Network databases

The TIMER database (https://cistrome.shinyapps.io/timer/) was used to analyze the differences in PRDX2 expression and the correlation between PRDX2 expression and the infiltration of six types of immune cells (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) in pan-cancer. The TIMER2.0 database (http://timer.cistrome.org) were used to analyze the correlation between PRDX2 expression and CD8+ T cell infiltration in pan-cancer. The KM-Plotter database (http://kmplot.com) was utilized to analyze PRDX2 expression levels in lung adenocarcinoma and lung squamous carcinoma and the prognosis of patients. RNAseq data of TCGA-LUAD were downloaded from the TCGA database (https://portal.gdc.cancer.gov) and collated to analyze PRDX2 expression disparities in LUAD paired and unpaired samples. The HPA database (https://www.Proteinatlas.org/) investigated PRDX2 protein expression levels in lung adenocarcinoma and normal lung tissues.

Human tissue samples

Clinical lung adenocarcinoma samples were provided by the Cancer Hospital affiliated with Anhui University of Science and Technology, and all collected samples were used for the study after the patients signed an informed consent form. The study was approved by the Medical Ethics Committee of Anhui University of Science and Technology and was conducted following the principles of the Declaration of Helsinki (NO. HX-002).

Cell culture

The human embryonic kidney cell line HEK293T, human lung adenocarcinoma cell lines (A549, 95-D, H292, and H1975), and the mouse Lewis lung cancer cell line (LLC) were obtained from ATCC. A549, 95-D, H292, and H1975 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Lonsera) and 1% penicillin/streptomycin (Beyotime) in RPMI-1640 medium (Gibco). HEK293T cells and LLC cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Beyotime).

Plasmids and vectors

To generate stable transfected cell lines, short hairpin RNAs (shRNAs) targeting PRDX2 were synthesized by Sangon Biotech (Shanghai, China) and inserted into the shRNA expression vector pLVX-shRNA (Sangon Biotech, Shanghai, China). For transient transfection, siRNAs targeting PRDX2 and LGALS9 were obtained from GenePharma (Shanghai, China). The oligonucleotide sequences used are shown in Supplementary Table 1.

Cell transfections

293T cells were transfected with Lipofectamine 2000 Reagent (Invitrogen), PsPax2 (Fenghui Biotechnology, Hunan, China), PMD-2 g (Fenghui Biotechnology), and shRNA-PRDX2 plasmids according to the manufacturer’s instructions, and the viral fluids were collected after 48 h.

For transfection of lentivirus-based constructs, 80% confluent H1975 and H292 cells were incubated for 24 h in a medium containing a concentrated viral solution and polyglutamine (Solarbio, H8761). Transfected cells were allowed to grow for a further 2 days and then selected with puromycin (1 µg/ml) (Sigma-Aldrich) for 1 week. Transfection efficiency was assessed by RT-qPCR and Western blot.

Cells with 60–80% confluence were transfected with Lipofectamine 2000 and siRNAs. Cells were incubated for 24–48 h and harvested for subsequent experiments.

RNA extraction and RT-qPCR

Total RNA was isolated using TRIzol (Invitrogen) and reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The cDNAs were subjected to RT-qPCR using the 2X Universal SYBR Green Fast QPCR Mix System. Primers were designed and synthesized in collaboration with Sangon Biotech (Shanghai). Relative mRNA expression was calculated using the 2−ΔΔCt method with GAPDH as a control. The sequences of the primers used are listed in Supplementary Table 2.

Western blot analysis

Total proteins were extracted by adding RIPA lysate (Beyotime, P0013C) containing protease inhibitors to the cells, and protein concentrations were determined using a BCA kit (Beyotime, P0012). Proteins were separated using SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, ISEQ00010). After transferring the membranes, they were blocked with a blocking solution containing 5% skimmed milk powder for 1 h at room temperature, incubated with primary antibodies at 4 °C overnight, and then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Membranes were developed using an Amersham ImageQuant™ 800 Imaging System (Cytiva) and ECL luminescent solution (Millipore, WBKLS100). The primary and secondary antibodies used are listed in Supplementary Table 3.

