## 4 Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy
### Tumor Therapy  
Historically, bacterial infection and bacterial products, including toxins, have been used as challenges in cancer treatment. For example, [PERSON]'s toxin, which is a cocktail of heat-killed bacteria, has been used to stimulate an anti-cancer immune response [101; 102]. The ClyA protein alone and the ClyA-OMV system have been investigated as potential anti-tumor agents or tools. [PERSON] et al. first demonstrated that the cytotoxic properties of ClyA, in combination with a hypoxia-inducible promoter, increased necrosis in hypoxic regions of tumors and inhibited tumor growth in mice, indicating the potential of ClyA as an anti-tumor agent [103]. It has been suggested that _E. coli_ OMVs have a potential role in tumor immunotherapy, as intravenous injection of these OMVs induced strong IFN-\(\gamma\)- and T cell-mediated anti-tumor responses [104]. In another study, the use of a ClyA-OMV system successfully achieved presentation of the ectodomain of the programmed cell death protein 1 (PD-1). The engineered OMVs bound to the PD-1 ligand 1 on tumor cell surfaces, thereby facilitating PD-1 ligand 1 internalization and reducing the expression levels on tumor cells [105]. A very recent study by [PERSON] et al. showed that ClyA served as an anchoring protein for hyaluronidase (Hy) in hypervesiculating _E. coli_ Nissle-produced OMVs and potentially exerted a cytotoxic effect against cancer cells [106]. These engineered OMVs helped distribute Hy more effectively and uniformly inside the tumors, permitting the penetration of drugs and potentiating their effects by considerably decreasing the levels of hyaluronic acid in the tumor tissues. These results indicated that the ClyA-OMV system could be an effective tool for stromal remodeling, cytolytic therapy, and improving the activity of anti-cancer targeted therapeutics. Similar to this approach, ClyA has been used as a leader protein for presenting human epidermal receptor 2 (HER2; expressed at high levels in breast cancer cells) affibodies on the surface of OMVs. The engineered OMVs delivered a loaded therapeutic siRNA cargo to ovarian and breast cancer cells expressing HER2, subsequently inducing cancer cell death without appreciable side effects [107]. In a recent study using advanced techniques, a flexible tumor vaccine platform based on OMVs expressing ClyA as a fusion partner for tumor antigens in conjunction with a \"plug-and-display\" system [108] comprising tag/catcher protein pairs [109] was established. This approach enabled the display of various tumor antigens linked to protein tags on the OMV surface, rapidly and simultaneously, as the protein catchers were fused to ClyA. The OMV-mediated tumor antigens were effectively delivered to lymph nodes and then presented to dendritic cells, resulting in the induction of antigen-specific T-lymphocyte-mediated anti-tumor immune responses in murine tumor models. Cancer is a global health issue, and researchers continue to explore strategies for cancer treatment from various perspectives. The ClyA-OMV system could further accelerate research in this field and help develop a new generation of more effective cancer therapies. ClyA can be fused to a foreign protein and stably anchor it on the surface of bacterial cells. This feature enables the facile functional display of a variety of different prokaryotic and eukaryotic proteins. In addition, ClyA elicits this ability to the fullest in combination with OMVs. For example, surface-exposed antigens facilitated using ClyA are more effectively immunogenic than cytoplasmic antigens and thus are useful for vaccine development [90; 95; 100]. In tumor therapy, ClyA serves as a fusion partner for tumor antigens and can form part of the OMV-based delivery platform for anti-tumor agents against specific cancer cells [105; 106; 107]. Although the ClyA protein has the potential for versatile applications, the biphasic activity of this toxin and its related physiological effects in vivo remain unclear.