Cell lines and cell culture
The cell lines we used in this study were derived from a mouse mammary tumor, and highly metastatic (66HM and 66Lu10) and low metastatic (66LM, 66 − 4, and 66Lu1) sublines were selected [2–4]. These cell lines were cultured in 5% CO2 at 37°C and grown in incomplete medium, which was composed of Dulbecco's Modified Eagle Medium (DMEM), low glucose (WAKO) and 10% fetal bovine serum (FBS; GIBCO).
Plasmids and stable transfection
The full-length murine Emid1 coding region was amplified from cDNA of 66HM cells using the forward primer 5′-ATGGGCGGCCCGCGGGCCTG-3′ and reverse primer 5′-ATGCACCACCACCATCACCATAGCTCCTCTCGCTGCGTCTCC-3′ and fused with a sequence encoding the His-tag. The construct pCIneo − Emid1 was made by subcloning this cDNA into the pCIneo expression vector (Promega). 66 − 4 cells were transfected with the pCIneo − Emid1 vector using Lipofectamine 3000 (Thermo Fisher Scientific). Stable transfectants were selected with 0.5 mg/ml G418 for 3 weeks, and strong expressers were cloned using the limiting dilution method. As a negative control, target cells were transfected with a pCIneo empty vector.
Small interfering RNA (siRNA) transfection
Three different siRNAs against mouse Emid1 and a negative control (Ambion) were used for transient gene knockdown in vitro. The sequences of the siRNAs are shown in Supplementary Table 4. These siRNAs were diluted in 0.1 ml Opti-MEM I medium to a final concentration of 10 nM in 24-well plates, and 1 µl lipofectamine RNAi MAX reagent (Thermo Fisher Scientific) was added to each well. After incubation for 5 min, the mixture was added to each well containing 66HM cells at 60% confluence in 0.5 ml DMEM with 10% FBS. The gene knockdown efficiency of siRNA was analyzed with quantitative real-time polymerase chain reaction (qRT-PCR).
XTT assay
The effect of EMID1 overexpression or knockdown on in vitro cell growth was examined using an XTT assay kit (Merck). XTT (50 µl per well in a 96-well plate) labeling mixture containing 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetorazolium-5-calboxanilide and phenazine methosulphate was added to 66 − 4 clones stably expressing EMID1 and 66HM cells at 48 hours after siRNA transfection. After incubation for 24 hours, absorbance at 490 nm was determined using a microtiter plate reader.
Cell cycle analysis
The cell cycle profile was determined with flow cytometry based on the cellular DNA content using the Cell Cycle Phase Determination Kit II (Cayman Chemicals). The siRNA-transfected cells were cultured for 24 hours, and 5 × 105 cells were stained with 0.1% propidium iodide (Sigma Aldrich) for 30 min at room temperature. Then, they were analyzed with a flow cytometer (Canto II; BD Biosciences) according to the manufacturer's instructions.
Wound healing assay
Cells were seeded in a 24-well plate and incubated until 90% confluent. Cell monolayers were then scratched with a p200 pipette tip. Cells migrating into the scratched region at the same points on the culture dish were visualized using microscopy at 0 and 24 hours. The extent of cell migration was evaluated as the speed of wound closure at 24 hours.
In vitro invasion assay
Invasion assay was performed in a biocoat Matrigel chamber (8-µm pore; BE Biosciences) in a 24-well tissue culture plate. The upper chamber was filled with 2 × 105 cells in culture medium with 10% FBS. The lower chamber was filled with 750 µl culture medium containing 10% FBS. After incubation at 37°C for 8 and 12 hours for 66 − 4 and 66HM transfectants, respectively, the membranes were removed, stained with hematoxylin, and mounted on slides, and the cells on the lower side of the membrane were counted in three randomly chosen fields of view.
Comprehensive gene expression analysis associated with EMID1 expression
Gene expression profiles associated with EMID1 were comprehensively analyzed using the microarray method. Total RNA was extracted from cultured cells using TRIZOL RNA Isolation Reagents (Thermo Fisher Scientific). Gene expression profiling was using 3D-Gene messenger RNA chip (Toray Industries) and an additional gene ontology was analyzed.
