Characteristics Of Side Population Cells And Their Effects On The Metastasis And Recurrence Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of the most malignant tumors in terms of numbers of cases in China(1,2).Despite enormous progress made in the cancer research field,which have improved quality of life,the prognosis of HCC remains dismal with 5-year survival rate of 5%,which was mainly attributed to the high rate of distant metastases and therapy-resistant local recurrences(2).Therefore,it is of importance to further explore the mechanism of metastasis and recurrence,identify those patients with high recurrence and metastasis in advance and thus allow intervening timely.Stem cells are defined as cells that have the ability to perpetuate themselves through self-renewal,to extensively proliferate and to generate mature cells of a particular tissue through differentiation(4).Recent studies(5) have shown several common characteristics between stem cell and tumor cell,thus cancer presumably is a disease of irregulated self-renewal owing to transforming mutation.Recently,a growing body of evidences(4,5)have supported the notion that tumours contain "cancer stem cells(CSCs)"- rare cells with indefinitely proliferative potential that drive the formation and growth of tumors,which are the root of tumor-initiating, metastasis and failure of therapy through acting as the role of stem cells.These cells have been identified in leukemia(6)and some solid tumors,including breast cancer(7), colon cancer(8,9) and prostate cancer(10).Although CD133⁺ HCC cells were also reported to initiate tumors in vivo,CD133 showed negative in several HCC cell lines and most HCC specimens(11-13).Therefore,this marker may not be successful for the isolation of CSCs in most HCC samples,which has hampered researches into the root of carcinogenesis and recurrence in HCC.Fortunately,other properties of stem cells,such as the ability of effiux to chemotherapeutic drugs and Hoechst 33342,can be used as an alternative mean to isolate cells with progenitors/CSCs-like cells from HCC samples. Side populations(SP) cells were coined as a subset of weak or negative staining cells with the ability to efflux Hoechst 33342 by flow cytometry(FCM)(14).Recently data have proved that some cancer cell lines and primary tumors,including glioma(15),ovarian cancer(16),head and neck cancer(17) and hepatocellular carcinoma(18),contain SP cells with high expression of ATP-binding cassette transporter family G2(ABCG₂/BCRP) and marker of CSCs.These cells possess the ability of high initiating-tumor and drug resistance through expulsion of most cytotoxic drugs.Some authors consider that SP cell is an enriched source of stem cells as well as an alternative source that is particularly useful in situation where stem cells molecular markers are unknown(19).Clinical observations and experimental data showed that primary tumors or cancer cell lines had the heterogeneous characteristics in term of tumorgenicity and aggressive potential(1),suggesting that SP cells might be different in terms of numbers or tumor-initiating abilities.However,up to date whether SP cells derived from different cell lines/tumors possess identically tumorgenie capability still remains elusive.The aim of our study is to explore similarities and disparities in biological characteristics of SP cells sorted from 4 HCC cell lines(HCCLM3,MHCC97-H, MHCC97-L and Hep3B) with different metastatic potentials,to investigate the roles of SP cells in invasion and metastasis of HCC using molecular biological technology and a xenograft model and to investigate the roles of SP cells in predicting the prognosis of HCC through analysis of SP cells in freshly clinical samples.Partâ… Sorting and identification of side population cells in hepatocellular carcinoma cell linesThe aim of this study is to determine whether HCC cell lines(HCCLM3, MHCC97-H,MHCC97-L and Hep3B) with different metastatic potentials contain SP cells and to investigate the disparities of phenotype and ability of proliferation, self-renewal and tumor-initiating in 4 SP cells.We sorted SP cells from 4 HCC cell lines by FCM,then investigated whether ABCG₂ expression was necessary for the SP phenotype by immunocytochemistry (ICC),Western blotting and real-time quantitative PCR(qRT-PCR),explored the relation between SP cell and putative stem cell markers by FCM,then identified the ability of proliferation and self-renewal by clonogenic and differentiation assay,of chemo-resistance by MTT assay and of initiating-tumor by tumorigenicity assay in non-obese diabetic/severe combined immunodeficiency(NOD/SCID) mice.In this study,we found that the proportions of SP cells from Hep3B,MHCC97-L, MHCC97-H and HCCLM3 were 0.9±0.1%,4.2±0.4%,14.5±0.6%and 28.7±1.6%, respectively,that SP cells had the characteristic of high expression of ABCG₂,high clonogenicity,and similarily remarkable chemoresistance.All these SP cells generated SP cells as well as MP cells.The ratio of SP to MP cells decreased to normal as in parent cell lines when cultured over five passages in vitro.As low as 2×10³ SP cells could initiate tumors in NOD/SCID mice successfully.However,the expressions of putative stem cell markers appeared diverse in different SP cells.In conclusion,the proportion of SP cells was in lines with metastatic potential of their parent cell lines,which maybe correlate with invasive ability in HCC.Different SP cells harbored similarily CSCs-like characteristic of proliferation,self-renewal, chemoresistance and high tumor-initiating.ABCG₂ was a determinant of phenotype of HCC SP cells.Partâ…¡Roles of side population cells in invasion and metastasis of hepatoeellular carcinomaWe have successfully identified that SP cells from HCC