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Galletti et al. Using CTCs in prostate cancer

biological information about a patient’s disease such as EpCAM could define a more mesenchymal
state. On the other hand, a potential downside of the subpopulation of CTCs, whose increase has been
size-based technologies is represented by the high associated with tumor relapse during treatment in solid
heterogeneity in diameter CTCs can display, with tumors . In addition, the presence of anti-EpCAM
[36]
subsets of CTCs with diameters as low as 11 µm, coated beads, which remain bound to the captured
which is virtually indistinguishable from the diameter CTCs, can interfere with functional applications of
of leukocytes [28,29] . These smaller CTCs may be of the isolated cells. Finally, the added steps of fixation
clinical importance but they are likely missed by the and immunofluorescence staining performed for cell
size based filtration techniques. enumeration purposes limits the molecular analysis
that can be performed.
CTC enrichment based on cell-surface antigen
expression or other biological properties Another antigen-based CTC isolation method is
Many CTC isolation techniques exploit the expression the AdnaTest (QIAGEN ), which uses anti-EpCAM
©
of tumor-selective cell surface antigens by using an and -HER2 antibodies conjugated to Dynabeads
TM
antibody-antigen interaction for CTC enrichment. magnetic particles [37,38] . CTCs with bound beads
The most established and the only FDA-cleared are magnetically separated and subsequently
method for CTC isolation is the CellSearch CTC processed for gene expression profiling (Prostate
®
Test system. Starting with 7.5 mL of peripheral blood, Cancer Select) or for PC-specific gene quantification
CTCs are positively selected using anti-EpCAM- (Prostate Cancer Detect). A major advantage of the
coated magnetic microbeads and subsequently AdnaTest is the combination of two isolation markers,
embedded and fixed onto a magnetic cartridge EpCAM and HER2, to capture CTCs; the addition
for subsequent immunofluorescence staining with of HER2, which can be found expressed in up to
antibodies against CK, CD45, and DAPI [30] . CTC 78% of metastatic castrate prostate cancers, could
enumeration by CellSearch has been established potentially include the EpCAM-/HER2+ CTCs in the
®
as a test predictive of patient outcomes in metastatic enrichment process, a subpopulation of cells, which
breast [31] , colorectal [32] and prostate cancer patients [33] . an EpCAM only based approach may easily miss [39,40] .
Following the initial excitement for a prognostic test Interestingly, pretreatment CTC identification by using
based on a liquid-biopsy, it soon became apparent the AdnaTest correlated with radiologic progression to
that enumeration alone could not guide therapy, as chemotherapy in a small cohort of Castration-Resistant
there was no information regarding the molecular PC (CRPC) patients, supporting the clinical relevance
CTC profiles. Nevertheless, these early clinical of the test . The main property of the AdnaTest is
[38]
studies using CellSearch have provided proof- having nucleic acids as a direct output of the assay
®
of-principle regarding the use of EpCAM for the for specific downstream analyses (e.g. RT-PCR, DNA
detection of epithelial-type cells in the bloodstream. sequencing, gene expression profile) . Even if this
[41]
To date, the majority of antigen-specific CTC characteristic precludes the direct visualization of the
enrichment techniques rely on the use of EpCAM. The captured cells, it provides flexibility to incorporate and
MagSweeper CTC enrichment represents another explore additional markers, like androgen receptor
example of platforms, which also utilize EpCAM- (AR) splice variants or stem cell markers [38,42] . Although
based immunomagnetic cell isolation [34] . potentially more comprehensive in CTC isolation than
EpCAM only strategies, this method is still based on the
These two methodologies have been extensively expression of tumor-selective antigens, an approach
used in several studies for the purposes of CTC that may still omit certain CTC subpopulations.
enumeration and identification of tumor-specific
biomarkers using immunofluorescence or FISH To take advantage of both the physical and biological
analyses [Table 1]. MagSweeper-based single CTC properties of CTCs, several microfluidic devices
mRNA seq has also been performed and provided a have been developed to capture CTCs from whole
detectable transcriptional signature of PC tissue . blood. The design of many of these devices takes
[35]
Despite their extensive use, there are significant advantage of the blood flow through the device
concerns associated with the EpCAM-based CTC that is engineered to facilitate the collisions of
enrichment techniques. Importantly, the dependence the larger CTCs with microposts, eliminating the
on EpCAM expression for CTC capture could smaller leukocytes; the posts are coated with an
specifically select for only a subset of CTCs resulting anti-epithelial marker antibody to specifically retain
in the potential loss of EpCAM-low or -negative CTCs tumor epithelial cells. The CTC-Chip utilizes an
and thus underestimate CTC numbers; it has been array of microposts coated with an antibody against
indeed shown that the loss of epithelial markers an epithelial marker (EpCAM, HER2, EGFR) to
194 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ September 27, 2017