CNN3 promotes angiogenesis in osteosarcoma, associated with upregulating VEGF-A and enhancing endothelial cell activity

Introduction

Osteosarcoma is characterised by high aggressiveness and metastatic potential. It typically arises during adolescence, a period of vigorous skeletal growth (1). According to epidemiological studies, the incidence of osteosarcoma is more than 4.4 per million individuals for the range 0–24 years, ranking highest among malignant bone tumours (2-4). Although advances in surgery, chemotherapy, and other treatment modalities have improved outcomes, the prognosis for osteosarcoma patients continues to be unfavourable, with metastatic or recurrent cases showing low survival rates of merely 25% at 5 years (5). Therefore, developing targeted therapies against metastatic progression is critically needed to improve survival.

Angiogenesis supplies the biological infrastructure for tumour growth, invasion, and metastasis. Anti-angiogenic therapy inhibits tumours by reducing angiogenesis in the surrounding microenvironment, thereby maintaining tumour cells in a hypoxic and nutrient-deprived state (6). It has emerged as a promising therapeutic approach alongside surgery, radiotherapy, and chemotherapy (7).

Calponin 3 (CNN3) is an actin-binding protein belonging to the calponin family (8). It promotes the progression of glioma, diffuse large B-cell lymphoma and colon cancer (9-11). Our previous study showed that CNN3 is aberrantly expressed in osteosarcoma specimens and that its expression correlates with tumour size, stage, and metastasis (12). However, its regulatory role in osteosarcoma angiogenesis remains unclear.

Therefore, this study aimed to investigate the role of CNN3 on angiogenesis in osteosarcoma and to explore the underlying mechanisms involved to provide a theoretical foundation for its potential clinical application. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1846/rc).

Methods

Sample collection

Twenty osteosarcoma specimens were obtained from patients diagnosed in our hospital. The clinicopathological and demographic characteristics of all enrolled osteosarcoma patients and tumour samples are shown in Table S1. Pathological tissue samples were obtained via surgery from January 2020 to December 2021, fixed with 4% formaldehyde, and retained after paraffin embedding. None of the patients had received chemotherapy or radiotherapy prior to surgery. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the First Affiliated Hospital, Army Medical University (Approval No. KY201927) and informed consent was obtained from all individual participants.

CNN3 and CD31 immunohistochemistry (IHC)

CNN3 and CD31 levels in twenty paraffin-embedded osteosarcoma specimens were detected using IHC using the same method described in our previous study (12). Rabbit polyclonal anti-CNN3 antibody (1:25; OM273307, Omnimabs, Alhambra, CA, USA) and rabbit monoclonal anti-CD31 antibody (1:200; ab76533, Abcam, Cambridge, MA, USA) In order to quantify CNN3 expression, five random fields were selected per section and scored according to the percentage of positive cells (zero for <1%, one for 1–25%, two for 26–50%, three for 51–75%, and four for >75%) and staining intensity (zero for unstained, one for yellow, two for tan, and three for brown). The total score of CNN3 staining was quantified by multiplying the positive cell score with the staining intensity score. The quantification of CD31 staining were expressed as microvessel density (MVD). IHC results were independently assessed by three pathologists, and the average score was used for analysis.

Cell culture and transfection

Osteosarcoma cell lines Saos-2 (CL-0202) and MG-63 (CL-0157) cells and human umbilical vein endothelial cells (HUVECs, CP-H082) were purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China). All cells were cultured according to the methods of Procell. During the logarithmic growth phase, Saos-2 and MG-63 cells were transfected with either negative control small interfering RNA (siRNA, NC group), siRNA targeting CNN3 (si-CNN3, target sequence: CCCTACAGATGGGTACCAA), empty vector pcDNA3.1+ (vector group), or a CNN3 overexpression vector (CNN3-OE) using Lipofectamine 2000. The CNN3 overexpression vector was constructed by inserting CNN3 coding sequence into the pcDNA3.1+ plasmid between the HindIII and EcoRI restriction sites using synthetic DNA (IGEbio, Guangzhou, China).

Cell counting assay

In the Transwell co-culture system, HUVECs were plated in the lower chamber. MG-63 and Saos-2 cells from the CNN3-OE, vector, si-CNN3, and NC groups were added to the upper chamber. Co-culture was conducted in a 5% CO₂ incubator for 0, 24, 48, or 72 h. Cell numbers were determined using a haemocytometer. Three independent experiments were performed with three technical replicates.

