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Kodama et al. J Cancer Metastasis Treat 2018;4:56 I http://dx.doi.org/10.20517/2394-4722.2018.61 Page 9 of 11

entered the region close to the subcapsular vein and invaded the vessel [Figure 4E]. The schematic diagram
in Figure 4F illustrates the internal features of the marginal sinus with metastatic tumor cells invading the
subcapsular vein.

DISCUSSION
The present study demonstrates that tumor cells in the marginal sinus of a LN can invade extranodal
veins via branches that communicate with intranodal veins. We suggest that this may be the first step in
hematogenous metastasis from LNs. Importantly, this form of metastasis occurs before tumor cells have
[5,6]
interfered with well-developed vascular networks and HEVs within the LN . Veins are ubiquitously
[22]
present on the surface layer of the LN [Figure 1] and communicate with intranodal veins, allowing
blood to flow to the systemic circulation from intranodal vessels. This anatomical arrangement and flow
[2]
characteristics have not been described previously. Kelch et al. surgically excised individual mesenteric
LNs from the mouse and clarified the topology of the entire intranodal vascular network using an
automated confocal imaging system and custom-written software. However, unlike the present study,
their analysis was limited to the vascular network within the LN. This study utilized malignant fibrous
histiocytoma-like KM-Luc/GFP cells and breast cancer FM3A-Luc cells, both of which are syngeneic to
the recipient mice. The invasion of tumor cells from the afferent lymphatic vessel into the intranodal veins
was confirmed for both cell types [Figure 4, Supplementary Figure 1]. This implies that LN-mediated
hematogenous metastasis may be a mechanism relevant to a wide variety of cancer types.

Lymphadenopathy in MXH10/Mo/lpr mice is due to the lpr gene and is characterized by the accumulation
of lpr-T cells in the paracortical region. The metastatic lesions shown in Figure 4 are located in the margin-
al sinuses of the LNs. Thus, it is unlikely that there is a direct relationship between abnormal lymphocyte
proliferation in the LN parenchyma and tumor cell proliferation and vascular invasion.

MXH10/Mo/lpr mice do not express the fas gene involved in apoptosis, since the lpr gene is a fas-deletion
mutant gene. Thus, the immune system in MXH10/Mo/lpr mice is functional except for the signaling path-
way related to fas. This precludes us from using human cell lines for our metastasis experiments, which
would require immune-deficient mice such as SCID or nude mice. We selected MXH10/Mo/lpr mice for
the present experiments because their characteristics make them better suited for use as a model of metas-
tasis than immune-deficient mice implanted with human cell lines.

[23]
Fisher et al. reported that LNs were not an effective barrier against tumor cell progression along the
lymphatic system based on the observation that tumor cells were confirmed in the efferent lymphatic
vessels after their injection into the lymphatic vessels of the rabbit. This means that tumor cells can flow
through the lymphatic system via the marginal sinuses of LNs [17,24] , suggesting that lymphatic flow based on
the anatomical structure of LNs should be distinguished from the mechanism of tumor metastasis.

Our findings suggest the possibility that tumor cells may undergo hematogenous metastasis at an early
stage of LN metastasis, i.e., even before the stage when the infiltration of tumor into the LN is detectable by
imaging or aspiration cytology. In other words, false-N0 metastatic LNs can be a source of hematogenous
metastasis. The importance of false-N0 LNs in distant metastasis is supported by the results of clinical
[27]
trials suggesting that LN dissection [25,26] and sentinel LN biopsy do not always improve survival in
patients with cancer.

The development of new methods to detect and treat metastasis in false-N0 LNs will be extremely
important. In previous studies, we have shown that a lymphatic drug delivery system has great potential in
the therapeutic and prophylactic treatment of false-N0 LNs [25,26] , and we expect this technique to be applied
in the clinical setting in future.