Mechanistic study of the role of emodin in targeting and inhibiting the Rap1 signaling pathway to regulate epithelial-mesenchymal transition and reverse cisplatin resistance in hepatocellular carcinoma

Introduction

Primary liver cancer (PLC) ranks as the sixth most prevalent malignancy globally and the third leading cause of cancer-related mortality (1). With its incidence demonstrating a persistent upward trajectory, recent projections suggest that by 2040, the global burden will escalate to 1.4 million new cases and approximately 1.3 million deaths annually (2). Notably, hepatocellular carcinoma (HCC) constitutes 75–85% of PLC cases, posing a major public health challenge that demands urgent intervention strategies (1). Current treatment options for HCC include surgical resection, liver transplantation, ablation therapy, and systemic chemotherapy. Nevertheless, the challenges associated with screening for early-stage liver cancer are significant, primarily due to the low sensitivity of available tests and the high cost of the procedure. Consequently, patients are frequently diagnosed at advanced stages, necessitating systemic chemotherapy as the sole viable treatment option for many (3,4). Despite being a first-line chemotherapeutic for advanced HCC, cisplatin faces significant clinical limitations due to the rapid development of tumor resistance. Therefore, developing new therapeutic strategies to reverse cisplatin resistance has become an urgent clinical challenge.

Natural products have demonstrated significant potential in the development of antitumor drugs due to their diverse biological activities. Among them, emodin, a natural anthraquinone compound, is abundantly distributed in the rhizomes of Polygonum cuspidatum (e.g., rhubarb, tiger balm) (5,6). Studies have shown that emodin exhibits antitumor effects against various malignant tumors, including rectal, pancreatic, and breast cancers (7-9). Similarly, several studies have confirmed that emodin plays a crucial role in the treatment of HCC. Research has found that emodin reduces HCC cell migration and invasion by downregulating the expression of epithelial-mesenchymal transition (EMT) markers while simultaneously increasing E-cadherin expression (10). Qin et al. (11) demonstrated that emodin inhibits HCC cell invasion and migration by affecting the autophagy pathway, which mediates the protein degradation of Snail and β-catenin. Yang et al. (12) found that emodin promotes GSK-3β-mediated programmed death ligand-1 (PD-L1) proteasomal degradation and enhances the antitumour activity of CD8⁺ T cells. Notably, our team’s previous study found that the combination of emodin and cisplatin inhibits the EMT process and reduces cisplatin resistance, but the underlying mechanism remains unclear (13).

Building on this, this study aims to analyze the mechanism of action of emodin in regulating EMT and enhancing cisplatin efficacy through transcriptomic analysis combined with WB validation, offering a new approach to overcoming chemotherapy resistance in HCC. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1404/rc).

Methods

Experimental drugs, main reagents, and instruments

Table 1 provides more information on the materials used in the experiment. Laboratory equipment included the Ultra-clean bench SW-CJ-2FD from Suzhou Antai; Ultra-low-temperature refrigerator 905-µLTS from Thermo; CO₂ incubator 3111 from Thermo; Microscope XD202 from Jiangnan Yongxin; Fluorescence microscope MF52 from Guangzhou. Centrifuge Pico17 from Thermo; Electrophoresis apparatus DYY-11 from Beijing Liuyi Instrument Factory Co., Ltd.

Experimental cells and cell culture

HepG2 cells (purchased from Saibai Kang, reagent catalog number: iCell-h092) were authenticated using short tandem repeats (STR) identification. Cells were resuscitated in MEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and routinely passaged in a 37 ℃, 5% CO₂ incubator.

Drug concentration screening

The experimental cells were divided into the blank control group, emodin groups with different concentrations (0, 25, 50, 100, 150 µM), and cisplatin groups with different concentrations (0, 2.5, 5, 10, 20, 40, 80 µM). HepG2 cells from each group were seeded into 96-well plates at a density of 1×10⁴ cells per well, with five replicates per group. After 24 hours of culture, drug treatment was applied according to the experimental groups, and the CCK8 assay was performed after an additional 24 hours of culture to determine the optimal drug concentration (Figure 1).

