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Page 2 of 11 Kodama et al. J Cancer Metastasis Treat 2018;4:56 I http://dx.doi.org/10.20517/2394-4722.2018.61

Conclusion: Metastatic LNs that would be considered by clinical imaging to be stage N0 can be a starting point
for hematogenous metastasis. The study findings highlight the need for the development of novel techniques for
the diagnosis and treatment of early-stage LN metastasis, i.e., when standard diagnostic imaging might incorrectly
classify the LN as stage N0.

Keywords: Lymph node, metastasis, N0, lymph node-mediated hematogenous metastasis

INTRODUCTION
Tumor cells reach the marginal sinus of a sentinel lymph node (LN) via afferent lymphatic vessels, after
[1]
which they proliferate along the lymphoid sinus, invade the cortex and reach the medulla . The abundant
[3,4]
[2]
vascular network in a LN allows tumor cells to grow without the induction of tumor neovasculature .
Since tumor cells growing in a LN can infiltrate both the lymphatic channel and the vascular network,
a sentinel LN can be the origin of lymphatic metastasis to downstream LNs as well as hematogenous
[5]
metastasis . It has been suggested that high endothelial venules (HEVs) may be involved in the
[5,6]
mechanisms underlying systemic metastasis , but the details remain unknown. Clinically, a LN is judged
as positive for metastasis (> N1) if tumor invasion is detected by diagnostic imaging or aspiration cytology.
However, since a LN can be erroneously classified as stage N0 during the early stages of tumor invasion,
[7]
a false-N0 LN can potentially be a source of systemic metastasis. For example, in the NSABP-32 trial ,
patients with breast cancer judged incorrectly to be stage N0 had no difference in overall survival, disease-
free survival and distant disease-free interval to patients judged to be stage N0. In other words, tumor cells
may have undergone systemic metastasis at a stage when LNs were incorrectly classified as N0.

Recently, we demonstrated that a fluorescent dye injected locally into a LN flowed into both the efferent
lymphatic vessel and extranodal veins and that intranodal and extranodal veins communicated via branches
[8,9]
that passed through the capsule , a feature not described in conventional textbooks of anatomy. Thus,
tumor cells can undergo both lymphatic and hematogenous metastasis. Based on these results, we proposed
[8,9]
a theory of LN-mediated hematogenous metastasis, whereby LNs can be the origin of systemic metastasis .

In this study, we used a mouse model in which metastasis to the proper axillary LN (PALN) was induced
by the inoculation of tumor cells into the subiliac LN (SiLN). We found that during early-stage PALN
metastasis (confirmed by pathological imaging), the invasion of tumor cells from the marginal sinus
into intranodal veins and then extranodal veins may be a first step in the mechanism of hematogenous
metastasis from a LN.

METHODS
Experiments were carried out in accordance with published guidelines and approved by the Institutional
Animal Care and Use Committee of Tohoku University.

Mice
MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice (12-17 weeks old), which are a congenic strain of MRL/Mp-lpr/lpr
[10]
and C3H/HeJ-lpr/lpr mice , were bred under specific pathogen-free conditions in the Animal Research
Institute, Graduate School of Medicine, Tohoku University, Sendai, Japan. The LNs enlarge to about 10 mm
+
-
[11]
+
-
in diameter at 12 weeks of age due to invasion by lpr-T (CD4 CD8 B220 Thy ) cells . The anatomical loca-
tions and nomenclatures of murine LNs have often been ignored or assigned incorrectly; in this study, we
[12]
used the term “subiliac LN” instead of “inguinal LN” .
Micro-computed tomography imaging
Specimens were analyzed using high-resolution micro-computed tomography (micro-CT) scanning
(scanXmate/E090, Comscan Tecno). Barium contrast agent (mean size, 935.7 nm) was prepared as