| [Background and Objective]Hepatocellular carcinoma (HCC) is one of the most common digestive system neoplasms. For nearly30years, despite the management of HCC have achieved great progresses, its prognosis has not been fundamentally improved yet. Therefore, in-depth studies on the occurrence and development of HCC, as well as its resistance mechanisms, in combination with searching effective therapeutic targets is a problem needs to be overcome by researchers.Along with the continuously deepening in cancer researches, the tumor cells are found not "alone", instead, they can recruit the surrounding stroma cells to form a new integration called tumor microenvironment (TME). The tumor microenvironment is consisted of tumor cells, extracellular matrix (ECM), multiple stromal cells and their secreted proteins. Stromal cell components are mainly divided into three categories, including angiogenic cells, immune cells and tumor-associated fibroblasts (TAFs). The increasing studies demonstrate that TAFs are involved in the maintenance of tumor hallmarkers such as sustained activation of proliferative signals, resistance to cell death, maintenance of cell continuous replication, inducing angiogenesis, activating invasion and metastasis and changing energy metabolism, etc. Meanwhile, TAFs increase the resistance of tumor cells to chemotherapeutic drugs. Therefore, targeting TAFs to block its effect on tumor "supporting" may provide a new strategy for HCC treatment.Based on the mentioned research background, the present study mainly intends to explore the role of TAFs in the occurrence and development of HCC. The research will be expanded from three aspects. Firstly, TAFs and peri-tumor fibroblasts (PTFs) are isolated and purified from human HCC tissue. The expression of TAFs and PTFs-associated markers are detected by immunofluorescent and Real-time PCR for investigating the differences in the biological characteristic between them. Secondly, on this basis, an in vitro co-culture system of TAFs and HCC cell lines is established to observe the effect of TAFs on the proliferation, invasion, metastasis, sphere formation, epithelial-mesenchymal transition (EMT), subcutaneous tumorigenesis and other biological effects in HCC cell lines. Thirdly, the mechanism of TAFs in promoting tumor malignant transformation is further studied. The microarray inspection is used for screening the chemokines with differences in the secretion by TAFs and PTFs, in order to investigate the role of chemokines in TAFs and Cross-talk in HCC cells and find the related signal transduction pathways, providing new ideas and methods for clinical treatment of HCC.[Methods]1. Isolation and identification of TAFs(1) Isolation and purification of TAFsHuman peritumoral and HCC tissues were minced with scalpels and enzymatically dissociated by collagenase Ⅳ. The suspension was then centrifugated to separate the epithelial and fibroblast cells. The primarily isolated fibroblasts were purified by using Anti-fibroblast beads.(2) Identification of TAFs and PTFsThe expressions of a-SMA, Vimentin, FSP-1and EMT related indicators in TAFs and PTFs were detected by immunofluorescent and Real-Time PCR.(3) Characterization of TAFs and PTFsTo evaluate proliferation ability, TAFs and PTFs were inoculated into a96-well plate with the concentration of3×103per well. Each group contained three wells. The proliferations of TAFs and PTFs were detected daily by CCK-8. TAFs and PTFs were enzymatically dissociated and suspended into serum-free DMEM, and then inoculated into the upper migration chamber with the concentration of4×104. The migrations of TAFs and PTFs were detected after24hours.2. Effect of TAFs on the biological characteristics of HCC cell lines(1) Establishment of the co-culture system of TAFs, PTFs and liver tumor cellsTAFs and PTFs were transferred into the0.4μm co-culture chamber and co-cultured with the lower tumor cells. Alternatively, TAFs and PTFs culture supernatants (conditioned medium, CM) were collected for direct coculture with HCC cells.