Patients and specimens
For phenotypic assays, 23 fresh paired gastric cancerous, tumor margin and tumor-free gastric tissues (greater than 1-cm distance from the tumor), routinely paraffin-embedded for immunohistochemistry(IHC)and immunofluorescence (IF), were collected from 23 patients with GC who underwent surgery at our hospital between May 2019 and July 2020. Tumor infiltrating lymphocytes (TILs) isolated from above 23 fresh gastric cancerous tissues for flow cytometry, were collected. The clinical characteristics of the patients for phenotypic assay are listed in Table 1.
For functional assays, peripheral blood from 18 patients with GC was collected before surgery. Paired 18 fresh gastric cancerous tissues were collected during surgery. The clinical characteristics of the patients for functional assays are listed in Table 2.
None of the patients who provided samples received preoperative radiotherapy or chemotherapy and were confirmed to have GC on postoperative pathology. The present study was performed in accordance with ethical standards and according to the declaration of the national and international guidelines. Written informed consent for publication was obtained from all participants. All the assays performed involving human blood and tissue samples were approved by the Ethics Committee of Jiangnan University (No.LS2018021).
Antibodies and reagents
RNAlater® was purchased from Ambion, USA. TRIzol was purchased from Invitrogen, USA. DEPC was purchased from Bio Basic Inc, Canada. The SYBR® PrimeScript® RT-PCR Kit was purchased from TaKaRa, Japan for two-step RT-PCR. PCR primers were designed by TaKaRa, Japan and synthesized by Yingjun Biotechnology Co., Ltd, China. An anti-CD137 rabbit mAb (#34549) used for IHC and IF and was purchased Cell Signaling Technology (CST, USA). An IHC detection reagent (HRP, rabbit, #8114) was purchased from CST, USA. An agonistic anti-CD137 mAb (#79097) was purchased from BPS Bioscience, USA. An anti-Foxp3 rabbit mAb (#12653) used for IHC was purchased from CST, USA. Anti-CD8 mouse antibody (#66868-1-Ig) for IHC and IF was purchased from the Proteintech group, China. MojoSortTM Magnet, MojoSortTM Human CD8 Nanobeads and MojoSortTM Human CD8 Cell Isolation Kit were purchased from BioLegend, USA. An NF-κB p65 rabbit mAb (#8242) for flow cytometry and IF was purchased from CST, USA. An anti-cytokeratin mouse mAb (#ab756) used for IHC was purchased from Abcam, England. A purified anti-human CD3 mAb (OKT3, #317326) for cell incubation and anti-CD45-PerCP (#368506), anti-CD3-FITC (#300406), anti-CD8-APC (#301014) and anti-CD137-APC (#309809) antibodies for flow cytometry were purchased from BioLegend, USA.
IHC assay
Fresh tissues for phenotypic assays or collected primary GC cells for functional assays to test separation purity were fixed, dehydrated and paraffin embedded. Paraffin sections were dewaxed and rehydrated using a routine protocol [22]. The cells underwent antigen repair, neutralization of endogenous catalases, serum blocking, incubation with anti-CD137 rabbit mAb antibody (1:100, CST, USA), anti-Foxp3 rabbit mAb antibody (1:100, CST, USA), anti-cytokeratin mouse mAb antibody (1:100, Abcam, USA) and anti-CD8 mouse antibody (1:100, Proteintech group, China) at 4°C overnight. Cells were incubated with a secondary antibody, and DAB was used for color development. Cells were counterstained, neutral gum sealed and observed according to a standard immunohistochemical operation procedure. PBS was used as a negative control. The stained sections were scanned using Panoramic MIDI. Image J was used to count positively stained cells. Two senior pathologists independently confirmed the results.
IF assay
Paraffin sections of a specimen for phenotypic assays were dewaxed and sealed with 3% H2O2 for 10 min and heat-retrieved with 0.01 mmol/l citrate buffer (pH=6.0) for 10 min at 95 ℃. After natural cooling, the sections were blocked with goat serum for 30 min and incubated with an anti-CD137 rabbit mAb (1:100, CST, USA) and anti-CD8 mouse mAb (1:100, Proteintech group, China) overnight in a water tank at 4 ℃. After 1 h of rewarming, antigens were detected with an anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) and anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) (both 1:500, CST, USA). The sections were incubated at 37 ℃ for 1 h, and DAPI was added. The sections were incubated in the dark for 5 min, sealed with 50% glycerol, and observed under a confocal microscope.
After slide preparation, cells for IF in NF-κB p65 nuclear translocation assay were fixed in 4% paraformaldehyde and penetrated using 0.5% Triton X-100 at room temperature for 20 min. The slides were blocked with goat serum for 30 min and incubated with an anti-NF-κB p65 rabbit mAb (1:100, CST, USA) overnight in a water tank at 4 ℃. After 1 h of rewarming, the primary antibodies were detected using an anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST, USA) for 1 h. DAPI was added, and the sections were incubated in the dark for 5 min. Stained sections were observed under a fluorescence microscope.
Isolation of TILs
Gastric cancerous tissues were cut into 1-mm-diameter pieces using ophthalmic surgical scissors, and the appropriate amount of tissue digestive solution containing 2 mg/ml type IV collagenase and 0.25 mg/ml hyaluronidase was added. The samples were transferred to a 15-ml centrifuge tube and digested in a shaker at 37 ℃ for 30 min. The cell suspension obtained from digestion was filtered with a 70-µm sieve, and the filtered liquid was collected in a 50-ml centrifuge tube. Ten milliliters of 40% Percoll was added, then 10 ml of 80% Percoll was added below the 40% Percoll. The tubes were centrifuged at 716 g for 20 min. TILs were isolated between the 40% Percoll and 80% Percoll.
