## 2 Results
### Engineered MCPIP1-Expressing Bacteria Induce Robust Anticancer Activity in Cervical Cancer  
Attenuated _S. typhimurium_ strains have been widely applied in various types of cancer (such as colon cancer, melanoma cancer, pancreatic cancer, etc.) [21; 22; 32; 33; 34; 35; 36]. Here, we used a targeted cancer therapy with attenuated _S. typhimurium_ engineered to express MCPIP1 to treat HeLa cancer in a murine model. The MCPIP1 gene was cloned into the L-arabinose inducible pBAD expression system (termed pMCPIP1 or pEmpty as a control), as described previously [21; 32]. Western blotting analysis revealed that the MCPIP1 protein was only detected in both the cell pellet and culture supernatant from pMCPIP1-carrying attenuated _S. typhimurium_ (SLpMCPIP1) after L-arabinose induction (Figure 7A). To explore the cytotoxicity of SLpMCPIP1 in vitro, we performed crystal violet staining and lactate dehydrogenase release (LDH) assay experiments with the HeLa cell line. It was shown that SLpMCPIP1 (+) exhibited more effective cell killing than SLpEmpty (+), whereas considerable cell-killing effects were induced after SLpMCPIP1 (-), SLpEmpty (-), and SLpEmpty (+) treatment (Figure 7B). Furthermore, Western blot analysis showed that cleaved caspase-3 and cleaved PARP1 were increased after SLpMCPIP1 (+) treatment, whereas XIAP, which inhibits cell death, was decreased (Figure 7C). To evaluate the antitumor effects of engineered _Salmonella typhimurium_, we intravenously injected the BALB/c nude mice implanted subcutaneously with HeLa tumors with PBS, SLpEmpty, or SLpMCPIP1 to examine the antitumor activity of engineered _S. typhimurium_. The volume and weight of tumors were decreased after SlpEmpty treatment, while they were greatly enhanced after SLpMCPIP1 (+) treatment (Figure 7D-F). Furthermore, Western blot analysis of excised HeLa tumor tissues demonstrated the overexpression of MCPIP1 in tumors treated with SLpMCPIP1 (+), and SLpMCPIP1 (+) further reduced XIAP but increased the expression of cleaved caspase-3 and cleaved PARP1 proteins compared with SLpEmpty (+) treatment (Supplementary Figure S9). Additionally, the cell proliferation marker Ki67 was more abundant in tumor tissues without bacterial colonization, and Ki67 was significantly decreased after SLpMCPIP1 (+) treatment compared with SLpEmpty (+) treatment (Figure 7G). Moreover, the cell death marker TUNEL was increased after bacterial treatment, and TUNEL signaling was stronger in the SLpMCPIP1 (+) group than in the SLpEmpty (+) group (Figure 7H). These results demonstrated that MCPIP1 could be used as a therapeutic target for cervical cancer, and attenuated _S. typhimurium_ expressing MCPIP1 provides a new strategy for anticancer therapy. Figure 7: Cont. Figure 7: Anticancer efficiency of engineered MCPIP1-secreting _Salmonella typhimurium._ (**A**) Western blotting analysis to check the bacterial expression of MCPIP1 in vitro. Bacterial samples were prepared with (+) or without (\(-\)) 0.2% L-Arabinose induction and separated into pellet (P) and supernatant (S) fractions. (**B**) Cell killing of HeLa by MCPIP1-expressing bacteria in vitro. Crystal violet staining was performed to check the viable cells, and the lactate dehydrogenase (LDH) released from HeLa cell co-culture with different treatments was determined with an LDH kit. The cell lysis of the positive control SLpMCPIP1 (+)-treated group was defined as 100%, n = 3. (**C**) HeLa cells were co-cultured with 1000 MOI SLpEmpty and SLpMCPIP1 with (+) or without (\(-\)) 24 h induction of 0.2% L-arabinose. All cell lysates were collected and detected by immunoblotting with the indicated antibodies. \(\beta\)-actin was used as an internal control. (**D**–**F**) Anticancer efficacy in the HeLa cancer model. BALB/c nude mice were implanted subcutaneously with HeLa cells. When the tumor reached 100–120 mm\({}^{3}\), mice were treated with 1 \(\times\) 10\({}^{7}\) CFU-engineered bacteria (SLpEmpty or SLpMCPIP1) (n = 5 per group). (**D**) Average tumor growth in the HeLa cancer model.