# Integration of _Salmonella_ into Combination Cancer Therapy
## 3 _Salmonella_ in Combination with Other Approaches to Cancer Therapy
### Combination with Immune Checkpoint Inhibitors  
There is no doubt that the development of immune checkpoint inhibitors is considered a paramount achievement in cancer immunotherapy. Checkpoint inhibitors mainly work by targeting the negative regulators of T cell function, such as CTLA-4, PD-1, and PD-L1, unleashing T-cells and allowing them to induce tumor cell death. The past few years have witnessed unprecedented and promising therapeutic outcomes in different types of cancer, including advanced melanoma [121; 122] and non-small cell lung carcinoma [123; 124]. Despite the outstanding success reported with immune checkpoint inhibitors, the percentage of cancer patients who respond to this approach remains rather modest [125], and serious immune-related adverse events were observed in some patients receiving checkpoint inhibitors therapy [126; 127]. This observation has led to the identification of different predictive biomarkers that help in patient selection, such as (a) the presence of tumor-infiltrating lymphocytes [128; 129; 130; 131], (b) the expression level of checkpoint inhibitors [132; 133], and (c) the tumor mutational load [134; 135]. Different studies have evaluated the effect of _Salmonella_ on the expression of inhibitory checkpoint proteins in different tumor and non-tumor cells. [PERSON] et al. reported that _Salmonella choleraesuis_ dose-dependently downregulated the expression of PD-L1 on different murine and human cancer cell lines through inhibiting the AKT/mTOR/p70s6K signaling pathway [136]. They also showed that the systemic administration of _Salmonella_ decreased the expression of PD-L1 in B16F10 and LL2 tumor tissues [136]. This downregulation enhanced T cell infiltration and thereby resulted in tumor growth inhibition [136]. Another study documented the role of attenuated shIDO-ST (_S. typhimurium_ delivering an shRNA plasmid targeting IDO) in inhibiting the expression of several inhibitory checkpoint proteins, including PD-L1, PD-1, and CTLA4, in different splenic immune cells [137] which highlights the capacity of _Salmonella_ to restructure the immune component of the tumor microenvironment in favor of tumor inhibition. On the other hand, different studies reported _Salmonella_-mediated upregulation of PD-L1 in different cell types. [PERSON] et al. utilized transcriptome analysis and flowcytometry to study the effect of _S. typhimurium_ on PD-L1 expression in NOD mice [138]. In their model, the intravenous administration of _Salmonella_ significantly increased the proportion and surface expression of PD-L1 among dendritic cells and macrophages [138]. Others also reported an increase in PD-L1 expression in B cells and CD4+T cells following _Salmonella_ treatment [139; 140; 141]. It is worth mentioning that none of these studies correlated the increased PD-L1 expression with tumor growth inhibition. [PERSON] et al. elegantly demonstrated the capacity of ovalbumin-producing _S. typhimurium_ A1-R and anti PD-L1 combination to rescue the function of peripheral and tumor infiltrating CD8+ T cells against B16-OVA melanoma [142]. This led to an increase in tumor rejection compared to bacterial therapy alone or the combination of anti PD-L1 and anti-CTLA-4 [142]. The same study reported the failure of _Salmonella_ and anti CTLA-4 combination in treating tumor-bearing mice. [PERSON] et al. combined bacterial therapy with immune checkpoint inhibitors by transforming attenuated _S. typhimurium_ with a plasmid for RNA interference targeting the inhibitory receptor PD-1 (siRNA-PD-1), and used it in B16 melanoma [143] and CT26 colon cancer [144] models. They reported a preferential accumulation of transformed _Salmonella_ in tumor tissues [143] and found that the intratumoral injection of siRNA-PD-1-carrying _Salmonella_ resulted in (a) a remarkable decrease in tumor weight, (b) prolonged survival rate of tumor bearing mice, in addition to (c) an increase in CD4+ and CD8+ TILs compared to treatment with _Salmonella_ containing a plasmid for scrambled si-RNA [143; 144].