Figure 1 Experimental flowchart. DDP, cisplatin; EdU, 5-ethynyl-2'-deoxyuridine; emoCP, emodin combined with cisplatin; WB, Western blot.

Transwell assay for migration ability

The cell culture protocol was identical to that in Section “Experimental cells and cell culture”. Cells were divided into the following groups: blank control, emodin, cisplatin, emodin + cisplatin, Rap1 inhibitor + emodin, Rap1 inhibitor + cisplatin, and Rap1 inhibitor + emodin + cisplatin. Cells were detached, centrifuged, and washed twice with phosphate-buffered saline (PBS). Serum-free medium was added to resuspend the cells, which were then counted, and the cell concentration was adjusted to 2×10⁵ cells/mL. Drug treatment was carried out according to the experimental protocol (cisplatin at 10 µM, emodin at 50 µM). A 24-well cell culture plate was used, and 600 µL of complete culture medium containing 10% fetal bovine serum (FBS) was added to each well. An 8 µM pore-size polyethylene terephthalate (PET) membrane of the Transwell chambers was placed in each well. A total of 200 µL of the drug-cell mixture prepared in the previous step was added to each upper chamber, and the culture plate was placed in a cell culture incubator. After 24 hours, the culture plate was removed, the chambers were taken out, and the upper culture medium was discarded. The cells in the upper layer were wiped off with a cotton swab, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet solution for 20 minutes, and then counted and photographed for preservation (Figure 1).

Invasion assay

Cell invasion was assessed using the Transwell assay. The Matrigel stored at −20 ℃ was thawed on ice and diluted 1:8 with serum-free culture medium. Then, 80 µL of matrix gel solution was added to the upper layer of the PET membrane and incubated in a cell incubator for 1 hour. Afterward, the matrix gel solution was aspirated, and the cell mixture was added. The remaining steps were identical to those in the migration assay (Figure 1).

EdU assay for assessing proliferation in HCC

In this experiment, cells were grouped as in Section “Transwell assay for migration ability”. Cells were seeded in 24-well plates at a density of 1×10⁵ cells/well. Following pharmacological interventions as per the experimental design, cellular proliferation was quantified using the 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. A double-concentration EdU working solution (40 µM, Click-iT^® EdU kit, Thermo Fisher) was equilibrated to 37 ℃ and introduced into the culture medium at 1:1 (v/v) ratio, achieving a final concentration of 20 µM. Cells were subsequently maintained under standard culture conditions (37 ℃, 5% CO₂) for precisely 2 hours to allow nucleotide incorporation. Upon completion of EdU labeling, cells were fixed with freshly prepared 4% paraformaldehyde in PBS (0.5 mL/well) for 15 minutes at room temperature. Following fixation, cells were washed thrice with phosphate-buffered saline (PBS, 0.5 mL/well, 5 min/wash) and permeabilized with 0.5% Triton X-100 in PBS (0.5 mL/well) for 10 minutes at room temperature. The click reaction cocktail, containing 20 µM Alexa Fluor 488-azide and 2 mM CuSO₄ in reaction buffer, was prepared according to manufacturer specifications. Cells were incubated with 0.5 mL/well of the reaction mixture for 30 minutes under light-protected conditions. After three PBS washes (5 min each), nuclear counterstaining was performed using 300 µL/well of 4',6-diamidino-2-phenylindole (DAPI) (1 µg/mL in PBS) for 10 minutes at room temperature. Post-staining washes were performed with PBS (3×5 min) to remove unbound fluorophores. Finally, anti-fluorescence quenching sealer was added, and the images were captured under a fluorescence microscope and photographed for storage (Figure 1).

