A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity

Abstract

Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike‐in experiments using MCF‐7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI‐g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient‐derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient‐derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

Breast cancer heterogeneity and its implication in personalized precision therapy

“…At the genomic level, the highly multiplexed single nucleus sequencing (HM-SNS) has been demonstrated as a good tool to explore the heterogeneity of copy number profiling in the breast cancer [ 201 ], which has been applied to explain the chemoresistance revolution in TNBC [ 16 ]. Deger et al reported a modular pipeline constructed of five individually adjustable steps, including enrichment, isolation, whole-genome amplification, low pass sequencing, and bioinformatical analysis, to perform single CTCs genomics at a high resolution by copy number profiling [ 60 ]. Each step can be adjusted to use different methods according to specific research needs.…”

“…High throughput Loss of spatial information [ 16 , 201 ] Modular single CTC analysis pipeline Each step is adjustable. Preserving single-cell information Loss of spatial information [ 60 ] At the epigenomic level scATAC-seq Preserving single-cell information Loss of spatial information [ 10 ] scDNase-seq Preserving single-cell information Loss of spatial information [ 11 ] scNOMe–seq Preserving single-cell information Loss of spatial information [ 12 ] scChIP-seq Preserving single-cell information. High throughput Chip design is highly complex.…”

“…Therefore, the analysis of CTCs can be an essential tool for studying the heterogeneity between cells from the primary and metastatic sites. Because CTCs can be obtained by minimally invasive peripheral blood extraction, they can be collected continuously to monitor molecular changes as the disease progresses and, significantly, under therapeutic pressure [ 60 ]. Thus, analysis of a single CTC enhances the assessment of heterogeneity.…”

Circulating Tumor Cells in Cancer Diagnostics and Prognostics by Single-Molecule and Single-Cell Characterization

“…For example, the VyCAP system (VyCAP BV, Enschede, The Netherlands) first distributes previously isolated CTCs onto a microwell chip, and following fluorescent image acquisition, the cells of interest are punched and collected one at a time through the chip . This system has been coupled with CellSearch for copy number profiling of single CTCs and as a pipeline has been validated in 2 mBRCA and 2 mPrCa patients . CellCelector (Sartorius AG, Göttingen, Germany) combines automated micromanipulation and microfluidic cell picking.…”

“…41 This system has been coupled with CellSearch for copy number profiling of single CTCs and as a pipeline has been validated in 2 mBRCA and 2 mPrCa patients. 42 CellCelector (Sartorius AG, Goẗtingen, Germany) combines automated micromanipulation and microfluidic cell picking. Single CTCs isolated with CellCelector following their capture by Parsortix were successfully used for tumor engraftment in mice and single-cell transcriptomic analysis.…”