Bacteria are found as integral component of numerous kinds of cancer, including colorectal, pancreatic, lung, and breast cancers [[1], [2], [3]]. These bacteria, known as intracellular bacteria, mainly colonize intracellularly. Recent studies reveal that tumor regions colonized by bacteria are highly immunosuppressive and have fewer cytotoxic T lymphocytes [4]. Fusobacterium nucleatum (Fn.), one kind of anaerobic bacteria, can colonize various kinds of tumor tissues. Fn. can remodel tumor environment to protect tumor cells from immune clearance [5]. Recent research demonstrates that in human breast cancer samples, Fn. can bind and invade breast cancer cells [6], promoting tumor progression and immune suppression [7,8]. Other studies further revealed the mechanism is that Fn. existing in tumors can inhibit the infiltration of CD8+ T cells at tumor sites, therefore weakening anti-tumor immune response [9,10]. All these findings highlight the critical role of intratumoral bacteria in breast tumor progression and immune resistance [11]. Bacterial clearance may be a novel therapeutic target for activating anti-tumor immunity.

Ferroptosis, an iron-dependent cell programmed death pathway, is characterized by lipid peroxidation (LPO) on cell membranes. Usually, the accumulation of LPO is driven by excessive reactive oxygen species (ROS) generation and suppression of glutathione peroxidase 4 (GPX4) activity. Induction of ferroptosis in tumor cells has been extensively studied, with GPX4 inhibitors and iron-based nanoparticles developed as ferroptosis inducers [12]. Recently, the concept of bacterial ferroptosis has emerged. Studies show that ferrous ions (Fe2+) can destroy bacterial integrity, resulting in content leakage and DNA degradation [13,14]. Most importantly, Fe2+ can also trigger bacterial lipid peroxidation to exert bactericidal effects. Notably, ferroptosis is still available in the bacteria-infected cell model [15]. In this model, the stress of ferroptosis is transmitted via iron transporters from the cytoplasm to bacterial vesicles, therefore inducing bacterial ferroptosis. Based on these researches and findings, we proposed that induction of ferroptosis may simultaneously kill tumor cells and eliminate intratumoral bacteria, offering a ‘two-pronged’ strategy with potential value in inhibiting tumor progression and recovering anti-tumor immunity.

Sustained and stable Fe2+ supply is essential for ferroptosis activation. Ferrocene (Fc), a stable Fe2+ donor, together with β-cyclodextrin (β-CD), can form a ROS-responsive β-CD@Fc complex via hydrophobic interactions [16]. ROS can oxidize Fe2+ in Fc to Fe3+, disrupting the hydrophobic structure and releasing Fc. As an iron source, Fc can drive continuous Fenton reactions to fuel ferroptosis. However, ferroptosis relying on Fe2+ is easy to be resisted by tumor cells, as tumor cells can upregulate GPX4 and produce glutathione (GSH) [17]. GSH can reduce cytotoxic lipid peroxides to inhibit ferroptosis. Depleting intratumoral GSH is thus a critical strategy to overcome ferroptosis resistance. We noticed that cinnamaldehyde (CA), a natural compound with an α, β-unsaturated ketone structure, can bind with GSH and deplete intracellular GSH [18]. CA-mediated GSH depletion can minimize LPO reduction, synergistically enhancing ferroptosis. Additionally, cinnamaldehyde is reported as a potent antimicrobial agent by destroying bacterial walls, inhibiting enzymatic activity, and interfering with DNA replication [19]. Thereby, CA can suppress intratumoral bacteria while promoting ferroptosis.

Recently, bacterial-derived carriers have emerged as promising delivery platforms [20]. Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria, with their ideal size and surface proteins, are widely explored in drug delivery application [21,22]. OMVs secreted by Fusobacterium nucleatum (FMV) target breast cancer cells. Thus, FMV-coated nanoparticles can be delivered to tumor sites specifically. Furthermore, OMVs provide pathogen-associated molecular patterns (PAMPs) [23], such as immunogenic lipopolysaccharides (LPS), to activate adaptive immunity [24].

In this study, a host-guest complex of hemirotaxane (mPEG-β-CD) and polyethyleneimine-ferrocene (Fc-PEI) is constructed and designated as PCFP. Antibacterial drug cinnamaldehyde (CA) is loaded into PCFP, and OMVs secreted by Fusobacterium nucleatum (FMVs) are coated to form the drug delivery system (FMV@PCFPC). FMV@PCPFC can effectively induce dual ferroptosis to clear immunosuppressive Fn. in tumor and strengthen antitumor immunity through tumor ICD activation (Scheme 1). In this system, FMV facilitates PCFPC targeting tumor to release ferrocene and cinnamaldehyde. Fe2+ in ferrocene can effectively clear intratumoral bacteria, and meanwhile induce ICD activation through ferroptosis. Cinnamaldehyde can disturb bacterial membranes and inhibit biofilms, it also depletes GSH to assist ferroptosis simultaneously. Therefore, Fe2+ and cinnamaldehyde work together in intratumoral bacteria, effectively generating ROS to kill intratumoral Fn. through ferroptosis. When intratumoral bacteria are cleared, immune suppression at tumor sites is relieved [25]. Concurrently, oxidative stress triggered by tumor ferroptosis significantly activates immunogenic cell death (ICD) to promote DC maturation. The dead Fn. fragments and PAMPs on FMVs also function as immunologic adjuvants in DC maturation. This biomimetic system effectively matures DC cells and activates T cells for tumor immunotherapy, which was verified by RNA sequencing. Relying on dual ferroptosis in intratumoral bacteria and tumor, FMV@PCFPC combines bacteria-mediated immunosuppression relief and tumor ICD activation together, effectively suppressing tumor progression through strong immunotherapy. This strategy shows great potential in tumor immunotherapy.