Thimerosal Inhibits Tumor Malignant Progression Through Direct Action and Enhancing the Efficacy of PD-1-Based Immunotherapy
Background: Thimerosal is a mercury-containing preservative widely used in vaccines.. This study aimed to investigate its potential antitumor effects and mechanisms in solid malignancies, particularly colorectal cancer and melanoma. Methods: A combination of in vitro and in vivo approaches was employed. Cell proliferation, apoptosis, migration, and invasion were assessed using Cell Counting Kit-8 (CCK-8), colony for-mation, ATP viability, Western blotting, flow cytometry, wound-healing and transwell assays. Sub-cutaneous, lung metastases, and Azoxymethane/Dextran Sulfate Sodium Salt (AOM/DSS)-induced colitis-associated colorectal cancer (CRC) models were established to examine antitumor efficacy and safety. The functional role of mercury ions was validated using structural analogues. Mechanistic studies included RNA sequencing, Western blot, and immunohistochemical analysis of CD8+ T cell infiltration. The synergistic effect with programmed cell death protein 1 (PD-1) antibodies therapy was also evaluated. Results: Thimerosal potently inhibited tumor growth (with IC50 values ranging from 0.1 to 1 µM in vitro) and significantly prolonged survival without overt toxicity in vivo. Mechanistically, mercury ions were critical functional sites mediating Thimerosal's antitumor effects. Specifically, Thimerosal inhibited the phosphorylation of Janus kinase 1(JAK1) and signal transducer and activator of tran-scription 3(STAT3). Furthermore, Thimerosal enhanced the infiltration of CD8+ T cells into the tumor microenvironment and synergistically augmented the efficacy of PD-1 antibody therapy. Conclusion: Thimerosal exerts dual antitumor roles by direct JAK1/STAT3 inhibition and immune modulation via CD8+ T cell recruitment. It represents a promising repurposed drug and immuno-therapeutic adjuvant for CRC and melanoma. Overall design: Based on the provided background, the experimental design was structured to comprehensively evaluate thimerosal's antitumor potential. It utilized a combination of in vitro assays (CCK-8, colony formation, flow cytometry, wound-healing, transwell) on cancer cell lines and in vivo models (subcutaneous, lung metastasis, and AOM/DSS-induced CRC) in mice to assess efficacy and safety. The role of mercury ions was specifically tested using structural analogs. Mechanistic studies involved RNA sequencing, western blotting to analyze the JAK1/STAT3 pathway, and immunohistochemistry to quantify CD8+ T cell infiltration. Finally, the synergistic effect of thimerosal with anti-PD-1 therapy was investigated.