Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting

Abstract

Background:Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.Methods:Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis.Results:On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines.Conclusion:We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.

Circulating tumor cell technologies

“…(Carpenter et al, 2014;Fabbri et al, 2013;Fernandez et al, 2014;Manaresi et al, 2003;Peeters et al, 2013;Polzer et al, 2014) Biophysical e Acoustophoresis Separates cells based on acoustophoretic mobility, which is size dependent, by exposing them to acoustic waves.…”

“…DEPArrayä is designed for single-cell recovery and not bulk enrichment of CTCs. Multiple clinical studies have used DEPArrayä to recover single CTCs for subsequent genetic analyses following enrichment using centrifugation or immunoaffinity (i.e., CellSearch) (Carpenter et al, 2014;Fabbri et al, 2013;Fernandez et al, 2014;Peeters et al, 2013;Polzer et al, 2014). The use of DEPArrayä technology for CTC recovery will likely be limited to samples with a relatively high number of CTCs (e.g., metastatic carcinomas) due to a cell-loss of approximately 40% during sample loading (Peeters et al, 2013).…”

Profiling of circulating tumor cells in liquid biopsies from metastatic cancer patients

“…[25] Many techniques have been developed and are under continuous improvement to enhance the efficacy of CTC isolation and enumeration. [16] Comparison of the OncoQuick density gradient method with FicollHypaque density gradients for separation of tumor cells from bone marrow and peripheral blood aspirates indicated that CTCs can be easily aspirated by both methods and analyzed further to determine their presence and quantity. [30] In the present study, we used the simple and cost-effective Ficoll-Hypaque density gradient method to separate PBMCs from whole plasma of cancer patients.…”

“…The CELLSEARCH ® system [34] is the only method approved by the US FDA for clinical use in metastatic breast, colorectal, and prostate cancer. [13,17,35] Patients with > 80 CTCs in 7.5 mL of blood were reported to die within one year of diagnosis from metastatic disease, [16] and CTCs continue to be detected after the first round of chemotherapy in some breast cancer patients with rapid progression of the disease to metastasis; [17] it has been suggested that, in such cases, it may be preferable to opt for an alternative treatment with novel therapeutic agents, rather than continuing with the same chemotherapy.…”

“…In one study, patients with > 80 CTCs in 7.5 mL of blood died within one year of diagnosis due to metastases. [16] In another study of patients with metastatic breast cancer, when CTCs continued to be detected after the first round of chemotherapy, there was rapid progression of the disease; it was suggested that, in such cases, it would be preferable to opt for alternative treatment, rather than continuing with the same chemotherapy. [17] A review of CTCs in patients with breast cancer in 2013 considered the clinical application of CTCs in breast cancer therapy and assessed the use of HER2 treatment in HER2-negative breast cancer patients, selected on the basis of CTC detection.…”