Antibody-independent capture of circulating tumor cells of non-epithelial origin with the ApoStream® system

Circulating tumor cells (CTCs) are increasingly employed for research and clinical monitoring of cancer, though most current methods do not permit the isolation of non-epithelial tumor cells. Furthermore, CTCs isolated with antibody-dependent methods are not suitable for downstream experimental uses, including in vitro culturing and implantation in vivo. In the present study, we describe the development, validation, and transfer across laboratories of a new antibody-independent device for the enrichment of CTCs from blood samples of patients with various cancer diagnoses. The ApoStream® device uses dielectrophoresis (DEP) field-flow assist to separate non-hematopoietic cells from the peripheral blood mononuclear fraction by exposing cells in a laminar flow stream to a critical alternating current frequency. The ApoStream® device was calibrated and validated in a formal cross-laboratory protocol using 3 different cancer cell lines spanning a range of distinct phenotypes (A549, MDA-MB-231, and ASPS-1). In spike-recovery experiments, cancer cell recovery efficiencies appeared independent of spiking level and averaged between 68% and 55%, depending on the cell line. No inter-run carryover was detected in control samples. Moreover, the clinical-readiness of the device in the context of non-epithelial cancers was evaluated with blood specimens from fifteen patients with metastatic sarcoma. The ApoStream® device successfully isolated CTCs from all patients with sarcomas examined, and the phenotypic heterogeneity of the enriched cells was demonstrated by fluorescence in situ hybridization or with multiplex immunophenotyping panels. Therefore, the ApoStream® technology expands the clinical utility of CTC evaluation to mesenchymal cancers.循环肿瘤细胞（Circulating Tumor Cells, CTCs）如今日益广泛地应用于癌症的研究与临床监测，但当前绝大多数方法无法实现非上皮性肿瘤细胞的分离。此外，通过抗体依赖型方法分离得到的循环肿瘤细胞，并不适用于体外培养、体内移植等下游实验应用。本研究详述了一款新型非抗体依赖装置的开发、验证及跨实验室转移流程，该装置可从多种癌症确诊患者的血液样本中富集循环肿瘤细胞。ApoStream®装置采用介电泳（dielectrophoresis, DEP）场流辅助技术，通过将层流中的细胞暴露于特定交变电流频率下，将非造血细胞从外周血单核细胞组分中分离出来。该装置通过正式的跨实验室方案完成校准与验证，实验采用了3种具有不同表型的癌细胞系：A549、MDA-MB-231及ASPS-1。在加标回收实验中，癌细胞的回收效率与加标水平无关，且因细胞系不同，平均回收率介于68%至55%之间。对照样本中未检测到批次间交叉残留。此外，研究纳入15例转移性肉瘤患者的血液样本，评估了该装置在非上皮性癌症场景中的临床就绪性。ApoStream®装置成功从所有纳入检测的肉瘤患者体内分离出循环肿瘤细胞，并通过荧光原位杂交（fluorescence in situ hybridization, FISH）与多重免疫表型检测组合，证实了富集细胞的表型异质性。因此，ApoStream®技术将循环肿瘤细胞评估的临床应用范围扩展至间叶源性癌症。