Clinical sample and IHC. Our study collected 25 paraffin-embedded, archived UM tumor samples from Xuanwu Hospital between 2010 and 2016, with the approval of the Institutional Review Committees of Xuanwu Hospital. Informed consent was obtained from all patients. All specimens were histologically confirmed as UM, without previous chemotherapy or radiotherapy and prior history of other malignancies. For IHC, the samples were deparaffinized with dimethylbenzene, rehydrated with gradient ethanol and then treated with 3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. Microwave treatment was used to retrieve the antibody-binding epitopes of antigens, and the specimens were subsequently incubated with 10% normal serum to reduce nonspecific binding. The samples were incubated with the primary antibodies, such as rabbit anti-PTK2 (1:100, ab40794, Abcam, Cambridge, MA, USA) and rabbit anti-phospho-PTK2 (Tyr397) (1:50, 44-624G, Invitrogen, Carlsbad, CA, USA), for 1h at room temperature. Then biotinylated anti-rabbit secondary antibody was added. 3,3 '- diaminobenzidine was used as chromogenic agent and hematoxylin was used for counterstaining. Finally, the score was calculated by multiplying the intensity of the staining (low, 1+; medium, 2+; strong, 3+) by the percentage of stained cells (0–100%).
Cell lines and culture. Human Uveal melanoma cell lines (OCM-1 and MUM-2B) were purchased from American Type Culture Collection (ATCC), and mycoplasma contamination was excluded. Routinely, UM cells were cultured in DMEM medium (Invitrogen) or RPMI 1640 medium (Invitrogen) supplemented with 1% penicillin/streptomycin (Invitrogen) and 10% FBS (HyClone, Logan, UT, USA).
Lentiviruses and reagents. Lentiviruses carried PTK2 shRNA vector were purchased form Genecreate (Wuhan, China). Cell lines with PTK2 stable knock-down were selected in 1µg ml− 1 puromycin for ⁓2 months. And pooled clones were screened using standard immunoblot protocols.
Gene set enrichment analysis. GSEA was performed using GSEA 2.2.3 software. PTK2 expression was regarded as a numerical variable. We applied the continuous CLS file of PTK2 spectrum to the phenotypic tag in GSEA. ‘Pearson’ was used for ranking genes and the other parameters were set to their default values.
Western blot. Western blot analysis was conducted according to the standard procedure. Both anti-PTK2 (Abcam) and anti-phospho-PTK2 (Tyr397) (Invitrogen) was used at dilutions of 1:500. Anti-GAPDH (1:2,000, Sigma) was used as a loading control.
RNA extraction and RT-PCR. According to the manufacturer’s protocol, total mRNA was extracted with RNA Extraction Kit (Takara Bio Inc., Shiga, Japan). For mRNAs detection, RNA was firstly reversely transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara Bio Inc.), and the real-time PCR was then performed with SYBR Premix Ex Taq™ (Takara Bio Inc.). Primers were listed in Supplementary Table 1.
Cell migration and invasion assays. Cell migration ability was determined using wound healing assays. When UM cells grown in 6-well plates attached as confluent monolayers, a 1-ml pipette tip was used to create the wound. Then, cells were washed with PBS and cultured in the medium with 1% FBS for 24 hours to allow the wound healing. For transwell invasion assay, chambers (Corning Inc.) were coated with Matrigel (BD Biosciences) on the upper surface. Equal cells were seeded into the upper chambers and cultured with medium containing 0.1% FBS, while cell medium containing 20% FBS was placed in the lower well as a chemoattractant. After 24 hours, cells that successfully invaded through the Matrigel were fixed with 4% paraformaldehyde and then stained with crystal violet before taking photographs.
Tumor xenografts in vivo. Animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at Xuanwu Hospital. BALB/c nu/nu mice (6-week-old, male) were purchased and randomly divided into different groups (5 mice per group). 1 × 106 MUM-2B cells with PTK2 knockdown were injected intravenously through the lateral tail vein of NOD-SCID mice (6-week-old, male, 6 mice per group) to establish the metastasis model. And all mice were maintained for 50 days until the analyses by the micro-PET.
PET imaging of glucose uptake in mice. In our study, the PET imaging of mice was conducted using an animal PET scanner (Philips Corp.) according to indications. Briefly, mice were anesthetized with pentobarbital followed by the intravenous injection with 3.7 MBq (100 µCi) of 18F radio-labeled fluorodeoxyglucose (18FDG). To obtain attenuation correction data, a 5-minute emission scan was performed in the prone position at 60 minutes after injection, and a 10-minute delay scan was then acquired at 2 hours after injection. The tumor radioactivity of each mouse was calibrated against a known aliquot of the injected tracer and presented as the percentage of the injected dose of tissue.
Statistical Analysis. All statistical analyses were performed using SPSS 22.0. All data were representative of at least three independent experiments and illustrated as the means ± standard deviation (SD). The Student’s t test was used for two-group comparisons. Survival curves according to PTK2 expression were estimated with the Kaplan-Meier method, and the log-rank test was used to assess significance. The univariate and multivariate analyses of risk factors were performed with Cox Regression analysis or Logistic Regression analysis. All statistical tests were two-sided. In all assays, a P value < 0.05 was considered statistically significant.