EGFR-Based Immunoisolation as a Recovery Target for Low-EpCAM CTC Subpopulation

Circulating tumour cells (CTCs) play a key role in the metastasis process, as they are responsible for micrometastasis and are a valuable tool for monitoring patients in real-time. Moreover, efforts to develop new strategies for CTCs isolation and characterisation, and the translation of CTCs into clinical practice needs to overcome the limitation associated with the sole use of Epithelial Cell Adhesion Molecule (EpCAM) expression to purify this tumour cell subpopulation. CTCs are rare events in the blood of patients and are believed to represent the epithelial population from a primary tumour of epithelial origin, thus EpCAM immunoisolation is considered an appropriate strategy. The controversy stems from the impact that the more aggressive mesenchymal tumour phenotypes might have on the whole CTC population. In this work, we first characterised a panel of cell lines representative of tumour heterogeneity, confirming the existence of tumour cell subpopulations with restricted epithelial features and supporting the limitations of EpCAM-based technologies. We next developed customised polystyrene magnetic beads coated with antibodies to efficiently isolate the phenotypically different subpopulations of CTCs from the peripheral blood mononuclear cells (PBMCs) of patients with metastatic cancer. Besides EpCAM, we propose Epidermal Growth Factor Receptor (EGFR) as an additional isolation marker for efficient CTCs detection.循环肿瘤细胞（Circulating Tumour Cells, CTCs）在肿瘤转移进程中发挥核心作用，其介导微转移发生，同时也是实现患者病情实时监测的重要工具。此外，开发CTCs分离与鉴定的全新策略，以及推动CTCs向临床实践转化，均需克服仅以上皮细胞黏附分子（Epithelial Cell Adhesion Molecule, EpCAM）的表达作为唯一标记来纯化该肿瘤细胞亚群所带来的局限性。CTCs在患者外周血中极为罕见，且被认为源自上皮源性原发肿瘤的上皮细胞群体，因此基于EpCAM的免疫分离策略曾被认为是合理的分选方案。该领域的争议核心在于，侵袭性更强的间质性肿瘤表型可能对整体CTC群体产生的影响。本研究首先对一组能够代表肿瘤异质性的细胞系开展系统表征，证实了具有受限上皮特征的肿瘤细胞亚群的存在，并佐证了基于EpCAM的分选技术所存在的局限性。随后我们开发了经抗体修饰的定制化聚苯乙烯磁珠，可高效从转移性癌症患者的外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMCs）中分离表型各异的CTC亚群。除EpCAM外，我们还提出将表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）作为额外分选标记，以实现CTCs的高效检测。