Reagents
Olaparib and niraparib were purchased from Selleckchem (China). Fluzoparib was a gift from Jiangsu Hengrui Pharmaceuticals Co., Ltd. (Jiangsu, China). Pamiparib was a gift from Beigene (Beijing) Biotechnology Co., Ltd. (Beijing, China). All of the drugs were dissolved in dimethyl sulfoxide to obtain a stock solution of 10 mM and stored at − 80 °C. Cisplatin was obtained from Jiangsu Hausen Pharmaceutical Co., Ltd. (Jiangsu, China) and stored at 5 mg/mL at room temperature (RT). D-Luciferin (sodium salt) was purchased from Glpbiochem (China), dissolved in PBS stored at − 80 °C in the dark.
Cell lines and culture
NKYS, KHYG1, YT, and NKL were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Clack, USA) and 1% penicillin/streptomycin (Invitrogen, California, USA). Additionally, NKYS, KHYG1, and NKL were recombinant human interleukin-2-dependent (100 IU/mL, PeproTech, Rocky Hill, NJ, USA). SNK6 was cultured in X-VIVO medium (Lonza, USA) with 500 IU/mL recombinant human interleukin-2. NKYS, KHYG1, and YT were kindly obtained from Dr. Wing C. Chan (City of Hope Medical Center). SNK6 was kindly provided by Dr. Norio Shimizu and Yu Zhang of Chiba University, and NKL was purchased from the cell bank of the Bena Culture Collection (Beijing, China).
Cell proliferation, cell apoptosis, and cell cycle
A total of 2 × 103 cells were seeded into 96-well plates and treated with different concentrations of different agents. After 72 h, CCK-8 solution (US Everbright Inc., Jiangsu, China) was added to each well and allowed to react for another 1 h at 37 °C. Then, the absorbance value at optical density (OD) 450 nm was measured by a Multiskan FC microplate reader (Thermo Scientific, Waltham, MA, USA). The half maximal inhibitory concentration (IC50) values were predicted using SPSS software version 25.0 (IBM Corp.). The synergistic effects were estimated by combination index (CI) value using the Chou–Talalay method and CompuSyn software (CompuSyn Inc.). CI < 1: synergism; CI = 1: additive effect; CI > 1: antagonism. Independent experiments were repeated at least three times.
A total of 2 × 105 cells were seeded in 24-well plates and treated with different concentrations of different agents for 48 h or 72 h. For cell apoptosis analysis, cells were stained with Annexin V-APC and PI/7-AAD (Keygen Biotech, Jiangsu, China) in the dark for 15 min at RT. For cell cycle analysis, cells were fixed in 75% ethanol overnight at 4 °C and then digested with RNase A and PI (Keygen Biotech, Jiangsu, China) in the dark for 30 min at RT. Then, all these cells were analyzed by a FACS Calibur flow cytometer (BD Biosciences, NJ, USA). Independent experiments were repeated at least three times.
mRNA-seq (mRNA-sequencing) analysis
Total RNA extraction, mRNA library construction, sequencing, and data analysis were performed by Shanghai Yuanqi Biomedical Technology Co., Ltd. (Shanghai, China) according to the standard procedure. Brief procedures are listed in Additional file 1. The raw sequencing data are available from the NCBI and are archived under accession number PRJNA884169.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from NKTCL cells according to the manufacturer’s instructions. The concentration and purity of these samples were measured by Nanodrop 1000 spectrophotometry (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized by reverse transcription using UEIris RT mix with DNase (US Everbright Inc., Jiangsu, China), and qRT-PCR was performed using Universal SYBR Green qPCR Supermix (US Everbright Inc., Jiangsu, China). The primers were synthesized by Hangzhou Shangyasai Biotechnology Co., Ltd. (Hangzhou, China), and the sequences are listed in Additional file 2: Table S1. GAPDH was used as a reference gene for mRNA quantitation. The relative expression level was calculated with the 2−ΔΔCt method.
Western blotting
Cells were lysed in cold RIPA lysis buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) for 30 min on ice. The cell lysates were clarified by centrifugation at 13,000 × g for 30 min. Proteins were resolved on gels with different concentrations by SDS-PAGE and transferred onto PVDF membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked in TBST buffer containing 5% non-fat milk for 1 h at RT. Then, the membranes were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at RT. The antibodies are listed in Additional file 2: Table S2. The band images were digitally captured and quantified with a ChemiDoc™ XRS + system (Bio-Rad Laboratories, Hercules, CA, USA).
Immunofluorescence
Cells were washed with PBS, fixed with 4% formaldehyde for 15 min at RT, and permeabilized with 0.5% Triton X-100 for another 15 min at RT. The cells were incubated with primary antibodies at 4 °C overnight and secondary antibodies in the dark for 1 h at RT. Then, the cells were stained with DAPI (US Everbright Inc, Jiangsu, China) in the dark for 5 min. Images were captured using a Zeiss Axio Imager M2 microscope (Carl Zeiss Corporation, Germany). The antibodies are listed in Additional file 2: Table S2.
