Upregulation of spondin-2 indicates poorer clinical outcomes and induces radioresistance in gastric cancer

Introduction

Gastric cancer (GC) ranks as the fourth leading cause of cancer-related deaths worldwide and is the fifth most prevalent malignancy, imposing a significant healthcare burden globally. In 2020, over one million new cases were diagnosed, resulting in approximately 768,000 deaths (1,2). Advances in diagnostic techniques and treatment strategies, including surgery, chemotherapy, radiotherapy, and molecular targeting, have significantly improved survival rates. For early-stage GC patients who undergo surgery, the 5-year survival rate can reach 90% to 100% (3). However, for patients with advanced GC, the 5-year survival rate remains below 30% (3). Among molecular targeted therapies, ERBB2 (HER2) is the first well-validated target: approximately 15–20% of advanced GC patients harbor ERBB2 amplification, and anti-ERBB2 monoclonal antibodies (e.g., trastuzumab) combined with chemotherapy have become the standard first-line treatment for this subgroup, significantly prolonging overall survival (4,5). Another emerging target is Claudin18.2, a tight junction protein overexpressed in ~40–60% of GC patients; recent phase III trials have confirmed that Claudin18.2-targeted antibodies (e.g., zolbetuximab) improve survival in Claudin18.2-positive advanced GC, expanding treatment options for patients without ERBB2 amplification (6,7). Radiotherapy plays a crucial role in treating unresectable GC, serves as an essential adjuvant therapy after surgery in cases with R1 margins, and helps alleviate local symptoms in patients with locally advanced GC (8-12). Studies have confirmed that radiotherapy can reduce the local recurrence rate for patients with GC. However, its effectiveness is often hindered by radioresistance (13-16). Understanding the biological and molecular mechanisms underlying GC progression, invasion, metastasis, and radioresistance is essential for improving prognosis and treatment outcomes.

Spondin-2 (SPON2) encodes a secretory extracellular matrix (ECM) protein that shares homology with the mindin/F-spondin family. Initially, SPON2 was isolated from non-cancerous lung cells, where it was found to be downregulated in lung tumor cells (17). Functioning as a host innate immune modulator, SPON2 acts as a pattern-recognition molecule for microbial pathogens (18). A previous study has highlighted SPON2’s complex role in tumor progression and metastasis (19). However, its physiological function and molecular mechanisms in tumors remain controversial. A study suggests that SPON2 regulates the recruitment of M1-like macrophages and the Hippo pathway, participating in the endothelial-to-mesenchymal transition. This inhibits hepatocellular carcinoma (HCC) cell invasion and distant metastasis, thereby improving prognosis (19). Conversely, other studies have reported that SPON2 is upregulated in various carcinomas, including liver, colorectal, prostate, and lung cancers, and is associated with poor prognosis (17,20-22). In colorectal carcinoma, SPON2 expression was significantly higher in colorectal carcinoma tissues than in colorectal adenoma tissues (18).

Recent studies have also shown that SPON2 is overexpressed in patients with GC, where its overexpression correlates with poor prognosis (23-25). However, despite these findings, the exact mechanisms and functions of SPON2 in GC cells remain incompletely understood (26). Furthermore, the association between SPON2 and radiosensitivity in patients with GC has not been explored. Therefore, this study aimed to investigate the relationship between SPON2 expression and clinical outcomes in patients with GC. Additionally, we explored the association between SPON2 and radiosensitivity, as well as its potential underlying mechanisms. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1706/rc).

Methods

Bioinformatics analysis

cBioPortal (https://cbioportal.org) is an open-access, web-based platform designed for exploring cancer genomics data. It integrates data from public resources such as The Cancer Genome Atlas (TCGA), ensuring access to high-quality cancer genomics information. The TCGA-stomach adenocarcinoma (STAD) cohort data used in this study was accessed on March 15, 2024. cBioPortal stores data at the gene level and can combine it with de-identified clinical data, including overall survival (OS) and disease-free survival (DFS) intervals, facilitating various types of analyses (27,28). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

To evaluate the association between SPON2 expression and the clinicopathological characteristics and survival outcomes of patients with GC, we obtained publicly available data from cBioPortal. This dataset included SPON2 expression levels, demographic information, clinical data, and post-surgical survival outcomes for 415 patients with GC. Of these, 412 had complete demographic and clinical data. The clinical characteristics of these patients, including SPON2 expression levels, are presented in Table 1.