Western blot (WB) assay for vimentin, N-cadherin, Epac1, Rap1b, AKT, P-AKT, and E-cadherin expression

The cells were divided into groups according to the experimental protocol described in Section “Transwell assay for migration ability”. Cells were seeded at a density of 1×10⁶ cells per well in 6-well plates, followed by drug treatment prior to WB analysis. Cells were lysed using ice-cold RIPA buffer containing protease inhibitors, incubated on ice for 30 min, then subjected to ultrasonication (5 cycles of 10 s pulses) and centrifugation at 12,000 ×g for 15 min at 4 ℃. The supernatant was collected and protein concentration was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were denatured by mixing with 5× LaemmLi loading buffer and boiling at 95 ℃ for 5 min. Proteins were separated on 10% SDS-PAGE gels and subsequently transferred to PVDF membranes using wet transfer system at 100 V for 90 min. The membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature. Primary antibody was incubated overnight at 4 ℃, followed by washing. The secondary antibody was added and incubated at room temperature for 2 h, followed by washing, signal development, and image acquisition.

Transcriptomics analysis

Cells were grouped into the blank control group, emodin group, cisplatin group, and emodin + cisplatin group. After standard cell culture, the cells were passaged into eight 10 cm Petri dishes. Drug treatment was initiated according to the experimental groups when the cell confluence reached approximately 70%. Cell samples were collected after 24 hours of continued incubation for transcriptome sequencing (Figure 1).

Statistical analyses

All data are expressed as mean ± standard deviation and were analyzed using GraphPad Prism 9.5, with differences between groups assessed using one-way ANOVA, with P values <0.05 considered significant (*, P<0.05; **, P<0.01; ***, P<0.001).

Results

Inhibitory effects of different concentrations of emodin and cisplatin on HepG2 cell activity

To investigate the inhibitory effects of different concentrations of emodin and cisplatin on HepG2 cell activity, HepG2 cells were treated with various concentrations of emodin (0, 25, 50, 100, 150 µM) and cisplatin (0, 2.5, 5, 10, 20, 40, 80 µM) for 24 hours. Cell viability was assessed using the CCK-8 assay. The study revealed that the activity of HepG2 cells was inhibited by 50 µM emodin (Figure 2A) and by 10 µM cisplatin (Figure 2B). Based on these results, the aforementioned concentrations were selected for subsequent experiments.

Figure 2 Assessment of cell viability by CCK-8 assay in HCC cells treated with different concentrations of emodin and DDP. (A) CCK-8 assay detecting the activity of HCC cells at different concentrations of emodin. (B) CCK-8 assay detecting the activity of HCC cells at different concentrations of DDP. Each experiment was repeated at least three times. Results are expressed as mean ± standard deviation (n=3). **, P<0.01. DDP, cisplatin; HCC, hepatocellular carcinoma.

Exploring the mechanism by which emodin enhances the anti-HCC effect of cisplatin through transcriptomic analysis

To investigate the potentiating effect of emodin on cisplatin and its underlying mechanism, transcriptomic sequencing was performed on HepG2 cells. The concentrations were selected based on the results of the CCK-8 assay and the cells were divided into four groups: blank control (HepG2 group), emodin (emodin group), cisplatin (DDP group), and emodin combined with cisplatin (emoCP group), all of which were subjected to transcriptomic sequencing. The results showed that the DDP group had 2,457 upregulated and 2,891 downregulated genes compared with the HepG2 group (Figure 3A); the emodin group had 1,601 upregulated and 1,689 downregulated genes compared with the HepG2 group (Figure 3B); and the emoCP group had 2,191 upregulated and 1,542 downregulated genes compared with the DDP group (Figure 3C). Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the differentially expressed genes between the groups (Figure 3D-3I).

