Thimerosal (C₉H₉HgNaO₂S, CAS: 54-64-8, purity ≥99%, S3646, Selleck, Shanghai, China) was dissolved in Phosphate Buffer Saline (PBS) to prepare a 0.1 M stock solution. This stock solution was aliquoted and stored at −80°C, with a demonstrated stability of up to 6 months. For use, aliquots were thawed on ice and diluted with PBS (pH 7.2–7.4, 0.01 M) to the desired concentrations; repeated freeze-thaw cycles were avoided.

To evaluate the role of mercury ions in Thimerosal’s antitumor activity, two structural analogues, Sodium 2-((sec-butyl) thio) benzoate (CPD1) and Sodium 2-((phenyl) thio) benzoate (CPD2), were custom-synthesized by BirdoTech (Shanghai, China). These compounds were designed to replace the mercury-containing moiety with methyl or benzene groups while maintaining molecular spatial similarity. Both compounds showed purities >95%, as determined by high-performance liquid chromatography.

Murine CRC cell lines (MC38, RRID: CVCL_B288; CT26, RRID: CVCL_7254), murine melanoma cell line (B16F10, RRID: CVCL_0159), human intestinal epithelial cell line (NCM460, RRID: CVCL_ 0460) and human CRC cell lines (SW620, RRID: CVCL_0547; SW480, RRID: CVCL_0546; LS-174T, RRID: CVCL_1384; DLD1, RRID: CVCL_0248; HCT8, RRID: CVCL_2478; RKO, RRID: CVCL_0504; HT29, RRID: CVCL_0320; LOVO, RRID: CVCL_0399; HCT15, RRID: CVCL_0292; HCT116, RRID: CVCL_0291) were obtained from the Cell Resource Center, Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China) and were maintained in the Laboratory of Pathology, Southern Medical University (Guangzhou, China). Murine cell lines were cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA), and human cell lines in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), both supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C, 5% CO₂. All cell lines were used within 8 passages authenticated by short tandem repeat (STR) profiling and confirmed mycoplasma-free via monthly PCR testing.

For cell proliferation, 500 cells per well were seeded in 96-well plates and treated with Thimerosal (MC38 0.5 μM, CT26 1 μM, B16F10 0.1 μM, NCM460 1 μM) or its analogues CPD1 and CPD2 (CT26 1 μM, B16F10 0.1 μM) for up to 5 days. Cell viability was assessed every 24 h using Cell Counting Kit 8 (CCK-8, CK04, Dojindo, Kumamoto, Japan) with 1 h incubation, and absorbance was measured at 450 nm using a microplate reader (EnVision, PerkinElmer, Waltham, MA, USA). For viability assay, 800 cells per well (including MC38, CT26, B16F10, NCM460, SW620, SW480, LS-174T, DLD1, HCT8, RKO, HT29, LOVO, HCT15, and HCT116) were seeded in 96-well plates. Cells were treated with Thimerosal at varying doses (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1562, 0.078, 0.039, and 0.02 μM), using PBS as the control, for 48 h. After CCK-8 incubation, absorbance was measured at 450 nm. The half-maximal inhibitory concentration (IC₅₀) was calculated by fitting dose-response data to a nonlinear regression model [log (inhibitor) vs. normalized response (Variable slope)] using GraphPad Prism software (Version 10.1, GraphPad Software, San Diego, CA, USA).

200 Cells per well were seeded in 6-well plates. Thimerosal was initially added at a specific concentration (CT26 1 μM, B16F10 0.1 μM, DLD1 1 μM, HCT15 1 μM). After 10–14 days, colonies formed by single cells were fixed with methanol (0.791 g/mL) and were stained with 0.1% crystal violet (Sigma, St. Louis, MO, USA) for 20 min. The plates were photographed (BX53, Olympus, Tokyo, Japan), and the number of colonies was quantified.

Cells (CT26, B16F10, DLD1, HCT15) were seeded into 6-well plates and grown to 90% confluence. A scratch wound was created in each well using a 10 μL pipette tip. The wells were then gently washed twice with 2 mL of PBS to remove detached cells and debris. Migration was monitored by microscope (CKX31, Olympus, Tokyo, Japan) at 0, 24, 48, and 72 h, and image were captured.

