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Page 4 of 11 Tran et al. J Cancer Metastasis Treat 2020;6:47 I http://dx.doi.org/10.20517/2394-4722.2020.104

His vector (Invitrogen, Carlsbad, CA) at the EcoRV site using the Infusion Cloning Enhancer kit (Takara
Bio). LM8-L and H/Ror2-KO cells were transfected with the plasmid by the NEPA21 type II electroporator
(NEPAGENE, Chiba, Japan), and appropriate transfectants were cloned to establish L/ROR2 and KO/ROR2
after culturing with G418 (2 mg/mL) selection medium.

Proliferation assay
Cell proliferation was evaluated with the water-soluble tetrazolium salt 1 (WST-1) reagent (Sigma Aldrich,
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St. Louis, MO) according to the manufacturer’s instructions. Cells (1 × 10 cells/100 µL culture medium)
were seeded in 96-well plates. After culturing for 24, 48, or 72 h, the medium was removed and 100 µL of
WST-1-containing medium (10-fold dilution) was added to each well. The cells were further incubated for
3 h, and then the absorbance of each well was measured at 450 nm with a reference wavelength of 750 nm
after shaking the plates for 1 min with the microplate reader Model 680XR (Bio-Rad, Hercules CA).

Mice
Male C3H mice were obtained from the Charles River Laboratory (Yokohama, Japan). All mice used were
6-8 weeks of age and were housed in the animal facilities at the Tokyo Institute of Technology. Animal
experiments were performed with the approval of the Animal Ethics Committees of the Tokyo Institute of
Technology (no. D20170004-2) and in accordance with the Ethical Guidelines for Animal Experimentation
of the Tokyo Institute of Technology.

Analysis of OS lung metastasis model
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The mice were injected intravenously with 1 × 10 LM8 cells in 100 μL of PBS. Twenty days after
inoculation, isolated lungs were embedded in an optimal cutting temperature compound (Sakura
Finetechnical Co., Ltd, Tokyo, Japan) and stored at -80 °C overnight. Lungs were divided into cryosections
of 10 µm in thickness and then fixed in 4% paraformaldehyde. Fixed lung cryosections were then stained
with hematoxylin and eosin (HE) and observed under a microscope BZ-X710 (Keyence, Osaka, Japan).
A whole lung image was obtained by stitching together partial lung images using BZ-X analyzer software
(Keyence).

Analysis of tumor cells in lungs
Cells were labeled with 100 μmol/L CellTracker® Green (Thermo Fisher Scientific) and intravenously
injected into C3H mice (1 × 10 cells/100 μL PBS). DyLight® 594-labeled isolectin B4 (6 mg/kg; Vector
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Laboratories, Burlingame, CA) was injected intravenously to stain endothelial cells 5 min before dissecting
the mice. The lungs were collected and observed under confocal microscopy (Carl Zeiss, Oberkochen,
Germany). The number of fluorescently labeled cells in the three microscope fields of each lung was
[18]
quantified using ImageJ software (http://rsb.info.nih.gov/ij/) . Results are shown as the average number of
cells per field. Each group was analyzed in triplicates. For measuring the fluorescence intensity of the lungs,
lung lysate was prepared with RIPA buffer and well-homogenized. The supernatant was then collected, and
the fluorescence intensity was measured using Infinite F500 (Tecan, Männedorf, Switzerland) with a filter
set for CellTracker® Green (Ex/Em = 480 nm/517 nm).

Anoikis assay
Poly(2-hydroxyethyl methacrylate) (poly-HEMA; Thermo Fisher Scientific) was completely dissolved
in 95% ethanol (20 mg/mL). Plates were coated with poly-HEMA solution and dried on a clean bench
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overnight. Cells (5 × 10 ) in serum-free medium were seeded in a poly-HEMA-coated 24-well plate. After
24 h, calcein AM (Thermo Fisher Scientific) and ethidium homodimer-1 (Thermo Fisher Scientific) were
added at final concentrations of 2 µmol/L and 4 µmol/L, respectively, and the cells were further incubated
for 30 min. The fluorescent signal was observed under fluorescence microscopy and quantified with the
BZ-X Analyzer (Keyence). To investigate the effect of AKT on LM8 anoikis, the AKT inhibitor MK2206