Type Conversion Of Secretomes In A 3D Tams And HCC Cell Co-culture System And Functional Importance Of CXCL2 In HCC

Part One Induce and identify different activated macrophages and evaluatetheir effect on HCC cellsHepatocellular carcinoma(HCC)is a classic inflammation-related tumor,the tumor microenvironment of HCC infiltrates with a large amount of tumor associated macrophages(TAMs),and the M2 phenotype has been assigned as the dominating TAMs.The aim of this part was to Induce and identify different activated macrophages in vitro and evaluate their effect on HCC cells.Firstly,human monocytic cell line THP-1 were induced into undifferentiated macrophages(UM0),UM0 were then stimulated into the classically activated macrophage M1 and alternatively activated macrophage M2 with LPS and interleukin(IL)-4 + IL-13,respectively.The characteristics of different activated macrophages were then identified by morphology,q RT-PCR and ELISA;finally,the effect of different activated macrophages on HCC cells were evaluated by using cell proliferative,migration and invasion assays,and the expression of several markers of EMT in HCC cells was also detected.Our results shown that:(1)When compared with UM0,the addition of LPS to UM0 lead to elongated,fibroblast-like shaped M1,and the m RNA expression level of TNF-α and CCL3,the secretion of IL-12 of M1 were all increased significantly;whereas the stimulation by IL-4 and IL-13 did not induce visible changes in macrophage morphology but led to a longer and disorganized pseudopodium of M2,and the m RNA expression level of AMAC-1,CCL22 and Arg-1,the secretion of IL-10 of M2 were all increased significantly.These results were consistent with others,indicating that we have successfully obtained the different activated macrophages.(2)Compared with the HCC cells treated with UM0 cultured conditional medium(CM),M2 CM was found could enhance the proliferative,migratory,and invasive capacity of HCC cells,while M1 CM shown no different effects,suggesting that M2 CM could significantly promote the malignant phenotype of HCC cells;(3)HCC cells treated with M2 CM also shown significant changes in epithelial-mesenchymal transition(EMT)marker,with epithelial marker E-cadherin significantly decreased and mesenchymal marker N-cadherin,Vimentin and α-SMA significantly increased;these results demonstrated that M2 CM stimulation could lead to EMT in HCC cells.Part Two Identifying the alteration of secreted proteins in the 3Dco-culture systemTumors in vivo exist in a three-dimensional(3D),complex and open microenvironment,and secreted proteins is the bridge of the cell-cell interaction between tumor cell and TAMs in this microenvironment.In the present part,agarose was used to establish the 3D co-culture system of M2 and SMMC7721 cells,as well as their single 3D culture systems.The conditional medium of these 3D culture systems were then collected,identified and quantified via i TRAQ based quantitative proteome analysis.Western blot was further used tovalidate the i TRAQ result.Our results shown:(1)After i TRAQ quantification and bioinformatic analysis of the secretion protein in different 3D culture systems,a total of 26677 peptides and 1640 proteins were identified(99%confidence Interval).According to the cutoff stander of ratios >1.3 or < 0.7considered up-or down-regulated,the differentially expressed proteins were screened,with 333 differentially expressed in the co-culture system when compared with the M2 culture system,and 357 when compared with the SMMC7721 culture system,159 overlaps were found when compared with both of them;among them,63 were up regulated and 96 were down regulated.(2)Western blot result show that all six proteins were expressed highest in the co-culture system,four(IL-8,IL-1,HBEGF,and IBP3)were completely coincident with the i TRAQ quantification trend,while MMP3 and CXCL2 were found to be more highly expressed in the M2 culture system than in the SMMC7721 culture system.Part Three The function and mechanism of the differentially expressedprotein CXCL2 in HCC metastasisCXCL2 was found as one of the most elevated differentially expressed protein in the co-culture system,and is higher in M2 CM than in SMMC7721 CM,which means it’s mainly secreted by M2 CM.The function of CXCL2 on HCC metastasis and mechanism remains unknown.In part three,we firstly treated HCC cells with different doses of CXCL2 and then evaluated the effects of CXCL2 on HCC cells via Cell migration,invasion and adhesion assays;cell signaling microarray and related biological inhibits techniques were further used to explore the possible mechanism.Our data shown:(1)The expression of CXCL2 was much higher in all 10 HCC tissues compared to the correspondingnon-tumor normal tissues;(2)HCC cells treated with CXCL2 were all found to have significantly increased migration ability as compared to the untreated cells,while have significantly enhanced matrigel invasion ability under 1ng/m L CXCL2 stimulation,and exhibited a significantly lower adhesion capacity on FN-coated surfaces after treatment with 10 and 100ng/m L CXCL2;(3)CXCL2neutralization weaken the migration and invasion ability of HCC cells;(4)By using specific inhibitor to block the CXCR2 activity of HCC cells,the effects of CXCL2 on the migration,invasion and adhesion ability of HCC cells were all inhibited,indicating that CXCL2 exerted its role in promoting HCC metastasis by binding to CXCR2;(5)Cell signaling microarrays were used to detected the possible activated signaling pathway after the overexpression of CXCL2 in HCC cells,and we found that the phosphorylation level of HSP27,PRAS40,bad,Chk1,Ik Bα(Ser32/36)and total level of Ik Bα were significantly increased;the result of western blot also validated that the increased phosphorylation result of p-Ik Bα;these results suggested that NF-k B pathway in HCC cells was activated after CXCL2 overexpression.(6)By using specific NF-k B inhibitor BAY117082 to block NF-k B pathway of HCC cells,the impacts of CXCL2 on the migration and invasion ability of HCC cells were significantly decreased.All these data indicating that CXCL2 mainly from M2 exerted its effects on HCC metastasis by binding to CXCR2 and activating NF-k B pathway.Conclusions1.We successfully induced and obtained different activated macrophages with typical characteristic of M1 and M2 in vitro;M2CM was found could significantly enhance the proliferative,migratory,and invasive capacity of HCC cells.2.Agarose with low melting temperature was successfully used to establish the 3D co-culture system of M2 and SMMC7721 cells;by using i TRAQ based quantitative proteome analysis,the profiles of secretion protein in different 3D culture systems were established,and 159 differentially expressed secretion proteins were found in the co-culture system;3.CXCL2 was found as one of the most elevated differentially expressed protein in the co-culture system,and it could promoted HCC cell migration and invasion and suppressed cell adhesion,such effects on HCC metastasis were exerted by binding to CXCR2 and activating NF-k B signaling pathway.