4 × 10⁴ cells (CT26, B16F10, DLD1, HCT15) were seeded with 200 μL serum-free medium into 8.0-mm filter transwell upper chamber (Corning, Tewksbury, MA, USA), which was inserted into 24 well transwell plate. Medium containing 10% FBS was added to the bottom chamber. After 48 h of incubation, cells that migrated to the lower chamber were fixed with methanol (0.791 g/mL), stained with 0.1% crystal violet and counted under a microscope (BX53, Olympus, Tokyo, Japan) at 20× magnification. For transwell invasion assay, the chamber was pre-coated with Matrigel, and cells were incubated for 72 h.

1 × 10⁴ cells per well were seeded in 96-well plates and treated with Thimerosal at specified concentrations (MC38 0.5 μM, CT26 1 μM, B16F10 0.1 μM, DLD1 1 μM, HCT15 1 μM). After 24 h incubation, ATP levels were measured by the CellTiter-Glo kit (G7570, Promega, Shanghai, China). Briefly, luciferin and luciferase were added to cell lysates to react with ATP. The ATP levels in treated cells were normalized to those in control cells. Luminescence was measured using a Multiscan Spectrum microplate reader (Cytation5, Bio-Tek, Agilent, Winooski, VT, USA).

Cells (MC38, CT26, B16F10, DLD1, HCT15) were treated with Thimerosal, CPD1, or CPD2 for 48 h, then lysed using SDS lysis buffer (KeyGEN BioTECH, Nanjing, China). Protein concentration was quantified with a BCA protein assay kit (23225, Pierce, Thermo Fisher Scientific, Waltham, MA, USA) in RIPA buffer. Equal amounts of protein were separated by 10% polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Merck Millipore, Burlington, MA, USA). After blocking with 5% skim milk, the membrane was incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were detected using SuperSignal West Femto Chemiluminescent Substrate (34096, Thermo Fisher Scientific, Waltham, MA, USA), and protein bands were visualized with Image Lab Software (Tanon 5200, Shanghai, China).

Primary antibodies included: Anti-PARP (1:1000, 9532T, RRID: AB_659884, Cell Signaling Technology, Danvers, MA, USA); anti-Cleaved Caspase-3 (1:1000, 9664T, RRID: AB_2070042, Cell Signaling Technology, Danvers, MA, USA); anti-Caspase-3 (1:1000, 9662S, RRID: AB_331439, Cell Signaling Technology, Danvers, MA, USA); anti-α-tubulin (1:10,000, HRP-66031, RRID: AB_2687491, Proteintech, Chicago, IL, USA); anti-JAK1 (1:1000, 3344T, RRID: AB_2265054, Cell Signaling Technology, Danvers, MA, USA); anti-p-JAK1 (1:200, 3331S, RRID: AB_2265057, Cell Signaling Technology, Danvers, MA, USA); anti-STAT3 (1:1000, 12640, RRID: AB_2629499, Cell Signaling Technology, Danvers, MA, USA); anti-p-STAT3 (1:1000, 9145S, RRID: AB_2491009, Cell Signaling Technology, Danvers, MA, USA); anti-GAPDH (1:10,000, 60004-1-Ig, RRID: AB_2107436, Proteintech, Chicago, IL, USA).

Cancer cells treated with Thimerosal (MC38 0.5 μM, CT26 1 μM, DLD1 1 μM, HCT15 1 μM) for 48 h were harvested, washed, and resuspended in PBS. The cell suspension was centrifuged (5702R, Eppendorf, Hamburg, Germany) at 1500 rpm for 5 min; this washing step was repeated twice. Apoptosis was assessed using the Annexin V-FITC assay kit (KGA107, KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. Cells were stained with Annexin V-FITC and propidium iodide for 10 min in the dark and analyzed by flow cytometry (BD, Franklin Lakes, NJ, USA).