Cell culture and viral transfection

The human GC cell line HGC-27 was obtained from the Cell Bank of the Shanghai Institute of Biological Sciences (Shanghai, China) and authenticated via short tandem repeat profiling. HGC-27 is a poorly differentiated GC cell line that is widely used in GC radioresistance and oncogene function, as it recapitulates the aggressive biological features of advanced GC that are relevant to our research on SPON2-mediated tumor progression. To mitigate its inherent floating tendency, cells were cultured in RPMI-1640 medium (KeyGEN, Nanjing, China) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Grand Island, NY, USA) and 1% penicillin-streptomycin (Beyotime Institute of Biotechnology, Shanghai, China), with medium refreshed every 24 hours and subcultured at 70–80% confluence (to avoid overgrowth-induced floating). Cells were maintained in a humidified incubator at 37 ℃ with 5% CO₂.

HGC-27 cells were transfected with lentiviruses carrying either the human SPON2 overexpression cassette or an empty control cassette. The lentiviral vector backbone was pLVX-IRES-Puro (Clontech Laboratories, Inc., Mountain View, CA, USA), with the SPON2 insert corresponding to the full-length human SPON2 cDNA (GenBank accession: NM_001130089.3). Lentiviruses were packaged by GeneChem Co., Ltd. (Shanghai, China) with a final titer of 1×10⁸ transducing units (TU)/mL. For transfection, cells were seeded at 5×10⁴ cells/well in 6-well plates and infected with lentiviruses at a multiplicity of infection of 10, supplemented with 5 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA) to enhance transduction efficiency. After 48 hours of infection, stable cell clones were selected by culturing in medium containing 2 µg/mL puromycin (Sigma-Aldrich, cat. no. P8833) for 14 days. Cells transfected with the SPON2 overexpression lentivirus were designated as the Lenti-SPON2 group, while those transfected with the empty vector lentivirus were designated as the Lenti-control group. Twenty-four hours post-selection, SPON2 mRNA and protein expression levels in both groups were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively.

Cell proliferation assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology) following the manufacturer’s instructions. HGC-27 cells (5×10³ cells per well) were seeded in triplicate in 96-well plates and incubated for 0, 1, 2, 3, 4, 5, or 6 days post-transfection. At each time point, 10 µL of CCK-8 solution was added to each well, and cells were incubated at 37 ℃ for 1–4 hours. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Cell viability was calculated relative to the vehicle control (set at 100%).

Cell colony-forming test

Seventy-two hours post-transfection, the proliferative and clonogenic potential of HGC-27 cells was evaluated using a colony formation assay. To assess radiation sensitivity, both groups of cells were exposed to various doses of ionizing radiation (IR). After 10–14 days, colonies were stained with crystal violet (Beyotime Institute of Biotechnology), and colonies containing ≥50 cells were counted.

Plating efficiency (PE) was calculated as follows:

PE=numberofcoloniesformednumberofcellsplated×100%[1]

The survival fraction (SF) was determined using the formula:

SF=colonyformationrateofirradiatedcellscolonyformationrateofunirradiatedcells×100%[2]

A single-hit multitarget model was applied to generate the cell survival curve, and the sensitizing enhancement ratio (SER), mean lethal dose (D₀), and quasi-threshold dose (Dq) were calculated.

Wound healing assays

A wound healing assay was performed to assess the migration capacity of HGC-27 cells. Cells were seeded at 5×10⁵ cells per well in a 6-well plate. A uniform scratch was made in the monolayer using a 200-µL pipette tip. Wound closure was observed at 0 and 48 hours using an inverted microscope (TE-2000S, Nikon, Japan).

Cell cycle and apoptosis analysis via flow cytometry

Flow cytometry was used to evaluate the relationship between SPON2 and apoptosis, cell cycle arrest, and DNA damage repair (DDR) in HGC-27 cells.

