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Page 6 of 18 Malentacchi et al. J Cancer Metastasis Treat 2020;6:34 I http://dx.doi.org/10.20517/2394-4722.2020.34

with high recovery rates. However, it requires very specific parameters such as cell type and electric field
[20]
frequency (e.g., DEPArray) .
(5) Density based: This procedure is based on centrifugation, which uses the specific density of RBCs,
leukocytes, and cancer cells on specific buffer. This method is one of the first reported.
(6) Reactive ion etching: This procedure is based on photolithography to make specific patterns on the glass
[20]
surface that favor CTC attachment to normal cell attachment on the basis of adhesion preferences .
(7) Acoustic-wave fields inside microchannels are used to capture CTCs based on the principle that all
cells experience different acoustic radiation forces that result in varying movement trajectories, ultimately
separating the cells. In most cases, this procedure is associated with others, including size, density, and
compressibility.
(8) Combined method: It is the combination of more than one of the above-listed procedures (e.g., CTC-
iChip1 and CTC-iChip2) [21,22] .

All the aforementioned technologies have been designed for cell capture ex vivo. Nowadays, a new
technology, known as GILUPICellCollector®, applies an anti-EpCAM wire directly into the peripheral arm
[20]
vein and captures CTCs with remarkable efficiency, processing approximately 1.5 L of blood in 30 min .
A new definition was recently introduced related to classical CTCs. It is not known if all CTCs are able
to induce relapse or metastasis. Consequently, the term CTCs refers to tumor cells that can be found in
circulation. Those CTCs able to induce metastasis and disseminate are defined disseminated tumor cells
(DTCs) based on the presence of specific antigens on their surface, identified by immunocytochemistry
(ICC) or via their corresponding mRNA by reverse transcription quantitative polymerase chain reaction
(RT-qPCR) [21,23] . Generally, CTCs, after enrichment, are evaluated to identify specific cancer-related profile
biomarkers by fluorescence in situ hybridization for genome amplification detection, ICC for protein
markers identification, and RT-PCR/RT-qPCR/NGS for quantifying specific RNA and DNA sequence
[20]
analysis . It is necessary to underline that most of the procedures for CTC isolation, characterization,
and count were designed for cancers other than EC, therefore they may not be appropriate for recognizing
specific antigens related to EC-CTC.

Only some of the above-mentioned technologies for CTC isolation are currently approved for in vitro
diagnostics and are mainly not specific for EC. These technologies may not be suitable to properly identify
EC-CTCs and could give different results. Moreover, these techniques may be inappropriate to identify
[25]
[24]
different biomarker profiles based on DNA mutations , DNA methylation , mRNA expression [26-28] , or
protein .
[28]
Count of CTCs
There are few articles related to CTC count in EC. One is based on CTC enumeration by MetaCell®
technology, which has been detected in 75% of patients with EC . Another one is based on the
[29]
identification of circulating progenitor cell number (characterized by CD34, VEGFR2, and KDR
expression) in the PB of women with early EC. Those numbers result significantly augmented compared
[24]
with the ones coming from healthy control women . Another study evidenced that the detection of more
than two CTCs is useful for a preoperative diagnosis of grade 3 EC: patients with poorly differentiated
[30]
endometrioid EC have higher CTC number. As suggested by Bogani et al. , the discrepant results are
mainly due to different biomarkers used for CTC characterization. Regarding DTCs, it has been reported
[31]
that they are present in 16% of bone marrow aspirates of women affected by EC .
Biomarkers of CTC
Some studies focused on the identification of the expression of single specific biomarkers such as Mig7,
[27]
CK19 , and thyroid transcription factor-1, which were found to be correlated with TNM staging, vascular