Female C57BL/6, BALB/c and BALB/c nude mice (Guangdong Medical Laboratory Animal Center) aged 5–6 or 8 weeks were used. Mice were group-housed (4–5 mice per cage) in individually ventilated cages under specific pathogen-free conditions, with a 12-h light/dark cycle, and had adlibitum access to autoclaved water and standard irradiated rodent chow. All animal experiments were approved by the Animal Care and Use Committee of Southern Medical University (Approval No.: NFYY-2018-031; Date: 10 February 2018) and conducted in accordance with National Institutes of Health (NIH; Bethesda, MD, USA) guidelines. Humane endpoints were strictly observed beyond the primary tumor volume (≥2000 mm³) and ulceration. Animals were humanely euthanized immediately if they exhibited any of the following: (1) body weight loss exceeding 20% of the initial weight; (2) lethargy, anorexia, or hunched posture for >24 h; (3) inability to access food or water normally.

Initially, all tumor cells were resuspended in PBS (pH 7.2–7.4, 0.01 M). MC38 cells (1 × 10⁶/100 μL) were injected subcutaneously into the right flank of 5–6-week-old C57BL/6 or BALB/c nude mice. CT26 cells (1 × 10⁶/100 μL) were injected into BALB/c or BALB/c nude mice of the same age. B16F10 cells (5 × 10⁵/100 μL) were injected into the right flank of 5–6-week C57BL/6 mice. When the tumor volume reached 50–100 mm³, mice were randomly assigned to various experimental groups, including vehicle control and Thimerosal (CPD1, CPD2) treatment groups (with specific group sizes of n = 6–10 mice per group as detailed in the figure legends for each experiment). Mice received Thimerosal, CPD1, CPD2, anti-PD-1 antibody. Tumor size was measured using vernier calipers, and volume was calculated as 0.5 × length × width². Mice were euthanized when volume tumor approached 2000 mm³ or upon observation of ulceration. Tumors were excised, recorded, fixed in 10% neutral buffered formalin (GHRHCH, Guangzhou, China), and embedded in paraffin.

B16F10 cells (1 × 10⁵/100 μL) were injected intravenously via the tail vein into 5–6 weeks C57BL/6 mice. Thimerosal treatment was initiated on day 1. After 21 days, mice were euthanized, and lungs were harvested, rinsed in PBS, and placed on a white background. The number of surface metastatic nodules was counted manually under a dissecting microscope by an investigator blinded to group assignments. Lungs were then fixed in 10% neutral buffered formalin (GHRHCH, Guangzhou, China) for 24–48 h at room temperature for further histological analysis.

8-week-old mice (Thimerosal n = 9 and PBS n = 8) were intraperitoneally injected (ip) with 5 mg/kg Azoxymethane (AOM, Sigma-Aldrich, St. Louis, MO, USA) on days 1 and 4, and received regular drinking water on days 1–7. Mice were then underwent three cycles of dextran sulfate sodium (DSS, MW 36,000–50,000 Da, MP Biomedical, Santa Ana, CA, USA) administration (1.8%, 2%, and 2%, each for 7 days) interspersed with 14-day periods of regular drinking water. After one month under standard conditions, mice were euthanized on day 101. Thimerosal (16.25 μg/kg) was administered during the late stage (after three DSS cycles). Colons were excised, and tumor number and size were recorded.

Tissues obtained from the subcutaneous, lung metastasis and AOM/DSS colitis-associated CRC model described in Sections 2.11–2.13 were fixed, embedded, and sliced into 3 μm slices. The slices were stained using 0.5% hematoxylin and 1% eosin (H&E) (C0105S, Beyotime, Shanghai, China) stains. For immunohistochemistry, the slices were incubated with CD31 (1:100, 77699s, Cell Signaling Technology, Shanghai, China), anti-CD4 (1:1000, ab183685, Abcam, Shanghai, China), anti-CD8 (1:1000, ab209775, Abcam, Shanghai, China), and anti-Ki-67 (1:1000, ab21700, Abcam, Cambridge, UK) overnight at 4°C, appropriate secondary antibodies were applied. Finally, the slices were stained with DAB (ZLI-9019, Zsbio, Beijing, China) at a ratio of reagent A to B = 1:20 and counterstained with 0.5% hematoxylin.

