CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM.

Guillaume Belthier,Jihane Vitre-Boubaker,Gérald Lossaint,Jérôme Solassol,Jean-Christophe Boyer,Fabienne Portales,Ilona Krol,Diana Heaug-Wane,Nicola Aceto,Fanny Grillet,Matthieu Merlot,Zeinab Homayed,Sophie Bravo,Olivia Villeronce,Julie Pannequin,Christophe Duperray,Jai Smith,François Bertucci,Marc Ychou,Jean Marc Pascussi,Thibault Mazard,Frédéric Hollande,Emmanuelle Samalin,Émilie Mamessier ,Julie A Vendrell ,Françoise Macari

doi:10.3390/cancers13194966

Abstract

Simple SummaryIn the present work, we describe (for the first time) the use of the transmembrane protein, CD44v6, to detect CTCs from blood samples of several patients with colorectal or breast cancer. We used CD44v6 antibodies to demonstrate that live CTCs can be specifically purified from CRC patient blood samples via magnetic bead- or FACS-based isolation techniques. Finally, we demonstrated that CD44v6-positive CTCs rarely expressed EpCam, which is currently the gold standard to enumerate CTCs, suggesting the need to use a combination of markers for a more comprehensive view of CTC heterogeneity.Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

Highlights

According to recent data, 9.6 million people die each year from cancer, with the vast majority of these deaths being due to metastasis to distant organs [1]
A tumor was formed following injection of these cells into the flank of nude mice, confirming their tumoral origin (Figure 2D). These results demonstrate that Circulating tumor cells (CTCs) can be purified from colorectal cancer (CRC) patient blood samples with magnetic beads based on the expression of CD44 variable alternative exon 6 (CD44v6)
In the present work, we detected CD45−/CD44V6high cells in all colorectal cancer (CRC) patient blood samples, irrespective of clinical data (Table 1), suggesting that this antigen could be a precious tool for providing a better overview of tumor heterogeneity

Summary

Introduction

9.6 million people die each year from cancer, with the vast majority of these deaths being due to metastasis to distant organs [1]. We extracted CD44v6-positive blood samples from 20 colorectal cancer patients A tumor was formed following injection of these cells into the flank of nude mice, confirming their tumoral origin (Figure 2D) All together, these results demonstrate that CTCs can be purified from CRC patient blood samples with magnetic beads based on the expression of CD44v6. To test if CD44v6 allows the isolation of CTCs, the number of CD45negative/CD44V6high (CD45−/CD44V6high) cells was quantified by flow cytometry analysis in CRC patient blood samples.

Method

Discussion

Findings

Material and Methods