Isolation of PBMCs and CD8+ T cells
After transferring 20 ml of blood from patients with GC into 50-ml centrifuge tubes, 10 ml of PBS was added to dilute the blood. The solution was mixed gently, and 10 ml of Ficoll lymphocyte separation solution was added to the bottom of 50-ml centrifuge tubes. Samples were centrifuged at 716 g for 20 min, and lymphocytes were collected and washed twice with PBS for 5 min each time. Isolated PBMCs were washed with MojoSortTM buffer once. The experimental procedure for CD8 isolation protocol was performed according to the MojoSort™ Human CD8 T Cell Isolation Kit provided by BioLegend, USA.
Isolation of primary GC cells
Fresh gastric cancerous tissues for functional assays were immersed in sterilized PBS containing 200 U/ml penicillin and streptomycin for 10 min, then washed with sterilized PBS containing 1000 U/ml penicillin and streptomycin 5 times. The specimens were immersed in sterilized PBS containing 200 U/ml penicillin and streptomycin for 10 min to remove blood and bacteria on the surface of the specimens. The tissue specimens were cut into 1-mm-diameter pieces using ophthalmic surgical scissors, and the appropriate amount of tissue digestive solution containing 2 mg/ml type IV collagenase and 0.25 mg/ml hyaluronidase was added. The samples were transferred to a 15-ml centrifuge tube And digested in a shaker at 37 ℃ for 30 min. The cell suspension obtained from digestion was filtered with a 70-µm sieve, and the filtered liquid was collected in a 50-ml centrifuge tube. Ten milliliters of Ficoll lymphocyte separation solution was added to the bottom of 50-ml centrifuge tubes, and the tubes were centrifuged at 716 g for 20 min for lymphocyte removal. Cells at the bottom of 50-ml centrifuge tubes were collected, and erythrocyte lysate was added for 10 min to remove red cells. The cells were washed with sterile PBS containing 1000 U/ml penicillin and streptomycin 5 times.
Primary GC cells and CD8+ T cells stained with CFSE
Primary GC cells and CD8+ T cells were collected and washed with PBS 3 times for 5 min each wash. Primary GC cells were treated with 1 ml of 5 µM CFSE and cultured in a 37 ℃ CO2 incubator for 15 min. One milliliter of fetal bovine serum was added to stop the staining for 1 minute, and the cells were washed twice with PBS.
Cell culture
CFSE-labeled CD8+T cells, PBMCs or/and CFSE-labeled primary GC cells isolated from GC patients were added to 96-well plates coated with a purified anti-human CD3 antibody (BioLegend, USA) at 5 µg/ml overnight to upregulate CD137 expression and cultured in DMEM with 10% FBS.
Flow cytometry
For CD137 detection, the PBMCs or TILs of GC patients were placed in flow tubes, and 5 µL each of an anti-CD45-PerCP antibody (BioLegend, USA), anti-CD3-FITC antibody (BioLegend, USA) and anti-CD137-APC antibody (BioLegend, USA) was added. The cells were incubated in the dark for 10 min and washed with PBS once. PBS (200 µL) was added for flow cytometry detection.
For examination of CD8+ T cells proliferation, CD8+ T cells of GC patients were placed in flow tubes and washed once with PBS. PBS (200 µL) was added for flow cytometry detection.
For NF-κB detection, CD8+ T cells from GC patients were treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA) for 72 h and placed in flow tubes. After washing once with PBS, a Fixation/Permeabilization Solution (BD Cytofix/CytopermTM) was added at room temperature for 30 min. After washing once with PBS, an NF-κB p65 rabbit mAb (1:1000, CST, USA) was added. The cells were incubated in the dark for 1 h and washed once with PBS. Five microliters of anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (1:500, CST, USA) was added, and the cells were incubated in the dark for 30 min. After washing once with PBS, 200 µL of PBS was added for flow cytometry detection.
Primary GC cell apoptosis detection using flow cytometry
PBMCs (1×105) and primary GC cells (2×104, CFSE stained) were mixed, placed in anti-human CD3 antibody-coated 96-well plates containing 200 µl of 10% FBS DMEM, and treated with 10 µg/ml anti-CD137 mAb (BPS Bioscience, USA). Apoptosis in the GC cells was detected using flow cytometry after 72 h.
Real-time quantitative PCR (real-time PCR)
Total RNA was extracted by TRIzol reagent (Invitrogen, USA) according to the manufacturer's instruction, and cDNA was generated using a TaKaRa PrimeScript RT Reagent Kit (TaKaRa, Japan) according to manufacturer’s instructions. Quantitative real-time PCR was performed on ABI step-one plus (Applied Biosystems, USA) using the TB Green Premix Ex Taq (TaKaRa, Japan). Data were normalized to the expression of β-actin. Primer pairs used in this study are presented in Table 3.
Statistical analysis
Statistical analyses were performed using SPSS 26.0 software. The figures were plotted by GraphPad Prism 6 software. Continuous variables are shown as means ± standard deviations (SD). Categorical variables were shown as counts and percentages. Descriptive statistics were shown as mean (standard deviations, SD) or median (interquartile range) according to data distribution. Statistical analyses between different groups were performed by one-way ANOVA, with S-N-K for post hoc multiple comparisons. An unpaired two-tailed Student’s t test was used for comparisons between two groups. Significant p-values are labeled with one or more ‘*’, denoting *p < 0.05, **p <0 .01, and ***p <0 .001. A threshold of P < 0.05 was defined as statistically significant.