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Page 6 of 14 Di Raimo et al. J Cancer Metastasis Treat 2018;4:54 I http://dx.doi.org/10.20517/2394-4722.2018.50

Tumor-secreted factors, such as VEGF-A, TNF-α and TGF-β, are able to promote bone marrow-derived he-
matopoietic progenitor cells (BMDCs) recruitment in the secondary organ. Accordingly, BMDCs recruit-
ment results in an ECM remodeling, upregulating fibronectin (FN) and other molecules, such as MMPs,
[121]
and stimulate angiogenesis . Hypoxia-inducible factor (HIF) represents a major effector and adaptor
in BC cells that, due to a massive and unregulated proliferation in association with vasculature dysfunc-
tions, are exposed to a hypoxic microenvironment [122-124] . Lysil oxidase (LOX), one of the principal HIF-
dependent BC secreted factor, is strictly correlated with tumor invasiveness and lung and bone metastasis
formation. In the pre-metastatic organ, LOX is able to co-localize with fibronectin and to modulate cell-
[125]
ECM interactions . Furthermore, through the interaction with type IV collagen, LOX recruits BMDCs
and, in a second attempt, promotes the colonization of metastatic tumor cells [126-128] . In the matrix remodel-
ing scenario, it has been demonstrated that the secretion of lysil oxidase-like 2 (LOX-2) is also able to induce
αSMA expression in pre-metastatic fibroblasts, inducing their activation and the secretion of FN and LOX,
generating a fibrotic microenvironment capable of supporting tumor cell persistence and survival [129,130] .
Finally, the primary cancer secretion of VEGF, TGF-β and TNF-α stimulates Angiopoietin-2 expression in
the pre-metastatic niche increasing vascular permeability and, consequently, promoting the extravasation
of CTCs so that metastatic process can move forward [131-133] .

STATE OF THE ART IN CTCs ANALYSES
The intrinsic mark of rarity of CTCs, in addition to their highly heterogeneous nature, represents an ob-
stacle to study their biology [134,135] . Nevertheless, several technologies are being developed for CTCs detec-
[136]
tion in patients’ peripheral blood sample based on their knowing biological properties . The most com-
monly used techniques are based on a combination of enrichment/isolation and detection procedures. In
the first phase, CTCs are separated from hematologic cells, especially leukocytes that, due to their similar
[134]
physiochemical and biological properties, could contaminate tumor cell pool . The enrichment proce-
dures exploit physical (size, deformability, density and electrical charge) or biological characteristics (cell
surface protein expression, viability and invasive capacity) of CTCs [137,138] . The detection step consists of im-
munostaining methods ranging from classic immunocytochemistry (ICC) or immunofluorescence to flow
[138]
cytometry . Furthermore, RT-PCR approach represents another option to detect tumor related mRNA
transcripts in patients’ blood. Although this method does not require a prior CTCs enrichment, the inabil-
[138]
ity to provide CTCs enumeration deeply restricts its utilisation . Regarding CTCs isolation from blood
components, density gradient centrifugation, such as Ficoll-Hypaque, Percoll (GEHealthcare Life sciences),
OncoQuick (Greiner Bio-One), Cytotrack, Accucyte-cytefinder, represents the most commonly physical
properties-based technique [139-141] . Other exploited approaches are based on cell-size separation, such as
microfiltration (Screen Cell, CellSieve, ISET, Parylene filter, Filtration/Sequential ICC) or microfluidic test
that combines size and deformability properties of CTCs (Ephesia, HB-CTC-chip, Iso-Flux, OncoCEE,
Parsortix system , the ClearCell FX or Vortex) [135,142-151] . Nevertheless, even if all the described isolation
methods represent rapid and less expensive alternatives, they are generally hampered by blood cells-related
false-positive results, thus making necessary the combination with other enrichment methods and the loss
of large CTCs and CTC clusters due to the high heterogeneity of CTC size [136,152] . Immunological assays,
based on the extremely specific reaction between antibodies and the target antigens on the cell surface,
provide a high purity rate of isolated CTCs [145,153-160] . Several of these techniques are based on EpCAM posi-
tive selection and, actually, the most standardized method is the CellSearch® system (Janssen Diagnostics),
the only one approved by the U.S. Food and Drug Administration for CTCs enumeration in BC and other
type of cancer [25,27,157,161] . Nevertheless, as reported by several clinical trials, in patients in which EMT oc-
curring with the downregulation of EpCAM and other epithelial markers, this system may fail to capture
the entire pool of CTCs and may result in false negative findings [74,134,162-165] . Furthermore, it has been dem-
+
onstrated that the lack of EpCAM CTCs detection does not reflect a status of benign prognosis. In fact,
it could be directly related with negative hormone receptors, high tumor grade, triple-negative disease,
inflammatory BC and brain metastasis (OR = 6.17, 95%CI: 2.14-17.79; P = 0.001) or conversely with bone