Clinical samples
60 cases of nasopharyngeal carcinoma tissue samples were collected from the Southern Hospital of Southern Medical University. The research subjects selected nasopharyngeal carcinoma patients with pathologically confirmed nasopharyngeal carcinoma from 2007 to 2019. Detailed pathological, clinical data and survival time of all NPC patients were collected through outpatient and telephone follow-up. The TNM grading is based on the definition of the UICC American Joint Committee on Cancer Staging Criteria, 7th edition.
Immunohistochemical And Cd31-pas Dual Staining
The tissue was fixed with formalin, embedded in paraffin, and sliced at a thickness of 4 mm. After collection, the tissue was fixed with 4% paraformaldehyde at 4°C overnight. Antigen blocking was performed using 10% goat serum (AR0009, Boster, China). Anti-e-cadherin ((24E10) Rabbit mAb #3195, CST), anti-cd31 ((PECAM-1) (D8V9E) XP Rabbit mAb #77699, CST), anti-Ve-cadherin ((D87F2) XP Rabbit mAb #2500, CST) and antibodies against vimentin ((D21H3) XP Rabbit mAb #5741, CST) were incubated overnight at 4°C. DAB system (ZLI-9017, Zsbio, China) was used to detect staining. Vasculogenic mimicry structures were detected using PAS staining kit (G1281, Solarbio, China) and anti-cd31 ((PECAM-1) (D8V9E) XP Rabbit mAb #77699, CST). The number of positive cells was obtained from 5 randomly selected fields and 400x magnification.
Immunohistochemical Score
Immunohistochemical scoring criteria: comprehensive score = staining intensity × positive area. Staining intensity score: strong positive (3 points), positive (2 points), weak positive (1 point), negative (0 points). The proportion of positive (including strong positive) regions: 100%-76% (4 points), 75%-51% (3 points), 50%-26% (2 points), 0–25% (1 point). The above scores were averaged by two pathologists independently.
Cell Culture
All nasopharyngeal carcinoma cells were obtained from the Cancer Research Center of Southern Medical University, and were cultured in RPMI-1640 medium (Thermo Fisher Scientific Corporation PM15101) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Corporation 10270- 106), 100u/ml penicillin (15140-122, Thermo Fisher Scientific, USA), 100 mg/ml streptomycin (15140-122, Thermo Fisher Scientific, USA), and humidified in 5% CO2 The environment was maintained at 37°C.
Three-dimensional Culture
24-well plates coated with 100µL Matrigel (354230, BD Biosciences, USA) reduced growth factor for each well, incubated at 37℃ for 1 h, Take 500µL medium containing 10% FBS (1*105 cells), spread it on the gel surface, and incubate at 37 ℃ for 24 h. Each group provides three holes. The cells were then photographed under an inverted microscope (IX71, OLYMPUS, Japan). ImageJ calculates the average number of tubular structures.
Rna Isolation, Reverse Transcription, And Quantitative Realtime Pcr
Total RNA was extracted from samples using RNA iso Plus (R401-01, Vazyme, China) and reverse transcribed using HiScipt III RT SuperMix for Quantitative Real-time PCR (+ gDNA wiper) (R323-01, Vazyme, China) as cDNA. Quantitative reverse transcription PCR (qRT-PCR) was performed on ABI QuantStudio5 system using ChamQ SYBR qRT-PCR Master Mix (Low ROX master mix) (Q331-02, Vazyme, China). GAPDH served as an mRNA endogenous control. All samples were normalized to an internal control and relative expression levels were calculated by using relative quantification.
Western Blot
Western blot
The proteins extracted from samples were assayed using lysis buffer (P0013B, Beyotime, China) containing protease inhibitor cocktail (HY-K0010, MCE, USA) using radioimmunoprecipitation. Proteins were solubilized in SDS loading buffer (FD006, Fdbio, China), and the lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, USA). Anti-E-Cadherin (24E10, CST, USA), Vimentin (D21H3, CST, USA), VE-Cadherin (D87F2, CST, USA), N-Cadherin (D4R1H, CST, USA), β-Catenin (D10A8, CST, USA) or GAPDH (D16H11, CST, USA) polyclonal antibodies were incubated at 4°C overnight at a dilution of 1:1000, and then incubated with species-specific enzyme-labeled secondary antibodies (1:5000 dilution) for 2 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (WBKLS0100, Millipore, USA).
Statistical Analyses
Statistical analysis was performed using SPSS 25.0 software. All data are from at least three independent experiments. Unless otherwise stated, data are presented as SEM means. A p-value < 0.05 was considered statistically significant.