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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 3 of 11
[13]
previously described . With the mouse under deep general anesthesia, 0.1 mL heparin (Novo-Heparin, 1000
units/mL, Mochida Pharmaceutical) was administered intravenously and 0.05 mL papaverine hydrochloride
(40 mg/mL, Nichi-Iko) was given subcutaneously. Ten min later, a syringe pump (Legato100, KD Scientific)
was used to infuse 4 mL of saline (18 mL/h) into the left ventricle through a thoracotomy. After cutting of
the caudal vena cava and draining of the blood from the body, 4 mL of contrast medium was injected into
the left ventricle (18 mL/h) via a T-shaped stopcock. Following perfusion with contrast medium, the LNs
were dissected and kept at 4 ℃ for > 2 h to allow fixation to occur. The samples were placed on the stage of a
micro-CT scanner (the gutta percha was used as a landmark for positioning) and scanned (angiography) at
resolutions of 5-30 μm and a slice thickness of 100 μm. Acquired slice data were rendered as 3D images using
a 3D analysis suite (Amira, Maxnet).

Cell culture
KM-Luc/GFP cells (mouse malignant fibrous histiocytoma-like cells derived from an MRL/Mp-gld/gld
mouse expressing a fusion of the luciferase and enhanced-green fluorescent protein genes) were cultured
[14]
as previously described . FM3A-Luc cells (C3H/He mouse mammary carcinoma cells expressing the
[10]
luciferase gene) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum,
k
1% L-glutamine-penicillin-streptomycin and 1 mg/mL G418 (Sigma-Aldrich). Both cell types had an H-2
haplotype, which is the same as that of MXH10/Mo/lpr mice, and expressed vascular endothelial growth
factor (VEGF)-A and VEGF-B but not VEGF-C; KM-Luc/GFP but not FM3A-Luc cells showed slight VEGF-
[15]
D expression . The relative growth rates of KM-Luc/GFP and FM3A-Luc cells were 3.8/day and 1.1/day, re-
[8]
spectively . Cell lines were incubated (37 ℃, 5% CO /95% O ) until 80% confluence was achieved. Lack of
2
2
Mycoplasma contamination was confirmed on the inoculation day (MycoAlert Mycoplasma Detection Kit;
Lonza Rockland).
NBD-liposomes
NBD-liposomes were synthesized from 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC; MC8080,
NOF Co.), 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-methoxy- polyethyleneglycol (DSPE-
PEG[2000-OMe]; DSPE-020CN, NOF Co.), and 1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine-N-
(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DPPE; FE6060, NOF Co.) [9,16] . The size and zeta potential of the
NBD-liposomes was 642 nm and -1.5 mV, respectively, as measured using a particle size and zeta potential
analyzer (ELSZ-2; Otsuka Electronics).

Visualization of venous flows internal and external to a LN
Under deep general anesthesia, an arc-shaped incision was made in the abdominal skin of a mouse (n = 1, 12 weeks
old) from the subiliac region to the proper axillary region, and 100 µL of 0.01 µmol/L NBD-liposomes was
injected into the tail vein (100 µL/min, 60 s). Fluid flow in the veins was visualized using a fluorescence
stereomicroscope (M165-FC; fluorescence filter: GFP2; excitation: 460-500 nm; emission: > 510 nm; Leica)
[8]
connected to a high-speed camera (10 frames/s; CoolSNAP HQ2; Photometrics) .

Induction of metastasis to the PALN by injection of tumor cells into the SiLN
6
5
KM-Luc/GFP (1.5 × 10 cells/mL) or FM3A-Luc (3.3 × 10 cells/mL) cells (passaged three times) were
suspended in a mixture of 20 µL phosphate-buffered saline (PBS) and 40 µL of 400 mg/mL Matrigel
(Collaborative Biomedical Products). The prepared cells were injected into the center of the SiLN of a
mouse (aged 14-16 weeks) using a 27G needle, which was maintained in the same position for 5 min to
allow solidification of the Matrigel. Tumor development in the SiLN and metastasis to the PALN (the rates
of which depended on the tumor cell type) were detected using an in vivo bioluminescence imaging system
(IVIS; Xenogen) at 4 and 7 days post-inoculation of KM-Luc/GFP cells and at 6, 13, 20 and 27 days post-
inoculation of FM3A-Luc cells. Injection of tumor cells into the SiLN induces metastasis to the PALN via
lymphatic vessels as well as hematogenous metastasis via the thoracoepigastric vein (TEV) [9,17] ; the cell
[14]
number is highest in the SiLN and second highest in the PALN .