Immunofluorescence and immunohistochemical

For multiplex immunofluorescence, paraffin sections were deparaffinized to water and blocked with 3% BSA after antigen repair in the EDTA antigen repair buffer (pH 8.0). The sections were then incubated overnight at 4 ℃ with the corresponding primary antibody in a humidified chamber, followed by incubation with the corresponding fluorescent secondary antibody for 1 h at room temperature. Each primary antibody was stained separately, and between each staining, the tissue sections were heated in a microwave oven in a repair cassette filled with EDTA antigen repair buffer (pH 8.0) to repair the antigen again. The nuclei were then restained by incubation with DAPI solution for 10 min at room temperature, protected from light, Finally, the sections were sealed with an anti-fluorescence quenching mounting medium. The images were detected and captured by inverted fluorescence microscopy (Leica 3000) and analyzed by Image J.

For immunohistochemistry, the paraffin sections were deparaffinized to water, the antigen was repaired with EDTA antigen repair solution, endogenous peroxidase was blocked with 3% hydrogen peroxide, and washed and blocked with 3% BSA. After adding the corresponding primary antibody, they were incubated at 4 ℃ overnight, followed by adding the corresponding secondary antibody (HRP labeled) and incubating at room temperature for 1 h. Subsequently, DAB was added to develop the color, hematoxylin re-staining of the nucleus was performed, and finally, the sections were dehydrated and sealed. The images were captured using an inverted fluorescence microscope (Leica 3000) and analyzed using Image J.

T cell-mediated tumor cell killing assay

To isolate PBMCs, 5 mL of venous blood was collected from healthy volunteers. Human blood collection was approved by the Medical Ethics Committee of Anhui University of Science and Technology, and written informed consent was obtained from all volunteers. PBMCs were obtained from peripheral blood using human peripheral blood lymphocyte isolate (TBD, LTS1077). To obtain activated T cells, PBMC were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin with ImmunoCult Human CD3/CD28/CD2 T cell activator (STEMCELL Technologies, 10970) and recombinant human IL-2 (PPL, PK0119) were cultured together and activated for one week according to the manufacturer’s protocol. NSCLC cells were inoculated into 6-well plates at the appropriate concentration. After 24 h, activated PBMCs were co-cultured with adherent NSCLC cells at a 3:1 ratio for 48 h. After 48 h of incubation, cell debris was removed, and T cells and NSCLC cells were collected separately for subsequent experiments.

Flow cytometry

To detect apoptosis, NSCLC tumor cells obtained by sorting with Dynabeads™ CD3 magnetic beads (Invitrogen, 1151D) after co-culture with T-cells, we utilized the Annexin V-FITC/PI Apoptosis Detection Kit (keyGEN BioTECH, KGA108) for double staining. Subsequently, the stained cells were examined using the FACSCanto II flow cytometer (BD, USA).

For the determination of Granzyme B expression in T cells, CD3 and CD8 antibodies were used to label cell surface expression of CD3 and CD8, and after fixation and permeabilization by Intracellular Fixation & Permeabilization Buffer Set (eBioscience,88-8824-00), followed by staining for intracellular Granzyme B using the GZMB antibody. The stained cells were then analyzed by FACSCanto II flow cytometer, and the data were further analyzed using Flow Jo 10.0 software.

RNA sequencing

For Sh-PRDX2 H1975 lung adenocarcinoma cells and control, total RNA was extracted using TRIzol reagent (Invitrogen). After establishing sequencing libraries, we performed the 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor’s recommended protocol.

In vivo study

All animals were housed in a 12-h light/12-h dark environment with controlled temperature cycles (22 ± 1 °C) and humidity (40 ± 5%). LLC cells were injected subcutaneously in BALB/c nude mice for the immunodeficient mouse model and in C57BL/6 mice for the immune-competent mouse model. When the tumor volume reached 100 mm3, saline-treated mice were used as the control group, and the experimental group was treated with Conoidin A (5 mg/kg or 10 mg/kg), once a day for 15 consecutive days. The tumor volume was measured with calipers every 3 days during the administration period and was calculated as follows: length × width2 × 0.5. On the next day of the last administration, the mice were artificially executed by cervical dislocation, and the tumors were isolated and measured. All animal experiments were performed strictly with the NIH Guide for the Care and Use of Laboratory Animals, and the Medical Ethics Committee of Anhui University of Science and Technology approved the animal experiments.

Statistical analysis

The data are presented as the mean ± SD. Data analysis was performed using GraphPad Prism 9. Unpaired or paired two-tailed Student’s t-tests were conducted to compare the two groups. All experiments were repeated at least three times. Statistical significance was set at P < 0.05.