Scratch-wound healing assay

A Transwell co-culture system was used to perform scratch-wound healing assay. HUVECs (lower chamber) were grown to confluence, and a standardized wound was created with a 10 µL pipette tip. After phosphate-buffered saline washing, differentially transfected MG-63/Saos-2 cells (CNN3-OE, vector, si-CNN3, or NC; upper chamber) were co-cultured with HUVECs for 24 h in 5% CO₂. The scratch width at 0 (W₀) and 24 h (W₂₄) was observed under a microscope, and the extent of wound healing was calculated using the formula: Average healing (%) = [(W₀ – W₂₄)/W₀] × 100. Three independent experiments were performed with one technical replicate.

Transwell-Matrigel invasion assay

Transfected MG-63 and Saos-2 cells (CNN3-OE, vector, si-CNN3, or NC) were seeded into the lower chamber. After cell attachment, culture medium was replaced with 600 µL of serum-free medium. HUVECs (2×10⁴ cells) were seeded into the upper chamber pre-coated with Matrigel and cultured in complete medium. Transwell co-culture system was cultured and invading HUVECs were stained using methods previously described (12). The number of invading HUVECs was counted in three fields at 200× magnification. Data represent the mean invading cells per field. Three independent experiments were performed with one technical replicate.

Vascular mimicry assay

Matrigel-coated lower chambers were seeded with HUVECs, while transfected MG-63/Saos-2 cells (CNN3-OE, vector, si-CNN3, or NC) were plated in upper chambers. After 6 h co-culture (37 ℃, 5% CO₂), micrographs were captured from randomly selected fields of view. The number of branch nodes in five random fields was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Three independent experiments were performed with one technical replicate.

Western blotting

Total protein was extracted and target protein expression levels were analysed by Western blotting using the following primary antibodies: anti-CNN3 (1:500, OM273307, Omnimabs), anti-vimentin (1:1,000, ab16700, Abcam), anti-vascular endothelial (VE)-cadherin (1:1,000, ab318152, Abcam), anti-Neuronal (N)-cadherin (1:1,000, ab207608, Abcam), anti-phosphorylated Akt (p-Akt, 1:2,000, #4060, Cell Signaling Technology, Danvers, MA, USA), anti-Akt (1:1,000, #9272, Cell Signaling Technology) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2,000, ab181602, Abcam) as a loading control, following our established protocol (12). Three independent experiments were performed with one technical replicate.

Enzyme-linked immunosorbent assay (ELISA)

Following transfection, cell culture supernatants were harvested, centrifuged at 12,000 rpm for 10 min to remove debris, and assessed for vascular endothelial growth factor-A (VEGF-A) concentration using an ELISA kit (D711056-0048, Sangon Biotech, Shanghai, China). Three independent experiments were performed with three technical replicates.

Statistical analysis

Quantitative data represent mean ± standard deviation. The relationship between CNN3 expression and MVD in osteosarcoma tissues was assessed using linear regression. Differences between experimental groups (si-CNN3 vs. NC; CNN3-OE vs. vector) were analysed with independent samples t-tests. HUVEC proliferation was compared by two-way ANOVA. Statistical significance was defined as P<0.05. All computations were executed in SPSS 22.0 (IBM Corp., Armonk, NY, USA).

Results

Correlation between CNN3 and CD31 expression

CNN3 and CD31 levels in osteosarcoma specimens was detected and the representative IHC images are shown in Figure 1A. To analyse the correlation between CNN3 and CD31. Their expression levels were quantified as CNN3 expression score (8.55±2.89; Figure 1B) or MVD (48.50±17.37; Figure 1C), respectively. Pearson’s correlation analysis (Figure 1D) showed a positive association between CNN3 expression and MVD (r=0.7264, P=0.0003), suggesting that CNN3 may contribute to vascularization in osteosarcoma.

Figure 1 Expressions of CNN3 and CD31 in osteosarcoma tissue. (A) Representative immunohistochemical images showing CNN3 and CD31 at original magnifications of 40× and 200× (the 200× areas are outlined by red boxes in the 40× views); (B) CNN3 expression scores in 20 osteosarcoma samples. (C) MVD (assessed by CD31 staining) in 20 osteosarcoma samples. (D) Correlation analysis between CNN3 expression score and MVD. CNN3, calponin 3; MVD, microvessel density.

Expression of CNN3 in osteosarcoma cells after transfection

CNN3 expression was lower in the si-CNN3 group than in the NC group and higher in the CNN3-OE group than in the vector group (Figure 2). These results confirmed that CNN3 in both MG-63 and Saos-2 cells was silenced or overexpressed.

