On April 18, the latest achievement by the team led by Researcher Yan Fei from the SIAT, in collaboration with Professor Chen Zhiyi's team from the Changsha Central Hospital Affiliated to University of South China, was published online in the Cell Reports Medicine, a sub-journal of Cell. The study is titled "Ultrasound-Visualized Engineered Bacteria for Tumor Chemo-immunotherapy." In this project, the research team constructed an Ultrasound-Visualized engineered bacterium (Ec@DIG-GVs) containing acoustic reporter genes and temperature-controlled gene expression circuits, with its surface modified with the chemotherapeutic drug doxorubicin (DOX). These engineered tumor-targeting bacteria can express acoustic reporter genes to produce gas vesicles (GVs), providing real-time imaging guidance for focused ultrasound (hHIFU), enabling precise localization of the ultrasound focus within the tumor at the engineered bacteria, inducing local expression and secretion of IFN-γ in the tumor. The production of IFN-γ not only kills tumor cells, but also induces macrophages to polarize from M2 to M1 phenotypes, promoting dendritic cell maturation. Additionally, DOX on the surface of the engineered bacteria can be released in the acidic microenvironment of the tumor, leading to immunogenic death of tumor cells. The combined action of IFN-γ and DOX activates tumor-specific T-cell responses, producing a synergistic effect that significantly enhances antitumor efficacy. This research has developed a new strategy for visually regulating gene expression of tumor-targeting bacteria in vivo, holding great potential applications in the regulation of gene expression in bacteria, immune cells, stem cells, and other living cells in vivo. Researchers Yan Fei from the SIAT, and Professor Chen Zhiyi from the Changsha Central Hospital Affiliated to University of South China are co-corresponding authors of the paper. Yang Yaozhang, a former visiting student at the SIAT, is the first author.
Screenshot of the article published online
Link to the article: https://doi.org/10.1016/j.xcrm.2024.101512
The integration of synthetic biology technology with tumor-targeting bacteria has significantly enhanced the responsiveness of bacteria to the internal microenvironment or external physical stimuli within tumors, making precise in vivo regulation of therapeutic gene expression possible. Currently, researchers have developed various chemical, physical, and biological methods by combining different promoter elements to achieve precise regulation of therapeutic gene expression by engineered bacteria at tumor sites. However, the intravenous injection of chemical inducers has side effects and difficulty targeting tumor sites; quorum sensing induction systems have uncontrollable gene induction times. In contrast, physical stimuli such as optogenetic or sonogenetic control have advantages such as targeted precision, high spatiotemporal resolution, and convenient switching on/off. However, optogenetic stimulation is insufficient in tissue penetration, limiting its application in deep tumors. In comparison, ultrasound offers high tissue penetration, excellent spatiotemporal resolution, which can not only achieve real-time imaging, but also focus sound waves on deep tumor sites to produce thermal effects for gene expression regulation. In previous research (Nat Commun. 2022;13:4468), the research team developed an ultrasound-responsive genetic circuit integrated into tumor-targeting bacteria to achieve spatiotemporally controlled expression of therapeutic genes at tumor sites and improve the effectiveness and safety of bacterial gene therapy for tumors. However, due to uneven distribution of bacteria within the tumor and difficulty in monitoring the ultrasound focus, it is still not possible to visualize these engineered bacteria inside the tumor and achieve image-guided ultraprecise localization of bacteria within the tumor to regulate therapeutic gene expression. To address this problem, Researcher Yan Fei's team and Professor Chen Zhiyi's team developed an Ultrasound-Visualized engineered bacterium (Ec@DIG-GVs) that can precisely regulate the gene expression of engineered bacteria within tumors under ultrasound imaging guidance, providing a new strategy for cancer treatment.
The research team first introduced the acoustic reporter gene (ARG1) plasmid and temperature-controlled gene circuit (IFN-γ) plasmid into Escherichia coli MG1655 successively to obtain Ec@IG. Then, they induced the expression of the acoustic reporter gene in Ec@IG in vitro to produce gas vesicles (GVs) containing bio-nano bubbles Ec@IG-GVs. Finally, using a chemical modification strategy, doxorubicin was conjugated to the surface of Ec@IG-GVs to prepare the Ultrasound-Visualized engineered bacterium Ec@DIG-GVs. The results of transmission electron microscopy and phase contrast microscope confirmed that Ec@DIG-GVs bacteria expressed a large number of bio-nano bubbles (GVs) internally, and immunofluorescence labeling and absorbance measurements indicated successful coupling of doxorubicin to the bacterial surface (Fig. 1).