(2) Proliferation assay, Invasion assay and migration assayThe co-culture system of TAFs, PTFs and HCC cells was established. The effects of TAFs and PTFs or their conditioned medium (containing3%FBS in DMEM, fibroblasts culture for24hours) on the proliferations of different HCC cells were detected by CCK-8. TAFs and PTFs (1×104) were inoculated into the bottom layer of a24-well plate. HCC cell line LM3was inoculated into the upper invasion chamber.24hours later, the invasive cells were stained with crystal violet, which were then observed and photographed under the microscope. TAFs and PTFs or their conditioned medium (containing3%FBS in DMEM, fibroblasts culture for24hours) were inoculated into the bottom layer of a24-well plate. The HCC cell line was inoculated into the upper invasion chamber.24hours later, the invasive cells were stained with crystal violet, which were then observed and photographed under the microscope.(3) Effect of TAFs on Epithelial-mesenchymal transition (EMT)Huh7cells (2×105) were inoculated into a35mm culture dish. When the cells were adherent to the wall, the medium was changed to TAFs/PTFs conditioned medium (containing3%FBS in DMEM, fibroblasts culture for24hours). The expression of EMT-related indicators was detected by Real-time PCR and immunofluorescence(4) Effect of TAFs on the sternness of cancer cellsHuh7cells were treated with TAFs/PTFs conditioned medium (stem cell-dedicated culture medium, fibroblasts culture for24hours) for48hours. The changes in mRNA expression levels of cancer stem cells (CSCs) and self-renewal-related genes were detected by Real-time PCR. Huh7cells were sorted into Epcam-positive and Epcam-negative cells with the fluorescence activated cell sorter. The sorted cells were added into an ultra-low attachment96-well plate with ten cells per well (the unsorted cells were considered as the controls). The cells were treated with TAFs/PTFs conditioned medium (stem cell-dedicated culture medium, fibroblasts culture for24hours). The pelletized cells were counted after seven and fourteen days for calculating the sphere formation efficiency rate.(5) Effect of TAFs on tumor xenografts and migrationSubcutaneous tumor experiments:TAFs/PTFs were mixed with human HCC cell line LM3at1:5for preparation of the mixed cell suspension. It was then subcutaneously inoculated into the naked mice for establishment of the mouse model of subcutaneous tumor. The subcutaneous tumorigenicity was observed.In vivo metastasis assay:for in vivo metastasis assay,1×106Huh7cells stably expressing luciferase and2×105PTFs/TAFs were coinjected into NOD-SCID mice subcutaneously. Seven weeks post inoculation, the mice were subjected to in vivo luciferase analysis to monitor metastasis of the left arm bone, lung, spleen, liver, kidney and brain.3. Mechanism of TAFs in promoting HCC malignant transformation(1) Detection of chemokine secretion in TAFs and PTFs Three pairs of TAFs and PTFs culture supernatants were (serum-free DMEM, culture for24hours) collected for detection of the chemokine expression profiles by Raybiotech Kit. The chemokines with significant different expressions were screened out. TAFs/PTFs RNA from different sources was collected (serum-free DMEM, culture for24hours).(2) Effect of chemokines (CCL2,CCL5,CXCL16) on migration ability of HCC cellsThe chemokines CCL2, CCL5and CXCL16were added into the lower medium (500μl, containing1%FBS in DMEM) of a24-well culture with the concentration of0,10,20and40ng/ml, respectively. Huh7cells (5×104) were inoculated into the upper migration chamber and incubated for24hours, followed by staining and photographing. TAFs/PTFs conditioned medium (containing1%FBS in DMEM, fibroblasts culture for24hours) was collected. The neutralizing antibodies of chemokines CCL2, CCL5and CXCL16were added into the TAFs/PTFs conditioned medium with the concentration of0,1and5μg/ml, respectively. The solution was mixed and added into a24-well plate with500μl per well. Huh7cells (5x104) were inoculated into the upper migration chamber and incubated for24hours, followed by staining and photographing.(3) Effect of TAFs on tumor signaling pathwayHuh7cells were transfected with reporter gene plasmids (Wnt, Hippo, NF-κB, Hedgehog and TGF-β). Six hours later, the culture medium was changed to TAFs/PTFs conditioned medium (containing1%FBS in DMEM, fibroblasts culture for24hours). The luciferase expression was detected after co-culture for24hours. The changed signal pathway was screened out. Huh7cells (1×105) were inoculated in a35mm culture dish. When the cells were adherent to the wall, the culture medium was replaced by TAFs/TFs conditioned medium (containing1%FBS in DMEM, fibroblasts culture for24hours).24hours later, the RNA was collected for detection of the expression of Hedgehog and TGF-β pathway-related genes by Real-time PCR.