| Bacteria mediated cancer therapy（BMCT）has unique advantages over traditional cancer treatments.For example,it can specifically target and penetrate tumor tissues to activate the host’s immune response,so it can be used as the ideal carrier of immune regulatory factors and drugs.In BMCT,attenuated Salmonella typhimurium has been widely studied by researchers due to its high tumor targeting,good anticancer effect and bios afety.However,at present,the anticancer molecular mechanisms of BMCT is still rarely studied.Meanwhile,BMCT alone is not enough to effectively kill or completely eliminate tumor cells,and it needs to be combined with other treatment methods to improve the anticancer effect.In this dissertation,we found that the antitumor effect induced by attenuated Salmonella typhimurium was weakened in system knockout of the LPS recognition receptor TLR4(TLR4-/-)tumor-bearing mice compared with that in wild-type（WT）mice group.Tissue homogenate for bacterial counting and bacterial bioluminescence imaging showed that attenuated Salmonella typhimurium was significantly reduced in tumor tissues of TLR4-/-mice,which was accompanied by liver injury and splenomegaly caused by increased bacterial content in spleen and liver.With H&E staining of normal organs and immunofluorescence staining of liver and spleen,we found that TLR4-/-decreased the number of immune cells in organs compared to WT group post bacterial infection,but the bacterial content in liver and spleen was significantly increased compared with WT treatment group.We believed that TLR4-/-weakened the ability of immune cells to remove bacteria,leading to liver damage and splenomegaly.After targeting tumor tissue,attenuated Salmonella typhimurium recruit a large number of neutrophils a nd macrophages and other immune cells,and then secrete pro-inflammatory cytokines,such as TNF-αand IL-1β,so as to kill tumor cells and inhibit tumor growth.Through immunofluorescence staining of tumor tissues and flow analysis of immune cells in tumor-draining lymph nodes,we found that the content of immune cells in tumor tissues in TLR4-/-group was significantly reduced compared with that in WT treatment group after bacterial treatment,but the number of immune cells in tumor-draining lymph nodes had no significant change.We found that TNF-αand IL-1βwere significantly decreased and the anti-inflammatory cytokine IL-10 was significantly increased in tumor tissue s of TLR4-/-mice after attenuated Salmonella typhimurium treatment by ELISA analysis.In vitro infection experiments,we demonstrated that TLR4-/-reduces the function of bone marrow-derived neutrophils（BMDNs）and macrophages（BMDMs）to secrete cytokines when attenuated Salmonella typhimurium infection.To explore the molecular mechanisms of TLR4 in attenuated Salmonella typhimurium mediated tumor therapy,we performed RNA sequencing of tumor tissues.Based on the analysis of RNA sequencing data,we deduced that calcium-binding proteins S100a8/a9 may be involved in the LPS/TLR4 signaling pathway regulated the antitumor effect mechanism in attenuated Salmonella typhimurium mediated tumor therapy.By using the S100a8/a9 inhibitor paquinimod,the antitumor effect of Salmonella was significantly inhibited,the bacterial content in tumor tissue s was significantly decreased.However,the bacterial content in liver and spleen were increased,accompanied with organ enlargements,and decreased in pro-inflammatory cytokines（TNF-αand IL-1β）in tumor tissues.These results indicated that attenuated Salmonella typhimurium upregulated S100a8/a9 through LPS/TLR4pathway,which further promoted the expression of pro-inflammatory cytokines（TNF-αand IL-1β）and exerting antitumor effects,which provided new ideas for the mechanism exploration and treatment direction of BMCT in the future,and establishes mechanism basis for the clinical trans lation of BMCT.The specificity of tumor targeting and deep tissue penetration of attenuated Salmonella typhimurium makes it an ideal delivery vehicle for anticancer drugs.In this dissertation,we used the strategy of BMCT combined with photodynamic therapy（PDT）（BMCT-PDT）to realize the integration of tumor diagnosis and treatment.PDT is a photosensitizer that produces toxic reactiv e oxygen species（ROS）under specific wavelengths of light to kill tumor cells.It is a precise and effective treatment with low side effects.We used attenuated Salmonella typhimurium expressing fluorescence-activated protein（FAP）dL5**in combination with PDT for the treatment of colon cancer.When FAPs are combined with fluoride,they can be activated to produce fluorescence and ROS at specific wavelengths.Tumor imaging based on attenuated Salmonella typhimurium expressing dL5**was sensitive and rapid with very low non-specific background.In addition,BMCT-PDT showed enhanced tumor inhibition and prolonged animal survival.Mechanically,ROS produced by PDT kills tumor cells and over-accumulated bacteria.The pathogen-associated molecular patterns（PAMPs）and damage-associated molecular patterns（DAMPs）released from the destroyed bacteria and cancer cells recruited and activated immune cells（macrophages,neutrophils,and dendritic cells,etc.）,which released additional pro-inflammatory cytokines（TNF-αand IL-1β）and reduced anti-inflammatory cytokines（IL-10）,and further enhanced immune cell infiltration in a positive-feedback manner,thus reducing bacterium-induced side effects and improving anticancer activities.This synergistic therapy has promi sing potential for cancer immunotherapy. |