cell lines with different metastatic potentials have similarily tumor-initiating ability.In this study,our aim is to investigate the role and mechanism of SP cell in invasion and metastasis of HCC. We observed the invasive abilities of different SP and MP cells using Matrigel invasion assay,detected the concentration of vascular endothelial growth factor (VEGF),matrix metallopeptidase 2(MMP₂),matrix metallopeptidase 9(MMP₉) in supematant of SP cells culture by enzyme linked immunosorbent assay(ELISA), investigated the metastastic potential of SP cells using nude mice orthotropic transplantation and observed dynamic process of tumor-initiating and metastasis of HCC with the help of bioluminescence imaging after inoculation of HCCLM3-green fluorescent protein(GFP).According to the Matrigel invasion assay,the numbers of invaded SP cells from MHCC97-L,MHCC97-H and HCCLM3 increased stepwise in accordance with the metastatic potentials of their parent cell lines(18.7±2.1%,35.7±5.1%and 48.0±3.6% respectively,p＜0.05),which were significantly higher than their corresponding MP cells(9.3±4.2%,15.0±2.0%and 15.7±1.1%respectively,p＜0.05).However,there was no significant difference between SP and MP cells in Hep3B cells(3.0±1.0%vs. 1.3±0.6%,p=0.068).ELISA showed that the concentration of VEGF in Hep3B SP cells,MHCC97-L SP cells,MHCC97-H SP cells and HCCLM3 SP cells were 40±16 pg/ml,87±4 pg/ml,135±32 pg/ml and 70±31 pg/ml,with significant difference (p＜0.05) other than between MHCC97-H cells and HCCLM3 cells.However,neither MMP₂ nor MMP₉ was found to be statistical significance among 4 HCC SP cells. Metastasis assays demonstrated that the pulmonary metastasis rates and metastatic tumor clusters per mouse were 100%(5/5) and 2168.2±926.1,100%(5/5) and 726.4±446.5,40%(2/5) and 132.0±195.7,20%(1/5) and 22.0±49.2 in HCCLM3 SP cells,MHCC97-H SP cells,MHCC97-L SP cells and Hep3B SP cells group respectively(p＜0.05),which were in accordance with the metastatic potentials of their parent cell lines.Bioluminescence imaging showed metastatic pulmonary tumor clusters were found at 21 day after inoculation of HCCLM3-GFP.In conclusion,SP cells correlated with the invasive and metastatic potential of HCC in vivo and in vitro,while different derived SP cells remained significant difference in metastatic potentials,in line with that of their parent cells.Partâ…¢Analysis of side population cells in hepatocellular carcinoma samples and their relationship with prognosisThe purpose of the present study is to investigate whether hepatocellular carcinoma tissues contain SP cells as well and to assay relationships between SP cell and prognosis of HCC after curative resection.95 cases of patients with definite HCC diagnosis by pathology were enrolled in this study.They all underwent surgical resection from March 2007 to July 2007 in Zhongshan Hospital,Liver Cancer Institute,Fudan University.Freshly tumor specimen were digested with 0.1%ultrapure collagenase,then purified using uncontinuous gradient percoll^TM centrifugation and standardized protocol for SP analysis in HCC samples.Kaplan-Meier method,log-rank test and chi-square test were used to analyze the relationship of the percentage of SP cells with prognosis of HCC.Multivariate study with Cox's proportional hazard model was used to evaluate the prognosis-relative aspects.To avoid the bias from the process of SP sorting,we developed an optimized protocol for SP analysis in HCC samples.About 88.4%(84/95) of HCC samples contained SP cells(0-23.0%,mean 1.1%).The proportion of SP cells was found to be associated with aggressive phenotypes of HCC,such as TNM stage(p=0.037),tumor encapsulation(p=0.011) and vascular/bile duct invasion(p=0.002).The overall survival(OS) and disease-free survival(DFS) rate of all the patients were 82.5%and 57.4%,respectively.Univariate analysis showed that SP cells proportion correlated with DFS.The DFS of high SP group was significantly lower than that of low SP group(46.2%vs.68.1%,p=0.035).Tumor size,vascular/bile duet invasion and TNM stage were also found to be associated with DFS.Multivariate Cox proportional hazards model showed that vascular/bile duct invasion and tumor size were independent prognostic factors for DFS.Only vascular/bile duct invasion was independent predictor for OS.We further investigated the role of prophylactic TACE in HCC.There was no significant difference in OS and DFS between prophylactic TACE group and non-TACE group.When stratified all the patients with SP cells proportion,significant difference in OS between prophylactic TACE and non-TACE cases can only be found in low SP group,but not in high SP group(p=0.033).In conclusion,HCC samples contained SP cells that correlated with malignant phenotype and early recurrence/metastasis of HCC after operation.Conclusions1 Four HCC cell lines with different metastatic potentials contained SP cells.The proportion of SP cells was positively correlated with the invasive ability of HCC. 2 Different SP cells harbored similar liver CSCs-like characteristics of proliferation, self-renewal,chemoresistance and high tumor-initiating ability.ABCG₂ may be a determinant phenotype of HCC SP cells.3 SP cells correlated with the metastatic potential of HCC,which may be a novel indicator of poor prognosis for HCC.4 Most HCC samples contained SP cells that correlated with malignant phenotype and attributed to early recurrence/metastasis of HCC.Novelty1 We firstly demonstrated that SP cells sorted from HCC cell lines with different metastatic potentials had similar tumor-initiating ability but significantly different invasive ability.2 We firstly confirmed that HCC samples contained SP cells which were correlated with malignant phenotype and early recurrence/metastasis of HCC.