Andrographolide potentiates anti-tumor immunity in colorectal cancer (CRC) by targeting voltage-dependent anion channel (VDAC) and activating the cGAS-STING axis

Introduction

Colorectal cancer (CRC) is the third most commonprevalent cancer globally. There are marked geographic disparities in the epidemiological patterns of CRC, particularly in industrialized nations and economically transitioning regions. In 2023, there were more than 1.9 million new cases of CRC and approximately 930,000 CRC-related deaths worldwide (1,2). The clinical course of CRC remains concerning. Many patients present with metastatic disease at the time of diagnosis and have a 5-year survival rate of less than 15% (3).

The pathogenesis of CRC involves multi-omics dysregulation, such as germline susceptibility (e.g., adenomatous polyposis coli/tumor protein p53 tumor suppressor gene defects) and epigenomic instability (e.g., MutL homolog 1 promoter hypermethylation and histone H3K27me3 modification), and dynamic tumor microenvironment (TME) remodeling through metabolic-immune network alterations (4-6). Current therapeutic frameworks combine surgical resection with folinic acid, Fluorouracil, and Oxaliplatin (FOLFOX)/folinic acid, fluorouracil, and irinotecan (FOLFIRI) regimens for localized disease management, while immune checkpoint blockade has revolutionized therapeutic paradigms for advanced-stage CRC (7,8). However, over 80% of microsatellite stable (MSS)-CRC subtype patients exhibit primary resistance to immune checkpoint inhibitors (ICIs), highlighting the urgent need for innovative approaches (9). Emerging evidence suggests that the development of precision strategies targeting the epigenetic-immunomodulatory crosstalk via combinatorial epigenetic modulators (e.g., DNA methyltransferase/histone deacetylase) could overcome therapeutic resistance and reshape TME functionality (10,11).

Natural products have emerged as promising pleiotropic therapeutic agents in CRC management (12). For example, one study showed that Qingjie Fuzheng granules suppress colitis-associated carcinogenesis via Toll-like receptor 4/Interleukin-4 receptor-mediated macrophage polarization (13). While another study showed that Bufalin overcomes chemoresistance-mediated metastasis via SRC-3/c-Myc axis inhibition in 5-fluorouracil (5-FU)-resistant LoVo cells (14). Among these compounds, the labdane diterpenoid andrographolide was shown to induce mitochondrial oxidative stress-mediated apoptosis in diffuse large B-cell lymphoma via proteinase-activated receptor-1 (PARP-1) cleavage (15). In CRC models, andrographolide has shown dose-dependent cytotoxicity via reactive oxygen species (ROS)-mediated endoplasmic reticulum stress inactivation, while sparing normal colon epithelial cells (16). Emerging evidence links mitochondrial dysregulation, particularly voltage-dependent anion channel (VDAC) oligomerization, with immunogenic cell death (ICD) through oxidized-mitochondrial DNA (ox-mtDNA)-dependent cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) activation, converting “cold” tumors into immunologically active microenvironments, as demonstrated in breast cancer and discussed in the context of multiple malignancies (17,18). However, the potential of andrographolide to reprogram mitochondrial-immune crosstalk through VDAC-mediated mitochondrial DNA (mtDNA) release remains unexplored, particularly in MSS-CRC subtypes resistant to current immunotherapies.

In this study, we investigated the anti-tumor efficacy of andrographolide in CRC and its immune-metabolic regulatory mechanisms. First, we evaluated the anti-tumor activity of andrographolide using CRC cell lines (CT 26 cells). Second, we performed molecular dynamics simulations (using AutoDock Vina) to predict the way in which it binds with downstream targets. Finally, we employed both in vitro and in vivo models to elucidate the mechanism by which andrographolide modulates the CRC immune TME, thereby providing a theoretical foundation for the development of natural product-based immunotherapy sensitizing strategies. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-592/rc).