Figure 1. Preparation of Ultrasound-Visualized Bacteria
The research team further validated the various functions of Ec@DIG-GVs. Firstly, they confirmed that Ec@DIG-GVs containing GVs had ultrasound contrast imaging signals with concentration-dependent enhancement. Meanwhile, to verify the feasibility of using ultrasound imaging to guide hHIFU stimulation for the expression of exogenous genes in engineered bacteria with temperature-controlled gene circuits, the research team replaced the IFN-γ gene in Ec@IG-GVs with a mCherry reporter gene (Ec@MG-GVs) and embedded the bacteria in agar gel (the upper layer contained bubble bacteria, and the lower layer did not contain bubble bacteria). Under ultrasound imaging guidance, the hHIFU focus was positioned at the Ec@MG-GVs site for irradiation for 25 minutes. The results showed that the area irradiated by hHIFU exhibited that ultrasound signals disappeared due to the bursting of GVs. After being placed at 37°C for 6 hours, it was observed that only the area receiving hHIFU exhibited significant red fluorescence, indicating that the engineered bacteria could achieve ultrasound contrast-guided hHIFU-induced localized expression of mCherry protein. Additionally, by placing Ec@DIG-GVs under different pH conditions, it was confirmed that DOX could achieve acid-responsive release and effectively enter tumor cells (Fig. 2).
Figure 2. Functional Characterization of Ultrasound-Visualized Bacteria
Subsequently, the research team tested the tumor cell killing activity and immune activation of Ec@DIG-GVs. They subjected Ec@DIG-GVs to acidic treatment and induced the expression of IFN-γ with hHIFU irradiation. After centrifuging to remove the bacteria, a supernatant containing DOX and IFN-γ was obtained. Incubating this supernatant with tumor cells resulted in significant apoptosis and immunogenic cell death (as detected by CRT and HMGB1 protein markers). Co-incubation of the supernatant with macrophages showed a significant increase in the proportion of M1-type macrophages, indicating that the released IFN-γ and DOX from Ec@DIG-GVs have the ability to kill tumor cells and induce polarization of M1-type macrophages (Fig. 3).
Figure 3. Ultrasound-Visualized Bacteria Express IFN-γ and Release DOX to Kill Tumor Cells and Induce Macrophage Polarization
Next, the research team applied the engineered bacteria to tumor-bearing mice by intratumoral injection of Ec@MG or Ec@MG-GVs. It was found that Ec@MG-GVs exhibited good ultrasound imaging capabilities within the tumor, allowing for the localization of bacterial positions within the tumor and guiding hHIFU irradiation. The results showed that in the same tumor, only the area irradiated by hHIFU exhibited disappearance of ultrasound signals, and red fluorescence (mCherry protein expression) appeared 4 hours after irradiation, while the non-irradiated areas did not show red fluorescence. Sectioning results revealed that the red fluorescence produced in the irradiated area was approximately circular, corresponding to the hHIFU irradiation focus. Using this method for ultrasound imaging-guided hHIFU irradiation of Ec@DIG-GVs for tumor treatment, significant inhibition of tumor growth was observed, and the survival period of tumor-bearing mice was extended (Fig. 4).
Figure 4. Verification of Antitumor Efficacy of Intratumoral Injection of Ultrasound-Visualized Bacteria
After confirming that intratumoral injection of engineered bacteria combined with ultrasound irradiation could achieve good therapeutic effects, the research team further tested the tumor targeting ability of the engineered bacteria after intravenous injection. To facilitate observation, ICG was replaced with DOX to modify the surface of Ec@IG-GVs to obtain Ec@IIG-GVs. After intravenous injection of Ec@IIG-GVs, fluorescence imaging of the tumor and major organs showed that Ec@IIG-GVs had good tumor targeting and aggregation ability. Importantly, the research team found that after intravenous injection of Ec@IIG-GVs, the ultrasound imaging signal in the tumor gradually increased over time, consistent with the fluorescence imaging targeting verification results (Fig. 5), indicating that the engineered bacteria could also achieve ultrasound imaging tracing within the tumor.
Figure 5. Tumor Targeting Verification of Ultrasound-Visualized Bacteria
Based on the good tumor targeting of the engineered bacteria, the research team further tested the effect of intravenous injection of Ec@DIG-GVs combined with ultrasound-induced IFN-γ gene expression for tumor treatment. After intravenous injection of Ec@DIG-GVs, the time and location of the engineered bacteria reaching the tumor were determined through ultrasound imaging, guiding hHIFU irradiation to induce IFN-γ expression in bacteria within the tumor. The results showed that under the combined action of IFN-γ and DOX, the Ec@DIG-GVs+hHIFU treatment group significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice. Further mechanism studies found that ultrasound imaging-guided hHIFU irradiation of Ec@DIG-GVs (Ec@DIG-GVs+hHIFU group) not only significantly promoted tumor cell apoptosis, but also facilitated the maturation of DC cells within the tumor and the polarization of M2 macrophages to M1 type, effectively activating the body's anti-tumor immune response (Fig. 6).
Figure 6. Antitumor and Immunostimulation Effects of Intravenous Injection of Ultrasound-Visualized Bacteria
This project was supported by the National Key R&D Program of the Ministry of Science and Technology, the National Natural Science Foundation of China, and the Shenzhen Institute of Synthetic Biology, among others.
Yang Yaozhang; Wang Yuanyuan; Feng Fengyi; Chen Yuhao; Chen Zhiyi; Yan Fei. Ultrasound-Visualized Engineered Bacteria for Tumor Chemo-immunotherapy. Cell Reports Medicine, 2024.