Clinical samples and immunohistochemistry (IHC)
In this study, we collected 67 tumor tissues from NKTCL patients who were histologically and clinically diagnosed by the department of pathology and oncology between 2014 and 2019 at the First Affiliated Hospital of Zhengzhou University. The IHC protocol was performed as the standard streptavidin–biotin-peroxidase-immunostaining procedure. The antibodies are listed in Additional file 2: Table S2. We defined that LMO2 expression was scored as negative or positive based on a 30% cut-off, which is based on previous studies in DLBCL and T-ALL [12, 13]. The scores were assessed by pathologists without prior knowledge of the patients’ information. Moreover, the detailed clinical characteristics of these patients are listed in Additional file 2: Table S3. The protocols were approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Lot No.2022-KY-1061–002).
Construction of stable cell lines
Stable LMO2 knockdown cell lines were generated using lentiviral constructs expressing short hairpin RNA (shRNA) of LMO2 (shLMO2, target sequence: AGGTGACAGATACCTCCTCAT) and negative control (shNC, target sequence: TTCTCCGAACGTGTCACGT). Stable 53BP1 knockdown cell lines were generated using lentiviral constructs expressing shRNA of 53BP1 (sh53BP1, target sequence: GATACTGCCTCATCACAGT) and negative control (shNC, target sequence: TTCTCCGAACGTGTCACGT). All of these were designed and provided by Shanghai Genechem Co., Ltd. (Shanghai, China).
A total of 2 × 105 cells were seeded in 24-well plates, which were infected with 10μL HitransG P solution and lentivirus including shNC, shLMO2, or sh53BP1 (MOI = 1:10). After more than 48 h, the cells were transferred to a fresh complete medium, which included 1 mg/mL puromycin. Subsequently, the transfection efficiency was measured by flow cytometry and western blotting, and stable cell lines were established for the following experiments.
Co-immunoprecipitation (Co-IP)
Cells were lysed in cold IP lysis buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) for 30 min on ice. Cell debris was removed by centrifugation at 13,000 × g for 30 min. Cell lysate (1 mg) was combined with antibody and incubated overnight at 4 °C with rotation. The antigen–antibody mixture was added to the tube containing prewashed agarose beads and incubated at RT for 1 h with mixing. After washing five times with IP lysis buffer, the antigen–antibody complex was eluted before SDS-PAGE. The antibodies are listed in Additional file 2: Table S2.
In vivo therapy on NKTCL-cell-drived xenograft models
Twenty NSG mice (3–4 weeks old, female, 15–20 g) were purchased from the Shanghai Model Organisms Center (Shanghai, China). A total of 1 × 107 NKYS-Luciferase cells dissolved in a mixture of 200 µL medium and 200 µL matrigel (Corning Incorporated, USA) were injected into the right axillary region by subcutaneous inoculation to establish the tumor xenograft models. After one week, these mice were randomly divided into four groups (n = 5) and given different treatments by intraperitoneal injections (i.p.): control, fluzoparib (40 mg/kg, twice every 5 days), cisplatin (2 mg/kg, three times in the first week), and fluzoparib (40 mg/kg, twice every 5 days) plus cisplatin (2 mg/kg, three times in the first week).
Tumor size was measured every week, and tumor volume (V) was calculated using the following formula: V = ab2/2 (a: the long diameter and b: the short diameter). Mice were given D-Luciferin (150 mg/kg, i.p.) and anesthetized with isoflurane. After 5 min, luminescence was detected using the in vivo imaging system (IVIS), and the intensity was quantitated and normalized by Living Image software (PerkinElmer, Massachusetts, USA).
The protocol was approved by the Institutional Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Lot No.2022-KY-1061–002).
Hematoxylin–eosin (HE) staining
First, we executed mice using the cervical dislocation method on day 49 after tumor cells inoculation. The fresh tissues (hearts, livers, kidneys, and lungs) were collected from mice, then put into 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm-thick sections. The next procedures were as follows: coating, dewaxing, dehydration, hematoxylin, differentiation, bluing, eosin, dehydration, clearing, and cover-slipping. Two senior pathologists viewed cellular and tissue structure using a Zeiss Axio Imager M2 microscope (Carl Zeiss Corporation, Germany).
NK cell isolation and culture
First, peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Paque density gradient centrifugation from the blood of healthy donors. NK cells were isolated from PBMCs using CD56 microbeads (Miltenyi Biotec, Germany). CD3 − CD56 + NK cells (more than 95%) were selected and cultured in RPMI 1640 with 15% fetal bovine serum and 100 IU/mL interleukin-2 for the next experiments.
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as the mean ± standard deviation (SD). Comparisons between groups were performed using Student’s t test and analysis of variance (ANOVA), respectively. PFS and OS were calculated using the Kaplan–Meier method and log-rank test. The correlation between LMO2 expression and clinicopathologic features was assessed using χ2-test. A value of p < 0.05 was considered statistically significant.