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Tran et al. J Cancer Metastasis Treat 2020;6:47 I http://dx.doi.org/10.20517/2394-4722.2020.104 Page 3 of 11

METHODS
Gene-set enrichment analysis
Differentially expressed genes were selected from the microarray data of LM8 sublines and subjected
to gene set enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes. The analysis was
performed with the clusterProfile package using R/Bioconductor. The reference gene set used was the Wnt
signaling pathway (mm0431) set.

Cell culture
The LM8 cell subline was provided by Dr. Hideki Yoshikawa (Osaka University). LM8 cell line with high
(LM8-H) and low (LM8-L) was established from LM8 in a previous study through the in vivo image-
[16]
guided screening system . Murine vascular endothelium cells, bEnd3, were obtained from the American
Type Culture Collection. All cells used in this study were cultured in 5% fetal bovine serum (FBS) Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL).

Reverse transcription PCR and quantitative PCR
Total RNA was extracted using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the
manufacturer’s protocol. One microgram of total RNA was reverse-transcribed using the Oligo(dT)20
primer (Toyobo, Osaka, Japan) and ReverTra Ace (Toyobo). Quantitative PCR (qPCR) and reverse
transcription PCR (RT-PCR) were carried out using the Thunderbird® SYBR qPCR Mix (Toyobo) and
EmeraldAmp® GT PCR Master Mix (Takara Bio, Shiga Japan), respectively.

Western blotting
Cells were lysed in RIPA buffer (50 mmol/L Tris HCl, pH 8.0; 150 mmol/L NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS) containing a protease cocktail inhibitor (Nacalai Tesque, Kyoto, Japan), and
protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific,
Waltham, MA). Proteins were separated by electrophoresis on a 10% acrylamide gel, transferred to a
hydrophilic polyvinylidene fluoride membrane (Merck, Kenilworth, NJ) and blocked with 5% skim milk
or 5% bovine serum albumin in TBST (20 mmol/L Tris, pH 7.5; 150 mmol/L NaCl, 0.1% Tween 20). The
membrane was then probed with relevant primary antibodies [anti-AKT (#2920S, 1:5000), anti-phospho-
AKT (Ser473; #4060s, 1:5000), anti-ROR2 (#88639s, 1:5000), and anti-GAPDH (#2118s; Cell Signaling
Technology, 1:5000)], and secondary antibodies [anti-mouse IgG HRP-linked antibody (#7076, 1:5000) and
anti-rabbit IgG HRP-linked antibody (#7074, 1:5000, Cell Signaling Technology)]. The resultant membranes
were washed with TBST and detected by ImageQuant LAS 4000 (GE Healthcare Life Science, Marlborough,
MA) after processing with Chemi-Lumi One (Nacalai Tesque). To investigate the effect of AKT inhibition,
cells were treated with 50 nmol/L MK2206 in 5% FBS DMEM for 24 h.

Gene knockout using the CRISPR-Cas9 system
Ror2 gene knockout (KO) was performed in LM8-H cells to establish the cell line H/Ror2-KO by using the
CRISPR-Cas9 system. The sequence of the guide RNA (5’-caccgTCGTGGCTCTTGCACAACCG-3’) was
used for targeting Ror2. The Ror2 guide RNA was inserted into a unique BbsI site of the pX330 plasmid
(42230; Addgene). The cells whose genomes were correctly edited by the CRISPR-Cas9 system were
[17]
selected by using a fluorescence indicator system with the pCAG/EGxxFP plasmid , provided by Dr.
Masahito Ikawa (Osaka University). GFP-positive cells were picked up, and ROR2 protein expression level
was validated by western blotting.

Establishment of cell lines with ROR2-overexpression
The cDNA of Ror2 (NM_013846.4) was amplified using the KOD® FX Kit (Toyobo) using the following
primer set: 5’-TGGAATTCTGCAGATATGGCTCGGGGCTGGGTG-3’ and 5’-GCCACTGTGCTGGA
TTCAGGCTTCAAGCTGGACATG-3’. The Ror2 cDNA fragment was cloned into the pcDNA3.1-myc-