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

Contrast-enhanced ultrasound imaging and spatiotemporal analysis of pixel intensity variations
A 2D image of the PALN microvasculature was reconstructed on days 6 and 9 post-inoculation of KM-
Luc/GFP cells and on days 8, 15, 22 and 29 post-inoculation of FM3A-Luc cells using a contrast-enhanced
high-frequency ultrasound (CE-HFUS) system with a 37.5-MHz transducer (RMV-710B, VisualSonics).
Each transducer was fixed to a 3D stage control system (Mark-204-MS; Sigma Koki). Contrast images (slice
thickness, 100 µm) were captured before and 60 s after an intravenous bolus injection of 100 µL microbubbles
(Sonazoid, Daiichi Sankyo) into the tail vein. During imaging, the mouse was positioned on a heated stage
[18]
and anesthetized with 2% isoflurane in oxygen . Acquired images were analyzed using ultrasound contrast
[19]
agent-detecting software to measure temporal changes in the PALN microvessel density.

Histological analysis
PALNs and SiLNs were harvested at the indicated time points, fixed in 10% formaldehyde in PBS for 3 days,
placed on a shaker for 1 day at room temperature and then stored at 4 ℃ for 2 days. Next, the samples were
dehydrated in 100% ethanol, placed into a tissue processor, embedded in paraffin and cut into 3-µm serial
sections using a fully-automated tissue-sectioning device (AS-400, Kurano). Samples were stained with
hematoxylin and eosin (HE) or immunostained for detection of CD31-positive vascular endothelial cells.

Statistical analysis
Data are presented as mean ± SD or mean ± SEM. Differences between groups were determined by one-
way analysis of variance followed by Tukey’s test or an unpaired t-test (GraphPad Prism 6J). P < 0.05 was
considered to be statistically significant.

RESULTS
Connection of the TEV to SiLN blood vessels
First, we examined the anatomical positions of the PALN, SiLN, accessory axillary LN (AALN) and TEV.
An arc-shaped incision was made in the abdominal skin of a mouse from the subiliac region to the proper
[16]
axillary region [Figure 1AI]. The axillary area contains two LNs, the PALN and AALN . The SiLN and
AALN lie upstream of the PALN in the lymphatic network. The TEV, which connects the subclavian vein
and inferior vena cava, runs adjacent to the PALN [Figure 1AII] and SiLN [Figure 1AIII] and along the
lymphatic vessels (not visualized in Figure 1AI) between these LNs. The TEV receives venous blood from
the PALN and SiLN via small branches [Figure 1AII and AIII] [9,16] . There were many vessels on the LN that
connected to intranodal vessels [Figure 1AII and AIII]. The hilum of the PALN [Figure 1AII] and of the
SiLN [Figure 1AIII] was located behind the image.

A series of 3D micro-CT images, rotated every 60º, revealed a complex vascular network in the PALN
[Figure 1B], with many small branches penetrating the LN capsule and connecting the TEV to intranodal
blood vessels. There were no similar networks on the reverse side [Figure 1B]. Immunolabeling of CD31
[Figure 1C] showed that the TEV ran along and penetrated the capsule of the SiLN [Figure 1CI-IV]. The
TEV ran along the capsule (Cap; image I) and connected with intranodal veins penetrating the marginal
sinus (Mgs; image II). The branches of TEV ran in the cortex (Cor) under the marginal sinus (image III)
and branched into two vessels in the cortex (image IV).

Flow dynamics between the PALN blood vessel network and TEV
The flow dynamics between the PALN blood vessel network and TEV were visualized under a fluorescence
stereomicroscope after intravenous injection of NBD-liposomes [Figure 2]. Consistent with the results shown
in Figure 1, the TEV communicated with intranodal blood vessels via many small branches [Figure 2]. In the
region excluding the TEV [Figure 2I and Video 1], a large vein running along the PALN made connections
with the intranodal veins, and the combined blood flow drained into the TEV. In the region including the
TEV [Figure 2II and Video 2], the venous network inside the LN connected to the TEV. The blood flow