For cell cycle analysis, DNA content was assessed. HGC-27 cells were seeded at 2×10⁵ cells per well in 6-well plates and incubated for 24 hours. Cells were then exposed to IR (4 Gy) or left untreated. After 24 hours, cells were washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol for at least 2 hours. Fixed cells were resuspended in 0.5 mL propidium iodide (PI)/RNase staining buffer (BD Biosciences, San Jose, CA, USA) and incubated in the dark at room temperature for 15 minutes before analysis via flow cytometry (BD Biosciences).

For apoptosis analysis, the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) was used according to the manufacturer’s instructions. Briefly, HGC-27 cells were seeded at 2×10⁵ cells per well in 6-well plates and incubated for 24 hours before being treated with or without IR (4 Gy). After 24 hours, both adherent and floating cells were collected, stained, and analyzed via flow cytometry to quantify apoptosis levels.

4',6-diamidino-2-phenylindole (DAPI) staining

To observe apoptotic nuclear morphology, DAPI staining was performed. HGC-27 cells were seeded at 2×10⁵ cells per well in 6-well plates and incubated for 24 hours. After incubation, cells were washed with PBS and fixed with 4% paraformaldehyde (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China) for 20 minutes. Fixed cells were stained with DAPI (Beyotime Institute of Biotechnology) in the dark at room temperature for 10 minutes. After washing with PBS, stained cells were observed under a fluorescence microscope.

Western blot analysis

Cells were washed with cold PBS and lysed in sodium dodecyl sulfate (SDS) lysis buffer containing phenylmethylsulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology). The lysates were centrifuged at 18,000 ×g for 20 minutes at 4 ℃, and protein concentrations were determined using a BCA assay (Beyotime Institute of Biotechnology). Equal amounts of protein (20 µg) were separated via 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 ℃ with primary antibodies against: SPON2 (Abcam, Cambridge, UK; cat. no. ab12345; 1:500), BAX (Cell Signaling Technology, Danvers, MA, USA; cat. no. 5023; 1:5,000), cytochrome c (Cell Signaling Technology; cat. no. 11940; 1:1,000), SMAC (Cell Signaling Technology; cat. no. 12482; 1:3,000), BCL-2 (Cell Signaling Technology; cat. no. 2870; 1:1,000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology; cat. no. 5174; 1:10,000; used as internal control). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000) were applied for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) detection (WBKLS0500, Millipore, Bedford, MA, USA).

Statistical analysis

Statistical analysis was performed using SAS 8.1 software, and GraphPad Prism 8.0 was used to generate figures. Continuous variables were reported as mean ± standard deviation (SD) or median with interquartile range (IQR). Categorical variables were expressed as counts and percentages. Comparisons between two groups were conducted using the Student’s t-test, Wilcoxon rank sum test, or Chi-squared test, as appropriate. Kaplan-Meier survival curves were used to analyze progression-free survival (PFS), disease-specific survival (DSS), DFS, and OS, with group differences assessed using the log-rank test. Cox proportional hazards regression models were used for univariate and multivariate survival analyses. All functional cell experiments included at least three replicates per group. A P value <0.05 was considered statistically significant.

Results

SPON2 expression is upregulated in human GC tissues

Using data from the TCGA database, we analyzed 415 GC specimens and 34 normal gastric tissues from patients with GC. SPON2 expression was significantly elevated in primary tumor specimens compared with normal gastric tissues (P<0.001, Figure 1).

Figure 1 SPON2 expression in normal and primary tumor specimens. STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas.

Correlation between SPON2 expression and GC clinical characteristics

To further examine the relationship between SPON2 expression and clinical features, patients with GC were classified into SPON2-high and SPON2-low groups based on the median expression value. The differences in clinical characteristics between these two groups are summarized in Table 1.

The results indicate a significant correlation between SPON2 expression and tumor size—patients with higher SPON2 expression exhibited larger tumors (P<0.001).

High SPON2 expression predicts poor clinical outcomes of patients with GC

A prognostic evaluation of SPON2 expression was performed using TCGA data. The results showed that patients with high SPON2 expression had significantly poorer OS than those with low SPON2 expression (P=0.01, Figure 2A). Specifically, patients with low SPON2 expression had a longer median survival time compared to those with high SPON2 expression (15.6 vs. 13.2 months).

