Chemokines Secreted By Cancer Associated Fibroblasts Promote Hepatocellular Carcinoma Metastasis

【Background and Objective】HCC lives in a complex microenvironment that includes the extracellular matrix(ECM),diffusible growth factors and cytokines,and a variety of non-epithelial cell types.A specialized group of fibroblasts called CAFs is believed to actively participate in the metastasis and invasion of the tumor cells by providing a unique tumor microenvironment.Evidences from clinical and epidemiological studies have shown a strong association between CAFs and poor prognosis in several types of cancer,including HCC.Although CAFs demonstrate a high degree of heterogeneity due to their various origins,their immunophenotypical characterization primarily excludes epithelial and endothelial common markers but include certain mesenchymal markers.CAFs are greatly different from PTFs in both molecular constitution as well as their functional impact on the neighboring epithelial cells.As α-SMA is a marker of fibroblasts,it is expressed in the CAFs at higher levels than in normal fibrobalsts in adenocarcinoma of the esophagogastric junction and HCC.Further researches found the number of α-SMA positive cells was much higher in CAFs compared with PTFs and CAFs proliferated more efficiently compared with PTFs in HCC.In oral squamous cell carcinoma,compared with normal oral fibroblasts,CAFs show increased expression of TGF-β target genes.However,the paracrine cross-talk between HCC and stromal fibroblasts such as CAFs or PTFs in still poorly understood.Further mechanistic studies reveal that CAFs promote cancer progression and metastasis by releasing a variety of cytokines,including inflammatory factors,growth factors and chemokines.Secretion of soluble factors such as transforming growth factor-β(TGF-β)and hepatocyte growth factor by CAFs was shown to induce malignant transformation.When CAF-secreted fibroblast growth factor-2(FGF-2)is inhibited,angiogenesis is reduced.CAF-derived hepatocyte growth factor(HGF)can active c-Met pathway in pre-invasive DCIS and enhance the transition to invasive carcinomas in breast cancer.CAFs from the prostate cancer also can secrete CXCL-1,CXCL-2,CXCL-3 and interleukin(IL)-8 in response to the stimulation of prostate epithelial cells.As mediators of inflammation,chemokines may modulate the recruitment of inflammatory cells and thus modify the environment in which cancer eventually develops.Fibroblast expression of stromal cell-derived factor-1(SDF-1/ CXCL12)acts as a systemic chemotactic signal for circulating immature endothelial cells(ECs),leading to breast cancer vascularization and metastasis.CAF promoted CXCR2/CXCL5 together promote HCC spreading by inducing the epithelial-mesenchymal transition(EMT)through activation of the PI3K/Akt/GSK-3β/Snail pathway.Elevated expression of CXCR6 promotes HCC invasiveness and a pro-tumor inflammatory environment and is associated with poor patient outcome.These results suggest that CAF and chemokines can promote HCC progression by modulating tumor microenvironment.Based on the research above,we foucs on the mechasim of CAF on promoting HCC’s the metastasis and invasion.Firstly,as the upregulation of abilities of invasion and metastasis of HCC coincide with the process of EMT,we tried to prove that HCC would underwent EMT after coculturing with CAF’s CM by Real-Time PCR,Westerning blotting and cellular Immunofluorescence.Secondly,in order to investigate the pro-metastsis effect of CAFs in vivo,HCC labeled with the luciferase gene(LUC)by lentivirus infection with CAF/PTF were injected subcutaneously into NOD/SCID mice.When obvious tumors in the hypodermal tissue were observed,the mice were subjected to an in vivo luciferase assay to monitor metastasis in organs.Thirdly,by profiling the distinctive chemokines secreted by PTFs and CAFs,we investigated the role of chemokines in the crosstalk of CAF and HCC cells and find the possibly involved pathway,thus providing new strategies for the HCC treatment.【Methods】1.CAFs promote process of EMT in HCC(1)Conditional medium(CM):CAFs or PTFs were plated in 35 mm culture dishes at 1×10~5 cells per dish and cultured with DMEM containing 10% FBS overnight.Then,the medium was changed to DMEM containing 3% FBS,The conditional medium(CM)was collected after culturing for 24 hours and filtered through a 0.22 μm membrane.(2)Huh7 cells were inoculated into a 35 mm culture dish.The medium was changed to CAF/PTF CM when Huh7 were adherent to the wall.The EMT related indicators were detected by Real-Time PCR,morphological shape,cellular immunofluorescence and Westerning blotting.2.Animal modelsFour-week-old male NOD/SCID mice were purchased from Shanghai Experimental Animal Center and housed under pathogen-free conditions.The HCC metastatic mouse models were established as previously described.Briefly,2×10~6 PLC cells labeled with the luciferase gene(LUC)by lentivirus infection were injected subcutaneously into NOD/SCID mice along with PTFs or CAFs at ratio of 4:1.When obvious tumors in the hypodermal tissue were observed at 8 weeks after inoculation,the mice were subjected to an in vivo luciferase assay to monitor metastasis of the left arm bone,lung,spleen,liver,kidney and brain.3.Molecular mechanism of CAF’s promoting HCC metastsis(1)Chemokine array and chemokine ELISAThe profile of chemokines secreted by CAFs and PTFs were detected with the culture supernatants of three pairs of CAFs and PTFs using a Human Chemokine Array according to the manufacturer’s instructions.The chemokines with significant differences in expression were screened out.By using CXCL16 ELISA kit,we determined the amount of CXCL16 secreted by 13 paired CAFs/PTFs and five HCC cells.