Figure 3 Transcriptomics, GO enrichment analysis, and KEGG enrichment analysis for each group of cells. (A) Differential transcriptome analysis between the control and DDP group. (B) Differential gene transcriptome analysis between the control and emodin group. (C) Differential transcriptome analysis between DDP and emoCP group. (D) GO enrichment analysis of DEG between control and DDP groups. (E) KEGG enrichment analysis of DEG between control and DDP groups. (F) GO enrichment analysis of DEG between control and emodin groups. (G) KEGG enrichment analysis of DEG between control and emodin groups. (H) GO enrichment analysis of DEG between DDP and emoCP groups. (I) KEGG enrichment analysis of DEG between DDP and emoCP groups. Each experiment was repeated at two times. DDP, cisplatin; DEG, differentially expressed genes; emoCP, emodin combined with cisplatin; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Next, we identified the intersection of differential genes between the HepG2 and DDP groups, the DDP and emoCP groups, and migration-related genes to demonstrate the effect of cisplatin on HCC and confirm the potentiation of cisplatin by emodin. The results revealed a total of 232 genes (Figure 4A), which were then subjected to GO and KEGG analysis (Figure 4B,4C). KEGG pathway analysis revealed significant enrichment in choline metabolism, ErbB signaling pathway, epidermal growth factor receptor tyrosine kinase, HCC, mTOR signaling pathway, gastric cancer, migration, Rap1 signaling pathway, and PI3K-Akt signaling pathway in cancers. GO enrichment analysis primarily identified pathways involved in protein phosphorylation, apoptosis, cell migration, and amino acid transport. The analysis indicated that the Rap1 signaling pathway plays a critical role in the potentiation of cisplatin by emodin in HCC cells. Emodin enhanced the anticancer effect of cisplatin by inhibiting cell migration.

Figure 4 Screening of the mechanism underlying the synergistic effect of emodin on cisplatin. (A) Venn diagram showing the relationship between three gene sets: HepG2 vs. DDP differential genes, DDP vs. emoCP differential genes, and migration-associated genes. (B) Gene sets identified by GO enrichment analysis. (C) Gene sets identified by KEGG analysis. DDP, cisplatin; emoCP, emodin combined with cisplatin; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Emodin enhances the inhibitory effect of cisplatin on HCC cell proliferation, migration, and invasion by inhibiting the Rap1 signaling pathway

To verify the potentiating effect of emodin on cisplatin, we assessed the proliferation, migration, and invasion of HCC cells in each group. The results of the proliferation assay showed that emodin inhibited the proliferation of HCC cells more effectively than cisplatin alone (Figure 5A,5B). Next, we evaluated the effects of emodin and cisplatin on the migration and invasion of HCC cells using the Transwell assay. Similarly, when emodin was combined with cisplatin, the migration and invasion of HCC cells were inhibited, with a greater effect than cisplatin treatment alone (Figure 5C-5F). To elucidate the effect of emodin and cisplatin on the Rap1 signaling pathway, GGTI298, a Rap1-specific inhibitor, was introduced to block pathway activity. The results showed that the proliferation, migration, and invasion of HCC cells were restored after the combination with the Rap1 pathway inhibitor GGTI298 (Figure 5A-5F). These results suggest that emodin enhances the inhibitory effect of cisplatin on the proliferation, migration, and invasion of HCC cells, an effect that was further reversed by the combination with the Rap1 pathway inhibitor GGTI298.

Figure 5 Effects of emodin on HCC proliferation, migration, and invasion via the Rap signaling pathway. (A) EdU assay detecting cell proliferation ability in each group. (B) Percentage of proliferating cells in each group. (C) Transwell assay detecting cell migration ability. (D) Number of migrated cells. (E) Effect on cell invasion ability. (F) Number of invaded cells. Following combined administration of DDP and emodin, liver cancer cell invasion, migration, and proliferation were all significantly reduced. It is a key finding of this study that these processes were further attenuated upon the addition of Rap1 pathway inhibitors. Each experiment was repeated three times (n=3). Staining method: (A) EdU proliferation fluorescence staining and DAPI nuclear staining, (C,E) crystal violet staining. Scale bar: 100 µM. **, P<0.01. DAPI, 4',6-diamidino-2-phenylindole; DDP, cisplatin; EdU, 5-ethynyl-2'-deoxyuridine; HCC, hepatocellular carcinoma.

WB analysis was performed to investigate dynamic alterations in Rap1 signaling pathway components and EMT-associated protein expression levels following specific inhibition of Rap1 activity

To further elucidate the mechanism by which emodin enhances the effects of cisplatin through modulation of the Rap1 signaling pathway, we performed WB analysis of migration, invasion, and Rap1 signaling pathway-related proteins. The results showed that Rap1 activity in the emoCP group was significantly reduced compared to the DDP group. In addition, the expression levels of vimentin, N-cadherin, Epac1, Rap1b, and P-AKT/AKT were downregulated and E-cadherin expression exhibited marked upregulation (Figure 6A-6G). After the addition of pathway inhibitors, we found that the expression trend of the remaining proteins was the same as before, except for Epac1, which did not differ significantly between the emoCP group and the DDP group (Figure 6A-6G).

