The study was approval by Institutional Reviewer Board of The Affiliated Huai’an Hospital of Xuzhou Medical University (Approval number: HEYLL202315); and all animal experiments conducted at The Affiliated Huai’an Hospital of Xuzhou Medical University were approved by the Institutional Animal Care and Use Committee (Approval number: HEYLL202315).

The predictive significance of TNFRSF21 expression in human malignancies was assessed using the GEPIA and the Kaplan-Meier plotter database.

A549, H1299, H460, SPC-A1, and BES-2B normal lung epithelial cells were sourced from ATCC. All cells were cultured in DMEM supplemented with 10% FBS. DDP was purchased from Sigma in St. Louis, Missouri, United States. A549/DDP and H1299/DDP cells were established following the procedure outlined in a previous study [17]. These DDP-resistant cell lines were maintained in 1 μg/mL DDP. All cells were incubated in a humidified environment with 5% CO₂ at 37°C.

The pcDNA3.1(+)-TNFRSF21 plasmid was constructed as follows: Sangon Biotech (Shanghai, China) synthesized the TNFRSF21 mRNA fragment, which was then incorporated into pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA). For cell transfection, lung cancer cells were exposed to lentivirus containing either TNFRSF21 shRNA or sh-control (Genechem, Shanghai, China). After 6 h, the medium was changed using a solution containing 1 μg/mL puromycin (Sigma, USA) for 2 days to obtain TNFRSF21 shRNA or sh-control cells.

The resistance of DDP was assessed using a CCK8 kit (Beyotime, Haimen, China). Transfected cells were cultured in 96-well plates and treated with various concentrations of DDP. After treatment, 20 μL of CCK8 solution was added for 4 h, and cell viability was determined by measuring the absorbance at 450 nm.

The stem cell medium consisted of DMEM/F12, 2% B27, 20 ng/mL EGF, 10 ng/mL bFGF, 0.4% BSA, and 5 μg/mL insulin. Tumorspheres measuring 100 μm or larger were counted after 7 days. Tumorspheres were imaged using a ZOE™ Fluorescent Cell Imager (Bio-Rad, CA, USA), and their sizes were measured using ImageJ software.

The EdU Cell Proliferation Kit (Beyotime) was utilized for the EdU assay. A549/DDP and H1299/DDP cells were seeded onto 12-well dishes. After a 2-h incubation with 10 μM EdU, the cells were fixed using 4% paraformaldehyde for 10 min. 0.3% Triton X-100 for another 10 min was used to permeabilize the cells. Subsequently, EdU was detected by performing a click reaction for 30 min. Nuclear Proliferating cells were observed using fluorescence microscopy.

Lung cancer cells (400 per well) were seeded onto 60 mm diameter plates. After transfection, the growth medium was changed every 3 days. Colonies were fixed using methanol for 10 min and stained using 0.1% crystal violet solution for 10 min after a 2-week incubation period. Images of colonies were captured using a digital camera, and ImageJ software was used for colony quantification.

Following transfection, cells were harvested and treated with APC-linked monoclonal CD44 antibody (Biolegend, San Diego, CA, USA, Cat#163604), PE-linked monoclonal CD133 antibody (Biolegend, Cat#372804), or PE-linked monoclonal Ki-67 antibody (Biolegend, Cat# 350504) for 30 min at 4°C. Live cells were labeled using live-dead BV421 (Biolegend, Cat# 423114). Flow cytometry (BD, USA) was used to test the proportion of cells positive for CD44, CD133, or Ki-67.

Cells were suspended at a concentration of 5 × 10⁴ cells per well (200 μL) and placed in the upper chambers (24-well migration chambers, 8.0 μm pore membrane, Corning) using serum-free medium. Matrigel (1:5; Corning, New York, NY, USA) was applied to the upper chambers to facilitate cell invasion, while cell migration was unaided. 800 μL medium with 10% FBS was added to the lower chambers. Non-migrated or non-invaded cells in the upper chambers were removed using cotton swabs. Methanol was used to fix the cells adhering to the lower membrane surface and they were stained by 0.1% crystal violet. Images were captured using a microscope in five randomly chosen microscopic fields.

