Based on our experimental findings, we have envisioned a series of future optimization strategies centered on engineered bacterial intracellular delivery, safety control, and therapeutic responsiveness, aiming to advance this system toward clinical translation. Now, let us consider a hypothetical triple-negative breast cancer (TNBC) patient prepared to undergo this treatment.
Safety Considerations
TNBC patients may have compromised immune function due to disease progression or prior treatments, making thorough safety evaluation necessary when administering a commensal bacterial chassis. We selected the intratumoral commensal and opportunistic bacterium Staphylococcus xylosus ATCC 29971. Compared to traditional attenuated pathogenic strains, this commensal bacterium offers advantages such as long-term coexistence within the host, stable persistence, and absence of major virulence factors. Furthermore, the engineered bacteria’s adaptive cohabitation features enable maintenance of stable bacterial densities without triggering significant immune responses, thus balancing safety and sustained therapeutic efficacy.
To further minimize potential off-target effects on normal tissues and reduce risks, we designed a suicide switch and tumor-specific anticancer drug delivery system as additional safeguards.
Administration Routes
After evaluating the safety of the treatment, we consider the patient’s administration route. Clinically, common routes include oral, intravenous, and intramuscular administration, each with specific indications and considerations. The optimal route should be selected based on the patient’s individual condition.
1. Intratumoral Injection
If the tumor can be clearly and precisely localized, intratumoral injection is preferred. This approach ensures high local concentrations of engineered bacteria at the injection site, maximizing precise delivery and efficient tumor cell invasion while minimizing bacterial dissemination to other tissues.
2. Intravenous Injection
For patients with poorly defined tumor boundaries or multiple lesions, intravenous injection may be chosen.
In tumor tissues or necrotic regions, interstitial gaps can expand to 1–2 μm, exhibiting the enhanced permeability and retention (EPR) effect. Combined with microvascular rupture and incomplete basement membranes, tumor tissue permeability is significantly increased, providing a passive entry route for particles of a size similar to Staphylococcus.
Beyond passive accumulation, S. xylosus ATCC 29971 demonstrates natural tumor tissue selectivity. Although the precise mechanism remains unclear, existing studies and our experimental results show that this strain exhibits tissue tropism and high invasion efficiency during host infection. This suggests that S. xylosus ATCC 29971 may possess unique host-recognition and invasion mechanisms. Future genomic and functional factor analyses may elucidate its tumor affinity, enabling optimization of the engineered bacterial chassis for targeted cancer therapy.
Administration Timing
Once the route is determined, the dosing interval must be designed considering the residual levels of antibiotics in the body to ensure effective function of the suicide switch.
Since antibiotics are gradually metabolized and cleared, we selected azithromycin as an auxiliary drug due to its relatively long half-life, which prolongs the dosing interval while maintaining stable antibiotic concentrations. Based on pharmacokinetics, as long as the area under the concentration-time curve (AUC) remains sufficiently high, transient drug concentrations below the minimum inhibitory concentration (MIC) do not compromise therapeutic efficacy.
Additionally, using intratumoral commensal bacteria offers higher safety compared to traditional attenuated strains. This means that even if engineered bacteria transiently colonize normal tissues, the risk of severe adverse effects is minimal, allowing for extended antibiotic dosing intervals. Under these conditions, we hypothesize that administering antibiotics every two weeks without repeated injections of engineered bacteria may maintain both safety and therapeutic efficacy.
Drug Types
Based on the TNBC patient’s characteristics and resistance profile, multiple drugs can be deployed. In our project, we selected the Apoptin protein derived from chicken anemia virus as a representative payload. Additionally, the system allows flexible loading of the following therapeutics to achieve precise intracellular delivery and controlled release, enhancing efficacy while reducing systemic toxicity:
- Small-molecule drugs requiring intracellular release (e.g., prodrugs);
- Protein therapeutics targeting the cytoplasm (e.g., Granzyme B, Cas9);
- Nucleic acid-based therapies (e.g., siRNA, CRISPR systems) or DNA-targeting drugs.
In addition to therapeutics targeting triple-negative breast cancer, we recommend—under strict ethical and regulatory oversight—conducting further investigations in vitro and in preclinical animal models to determine whether tumor-type-specific nitrate treatment, tailored to the degree of hypoxia, can modulate engineered bacterial metabolism under hypoxic conditions toward a more aerobic-like phenotype, thereby improving bacterial colonization or invasiveness.
Potential Risks
In certain circumstances, accidental release of engineered bacteria into the external environment may occur, for example due to improper handling or container damage. Although the external environment is typically aerobic, the absence of antibiotic selection pressure means that the leaked bacteria would not be immediately inactivated or eliminated, potentially posing a risk to medical personnel and patients. However, compared with traditional attenuated bacterial strains, intratumoral commensal bacteria exhibit a coexistence relationship with the host, thereby conferring a higher level of safety. Moreover, such symbiotic bacteria often display host dependency and relatively low environmental adaptability, making it difficult for them to survive or propagate in external environments over extended periods. This characteristic helps reduce the potential risks of environmental dissemination and ecological disturbance to a certain extent.
Expected Outcomes and Future Perspectives
We plan to further enhance the system’s responsiveness, enabling engineered bacteria to sense tumor microenvironmental signals (e.g., hypoxia, lactate) and modulate drug release accordingly, achieving truly “adaptive therapy.”
By incorporating immune-modulatory modules, engineered bacteria can trigger localized cell clearance responses and activate systemic immune responses, moving beyond purely local treatment toward full-body immune defense and durable immunological memory.
Beyond TNBC, this platform can be adapted to other tumors. By designing tumor-specific targeting elements and drug modules tailored to different microenvironments, we aim to develop a customizable precision cancer therapy platform, achieving a genuinely adaptive treatment paradigm.