Methods

Reagents and cells

Andrographolide (purity ≥98%, MedChemExpress, New Jersey, USA), Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan), the live and dead cell staining kit (DCFH-DA, Beyotime, Shanghai, China), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-10) for measuring mitochondrial function (BD Biosciences, CA, USA), the MitoSOX™ Red kit (MitoSox, CA, USA), and the mitochondrial permeability transition pore (mPTP) kit (Invitrogen, San Diego, CA, USA) were purchased and used according to the instructions provided. Goat antibodies against 8-OHdG (NB600-1508) were obtained from Novus Biologicals (Novus Biologicals, CA, USA). Mouse monoclonal antibodies (564879) were obtained from BD Pharmingen (San Diego, CA, USA). The mouse CRC cell line CT26 was purchased from ATCC (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Corning, New York, USA) containing 10% heat-inactivated fetal bovine serum (Gibco, New York, USA) and 1% penicillin-streptomycin (HyClone, UT, USA) at 37 ℃ with 5% carbon dioxide.

Cell activity assay (CCK-8)

Cells (5×10³) in the logarithmic growth phase were inoculated into a 96-well plate (Corning, New York, USA), and for 24 hours until they adhered to the well. The cells were then treated with different concentrations of andrographolide for 24 hours. CCK-8 (10 µL) reagent was added to each well, and the cells were incubated at 37 ℃ for 1 hour in the dark. Absorbance (reference wavelength 630 nm) was detected at 450 nm.

Staining of live and dead cells

The treated cells were labeled using the Calcein-AM/PI double staining kit (Beyotime, Shanghai, China), Calcein-AM (2 µM) was used to label the live cells (green fluorescence, Ex/Em =488/515 nm), and PI (4 µM) was used to label the dead cells (red fluorescence, Ex/Em =561/617 nm). A fluorescence microscope (Olympus, Tokyo, Japan) was used to acquire images.

ROS detection

The cells were incubated with 10 µM of DCFH-DA for 30 min. After washing with phosphate-buffered saline, fluorescence intensity (Ex/Em =488/525 nm) was measured by fluorescence microscopy (Olympus IX73, Tokyo, Japan).

Molecular docking verification

The three-dimensional (3D) structure of andrographolide (from PubChem) and the 3D structure of VDAC (from Uniprot) were used in this study. AutoDock was used for various steps, including the removal of protein ligands and water molecules, hydrogenation, charge calculation, molecular docking, and result processing. The results were visualized using Pymol. The binding energy was used to evaluate the binding activity and docking effect of the light protocol interaction. A binding energy of less than −5 kcal/mol indicated high binding activity and efficiency.

Mitochondrial function test

JC-10 staining

JC-10 is a cationic dye used to assess mitochondrial membrane potential (ΔΨm). In healthy cells with high mitochondrial membrane potential, JC-10 accumulates in the mitochondria and forms aggregates that emit red fluorescence (emission ~590 nm). In cells with low mitochondrial membrane potential, JC-10 remains in monomeric form and emits green fluorescence (emission ~525 nm). The red/green fluorescence ratio is therefore used as an indicator of mitochondrial membrane potential: a higher ratio reflects higher (more polarized) mitochondrial membrane potential, while a lower ratio reflects depolarization. The CT26 cells were incubated with 2 µM of JC-10 (BD Biosciences) for 30 minutes, and the fluorescence ratio of red (Ex/Em =585/590 nm) and green (Ex/Em =514/529 nm) was detected by fluorescence microscopy.

MitoSOX assay

The CT26 cells were incubated with 5 µM of MitoSOX™ Red for 30 minutes, and red fluorescence intensity was measured by fluorescence microscopy.

MtDNA assay

The CRC cells were seeded in a Confocal Laser Scanning Microscopy (CLSM) dish and after 24 hours, the cells were treated with DAPI staining (to represent the nucleus) and 8-OHdG (to represent mtDNA) after fixation in 4% paraformaldehyde and permeabilization in 0.3% Triton X-100. Finally, CLSM imaging was used to observed the content of mtDNA.

cGAS-STING pathway detection

For Western blot, after cell lysis, 30 µg of total protein was isolated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, USA). The following primary antibodies were used: anti-CGAS (#15102, 1:1,000, CST, Danvers, USA), STING (#13647, 1:1,000, CST, Danvers, USA), IRF3 (#11904), p-IRF3 (#29047) (1:1,000, CST, Danvers, USA), and Tublin (ab15251, 1:3,000, Abcam, Cambridge, UK). The following secondary antibody was used: HRP labeled goat against rabbit (#7074, 1:5,000, CST, Danvers, USA). ECL development (Bio-Rad ChemiDoc™ MP, Hercules, USA) was carried out, along with image analysis of gray values.

