【作者】 赵一鸣；

【导师】 樊嘉；

【作者基本信息】 复旦大学 ， 外科学， 2011， 博士

【摘要】 肝细胞癌(以下称肝癌,hepatocellular carcinoma, HCC)是中国第二和世界第三大肿瘤相关死亡的病因[1,2]。尽管外科手术和肝移植为肝癌提供了有效的治疗措施,但肝癌根治性切除术后的5年复发率仍高达54.1-61.5%,其中62.4-77.8%发生在术后两年以内,这些导致了肝癌患者的预后仍然很差[3]。人们一直以来都认为肝癌复发和转移主要和肿瘤细胞本身的生物学特性有关。近些年的研究发现,肿瘤细胞并不是孤立存在,而是处在一个包括炎症免疫细胞、基质细胞、血管内皮细胞、细胞因子、趋化因子等共同组成的复杂环境之中,这种微观环境被称为“肿瘤微环境”[4]。‘炎症破坏一组织修复”的恶性循环促进肿瘤发生发展的重要原因[5]。临床上大部分肝癌患者有乙型或(和)丙型肝炎肝硬化背景。炎症和肝硬化导致的细胞增殖异常可以促进肿瘤的演进和进展[6]。因此,肿瘤的免疫微环境在肝癌的侵袭和转移过程中发挥着重要作用。肿瘤相关巨噬细胞(tumor associated macrophages, TAMs)是活跃于肿瘤为环境中的炎症细胞的重要成分。它们的主要特点是诱导宿主免疫抑制,刺激血管生成,分泌肿瘤生长因子和诱导ll型炎症反应[7]。-TAMs被认为是替代途径激活的巨噬细胞(alternatively activated macrophages, AAMs)或M2型巨噬细胞亚型[8]。以前的研究多利用泛巨噬细胞标志物(pan-macrophage marker) CD68作为标记识别肿瘤组织中的TAMs[9-11]。由于肿瘤组织中浸润的巨噬细胞是由多种不同的巨噬细胞亚群组成,各自在不同的微解剖位置发挥不同甚至相反的作用。因此寻找更加特异的标志物对于识别肝癌组织中TAMs并探索其功能具有重要的意义。由于肝癌细胞本身及肝硬化环境等原因,至今临床上对肝癌治疗有效的药物很少。sorafenib作为酪氨酸激酶受体抑制剂,可以通过抑制肝癌组织中的血管内皮生长因子受体(vascular endothelial growth factor receptor, VEGFR)、血小板衍生生长因子受体(platelet derived growth factor receptor, PDGFR)阻断肿瘤血管生成及通过抑制RAF/MEK/ERK通路直接影响肿瘤的凋亡过程进而抑制肿瘤生长的多点靶向治疗药物[12]。临床实验证明,sorafenib可以使晚期肝癌患者生存期提高3.8个月[13]。我所(复旦大学肝癌研究所)利用自主建立的人肝癌裸鼠模型筛选药物发现a干扰素(Interferon-α、IFN-α)可以有效抑制肝癌的生长[14]。临床试验也证实IFN-α可以有效预防肝癌患者术后的复发与转移[15]。即便如此,两种药物对肝癌的疗效仍不满意。而且,以上两种药物临床上均出现药物耐受和副作用[16]。动物实验证明sorafenib治疗可能促进肝癌组织的血管生成的恶性反弹及肿瘤的转移与复发,此种副作用的出现可能与肿瘤组织中的TAMs的诱导有关[17]。因此本研究以’TAMs为中心,寻找人肝癌组织中‘TAMs特异性标志物阐述其重要性。观察临床有效的抗肝癌药物对TAMs的影响并分析其机制。第一部分CD206鉴定肝癌组织中的肿瘤相关巨噬细胞的可行性研究CD206作为体外实验中AAMs或M2型巨噬细胞的特异性标志物,其在肿瘤组织中的表达还未见相关报道。相对于体外细胞实验,原位观察人肝癌组织中TAMs的分布及其鉴定具有重要意义。因此本部分利用人肝癌和癌周组织标本,分离原代细胞,直观反映炎症CD206+细胞在肝癌组中的分布,分析CD206作为特异性标志鉴定识别肝癌组织中TAMs的可行性。收集人肝癌和癌周组织标本,制作连续切片,免疫组织化学方法显示,CD206在肝癌组织和癌周组织的表达完全不同。在肝癌组织中CD206表达在肿瘤间质的单核巨噬细胞中并与CD68有共表达现象,而在癌周其呈管状表达,贴附于肝血窦内皮内壁上与CD68的表达无共表达现象。为进一步证实免疫组化的发现,我们分离新鲜肝癌组织和癌周组织的CD206+细胞、巨噬细胞及肝血窦内皮细胞(liver sinusoidal endothelial cells, LSECs)。流式细胞术(flow cytometry, FCM)检测以上各群原代细胞的CD206和CD68的表达发现。癌内和癌周CD206+细胞的CD68中位阳性率分别是96.6%和0.32%(范围,94.5-98.2%和0.16-0.92%)。癌内和癌周巨噬细胞的CD206中位阳性率分别是33.8%和0.57%(范围,0-73.4%和0.12-0.96%)。癌内和癌周LSECs的CD206中位阳性率分别是0.64%和56.8%(范围,0.37-0.89%和14.1-87.3%)。综合免疫组化和流式细胞术结果,经逻辑分析,癌内CD206+细胞是巨噬细胞的亚群,而癌周CD206+细胞是LSECs的亚群。为进一步验证CD206+巨噬细胞的真实身份,我们将分离的原代细胞体外培养。酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测上清液中的细胞因子水平发现,和CD206-巨噬细胞相比,CD206+巨噬细胞分泌更高水平的M2型细胞因子(IL-10和TGF-p)和更低水平的M1型细胞因子(IL-6和IL-12)。将原代细胞与肝癌细胞系共培养后发现,CD206+巨噬细胞可以提高肝癌细胞系的移动和侵袭能力,而CD206一巨噬细胞对其无明显影响。因此得出结论,癌内CD206+细胞是一群表型和功能上接近TAMs的巨噬细胞亚群,癌周CD206+细胞则是LSECs的亚群。CD206可以作为特异性标志识别鉴定肝癌组织中的TAMS。另外,我们也揭示之前未被发现的肝癌组织中巨噬细胞分布的特点,即TAMs只出现在癌内组织中而非癌周组织。第二部分肿瘤相关巨噬细胞与肝癌患者预后及IFNα疗效的关系在第一部分的基础上,本部分利用临床患者的肝癌组织标本制作组织芯片,分析以CD206作为标志鉴定的巨噬细胞与肝癌患者根治性切除后的预后及术后应用IFN-α疗效的关系。首先,我们收集了两群(训练集(n=323)和验证集(n=401))在复旦大学附属中山医院肝外科经根治性切除治疗的肝癌患者组织标本,制作组织芯片,经免疫组化CD206和CD68染色,利用图像分析系统分析CD206在肝癌组中的表达。免疫组化发现癌内的CD206表达在单核-巨噬细胞上与CD68有部分共表达现象,而癌周CD206主要表达在肝血窦内壁。这和第一部分的观察结果一致。之后,我们分析了CD206+细胞和患者预后的关系。验证集中,癌内CD68+细胞的表达与肝癌患者的预后无关。癌周CD68+细胞与患者总体生存时间(overall survival, OS)和无瘤生存时间(disease-free survival,DFS)均呈负性相关。