TRIzol reagent (Invitrogen) was used to obtain total RNA. cDNA synthesis was performed using a cDNA synthesis kit (Applied Biological Materials, Richmond, British Columbia, Canada). EvaGreen 2 × qPCR MasterMix was used with a LightCycler96 real-time detection system (Roche, Switzerland). Amplification included an initial step at 95°C for 5 min, followed by 40 cycles at 95°C for 15 s, optimal annealing temperature for 30 s, and 72°C for 60 s. Primer sequences for PCR were shown in Table 1. RAN expression levels were calculated using 2^−ΔΔCt method.

5 × 10⁶ A549/DDP cells, either infected with TNFRSF21 shRNA or not, were injected subcutaneously in the right armpit of male nude mice (12 in total, 6 weeks old). Tumor size was measured every three days starting from day 14. After the experiment was completed, the mice were euthanized, and tumors were excised and weighed. Tumor tissue samples were collected for qRT-PCR, western blot analysis, or flow cytometry. BALB/c nude mice were obtained from the Animal Research Center at Nanjing Medical University. All animal experiments conducted at The Affiliated Huai’an Hospital of Xuzhou Medical University were approved by the Institutional Animal Care and Use Committee (Approval number: HEYLL202315).

After treatment, cells were collected. MDA and SOD levels in lung cancer cells were evaluated using MDA Detection Kit (#BC0020, Beyotime) and SOD Activity Detection Kit (#BC0175, Beyotime). GSH levels were assessed using the GSH Content Detection Kit from Jiancheng Biotech Co. in Nanjing, China. Additionally, the Reactive Oxygen Species Detection Kit (#S0033, Beyotime) was used. DCFH-DA (2′,7′-Dichlorodihydrofluorescein diacetate) was incubated with cells at 37°C for 20 min. Cells were then examined using a fluorescence microscope, and cellular ROS fluorescence intensity was quantified.

Cell apoptosis was assessed by FITC Annexin V Apoptosis Detection Kit (BD, USA). In a six-well plate, 5 × 10⁵ cells were seeded and incubated at 37°C. Adherent cells were detached using trypsin-EDTA, then centrifuged, washed twice with PBS, and suspended in 1 × binding buffer. Subsequently, 100 μL of the cell suspension was transferred to a 5 mL culture tube and incubated in the dark for 30 min with 5 μL FITC Annexin V and 5 μL PI. An additional 400 microliters of binding buffer were added. Flow cytometry (BD, USA) was used to measure fluorescence intensity.

MitoSOX Red Dye (Invitrogen; M36008) was used to test mitochondrial reactive oxygen species (mtROS). Cells were indicated with 5 μM MitoSOX Red Dye and incubated for 10 min. After staining, cells were washed with PBS and examined using a Nikon A1R confocal laser scanning microscope system (Nikon, Tokyo, Japan).

GraphPad Prism 9.5 was used to do statistical analysis. Data were presented as mean ± standard deviation. One-way ANOVA was employed for comparing statistical analysis among multiple groups. Unpaired two-tailed Student’s t-test was used to compare statistical analysis between two groups, Statistical significance was considered at p < 0.05.

We utilized the GEPIA database to evaluate TNFRSF21 expression in LUAD, revealing an elevation in LUAD tissues (Fig. 1A). Subsequently, Kaplan-Meier analysis (Fig. 1B,C) indicated that elevated TNFRSF21 levels correlated with unfavorable OS and FP in LUAD patients. Moreover, mRNA of TNFRSF21 was increased in A549, H1299, H460, and SPC-A1 cell, while BES-2B exhibited decreased TNFRSF21 expression (Fig. 1D). Additionally, Fig. 1E demonstrates higher TNFRSF21 levels in A549/DDP and H1299/DDP cells. Taken together, these findings suggest TNFRSF21’s potential as a promising prognostic biomarker for LUAD.