Figure 2 Kaplan-Meier survival curves for different clinical endpoints stratified by SPON2 expression in gastric cancer patients from the TCGA-STAD cohort. (A) Kaplan-Meier curve showing OS of GC patients grouped by SPON2 expression. (B) Kaplan-Meier curve analyzing PFS of GC patients stratified by SPON2 expression. (C) Kaplan-Meier curve evaluating DFS of GC patients grouped by SPON2 expression. (D) Kaplan-Meier curve assessing DSS of GC patients stratified by SPON2 expression. DFS, disease-free survival; DSS, disease-specific survival; GC, gastric cancer; OS, overall survival; PFS, progression-free survival; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas.

No statistically significant correlations were observed between SPON2 expression and PFS, DSS, and DFS (Figure 2B-2D).

Univariate Cox regression analysis identified SPON2 expression, tumor stage, T stage, and N stage as significant risk factors for OS (Table 2). Multivariate Cox regression analysis further confirmed that SPON2 expression, T stage, N stage, and age were independent predictors of OS. Specifically, SPON2 expression was identified as an independent prognostic indicator for OS in patients with GC [hazard ratio (HR) =1.435, 95% confidence interval (CI): 1.023–2.013].

SPON2 overexpression promotes GC cells proliferation and migration

SPON2 is upregulated in various cancer types (17,20-22), suggesting that its overexpression may accelerate GC progression. To investigate this hypothesis, we examined the biological function of SPON2 in HGC-27 cells by inducing overexpression using a lentiviral vector (Figure 3A,3B).

Figure 3 Effect of SPON2 overexpression on GC cell proliferation and migration. (A) Western blot analysis of SPON2 expression in HGC-27 cells following lentiviral transfection. (B) RT-qPCR analysis of SPON2 expression. (C) SPON2 overexpression increased HGC-27 cell proliferation. (D,E) SPON2 overexpression increased colony formation. (F,G) SPON2 overexpression enhanced cell migration. *, P<0.05; ***, P<0.001. GC, gastric cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

SPON2 overexpression significantly enhanced proliferation compared to control HGC-27 cells (Figure 3C). Additionally, the colony formation assay demonstrated that upregulation of SPON2 markedly increased the number of colonies formed by HGC-27 cells (Figure 3D,3E).

To assess the impact of SPON2 on cell migration, a wound healing assay was performed. The results indicated that SPON2 overexpression enhanced cell migration (Figure 3F,3G).

SPON2 overexpression induces radioresistance in HGC-27 cells

To investigate the impact of SPON2 overexpression on radiosensitivity, a colony formation assay was performed. HGC-27 cells in the Lenti-control and Lenti-SPON2 groups were exposed to increasing doses of IR (0–8 Gy). The survival rate of HGC-27 cells decreased with increasing radiation doses, with minimal survival observed at 6 Gy. However, the survival curve indicated that SPON2-overexpressing cells exhibited higher survival rates following irradiation (Figure 4).

Figure 4 Effect of SPON2 overexpression on radiosensitivity. (A) Representative colony formation assay images. (B) SPON2 overexpression increased the survival proportion of HGC-27 cells following radiation exposure.

The SER in the SPON2-overexpression group was 0.68-fold lower than that of the control group (Table 3), indicating that SPON2 overexpression contributes to radioresistance in HGC-27 cells. These findings suggest that SPON2 may play a role in GC radioresistance and its upregulation could potentially counteract the effects of radiotherapy.

Upregulation of SPON2 is associated with apoptosis, cell cycle arrest and DDR in HGC-27 cells

To further explore the role of SPON2 in radioresistance, flow cytometry was performed to assess apoptosis in HGC-27 cells with or without radiation treatment.

The results showed that irradiation (6 Gy) significantly increased apoptosis in the Lenti-control group. However, the increase in apoptosis was significantly lower in the Lenti-SPON2 group, suggesting that SPON2 overexpression promotes cellular resistance to radiation-induced apoptosis (Figure 5A). In the SPON2-overexpression group, apoptosis rates were significantly lower following radiation treatment (7.33%±0.51% vs. 25.37%±0.83%, P<0.001, Figure 5B).

