Low-frequency Ultrasound Promotes An Anti-tumor Immunological Response By Inducing Myeloid-derived Suppressor Cell Apoptosis

Background and Objective:Cancer poses a major threat to public health throughout the world.Since 1990,the incidence of cancer in most countries has increased.Cancer is one of the main causes of death.It has seriously affected the health of people in both developed and developing countries.Traditional cancer treatments include surgery,chemotherapy and radiotherapy treatment for cancer is still the main choice of most people.However,these conventional treatment methods have disadvantages such as systemic toxicity,low selectivity,and drug resistance.Therefore,there is an urgent need for new methods for tumor treatment that are highly selective,minimally invasive,and have few side effects.In recent years,with the development of ultrasound technology,the application of low-frequency ultrasound(LFU)in the treatment of tumors has gradually been paid attention.Studies have shown that with low-frequency ultrasound irradiation,the cavitation effects can directly damage cells,promote tumor cell apoptosis,and can damage the tumor blood vessels,so that the blood supply in the tumor area is significantly reduced.Meanwhile,it has been reported that low-frequency ultrasound exhibits anti-tumor effects by inhibiting tumor angiogenesis and reversing tumor-induced immune tolerance.In recent years,myeloid-derived suppressor cells(MDSCs)have been the focus of numerous studies on anti-tumor immune mechanisms.MDSCs are heterogeneous cell populations of bone marrow cells at different stages of differentiation,which play an important role in tumor progression.MDSCs have been considered an important factor in tumor evasion of immune surveillance and tumor progression.The observed MDSC-mediated immunosuppression is reflected by the impaired function of the adaptive(T cells)and innate(NK cells)immune systems.It is an important factor for tumors to evade immune surveillance and tumor progression.Studies have shown that chemotherapeutic drugs such as gemcitabine(GEM)and polyclonal antibodies such as anti-Gr-1 antibodies can also enhance the anti-tumor immune response by selective depletion of MDSCs to inhibit tumor growth.However,these chemotherapeutic drugs cannot target tumor cells or MDSCs,and at the same time,cause damage to normal tissues and cells around the tumor,and may even cause severe side effects such as myelosuppression.Yet it has not been reported whether low-frequency ultrasound can enhance anti-tumor immune responses by inhibiting the proliferation and function of MDSCs.In this study,we established a mammary carcinoma mouse model.With treatment of tumor-bearing mice with LFU,anti-Gr-1 or GEM,we evaluated the growth of the tumor,the condition of tumor neovascularization,and the proliferation of immune cells such as CD8~+T cells and NK cells.Meanwhile,we detected the number of MDSCs in tumor-bearing mice,and whether low-frequency ultrasonography could induce apoptosis of MDSCs both in vitro and in vivo,to explore the role of MDSCs in the treatment of tumors with Low-frequency ultrasound and its related mechanisms.Methods:The mouse breast cancer 4T1 cells derived from Balb/c mice were cultured and inoculated into the second pair of mammary glands on the left side of the mice to establish a mouse breast cancer xenograft model.Then,tumor-bearing mice were randomly divided into four groups:control group,anti-Gr-1 antibody group(anti-Gr-1),gemcitabine group(GEM),and low-frequency ultrasound group(LFU).After treatment,the body weight of mice and tumor volume and tumor weight were measured.The expression of VEGFA,Flk-1,CD34 and MMP-9 was measured by immunofluorescence and Western blotting to evaluate the effect of Low-frequency ultrasound on tumor neovascularization.Immunofluorescence was used to detect the expression of CD8 and NKp46 to evaluate the effect of low-frequency ultrasound on the number of CD8~+T cells and NK cells in tumor-bearing mice.Immunof-luorescence and flow cytometry were used to analysis the proportion of Gr-1~+CD11b~+cells and apoptosis of MDSCs in vivo.Then,MDSCs were isolated and purified and randomly divided into four groups:control group,anti-Gr-1 antibody group(anti-Gr-1),gemcitabine group(GEM),low-frequency ultrasound group(LFU).After treatment,the rate of apoptosis of MDSCs was detected by flow cytometry to clarify the mechanism of low-frequency ultrasound-mediated anti-tumor immunological response.Results:1.After treatment,anti-Gr-1,GEM and LFU all significantly reduced tumor volume and tumor weight compared with the control(P<0.01).The tumor volume and quality of mice in the LFU group were significantly reduced compared with the anti-Gr-1 group,but there was no statistically significant change in tumor volume and quality compared with the GEM group(P>0.05).With low-frequency ultrasound irradiation,treatment of anti-Gr-1 antibody and GEM,the body weights of mice in each group showed a tendency to decrease,there was no significant difference among the groups(P>0.05).However,the weight of mice in the GEM group was reduced more compared with the other groups.2.The levels of VEGFA,its receptors Flk-1,CD34,and MMP-9 were significantly decreased in the anti-Gr-1,GEM and LFU groups compared with those in the untreated control group(P<0.001).Compared with the anti-Gr-1 group,the expression levels of VEGFA,Flk-1,and CD34 were significantly decreased in the LFU group,but the difference was not statistically significant compared with the GEM group(P>0.05).3.We found significantly enhanced CD8 and NKp46 expression in the anti-Gr-1,GEM and LFU groups compared with that in the untreated control group(P<0.001).LFU treatment significantly increased the expression of CD8 and NKp46 compared with that in the anti-Gr-1 group or the GEM group,suggesting that the quantities of CD8~+T cells and NK cells were enhanced by LFU treatment.4.The proportion of Gr-1~+CD11b~+cells in tumor tissues and bone marrow was significantly decreased in the anti-Gr-1,GEM and LFU groups compared with that in the untreated control group(P<0.001).Compared with the anti-Gr-1 antibody group,low-frequency ultrasound significantly reduced the content of MDSCs.However,there were no differences between the GEM group and the LFU group(P>0.05).5.After isolation and purification of MDSCs,the percentage of apoptosis of MDSCs in the anti-Gr-1 group,GEM group,and LFU group was significantly increased compared to the control group(P<0.001).Compared with the anti-Gr-1group,the apoptosis rate of MDSCs was significantly increased in the LFU group,but there was no significant difference(P>0.05)compared with the GEM group.6.The expression of caspase-3 in Gr-1~+cells was significantly increased in the LFU group compared with that in the untreated control group(P<0.001).LFU treatment significantly increased the expression of caspase-3 in Gr-1~+cells compared with that in the anti-Gr-1 group.However,there were no differences between the GEM group and the LFU group.Conclusion:1.Low-frequency ultrasound can inhibit tumor growth and tumor neovascularization.2.Low-frequency ultrasound can enhance anti-tumor immune response by promoting the proliferation of lymphocytes such as CD8~+T cell and NK cell.3.Low-frequency ultrasound reduces MDSCs in tumor-bearing animals and promotes anti-tumor immunological response by inducing MDSCs apoptosis.