Antibodies against mouse and human EMID1 proteins
We used three specific antibodies against mouse and human EMID1/EMU1 (IBL). The antibodies EMU1-179 and 185 were generated using 20 amino acid peptides in the middle region of mouse and human EMID1, respectively, whereas antibody EMU1-413 was raised against a 20-amino acid sequence common to the C-terminal regions of mouse and human EMID1 proteins. EMU1-185 can be used for immunohistochemistry on formalin-fixed paraffin-embedded specimens of human tissues, whereas EMU1-179 and 413 can be used for immunohistochemistry only on paraformaldehyde-fixed frozen sections. The specificity of all antibodies against EMID1 was determined by absorption tests using each EMID1 recombinant peptide; the signal of each protein was diminished in immunohistochemistry.
The analysis of human tissues was approved by the Human Research Ethical Committee of Fukushima Medical University (registration number 1203).
All procedures conformed to the principles outlined in the Helsinki Declaration.
Immunofluorescence and western blot analyses
Cultured cells transfected with Emid1-His were plated on eight-well chamber slides for 24 to 48 hours. Cells were fixed with 4% paraformaldehyde and permeabilized for 10 min at room temperature with 0.1% Triton-X in phosphate buffer saline (PBS). Cells were stained with Anti-His-tag mAb was conjugated with Alexa Fluor 488 (clone OGHis, 1/1000, MBL) for 30 min. Phalloidin-iFluor 594 reagent (Ab176757, 1/1000, Abcam) was used for filamentous actin (F-actin) staining. The slides were mounted in mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Southern biotech) and observed with fluorescence microscopy (Olympus).
Western blotting was performed using cellular protein extracted with cell lysis reagent, deposited protein on the dish collected with a cell scraper, and secreted protein in serum-free medium concentrated with an iCON Concentrator 20K Pierce. Proteins (10 µg) were electrophoresed with standard sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in non-reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). After blocking with 5% skimmed milk for 1 hour, the membrane was incubated with polyclonal rabbit antibody EMU1-179, and then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich). The signals were visualized with enhanced chemiluminescence (ECL Advance Cytiva).
In vivo metastasis assay
Cultured tumor cells (1 × 107) suspended in 200 µl of PBS were inoculated into the mammary fat pad of 8-week-old female C3H/He mice. At 8 weeks after inoculation, animals were sacrificed, and tumor tissues and major organs were excised for counting metastatic colonies macroscopically and microscopically.
All animal studies were carried out in accordance with ARRIVE guidelines and approved by the Animal Care and Use Committee of Shizuoka Cancer Center (approval number 30 − 5).
Cell adhesion assay
At 12 hours after seeding 1 × 105 tumor cells in a 96-well plate, the cells in a confluent culture were completely removed with 0.2% Triton X-100 in PBS for 15 min at room temperature, and the well was rinsed three times with PBS. Then, 5 × 103 66 − 4 parent cells were added to the plate and incubated in culture medium with 10% FBS for 6 hours at 37°C. After gently rinsing the well with PBS to remove floating cells, the remaining cells were stained with crystal violet. Absorbance at 570 nm was determined using a microtiter plate reader after elution with dimethyl sulfoxide (DMSO).
Immunohistochemical analysis
Fresh frozen mouse sections fixed with paraformaldehyde and formalin-fixed paraffin-embedded human sections were used. Immunostaining was performed using an indirect streptavidin–biotin immunoperoxidase method (SAB-PO (M) kit; Nichirei Corp.). Formalin-fixed paraffin-embedded sections were pretreated with proteinase K (0.4 mg/ml) for 5 min for antigen retrieval. After blocking endogenous peroxidase activity with a 3% H2O2-methanol solution, the slides were incubated with primary antibodies (1/100) overnight at 4°C, washed with PBS, and then incubated with secondary antibodies for 30 min at room temperature. Antibody localization was visualized by incubating with a secondary antibody conjugated to horseradish peroxidase for 30 min at room temperature, followed by diaminobenzidine reaction. The slides were counterstained with hematoxylin.
Human tissue samples
An immunohistochemical study was performed using formalin-fixed, paraffin-embedded tissue specimens obtained from Shizuoka Cancer Center Hospital. Primary tumor specimens originated from the breast, lung, stomach, colon, liver, and kidney, and the corresponding normal tissues. Ten cases from each cancer type were selected.
All procedures were followed in accordance with the ethical standards of the Institutional Review Board of Shizuoka Cancer Center (approval number: J2020-54-2020-1).
Informed consent from enrolled patients was waived by the requirement of the approving authority.
Statistical analysis
Unless otherwise specified, data represent the mean ± standard deviation (SD) and are representative of three independent experiments. To test for significant differences between two groups, unpaired Student’s t tests were used. Two-sided p values < 0.05 were considered significant.