【作者】 施国明；

【导师】 樊嘉；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 原发性肝细胞肝癌（Hepatocellular carcinoma,HCC,简称肝癌）是我国最常见的恶性肿瘤之一（1,2）。虽经几十年的努力,肝癌研究已取得很大进展,但由于肝癌极高的转移/复发率,肝癌病人总体5年生存率仍只有5%左右（2）。因此,探索肝癌转移/复发的机制,寻找有效的抑制途径,已成为进一步提高肝癌生存率的关键（1,3）。干细胞（stem cell）是一类具有自我更新、无限增殖和多向分化潜能的原始细胞（4）。研究发现,干细胞和肿瘤细胞有很多共同特点（5）,肿瘤很可能是干细胞在自我更新过程中由于基因突变导致生长失控的疾病。有学者（5）提出肿瘤中存在含量极少、具有无限增殖潜能的肿瘤干细胞,这些高致瘤性细胞亚群在肿瘤中充当干细胞的角色,是肿瘤的发生、转移和治疗失败的根源。这些细胞已经在白血病（6）和一些实体肿瘤,如乳腺癌（7）、结肠癌（8,9）和前列腺癌（10）中得到证实。虽然研究发现CD133⁺肝癌细胞能在体内引起肿瘤,然后在大多数细胞株和肝癌临床标本不存在这种细胞（11-13）。因此,CD133不适合于大多数肝癌肿瘤样本分离肿瘤干细胞,这阻碍了肝癌形成和复发机制的研究。幸运的是,干细胞的其他特性,如能外排化疗药物和Hoechst 33342能力,可以作为肝癌样本中始祖细胞和肿瘤干细胞样细胞分离的另一种选择。侧群细胞（side populmion,SP）是指能将细胞核染料Hoechst33342泵出细胞外,而使自身着色较浅或不着色的一类细胞（14）。研究发现胶质母细胞瘤（15）、卵巢癌（16）、甲状腺癌（17）和肝癌（18）等细胞株中存在SP细胞,这类细胞常高表达ATP-binding cassette transporter family G2（ABCG₂/BCRP₁）和肿瘤干细胞标记,拥有肿瘤干细胞样的高成瘤能力,并可将大多数细胞毒化疗药排出细胞外而耐药。大多学者认为SP细胞富含肿瘤干细胞,在缺乏肿瘤干细胞特异表面标记情况下SP细胞可以作为研究肿瘤干细胞的重要手段（19）。临床观察和实验研究资料显示,临床肿瘤和肿瘤细胞株在成瘤和侵袭能力存在异质性（1）,意味着不同肝癌组织和细胞株中SP细胞可能存在质和量的差异。然而,不同转移潜能肝癌细胞株SP细胞在成瘤能力及其在肝癌侵袭、转移中的作用尚未明确。本研究借助于流式细胞仪对4种不同转移潜能的肝癌细胞株HCCLM3、MHCC97-H、MHCC97-L和Hep3B中的SP细胞进行分选,应用分子生物学和动物模型探索不同转移潜能肝癌细胞株SP细胞在干细胞特性方面的异同及其在肝癌侵袭和转移中的作用,应用临床新鲜肝癌组织测定SP细胞比例,研究SP细胞比例作为判断肝癌侵袭性和患者预后的价值。第一部分肝癌侧群细胞的分选及其特性研究本研究旨在探索不同转移潜能人肝癌细胞株是否存在SP细胞及不同来源SP细胞在表型、增殖潜能、自我更新和成瘤能力等生物学特性方面的差异。借助于流式细胞仪对4种不同转移潜能肝癌细胞株HCCLM3、MHCC97-H、MHCC97-L和Hep3B中SP细胞进行检测和分选;利用免疫组化、免疫印迹和定量PCR研究ABCG₂在维持SP细胞表型中的作用;借助于流式细胞仪分析SP细胞与常见干细胞标记的关系:借助克隆形成实验和体外培养观察不同SP细胞体外增殖和自我更新能力;利用MTT法观察常用化疗药物对其生长抑制影响;利用Non-obese Diabetic/Severe Combined Immunodeficiency（NOD/SCID）小鼠接种实验观察SP细胞成瘤和自我更新能力。结果发现4种肝癌细胞株HCCLM3、MHCC97-H、MHCC97-L和Hep3B中SP细胞比例分别为（28.7±1.6）%,（14.5±0.6）%,（4.2±0.4）%和（0.9±0.1）%;SP细胞高表达ABCG₂;SP细胞表达部分干细胞标记;克隆形成实验显示SP细胞具有较强克隆形成能力,在体外培养时,SP细胞能产生SP和主群（main population,MP）细胞,体外培养到第五代,SP细胞比例逐渐恢复到分选前水平;MTT实验显示不同来源SP细胞具有相似的抵抗化疗药物作用:成瘤实验显示2000个SP细胞即能在NOD/SCID小鼠内成瘤。本实验证明了不同转移潜能的肝癌细胞株SP细胞比例不同,可能与肝癌侵袭转移潜能相关,不同来源SP细胞具有相似增殖潜能、自我更新、化疗抵抗和高成瘤能力等肝癌干细胞样作用,ABCG₂是决定SP细胞表型的重要因素之一。第二部分肝癌侧群细胞与肝癌侵袭性的关系研究前研究明确了不同转移潜能人肝癌细胞株SP细胞具有相似成瘤能力。本部分探索肝癌SP细胞在肝癌侵袭和转移中的作用,并初步探讨其可能的分子机制。