(4) Effect of chemokines on Hedgehog and TGF-β pathwayHuh7cells were transfected with Hedgehog and TGF-P reporter gene plasmids. Six hours later, the culture medium was changed to serum-free DMEM. The chemokines CCL2, CCL5and CXCL16were added into the culture medium with the concentrations of0,10and20ng/ml. The activations of Hedgehog and TGF-β pathways were detected. TAFs/PTFs conditioned medium (containing1%FBS in DMEM, fibroblasts culture for24hours) was used for treatment of transfected cells. The neutralizing antibodies of CCL2, CCL5and CXCL16were added (0,1,5μg/ml) and co-culture for24hours. The activations of Hedgehog and TGF-β pathways were detected. Huh7cells (1×105) were inoculated in a35mm culture dish. When the cells were adherent to the wall, the culture medium was replaced by serum-free DMEM for12hours of starvation. The chemokines CCL2, CCL5and CXCL16were added into the culture medium with the concentrations of0.10and20ng/ml.24hours later, the RNA was collected for detection of the expression of Hedgehog and TGF-β pathway-related genes by Real-time PCR.4. Statistical analysisSPSS16.0software package was used for statistical analysis. The results were expressed as with X±SD. One-Way ANOVA was used for inter-group analysis. P<0.05was considered significantly different and P<0.01was considered extremely significantly different.[Results]1. Primary TAFs and PTFs present different biological characters(1) Identification of TAFs and PTFsThe immunofluorescence results showed that a-SMA, Vimentin and FSP-1were all expressed in PTFs and TAFs, in which a-SMA and FSP-1expressions in TAFs were significantly stronger than those in PTFs. However, there was no significant difference in Vimentin expression between TAFs and PTFs.Real-Time PCR results showed that a-SMA, FAP and PDGF expressions in TAFs were higher than those in PTFs (P<0.05). The expressions of N-cadherin, Snail and Twist in TAFs were higher than those in PTFs (P<0.05). The expression of E-cadherin, epithelial-related indicator, in PTFs was higher than that in PTFs (P<0.05).(2) Characterization of TAFs and PTFsAll TAFs showed higher proliferation and migration abilities. Different TAFs present different abilities of proliferation and migration.2. TAFs promotes the malignancy properties of HCC cells(1) TAFs promoting the proliferation, invasion and migration of HCC cellsLM3and Huh7cells were treated with fibroblast conditioned medium. It was found that the promoting effect of TAFs on the proliferation of HCC cell line was significantly higher than that in PTFs and the normal control group. In addition, different TAFs conditioned medium was co-cultured with Huh7cells, leading to varying degrees of increase in Huh7cell proliferation. TAF cells were co-cultured with MHCC-H, SK-Hepl, Huh7and PLC, which could significantly promote the proliferation of different tumor cell lines. The results of invasion experiment showed that the invasion rate of LM3cells (89.7%) in the TAFs treatment group was higher than that in the PTFs treatment group (66.3%) and the control group (39.1%)(P<0.05). The migration experiment showed that the TAFs conditioned media from different sources could promote Huh7cell migration, and the promoting ability was higher than in the PTF group and control group (P<0.05). Furthermore, TAFs enhanced the migration ability of MHCC-H, SK-Hep1, Huh7and PLC cells in HCC cells.(2) TAFs facilitate Epithelial-mesenchymal transition of HCC cellsHuh7cells were treated with TAFs conditioned medium. The results of Real-time PCR showed that the expressions of representatives genes of Huh7intercellular matrix N-cadherin, Snail, Twist and α-SMA were all up-regulated (compared with the PTFs group, P<0.01), and the expression of representatives gene E-cadherin was down-regulated (P<0.01). The immuno fluorescence results showed that there was no significant difference in E-cadherin and Vimentin fluorescence intensities between the control group, PTFs group and TAFs group; however, the ratio of Vimentin-positive cells was significantly increased in TAFs conditioned medium treatment group.