To evaluate Thimerosal toxicity, 5–6 weeks old C57BL/6 mice were intraperitoneally injected with Thimerosal (16.25 μg/kg/qod) for a total of 10 administrations and then euthanized. The heart, liver and kidney were examined by H&E. For biochemical tests, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were measured using an Automatic Biochemical Analyzer (BS-240VET, Mindray, Shenzhen, China).

In initial dose-finding studies, Thimerosal was administered at 4.0625, 8.125, 16.25, and 32.5 μg/kg, qod/ip. Based on the results, 16.25 μg/kg was selected for subsequent experiments. The following treatments were used: Thimerosal at 16.25 μg/kg. Its analogues CPD1 and CPD2 are at 16.25 μg/kg. Colivelin (TP1856, TargetMol, Shanghai, China), a synthetic peptide activator of the STAT3 signaling pathway, at 1 mg/kg, qod/ip. For combination therapy, 200 μg anti-PD-1 (BE0146, RMP1-14, BioXcell, Lebanon, NH, USA) or its Isotype control (BE0089, 2A3, BioXcell, Lebanon, NH, USA), diluted in PBS to a final volume of 100 μL, was administered intraperitoneally on days 8, 11, 13, 15, and 17.

Total RNA was extracted from PBS-or Thimerosal-treated CT26 cells using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) and assessed for purity, concentration, and integrity. Libraries were prepared from 1.3 μg total RNA using the TruSeq™ RNA Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq4000 (150-bp paired-end reads, ~40 million reads/sample).

After sequencing and quality control, clean reads were mapped to the mouse reference genome GRCm39 using HISAT2 (v2.2.0) [27], and gene expression levels were calculated using StringTie (v2.1.7) [28]. Differentially gene expression analysis was performed using DESeq2 (v1.44.0) [29] with significance thresholds set at |log₂ fold change| > 1.5 and FDR < 0.05.

Functional enrichment analysis of differentially expressed genes was performed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using the enrich GO and enrich KEGG functions from the clusterProfiler (v4.0) [30] R package.

Data were presented as mean ± Standard Error of the Mean (SEM). The nonparametric two-tailed t-test was used for two-group comparisons; two-way ANOVA was applied for multi-group comparisons with two variables. The log-rank test was applied to analyze survival data. For the analysis of the distribution of tumor grades, the Chi-square test was applied. For in vivo studies, animals were randomly assigned to treatment groups, and tumor measurements were performed by an investigator blinded to the group allocation. All data were derived from at least three independent experiments. p < 0.05 was defined statistically significant.

The chemical structure of Thimerosal is shown in Fig. 1A. To evaluate its cytotoxicity and inhibitory effects, mouse CRC cell lines MC38 and CT26, mouse melanoma cell line B16F10, and multiple human CRC cell lines were treated with Thimerosal. Cells were exposed to various concentrations for different durations, and viability was assessed using CCK-8 assay. Thimerosal inhibited tumor cell growth in a time-and dose-dependent manner (Figs. 1B,C and A1A). Importantly, this growth inhibition was not observed in normal human intestinal epithelial (NCM460) cells (Fig. A1B), indicating selective cytotoxicity. Thimerosal concentrations were selected based on IC₅₀ values for each cell line. Microscopic examination further confirmed the growth-inhibitory effects, with treated cells showing reduced density and morphological changes indicative of impaired viability (Fig. 1D).

To further characterize the antiproliferative properties of Thimerosal, colony formation assays were performed. Thimerosal treatment significantly suppressed the colony-forming ability of tumor cells (Figs. 1E and A1C). This finding was supported by ATP assays, which revealed markedly reduced ATP levels in MC38, CT26, B16F10, DLD1, and HCT15 cells after treatment (Figs. 1F and A1D).

