# Integration of _Salmonella_ into Combination Cancer Therapy
### Ease of Gene Modification  
Although _Salmonella_ possesses immunomodulatory and intrinsic encolytic capacities, these advantages are not sufficient for achieving optimum therapeutic outcomes. In order to enhance the overall therapeutic index of _Salmonella_ treatment alone or in combination with other therapies, several strains were engineered to improve the safety and potentiate anti-tumor efficacy. _Salmonella_'s amenable gene modification helped in improving the safety of bacterial therapy through the deletion of major virulence factors or the generation of auxotrophic mutants that are incapable to replicate efficiently in an environment deficient in specific nutrients [21]. For example, deletion of msbB gene, responsible for myristoylation of an essential component of LPS, reduced _Salmonella_'s toxicity by 10,000-fold while retaining its anti-tumor efficacy in mice [83]. It is worth bearing in mind that bacterial attenuation might occur at the expense of its ability to inhibit tumor growth, because some virulence factors underlie _Salmonella_'s intrinsic anti-tumor properties [21]. The _Salmonella_ A1-R strain, an attenuated leucine-arginine auxotrophic mutant, exhibited enhanced preferential colonization in tumor tissues due to the abundance of these nutrients in the tumor microenvironment but not in normal tissues [84; 85; 86]. Besides improving the safety and minimizing toxicity to normal organs, _Salmonella_ was also engineered to enhance the antigenicity of the tumor by delivering tumor-associated or specific antigens [74]. This in turn potentiates the anti-tumor immune response in favor of tumor regression. In addition, several studies documented the unprecedented success of engineered _Salmonella_ in transporting various anti-tumor therapeutic agents specifically to the tumor site. _Salmonella_ has been utilized as a delivery vehicle of various cytotoxic agents (e.g., cytolysin A, PE38, and diphtheria toxin) and apoptosis-inducing proteins (e.g., Fas ligand, TRAIL, and apoptin), as well as immunomodulatory cytokines (e.g., IL-2, IL-12, IL-18, and IFN-\(\gamma\)) [20; 56; 87]. Engineering of prodrug-converting enzymes into tumor-targeting _Salmonella_ was also reported. The payload enzyme released by _Salmonella_ metabolizes the systemically administered pro-drug into cytotoxic agent at the tumor site only, thereby minimizing the drug-related toxic effects on normal tissues [21]. An example of prodrug-converting enzyme is cytosine deaminase (CD), which converts the non-toxic 5-fluorouracilocytosine (5-FC) into anti-tumor 5-fluorouracil (5-FU). The enhanced anti-tumor efficacy of CD-expressing _Salmonella_ was reported using _in vitro_ and _in vivo_ studies [88; 89]. In this context, it is worth mentioning that the application of this approach in cancer patients could have some limitations because of the ability of 5-FU to freely diffuse across the cell membrane and exert its cytotoxic activity in neighboring cells [90]. Moreover, increased tumor targeting could be achieved by utilizing engineered _Salmonella_ in order to minimize bacterial-related toxicity or enhance its therapeutic potential while keeping toxicity to minimal. For example, the surface expression of antibody fragments against specific tumor-associated antigens, including carcinoembryonic-onic antigen (CEA) and CD20, was shown to increase _Salmonella_'s tumor localization in murine adenocarcinoma and human lymphoma models, respectively [91; 92]. In addition, _Salmonella_'s engineering plasticity helped to integrate controlled gene expression systems into tumor-targeting bacteria. This could be achieved through either the incorporation of gene promoters responsive to tumor-associated signals, such as hypoxia-inducible promoter systems (HIP-1 and NirB) [93; 94; 95], or remotely through inducible promoters (e.g., as pBAD, pTet, and Pm) in response to exogenous transcriptional factors (e.g., L-arabinose, tetracyclines, and acetyl salicylic acid) [96; 97; 98; 99].