Woelfle Ute, Breit Elisabeth, Zafrakas Kristine, Otte Marcus, Schubert Falk, Müller Volkmar, Izbicki Jakob R, Löning Thomas, Pantel Klaus
Institute of Tumour Biology, Center of Experimental Medicine, University Hospital Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
J Immunol Methods. 2005 May;300(1-2):136-45. doi: 10.1016/j.jim.2005.03.006. Epub 2005 Apr 18.
Metastasis-the spread of tumour cells from a primary lesion to distant organs-is the main cause of cancer-related death, and bone marrow (BM) is a frequent site for the settlement of disseminated tumour cells. Many BM samples harbour isolated tumour cells, whereas tumour cell clusters, as the potential precursors of solid distant metastases, are rarely detected after current enrichment procedures. We have analysed BM samples from 43 patients with carcinomas of the breast, colon and ovaries; 41 of these patients had no clinical signs of overt metastases (stage M0). Tumour cells in BM were enriched with immunomagnetic beads coupled to monoclonal antibodies against both EpCAM and HER2/neu. After enrichment, tumour cells were identified by immunostaining with the anti-cytokeratin antibody A45-B/B3. In total, 886 CK-positive cells were detected in 16 (35%) samples after immunomagnetic enrichment as compared to 34 cells in 9 (21%) samples using Ficoll density centrifugation previously used as the standard enrichment technique. Most remarkably, clusters of 2 to 10 CK-positive cells were found in 75% of CK-positive samples enriched by immunobeads, whereas no CK-positive cell clusters were detected after Ficoll enrichment. The method described offers an excellent tool for the enrichment of micrometastatic tumour cell clusters; these clusters may represent the initial stage of development from a single disseminated tumour cell towards an overt metastasis.
转移（即肿瘤细胞从原发性病灶扩散至远处器官）是癌症相关死亡的主要原因，而骨髓（BM）是播散性肿瘤细胞定居的常见部位。许多骨髓样本含有孤立的肿瘤细胞，然而，作为实体远处转移潜在前体的肿瘤细胞簇，在当前的富集程序后很少被检测到。我们分析了43例乳腺癌、结肠癌和卵巢癌患者的骨髓样本；其中41例患者没有明显转移的临床症状（M0期）。通过与抗EpCAM和HER2/neu单克隆抗体偶联的免疫磁珠富集骨髓中的肿瘤细胞。富集后，用抗细胞角蛋白抗体A45-B/B3进行免疫染色鉴定肿瘤细胞。免疫磁珠富集后，在16份（35%）样本中总共检测到886个细胞角蛋白阳性细胞，而使用之前作为标准富集技术的Ficoll密度离心法，在9份（21%）样本中仅检测到34个细胞角蛋白阳性细胞。最值得注意的是，在通过免疫磁珠富集的细胞角蛋白阳性样本中，75%发现了2至10个细胞角蛋白阳性细胞的细胞簇，而Ficoll富集后未检测到细胞角蛋白阳性细胞簇。所描述的方法为富集微转移肿瘤细胞簇提供了一个极好的工具；这些细胞簇可能代表了从单个播散性肿瘤细胞发展为明显转移的初始阶段。