For enzyme-linked immunosorbent assay (ELISA), CT26 cells were treated with drugs for 24 hours, and the cell supernatant was collected (via 300 ×g centrifugation for 10 min), and the IFN-β (SEKM-0032(OS), Solarbio, Beijing, China) concentration was detected according to the kit instructions.

Animal experiment

Six-week-old C57 female mice (18–22 g) (purchased through the Affiliated Hospital of Jiaxing University) were subcutaneously inoculated with CT26 cells (5×10⁵/mice) and randomly divided into a control group (saline) and an andrographolide group {20 mg/kg, dissolved in 5% DMSO + 95% saline [intraperitoneal (i.p.)], administered intraperitoneally every other day (qod)}, with four mice in each group. After 15 days of treatment, the mice were sacrificed, and the tumor tissues were weighed. The tissues were then prepared in single-cell suspension, and the proportion of relevant immune cells was detected by flow cytometry, CD80⁺ (#560526, BD, New Jersey, USA), CD86⁺ (#561963, BD, New Jersey, USA), CD4⁺ (#553651, BD, New Jersey, USA), CD3⁺ (#561798, BD, New Jersey, USA), CD8⁺ (#563332, BD, New Jersey, USA), F4/80⁺ (#565410, BD, New Jersey, USA), CD25⁺ (#563354, BD, New Jersey, USA), FOXP3⁺ (#563101, BD, New Jersey, USA).

A protocol was prepared before the study without registration. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition and were approved by the Medical Ethics Committee of The Affiliated Hospital of Jiaxing University (approval No. 2022-LY-008).

Statistical analysis

Three independent experiments were performed, with three images captured per experimental group in each experiment. Mean fluorescence intensity (MFI) was calculated from these images. The data are expressed as the mean ± standard deviation, and were statistically analyzed using GraphPad Prism 8.0. The unpaired t-test was used for comparisons of two groups, and univariate analysis of variance (Tukey’s post-hoc test) was used for comparisons of multiple groups. P<0.05 were set as the thresholds of significance.

Results

Andrographolide inhibits the proliferation of CRC cells and induces ROS production

In order to investigate the anti-proliferative effects of andrographolide on CT26 cells, we first performed the CCK-8 assay, which demonstrated that andrographolide significantly inhibited CT26 cell proliferation in a dose-dependent manner. The half-maximal inhibitory concentration (IC₅₀) ability of the CT26 cells was 19.38 µM (Figure 1A). Based on the IC₅₀, the following three concentrations were selected for subsequent experiments: low (4 µM), medium (8 µM), and high (10 µM). The live and death staining indicated that in the CT26 cells, the rate of dead cells (red light) increased as the concentration increased (Figure 1B,1C). The DCFH-DA test showed that after andrographolide treatment for 24 hours, the low, medium, and high concentrations induced a 1-, 1.8- and 3.6-fold increase in ROS fluorescence intensity in the CT26 model, respectively (Figure 1D,1E).

Figure 1 Dose-dependent cytotoxicity and ROS generation. (A) Cell viability of the CT26 cells treated with different concentrations (0, 6, 12, 24, 48, and 96 µmol/mL) showing dose-dependent cytotoxic effects. (B) Live/dead fluorescence microscopy images showing live cells (green) and dead cells (red) across different treatment concentrations: control, low, medium, and high. Scale bar =250 µm. (C) Quantitative analysis of the ratio of red (dead)/green (live) cells in each treatment group. (D) Quantitative analysis of the mean fluorescence intensity of the ROS in the treated cells across different concentration groups. (E) Fluorescence microscopy images showing ROS generation (green fluorescence) in the cells treated with control, low, medium, and high concentrations. Scale bar =250 µm. Data are presented as the mean ± standard deviation. ***, P<0.001. ROS, reactive oxygen species.