Figure 5 SPON2 is associated with apoptosis, cell cycle arrest, and DDR induced by irradiation in HGC-27 cells. (A,B) Apoptotic rates were detected using flow cytometry. The apoptosis rate in the SPON2 overexpression group was lower than that in the control groups. (C,D) Cell cycle distribution was analyzed using flow cytometry, showing a decrease in the G2/M phase proportion in the SPON2 overexpression group. (E) Confocal microscopy images of HGC-27 cells stained with DAPI (blue) for the nucleus and an antibody against γH2AX (green) for DNA damage. *, P<0.05; ***, P<0.001. DAPI, 4',6-diamidino-2-phenylindole; DDR, DNA damage repair.

Figure 5C,5D further revealed that radiation treatment (6 Gy) significantly altered cell cycle distribution, causing a decrease in the G1 phase population and an increase in the G2 phase population. In contrast, SPON2 overexpression decreased the G2 cell population in HGC-27 cells. The proportion of cells in the G2/M phase in the SPON2 overexpression group was 7.1%±0.40%, significantly lower than that in the control group (17.23%±0.83%). Additionally, SPON2 overexpression reduced radiation-induced G2/M cell accumulation. The G2/M cell proportion in the Radiotherapy + SPON2 overexpression group was 22.73%±0.61%, which was significantly lower than that in the Radiotherapy-alone group (37.47%±0.67%) (Figure 5D, P<0.01). To investigate the mechanisms through which SPON2 contributes to radioresistance, immunofluorescence staining was performed to assess phosphorylated histone H2AX (γ-H2AX), a marker of DNA damage. The γ-H2AX foci count in SPON2-overexpressing cells was lower than that in control cells after irradiation (Figure 5E). These findings indicate that SPON2 enhances the repair of radiation-induced DNA damage in HGC-27 cells.

SPON2 overexpression influenced apoptosis-related biomarker expressions

The expression levels of pro-apoptotic proteins Bax, Cytc, and SMAC, as well as the anti-apoptotic protein Bcl-2, were analyzed using western blotting. Before and after irradiation, SPON2 overexpression led to a reduction in the levels of pro-apoptotic proteins Bax, Cytc, and SMAC, while Bcl-2 expression was increased in HGC-27 cells (Figure 6).

Figure 6 SPON2 overexpression reduces the expression levels of Bax, Cytc, and SMAC while increasing Bcl-2 expression before and after irradiation.

Discussion

In this study, we identified SPON2 as a potential prognostic marker for patients with GC. By comparing SPON2 expression levels in GC tissues and normal gastric tissues, we found that SPON2 was significantly upregulated in GC tissues. A strong correlation was observed between high SPON2 expression and larger tumor size in patients with GC, which was associated with poorer OS. Additionally, SPON2 plays a crucial role in radiation resistance, as its overexpression induces cellular resistance to radiation, potentially affecting cell survival and DNA repair mechanisms following radiation exposure.

SPON2 (DIL-1, mindin, spondin-2), a member of the F-spondin family of secreted ECM proteins (29), is essential for malignancy development. While SPON2 dysregulation has been reported to influence the diagnosis and progression of various cancers (21,22,30), its physiological function and molecular mechanisms in tumors remain controversial. A previous study reported that SPON2 inhibits HCC cells from invading adjacent tissues and migrating to distant sites, thereby improving prognosis (19). However, another study indicated that patients with HCC who exhibited elevated SPON2 protein expression experienced worse survival outcomes compared to those with lower SPON2 levels (20). In this study, we found that SPON2 was upregulated in GC tissues compared to normal gastric tissues. Furthermore, SPON2 expression was significantly correlated with tumor size, and patients with high SPON2 expression had worse survival outcomes than those with lower SPON2 levels. These findings are consistent with previous studies on GC (23-25). No prior research has explored whether SPON2 serves as a prognostic predictor for patients with GC. In our study, we employed multivariate Cox regression analysis and confirmed that high SPON2 expression is an independent risk factor for OS in patients with GC, aligning with findings in other cancers (20,21,31). These results support our hypothesis that SPON2 plays a role in GC progression and could serve as a prognostic marker.

