| Malignant tumors are complex organs consisting of tumor cells and theirmicroenvironment. Increasing evidence has shown that the tumor microenvironment iscritical to the initiation and progression of tumors. The tumor microenvironment plays adecisive role in regulating the process of hepatocarcinogenesis, epithelial-mesenchymaltransition (EMT), tumor invasion and metastasis. Some investigations on the tumormicroenvironment have made significant advancements, but focused mostly on theeffect(s) of single component on tumor cells. The interaction between tumor cells andall surrounding components have not been comprehensively and systematically shown.Thus, the actual progress and mechanism of metastasis are difficult to discover.Objective: In order to systematically evaluate the influence of lymph nodes (LNs)in lymph node metastases (LNM) of hepatocellular carcinoma (HCC), we set up a newin vitro model in which HcaF and HcaP cells were cultured in medium containinglymph node homogenates (LNHs).Methods:Hca-F and Hca-P cells were treated with1.5mg/ml lymph node homogenate in1.0ml serum-free RPMI1640, incubated at37oC in a5%CO2humidified atmosphere for24h. Cells were harvested, centrifuged and supernatants collected for gelatinzymography analysis in7.5%SDS-PAGE gel. After electrophoresis, gels were washed,then incubated at37°C for24h. Gels were stained inCommassie brilliant blue R-250,and destained until clear light brands were visualized against a blue background. Theactivity of MMP-9and MMP-2were analysised by Gel-pro Analyzer4.0.Cell migration was measured with Costar transwell membrane. Hca-F and Hca-Pcells were collected and suspended the transwell chambers with0.6ml RPMI1640with10%FBS for control and with0.6ml RPMI1640with15mg/ml,1.5mg/ml, and0.15mg/ml lymph node homogenate for test. incubated at37oC with5%CO2for18h. After incubation, the filters were fixed in methanol and stained with20%Giemsa. Cellsthat migrated through the filter were counted in five random microscopic fields of400×magnification. The data were expressed as the average cell number of five randomfields. Experiments were repeated twice.Differential protein expression was measured by twodimensional gelelectrophoresis (2-DE) combined with matrixassisted laser desorption/ionizationtimeofflight/timeofflight mass spectrometry (MALDI TOF/TOF MS).The extracted proteins (40μg total protein) were subjected to12%sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted ontonitrocellulose membranes. After incubated in1%BSA for1h, the blotted membraneswere incubated with primary antibodies overnight at4oC. Membranes were washed,incubated with secondary antibodies for1h, and determined by Pierce ECL Westernblotting kit. The bands were analyzed with Gel-Pro Analyzer4.0.Results:Migration of Hca-F and Hca-P cells was increased to about4.75folds and2.59folds respectively, after incubation in LNH for18hours, by transwell assay. Resultsfrom gelatin zymography assay indicated that active matrix metalloproteinases(MMP)-9and MMP-2were increased5.2folds (p<0.05) and4.8folds (p<0.05) inHca-F cells respectively, but they weren’t increased obviously in Hca-P cells, afterincubating with LNH for24hours. Results from protein identification revealed twometastatic correlative proteins,78-kDa glucoseregulated protein (GRP78) andgalectin-3(GAL3). Western blotting confirmed that GRP78, a protein positivelycorrelated with metastasis, increased2.4fold in HcaF cells but decreased to almost ahalf in HcaP cells (P<0.05). However, GAL3, a protein negatively correlated withmetastasis, was decreased by a half in HcaF cells but slightly increased nonsignificantlyin HcaP cells.Conclusions:In our new model, we have used whole non-cellular components of lymph node tosimulate a relatively non-cellular metastatic LN microenvironment. They regulate theHCC cell line with high LN metastasis potential to express appropriate proteinsnecessary for LNM. Albeit profiling/fractionation of the LNH may serve to standardizeour experimental conditions, we propose, within the experimental limitations of thecurrent study, that GRP78and GAL3may be biomarkers for LN metastases diagnosisin HCC. These results reveal that some components of LNHs may facilitate a permissive environment for cancer cells with high metastasis potential to eventuallymetastasize. |
