A Workflow for the Enrichment, the Identification, and the Isolation of <scp>Non‐Apoptotic</scp> Single Circulating Tumor Cells for <scp>RNA</scp> Sequencing Analysis

doi:10.1002/cyto.a.24816

DOI: 10.1002/cyto.a.24816 ISSN: 1552-4922

A Workflow for the Enrichment, the Identification, and the Isolation of Non‐Apoptotic Single Circulating Tumor Cells for RNA Sequencing Analysis

Anna Abramova, Mahdi Rivandi, Liwen Yang, Nadia Stamm, Jan‐Philipp Cieslik, Ellen Honisch, Dieter Niederacher, Tanja Fehm, Hans Neubauer, André Franken

Cell Biology
Histology
Pathology and Forensic Medicine

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Abstract

Background

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome.

We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non‐apoptotic CTCs for further single CTC transcriptome analysis.

Methods

Viable tumor cells – we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients – were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used.

Results

Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA‐MB‐231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs.

Conclusion

This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non‐apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.