Molecular docking verification and mitochondrial dysfunction induced by andrographolide

Given the critical role of the VDAC in regulating mitochondrial membrane potential and its involvement in cancer cell metabolism and survival, we hypothesized that andrographolide might exert its effects on CRC cells by targeting VDAC. To test this, we performed molecular docking analysis using AutoDock Vina, which predicted that andrographolide binds strongly to VDAC with a binding free energy of ΔG =−7.3 kcal/mol (Figure 2A,2B). Based on these findings, we further examined the mitochondrial function of CRC cells to explore the downstream effects of this interaction. The mitochondrial membrane potential (JC-10) results showed that compared with the control group, the red/green fluorescence ratio of the CRC cells in the positive group [VDAC inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 20 µM] decreased after treatment. As a lower red/green fluorescence ratio reflects mitochondrial depolarization, this indicates a reduction in mitochondrial membrane potential. At the same time, the mitochondrial membrane potential of the cells treated with high, medium, and low concentrations also decreased in a dose-dependent manner (Figure 2C,2D). The mPTP opening results based on Calcin-CoCl₂ probe detection showed that DIDS and andrographolide significantly induced mPTP opening. Notably, the fluorescence intensity of the high concentration group decreased to ~30% of that of the control group (Figure 2E,2F). The mitochondrial ROS (MitoSOX) fluorescence test results indicated that under low, medium, and high concentrations, the MitoSOX of CT26 increased by 2-, 3.2-, and 4.0-fold, respectively (Figure 2G,2H). Notably, our study further found that DIDS and andrographolide significantly increased the expression of ox-mtDNA (green light, 8-OHdG) in the cytoplasm. Compared with the control group, the fluorescence intensity of ox-mtDNA in the andrographolide treated group increased by approximately 4-fold (Figure 2I,2J).

Figure 2 Molecular docking analysis and mitochondrial dysfunction assessment. (A) AutoDock Vina molecular docking analysis of andrographolide binding to VDAC showing the binding parameters and interaction modes. (B) 3D visualization of the andrographolide-VDAC molecular docking complex. (C) JC-10 fluorescence microscopy images showing mitochondrial membrane potential changes across the treatment (control, low, medium, high, and DIDS) groups, with green indicating JC-10 monomers and red indicating JC-10 aggregates (100× magnification). (D) Quantitative analysis of the JC-10 aggregate/monomer ratio indicating the mitochondrial membrane potential. (E) mPTP opening assessment using fluorescence microscopy across different treatment (200× magnification). (F) Quantitative analysis of mPTP fluorescence intensity. (G) MitoROS detection using fluorescence microscopy across the treatment groups (200× magnification). (H) Quantitative analysis of mitoROS fluorescence intensity. (I) Merged fluorescence images showing mtDNA (green) and nuclear staining (blue) across the treatment groups (400× magnification). (J) Quantitative analysis of 8-OHdG fluorescence intensity as an oxidative DNA damage marker in the treated cells. Data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001. 3D, three dimensional; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; mitoROS, mitochondrial reactive oxygen species; mPTP, mitochondrial permeability transition pore; VDAC, voltage-dependent anion channel.

Andrographolide activates the cGAS-STING pathway and promotes DC maturation

Recent studies have shown that once released into the cytosol, ox-mtDNA activates numerous immunostimulatory DNA sensors, such as cGAS (19,20). Research has shown that synthesized cGAMP activates transcription factors IRF3 and NF-kB by binding to the STING protein, stimulates type I IFN expression, and ultimately triggers innate immunity (21). Thus, we hypothesized that andrographolide may also activate the cGAS-STING pathway after inducing ox-mtDNA release. To test this hypothesis, we further detected changes in related proteins by Western blot. The results showed that compared with the control group, different concentrations of andrographolide caused the upregulation of cGAS, STING, and p-IRF3 proteins. Notably, in the high concentration group, cGAS was upregulated by ~4-fold, STING by ~5.5-fold, and p-IRF3 by ~5 fold (Figure 3A,3B). Additionally, supernatant IFN-β levels were measured by ELISA, and the expression levels of IFN-β were increased by 0.2-, 2-, and 3.8-fold by low, medium, and high concentrations of andrographolide, respectively (Figure 3C).

Figure 3 cGAS-STING pathway activation and IFN-β production. (A) Western blot analysis of cGAS, STING, p-IRF3, and IRF3 protein expression across the different treatment (control, low, medium, high, and DIDS) groups with Tubulin as the loading control. (B) Quantitative analysis of relative protein expression levels showing the cGAS/Tubulin, STING/Tubulin, and p-IRF3/IRF3 ratios. (C) ELISA measurements of the IFN-β secretion levels (pg/mL) in cell culture supernatants from the different treatment groups. Data are presented as the mean ± standard deviation. **, P<0.01; ***, P<0.001. cGAS, cyclic GMP-AMP synthase; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; ELISA, enzyme-linked immunosorbent assay; IFN-β, interferon β; STING, stimulator of interferon genes.

