# Integration of _Salmonella_ into Combination Cancer Therapy
## 4 Clinical Application of _Salmonella_ in Combination Cancer Therapy
### Efforts to Overcome Challenges  
_Salmonella_'s anti-cancer and immunomodulatory characteristics, along with its durable capacity to enhance the outcome of other modalities of cancer therapy, necessitate the development of strategies to overcome the limitations and facilitate the implementation of _Salmonella_ in clinical use. In the past decades, several strategies have been followed to maintain the balance of therapeutic efficacy and safety when utilizing _Salmonella_ in cancer therapy. The aim of the earliest approaches was to enhance tumor targeting by selection,and this was done by passaging _Salmonella_ through cancer cells either _in vitro_ or _in vivo_. The attenuated _Salmonella_ strains VNP20009 and A1-R were generated following such strategy of random selection [187; 188; 189]. The ineffectiveness of VNP20009 in treating cancer patients was due to the over-attenuation through uncontrolled introduction of multiple deletions during the process of selection [169; 174]. _Salmonella_ strain A1-R showed promise in treating different types of cancer in pre-clinical models but has not been utilized in clinical trials so far [187; 190]. Unexpected modifications were then avoided by designing _Salmonella_ strains through targeted gene deletions. [PERSON] et al. and his group investigated the role of LPS in establishing the balance between the therapeutic and harmful effects of _S. typhimurium._ They concluded that minor modifications of LPS (e.g., _ArfaP_, _ArfaL_) alone did not alter the toxicity of _Salmonella_, whereas the core deletion mutants _ArfaG_ and _ArfaD_ considerably enhanced their safety at the expense of the anti-tumor activity [184]. To this point, they succeeded in establishing a balance between therapeutic potency and attenuation through controlling the synthesis of LPS using the inducible arabinose promoter P\({}_{\text{BAD}}\)[8; 184]. Targeted gene engineering was also implemented to induce modifications in Lipid A and flagella synthesis, resulting in the auxotrophic _Salmonella_ vector strain SF200 (\(\Delta\)_pxR9_ \(\Delta\)_pagP1_ \(\Delta\)_pagP8_ \(\Delta\)_aroA_ \(\Delta\)_ydiV_ \(\Delta\)_fliF_) [191]. This attenuated strain showed enhanced immune-stimulatory capacity and overcame the limitation of pre-exposure of anti-_Salmonella_ immunity. The careful selection of genetic manipulations is critical for maintaining the balance between therapeutic potential and pathogenicity. Moreover, another study suggested an approach to override the non-specific invasiveness and intrinsic toxicity of _Salmonella_ through coupling the bacteria with a surface-expressed single-domain antibody directed against a tumor-associated antigen [92]. This approach showed success when CD20-targeted _Salmonella_ was employed against lymphomas with minimal non-specific invasiveness [92]. Other researchers utilized _Salmonella_ as a vector system for drug delivery in order to enhance its therapeutic potential and overcome the limitation in tumors that could not be resolved by the intrinsic and immunostimulatory effects of _Salmonella_ alone. Interestingly, [PERSON] et al. illustrated the potential of utilizing _Salmonella_ as a targeted delivery system for therapeutic agents. In their model, bacterial growth was controlled through engineering a bacterium that undergoes lysis at a threshold population density, allowing the repetitive release of genetically encoded anti-tumor therapeutic agents during lytic cycles [192]. This helps in delivering the therapeutic benefit while minimizing the systemic inflammatory response [192]. In this context, it would be ideal if bacterial presence could be controlled, allowing sufficient time to achieve the optimal anti-tumor effects and then eliminating it to avoid uncontrolled proliferation that may lead to unpredictable systemic inflammatory responses. Another innovative step in designing therapeutic vectors was taken by [PERSON] et al. through conjugating gold nanoparticles (AuNPs) with the effector protein SipA of _Salmonella_ SPI1 (_Salmonella_ pathogenicity island 1) [193].