Figure 2 CNN3 protein levels assessed by western blotting. (A) Western blotting image of CNN3 level in MG-63 cells from each group; (B) relative expression of CNN3 in MG-63 cells; (C) Western blotting image of CNN3 level in Saos-2 cells; (D) relative expression of CNN3 in Saos-2 cells. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, overexpression vector; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Effects of CNN3 on HUVEC proliferation

In the HUVECs + MG-63 co-culture system, HUVEC numbers were lower in the si-CNN3 group than in the NC group at 48 and 72 h (Figure 3A). Conversely, HUVEC numbers were higher in the CNN3-OE group than in the vector group at 48 and 72 h (Figure 3B). In the HUVECs + Saos-2 co-culture system, cell numbers followed the same trend (Figure 3C,3D). Two-way ANOVA indicated that the effects of both CNN3 silencing and overexpression were statistically significant. These findings indicate that CNN3 silencing in MG-63 and Saos-2 cells inhibited HUVEC proliferation, whereas CNN3 overexpression promoted it.

Figure 3 CNN3 modulates HUVEC proliferation in co-culture systems. (A,B) Number of HUVECs at different time points when co-cultured with MG-63 cells transfected with NC, si-CNN3, Vector, or CNN3-OE; (C,D) number of HUVECs at different time points when co-cultured with similarly manipulated Saos-2 cells. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; HUVECs, human umbilical vein endothelial cells; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Effect of CNN3 on the migration of HUVECs

In the HUVECs + MG-63 co-culture system, the healing rate of HUVECs in the si-CNN3 group after 24 h of culture was lower than that in the NC group, whereas the healing rate in the CNN3-OE group was higher than that in the vector group (Figure 4A,4B). In the HUVECs + Saos-2 co-culture system, the healing rate of HUVECs in each group showed the same trend as in the MG-63 + HUVECs co-culture system (Figure 4C,4D). These results indicate that silencing CNN3 in MG-63 and Saos-2 cells inhibited, while overexpressing CNN3 promoted, HUVEC migration.

Figure 4 CNN3 modulates HUVEC migration in co-culture systems. (A,B) Healing rate of HUVECs when co-cultured with MG-63 cells transfected with NC, si-CNN3, Vector, or CNN3-OE. (C,D) Healing rate of HUVECs when co-cultured with similarly manipulated Saos-2 cells. Panels (A,C) are the representative images of scratch wound healing assay at 0 and 24 h (magnification: ×100). Panels (B,D) are the bar graphs representing the average healing rate of each group. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; HUVECs, human umbilical vein endothelial cells; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Effect of CNN3 on the invasion of HUVECs

In the HUVECs + MG-63 co-culture system, the number of invading HUVECs in the si-CNN3 group was lower than in the NC group, whereas the number in the CNN3-OE group was higher than in the vector group (Figure 5A,5B). In the HUVECs + Saos-2 co-culture system, the number of invading HUVECs in each group followed the same trend as observed in the MG-63 + HUVECs co-culture system (Figure 5C,5D). The results of the Transwell invasion assay demonstrated that CNN3 silencing in MG-63 and Saos-2 cells reduced, whereas CNN3 overexpression increased, the invasive ability of HUVECs.

Figure 5 CNN3 modulates HUVEC invasion ability in co-culture systems assessed by Transwell-Matrigel invasion assay. (A,B) Number of invading HUVECs when co-cultured with MG-63 cells transfected with NC, si-CNN3, Vector, or CNN3-OE. (C,D) Number of invading HUVECs when co-cultured with similarly manipulated Saos-2 cells. Panels (A,C) are the representative images of Transwell-Matrigel invasion assay (magnification: ×200). Panels (B,D) are the bar graphs representing the average number of invading HUVECs of each group. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; HUVECs, human umbilical vein endothelial cells; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Effect of CNN3 on the expression of endothelial-to-mesenchymal transition (EndMT)-related proteins in HUVECs

In the HUVECs + MG-63 co-culture system, compared with the NC group, the relative expression of vimentin and N-cadherin decreased, whereas VE-cadherin expression increased, in the si-CNN3 group. By contrast, compared with the vector group, the relative expression of vimentin and N-cadherin increased, whereas VE-cadherin decreased, in the CNN3-OE group (Figure 6A,6B). In the HUVECs + Saos-2 co-culture system, the expression trends of vimentin, N-cadherin, and VE-cadherin were consistent with those observed in the MG-63 + HUVECs co-culture system (Figure 6C,6D). These results suggest that CNN3 modulates the expression of EndMT-related proteins in HUVECs.