CD206+细胞的表达和患者的OS(癌内,风险指数(HR)=2.790,P<.001；癌周,HR=1.774,P=.001)与DFS(癌内,HR=1.621,P=.003；癌周,HR=2.693,P<.001)呈负性相关,两者联合后可获得更显著的独立预后价值(OS,HR=3.745,P<.001; DFS, HR=3.061,P<.001)。验证集中此结果得到证实。之后,我们收集了术后IFN-α治疗随机对照临床试验中治疗组和对照组肝癌患者组织标本(治疗集),制作组织芯片。免疫组化CD206染色,分析与IFN-α术后治疗后肝癌患者预后的关系。结果显示,治疗组中,癌内CD206+细胞高表达者OS和DFS均高于癌内CD206+细胞低表达者。对照组中，癌内CD206+细胞高表达者OS和DFS均低于癌内CD206+细胞低表达者。治疗组中的癌内CD206+细胞高表达者OS和DFS均高于对照组中癌内CD206+细胞高表达者。经与临床病理资料比较和生存分析,癌内CD206+细胞与患者预后有关,而癌内CD68+细胞与患者预后无相关性。因此,从临床意义的角度,癌内CD206+细胞也更接近TAMS。癌周CD206+细胞高表达患者经IFN-α治疗后OS和DFS均得到延长,提示癌内CD206+细胞可能是IFN-α治疗肝癌的重要靶点之一。第三部分IFNα通过下调巨噬细胞HSP27的表达抑制肿瘤相关巨噬细胞的诱导本所的研究证实,在高肝转移肝癌裸鼠模型中,IFNα可以抑制肿瘤的生长和转移。初步的研究发现,其抗肿瘤作用可能通过抑制血管生成。但具体机制还不明确。我们收集经IFNα治疗和对照组的高转移肝癌裸鼠模型的组织标本,提取RNA,进行RT2 ProfilerTM PCR基因芯片分析后发现,和对照组相比,IFNa治疗组中热休克蛋白27(heat shock protein 27,HSP27)下调(P<.001)。为了寻找HSP27下调的原因,我们利用IFNa干预高转移肝癌细胞株,收集后提取蛋白和RNA,进行Western Blot与PCR验证,但我们发现,治疗组和对照组HSP27在RNA水平无显著性差异。因此我们推测,HSP下调的原因可能来源于肿瘤间质。近期有文献报道,HSP27可以促进M2型巨噬细胞的诱导分化。因此,我们检测了经IFN-α干预的人单核细胞系THP-1诱导的人巨噬细胞表型的变化及HSP27表达水平的影响。结果显示,IFN-α可以使M2型巨噬细胞的表型向M1型巨噬细胞转化,并抑制巨噬细胞HSP27的表达。最近的研究提示,HSP27可以促进M2型巨噬细胞的诱导分化。因此,该部分结果提示,IFN-α可能通过抑制巨噬细胞HSP27的表达进而抑制M2型巨噬细胞的诱导。更多还原

【Abstract】 Hepatocellualar carcinoma (HCC) is the second and the third tumor associated death cause in China and worldwide, respectively. Although the surgery and liver transplantation provided valid approaches for HCC, the 5-years recurrence rate are always up to 54.1-61.5%, of which 62.4-77.8% occurred in the first two years after resections and it will lead to the poorer prognosis. Previously, the recurrence and metastasis of HCC is mainly related to the tumor biological features. Recently, accumulating evidences indicated that tumor cells located in a complex microenvironment consisted of immune cells, matrix cells, vascular endothelial cells, cytokines and chemokines which were called "tumor microenvironment". A malignant cycle of inflammation and renovation promotes oncogenesis. With regard to HCC, most lesions develop in a milieu of chronic inflammation produced by hepatitis B or C viruses as well as dysregulated cellular proliferation due to cirrhosis. The "tumor immune-microenvironment" plays an important role in tumor invasion and metastasis.Tumor associated macrophages (TAMs) is key ingredient of infiltrated inflammatory cells activated in the tumor environment, of which the function are induction of host immune suppression, stimulating angiogenesis, secreting tumor growth factors and mediating typeⅡinflammation. TAMs are considered as alternatively activated macrophages (AAMs) or M2 macrohages. Previous in situ studies always use the pan-macrophage marker CD68 to recognize the TAMs in tumor tissue. The infiltrated macrophage in tumor location consisted of different cell subpopulations which play different even opposite roles on tumor cells. Therefore, finding a specific marker of TAMs is very important for TAMs studies.Due to liver cirrhosis and reason of HCC cells itself; there is no drug work effectively on HCC in