Molecular analysis of CTCs with different EpCAM and CD44 expression from metastatic breast cancer patients

Background Tumor cells that have undergone a mesenchymal-epithelial transition (EMT) show upregulated CD44 and downregulated EpCAM expression, which may lead to therapy resistance and metastatic tumor spread.

We aim to develop an appropriate method to enrich CTCs in an EpCAM-independent fashion and to analyze the role of CTCs, which express different levels of EpCAM and CD44.

Methods Spike-in experiments with MCF7 and MDA-MB-231 cells were performed and processed with the Parsortix system (ANGLE plc, UK), cells were then characterized by immunofluorescent staining on Sievewell slide (TOK Co. Ltd., Japan). Labelled cells were imaged and enumerated with the CellCelector (ALS GmbH, Germany) platform. In a small study, blood from 23 metastasized BC patients was analyzed, single CD44^pos CTCs were selected and genomic DNA was amplified (Ampli1, Menarini, Italy). Targeted sequencing analysis for PIK3CA, AKT1, ERBB2, and ESR1 was performed (Franken, et al. 2020).

Results In MCF7 cells, 4.5 µm cassette & 99mBar pressure reached 92% ± 8% recovery rate, while 4.5 µm cassette & 50mBar pressure achieved 76% ± 19% with MDA-MB-231 cells. From 23 patient samples, in total 47 CD44^pos and 120 CD44^low/neg CTCs were isolated from 9 patient samples. Amplified genomic DNA of 77.78% (21/27) CD44^pos CTCs and 59.52%–25/42) CD44^low/neg CTCs from 7 patients resulted in high quality. ERBB2 mutations were observed in 4 CD44^pos and 8 CD44^low/neg CTCs from 3 patients.

Conclusion With this workflow, CD44^pos CTCs – a CTC subpopulation associated with therapy resistance and metastatic tumor spread – can be detected and isolated to identify targets for therapies.

Publication History

Article published online:
11 October 2022