AniView Supports Tumor Immunotherapy Research: Thymidine-Auxotrophic Salmonella Enables Selective Tumor Colonization and Adenosine Depletion for Cancer Immunotherapy

New progress has been made in Salmonella-based selective tumor immunotherapy.

Professor Jinhui Wu from Nanjing University, in collaboration with Professor Xiaozhi Zhao from Nanjing Drum Tower Hospital and Professor Xiaoxiang Guan from The First Affiliated Hospital of Nanjing Medical University, published their findings in “Advanced Materials” (IF = 29.8).

This study provides a new strategy for metabolically gated bacterial immunotherapy, enabling tumor-specific bacterial colonization while significantly improving systemic safety and antitumor immune responses.

Bacterial therapy exploits the intrinsic tumor-targeting capability, immunogenicity, and genetic programmability of live bacteria, offering unique advantages for solid tumor treatment. Engineered bacterial strains such as attenuated Salmonella (e.g., VNP20009) and Escherichia coli (e.g., EcN Nissle1917) can selectively colonize tumors and deliver therapeutic molecules, including SGN1 that disrupts tumor methionine metabolism (currently in Phase I clinical trials) and engineered bacteria expressing Pin1 inhibitors for tumor microenvironment (TME) reprogramming. These approaches enhance antitumor immune responses through metabolic intervention and have demonstrated considerable therapeutic potential. However, the self-replicating nature of live bacteria in vivo raises serious safety concerns, as off-target colonization may trigger systemic inflammation or even sepsis. The clinical failure of VNP20009 due to excessive proliferation in the liver and spleen, as well as dose-limiting toxicity, highlights the key challenge in clinical translation: balancing tumor-targeting specificity with systemic safety. Existing strategies, including local administration (such as intravesical BCG therapy), are limited in application, while surface modification approaches suffer from poor stability. Engineered auxotrophic bacteria based on purine or D-alanine metabolism also show insufficient selectivity because these metabolites are widely distributed in normal tissues.

To address these challenges, the researchers proposed a novel metabolically gated strategy that controls bacterial proliferation through tumor microenvironment-specific nucleotide metabolic reprogramming. Pan-cancer analysis based on TCGA data revealed that thymidylate synthase (TYMS) is significantly overexpressed in 32 of 33 tumor types, with a 50% increase observed in colon adenocarcinoma (COAD) compared with normal tissues and a strong positive correlation with thymidine kinase 1 (TK1). By knocking down the thyA gene encoding TYMS in Salmonella VNP20009, the researchers constructed a thymidine-auxotrophic strain termed TK VNP, whose growth strictly depends on exogenous deoxythymidine monophosphate (dTMP). Due to rapid proliferation and cell lysis, tumors contain abundant dTMP, with MC38 tumors in mice showing 2.9-fold higher dTMP levels than normal tissues, whereas normal tissues fail to support bacterial growth because of insufficient dTMP availability. In vivo biodistribution studies demonstrated that TK VNP maintained tumor colonization efficiency comparable to the parental strain, reaching peak levels of 10⁹ CFU/g in CT26 tumors, while bacterial retention in organs such as the liver was reduced by approximately tenfold. On day 7, liver bacterial loads were 10⁵ CFU/g for VNP and only 10³ CFU/g for TK VNP, resulting in an almost tenfold improvement in tumor-targeting specificity. This design dramatically enhanced safety: after high-dose injection (10⁷ CFU), the mortality rate in the VNP group reached 100% within 5 days, whereas the survival rate in the TK VNP group exceeded 85%. At therapeutic doses, no liver abscess formation or ALT/AST elevation was observed, while robust antitumor efficacy was fully preserved, with tumor weight reduced by 75% on day 15 in the CT26 model. These findings effectively resolved the major safety bottleneck associated with live bacterial therapy.

Based on this safer platform, the researchers further introduced an immunometabolic reprogramming module. In the tumor microenvironment, CD73-mediated adenosine (ADO) accumulation activates A2A/A2B receptor signaling, suppressing T-cell function and inducing T-cell exhaustion. In this study, adenosine deaminase (ADD) was expressed in TK VNP to convert immunosuppressive ADO into immunostimulatory inosine (INO), which supports T-cell metabolism and resistance to exhaustion. Three engineered variants were constructed through subcellular localization optimization: cytoplasmic expression (cTKA VNP), secretory expression guided by the OmpA signal peptide (sTKA VNP), and surface-display expression anchored by Lpp-OmpA (wTKA VNP). In vitro experiments showed that sTKA VNP completely converted ADO into INO within 20 minutes with an efficiency exceeding 97%, significantly outperforming the intracellular variant, which achieved only 45% conversion after 60 minutes. This spatiotemporally coupled design precisely remodeled the metabolic landscape of the TME, simultaneously depleting ADO and replenishing INO to relieve immunosuppression. The strategy also provides a synergistic foundation for combination therapies such as immune checkpoint blockade (e.g., anti-PD-1 therapy) and radiotherapy, which induces CD73 upregulation, thereby offering a new paradigm for the clinical translation of next-generation tumor-targeting engineered bacteria.

Figure 1. Schematic illustration of TGA VNP based on thymidine auxotrophy and adenosine metabolism

Experiments using Aniview

To validate the tumor-targeting specificity and systemic safety of the engineered bacterial strain TK VNP, the AniView multimodal in vivo imaging system developed by Guangzhou Biolight Biotechnology Co., Ltd. was used to monitor bioluminescence signals in mice intravenously injected with luminescent bacterial strains. The results showed that in tumor-bearing mice (Figure E), both TK VNP and the parental VNP strain efficiently colonized CT26 tumors, with no significant difference in signal intensity observed at 48 hours, confirming that the engineered strain retained robust tumor-targeting capability. In contrast, in non–tumor-bearing mice (Figure F), the systemic bioluminescence signal of TK VNP was completely cleared within 48 hours, whereas persistent signals remained in the parental strain group. Moreover, in high-dose injection experiments (10⁷ CFU), the survival rate of the TK VNP group reached 88%, significantly higher than the complete mortality observed in the parental strain group (p < 0.0001). Collectively, these findings demonstrate that TK VNP achieves tumor-specific colonization through a thymidine metabolism-gated mechanism, while being rapidly eliminated from normal tissues because of its metabolic deficiency, thereby effectively avoiding the lethal systemic infection risk associated with the parental strain.

Figure 2. Tumor-targeting specificity and systemic safety of TK VNP compared with the parental VNP strain