2.1 Cell lines and cell culture
Cell lines, including HEK293T, A375, A875 and B16F10, were purchased from Cell Resource Center (IBMS, CAMS/PUMC, China). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, # SH30081.LS) supplemented with 10% FBS (ExCell, #FSP500) and Pen/Strep (100 mg/ml) (Gibco, #15140122) at 37°C in 5% CO2 incubator.
For transfection and creation of stable cell lines, the pBabe-puro empty vector and YKL-40 containing vector plasmids were extracted according to the instructions of the plasmid extraction kit (TIANGEN, #DP117). HEK293T cells were transfected with pBabe and packaging vector pCL-Ampho using the liposome transfection method (Lipofectamine 2000, Invitrogen, #11668019). Viral supernatant was collected after 72 hours, centrifuged, and added to the target cells at a 3:4 ratio with DMEM complete medium. After 24 hours, the medium was replaced with medium containing puromycin 0.5 µg/ml (MedChemExpress, # HY-K1057) for positive clone screening.
2.2 Cell lysate and immunoblotting
Cellular protein lysates were extracted using RIPA buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor cocktail). The total protein concentration was determined by a BCA protein assay kit (Thermo Fisher, #23225). Cell lysates were mixed with 5× Laemmli sample buffer, boiled for 4 minutes at 95°C, and then stored at -20°C. Equal amounts of total protein were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween (TBST) for 1 h at room temperature and incubated with the appropriate primary and secondary antibodies. Labeled proteins were visualized using ECL Western blotting detection reagents. Antibodies used for immunoblotting were YKL-40 (Cell Signaling, #47066), β-Actin (Cell Signaling, #4967S), and anti-rabbit HRP-conjugated secondary antibody (Cell Signaling, #7074S).
2.3 Cell viability assay
Cell viability was measured using an MTS assay kit (Promega, Madison, WI). For viability assays, cells (1×104) were plated in 96-well plates and allowed to adhere overnight. After 24 h of seeding, cells were treatmented with Doxorubicin (MedChemExpress, HY-15142A), Cisplatin (MeilunBio, #MB1055) and Temozolomide (MedChemExpress, HY-17364) for 72 hours, following treatment the medium was removed from the wells. PMS, MTS and DMEM colorless medium were mixed at a ratio of 1:20:100 to prepare MTS working solution. Subsequently, 100 µL of MTS working solution was added to each well, incubated for 10 min at 37°C, mixed thoroughly, and the absorbance at 490 nm was measured using a plate reader.
2.4 Colony formation assays
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 lg/ml) in a 5% CO2 incubator at 37°C. The medium was replaced every 3 days. After 14 days, colonies were stained with Crystalline Violet and counted.
2.5 Cell migration assays
For the wound healing assay, cells were seeded in 6-well plates and cultured at 37°C overnight until a confluent monolayer was formed. Subsequently, a wound was introduced by manually scratching the monolayer using a sterile 200 µl pipette tip. The wounded monolayer was then washed three times with PBS to remove cell debris and incubated with fresh medium containing 1% serum. Images of the scratched area were captured at 0 h, 20 h and 24 h post-scratching. The wound closure efficacy was quantified by calculating the percentage of area healed by the cells relative to the initial wound area.
For the invasion assay, a total of 1×106 cells in 300 µl of 1% serum medium were added to the Matrigel-coated upper chamber, and 700 µl of 10% serum medium was added to the lower chamber. After incubation in a humidified tissue culture incubator for 36 h, the noninvasive cells on the upper surface of the membrane were gently removed with a cotton swab. The invading cells that had traversed to the lower surface of the membrane were fixed in paraformaldehyde for 10 min, treated with 0.2% Triton for 10 min to increase cell membrane permeability, stained with DAPI for 10 min, and rinsed thrice with PBS. Cell counting was carried out by photographing the membrane with a fluorescence microscope. Five random fields of view were captured under the fluorescence microscope to quantify the invading cells.
2.6 Mouse tumor models
Male C57BL/6 mice (6 weeks old) were subcutaneously implanted with cells (5×105 cells in 200 µL saline) in the lateral aspect of the left hind leg. Tumor volumes were assessed every alternate day commencing on day 13 post-inoculation, and mice were dissected and sacrificed on day 19. After dissecting the mice, the tumors were weighed and measured for volume, half of the tumors were fixed in tissue fixative, while the other half were frozen for transcriptome sequencing analysis. The tumor volume was calculated by the following formula: tumor volume (mm3) = 0.5× (width [mm]) 2× (length [mm]).
For experimental lung metastasis analysis, female C57BL/6J mice (6 weeks) were intravenously injected with 4×105 cells in 500 µL saline cells into the lateral tail vein. After 15 days post-inoculation, the mice were euthanized and subjected to dissection, during which all organs were visually inspected for the existence of macroscopic melanoma metastases. Metastatic nodules were counted under a dissecting microscope.
2.7 Hematoxylin-eosin stain assay
The tumor tissue and lung tissue were removed and fixed in 4% paraformaldehyde solution at room temperature for 48 h. Subsequently, the tissues underwent dehydration, paraffin embedding, and sectioning. The sections were stained with hematoxylin-eosin dye and subsequently scanned using a pathology section scanner. Nodules present on organs displaying evident melanoma metastases were quantified under a dissecting microscope.
2.8 Quantitative RNA sequencing and data analysis
The tumor tissue dissected form mice model were lysed in 1 ml of TRIZOL™ Reagent (Invitrogen, #15596026). Total RNA was extracted and analyzed by Agilent 2100 Bioanalyzer. Following RNA qualification, the samples were subjected to quantitative RNA sequencing by Genesky Bio-Tech (Genesky Biotechnologies, Shanghai). Differential expression genes (DEGs) were identified with a threshold set at a log2 value greater than 1 and a p-value less than 0.05. For functional analysis, a gene set enrichment analysis (GSEA) (Broad Institute) tool kit was used to obtain the enrichment score for defined gene sets, such as hallmark gene sets, GO gene sets and KEGG pathway gene sets. The data discussed in this study have been deposited in NCBI's Gene Expression Omnibus and can be accessed via the GEO Series accession number GSE217590.
2.9. Statistical analysis
Data are represented as the mean ± SD in the in vitro experiments and represented as the mean ± SEM in the in vivo experiments. Differences in the results of the two groups were evaluated using either two-tailed Student’s t test or one-way ANOVA followed by post hoc Dunnett’s test. Statistical significance was defined as a level of P < 0.05.