【作者】 黄昕；

【导师】 郑树； Wu,Lily；

【作者基本信息】 浙江大学 ， 肿瘤学， 2010， 博士

【摘要】 研究背景肿瘤转移是恶性肿瘤致死的主要原因。近年来研究显示,淋巴系统对肿瘤向局部淋巴结和远端器官转移具有直接作用。本课题组前期研究结果提示,前列腺癌首先通过淋巴管向局部淋巴结扩散,而这一过程又为后续向远端器官转移提供了细胞富集库。淋巴内皮细胞生长因子-C (VEGF-C)是淋巴道新生及由此引起肿瘤细胞向淋巴结迁移的主要因素。趋化因子受体-配体间相互作用有助于肿瘤细胞实现向特定器官转移。研究目的本研究首要目标是构建一个由基质细胞来源VEGF-C诱导肿瘤淋巴道新生,引起前列腺癌淋巴结转移的动物模型。第二个目标是论证CCR7/CCL21趋化因子受体-配体作用轴是引导前列腺癌淋巴结转移的关键分子信号之一。实验方法1.为探索基质细胞来源VEGF-C在前列腺癌扩散中的作用,我们构建了一个由人类前列腺癌细胞LAPC-9与小鼠基质细胞株NIH 3T3以等比混合组成的皮下移植瘤动物模型。利用慢病毒载体在LAPC-9细胞标记编码Renilla luciferase (RL)的基因；在NIH 3T3细胞中标记编码人类VEGF-C (hVEGF-C)的基因,用含有标记GFP基因的慢病毒载体作为对照。LAPC-9/3T3混合移植瘤被植入重度联合免疫缺陷(SCID)小鼠皮下。用检测RL信号的CCD光学影像技术,在体和离体追踪移植瘤生长与扩散情况。原位移植瘤和肿瘤转移灶中趋化因子与受体的表达水平,分别由实时定量RT-PCR法、免疫组织化学法及western blot法进行分析。2.为了进一步分析由上述动物模型所得结果,我们采用两株人类前列腺癌细胞株CWR22Rv1 (CWR)和LNCaP作为体外替代模型。并用慢病毒载体CMV-CCR7-IRES-GFP标记上述细胞株。用含有编码CCL21基因的腺病毒载体(Ad-CCL21)感染A549细胞株以生产含有CCL21的条件培养基,用Ad-firefly腺病毒载体作为对照。用趋化跨膜迁移实验(chemotaxis assay)检测上述表达CCR7的细胞株与条件培养基之间的相互作用。用实时定量RT-PCR和western blot法检测相关基因和蛋白产物的表达情况。实验结果1. LAPC-9/3T3-hVEGF-C移植瘤的边缘呈现大量新生淋巴管,并因此促进被标记的肿瘤细胞向局部淋巴结和肺进行转移(该组出现淋巴转移概率为66.67%)。与之相异,GFP对照组移植瘤均未见明显转移。通过检测两组样本中一系列与转移相关基因表达水平,我们发现hVEGFR-3、hMMP9、CCR7和CCL21淋巴结转移灶表达增加,CXCR4在肺转移灶中表达上调。另外,将淋巴结中分理出的部分转移灶重新移植入SCID小鼠皮下进行扩增,并检测相关基因表达水平。与对应原位移植瘤相比,扩增后肿瘤的CCR7基因表达增加11.62倍(P=0.0007),而CCL21基因表达增加11.37倍(P=0.001)。免疫组织化学实验检测进一步证实了上述结果。此外,分别从淋巴转移病灶和原位移植瘤分离制备单细胞悬液,前者在体外响应CCL21条件培养基发生趋化迁移的能力较后者明显增强(P0.000009)。2.经基因修饰表达CCR7的CWR和LNCaP细胞株响应CCL21条件培养基进行趋化迁移的能力增强,并且这一现象可被抗-CCL21单克隆抗体拮抗。CCL21条件培养基刺激CWR/CCR7+细胞早期,其作用机制可能与激活促进存活的PI3K/Akt信号通路及NF-κB转录原件相关。结论上述结果提示,基质细胞来源的VEGF-C能引起肿瘤边缘功能性淋巴管新生,为前列腺癌转移提供途径。CCR7/CCL21作用在前列腺癌细胞上调,为肿瘤细胞经淋巴系统向局部淋巴结定向迁移提供趋化吸引信号。前列腺癌细胞在淋巴结的富集后经体内循环系统向远处转移与CXCR4受体上调有关。更多还原