应用Matrigel实验观察不同来源肝癌SP细胞体外侵袭能力:借助酶联免疫吸附实验（enzyme linked immunosorbem assay,ELISA）分析不同来源SP细胞条件培养上清液中血管内皮生长因子（Vascular endothelial growth factor,VEGF）、基质金属蛋白酶2（matrix metallopeptidase 2,MMP₂）、基质金属蛋白酶9（matrixmetallopeptidase 9,MMP₉）含量;通过原位接种实验观察不同来源SP细胞转移能力,并利用HCCLM3 SP-GFP（green fluorescent protein）细胞和小动物活体成像系统动态观察其形成和转移情况。Matrigel侵袭实验显示MHCC97-L SP细胞、MHCC97-H SP细胞和HCCLM3SP细胞组侵袭细胞数分别为（18.7±2.1）%,（35.7±5.1）%和（48.0±3.6）%,显著高于对应的MP细胞（9.3±4.2）%,（15.0±2.0）%和（15.7±1.1）%（p＜0.05）,然而Hep3B SP细胞与MP细胞组侵袭细胞数无明显差异（3.0±1.0%vs.1.3±0.6%,p=0.068）。各组SP细胞保持原细胞株的侵袭能力（p＜0.05）;ELISA检测显示Hep3B细胞、MHCC97-L细胞、MHCC97-H细胞和HCCLM3细胞来源的各组SP细胞培养上清液VEGF分别为40±16 pg/ml,87±4 pg/ml,135±32 pg/ml和170±31pg/ml,统计学检验显示除MHCC97-H SP和HCCLM3 SP组间未见显著差异外,其余各组均有显著差异（p＜0.05）;而各组SP细胞上清液中MMP₂和MMP₉未见显著差异;原位接种实验显示HCCLM3 SP细胞、MHCC97-H SP细胞、MHCC97-L SP细胞和Hep3B SP细胞肺转移率和肺转移灶分别是100%（5/5）和2168.2±926.1,100%（5/5）和726.4±446.5,40%（2/5）和132.0±195.7,20%（1/5）和22.0±49.2,各组SP细胞间存在显著差异（p＜0.05）;小动物活体成像系统动态观察显示HCCLM3 SP-GFP细胞平均原位接种后21天出现肺转移。本实验结果提示,肝癌SP细胞与肝癌体内、外侵袭能力密切相关,不同来源肝癌SP细胞侵袭潜能有显著差异。第三部分肝癌侧群细胞与临床肝癌预后的关系研究本部分实验观察肝癌临床标本中是否存在SP细胞,探讨肝癌SP细胞与临床病理、肝癌侵袭性和预后间的关系。选取2007年3月～2007年7月间在复旦大学附属中山医院肝外科行肝癌切除术,经组织病理学检查证实为肝细胞肝癌的95例病例的新鲜肿瘤标本,用胶原酶消化法分离肿瘤细胞,用不连续的Percoll^TM梯度离心纯化肿瘤细胞后行SP细胞分析,SP细胞分析方法进行标准化处理;统计学分析肝癌SP细胞比例与肝癌临床病理特征及预后的关系。结果发现,约88.4%（84/95）肝癌组织存在SP细胞,平均比例1.1%（0-23.0%）,SP细胞比例与肝癌侵袭表型,如TNM分期（p=0.037）,肿瘤包膜（p=0.011）、血管/胆管侵犯（p=0.002）密切相关。患者总体生存率和无瘤生存率分别为82.5%和57.4%;单因素分析显示SP细胞比例与无瘤生存时间有关,高SP组无瘤生存率显著低于低SP组（46.2%vs.68.1%,p=0.035）,肿瘤大小、血管/胆管侵犯和TNM分期也与无瘤生存率相关;采用Cox比例风险模型多因素分析显示血管/胆管侵犯、肿瘤大小是影响无瘤生存的独立预后指标,而只有血管/胆管侵犯是总体生存率的独立预后因素;预防性经肝动脉化疗栓塞（Transcatheter arterialchemoembolization,TACE）对低SP比例组的总体生存率有显著影响（p=0.033）。实验结果显示,肝癌临床标本中存在SP细胞,肝癌SP细胞比例与肝癌侵袭表型相关,可能是术后早期复发转移的重要原因。结论1不同转移潜能肝癌细胞株中均存在SP细胞,其比例与转移能力呈正相关。2不同来源肝癌SP细胞具有相似增殖潜能、自我更新、对抗化疗药物和高成瘤能力,而ABCG₂可能是决定肝癌SP细胞表型的主要因素。3肝癌SP细胞与肝癌侵袭转移密切相关,肝癌sP细胞可能可以作为判断肝癌侵袭能力的一个重要指标。4大部分肝癌临床标本中存在SP细胞,SP细胞比例与肝癌侵袭表型相关,可能是引起肝癌术后早期复发转移的重要原因。创新点1首次证明不同转移潜能肝癌细胞株SP细胞成瘤能力相似,但其侵袭性有显著差异。2首次证实肝癌临床标本中存在SP细胞,且SP细胞比例与肝癌侵袭表型和肝癌术后早期复发转移相关。更多还原

【Abstract】 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.PartⅠSorting 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.PartⅡRoles 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.PartⅢAnalysis 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.更多还原