2 MATERIALS AND METHODS
2.1 Mice, tumor model and agents
6-8 weeks old, male C57BL/6 mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. (Shanghai, China). Animals were housed and maintained under optimal conditions of light, temperature, and humidity with free access to food and water. For subcutaneous tumor model, a total of 1×105 LA795 cells were resuspended in 200 μL PBS and inoculated subcutaneously into the right flank of mice. Tumor dimensions were measured by caliper every other day, and tumor volume (mm3) was estimated using the formula: tumor volume = (length) × (width)2 × π/6. Different doses of anti-VEGFR2 antibody (DC101, BioXCell, BE0060) and IgG1 (BioXCell, BE0088) treatment were initiated 11 days after tumor cells inoculation and administered by intraperitoneal injection every 3 days. Anti-PD1 antibody (Anti-CD279, BioXCell, BE0146) and IgG2a (BioXCell, BE0089) treatment were initiated 12 days after tumor cells inoculation and was administered at 200 μg/mouse by intraperitoneal injection every 3 days. At indicated days later, the tumor-bearing mice were anesthetized and tissues were harvested for further analysis and measurement. Survival analysis was continued as independent experiments for indicated days.
2.2 Cell lines
LA795 cells were purchased from Qingqi Biotechnology Development Co., Ltd. (Shanghai, China) and cultured at 37°C in a humidified incubator with 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) with 100 units/mL penicillin and 100 ug/mL streptomycin. The cells were routinely tested to confirm the absence of mycoplasma contamination and were cultured for a limited number of generations.
2.3 Isolation of primary T cells from mouse models
Tumors from subcutaneous LA795 model were harvested on the days indicated. Tumor tissues were exhaustively flushed with PBS, minced into small pieces. Single cell suspensions were yielded through enzymatic digestion at 37°C for 40 minutes in RPMI 1640 medium containing collagenase type IV (1 mg/mL, Sigma) and DNase I (20 U/mL, Sigma). Triple volume FACS were added to stop the digestion. Filter the tissue digestion suspension into a new centrifuge tube with a 100 mesh filter, and centrifuge (350g, 24°C, 10min). Add 40% Percoll to resuspend, use a 300 mesh filter into 15ml centrifuge tube, insert 3ml 80% Percoll into the bottom of the filtered cell suspension, and centrifuge (2500 rpm, 24°C, 20 min). The lymphocyte layer was aspirated and transferred to a new 15ml centrifuge tube. FACS was added to the full volume, and centrifuged (400g, RT, 10 min). Discard the supernatant and resuspend the pellet with FACS.
2.4 Flow cytometry analysis
To determine intracellular cytokine expression, cells were stimulated with phorbol12-myristate 13-acetate (PMA), ionomycin, Golgistop for 4-6 h. At the end of stimulation, cells were stained with the indicated antibodies. Cells were stained in PBS containing 2% fetal bovine serum (FBS) with indicated antibodies for analysis of surface markers. For intracellular stain, cells were fixed in Fixation/Permeabilization according to the manufacturer’s instructions (eBioscience) for 40 min and then subjected to antibody staining. All samples were run on the BD LSRFortessa Flow Cytometer (BD Biosciences), and FlowJo software (TreeStar, Ashland, OR, USA) was used to analyze the data. Specific information on antibodies was listed in Supplementary Table 1. Tumor-infiltrating Immune cells were gated followed by Supplementary Figure 1.
2.5 Immunohistochemistry and immunofluorescence
Paraffin-embedded samples were cut into 5-mm consecutive sections and deparaffinized. Specimens were incubated at 4°C, overnight with antibodies against human LAYN, CD8, or against mouse CD8a, LAYN. Specimens were then washed by PBS and stained with anti-mouse/rabbit immunohistochemistry (IHC) secondary antibody kit. Double fluorescent staining with anti-CD8 and anti-LAYN was performed on snap-frozen sections embedded in OCT compound (8 mm), then 3–5 areas per tumor sample were randomly selected
and evaluated by two pathologists who were blinded to patient information at 400×magnification. The results were determined based on both the staining intensity and the percentage of positive cells. The staining intensity was defined as follows: 0 = none, 1 = weak, 2 = intermediate, 3 = strong. The percentage of positive cells was scored as follows: 0-5%=0, 6%–25%=1, 26–50%=2, 51–75%=3 and ≥76%=4. The final scores for the slides were determined by the two scores. A final score of <4 was defined as low expression group, and ≥4 was marked as high expression group. Specific information on antibodies was listed in Supplementary Table 1.
2.6 In vitro experiments
CD8+T cells isolated from human PBMC were cultured in the presence of plate-bound anti-CD3 (1µg/ml) with different dosage VEGF-A (0, 20, 50ng/ml, R&D Systems). Cells were harvested and analyzed by flow cytometry after 84 h or used to extract mRNA after 60h. T cells isolated from tumor of tumor-bearing mice were treated by the combination of antibody Anti-CD279 (BioXCell, BE0146) or IgG2a (BioXCell, BE0089) with different dosage anti-VEGFR2 antibody (DC101, BioXCell, BE0060) or IgG1 (BioXCell, BE0088). After 48h, cells were analyzed by flow cytometry.