clinic by now. Sorafenib, an inhibitor of tyrosine kinase receptors, could inhibit HCC growth by two pathways. One is decreasing the tumor angiogenesis by blocking vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor (PDGFR). The other is inhibiting the tumor apoptosis by blocking the pathway RAF/MEK/ERK. Randomized control trial (RCT) showed sorafenib prolonged the survival time of advanced stage HCC 3.8 months compared with the control group. On the human HCC nude mice model established by our institute (liver cancer institute, Fudan University), IFN-αwas found to suppress tumor growth and metastasis. RCT in our institute also indicated that IFN-a postponed recurrence and improved overall survival in HCC patients after curative resection. Nevertheless, both of the two drugs have limited effects in clinic and obtained unsatisfied outcome due to many reasons including drug resistance after long term administration. In preclinical trials, sorafenib induced tumor angiogenesis "malignant rebound" and stimulated tumor metastasis and these pro-tumor side effects could be related to the induction of TAMs.Therefore, this study focused on the TAMs, aimed to find specific marker of TAMs and the impact of effective drugs on the TAMs and its’mechanism.Part I CD206 is able to be a specific marker to identify TAMs in HCC tissueCD206 was considered as the specific marker to identify the AAMs and M2 macrophage in in vivo studies. However, its’expressions in situ tumor tissue have not reported. Compared with in vivo study, the observation the TAMs in in situ HCC tissue is more important. In this part, we collected tissue sample from HCC patients who underwent curative resections and isolated primary cells. The locations of CD206+ cells were directly demonstrated in HCC tissue samples. We also estimated whether CD206 could be used to identify real TAMsThe tissue samples collected from HCC patients was made into serial sections. The immunohistochemistry (IHC) in HCC tissue indicated that the profiles of CD206 in intratumoral and peritumoral tissue are totally different. In intratumoral tissue, CD206 expressed on the mono-macrophages in mesenchyme and co-located with part of CD68. However, in peritumoral tissue, the CD206 expressed as tubular pattern attached in the wall of liver sinusoid and not co-located with CD68. To confirm the findings of IHC, we isolated six cell populations (intratumoral and peritumoral CD206+cells, macrophages and liver sinusoidal endothelial cells (LSECs)). The expressions of CD206 and CD68 on primary cells mentioned above were tested by flow cytometry (FCM). This analysis revealed that the median CD68-positive rate of intratumoral CD206+ cells was 96.6%(range,94.5%-98.2%), which was strikingly higher than the 0.32% CD68-positive rate observed in peritumoral CD206+ cells (range,0.16%-0.92%). The CD206-positive rate was also higher in intratumoral macrophages (median,33.8%; range,0%-73.4%) than in peritumoral