Clinical participants
From September to December 2019, peripheral blood samples and tumor tissues were collected from 24 pathologically verified patients with colorectal cancer from the Third Affiliated Hospital of Soochow University (cohort 1). Peripheral blood was collected 1-3 days before the surgery. The exclusion criteria were(1) patients with infection and autoimmune diseases,(2) patients who had received chemotherapy or radiotherapy before surgery. Peripheral blood samples from 20 healthy donors (HDs) were used as controls. Informed consent was obtained from every patient. This study was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Soochow University and was conducted in accordance with ethical guidelines: Declaration of Helsinki.
Immunofluorescence
Tissue microarrays (HRec-Ade180Sur-03, Shanghai Outdo Biotech Co.) were constructed using formalin-fixed, paraffin-embedded carcinoma tissues matched to adjacent normal tissues collected from 90 colorectal cancer cases (cohort 2). Immunofluorescence was performed following an established protocol. Briefly, tissue microarrays were dewaxed, rehydrated through graded ethanol, and incubated with 3% hydrogen peroxide for 30 mins. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 M citrate buffer (pH6.0) for 15 mins. Slides were then washed in PBS for 15 mins, treated with 10% normal horse serum for 30 mins and incubated with the primary antibodies, including Alexa Fluor 549 mouse anti-CD3 (BioLegend, San Diego, USA) and DyLight 488rabbit anti-TIGIT (BioLegend, San Diego, USA). After staining, the slides were mounted with ProLong Gold Antifade Mountant with 4',6-diamidino-2-phenylindole (DAPI,Thermo Fisher, USA). The images were taken using the Leica Aperio System (Leica, USA).
Cell isolation
Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll–Hypaqueby density gradient centrifugation within 2 hours of peripheral blood sample collection. Fresh colorectal tumor tissues were minced and digested within 2h of surgery. Digested cells were filtered through a 70 mm nylon mesh and washed with PBS. Native CD3+ T cells were purified from PBMC by negative selection using the EasySep human total or native CD3+T Cell EnrichmentKits (STEMCELL Technologies Inc. USA). CD3+TIGIT+T cells and CD3+TIGIT-T cells were sorted using a BD FACS Influx (BD Biosciences, USA).
Flow cytometry
PBMCs and tumor tissues isolated from colorectal cancer patients or HDs were stained with the following antibodies: PC5 conjugated anti-CD3, Ac7 conjugated anti-CD4, PE-Cy7 conjugated anti-CD8, APC conjugated anti-TIGIT, PE conjugated anti-Tim-3, FITC conjugated anti-PD-1, PE-Cy7 conjugated anti-INF-γ, V670 conjugated anti-IL-2, PC7 conjugated anti-Granzyme B, and V450 conjugated anti-IL10 antibodies. Samples were analyzed using BD FACS ARIA (BD Biosciences).
Glucose metabolism
Glucose consumption reflects the metabolic activity of many cells. 2-deoxyglucose (2-DG) is a glucose analog and widely used to assess glucose uptake. Similar to glucose, 2-DG can be taken up by glucose transporters and then metabolized to 2-DG-6-phosphate (2-DG6P). 2-DG6P is oxidized, resulting in the generation of NADPH which can be determined by an enzymatic recycling amplification reaction. T cells (2x105/well, 50ul) were stimulated with aCD3/CD28 (5ug/mL) for 8 hours. The cells were washed 3 times with PBS and then glucose starved with 100 mL Krebs-Ringer-phosphate-HEPES buffer containing 2% BSA for 40 min. The cells were then stimulated with or without 1 mmol/L insulin for 20 minutes. 10uL 10umol/L 2-DG was added into the cells for 20 mins. Glucose metabolism was analyzed according to the manufacturer's instructions of the 2-DG Assay Kit (Sigma-Aldrich, USA).
T-cell proliferation assays
In T-cell proliferation assays, cells were seeded in 96-well plates, labeled with 5mmol/L carboxy fluorescein diacetate succinimidyl ester (CFSE), and stimulated with aCD3/CD28(5 ug/mL) at 37℃with 5% CO2 for 4 days. Cells were collected, and the dilution of intracellular CFSE caused by proliferation was calculated using BD FACS ARIA (BD Biosciences).
