# A Review of the Use of Native and Engineered Probiotics for Colorectal Cancer Therapy
## 3 Genetically Modified-Bacteria for Colorectal Cancer (CRC) Treatment
### Engineered Bacteria Mediate CRC Treatment via Outer Membrane Vesicles  
Several Gram-negative bacteria continuously release vesicles called outer membrane vesicles (OMVs) to the outside. The components of secreted OMVs include outer membrane proteins, lipopolysaccharide, phospholipids, and some periplasm [57]. OMVs are involved in both internal and external bacterial stress responses and play an important role in bacterial growth and survival [98]. OMVs possess nano-size and immunomodulatory properties [99], which may make them potential cancer immunotherapeutic agents. [PERSON] et al. reported that lipidA acyltransferase-deficient _E. coli_ (\(\Delta\)msbB)-derived \(\Delta\)msbB OMVs were used as cancer immunotherapeutic agents. In CRC mice, \(\Delta\)msbB OMVs targeted tumor tissues via enhanced permeability and retention, and the trypsin-sensitive surface proteins on OMVs then induced the production of interferon-\(\gamma\) to activate antitumor immune responses. This is an exciting attempt to apply OMVs to the treatment of cancer [100]. Through a bacterial sorting mechanism, several proteins were carried by OMVs, leading to the selective localization of proteins, such as the pore-forming toxin CyA, from the periplasmic and outer membranes. A study indicates that C-terminal ClyA fusion heterologous proteins can be displayed on the outer surface of _E. coli_ and the OMV surface and still retain activity. Interestingly, the hemolytic activity of ClyA is lost in this process. These results show that bacteria-derived-OMVs can be engineered as potential drug-delivery vehicles. In one example, a hyper-vesiculating E\(\kappa\) (\(\Delta\)ECHy) producing recombinant OMVs packaged with ClyA-hyaluronidase (Hy) was developed for tumor-targeting therapy [19]. AECHy can preferentially locate within tumors, followed by the release of OMVs with therapeutic molecules in situ. The recombinant OMVs easily reach deep into the tumor to induce tumor stromal changes due to their nanoscale size. Significantly, in MC38 tumor mice, \(\Delta\)ECHy treatment resulted in reductions in the hyaluronic acid synthesis and smooth muscle actin of tumor tissues. The combination therapy of \(\Delta\)ECHy+PDL1 antibody significantly suppressed tumor growth and improved survival by enhancing the efficacy of therapeutic antibodies and facilitating immune cell infiltration [19]. Another study involved the development of an oral tumor vaccine using engineered bacteria-producing OMVs [21]. Specifically, engineered _E. coli_ Top10 was constructed to express a designated tumor antigen (Ag) along with an Fc fragment from mouse immunoglobulin G (Ag-mFc), both regulated by a monosaccharide-inducible promoter. Ag-mFc was fused with the protein ClyA, which was located on the surfaces of OMVs released by the engineered bacteria. In mice, OMVs from engineered bacteria can cross the intestinal epithelium into the lamina propria to stimulate dendritic cell maturation after the oral administration of engineered bacteria. The results showed that engineered bacteria activated the tumor antigen-specific immune response and inhibited the tumor growth in MC38 colon cancer mice [21]. In summary, in contrast to delivering therapeutic proteins directly using engineered bacteria, delivery via OMVs may be even more advantageous. This is because OMVs have immunogenic properties that contribute to reducing the burden of the administration of bacteria, and their nano-size gives OMVs an excellent tumor penetration ability. However, in future studies, the safety can be meticulously evaluated due to their bacterial origin..