macrophages (median,0.57%; range,0.12%-0.96%). In contrast, the median CD206-positive rate was much lower in intratumoral LSECs (median, 0.64%; range,0.37%-0.89%) than in peritumoral LSECs (median,56.8%; range, 14.1%-87.3%).On the basis of above observations, we conclude that CD206+ cells in intratumoral tissue are macrophages, and CD206+ macrophages are present only in intratumoral tissue. Due to the lack of specific marker for LSECs, we were unable to directly identify peritumoral CD206+ cells. A previous study11 reported that CD206 was only expressed on human hepatic endothelium and liver macrophages. Because peritumoral CD206+ cells rarely expressed CD68, and some peritumoral LSECs expressed CD206, it is reasonable to propose that the peritumoral CD206+ cell group could be a subpopulation of LSECs.CD206 (+) macrophages (intratumoral CD206 (+) cells) secreted higher levels of anti-inflammatory (M2 specific) cytokines (IL-10 and TGF-β) (P<0.05) and lower levels of pro-inflammatory (M1 sepcific) cytokines (IL-6 and IL-12) (P<0.05) compared with CD206 (-) macrophages (intratumoral CD206 (-) macrophages and peritumoral macrophages).To determine the impact of CD206 (+) macrophages on the HCC cell lines, we respectively co-cultured intratumoral CD206 (+) cells, intratumoral CD206 (-) macrophages and peritumoral macrophages with HCC cell lines (HepG2, Huh7 and HCCL-M3). After co-cultured with CD206 (+) macrophages, all three HCC cell lines got more invasion and migration capability of HCC cell lines. Whereas, CD206 (-) macrophages have no significant impact on invasion and migration of HCC cell lines.In clnclusion, intratumoral CD206+cells are subpopulation of macrophage which resembles TAMs on phenotypes and functions. CD206 is a good and specific marker to identify TAMs in HCC tissue. Moreover, we found a previously un-recognized regularity of macrophages subpopulation distribution that TAMs only present in intratumoral tissue.Part II TAMs are related to the prognosis of HCC patients after curative resection and the outcome of postoperative IFN-a administrationOn the base of partⅠ, we collected HCC tissue samples from HCC patients who underwent curative resection and made them into tissue micro-arrays. The relation of CD206 identified TAMs and the prognosis of HCC patients and outcome of postoperative IFN-αadministration was also analysed. Firstly, two HCC patient cohorts—the training cohort and the validation cohort—were recruited. The training and validation cohorts respectively enlisted 323 (January 2003 to March 2004) and 401 (September 2000 to December 2002) consecutive Chinese patients who underwent curative resection of HCC in the Liver Cancer Institute, Zhongshan Hospital, Fudan University. Profiles of CD206 expressions were different in intratumoral and petitumoral liver tissue. In intratumoral tissue, CD206 expression was co-localized with part of CD68. In contrast, CD206 expression in peritumoral tissue was localized to