To determine whether growth inhibition was associated with apoptosis, cells were analyzed for apoptotic markers after 48 h of treatment. Flow cytometric analysis with Annexin V/PI staining showed a significant increase in the proportion of apoptotic cells (Fig. A1E). Western blot analysis demonstrated increased levels of cleaved caspase-3 and cleaved PARP (Fig. 1G). These results indicate that Thimerosal exerts potent antitumor effects by selectively inhibiting proliferation and inducing apoptosis.

Since cell migration and invasion are crucial for tumor metastasis, we evaluated the effects of Thimerosal using wound healing and Transwell assays. Thimerosal treatment significantly reduced the migratory capacity of tumor cells (Fig. 2A,B). Similarly, Transwell migration assays confirmed that Thimerosal markedly inhibited tumor cell migration (Fig. 2C,D). In Matrigel-coated Transwell assays, Thimerosal also effectively suppressed tumor cell invasion (Fig. 2E,F).

To evaluate the therapeutic potential of Thimerosal in vivo, MC38 cells were subcutaneously injected into C57BL/6 mice. When tumor volumes reached approximately 50–100 mm³, mice were randomly divided into five groups: a control group (PBS) and four experimental groups receiving different doses of Thimerosal via intraperitoneal injection (Fig. 3A). Notably, Thimerosal treatment significantly suppressed the growth of MC38-derived subcutaneous tumors at both low and high doses, as indicated by reduced tumor volumes compared with the PBS control group (Fig. 3B). A dose of 16.25 μg/kg was selected for subsequent experiments. Similar suppression of tumor growth was observed in B16F10 and CT26 subcutaneous models (Figs. 3C and A2A).

Metastasis is a major cause of tumor progression and cancer-related mortality. We next evaluated the efficacy of Thimerosal in a lung metastasis model established by intravenous injection of B16F10 cells via the tail vein (Fig. 3D). Thimerosal treatment reduced lung weight, decreased the number of lung metastases (Fig. 3E), and improved survival rates (Fig. A2B). CD31 staining revealed significantly reduced angiogenesis in Thimerosal-treated MC38 tumors (Fig. A2C).

In an AOM/DSS-induced colitis-associated CRC model (Fig. 3F), Thimerosal significantly reduced both tumor number and cumulative diameter (Fig. 3G,H). Histopathological analysis showed that most tumors in the Thimerosal-treated group were adenomas with low-or high-grade intraepithelial neoplasia, whereas tumors in the AOM/DSS group consisted mainly of high-grade intraepithelial neoplasia or adenocarcinoma (Fig. 3I). Tumors were blindly categorized as low-grade dysplasia, high-grade dysplasia, or adenocarcinoma. The distribution of tumor grades differed significantly between PBS-and Thimerosal-treated groups, as determined by the chi-square test (Fig. A2D).

Safety assessment in healthy C57BL/6 mice revealed no significant body weight changes (Fig. A3A). Histopathological examination of major organs (heart, liver, and kidney) by H&E staining revealed no pathological abnormalities (Fig. 3J). Furthermore, serum levels of ALT, AST, BUN, and CREA remained within normal ranges, with no significant differences between Thimerosal-treated and control groups (Fig. A3B). These results demonstrate that Thimerosal exerts potent antitumor effects, suppressing tumor growth and metastasis in CRC and melanoma models, with minimal toxicity.

Given the importance of metal ions in the biological activity of many drugs, we hypothesized that the mercury ions in Thimerosal were critical for its antitumor effects. To explore this, we utilized two structural analogues of Thimerosal (CPD1 and CPD2), in which the mercury group was replaced with an inert methyl group or benzene ring, respectively (Figs. 4A and A4A).

CCK-8 assays showed that CPD1 and CPD2 exerted no inhibitory effect on CT26 or B16F10 growth (Figs. 4B and A4B). Colony formation assays confirmed that both analogues failed to reduce colony numbers (Figs. 4C and A4C). ATP assays showed no significant differences in metabolic activity (Figs. 4D and A4D). Wound healing and Transwell assays demonstrated that neither analogue inhibited migration (Figs. 4E and A4E) or invasion (Figs. 4F and A4F).