Figure 6 CNN3 modulates the expression of endothelial-to-mesenchymal transition-related proteins in HUVECs in co-culture systems. (A,B) Expression of vimentin, N-cadherin, and VE-cadherin in HUVECs when co-cultured with MG-63 cells transfected with NC, si-CNN3, Vector, or CNN3-OE. (C,D) Expression of vimentin, N-cadherin, and VE-cadherin in HUVECs when co-cultured with similarly manipulated Saos-2 cells. Panels (A,C) are the representative images of Western blotting. Panels (B,D) are the bar graphs representing the relative expression of vimentin, N-cadherin, and VE-cadherin in HUVECs of each group. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; HUVECs, human umbilical vein endothelial cells; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Effect of CNN3 on the tube formation ability of HUVECs

In the HUVECs + MG-63 co-culture system, the number of branch nodes formed by HUVECs in the si-CNN3 group was lower than in the NC group, whereas the number in the CNN3-OE group was higher than in the vector group (Figure 7A,7B). In the HUVECs + Saos-2 co-culture system, the number of branch nodes formed by HUVECs in each group followed the same trend as observed in the MG-63 + HUVECs co-culture (Figure 7C,7D). Vascular mimicry assays demonstrated that CNN3 silencing inhibited, while CNN3 overexpression enhanced, the tube formation ability of HUVECs.

Figure 7 CNN3 modulates the tube formation ability of HUVECs in co-culture systems assessed by vascular mimicry assay. (A,B) The tube formation ability of HUVECs when co-cultured with MG-63 cells transfected with NC, si-CNN3, Vector, or CNN3-OE. (C,D) The tube formation ability of HUVECs when co-cultured with similarly manipulated Saos-2 cells. Panels (A,C) are the representative images of the vascular mimicry assay (magnification: ×100). Panels (B,D) are the bar graphs representing the number of branch nodes formed by HUVECs of each group. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; HUVECs, human umbilical vein endothelial cells; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Effects of CNN3 on VEGF-A expression in osteosarcoma cell culture medium

In the cell culture media, VEGF-A levels were lower in the si-CNN3 group than in the NC group and higher in the CNN3-OE group than in the vector group (Figure 8A,8B). These results indicated that silencing CNN3 inhibited, while overexpressing CNN3 enhanced, VEGF-A expression in both cell lines.

Figure 8 Effects of CNN3 silencing or overexpression on VEGF-A expression in MG-63 and Saos-2 cell culture medium. (A) VEGF-A expression in MG-63 cell culture medium; (B) VEGF-A expression in Saos-2 cell culture medium. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector; VEGF-A, vascular endothelial growth factor-A.

Effect of CNN3 on Akt phosphorylation in HUVECs

To further clarify how CNN3-modulated osteosarcoma cells affect endothelial cell activity, we measured the phosphorylation of Akt, a downstream target of VEGF-A signaling, in HUVECs. In the HUVECs + MG-63 co-culture system, HUVECs co-cultured with MG-63 cells of si-CNN3 group exhibited a lower p-AKT level than those co-cultured with NC group cells, whereas HUVECs co-cultured with CNN3-OE group cells showed a higher p-AKT level than those co-cultured with vector group cells (Figure 9A,9B). In the Saos-2 and HUVECs co-culture system, the level of Akt phosphorylation in HUVECs followed the same trend as in the MG-63 co-culture system (Figure 9C,9D).

Figure 9 The levels of Akt and p-Akt in HUVECs in co-culture systems. (A) Western blotting images of Akt and p-Akt level in MG-63 cells from each group; (B) relative expression of p-Akt level normalized to both Akt and GAPDH in MG-63 cells; (C) Western blotting images of Akt and p-Akt level in Saos-2 cells; (D) relative expression of p-Akt level normalized to both Akt and GAPDH in Saos-2 cells. ^#, P<0.05, si-CNN3 group vs. NC group; ^&, P<0.05, CNN3-OE group vs. vector control group. CNN3, calponin 3; CNN3-OE, CNN3 overexpression vector; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVECs, human umbilical vein endothelial cells; NC, negative control siRNA; si-CNN3, CNN3-targeting siRNA; Vector, empty vector.

Discussion

Angiogenesis is a key link in tumour growth, invasion, and metastasis. Through a series of physiological and pathological processes, tumour tissues establish a new vascular network with surrounding capillaries, providing a material basis for tumour cell growth and proliferation. Without neovascularisation, the tumour ceases to grow or undergoes degeneration. The structure of tumour neovascularisation differs from that of normal blood vessels, and its permeability is high, resulting in the frequent occurrence of haematogenous metastasis of tumour cells, which greatly increases the malignancy of bone tumours. Clinical studies have found that most patients with osteosarcoma already have metastases at the time of diagnosis, and approximately 90% of metastases occur in the lungs, which substantially affects patient survival rates (13,14). Therefore, anti-angiogenesis-targeted therapy has become an important strategy in the treatment of osteosarcoma and has improved prognosis.