the inner wall of liver sinusoids, where it exhibited a tubule-like staining pattern, but was not co-localized with CD68+ cells. That is analog with the part I.From January 1999 to December 2003,232 patients who underwent curative resection by the same surgical team for pathologically confirmed HCC at the Liver Cancer Institute (Fudan University, Shanghai, China) were enrolled into a randomized trial of postoperative IFN-a treatment to prevent recurrence. All patients were randomized into an IFN-a treatment group (5 MU 3 times weekly until recurrence was diagnosed; otherwise, for 18 months) or a control group (without any antitumor treatment until recurrence was diagnosed). Because some archived paraffin blocks were too small to analyze,80 samples (Group 1) the IFN-a group and 75 samples (Group 2) from the control group were retrieved from the Department of Pathology at Zhongshan Hospital. All samples were from patients who followed the treatment protocol. Disease-free survival (DFS) and overall survival (OS) in high CD206+ cells patients were better than that in low CD206+ cells patients in Group 1, however, DFS and OS did not differ between patients with high and low CD206+ cells in Group 2. OS in P48-positive patients from Group 1 was better than that in patients with high CD206+ cells patients from Group 2 but did not differ when low CD206+ cells.In conclusion, intratumoral CD206+ cells are closer to TAMs compared with CD68+ cells. Compared with CD206- cells, patients with high intratumoral CD206+ cells had longer OS and DFS that indicated intratumoral CD206+ cells is the target of IFN-αtreatment in HCC.Part III IFN-a inhibits the induction of TAMs by down-regulating the expression of HSP27 of macrophagesIt was demonstrated that IFN-a could inhibit the tumor growth and metastasis in human high metastasis potential nude mice model. Primarily, the underlying mechanism is possibly inhibiting tumor angiogenesis. Nevertheless, the exact mechanism is not well clarified.After collecting tumor samples from IFN-αtreatment group and control group, we extractd RNA from them and looked for the different genes by RT2 ProfilerTM PCR gene microarray. For defining the reason of the down regulation of HSP27 mRNA, we detected the HSP27 mRNA and protein levels in human high metastasis potential HCC cell line HCCL-M3 after treatment of IFN-α. However, there is no significant difference between IFN-αtreatment group and control group. Therefore, we postulate the origin of HSP27 down-regulation is from tumor mesenchyma. Recent study showed that HSP27 induced the M2 macrophages differentiations. So, we observed the impact of IFN-αon phenotype of macrophages in tumor and HSP27 expressions. As a result, we found that IFN-αcould lean the balance from the M2 to M1 macrophages and inhibited the expression of HSP27. By neutralized anti-HSP27 antibody, we showed that HSP27 is the key cytokine of impact of HSP27 on the macrophage transformations.更多还原