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| Therapeutic Efficacy Study Based on Listeria-based Live Attenuated Double Substitution (LADS) Expressing WT1 in a CT26-WT1 Tumor-bearing Mouse Model |
| SUN Jing, GAO Meng-Yuan, HU Lai-Yin, WANG Yi-Fan, LI Chao-Jie, YU Lu-Ying, QIAN Jia-Miao, SONG Hou-Hui, WU Yong-Shu* |
| College of Veterinary Medicine / Key Laboratory of Applied Biotechnology on Animal Science & Veterinary Medicine of Zhejiang Province/Zhejiang Engineering Research Center for Veterinary Diagnostics & Advanced Technology / Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management / Belt and Road International Joint Laboratory for One Health and Food Safety / China-Australia Joint Laboratory for Animal Health Big Data Analytics , Zhejiang A&F University, Hangzhou 311300, China |
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Abstract Colorectal cancer is one of the malignant tumors that severely threaten human health. Safe and effective therapeutic vaccines are urgently required to induce tumor-specific anti-tumor immunity. Wilms tumor antigen 1 (WT1) is highly expressed in various solid tumors but restricted in normal tissues, making it an ideal tumor-associated antigen. In this study, a LADS-WT1 vaccine was constructed using Listeria-based live attenuated double substitution (LADS) as a vector (LADS-WT1). Furthermore, a mouse (Mus musculus) colon cancer cell line (CT26-WT1) stably expressing WT1 was established to evaluate the specific therapeutic efficacy of LADS-WT1. In vitro growth analysis showed that the proliferation kinetics of the recombinant strain LADS-WT1 in brain heart infusion (BHI) broth were basically consistent with those of the parental LADS strain, indicating that the insertion of the heterologous antigen did not significantly impair its growth capacity. In addition, hemolysis assays demonstrated that the hemolytic activity of LADS-WT1 in liquid culture was significantly reduced, suggesting that the function of listeriolysin O (LLO), a virulence-related protein, was inhibited and its safety was further improved. The LADS-WT1 vaccine was administered via tail vein injection to CT26 and CT26-WT1 tumor-bearing BALB/c mice, respectively, and changes in tumor growth and immune responses were dynamically monitored. The results showed that following continuous LADS-WT1 vaccine treatment for 20 d, CT26-WT1 tumor-bearing mice exhibited a slower tumor growth rate and significantly smaller tumor volumes compared with CT26 tumor-bearing mice, indicating that the LADS-WT1 vaccine could inhibit the growth of WT1-expressing colorectal cancer cells. Flow cytometry analysis revealed an increased proportion of CD4+ T cells and CD8+ T cells in the spleens of CT26-WT1 tumor-bearing mice immunized with the LADS-WT1 vaccine, indicating that the vaccine effectively activated systemic cellular immune responses. In conclusion, the LADS-mediated delivery of the WT1 vaccine exerts anti-colon cancer effects through cellular immune responses, which provides a therapeutic strategy for other WT1-positive malignancies.
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Received: 19 November 2025
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Corresponding Authors:
*wuyongshu0224@zafu.edu.cn
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