We found that the expression of CNN3 in osteosarcoma specimens was positively correlated with MVD, indicating that CNN3 might be involved in the formation of new blood vessels in osteosarcoma. By establishing a co-culture system of osteosarcoma cells and HUVECs, we found that silencing CNN3 in osteosarcoma cells inhibited the proliferation, migration, invasion, and tube formation of HUVECs in co-culture systems, whereas overexpressing CNN3 in osteosarcoma cells promoted these functions in HUVECs in co-culture systems. These results further suggest that CNN3 affects angiogenesis in osteosarcoma. Therefore, CNN3 may serve as a potential therapeutic target, and its inhibition may suppress angiogenesis in osteosarcoma, providing a reliable foundation for the development of antitumour therapies.

EndMT is a process in which endothelial cells downregulate endothelial-specific proteins (including VE-cadherin and CD31) and upregulate mesenchymal-specific proteins (including α-SMA, N-cadherin, and vimentin), transforming into migratory mesenchymal cells (15,16). EndMT promotes vascular branching and remodelling by enhancing endothelial cell migration and invasiveness (17). This study showed that silencing CNN3 in osteosarcoma cells decreased the expression of vimentin and N-cadherin and increased the expression of VE-cadherin in HUVECs in co-culture systems. However, overexpressing CNN3 increased vimentin and N-cadherin expression and reduced VE-cadherin expression in HUVECs in co-culture systems. This suggests that CNN3 may induce EndMT in vascular endothelial cells co-cultured with osteosarcoma cells.

The expression of VEGF-A in osteosarcoma cells was reduced following CNN3 silencing. However, VEGF-A expression was increased in osteosarcoma cells overexpressing CNN3. VEGF is a key marker of angiogenesis, promoting vascular formation and inhibiting endothelial cell apoptosis. It plays an important role in tumour angiogenesis and constitutes a major signaling axis in the vascularisation of tumours (18). VEGF has a significant impact in osteosarcoma and may be used as an index to evaluate angiogenic prognosis (19). Therefore, we hypotheses that CNN3 may promote angiogenesis primarily by regulating VEGF-A secretion, thereby modulating vascular endothelial cell behaviour. In addition, we found that CNN3 expression in osteosarcoma cells promoted Akt phosphorylation in co-cultured HUVECs. Given that Akt is a principal downstream node that transduces VEGF-A signals to drive angiogenesis (20), this result further suggests that CNN3 functions through the VEGF-A pathway. Although this work identifies a significant “CNN3-VEGF-A” signaling axis in osteosarcoma, two key mechanistic limitations must be acknowledged. First, our experiments could not rule out the possibility that CNN3 functions through pathways other than VEGF-A. Second, the precise manner by which CNN3 regulates VEGF-A (such as transcription, secretion, or stability control) has not yet been clarified. These limitations indicate that the current data are insufficient to assert that CNN3 acts exclusively through VEGF-A. Future studies should employ strategies such as rescue experiments with recombinant VEGF-A protein supplementation to confirm the directness and exclusivity of this signaling axis and to further dissect its upstream and downstream molecular events.

Conclusions

In summary, CNN3 regulates the proliferation, migration, invasion, EndMT, and tube formation of vascular endothelial cells and may contribute to angiogenesis by promoting VEGF-A secretion. This study provides a theoretical basis for the development of anti-angiogenic therapies for osteosarcoma. However, evidence from in vivo experiments in animal models is currently lacking. In future studies, we aim to verify the oncogenic role of CNN3 in the pathological process of osteosarcoma using animal models and to explore the specific signaling pathways by which CNN3 mediates angiogenesis in osteosarcoma. These findings may offer new insights and directions for the clinical treatment of osteosarcoma.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1846/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1846/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1846/prf

Funding: This study was supported by the funding from the Chongqing Natural Science Foundation General Project (Nos. CSTB2022NSCQ-MSX0166 and cstc2020jcyj-msxmX0351) and the Open Topics from the Key Laboratory of Tumour Immunopathology, Ministry of Education (No. 2021jsz701).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1846/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. The study was approved by the First Affiliated Hospital, Army Medical University (Approval No. KY201927) and informed consent was obtained from all individual participants.

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/.

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