# 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 Treat CRC via Therapeutic Protein Delivery
#### 3.1.5 Immunoregulatory Factors  
Cytokines, secreted by various types of cells, are classic immunomodulators. Immune cells can be induced by cytokines to clear tumors by modulating innate and adaptive immune responses (Figure 6) [17]. However, monotherapy using cytokines is often accompanied by severe dose-limiting toxicity and the therapeutic outcome is affected by the route of administration, schedule, and strategies [87; 88]. The precise delivery of cytokines to tumor sites using bacteria as a vehicle can reduce off-target effects and systemic toxicity. This may be an alternative strategy for using cytokines to treat cancer with great potential. The use of engineered bacteria-carrying cytokines for the treatment of CRC has been extensively explored. As early as 1996, an engineered attenuated _S. typhimurium_ strain that enabled the production of the bioactive cytokine IL-2 was developed, and the administration of the engineered strain significantly reduced liver metastasis from MC38 colon adenocarcinoma in a mouse model of liver metastasis [89]. After that, one study also used attenuated _S. typhimurium_ to deliver another human cytokine, LIGHT, which is homologous to the TNF family cytokine lymphotoxin and can promote tumor rejection. The engineered _S. typhimurium_ expressing LIGHT significantly inhibited the growth of tumors and the dissemination of lung metastatic tumors in CT26 tumor-bearing mice [90]. Another example is the modification of attenuated _S. typhimurium_ to deliver the cytokine IL-18, an interleukin family member that enhances immune cell activity. These engineered _S. typhimurium_ strains expressing IL-18 (but not control strains) inhibited the growth of tumors in mice with CT26 tumor cells, without apparent toxicity within normal tissues [91]. Subsequently, other cytokines, such as Fas ligand (FasL) and CCL21, were also delivered to tumors by an attenuated strain of _S. typhimurium_. Among these factors, FasL, a cytokine belonging to the tumor necrosis factor (TNF) family, exhibits potential antitumor properties,  
Figure 5: Engineered bacteria harboring a shRNA plasmid targeting indoleamine 2,3-dioxygenase (IDO) can activate the CTNNB1 pathway and induce colorectal carcinogenesis, with the shRNA employing bacteria as a vector to transport therapeutic RNAi target cells to silence specific mRNAs. such as facilitating tumor rejection and stimulating the proliferation of CD8+ T-cells [92]. CCL21 belongs to the chemokine family. The activation of CCL21 can effectively induce an immune response to eliminate tumors [93]. It was observed that the systemic delivery of both of these engineered _S. typhimurium_ expressing these cytokines significantly suppresses tumor growth in BALB/c mice bearing CT26 colon carcinoma tumors with minimal toxicity [94; 92]. In addition, [PERSON] et al. engineered an attenuated _S. typhimurium_ strain to produce flagellin B (FlaB) derived from _Vibrio vulnificus_, which can also serve as an immunomodulatory agent. Lipopolysaccharide (LPS) from _S. typhimurium_ and FlaB expressed by _S. typhimurium_ can activate the TLR4 and TLR5 pathways, respectively, which results in the massive infiltration of immune cells into the TME and promotes the secretion of proinflammatory cytokines TNF-\(\alpha\) and IL-1\(\beta\) to fight tumor cells [95; 96]. Animal experiments showed that the engineered _S. typhimurium_ expressing FlaB effectively retarded tumor growth and metastasis and promoted survival in several different murine colon cancer models [96]. Currently, therapy targeting immune checkpoints is an important means of cancer immunotherapy. However, there are also toxic side effects caused by systemic administration. Therefore, [PERSON] et al. engineered a probiotic EcN system for the release of nanobodies targeting programmed cell death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4).