All procedures were ethically approved by the institutional Ethics Review Committee of Fudan University Shanghai Cancer center (FUSCC). Appropriate written informed consent was obtained from all patients prior to sample collection.
A retrospective cohort study was conducted in the Department of Gynecology Oncology, FUSCC, which included 180 patients with the 2009 FIGO stage IB1-IIA2 who underwent radical abdominal hysterectomy with or without bilateral salpingooophorectomy and pelvic ± para-aortic lymphadenectomy from 2009 to 2012. All the enrolled patients had undergone standard pelvic lymphadenectomy by experienced gynecological oncologist. All the microscopic slides including gene SPOP staining were reviewed and graded by the same professional gynecologic pathologist and were reconfirmed by another experienced gynecologic pathologist. All clinical records were retrospectively studied.
Proteomic Sequencing and Data Process
Proteomic sequencing was performed in 5 lymph node positive cervical cancer tissues and 5 lymph node negative cervical cancer tissues, and subsequent analysis was performed.
Immunohistochemical Staining (IHC)
IHC of SPOP was performed on the paraffin-embedded sections of 180 CC tissues. Slice thickness was set at 5 mm and 3 sections were selected from each specimen. Slides were rinsed and incubated with primary antibodies SPOP (1:100; Cell Signaling Technology). Subsequent antibody detection was carried out with a secondary antibody: Cy3-conjugated goat anti-rabbit (1:300; Wuhan Goodbio Technology CO., LTD.). The expression level of SPOP was determined by immunoreactive score (IRS) [32–34].
Cell Culture and Reagents
The human cervical cancer cell lines HeLa, SiHa, ME-180, MS751 were acquired from the American Type Culture Collection (ATCC), which were authenticated by short tandem repeat profiling. These cell lines were cultured at 5% CO2 and 37℃ in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA), containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA).
The SPOP overexpression plasmid was constructed by cloning the cDNA into the PGMLV-CMV-EF1-ZsGreen1-T2A-Puro vector (System Biosciences, CA, USA). Plasmids carrying shRNAs targeting SPOP were generated using the U6-MCS-CMV-ZsGreen1-PGK-Puro vector (System Biosciences, CA, USA). The siRNAs and matched empty vector controls were obtained from Lncbio (Shanghai, China). The SPOP shRNAs target sequence is as follows: shRNA1: GTAGCACCAACTCTCAGCTA, shRNA2: CCTCCGGCAGAAATGTCGAG, shRNA3: TGACTTCACCCATTTCCTCC.
RNA extraction and qRT-PCR
Total RNA was extracted from samples and cells using TRIzol reagent (Life Technologies, CA, USA), according to the manufacturer’s protocol. qRT-PCR was conducted using TB Green PCR Master Mix (TaKaRa, Dalian, China) inanABI7900HT Real-Time PCR system (Applied Biosystems, USA). The relative quantification was normalized to b-actin with the 2CT formula. The primers used for qRT-PCR are listed as follows: β-actin-F: AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG; β-actin-F: CTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCC; SPOP-F: GCCCCGTAGCTGAGAGTTG; SPOP-R: ACTCGCAAACACCATTTCAGT.
Western Blot Analysis
Western blotting was performed as our previously described .
Cells were seeded and cultured into 96-well plates overnight. At 1, 2, 3, 4, 5 day, 10ul cell counting kit-8 solution (MedChem Express, Monmouth Junction, NJ, USA) was added into each well, followed by further incubation at 37°C. After 1 h, the absorbance was measured at 450 nm wavelength to assess cell proliferation ability .
Colony Formation Assay
Cells were seeded and cultured in 6-well plates at a density of 1*103 cells/well. During this period, the medium should be changed as needs. After two weeks, cells were fixed with 4% paraformaldehyde (PFA, Sangon Biotech) and stained with 0.1% crystal violet (Beyotime) for 15 minutes .
Cell Cycle Assay
Cells were seeded and cultured into 6-cell plates at a density of 3*105 cells/well. After 2 days, cells for cell cycle analysis were digested with trypsin (Hyclone), washed with phosphate-buffered saline (PBS) for three times, and fixed into 70% ethanol overnight at -20 ℃. Then cells were centrifuged at 500g for 20 minutes, washed three times with cold PBS. After treated with RNase A (0.1mg/ml) and propidium iodide (PI, 0.05mg/ml) for 15min in the dark. Flow cytometry were applied in the tested .
Cell Migration and Invasion Assays
For wound healing assays, cells were cultured in 6-well plates and scratched with 1ml pipette tip and the wounds were photographed at 0 h and 36 h. The relative migration ratio was calculated [39, 40].
For migration assays, a 24-well plate with Transwell chambers (8um pore size, Coring) was applied. A serum concentration difference is formed between the upper and lower chambers (upper: 10%FBS; lower: FBS-free). 4*104 cells were cultured into the upper chamber containing solubilized extracellular matrix (ECM) -coated members, as previously described manufacturer’s instructions (Coring Matrigel invasion assay; USA) for 24h. The cells on the lower surface of the chambers were fixed with 4% PFA for 15 minutes and stained with 0.1% crystal violet for 15 minutes [41, 42].
Making Tissue Microarray (TMA)
The 180 CC patients’ tissues (in situ) were prepared into TMA as previously described .
M-IF Staining Protocol
Opal 7-colour kit (NEL811001KT, PerkinElmer) was used for mIF. TMAs were dewaxed and rehydrated. In the first step, antigen was retrieved at 125 ℃ for 3 min and then cooled to room temperature (RT). Washed with TBST three times for 5min, incubated in H2O2 for 10 min. Repeated washed and blocked with blocking buffer. Primary antibody, PDL-1 (ab237726, abcam, 1:500, dye 480) was incubated at RT for 30min. Slides were washed and an HRP-conjugated secondary antibody was incubated at RT for 10min. TSA dye (1:100) was applied for 10min after washes. The procedures were repeated six times using the following antibodies, CD3 (ab16669, abcam, 1:200, dye 690; used as T lymphocyte cell marker), CD8 (ab93278, abcam, 1:100, dye 570; used as cytotoxic T cell marker), CD56 (ab75813, abcam, 1:500, dye 620; used as NK cell marker), CD68 (ab213363, 1:1000, abcam, dye 780; used as pan-macrophage marker), programmed death-1 (PD-1) (ab237728, abcam, 1:300, dye 520), programmed death ligand-1 (PD-L1) (ab237726, 1:500, dye 480). Secondary antibodies anti-mouse (NEF822001EA, PerkinElmer) or anti-rabbit (NEF812001EA, PekinElmer) were used at a 1:1000 dilution [22, 44, 45].
Further analysis by HALO system, we can quantify the number of six immune targets and the spatial position relationship between them .
Half Maximal Inhibitory Concentration (IC50)
Cells (4*103 cells/well) were seeded and cultured into 96-well plates overnight. At 1 day, PD-1 was treated with different concentrations (0, 0.0625mg/ml, 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml, 8mg/ml). At 2 day, 10ul cell counting kit-8 solution (MedChem Express, Monmouth Junction, NJ, USA) was added into each well, followed by further incubation at 37°C. After 1 h, the absorbance was measured at 450 nm wavelength to assess cell proliferation ability .
Statistical analysis was performed with IBM SPSS 23.0, Graphpad Prism 8 and R language. Comparisons between two conditions were based on two-sided Student’s test. The results of all statistical analyses were reported as p values from two-tailed tests, and P ༜0.05 was judged to be statistically significant (*P ༜0.05, ** P ༜0.01, and *** P ༜0.001).