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Di Raimo et al. J Cancer Metastasis Treat 2018;4:54 I http://dx.doi.org/10.20517/2394-4722.2018.50 Page 5 of 14

press high levels of activated Met-RTK which are able to induce membrane blebbing and, as a consequence,
cell dissociation, amoeboid motility and invasion. Furthermore, they highlighted a Met-induced protection
from apoptosis and the ability of these Met-expressing cancer cells to promote the metastatic process. The
lymphatic vessel pathway, due to its discontinuous structure, the high concentration of hyaluronic acid and
the lymph fluid composition, which is able to improve cell survival and to reduced shear stress, represents
a better and safer dissemination vehicle for cancer cells than the blood stream. Thus, it could be reasonable
to consider the possibility that both epithelial and mesenchymal cancer cells migrate, preferably, through
the lymphatic system, spread first to lymph nodes and then drain into the blood [92-94] . Accordingly, another
mechanism of tumor EMT-independent metastasis, namely tumor-induced lymphangiogenesis, has been
[95]
proposed . Briefly, mesenchymal cancer cells, which are able to produce and release lymphangiogenic
factors, such as vascular endothelial growth factor C and D (VEGF-C and VEGF-D), promote an increase
of lymphatic vessel density in the peri- and intratumoral area, so that epithelial cells are able to colonize
lymphatic system and lymph nodes can facilitate their entry into the systemic circulation [96-100] . It has been
demonstrated that an increase in lymph vessel density, due to tumor-induced lymphangiogenesis, is cor-
related with a high amount of lymph node metastasis, VEGF-C expression and worse disease-free/overall
[101]
survival in BC patients .

Immune escape
An important issue related to the EMP of CTCs and their metastatic potential is the immune-escape, which
is the ability of tumor cells, during their migration, to counteract the elimination by the immune system and
to increase their possibility to survive and to colonize distant organs [102-104] . One of the most studied immune
evasion mechanism is the programmed death-ligand 1 (PD-L1)/programmed death receptor (PD-1) axis. In
normal conditions, the PD-L1 and its PD-1 represent a physiological checkpoint of the immune system.
Antigen-presenting cells express PD-L1 while PD-1 is detectable on the surface of activated T-cells. Once
ligand/receptor interaction occurred, a strong inhibitory signal promotes apoptosis and functional ex-
[105]
[106]
haustion in T-cells . In 2014, Chen et al. have identified, in lung tumor, a molecular link between the
overexpression of the EMT-effector ZEB1 and a more abundant presence of PDL1, able to promote the
exhaustion of intratumoral T lymphocytes and the development of metastasis [106-108] . Similarly, in breast
cancer, it has been demonstrated that PD-L1 expression is heterogeneous and it is generally associated with
the presence of poor-prognosis factors, high proliferative index and aggressive molecular subtypes [109,110] . In
[111]
2015, for the first time, Mazel et al. provided evidence that CTCs, isolated from the blood of BC patients,
frequently express PD-L1 on their surface. The Fas/FasL axis represents another EMP-dependent immune
escape mechanism based on the ligand/receptor interaction with a negative impact on the clinical outcome
[112]
of BC patients . Briefly, when the factor-associated suicide (Fas), a transmembrane receptor belonging to
the tumor necrosis factor (TNF) family, interacts with its ligand (FasL), expressed on the surface of acti-
vated T lymphocytes, Fas-expressing cells go through apoptosis. During BC progression, Fas was found to
be repressed in association with an increase of FasL level and TGF-β secretion in tumor cells, conferring to
[113]
CTCs the ability to induce cell death and escape immune recognition .

Metastatic niche
Despite the migration mechanism and the above-mentioned immune evasion systems adopted by cancer
cells, only a few percentage of cells that extravasate are able to survive in the unsuitable secondary organ
environment and promote metastatic growth. Thus, the microenvironment in the metastatic site repre-
sents a major challenge for invading cancer cells. Starting from the “seed and soil” hypothesis, postulated
by Paget, up to date, it is well known that cancer cells (the seed) require a specific and compatible “soil”
microenvironment, the pre-metastatic niche, which is able to evolve and to promote both cell engraftment,
creating the metastatic niche, and cell proliferation, leading to the micro- to macro- metastatic transi-
tion [114-119] . Many evidences demonstrate how primary tumor site is able to modify, before cancer cells’
arrival, the secondary organ microenvironment, stimulating the creation of the pre-metastatic niche [120] .