Background

Triple Negative Breast Cancer (TNBC)

Breast Cancer (BC) is the second leading cause of cancer-related deaths after lung cancer (Nik-Zainal et al., 2016). In 2018, approximately 2.089 million women were diagnosed with breast cancer, which predominantly occurs in females. The onset of breast cancer may be driven by multiple factors, including age, prolonged exposure to estrogen (longer exposure correlates with higher risk), mutations in tumor suppressor genes such as BRCA1/2, TP53, and PTEN, the use of hormonal contraceptives, and early exposure to ionizing radiation. Triple Negative Breast Cancer (TNBC) is defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression in immunohistochemical assays, and it is associated with a high risk of distant metastasis. The expression of these three genes is common in other types of breast cancer but rare in this subtype. Since conventional breast cancer treatments, such as hormone therapy, often target the two hormone receptors and the HER2 gene, these therapies are generally ineffective for patients with triple-negative breast cancer.

Current Treatment

Currently, chemotherapy remains the main treatment option for TNBC. Due to the lack of hormone receptors and HER2, broadly applicable targeted therapies are still unavailable. Researchers are actively exploring alternative strategies, including immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway, which enhance the immune system’s ability to recognize and attack tumors. However, their efficacy is significant only in a subset of TNBC patients, and the overall benefit remains limited. PARP inhibitors provide new treatment opportunities for patients carrying BRCA1/2 germline mutations. In addition, various novel targeted drugs and combination therapies are under investigation, including PI3K/AKT/mTOR pathway inhibitors, androgen receptor antagonists, and DNA damage response regulators (e.g., ATR and CHK1 inhibitors). Despite these advances, the applicability remains limited and resistance develops easily, making TNBC treatment a persistent challenge (Won & Spruck, 2020).

Key Research

In the study conducted by Cai Shang and his colleagues, a large number of intracellular bacteria were observed in the spontaneous murine breast tumor model MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen). These bacteria mainly colonized tumor epithelial cells, with fewer detected in stromal cells. High-resolution electron microscopy (EM) revealed that most bacteria-like structures (about 97.25%) were located in the cytoplasm rather than in the extracellular space. Quantitative EM results showed a bacteria-to-host cell ratio of about 3% (218 bacteria in 7,201 scanned cells). Importantly, these intracellular microbes remain viable even after treatment with cell-impermeable antibiotics such as ampicillin and gentamicin (Kumar et al., 2017).

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A commensal bacterial platform for intracellular drug delivery in TNBC

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Project Objectives and Experimental Strategy Explore

The study “Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer” published by Dr. Shang Cai and colleagues in Cell (2022) revealed that specific commensal bacteria exhibit pronounced selective enrichment within triple-negative breast cancer (TNBC) cells (Fu et al., 2022). Building on this discovery, we propose to exploit the intrinsic tumor-targeting potential and intracellular invasion capacity of these commensal bacteria to develop an engineered platform for targeted drug delivery in TNBC.

Guided by these findings, we selected Staphylococcus epidermidis and Staphylococcus xylosus as chassis strains for engineering and constructed a drug delivery system through genetic engineering. In parallel, we introduced analogous modifications into Escherichia coli Nissle 1917 (EcN), both to ensure stable heterologous gene expression and to evaluate the cross-species applicability of this strategy.

To enhance tumor cell invasion efficiency, we introduced exogenous expression of Staphylococcus aureus fibronectin-binding protein A (FnBPA) in the staphylococcal strains. By engaging integrin-mediated internalization (Hauck and Ohlsen, 2006), this protein substantially promotes bacterial internalization into TNBC cells.

Recognizing the potential risk of nonspecific invasion conferred by FnbpA, we incorporated a hypoxia-responsive module comprising the endogenous nreABC operon and its downstream responsive promoter P_narT. Leveraging this regulatory system, we engineered a suicide switch and a controlled drug release circuit to restricting bacterial survival and therapeutic payload secretion to the hypoxic tumor microenvironment, thereby ensuring enhanced biosafety and tumor specificity of the treatment.

Model Explore

Our modeling work consists of two parts: Integrated FnBP Screening and Intratumoral Bacterial Diffusion.

In the Integrated FnBP Screening part, we integrated evolutionary tree analysis with protein engineering to identify exogenous proteins that enhance bacterial invasion and colonization of tumor cells. Using AlphaFold-Multimer, molecular dynamics simulations, MM/PBSA binding free energy analysis, and phylogenetic tree construction, we systematically evaluated fibronectin-binding proteins (FnBPs). Combining molecular-level results with phylogenetic relationships, we identified optimal FnBP candidates for engineering Staphylococcus epidermidis and Staphylococcus xylosus.

In the Intratumoral Bacterial Diffusion part, we developed a COMSOL-based mathematical model to analyze and visualize bacterial proliferation and spatial dynamics within tumor cells. A coupled diffusion–convection–growth PDE framework was established, incorporating bacterial motility, interstitial fluid convection, nutrient-dependent growth, density limitation, and substrate consumption.

Human Practices Explore

In this year’s Human Practices, we have consistently aligned our research with real social needs.

In Integrated Human Practices (IHP), we began with the concerns of doctors and patients. Guided by experts’ feedback, we iterated on our project design, making it more feasible, practical, and socially relevant.

In Education, we developed two complementary curricula with distinct learning loops:

• For students, the “Integrated Synthetic Biology Curriculum”, following the cycle “Ignite curiosity → Build understanding → Strengthen thinking → Internalize habits → Give back to society.” This was realized through classroom teaching, hands-on experiments, and art-based science communication.
• For older adults, the “Reminder & Care-Based Education”, corresponding to “Acknowledge existing knowledge → Gentle reminders → Small accessible actions → Consolidate healthy habits → Community resonance.” These activities focused on everyday health practices and companionship, rather than technical science.

  

In Public Engagement, we combined science with art through exhibitions, social media, and team events, creating experiences that conveyed both the warmth and the beauty of science.

We believe that because our education and engagement have always remained in sync with society, our project never drifted away from reality—instead, it has continuously responded to genuine needs.

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