Preliminary Study On The Differences And Related Factors In Tumor Growth And Metastasis Of Three Oscc Orthotopic-Xenograft Models

Objective:In recent years,orthotopic-xenograft nude-mouse models established from oral-cancer cell lines have been widely used to study the biology of oral cancer.In some studies,to rapidly evaluate the initial effects of new targeted drugs on the proliferation of oral cancer in vivo,an orthotopic-xenograft model with a short tumor formation time and rapid tumor growth rate is needed.In other studies,such as those examining tumor metastasis,an orthotopic-xenograft model characterized by a high lymph node metastasis rate is needed.However,few studies have compared the differences in tumor formation time,growth rate,and tumor metastasis among orthotopic-xenograft nude-mouse models established from different oral cancer cell lines.Therefore,to explore which oral cancer cell lines can be used to establish the orthotopic-xenograft model with fast proliferation rate or high metastasis rate in a short time has become the primary problem need to be solved to improve the efficiency of orthotopic-xenograft nude-mouse model establishment of oral cancer.In addition,it is not clear why there are differences in tumor formation,proliferation and metastasis in orthotopic-xenograft models established by different oral cancer cell lines.In this study,we used three widely used tongue cancer cell lines(CAL27,SCC9,and SAS)to establish orthotopic-xenograft nude-mouse models.Differences in tumor growth and metastasis were compared and potential factors explaining these differences were predicted.The purpose of this study is to improve the efficiency of orthotopic-xenograft model establishment and to provide a selective reference for the researchers who need to establish orthotopic-xenograft model of oral cancer.Methods:(1)Forty-five nude mice were randomly divided into 9 groups with 5 mice in each group.CAL27,SCC9,and SAS cells were trypsinized and resuspended in PBS,and then adjusted to 3 different concentrations of 5×10~6/ml?1×10~7/ml and 2×10~7/ml.The cells were injected(50?l per tongue)into the tongue of nude mice.Tumor formation in the tongue of nude mice was observed to select appropriate cell concentration for subsequent experimental study.(2)Fifteen nude mice were divided into three groups randomly with five mice in each group.Three groups of mice were injected with tongue cancer cell line CAL27,SCC9,or SAS with 2×10~7/ml cell concentration into their tongue.(3)Observation was taken every day to determine the orthotopic tumor formation time.The volume of tongue tumors was measured every two days after tumor formation to evaluate the growth rate of orthotopic tongue tumors in vivo.(4)HE staining was used to determine the formation of the orthotopic-xenograft tumor in the tongue and the tumor cell proliferation level in vivo was determined by immunohistochemical Ki67 staining.(5)HE staining and immunohistochemical cytokeratin staining of submandibular lymph nodes were performed to determine the level of submandibular lymph node metastasis in three orthotopic-xenograft models.(6)A cell proliferation assay was conducted to determine the growth of CAL27,SCC9,and SAS cells in vitro.(7)The cell migration ability of CAL27,SCC9,and SAS cells was measured by scratch assay.(8)The mRNA expression levels of epidermal growth factor receptor(EGFR),matrix metalloproteinase 9(MMP9),E-cadherin,N-cadherin,CD44,cyclin D1,and intercellular cell adhesion molecule 1(ICAM-1)in these three cell lines were measured by real-time PCR.Results:(1)Three orthotopic-xenograft models of oral cancer were successfully established in nude mice via cell lines CAL27,SCC9,and SAS.When the cell concentration was 2×10~7/ml,tumor formation was observed in the tongue of mice in groups SAS and CAL27 three days after tumor cells were injected,and the tumor formation rate was 100%.Tumor formation in the SCC9group was seen approximately at 4 weeks after injection,and the tumor formation rate was 80%.(2)Three groups of orthotopic-xenograft models were successfully established in nude mice at 2×10~7/ml cell concentration.The order of the tumor volume and the growth speed of tongue tumors among the three groups was SAS>CAL27>SCC9.(3)The order of the expression of Ki67 in tumors among the three groups was SAS(65.13%±5.24%)>CAL27(21.02%±2.50%)>SCC9(5.24%±3.42%)(P<0.05).(4)The cell proliferation level in vitro and the expression level of EGFR mRNA in cell lines from high to low were SAS group,CAL27 group and SCC9 group whereas the expression levels of N-cadherin mRNA in cell lines from low to high were SAS group,CAL27 group and SCC9 group.The results were consistent with the trend of tumor proliferation in the three groups of orthotopic-xenograft models.(5)HE staining showed that the lymph node metastasis rates of the SAS group and the CAL27 group were relatively lower.The lymph node metastasis rate was 20%in the CAL27 group and 40%in the SAS group.The lymph node metastasis rate was relatively higher in the SCC9 group,at a metastasis rate of 90%(P<0.01).(6)The order of the rates of cytokeratin positive metastatic cells in lymph node among the three groups was SCC9(51.73%±17.59%)>SAS(19.64%±9.66%)>CAL27(8.79%±4.51%)(P<0.05).(7)The cell migration ability in vitro and the order of the expression of MMP9?E-cadherin mRNA(in cells)among the three groups was SCC9>SAS>CAL27.The results were consistent with the trend of lymph node metastasis in the three groups of orthotopic-xenograft models.(8)The expression levels of CD44,cyclin D1 and ICAM-1 mRNA in CAL27?SCC9 and SAS cells were not significantly correlated with the trend of tumor proliferation and metastasis in the three groups of orthotopic-xenograft models.Conclusions:(1)Orthotopic-xenograft nude-mouse models established via CAL27 and SAS cells have a higher speed of tumorigenicity and are suitable for researches related to rapid tumorigenicity.(2)The orthotopic-xenograft model established on the basis of SCC9 cells has a higher lymph node metastasis rate and is appropriate for researches related to metastasis.(3)In the orthotopic-xenograft model induced by CAL27,SCC9 and SAS cell lines,the proliferation level of a primary orthotopic-xenograft tumor may correlate with the cell proliferation level in vitro,the high expression of EGFR and low expression of N-cadherin mRNA in oral-cancer cell lines.The cell migration ability in vitro,the expression of MMP9 and E-cadherin mRNA in an oral cancer cell line may have a strong influence on lymph node metastasis in in vivo models.