M2-type TAMs(CD61~+).Meanwhile,we are going to establish a universal and effective method to evaluate the histological grading of necrosis in HCC specimens,and to evaluate the effect of tumor necrosis risk on the prognosis of HCC.Materials and methods:HCC tissue samples were obtained from the First Affiliated Hospital of Zhejiang University School of Medicine and the Second Affiliated Hospital of Zhejiang University School of Medicine.We got 179 tumor specimens and established an effective method for necrosis scoring by study the pathological features of HCC samples.We have analysed the related factors of tumor necrosis by collecting some clinical information of HCC patients,including tumor size,Body Mass Index,TBIL,AFP and so on.The analysis of gene expression of macrophages was accomplished by RNA sequencing.And the expression of CD206,CD61,Elastase,CXCL5 were detected by immunohistochemistry and cell cytometry.The expression of TLR4,NF-κb were tested by western blotting.And IL-1β,CXCL5 levels in the cultured cell supernatant were detected by enzyme linked immunosorbent assay(ELISA).And chemotaxis of neutrophils was detected by transwell migration assay and FACS counting.Results:We have collected 179 HCC cases,including 96 cases with necrosis and 83 cases without necrosis by pathological analysis.And we found that TNM classification(III-IV)is a risk factor for HCC necrosis(p<0.05).TBIL(>21umol/L),ALB(<40g/L),TNM classification(III-IV),tumor necrosis are independent risk factors for the prognosis of HCC.The OS of HCC without necrosis is significantly higher than that of HCC with necrosis(p<0.001),and the DFS of HCC without necrosis is higher than that of HCC with necrosis(p=0.0978).We have confirmed that there is a kind of CD61-positive M2-type macrophages induced by tumor necrosis,whose energy source depends on glycolysis.Meanwhile,we found the up-regulation of CXCL5 derived from CD61-positive M2-type TAMs could regulate the accumulation of neutrophils through TKR4/NF-κb pathway in vitro.Higher level of tumor-associated neutrophils in HCC implied a positive relationship with poor prognosis of HCC patients.Conclusions:We have established an effective method to evaluate HCC necrosis.The level of necrosis in HCC tumor,which is also named as HCC necrosis score(HNS),is categorized into 4 degrees according to SNS,including 0,1,2,3.TNM classification(III-IV)is a risk factor of HCC necrosis.HCC necrosis is significantly associated with poor prognosis.This model can be effectively used to predict the prognosis of HCC patients.And it can provide strong evidence in prevention of metastasis and relapse of HCC.There is a kind of CD61-positive M2-type macrophages induced by tumor necrosis in HCC.And CXCL5 derived from CD61-positive M2-type TAMs,induced by tumor necrosis could regulate the accumulation of neutrophils through TKR4/NF-κb pathway.To summarize,tumor necrosis is significantly correlated with poor prognosis of HCC patients.