Andrographolide inhibits tumor growth and remodels the immune microenvironment in animal experiments

The animal experiments showed that the tumor volume and final weight of the andrographolide monotherapy group (20 mg/kg) was significantly lower than that of the control group (Figure 4A-4C). Due to their participation in initiating, regulating, and maintaining the innate and adaptive immune response, dendritic cells (DCs) are critical decision-making cells in the immune response (22). Notably, our study showed that after the andrographolide treatment, the proportion of double positive DCs was statistically increased (Figure 4D,4E), indicating the induction of DC maturation. Flow cytometry analysis of immune cells isolated from tumor tissue suspensions showed a 2-fold increase in the density of the CD4⁺ T cells (Figure 4D,4F) and CD8⁺ T cell infiltration (Figure 4D,4G), the proportion of M1 type macrophages (CD80+++) was increased in the andrographolide-treated group (Figure 4D,4H), accompanied by a decreased ratio of regulatory T cells (Tregs) (Foxp3⁺CD25⁺) (Figure 4D,4I), and this was associated with the polarization of macrophages.

Figure 4 In vivo anti-tumor efficacy and tumor-infiltrating immune cell analysis. (A) Representative images of excised tumors from the control and andrographolide-treatment groups. (B) Tumor volume growth curves over time (15 days) comparing the control and andrographolide-treatment groups. (C) Tumor weight comparison between the control and andrographolide-treatment groups. (D) Quantitative analysis of the relative expression levels of the tumor-infiltrating immune cell markers, including DCs, CD4⁺ T cells, CD8⁺ T cells, M1 macrophages, and Tregs. (E-I) Flow cytometry analysis of tumor-infiltrating immune cells: (E) DCs (CD80⁺ CD86⁺), (F) CD4⁺ T cells (CD4⁺ CD3⁺), (G) CD8⁺ T cells (CD8⁺ CD3⁺), (H) M1 macrophages (CD86⁺ F4/80⁺), and (I) Tregs (CD25⁺ FOXP3⁺) in the control and andrographolide-treatment groups. Data are presented as the mean ± standard deviation. ***, P<0.001. DCs, dendritic cells.

Discussion

Targeting the TME to augment immunotherapy efficacy has emerged as a central focus in CRC research (23). We showed that andrographolide triggers mitochondrial oxidative stress through the specific targeting of VDAC, inducing ox-mtDNA release and the subsequent activation of the cGAS-STING signaling cascade, thereby converting the immunosuppressive TME into immunostimulatory niches and potentiating anti-tumor immunity. Notably, this mechanism differs fundamentally from existing natural product paradigms. Icariside I activates STING via the targeting of transient receptor potential cation channel subfamily V member 4 (TRPV4) (24). Conversely, andrographolide specifically modulates mitochondrial VDAC via ΔΨm depolarization, mPTP opening, and ox-mtDNA efflux, thereby initiating innate immune signaling at earlier regulatory nodes. This mitochondrial specificity confers distinct therapeutic advantages, as VDAC serves as a master regulator coordinating both metabolic homeostasis and ICD—dual processes frequently dysregulated in CRC progression. The dual-targeting capacity of andrographolide has novel therapeutic potential for converting immunologically “cold” tumors into “hot” microenvironments receptive to checkpoint blockade therapies.

As the primary channel protein in the mitochondrial outer membranes, VDAC coordinates metabolite transport and mitochondrial-cytoplasmic crosstalk. VDAC oligomerization induces mitochondrial permeability transition by promoting cytochrome c release and apoptosome activation (25). However, its immunoregulatory functions remain incompletely characterized, particularly in tumor immunity contexts. We showed that andrographolide binds to VDAC with high affinity. Similar to the VDAC-specific inhibitor DIDS, andrographolide caused mPTP-mediated ox-mtDNA efflux. This mechanism exhibits mechanistic divergence from POLG-dependent mtDNA release induced by oxaliplatin (26). Unlike the chemotherapeutic agents’ DNA repair enzyme inhibition strategy, andrographolide directly modulates VDAC gating activity, indicating target specificity. This mitochondrial membrane-targeting strategy represents a novel mechanistic paradigm, as andrographolide’s physical disruption of VDAC oligomerization has unprecedented multi-target potential in natural product pharmacology.