Experiments aimed at reducing TNFRSF21 expression were conducted using A549/DDP and H1299/DDP cells as the experimental models. TNFRSF21 expression was efficiently reduced in A549/DDP and H1299/DDP cells by applying shRNA, with sh1 showing the highest knockdown efficacy and thus chosen for subsequent assays (Fig. 2A,B). Notably, decreased cell viability was observed in A549/DDP and H1299/DDP cells when exposed to various DDP doses (Fig. 2C,D). Furthermore, the impact of TNFRSF21 on LUAD cell proliferation was evaluated using the EdU assay and colony formation experiment, revealing that suppressing TNFRSF21 significantly hindered the proliferation of A549 and H1299 cells (Fig. 2E,F).

Additionally, a cell colony formation assay demonstrated a down-regulation in the colony-forming ability upon TNFRSF21 depletion (Fig. 3A). TNFRSF21 shRNA treatment notably increased apoptosis in A549 and H1299 cells, as observed in the flow cytometry assay (Fig. 3A). Transwell assays were employed to investigate the effects of TNFRSF21 knockdown on the migration and invasion of LUAD cells. Suppression of TNFRSF21 led to a reduction in the migratory and invasive capacities of lung cancer cells (Fig. 3B,C), suggesting the involvement of TNFRSF21 in LUAD.

To understand the role of TNFRSF21, we conducted overexpression experiments on H460 and SPC-A1 cells, which exhibit low levels of TNFRSF21 expression. Using shRNA, we downregulated the expression of TNFRSF21 in H460 and SPC-A1 cells. qPCR data in Fig. 4A confirms TNFRSF21mRNA expression in H460 and SPC-A1 cells transfected with the overexpression plasmid-pcDNA3.1-TNFRSF21. Enhanced expression of TNFRSF21 significantly increased the capacity of H460 and SPC-A1 cells to form colonies, as depicted in Fig. 4B. Flow cytometry results revealed a significant proportion of CD44 and CD133 positive cells in H460 and SPC-A1 cells with increased TNFRSF21 expression (Fig. 4C–F). Moreover, transfected cells showed enhanced ability to form tumor spheres upon TNFRSF21 overexpression, as shown in Fig. 4G,H. TNFRSF21 also reduced the DDP sensitivity of H460 and SPC-A1 cells as illustrated in Fig. 4I. These findings suggest that increased TNFRSF21 expression enhances the maintenance of CSCs.

In LUAD, GEPIA findings revealed a favorable association between the CSC-like indicators CD44 and CD133 and TNFRSF21, as depicted in Fig. 5A,B. Fig. 5C,D demonstrates a notable increase in TNFRSF21 mRNA expression within CD44/CD133 positive A549 and H460 cells compared to CD44/CD133 negative LUAD cells. These findings indicate a positive correlation between increased TNFRSF21 expression and the level of stemness in LUAD.

ROS production plays a significant role in the inhibitory effects on lung CSCs. In Fig. 6A–D, it was observed that the use of TNFRSF21 shRNA resulted in a higher accumulation of MDA and ROS in DDP-resistant lung cancer cells compared to the control group. Furthermore, LUAD cells exhibited a decrease in the SOD and GSH. Moreover, we assessed the buildup of mitochondrial ROS by employing MitoSOX staining. This analysis unveiled that the depletion of TNFRSF21 triggered an elevation of mitochondrial ROS levels (Fig. 6E,F). Consistency in the staining of ROS levels was shown in DDP cells using MitoSOX (Fig. 6G,H). These findings suggest that the decrease of TNFRSF21 activates oxidative stress levels and mitochondrial ROS accumulation in LUAD cells.

In this investigation, a xenograft tumor mouse model of LUAD using A549/DDP cells allowed us to assess the impact of TNFRSF21 on the progression of lung carcinoma, as illustrated in Fig. 7A. Our study results revealed that mice injected with A549/DDP cells infected with TNFRSF21 exhibited reduced tumor sizes and decreased average tumor masses, as depicted in Fig. 7B,C. Furthermore, there was a gradual decrease in the rate of tumor growth over time, as shown in Fig. 7D. Fig. 7E demonstrates that the tumorigenicity was suppressed by the knockdown of TNFRSF21, further confirmed through flow cytometry analysis of Ki-67. These findings suggest that TNFRSF21 contributes to in vivo LUAD progression (Fig. 7F). Fig. 7G summarizes our overall research diagram.