Circulating tumor cells (CTCs) are shed by the solid tumor into the bloodstream. Consistently, single-cell sequencing of CTCs is a powerful tool to further decipher tumor spatiotemporal heterogeneity, plasticity and to identify new pharmacological targets influencing clinical outcome and response to treatment. The aim is to validate an original workflow allowing the isolation at the single-cell level of mimicking CTCs without interfering with single-cell RNAseq analysis.
The advances in microfluidic systems and isolation technologies have resulted in the enriched extraction of mimicking CTCs from healthy blood samples. The ClearCell Fx (Biolidics Limited) was used as a label-free microfluidic system for enrichment of wholly intact CTCs, while the cellenONE F1.4 system (Cellenion) was used to isolate single CTCs. The later allowed high-throughput automated isolation and dispensing of single CTCs in 96-well plates. ScRNA libraries were prepared with the NebNext Single Cell/Low Input kit (New England Biolabs).
The h-TERT-shp53/RAS HMEC breast cancer cell line was used as mimicking-CTC cell line to evaluate transcriptomic changes. We compared the transcriptomic profiles at each major step: (1) directly after trypsinization, (2) after spiking in whole blood and enrichment with the ClearCell Fx device, (3) after isolation with the cellenONE instrument in bulk of 200 cells and at the single-cell level. The preparation of the scRNAseq library was successful in over 90% of cases at the single-cell level and 100% for bulk samples. Minor effects of the enrichment and isolation methodology were observed at the single-cell level on the transcriptomic profiles. The quality of the libraries and the sequencing provided reliable scRNAseq analyses.
This protocol could be further improved by adding a pre-selection of CTCs based on negative sorting, using a fluorescent anti-CD45 immune labelling exclusion. Such knowledge of single-cell biology may lead to the development of specific therapies to limit tumor progression and seeding of tumoral cells into secondary healthy organs by blocking newly identified targets.
Léa Payen-Gay.
New England Biolabs.
All authors have declared no conflicts of interest.