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Galletti et al. Using CTCs in prostate cancer

Table 1: Overview of main methodologies for CTC isolation and potential research implementation in prostate
cancer
Isolation Technique Commercially Surface antigen Cell output Downstream analyses
principle available? requirement
Physical Density gradient Yes - Live cells IF, RT-PCR
centrifugation
EPIC Sciences™ Yes - Fixed cells IF, FISH, CTC enumeration
HD-CTC assay
®
ScreenCell filter Yes - Live cells IF, FISH, CTC enumeration
®
ISET filter Yes - Live cells IF, FISH, CTC enumeration
Biological CellSearch ® Yes EpCAM Fixed cells IF, FISH, CTC enumeration
MagSweeper No EpCAM Live cells with IF, FISH, CTC enumeration,
bound beads RT-PCR, RNA-seq
AdnaTest Yes EpCAM/HER2 RNA RT-PCR
RosetteSep™ Yes CD45 Live cells IF, FISH, CTC enumeration,
CD45 depletion RT-PCR, RNA-seq, CTC-organoid
culture and PDX models, ex-vivo
drug treatment
Microfluidics CTC-Chip No EpCAM Live cells on IF, FISH, CTC enumeration,
device RT-PCR, RNA-seq, ex-vivo drug
treatment
HB-Chip No EpCAM Live cells on IF, FISH, CTC enumeration,
device RT-PCR, RNA-seq, ex-vivo drug
treatment
iChip No - Live cells IF, FISH, CTC enumeration,
RT-PCR, RNA-seq, CTC-organoid
culture and PDX models, ex-vivo
drug treatment
GEDI Chip No PSMA Live cells on IF, CTC enumeration, RT-PCR,
device RNA-seq, ex-vivo drug treatment
VERSA No EpCAM Live cells IF, CTC enumeration, RT-PCR,
RNA-seq, ex-vivo drug treatment
In vivo GILUPI ® Yes EpCAM Live cells on IF, CTC enumeration, RT-PCR,
device RNA-seq, ex-vivo drug treatment

CTC: circulating tumor cell; IF: immunofluorescence; RT-PCR: real time polymerase chain reaction; PDX: patient-derived xenograft; HD:
high definition; HB: herringbone; GEDI: geometrically enhanced differential immunocapture; EpCAM: epithelial cell adhesion molecule;
PSMA: prostate specific membrane antigen
adhesion molecule (EpCAM) and E-cadherin [14,15] . cancer cells using an immunofluorescence-based
Importantly, these approaches yield live, unaltered algorithm, which measures CK and CD45 intensities,
cells, which can be then used in a wide variety of as well as cell physical properties including nuclear
downstream analyses. However, the low purity of the and cytoplasmic size and shape . This assay has
[17]
CTC population obtained, due to the presence of many been extensively used clinically and identifies CTCs
contaminating hematopoietic cells that outnumber the in several cancer types, including NSCLC, breast
CTCs by several logs, compromises the sensitivity of and prostate cancers [17,18] . Interestingly, the HD-CTC
this technique [16] . test recognizes distinct categories of CTCs based on
morphologic characteristics of the cells (traditional
To better differentiate CTCs from peripheral blood CTCs, small CTCs, CTC clusters and apoptotic CTCs),
cells, immunofluorescence is commonly used in whose clinical relevance has yet to be determined .
[19]
conjunction with density-based separation. CTCs The versatility of the method is exemplified by the
are typically identified as nucleated cells (positive
for DAPI staining) that express an epithelial possibility to assess a wide range of protein biomarkers
marker [e.g. cytokeratin (CK), or prostate specific via immunofluorescence (e.g. androgen receptor in
membrane antigen (PSMA) in the case of PC prostate cancer or PD-L1 in bladder cancer), specific
CTCs], and are negative for expression of the driver genomic alterations (by FISH) and genome-wide
[20]
hematopoietic marker, CD45. copy number alterations . Unfortunately, the lack of
a robust multiplexing of the technique does not allow
In practice, following a “no cell left behind” philosophy, for concomitant investigation of more than one or two
the Epic Sciences™ high definition (HD)-CTC assay biomarkers within the same slide and, together with
screens for CTCs amid all blood nucleated cells plated a low resolution used in image acquisition, limits the
onto custom glass slides and identifies epithelial clinical power of the assay.
192 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ September 27, 2017