| Interferon-gamma(IFN-γ)is the only member of Interferon II.It is a kind of glycoprotein with broad-spectrum antiviral effect.It also has the role of highly effective immune regulation and cancer treatment.Studies have shown that adjuvant anaerobic bacteria such as attenuated Salmonella typhimurium target tumors and accumulate and proliferate,and a large number of neutrophils infiltrate around tumors,leading to recurrence and metastasis of tumor treatment effect and poor prognosis.It has been reported that attenuated Salmonella typhimurium and IFN-γhave anti-tumor effects in the treatment of tumor respectively,but the mechanism of their combined therapeutic effect has not been reported.Therefore,this study focused on the combined immunomodulatory mechanism of attenuated Salmonella typhimurium against cancer and IFN-γ.To explore the effect of its combination therapy on immune cells and tumor therapy,and to provide a theoretical basis for the study of the synergistic effect of bacteria-mediated tumor therapy and other drugs.In this experiment,attenuated Salmonella typhimurium(S.tΔpp Gpp strain),IFN-γand BALB/c mice were used as the main experimental materials to study the following contents:1)Combination of S.tΔpp Gpp+IFN-γagainst colon cancer in mice.In this experiment,Balb/c male mice aged 5-6 weeks were injected with 1×10~6 CT26 cells each into the subcutaneous muscle of mice to establish a colon cancer mouse model.When the tumor size reached 120-180 mm~3,1μg/IFN-γwas injected into the caudal vein on the same day,and 100μl of bacteria were injected into the caudal vein on the next day(only once;3×10~7 CFU per mouse)and 1μg/mouse IFN-γwas injected through tail vein every day for 21 days.Tumor volume=(L×H×W)/2.It can be seen from the tumor growth curve that within 7 days after the injection of bacteria,the tumor volume gradually decreased and the scab fell off,but the tumor gradually recurred after the scab fell off.Compared with the single S.tΔpp Gpp+IFN-γgroup,the combined treatment group has significantly enhanced tumor inhibition effect and even completely shed tumors:Mice treated with the combination(27%)showed complete tumor regression and survived longer than the other groups,regrow hair on the right side of shaving and remain healthy until observation stopped on day 60.These results indicate that attenuated Salmonella typhimurium combined with IFN-γcan enhance the anti-tumor effect on the basis of a certain anti-tumor effect.2)Effect of S.tΔpp Gpp+IFN-γcombined action on bacterial targeting in tumors.In this experiment,mice in the single bacteria group and the combined treatment group were necked and died on the second and sixth days after bacterial injection.Tumors,livers,spleens and lungs of mice were collected for mechanical homogenization according to the ratio of weight(g):PBS volume(m L)=1:9,and cultured overnight at 37℃to calculate the number of bacteria contained in each gram of tissues.In order to study the effect of IFN-γon the ability of S.tΔpp Gpp(strain transferred to Lux fluorescent gene)engineering bacteria to target tumor and the survival time in tumor.In vivo luminescence imaging of mice showed that S.tΔpp Gpp+IFN-γcombined treatment did not affect the ability of bacteria to accumulate and proliferate in the tumor,and all bacteria were colonized in the tumor site 1-3 days after the injection.The statistical results of bacterial count on the 2nd and 6th days showed that the number of accumulated bacteria in the tumor of the combined treatment group and the single bacteria group was much higher than that of other organs,and the difference was extremely significant(P<0.001).Especially on the 6th day after bacterial injection,the number of bacteria in the tumor of the combined treatment group was significantly higher than that of the single treatment group(P<0.05).It can be seen that IFN-γcan enhance the internal targeting ability of S.tΔpp Gpp engineering bacteria for a certain period of time.3)Effect of S.tΔpp Gpp+IFN-γcombined action on tumor microenvironment.In this study,tumor tissues were collected 48h after bacterial injection for routine immunohistochemistry.The neutrophil primary antibody LY6G(rabbit anti-rat 1:100),macrophage M1-type primary antibody CD86(rabbit anti-rat 1:50)and macrophage M2-type primary antibody CD206(rabbit anti-rat 1:200)were incubated,and the biology-labeled sheep anti-rabbit/rat Ig G of the secondary antibody was incubated.IHC results showed that the number of M1/M2 type macrophages was significantly increased in the combined treatment group and IFN-γalone group compared with the control group and IFN-γalone group after bacterial infiltration in tumor tissue,and the difference was extremely significant(P<0.001).At the same time,the number of M1/M2 macrophages in the combined treatment group was significantly higher than that in the single bacteria group(P<0.05).In particular,the difference between M1 type macrophages and M2 type macrophages was significantly increased(P<0.001).For neutrophils,the bacteria alone group was significantly higher than the control group and IFN-γalone group,and the difference was extremely significant(P<0.001).These results indicated that IFN-γsignificantly induced M1/M2 polarization of macrophages and inhibited the recruitment of neutrophils after bacterial invasion.4)Changes of cytokines associated with S.tΔpp Gpp+IFN-γcombined therapy in tumors.Since attenuated Salmonella typhimurium induces different immune cell population infiltration after tumor colonization,the resulting effector molecules may be involved in anti-tumor effects.Therefore,we next investigated the immunological changes of the tumor.QRT-PCR was used to detect the changes in the expression of related cytokines after 48 hours of S.tΔpp Gpp+IFN-γtreatment.The results showed that compared with the control group and IFN-γgroup alone,the transcription levels of IL-1β,TNF-α,TGF-β,IL-6,IL-10,GM-CSF,and GM-CSF in the single bacteria group and the combined treatment group were significantly higher,and the differences were extremely significant(P<0.001).The transcription levels of IL-1β,TNF-αand GM-CSF in the combined treatment group were all highly expressed,and the differences were significant(P<0.05).The difference in G-CSF in the combination treatment group was significantly lower than that in the single bacteria group(P<0.05).Therefore,it can be speculated that the combined treatment of S.tΔpp Gpp+IFN-γcan reduce the transcription level of G-CSF(granulocyte colony stimulating factor)and effectively inhibit the recruitment of neutrophils compared with single bacteria group.At the same time,the expression of IL-1β(interleukin 1β),anti-inflammatory cytokine IL-10(interleukin 10),TNF-α(tumor necrosis factor-α),GM-CSF(granulocyte-macrophage colony stimulating factor)and other related inflammatory cytokines secreted by macrophages were increased,which enhanced the inhibition effect of tumor growth.In conclusion,the combination therapy of S.tΔpp Gpp+IFN-γcan inhibit the tumor by inducing M1/M2 polarization of macrophages and promoting the apoptosis of neutrophils to reduce the number of recruitment,which provides a strategy for the combination immunotherapy mode. |
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