A previous study has demonstrated that downregulation of SPON2 inhibits cell invasion and migration in vitro (32). SPON2 overexpression has been shown to promote invasion, epithelial-to-mesenchymal transition, and migration of adenocarcinoma cells in vitro (33). Additionally, SPON2 has been implicated in mediating metastasis, proliferation, and invasion in colorectal cancer cells both in vitro and in vivo (34). Treatment with recombinant SPON2 protein has been shown to enhance GC cell motility in vitro (25). Furthermore, SPON2 silencing has been associated with a reduction in laryngeal squamous cell carcinoma cell proliferation through the suppression of PI3K/AKT signaling activation (31). To examine the effect of SPON2 on GC progression, we conducted in vitro experiments using HGC-27 cells, and the results demonstrated that SPON2 overexpression led to increased proliferation and migration of GC cells. Our findings also indicate that SPON2 overexpression induced G1 phase arrest and reduced the number of cells in the G2 phase. Notably, upon SPON2 overexpression in HGC-27 cells, we observed an increased proportion of apoptotic cells. In addition, pro-apoptotic proteins Bax, Cytc, and SMAC were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. These findings suggest that SPON2 overexpression promotes tumor growth through the activation of multiple molecular pathways in HGC-27 cells.

Although SPON2 has been associated with poorer prognosis, whether it affects the radiosensitivity of patients with GC and subsequently influences their prognosis has not yet been explored. In this study, we found that SPON2 overexpression in HGC-27 cells significantly increased cell survival following radiation therapy, indicating a strong association between SPON2 expression and radioresistance in GC. Further investigations were conducted to elucidate the potential mechanisms underlying SPON2-induced radioresistance, revealing that SPON2 overexpression resulted in lower levels of DNA damage, a decreased proportion of apoptotic cells, and a reduction in G2-phase cell cycle arrest following radiation exposure. Cancer outcomes after radiation therapy are largely dependent on cellular responses to DNA damage, including cell cycle arrest and apoptosis (35). We observed that SPON2 overexpression desensitized HGC-27 cells to radiation and reduced radiation-induced DNA damage and apoptosis. These findings suggest that targeting SPON2 could be a potential therapeutic strategy to enhance the efficacy of radiation therapy and improve survival outcomes in patients with GC. Although this study, along with previous research, has demonstrated SPON2’s role in apoptosis and cell cycle regulation, further investigation is required to determine the specific signaling pathways through which SPON2 modulates the DDR following radiation exposure. In HGC-27 cells, with or without radiation treatment, SPON2 overexpression significantly reduced the levels of pro-apoptotic proteins Bax, Cytc, and SMAC, while increasing the expression of the anti-apoptotic protein Bcl-2.

Our study has several limitations that should be addressed in future research. First, cell line specificity: we solely used the HGC-27 cell line, a poorly differentiated GC model. While HGC-27 is validated for studying radioresistance, its biological behavior may not fully represent all GC subtypes. Results may vary across genetic backgrounds, highlighting the need for validation in multiple cell lines. Second, lack of in vivo validation: our in vitro findings were not confirmed in animal models. Xenograft studies combining SPON2 modulation with radiotherapy would better recapitulate the tumor microenvironment and clarify whether SPON2 affects radiation response in vivo. Third, clinical data gaps: the TCGA-STAD cohort lacks detailed radiotherapy records, preventing us from directly linking SPON2 expression to clinical radiotherapy outcomes in patients. Correlative studies using GC patient samples with known radiotherapy histories are needed to validate SPON2 as a predictive marker. Fourth, mechanistic incompleteness: while we observed SPON2 reduces γ-H2AX foci and apoptosis, we did not identify the upstream signaling or downstream effectors mediating these changes.

To address these gaps, future research should focus on three key areas: (I) investigate whether SPON2 directly binds to DDR components to inhibit DNA damage signaling, or indirectly regulates DDR via upstream pathways. Co-immunoprecipitation and proximity ligation assays could map these interactions; (II) clarify how SPON2 modulates Bcl-2 family proteins. Luciferase reporter assays and ubiquitination assays would help dissect these mechanisms; (III) explore SPON2’s function in different GC subtypes. Co-culture models or patient-derived organoids could address subtype specificity; and (IV) test whether SPON2 silencing enhances radiotherapy efficacy in vivo, and whether this effect is augmented when combined with DDR inhibitors.