(2)Effect of chemokines(CCL2,CCL5,CCL7 and CXCL16)on migration and invasion ability of HCC cellsCell migration and invasion assays were performed using transwell chambers with or without Matrigel according to the manufacturer’s instructions.HCC cells were plated into the upper chamber in serum-free medium.Conditional medium(CM)from CAFs or PTFs or the indicated chemokines were added to the lower chamber.After a 24-or 72-hour incubation at 37℃,the cells remaining in the upper chamber or on the upper membrane were removed with a cotton swab.The cells that had migrated or invaded to the lower surface of the membrane were fixed with a solution containing 0.1% crystal violet and 20% methanol and then photographed with an inverted microscope.The area of positive staining was measured using image analysis software(Image-Pro Plus 6.0).Migration and invasion were calculated as the percentages of the positive-staining areas.(3)Effect of antagonist of the four chemokines’ s receptors on CAF induced migrationAntagonist of the CCR2(INCB3284 100 nM),CCR5(Maraviroc 100 nM),CCR1/3(UCB35625 100 n M)mixed with HCC were added to the upper chamber of transwell.DMEM(contain 10%FBS)or 10%CAF’S CM were added to the lower chamber.After 24 hours,staining positive area were calculated.(4)Effect of CAF on tumor signaling pathwayTo detect the effect of CAFs on the Wnt,Hippo,NF-κB,Hedgehog and TGF-β pathways,the reporter plasmids of these pathways were co-transfected into Huh-7 cells with p RL-SV40 using Lipofectamine 2000 in serum-free DMEM.Twelve hours later,the culture medium was changed to CM from PTFs or CAFs.Luciferase activities were measured using Dual-Luciferase Reporter Assay 24 hours later with a luminometer.The luciferase score was calculated by normalizing the firefly luciferase signal to that of renilla luciferase.(5)CXCL16,CCL7 activate TGF-β pathwayWe collect the HCC protein after being stimulated by CXCL16,CCL7 for 0,15 min,30min,1 hour,2 hours and detected the phosphorylation of smads protein by Western blotting.(6)The role of chemokines in the process of EMTThe netrulizing antibodies of the four chemokines were added to the CM of CAF and cultured HCC for 24 hours,then the protein lysis and RNA were collected for the detection of EMT related markers.【Results】1.CAFs promote process of EMT in HCCWe investigated the effect of CAF on the EMT of HCC cells.As expected,Huh-7 cells cultured with CAF-CM for 6 days exhibited a striking morphological change,from a flat round shape to a spindle-like shape with the loss of cell-cell contact.Real-time PCR data showed that CAFs significantly increased the expression levels of the representative mesenchymal genes,including N-cadherin,Snail,Twist1,α-SMA and Vimentin in Huh-7cells,while PTFs had only a slight effect on these genes.In contrast,the expression levels of epithelial genes such as ZO-1 and E-cadherin were downregulated by CAFs.Western blotting and immunofluorescence analysis also confirmed that CAF could induce the EMT of Huh-7 cells.2.CAF promotes the metastasis of HCC in vivoWe examined the effect of CAFs on HCC metastasis in vivo.Luciferase-labelled PLC cells alone or mixed with CAFs or PTFs were subcutaneously inoculated into the NOD/SCID mice to establish the subcutaneous tumor mouse model.Obvious subcutaneous tumors were formed 8 weeks after inoculation in all mice.The mean tumor mass in the three groups of mice did not differ significantly at the endpoint.The mice were then subjected to an in vivo luciferase assay to monitor the metastasis of the left arm bone,lung,spleen,liver,kidney and brain.In the group of mice inoculated with PLC cells alone,two of the eight mice had metastasis,one in the kidney and bone and the other in the liver.In the group of mice co-injected with PLC cells and PTFs,two mice had bone metastasis,and one mouse had lung metastasis.However,most of the mice(7/8)inoculated with PLC cells and CAFs had HCC metastasis in the bone,lung or brain.The metastatic tumor nodules of the lung and bone were further confirmed by H&E staining.Bone lesions formed by metastatic tumors were mostly restricted to the bone marrow cavity,with little engagement of osteoclasts or bone matrix destruction.These data suggest that CAFs markedly enhance the tumor metastasis ability of PLC cells.3.CAF activate the Hedgehog and TGF-β pathway in HCC through secreting CCL2,CCL5,CXCL16 and CCL7Seven chemokines were more highly expressed in response to CAFs-CM compared with PTFs-CM.Particularly,CCL2,CCL5,CCL7 and CXCL16 were significantly increased in CAFs-CM compared with PTF CM.CXCL16 ELISA also shows that six CAFs secretd more CXCL16 than the paired PTF in thirteen CAFs/PTFs.Our experiments showed that the activity of the TGF-β luciferase reporter in Huh-7 cells increased almost 9-fold in response to CAFs-CM treatment compared with the control group.In addition,Hh signaling was also highly activated in Huh-7 cells cultured with CAFs-CM.As expected,these four chemokines all increased the migration of HCC cell.In addition,the neutralizing antibodies of the chemokines(CCL2,CCL5,CCL7 and CXCL16)partially blocked the CAF-induced HCC migration.However,only CCL7 and CXCL16,but not CCL2 and CCL5,significantly enhanced the invasion of HCC cells.Consistently,the neutralizing antibodies of CCL7 and CXCL16 attenuated the effect of CAFs on HCC invasion.The neutralizing antibodies of these four chemokines suppressed the effect of CAFs on EMT of HCC cell.【Conclusions】1.CAFs can promote EMT in HCC through CCL2,CCL5,CCL7 and CXCL16.2.CAFs can prmote HCC metastasis in vivo.3.CAFs secreted CCL2,CCL5,CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.