## 9 Clinical Trials, Challenges and Future Perspectives  
The first attempt of using _Salmonella_ in preclinical studies and clinical trials for the treatment of advanced or metastatic tumor can be dated back in the early 2000 where, in particular, the strain VNP20009 described earlier in this paper, was developed by Vion Pharmaceutics Inc [87]. Unfortunately, the phase 1 clinical trials of VNP20009 was discontinued due to low tumor regression and incidence of side effects at high dosage [85; 87]. Despite the outcome, VNP20009 has been safely administered via intravenous infusion resulting in increased circulation of proinflammatory cytokines and marked colonization in the tumor biopsies of three out of 25 patients in the trial [87; 88]; this means, it is safe to assume that _Salmonella_ can be further developed to improve the outcomes of future clinical trials. One hindrance affecting the tumor targeting ability of VNP20009, as reflected in the results of phase-1 clinical trials, is the point mutation in _CheY_ gene that forms part of the two-component system CheA/CheY associated with motility and bacterial distribution of _Salmonella_ in tumor tissue [6; 18]. The chemotactic ability of VNP20009 was enhanced, by 69% efficiency, with respect to the parental strain, after replacing the _CheY_ mutated copy with the wild-type sequence [18] and then optimized by restoring _msbB_ gene in VNP20009 _CheY\({}^{+}\)_ that further increased chemotactic mobility [20]. Another strategy that may better equip _Salmonella_ in targeting tumor sites is surface modification. Recently, [PERSON] et al. [89] modified the _S. typhimurium_ to display arginine-glycine-aspartate (RGD) peptide sequence on the external loop ofouter membrane protein A (OmpA). RGD peptide binds efficiently to \(\alpha\)v\(\beta\)3 which is overexpressed in most tumor cells; therefore, RGD-displaying _Salmonella_ can target tumor overexpressing \(\alpha\)v\(\beta\)3 at high efficiency while weakly binding to \(\alpha\)v\(\beta\)3-negative cells [89]. Majority of _Salmonella_ strains used as anticancer agents were developed by disabling genes associated with virulence which may be required for immune activation associated with antitumor response, an essential aspect that makes cancer immunotherapy efficient. Several reports have already provided solutions for the improvement of the antitumor potential of _Salmonella_, mostly by surface modification and/or engineering of various strains, such as VNP20009, A1-R and the avirulent \(\Delta\)ppGpp, among others, but not directly addressing over-attenuation [5,90]. Low tumor regression possibly due to over-attenuation can be addressed by altering a virulence factor to be expressed under an inducible promoter such as PBAD requiring arabinose as an inducer in an in vitro system [91]. The concentration of arabinose becomes heavily diluted after administration, which will eventually lead to a halted virulence factor expression. In turn, _Salmonella_ will become attenuated after a few rounds of replication without the inducer arabinose. For instance, the transcriptional regulator _phoP_ that is responsible for _Salmonella_ virulence and a regulator of LPS structural modifications, has been engineered to be expressed under PBAD promoter [92]. By so doing, a more vigorous anti-tumor activity may be prompted compared to an over-attenuated strain. This opens another area of investigation in cancer therapeutics that is focused on using anticancer _Salmonella_. In another phase 1 clinical trial, _S. typhi_ Ty21a was used as a vector to deliver the oral DNA vaccine VXM01 targeting the vascular endothelial growth factor receptor 2 (VEGFR-2) as an antiangiogenic intervention for advanced pancreatic cancer patients [93,94]. Preliminary findings suggest that Ty21a can be safely administered with only minimal adverse effect, relatively substantial tumor regression and a significant reduction of tumor perfusion along with elevated levels of serum anti-angiogenic biomarkers [94].