2.7 Co-culture experiment
Mouse layilin was cloned into PLVX-IRES-PURO retroviral vector. Retrovirus was generated by using lentivirus packaging system before infecting OTI CD8+T cells. OTI CD8+T cells were isolated from the lymph nodes of OTI mice by using CD8a cell isolation kit (Miltenyi) and activated with OVA antigen peptide for 48 hr. 48 hr later, activated OTI CD8+T cells were spin-infected with retrovirus at 2000 rpm for 1 hr under 32°C. Puromycin was added to screen infection successful OTI CD8+T cells. OTI CD8+T cells with overexpression of LAYN were co-cultured with LA795-OVA for 48h.After 48h, killed LA795-OVA cells were detected by flow cytometry.
2.8 Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from cells according to the protocol of RNAprep Pure Micro Kit (TIANGEN, China). PrimeScriptTM RT Reagent Kit (TaKaRa Bio, Shiga, Japan) was used to reverse transcribe mRNA into cDNA, and the SYBR Premix Ex Taq kit (Takara, RR420A) was used to quantitatively analyze the mRNA of the target gene. When testing, each sample was equipped with three replicate holes. β-Actin was used as an internal reference to calculate the relative expression of mRNA, and the formula was: RQ = 2-△△Ct. PCR primers were listed in Supplementary Table 2.
2.9 Western Blot
Samples lysed in RIPA buffer were cooked and then resolved by SDS-PAGE, followed by transferring to polyvinylidene fluoride membranes. Next, membranes were blocked with skimmed milk powder, incubated with the primary antibody overnight, and then incubated with the secondary antibody for 1 hour. All experiments were replicated three times.
2.10 Lentiviral Transduction
To overexpressing LAYN on CD8+T cells, lentiviral transduction was performed by transfecting HEK293T cells with PHR-SFFV-EGFP lentiviral vectors encoding WT or LAYN along with lentivirus packaging system. To knock down NR4A1 on CD8+T cells, lentiviral transduction was performed by transfecting HEK293T cells with pLKO.1-GFP lentiviral vectors encoding WT or NR4A1 along with lentivirus packaging system. Isolated human CD8+T cells were activated with plate-bound anti-CD3 (1mg/mL) and anti-CD28 (1 mg/mL) in 96-well plates for 72 hours and then infected with the packaged lentivirus in the presence of 10 mg/ml polybrene by spinning at 2000 rpm for 1 hr under 32°C. CD8+T cells transduced with indicated lentiviral vectors were used for flow cytometric analysis or other experiments.
2.11 Chip-qPCR
A total of 107 CD8+T cells, isolated from human PBMC using CD8 isolation kit (Miltenyi Biotec), was activated with plate-bound anti-CD3 (1 ug/ml) and anti-CD28 (1 ug/ml) in 96-well plates for 72h. Chromatin immunoprecipitation (ChIP) assays were performed according to the protocol of the SimpleChIP® Plus Sonication Chromatin IP Kit (56383). NR4A1 antibody used for ChIP was purchased from Novus Biologicals. DNA isolated was tested by real-time quantitative PCR. The enrichment of samples in chromatin was based on the calculation formula, △CT=CTChIP DNA – CTInput DNA. Specific information on antibodies was listed in Supplementary Table 1.
2.12 Patient samples
Written informed consent was obtained from all patients. Tumor samples were acquired from 8 patients with advanced lung adenocarcinoma (LUAD) who received pembrolizumab treatment (2 mg/kg body weight each time for every three weeks) combined with anlotinib treatment (12 mg, once daily for 2 weeks and stop the drug for 1 week) in the oncology department of Shanghai Changzheng Hospital. These patients were followed up from January 2014 to November 2021. The main selection criteria included patients with metastatic or unresectable recurrent lung adenocarcinoma (LUAD), measurable diseases in the Response Evaluation Criteria in Solid Tumors (RECIST 1.1), and had a representative tumor sample (paraffin). During the combination treatment, the tumor was evaluated every 8 weeks, and if the disease progresses, and we would change the treatment plan. Needle biopsy was used to collect tumor tissue before combination therapy. The analysis deadline for this study was November 2021.
2.13 Statistical analysis
Flow data were processed and analyzed using Flowjo10.0 software. GraphPad Prism software (version 8.0) was used for data statistical analysis and graphing. Data statistics were presented as mean±standard (Mean±SEM), and paired or unpaired T test (student t test) was used to test the significance of differences between two sets of data. The survival curve was drawn by the Kaplan-Meier method, and the difference was analyzed by the log-rank (Mantel-Cox) test. To compare the differences among the treatment groups in the in vivo study, one-way ANOVA was performed. P <0.05 was considered statistically significant. In the figure, the symbols were used as: *, P< 0.05, **, P< 0.01, ***, P< 0.001, ****, P< 0.0001 by Student t test.