Cell culture
Human HCT-116 colorectal cancer cells were cultured in RPMI-1640 medium (GIBCO, Grand Island, USA) supplemented with 10% FBS (fetal bovine serum) and streptomycin/penicillin at 37◦C with 5% CO2 under fully humidified conditions, while murine MC38 colorectal cancer cells were cultured in DMEM medium (GIBCO, Grand Island, USA) under the same conditions. CD3+ T cells stimulated with aCD3/CD28 were sorted and co-cultured with HCT-116 cells at a ratio of 5:1 in 24-well plates for 2 days. Then 5 mg/mL anti-TIGIT antibodies (BPS Biosciences, USA) or isotype controls were added to the co-culture system. T cells were collected to measure, their activity and the key markers in the metabolic pathways by flow cytometry, RT-PCR and western blot. HCT-116 cells were collected to determine the apoptosis and cell cycle distribution.
Real-time PCR
Total RNA was isolated using Trizol reagents (Thermo Fisher, Inc., USA) according to the manufacturer's protocol. cDNA was obtained using a 1μg total RNA reverse transcription kit (Life Technologies,USA). The qPCR reaction was performed using the SYBR Green PCR Master Mix kit (Takara, Japan) through the ABI 7900 PCR system (Applied Biosystems, USA). The data were quantified according to the 2-ΔΔCT method. The mRNA expression levels of cultured cells were normalized to GAPDH, respectively. Primer sequences are as follows:Glut1 (Glucose transporter 1),5’-ACTGCTGGAGCAGCTACCCT-3’ and 5’-GAAGCCTGCAACGGCAATGG-3’,HK1(hexokinase 1), 5’-CTGTTACGTCGGCGCTGCTA-3’ and 5’-AGAGCGCCATTGTCATCGGG-3’,HK2(hexokinase 2), 5’-CTGTTACGTCGGCGCTGCTA-3’ and 5’-AGAGCGCCATTGTCATCGGG-3’, PFK(phosphofructokinase), 5’-TCCGATTTCAGCATCGGGGC-3’ and 5’-CAGGTAGCCACAGTAGCCGC-3’, GAPDH,5’-GCGGGGCTCTCCAGAACAT-3’ and 5’-TCCACCACTGACACGTTGGC-3’.
Westernblot
The total protein was extracted using a mixture of RIPA lysis buffer and protease inhibitor. Proteins were quantitated using a BCA protein assay kit (Pierce, Keygen Biotech, China), then isolated with SDS-PAGE gel and transferred to PVDF membrane (Millipore, Darmstadt, Germany). The membrane was sealed with Tris buffer containing 0.1% Tween-20 and 5% skimmed milk at 4◦C. Rabbit anti-p-Akt (Ser473), Akt (1:1000, CST, Denver, MA, USA), mTOR (S2448) and p-mTOR (1:1000, CST, DANVERS, MA) were incubated with membrane overnight and then treated with HRB-conjugated secondary antibodies (1:10000, CST, Denver, MA, USA). The signal is detected by the Enhanced Chemiluminescence Detection System (Tennon5200, Shanghai, China), following the manufacturer’s instructions.
Xenotransplant Models
MC38 cells (5×106 cells/mouse) were injected subcutaneously into the right flank of B6J mice (n=6, male, 4-week). When the tumor volume reached 100 mm3, 20 tumor-bearing mice were evenly divided into 4 groups. The TIGIT antibody (10917-MM52, Sinobiology, Beijing) and PD-1 antibody (10377-M94, Sinobiology, Beijing) were both administrated in a dose of 10 mg/kg via tail-vein injection three times a week. The tumor volume of mice was measured every 5 days and mice were sacrificed after 30 days with the xenograft tumors harvested for further analysis. All experimental procedures were performed following the NIH Guide for Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee.
Apoptosis Assay and Cell-Cycle Analysis
Cells (1*106) were collected and incubated with the solutions of Annexin V and propidium iodide (PI) according to the apoptosis detection kit (BD Biosciences Pharmingen). For cell-cycle analysis,cells were fixed in 70% ice-cold ethanol at 4◦C overnight and then stained in a mixed solution (200 mg/mL PI, 0.1% sodium azide, 0.1% Triton X-100, and 10 mg/mL RNase). The apoptotic cells and cell-cycle distribution were detected by a FACSCalibur flow cytometer.
Statistical analysis
Three independent experiments were conducted for each treatment, and all data were expressed as mean ± standard deviation. The student’s t-test was only used for difference analysis between two groups, and one-way analysis of variance (ANOVA) was used when three or more groups were compared. P<0.05 was considered statistically significant. SPSS version 22.0 (SPSS, Chicago, IL, USA) software was used for statistical analysis of clinical samples.