Mechanistically, andrographolide-induced ox-mtDNA release potently activates the cGAS-STING axis, as evidenced by the 2.1-fold cGAS upregulation and the 3.0-fold STING phosphorylation (p-STING) enhancement, as well as the 5-fold increase in IFN-β secretion. This direct immunomodulation differs to Qingjie Fuzheng granules’ TLR4/IL-4R-mediated macrophage polarization mechanism (13). cGAS-STING activation holds particular therapeutic value for MSS-CRC, which constitutes 80% of cases and has response rates of ≤5% to ICIs (27). By leveraging endogenous mtDNA as DAMPs, andrographolide circumvents pharmacokinetic limitations inherent to synthetic STING agonists. Notably, while ADU-S100 demonstrates suboptimal pharmacokinetics and grade ≥3 adverse events in some patients (28), andrographolide sustains mitochondrial permeability via VDAC modulation, maintaining ox-mtDNA elevation in serum pharmacokinetic analyses. Our research also showed that andrographolide-treatment could promote DC maturation, mechanistically link to ox-mtDNA’s cGAS-STING-MHCI antigen presentation axis (17). Mature DCs orchestrate anti-tumor immunity through antigen cross-presentation, increasing CD8⁺ T cell infiltration, and IFN feedback amplification (29). This mitochondrial-driven immunomodulation differs to 5-fluorouracil’s dual effect, which induces tumor apoptosis while suppressing lymphocyte proliferation (30,31). Andrographolide uniquely coordinates mitochondrial stress-antigen presentation-immune activation cascades, achieving therapeutic synergy. Due to this multimodal immuno-metabolic reprogramming, andrographolide represents a promising candidate for MSS-CRC immunotherapy.

Several limitations of our study should be acknowledged. First, although we demonstrated robust cGAS-STING pathway activation in vitro, Western blot analyses confirming activation in vivo were not performed, limiting the direct evidence of mechanistic engagement in animal models. Thus, we cannot fully exclude the possibility that the observed anti-tumor responses in vivo may result, at least in part, from alternative mechanisms or off-target effects. Second, while increased tumor-infiltrating immune cells were observed following andrographolide treatment, we cannot definitively rule out the contribution of non-specific drug toxicity, such as apoptosis or necrosis, to these changes. Future investigations employing immunoblotting of tumor tissues and comprehensive toxicity evaluation will be required to address these issues.

Conclusions

Andrographolide induces mitochondrial stress by targeting VDAC, activates the cGAS-STING-IFN-β axis, and reshapes the TME in CRC. Our findings not only identify a new natural synergist for CRC immunotherapy, but also lay a theoretical foundation for the development of mitochondria-targeting drugs (see the Figure 5). However, differences in the sensitivity of different CRC molecular subtypes to andrographolide need to be further explored, and more models need to be validated in the future to try to complete the clinical conversion.

Figure 5 Andrographolide targets VDAC, induces mitochondrial damage, activates cGAS-STING, remodels CRC TME, recruits DC/CD4⁺/CD8⁺ T cells, suppressing tumor proliferation. cGAS, cyclic GMP-AMP synthase; CRC, colorectal cancer; DC, dendritic cells; DID; IFN-β, interferon β; LN, lymph node; STING, stimulator of interferon genes; TME, tumor microenvironment; VDAC, voltage-dependent anion channel.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-592/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-592/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-592/prf

Funding: This study was supported by the Jiaxing Science and Technology Plan Project-Research on Technological Innovation of People’s Livelihood (No. 2023AY31005), the Jiaxing City and Provinces to Build Medical Key Disciplines – Oncology (No. 2023-SSGJ-001), the National Oncology Clinical Key Specialty Program (No. 2023-GJZK-001), and the Medical Scientific Research Foundation of Guangdong (Nos. C202460 and 202405292036507807).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-592/coif). All authors report that this study was supported by the Jiaxing Science and Technology Plan Project-Research on Technological Innovation of People’s Livelihood (No. 2023AY31005), the Jiaxing City and Provinces to Build Medical Key Disciplines – Oncology (No. 2023-SSGJ-001), the National Oncology Clinical Key Specialty Program (No. 2023-GJZK-001), and the Medical Scientific Research Foundation of Guangdong (Nos. C202460 and 202405292036507807). The authors have no other 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.All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition and were approved by the Medical Ethics Committee of the Affiliated Hospital of Jiaxing University (approval No. 2022-LY-008).

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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