Zijlstra Andries, Mellor Rebecca, Panzarella Giano, Aimes Ronald T, Hooper John D, Marchenko Natalia D, Quigley James P
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Cancer Res. 2002 Dec 1;62(23):7083-92.
A quantitative assessment of rate-limiting steps in metastasis has always been challenging because of the difficulty of detecting small tumor cell populations. We have developed a highly sensitive assay for monitoring the metastatic dissemination of human tumor cells in the chick embryo and used this assay to investigate the relative efficacy of sequential stages in the metastatic cascade for two malignant human tumor cells lines, HEp3 and HT1080. This assay is based on the real-time PCR amplification of human alu sequences and exhibits a high sensitivity (25 cells/lung) with a large linear range (50-100,000 cell/lung). The assay is optimized for a high number of replicate in vivo assays (50-100 animals/assay) and can be applied in both experimental and spontaneous metastasis models. Using quantitative alu PCR, we determined that HEp3 spontaneously metastasizes very efficiently and rapidly, generating secondary growth in the lung exceeding 1-2 x 10(4) cells/lung in 7 days. In contrast, spontaneous HT1080 metastasis is 50-100-fold less efficient, resulting in only 200-400 cells/lung in 7 days. By taking advantage of the sensitivity and specificity of the real-time alu PCR assay we were also able to quantitatively assess multiple steps in metastasis including intravasation, arrest of tumor cells in secondary organs of the embryo, and the initial growth and expansion of the arrested tumor cells. A comparative analysis of HEp3 and HT1080 metastasis demonstrates that the relatively low-to-moderate metastatic rate of HT1080 is caused by two distinct deficiencies, an 8-10-fold lower rate of intravasation and a delayed onset of HT1080 growth expansion in the secondary organ. Thus, a very facile metastasis model system coupled with the sensitive, real-time PCR-based assay allows for the identification and quantification of rate-limiting steps in the metastatic cascade for select human tumor cell lines.
由于难以检测到少量肿瘤细胞群体，对转移过程中限速步骤进行定量评估一直具有挑战性。我们开发了一种高度灵敏的检测方法，用于监测人肿瘤细胞在鸡胚中的转移扩散，并利用该方法研究了两种恶性人肿瘤细胞系HEp3和HT1080在转移级联反应中各个连续阶段的相对效力。该检测方法基于人alu序列的实时PCR扩增，具有高灵敏度（每肺25个细胞）和大线性范围（每肺50 - 100,000个细胞）。该检测方法针对大量体内重复检测（每次检测50 - 100只动物）进行了优化，可应用于实验性和自发性转移模型。使用定量alu PCR，我们确定HEp3自发转移非常高效且迅速，在7天内肺部产生的二次生长超过每肺1 - 2×10⁴个细胞。相比之下，HT1080的自发转移效率低50 - 100倍，7天内每肺仅产生200 - 400个细胞。利用实时alu PCR检测的灵敏度和特异性，我们还能够对转移过程中的多个步骤进行定量评估，包括肿瘤细胞的血管内渗、在胚胎二级器官中的滞留以及滞留肿瘤细胞的初始生长和扩展。对HEp3和HT1080转移的比较分析表明，HT1080相对较低至中等的转移率是由两个明显的缺陷导致的，即血管内渗率低8 - 10倍以及HT1080在二级器官中生长扩展的延迟。因此，一个非常简便的转移模型系统与基于实时PCR的灵敏检测方法相结合，能够识别和定量特定人肿瘤细胞系在转移级联反应中的限速步骤。