Figure 6 Changes in cell migration, invasion-related proteins, and Rap1 pathway in each group as detected by WB. (A) WB detection of alterations in the Rap signaling pathway and EMT-related changes. (B) Vimentin protein expression in cells across groups. (C) N-cadherin protein expression in cells across groups. (D) E-cadherin protein expression in cells from each group. (E) Epac1 protein expression in cells from each group. (F) Rap1b protein expression in cells from each group. (G) p-AKT/AKT protein expression in cells from each group. Each experiment was repeated three times(n=3). *, P<0.05; **, P<0.01; ***, P<0.001. DDP, cisplatin; EMT, epithelial-mesenchymal transition; WB, Western blot.

Discussion

Chemotherapy is a commonly used treatment for unresectable advanced HCC; however, resistance to chemotherapeutic agents remains a significant challenge, severely affecting both the efficacy of chemotherapy and patient prognosis. Cisplatin is a platinum-based chemotherapeutic agent widely used for its broad-spectrum anticancer activity; however, its effectiveness is often hindered by the emergence of drug resistance (14,15). Therefore, reducing resistance and enhancing the effectiveness of cisplatin remain critical challenges.

EMT is associated with cancer progression, including recurrence, invasion, and migration (16,17). Particularly noteworthy is its close relationship with drug resistance, which can potentially be overcome by targeted therapies specific to EMT (18). Debaugnies et al. (19) found that knockdown of RHOJ, a small GTPase preferentially expressed in EMT cancer cells, reversed the EMT phenotype and restored chemosensitivity in a squamous cell carcinoma model. Meanwhile, Kantapan et al. (20) ascertained that pentagalloylglucose enhances the efficacy of the chemotherapy drug doxorubicin by impeding the process of EMT, thus facilitating the treatment of triple-negative breast cancer. All these studies suggest that the targeted inhibition or blockade of EMT is a key strategy to mitigate drug resistance.

In a previous study (13), we found that emodin inhibited HCC metastasis, and the combination of emodin and cisplatin had a more significant synergistic effect than either agent alone. Emodin enhances the sensitivity of HepG2 cells to cisplatin by inhibiting EMT. Building on the results of previous studies, this study further investigates the mechanism by which emodin enhances cisplatin sensitivity.

In this study, we used different concentrations of emodin and cisplatin in CCK-8 experiments to select the optimal concentration for inhibiting the proliferation of HCC cells. The experimental results showed that the viability of HepG2 cells was inhibited at 50 µM emodin, while the viability was similarly reduced with 10 µM cisplatin. Based on these results, the above concentrations were used in subsequent transcriptomic experiments, with groups divided as follows: HepG2 blank control, emodin-treated, cisplatin-treated, and emodin + cisplatin-treated for analysis. Comparative analysis was performed by identifying the intersection of migration-related genes between the HepG2 and DDP groups, and between the DDP and emoCP groups, to identify differential genes where emodin potentiated the effect of cisplatin and were associated with cell migration. The differential genes were analyzed for KEGG and GO enrichment, and the Rap1 signaling pathway was identified as playing a key role in this process.

Rap1, a small Rap-like GTPase, plays a crucial role in tumor cell migration, invasion, and metastasis. Aberrant activation of Rap1, which promotes EMT, accelerates tumor progression (21-23). Rap1 exists in two isoforms, Rap1a and Rap1b. Rap1b, specifically expressed by endothelial cells, plays a key role in the tumor microenvironment (24). Notably, Rap1b exhibits pro-carcinogenic properties in malignant tumors, with specific activation of Rap1b accelerating tumor progression in pancreatic and endometrial cancer models (25,26). Conversely, knocking down Rap1b expression effectively inhibits the invasive migratory ability of colorectal cancer cells and reduces their stromal adhesion and spreading activity (22). All of this evidence confirms that Rap1b acts as a pro-oncogenic factor driving the malignant phenotype and may also serve as a potential therapeutic target.

