Page 132 - Read Online

Page 4 of 11 Nakamura et al. J Cancer Metastasis Treat 2018;4:32 I http://dx.doi.org/10.20517/2394-4722.2017.78

cell markers are present on normal epithelial surface and epithelial tumors (i.e., carcinomas), but absent on
normal blood cells; therefore, they can be used to identify the cancer cells in the bloodstream apart from
normal blood cells. EpCAM and members of the CKs family (e.g., CK8, CK18, and CK19) are frequently
used for positive selection of epithelial CTCs. However, epithelial cells can undergo EMT, resulting in
downregulation of epithelial markers. To prevent false-negatives caused by EMT, N-cadherin and vimentin
are used for identification of mesenchymal CTCs. In addition, to enrich CTCs specifically, negative selection
is performed by using antibodies against CD45. CD45 is specifically expressed on the surface of leukocytes,
whereas it is not expressed on carcinoma cells; thus, anti-CD45 antibody can deplete unnecessary
leukocytes.

Tumor-specific makers, such as carcinoembryonic antigen (CEA), or α-fetoprotein (AFP), are also used
for biological CTC enrichment. In particular, human epidermal growth factor 2 (HER2) is suggested to be
[19]
important biomarkers in the context of recent targeted therapies .
On the basis of these techniques, there are various enrichment techniques. Magnetic activated cell sorting
[20]
(MACS) uses magnetically labeled antibodies to enrich EpCAM-positive CTCs . MagSweeper (Illumina,
[21]
Hayward, CA, USA) is an automated immunomagnetic cell isolator for separation of rare endothelial cells .
CellSearch System® (Veridex) captures CTCs using ferrofluid beads coated with anti-EpCAM antibody.
Then, captured EpCAM-positive CTCs are stained with anti-CK and anti-CD45 fluorescently-conjugated
dyes. Finally, enumeration of EpCAM-positive, CK-positive, and CD45-negative CTCs is completed by
[22]
immunofluorescence . The US Food and Drug Administration (FDA) has approved CellSearch for clinical
use in breast, colorectal, and prostate cancer patients [6,23,24] . However, CTC detection by this system is not
suitable for non-epithelial phenotype or EMT phenotype not expressing EpCAM and CK.

AdnaTest (AdnaGen AG, Langenhagen, Germany) is also an assay combining the enrichment and detection
processes; that is, enriched by the magnetic procedure and detected by RT-PCR for identification of tumor-
[25]
associated transcripts .

CTC-chip is based on a microfluidic platform that contains an assortment of microposts coated with anti-
EpCAM antibodies. Whole blood is pumped through this chip and EpCAM-positive cells are isolated and
[26]
detected by cameras identifying their morphology, viability and the expression of tumor markers .
Physical property-based techniques
Other enrichment techniques depend on physical properties of CTCs, such as size, diameter, density,
deformability, and electric charge. The tumor cells were previously thought to be larger (> 8 μm), and less
deformable than blood cells. Isolation by size of epithelial tumor cells (ISET; RareCells, Paris, France) isolates
epithelial cancer cells by using blood filtration with a membrane with 8 μm pores; thus, larger cancer cells
[27]
are filtered. ISET can detect a single CTC from 1 mL of peripheral blood .
Density-dependent cell separation uses an inert polysucrose called Ficoll (GE Healthcare Bio-Science,
Pittsburg, PA; BD Bioscience, San Jose, CA). Ficoll was originally developed to isolate intact mononuclear
blood cells from whole blood. Oncoquick™ (Greiner Bio One, Frickenhausen, Germany) based on Ficoll is a
[28]
density gradient centrifugation system that can separate CTCs from whole blood samples .
RosetteSep™ (StemCell Technologies, Vancouver, BC, Canada) is based on negative selection consisting of the
depletion of the majority of the leukocytes and erythrocytes. This method employs a complex of antibody-
targeted hematopoietic cells in human whole blood and crosslinks them to multiple erythrocytes, which
leads to immunorosette formation. A centrifugation over a buoyant density medium such as Ficoll-Paque®
allows for the precipitation of immunorosettes and unbound red blood cells, while CTC fractions can be