(3) TAFs increase the sternness of cancer cellsHuh7cells were treated with TAFs conditioned medium. The results of Real-time PCR showed that the expressions of CSC-related genes CD44, CD90, CD133and Epcam were all up-regulated (P<0.05). In addition, the expressions of self-renewal-related genes Sox2, Oct4and Klf4were also significantly increased (P<0.05). Huh7cells were sorted into Epcam-positive and Epcam-negative cells with the fluorescence activated cell sorter. The pelletizing testing was conducted with the unsorted cells considered as the controls. The results showed that TAFs conditioned medium could increase the pelletizing rates of Epcam-positive, Epcam-negative and unsorted Huh7cells, in which the pelletizion rate of positive cells was most significantly increased up to about12%(P<0.05). However, PTFs had no significant effect on the pelletizion ability.(4) TAFs increase tumor formation and metastasis abilities of HCC cellsTumors in TAFs group were larger than that in PTFs group at every time point. Nude mice were sacrificed at day21, The results showed that both the tumor weight and tumor volume in TAFs group were greater than the PTFs group, indicating that TAFs had a stronger ability of promoting the tumorigenicity than PTFs. TAFs significantly promoted the lung metastasis of Huh7cells, six mice presented metastasis in TAFs group (seven mice totally). By contrast, one mice occurred metastasis in PTFs group.3. Mechanism of TAFs in promoting HCC malignant transformation(1) Detection of chemokine secretion in TAFs and PTFsThe chemokine secretion profile of TAFs and PTFs were different, there were seven chemokines which express higher in TAFs. Real-time PCR confirmed that the expressions of CCL2(MCP-1), CCL5(RANTES) and CXCL16in TAFs were significantly higher those in PTFs. Meanwhile, the receptors of chemokines were increased in TAFs. The results were validated in other two pairs of TAFs and PTFs.(2) Chemokines CCL2, CCL5and CXCL16promoting Huh7cell migrationThe results of migration assay showed that CXCL16had the highest pro-migration ability, and CCL2had a similar pro-migration ability to CCL5, with a migration rate of about30%under the stimulus of20ng/ml concentration (P<0.001). Meanwhile, the neutralizing antibodies of chemokines CCL2, CCL5and CXCL16could partially block TAFs in promoting Huh7migration.(3) TAFs activate Hedgehog and TGF-β pathwayAmong five detected pathways including Hedgehog, TGF-β, NF-κB, Wnt and Hippo, only Hedgehog and TGF-β were activated after stimulation by TAFs. The TGF-β pathway was significantly activated in TAFs treatment group, in which the luciferase expression was nine times as the control group and the luciferase expression in Hedgegog pathway was2.5times as the control group (P<0.01). Real-time PCR was used to determine whether TAFs activated the downstream genes in Hedgehog and TGF-β pathways. The results showed that TAFs could up-regulate smad-5and c-myc in TGF-β pathway, and the downstream genes Gli-1, Shh and Bcl-2in Hedgehog pathway could be also activated by TAFs.(4) CCL2, CCL5, CXCL16activate Hedgehog and TGF-β pathwayThe reporter gene was used for assay of chemokines in activating the pathway. It was found that CXCL16could activate TGF-β pathway, and CCL2and CCL5could activate Hedgehog pathway. In addition, the neutralizing antibodies of CCL5and CXCL16could block the activation of TAFs on Hedgehog and TGF-β pathways. It was suggested that CCL5and CXCL16were conducive to the activation of TAFs. The effect of chemokines on the downstream gene in the pathway was detected by Real-time PCR. The results showed that CCL2and CCL5could activate Shh, Bcl-2and Gli-1, the downstream genes in Hedgehog pathway, and CXCL16could upregulate the expressions of c-myc and smad-5in TGF-P pathway.[Conclusions]1. TAFs present stronger expression in a-SMA and FSP1. TAFs show greater abilities to proliferate and migrate.2. TAFs promote proliferation, invasion, migration, tumorigenesis and tumor metastasis of HCC cells.3. TAFs accelerate EMT in tumor cells and maintain the "stemness" of tumor cells.4. The chemokines secreted by TAFs such as CCL2, CCL5and CXCL16promote tumor cell malignant transformation by activating TGF-β and Hedgehog pathways. |
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