Efficient leukocyte depletion by a novel microfluidic platform enables the molecular detection and characterization of circulating tumor cells.

Eva Obermayr,Michael Krainer,Elisabeth Maritschnegg,Nina Pecha,Paul Speiser,Christiane Agreiter,Christian Singer,Sabine Danzinger,Robert Zeillinger,Samir Helmy-Bader

doi:10.18632/oncotarget.22549

Abstract

RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix™ technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach.In this study, we established a workflow including the micro-fluidic Parsortix™ technology for the molecular detection of CTC related transcripts. Background levels of EpCAM, PPIC, TUSC3, and MAL2 were efficiently removed due to an up to 106-fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix™-enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%).The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.

Highlights

The analysis of circulating tumor cells (CTCs) in the blood of cancer patients represents an enormous technical challenge, due to their low absolute numbers and the extreme abundance of blood cells
First we evaluated whether the ParsortixTM technology and default separation conditions (2 ml blood, 23 mbar pressure, 10 μm final step size) can be applied for ovarian cancer cells
Our workflow meets the key prerequisite for reverse transcription quantitative polymerase chain reaction (RT-qPCR) based analysis of CTC-related gene markers in the obtained cell sample by efficiently removing unwanted leukocytes, which could contribute to false positives due to illegitimate transcription of the used markers

Summary

Introduction

The analysis of circulating tumor cells (CTCs) in the blood of cancer patients represents an enormous technical challenge, due to their low absolute numbers and the extreme abundance of blood cells. The standard recommendation is to increase the relative amount of CTCs. Density gradient www.impactjournals.com/oncotarget centrifugation is one approach to enrich the blood sample for CTCs: it is easy to perform, inexpensive, suitable for large blood volumes A further depletion of unwanted cells can be achieved by immune-magnetic capture of white blood cells using an antibody against the leukocyte-specific CD45. At first glance, this approach may be tempting, but with large blood volumes it is associated with high costs, CTCs may be trapped within the bulk of captured leukocytes, or may even bind to the magnetic beads [4]

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Conclusion