Conclusions

In summary, SPON2 upregulation promotes GC cell proliferation and is associated with poorer OS in patients with GC. SPON2 overexpression induces cellular resistance to radiation and decreases radiation-induced DNA damage and apoptosis. Thus, SPON2 may serve as a potential prognostic indicator for GC, and its inhibition could be a viable strategy for overcoming both intrinsic and acquired radioresistance in patients with GC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1706/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1706/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1706/prf

Funding: This work was supported by Research project of Jiangsu Provincial Health Commission (No. ZKY2019006).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1706/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-49. [Crossref] [PubMed]
2. World Health Organization. International Agency for Research on Cancer. GLOBOCAN 2020: stomach cancer fact sheet. 2020. Accessed July 9, 2021. Available at: https://gco.iarc.fr/today/data/factsheets/cancers/7-Stomach-fact-sheet.pdf
3. Song Z, Wu Y, Yang J, et al. Progress in the treatment of advanced gastric cancer. Tumour Biol 2017;39:1010428317714626. [Crossref] [PubMed]
4. Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010;376:687-97. [Crossref] [PubMed]
5. Shitara K, Bang YJ, Iwasa S, et al. Trastuzumab Deruxtecan in Previously Treated HER2-Positive Gastric Cancer. N Engl J Med 2020;382:2419-30. [Crossref] [PubMed]
6. Klempner SJ, Lee KW, Shitara K, et al. ILUSTRO: Phase II Multicohort Trial of Zolbetuximab in Patients with Advanced or Metastatic Claudin 18.2-Positive Gastric or Gastroesophageal Junction Adenocarcinoma. Clin Cancer Res 2023;29:3882-91.
7. Covert WM, Rogers JE. Future Landscape of Anti-Claudin 18.2 Antibodies in Gastric Adenocarcinoma. Antibodies (Basel) 2025;14:26. [Crossref] [PubMed]
8. Loi M, Verheij M, Nuyttens J, et al. From twilight to starlight? Debating the role of chemoradiotherapy in gastric cancer in the D2 dissection era. Radiol Med 2024;129:1710-9. [Crossref] [PubMed]
9. Dikken JL, Jansen EP, Cats A, et al. Impact of the extent of surgery and postoperative chemoradiotherapy on recurrence patterns in gastric cancer. J Clin Oncol 2010;28:2430-6. [Crossref] [PubMed]
10. Ho VKY, Jansen EPM, Wijnhoven BPL, et al. Adjuvant Chemoradiotherapy for Non-Pretreated Gastric Cancer. Ann Surg Oncol 2017;24:3647-57. [Crossref] [PubMed]
11. Merchant SJ, Kong W, Mahmud A, et al. Palliative Radiotherapy for Esophageal and Gastric Cancer: Population-Based Patterns of Utilization and Outcomes in Ontario, Canada. J Palliat Care 2023;38:157-66. [Crossref] [PubMed]
12. Takeda K, Sakayauchi T, Kubozono M, et al. Palliative radiotherapy for gastric cancer bleeding: a multi-institutional retrospective study. BMC Palliat Care 2022;21:52. [Crossref] [PubMed]
13. Park SH, Sohn TS, Lee J, et al. Phase III Trial to Compare Adjuvant Chemotherapy With Capecitabine and Cisplatin Versus Concurrent Chemoradiotherapy in Gastric Cancer: Final Report of the Adjuvant Chemoradiotherapy in Stomach Tumors Trial, Including Survival and Subset Analyses. J Clin Oncol 2015;33:3130-6. [Crossref] [PubMed]
14. Yu JI, Lim DH, Lee J, et al. Necessity of adjuvant concurrent chemo-radiotherapy in D2-resected LN-positive gastric cancer. Radiother Oncol 2018;129:306-12. [Crossref] [PubMed]
15. Meng K, Lu H. Clinical application of high-LET radiotherapy combined with immunotherapy in malignant tumors. Precis Radiat Oncol 2024;8:42-6. [Crossref] [PubMed]