Rap1 cycles between an inactive GDP-bound state and an active GTP-bound state. The GDP-GTP cycle is regulated by guanine nucleotide exchange factors (GEFs), which facilitate the release of GDP, allowing GTP to bind to Rap1. Epac, the GEF of Rap1, influences GDP-GTP exchange, thereby stimulating Rap1 activity and basal adhesion capacity. It was found (27) that Epac1 expression was abnormally elevated in human pancreatic cancer cells compared to normal pancreatic or surrounding tissues. Inhibition of Epac1 by gene knockdown or drugs effectively inhibited the migration and invasion of pancreatic cancer cells. In triple-negative breast cancer, the anti-angiogenic effects of Epac inhibitors have been demonstrated, thereby contributing to the retardation of disease progression (28). In primary melanoma, there is a close association between Epac and glycolysis, oxidative metabolism, and mitochondrial reactive oxygen species production. The present study hypothesises that Epac signalling promotes tumour progression and serves as an effective prognostic biomarker (29). Additionally, the Epac1/Rap1 signaling pathway promotes cancer progression by enhancing glucose uptake and metabolism (30). Rap1 may also contribute to tumor invasion and migration by affecting AKT, activating p-AKT, which inhibits E-cadherin expression, upregulates mesenchymal markers such as N-cadherin, and reduces cell-to-cell adhesion, thus influencing EMT and driving the process (31).

This study demonstrated that compared to cisplatin monotherapy, emodin combined with cisplatin exerted a more pronounced inhibitory effect on the proliferation, invasion, and migration of HCC cells. This combination downregulated the expression of vimentin, N-cadherin, Rap1b, Epac1, and p-AKT/AKT, while upregulating E-cadherin expression. These findings suggest that emodin may enhance cisplatin efficacy and mitigate drug resistance. To further elucidate this mechanism, we introduced a Rap1 inhibitor, which amplified these expression differences. Notably, no significant difference in Epac1 expression was observed between the emoCP and DDP groups in the presence of the inhibitor. Analysis of this phenomenon revealed the following contributing factors. First, literature analysis indicates that cisplatin intrinsically inhibits Epac1 (32). In this experiment, the DDP group alone suppressed Epac1; thus, the Rap1 inhibitor likely masked the differential effect of emoCP on Epac1 by synergizing with cisplatin’s inhibitory activity. Second, although Epac1 is primarily regulated by cyclic adenosine monophosphate, Rap1 activation can occur independently through alternative GEFs. Consequently, despite unchanged Epac1 levels, the significant downregulation of Rap1b and p-AKT/AKT confirmed overall Rap1 pathway inhibition. This implies that the emodin-cisplatin combination may operate through non-Epac1-dependent mechanisms, potentially via multi-GEF suppression or direct targeting of Rap1.

Conclusions

In this study, building on our previous work, we further validated the synergistic effect of emodin on cisplatin. The combined application of emodin and cisplatin significantly inhibited the proliferation, migration, and invasion of HCC cells. Through transcriptomic analysis, we explored the underlying molecular mechanisms and identified the Rap1 signaling pathway as a key player in this process. Further experimental validation demonstrated that the combination of emodin and cisplatin inhibited Rap1 signaling pathway-related and EMT-related proteins. This dual inhibition was characterized by specific downregulation of vimentin, N-cadherin, Rap1b, and p-AKT/AKT expression, along with upregulation of E-cadherin. These results suggest that emodin inhibits the EMT process in HCC cells by modulating the Rap signaling pathway, thereby enhancing cisplatin sensitivity and reducing the metastatic potential of HCC cells.

Acknowledgments

All the authors of this manuscript are very grateful to the various departments of Changchun University of Chinese Medicine for their support.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1404/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1404/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1404/prf

Funding: This work was supported by the Education Department of Jilin Province (No. JJKH20241071KJ).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1404/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.

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