【Abstract】 BackgroundMetastasis is the main cause of cancer mortality, including prostate cancer. Recent reports suggested a direct role of the lymphatic system in the tumor spread not only to loco-regional lymph nodes but also to distant organs. Preliminary findings from our group suggest that a preferred route of prostate cancer dissemination is via lymphatic vessels to the regional lymph nodes (LN), which in turn provide a reservoir for subsequent spread to distal vital organs. Vascular Endothelial Growth Factor sub-family member C, VEGF-C, has been demonstrated to play a dominant role in lymphatic vessels formation and thus facilitating tumor cells metastasizing to lymph nodes. Chemokine-receptor axis has been suggested helping drain tumor cells escaping to certain organs.ObjectivesOur first aim is to build a lymphatic metastasis model of prostate cancer by modulating lymphangiogenesis in the stroma. The secondary aim is to demonstrate the CCR7/CCL21 chemokine-receptor axis as the guidance signal in lymph node metastasis of prostate cancer.Methods1. In order to determine the role of stromal cell secreted VEGF-C in prostate tumor dissemination, we generated a recombinant human prostate xenograft model composed of Renilla luciferase (RL)-expressing LAPC-9 human prostate tumor cells accompanied with murine NIH 3T3 stromal cells that overexpressing human VEGF-C (hVEGF-C). The genetic modulation of both the tumor and stromal cells was accomplished by lentiviral vectors, and the lentivirus expresses only the Gfp gene was used as control. The LAPC-9/3T3 tumor-stroma combined xenografts model was established subcutaneously in severe combined immunodeficiency (SCID) mouse. Optical CCD imaging of RL was employed to monitor tumor growth and dissemination in vivo and ex vivo. Expression and secretion of chemokines and receptors by primary tumor and tumor metastases were determined by quantitative real-time polymerase chain reaction (real time RT-PCR), immunohistochemical staining and western blot assay, respectively.2. To refine our model system, we employed prostate cancer cell lines CWR22Rvl (CWR) and LNCaP as in vitro models, which were marked as highly expressing human CCR7 with CMV-CCR7-IRES-GFP lentiviruses. An adeno-CCL21 vector (Ad-CCL21) was used to generate the medium containing CCL21 for chemokine-receptor interaction study. The assays of gene profiles and protein production alterations were carried out using real time RT-PCR and western blot. Chemotaxis assay was performed to test the chemotactic activity of CCR7+prostate cancer cells.Results1. The LAPC-9/3T3-hVEGF-C tumors exhibited abundant peritumeral lymphatic vessels promoting regional LN metastases and lung metastasis with detectable luciferase signals, at an incidence of 4/6 animals in the cohort. Conversely, the LAPC-9/3T3-GFP control tumors displayed negligible metastatic potential in 0/6 mice. The expression of a selected set of metastasis-related genes between these 2 tumor samples were analyzed and compared by real time RT-PCR. We observed a consistent elevation of hVegfr-3, hMmp9, Ccr7 and Ccl21in the nodal metastatic lesion, and Cxcr4 in lung metastasis lesion. Moreover, we isolated and expanded the primary and LN metastatic lesions and propagated as subcutaneous tumors in SCID mice to profile their gene expression. We observed a consistent up-regulation of C-C chemokine receptor 7 (CCR7) (11.62 folds, P=0.0007) and its cognate ligand CCL21 (11.37 folds, P=0.001) at the gene expression level in the LN lesions over primary tumors, which were confirmed by immunohistochemical staining of LN and tumor sections. Furthermore, the disrupted single tumor cell derived from the LN metastases with higher expression of CCR7 responded more robustly to chemotactic attraction of CCL21 in an in vitro migration assay as compared to control primary tumor cells lacking CCR7 upregulation (P= 0.000009).2. The gene modified CWR and LNCaP cells chemotactically responded to Ad-CCL21 arose conditioned medium (CCL21-CM), and the chemotaxis pattern could be impaired by the anti-CCL21 antibody. The CCL21-CM also caused the activation of prosurvival PI3K/Akt signaling pathway and NF-κB.ConclusionsOur findings suggest that, stromal VEGF-C expression induced lymphangiogenesis and functional lymphatics that provide an escape route for the prostate tumor cells. And the nodal enrichment possibly induced the metastasis to lung by the upregulation of CXCR4 in prostate tumor cells. Moreover, the CCR7/CCL21 axis upregulated in the prostate tumor cells likely provided a chemotactic guidance aiding the tumor cells to navigate to regional lymph nodes through the lymphatics.更多还原