In CT26 and B16F10 subcutaneous models, CPD1 and CPD2 showed no significant effects on tumor growth (Figs. 4G,H and A4G,H). Ki-67 staining revealed no significant difference in proliferation index (Figs. 4I and A4I). These results confirm that mercury ions are essential for Thimerosal’s antitumor activity.

To investigate the mechanism underlying the antitumor effects of Thimerosal, transcriptome analysis was performed to identify differentially expressed genes between Thimerosal-treated and untreated CT26 cells. GO analysis showed that these differentially expressed genes were predominantly associated with biological processes such as response to interferon-beta, regulation of response to biological stimulation, and regulation of innate immune response (Fig. 5A). KEGG pathway enrichment analysis and Volcano plot indicated significant enrichment in pathways related to drug metabolism, JAK-STAT signaling, and TNF signaling (Fig. 5B,C). Notably, the interferon response identified by GO enrichment was closely linked to the JAK-STAT signaling pathway identified by KEGG analysis. Given that the JAK-STAT pathway is a key downstream effector of interferon signaling and plays a pivotal role in antitumor responses, we hypothesized that this pathway might mediate Thimerosal’s antitumor effects.

Western blot analysis confirmed that Thimerosal decreased JAK1 and STAT3 phosphorylation (Fig. 5D). In contrast, CPD1 and CPD2 did not alter JAK1/STAT3 pathway activity. In subcutaneous tumor models, Thimerosal monotherapy significantly suppressed tumor growth, while combination with the STAT3 activator Colivelin restored tumor growth to control levels (Fig. 5E–H). This rescue effect supports the conclusion that Thimerosal acts through JAK1/STAT3 inhibition.

Thimerosal has been reported to exhibit antigen-stabilizing properties, and transcriptome analysis has linked its activity to interferon response-related genes and the JAK/STAT signaling pathway. Given that the JAK/STAT pathway serves as a crucial downstream effector of interferon signaling and plays a pivotal role in tumor immune responses, we investigated whether Thimerosal modulates the tumor immune microenvironment to influence tumor growth.

Immunohistochemical analysis revealed significantly increased infiltration of CD4⁺ and CD8⁺ T cells in MC38 and CT26 tumors after Thimerosal treatment (Fig. 6A,B). To further evaluate the role of T cell infiltration in Thimerosal-mediated antitumor effects, a subcutaneous tumor model was established in BALB/c nude mice, which lack functional T cells (Fig. 6C). Thimerosal treatment resulted in a modest reduction in tumor growth rate and tumor weight in MC38 subcutaneous tumors in nude mice compared to the control group (Fig. 6D,E). However, the antitumor efficacy was markedly reduced compared to that observed in immune-competent C57BL/6 mice. Similarly, Thimerosal treatment partially inhibited the growth of CT26 subcutaneous tumors in nude mice, but the effects were less pronounced than those in immune-competent BALB/c mice (Fig. 6F,G). These results indicate that Thimerosal enhances the infiltration of CD4⁺ and CD8⁺ T cells in the tumor microenvironment, which contributes to its antitumor efficacy.

We evaluated the combination of Thimerosal with anti-PD-1 in subcutaneous tumor models. Both monotherapies suppressed tumor growth, while the combination showed enhanced efficacy, achieving complete tumor regression in 3 of 10 MC38-bearing mice (Fig. 7A–C). Similar synergy was observed in CT26 models, with the combination being more effective than either agent alone (Fig. 7D,E). Immunohistochemical analysis revealed that Thimerosal promoted the infiltration of CD8⁺ T cells within the tumor stroma (Fig. 7F,G).

The antitumor efficacy of the combination was further evaluated in a B16F10 lung metastasis model established via tail vein injection (Fig. 7H). Both Thimerosal and anti-PD-1 monotherapies partially suppressed metastatic tumor growth. In contrast, the combination therapy significantly reduced lung tumor burden, including metastatic growth and number (Fig. 7I–K), and prolonged the survival time of treated mice (Fig. 7L). These results indicate that the combination of Thimerosal and anti-PD-1 enhances antitumor immunity and improves therapeutic outcomes in both subcutaneous and metastatic tumor models.