16. Varghese TP, John A, Mathew J. Revolutionizing cancer treatment: The role of radiopharmaceuticals in modern cancer therapy. Precis Radiat Oncol 2024;8:145-52. [Crossref] [PubMed]
17. Yuan X, Bian T, Liu J, et al. Spondin2 is a new prognostic biomarker for lung adenocarcinoma. Oncotarget 2017;8:59324-32. [Crossref] [PubMed]
18. He YW, Li H, Zhang J, et al. The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens. Nat Immunol 2004;5:88-97. [Crossref] [PubMed]
19. Zhang YL, Li Q, Yang XM, et al. SPON2 Promotes M1-like Macrophage Recruitment and Inhibits Hepatocellular Carcinoma Metastasis by Distinct Integrin-Rho GTPase-Hippo Pathways. Cancer Res 2018;78:2305-17. [Crossref] [PubMed]
20. Feng Y, Hu Y, Mao Q, et al. Upregulation of Spondin-2 protein expression correlates with poor prognosis in hepatocellular carcinoma. J Int Med Res 2019;47:569-79. [Crossref] [PubMed]
21. Zhang Q, Wang XQ, Wang J, et al. Upregulation of spondin-2 predicts poor survival of colorectal carcinoma patients. Oncotarget 2015;6:15095-110. [Crossref] [PubMed]
22. Lucarelli G, Rutigliano M, Bettocchi C, et al. Spondin-2, a secreted extracellular matrix protein, is a novel diagnostic biomarker for prostate cancer. J Urol 2013;190:2271-7. [Crossref] [PubMed]
23. Kang HG, Kim WJ, Noh MG, et al. SPON2 Is Upregulated through Notch Signaling Pathway and Promotes Tumor Progression in Gastric Cancer. Cancers (Basel) 2020;12:1439. [Crossref] [PubMed]
24. Lu H, Feng Y, Hu Y, et al. Spondin 2 promotes the proliferation, migration and invasion of gastric cancer cells. J Cell Mol Med 2020;24:98-113. [Crossref] [PubMed]
25. Kuramitsu S, Masuda T, Hu Q, et al. Cancer-associated Fibroblast-derived Spondin-2 Promotes Motility of Gastric Cancer Cells. Cancer Genomics Proteomics 2021;18:521-9. [Crossref] [PubMed]
26. Rajkumar T, Vijayalakshmi N, Gopal G, et al. Identification and validation of genes involved in gastric tumorigenesis. Cancer Cell Int 2010;10:45. [Crossref] [PubMed]
27. Cerami E, Gao J, Dogrusoz U, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012;2:401-4. [Crossref] [PubMed]
28. Gao J, Aksoy BA, Dogrusoz U, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 2013;6:pl1. [Crossref] [PubMed]
29. Klar A, Baldassare M, Jessell TM. F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension. Cell 1992;69:95-110. [Crossref] [PubMed]
30. Simon I, Liu Y, Krall KL, et al. Evaluation of the novel serum markers B7-H4, Spondin 2, and DcR3 for diagnosis and early detection of ovarian cancer. Gynecol Oncol 2007;106:112-8. [Crossref] [PubMed]
31. Ni H, Ni T, Feng J, et al. Spondin-2 is a novel diagnostic biomarker for laryngeal squamous cell carcinoma. Pathol Res Pract 2019;215:286-91. [Crossref] [PubMed]
32. Ma HM, Yu M, Wu C, et al. Overexpression of Spondin-2 Is Associated with Recurrence-Free Survival in Patients with Localized Clear Cell Renal Cell Carcinoma. Dis Markers 2020;2020:5074239. [Crossref] [PubMed]
33. Wu M, Kong D, Zhang Y. SPON2 promotes the bone metastasis of lung adenocarcinoma via activation of the NF-κB signaling pathway. Bone 2023;167:116630. [Crossref] [PubMed]
34. Chandrasinghe P, Stebbing J, Warusavitarne J. The MACC1-SPON2 axis: a new biomarker and therapeutic target in colorectal cancer. Oncogene 2017;36:1474-5. [Crossref] [PubMed]
35. Goldstein M, Kastan MB. The DNA damage response: implications for tumor responses to radiation and chemotherapy. Annu Rev Med 2015;66:129-43. [Crossref] [PubMed]