Study On The Na-FUS Improves The Efficacy Of HIFUa In 4T1 Breast Cancer

In recent years,high intensity focused ultrasound ablation(HIFUa)is increasingly used in malignant tumors.However,like other malignant tumor treatments,HIFUa cannot achieve long-term tumor control in most cases.How to improve the efficacy of HIFUa treatment is a problem we want to solve.Different treatment modes of focused ultrasound(FUS)will lead to different anti-tumor immune effects.HIFUa leads to coagulative necrosis of tumors in the focal area,releasing antigens and signaling molecules.However,due to high fever,antigens and cytokines may be inactivated within a short period of time,and vascular endothelial cells may swell,blood flow slows,or even vascular occlusion.The tissue surrounding the focal region undergoes mild heat stress/hyperthermia,which can increase DNA damage,induce the production of new tumor antigens,promote apoptosis,release DAMPs,and induce ICD,thereby regulating the tumor microenvironment.Therefore,it is currently believed that HIFUa can stimulate immunity,but the stimulated immunity is not durable enough and not to be strong enough.However,as a non-ablation mode induced by FUS,non-ablation FUS(na-FUS)can promote apoptosis and release molecular models related to injury.Moreover,due to mechanical and cavitation effects,cell permeability is enhanced and physiological barrier will be opened.This will enhance the circulation of antigens and signal molecules,increase the infiltration of lymphocytes.So in theory,na-FUS can stimulate anti-tumor immunity better than HIFUa.However,na-FUS cannot kill tumor cells,and other treatment methods need to be combined to reduce the tumor load.Combining HIFUa can quickly reduce the tumor load.We therefore want to use na-FUS as a pre-treatment to stimulate the body’s immunity,and then use HIFUa to reduce the tumor load,so as to improve the anti-tumor effect of current HIFUa treatment.To explore a new non-invasive method for tumor treatment-"na-FUS+HIFUa".ObjectiveDetermine the specific parameters of FUS pretreatment and verify its nature.And verified the efficacy of"na-FUS+HIFUa"and its related immune enhancement by experimental study in the treatment of 4T1 breast cancer in BALB/c mice.To further specify the effects of gemcitabine(GEM)combined with"na-FUS+HIFUa".Methods1.Verify the specific parameters and properties of FUS pretreatmentFirstly,we using a simulation model calculated two sets of parameters that do not cause high temperature(<56℃)and low energy.Then two sets of parameters were selected for subsequent in vivo experimental.Based on simulation model results,mice were divided into four groups:Group A(control):sham irradiation;Group B(HIFUa):continuous wave,140W,1s/point;Group C:5%pulse wave,5Hz,50W,30s/point;Group D:5%pulse wave,5Hz,5W,30s/point.Validation of biological effects using in vitro bovine liver experiments and in vivo experiments on experimental animals.After FUS therapy,tumors were taken out for TTC,HE or other tests.2.To verify the efficacy and related immune mechanism of"na-FUS+HIFUa"in treating 4T1 breast cancer in BALB/c mice4T1 breast cancer bearing mice were randomly divided into three groups:(1)The"na-FUS+HIFUa"group received na-FUS treatment 12days after subcutaneous transplantation,and then received HIFUa treatment3 days later;(2)"HIFUa"group received HIFUa treatment 12 days after subcutaneous transplantation;(3)The control group received sham irradiation 12 days after subcutaneous transplantation.In unilateral tumor models,fourteen days after the FUS treatment,the tumors were removed for RNA-seq to detect the changes of immune microenvironment related genes.Immunofluorescence was used to detect the content of CD8 protein.CD3~+T lymphocytes,CD4~+T lymphocytes,CD8~+T cells,B lymphocytes and NK cells were detected by flow cytometry.In bilateral tumor models,fourteen days after FUS treatment,the treated side,untreated side tumors and spleens were taken out and the infiltrating lymphocytes were also detected by flow cytometry.Tumor growth monitoring and survival record were performed in both unilateral and bilateral tumor model mice.3.Clarify the effects of gemcitabine combined with"na-FUS+HIFUa"4T1 breast cancer bearing mice were randomly divided into four groups:(1)The"na-FUS+HIFUa+GEM"group received na-FUS therapy and intraperitoneal injection of gemcitabine 12 days after subcutaneous transplantation.And then received HIFUa treatment 3 days later.(2)"HIFUa+GEM"group received HIFUa therapy and intraperitoneal injection of gemcitabine 12 days after subcutaneous transplantation.(3)The GEM group received intraperitoneal injection of gemcitabine 12 days after subcutaneous transplantation;(4)The control group received sham irradiation 12 days after subcutaneous transplantation.Fourteen days after the treatment of unilateral and bilateral transplanted tumor model mice,the tumor was removed and the infiltrating lymphocytes in the tumor were detected by flow cytometry.All mice were monitored for tumor growth and survival records.And the number of lung metastases was counted.Meanwhile,the changes of PD-1~+T lymphocytes were explored in the four groups after treatment.Results1.Determine group D parameters(5%pulse wave,5Hz,5W,30s/point)as the subsequent experimental parameters of FUS pretreatmentThe simulation model results proposed two sets of pretreatment parameters:group C:5%pulse wave,5Hz,50W,30s/point;group D:5%pulse wave,5Hz,5W,30s/point.The gray value of ultrasound image after treatment in group A and group D did not change from that before treatment,while the gray value of group B and group C changed significantly more.TTC results showed that the intermediate treatment area of group B and group C was dyed white,and the group A and group D were dyed red.The HE results showed that there was no obvious necrosis area in Group A and Group D after FUS therapy,while there was tissue necrosis in the treatment area of Group B and Group C after treatment.From the result of TTC,HE,and ultrasound images,it can be concluded that group D parameters did not cause tumor tissue necrosis,while group C parameters caused necrosis.Ki-67 immunohistochemistry results showed that although there was no statistical difference in Ki-67 expression among the four groups after 7 days of treatment(P>0.05),the Ki-67 protein expression in Group C and Group D was higher than that in Group B(HIFUa group),indicating that Group C and Group D needed to combine stronger FUS treatment methods to better control tumors.There was no significant difference in tumor growth curve and survival curve among the four groups(P>0.05).Immunohistochemical results of CD8 suggest that group A parameters have the highest expression of CD8 after treatment,suggesting that group D parameters can induce more CD8 lymphocyte infiltration.The purpose of this section was to select a group of data with the best safety as the experimental parameters of FUS pretreatment.We ultimately selected group D parameters(5%pulse wave,5Hz,5W,30s/point)as the FUS pretreatment for subsequent experiments.The name of the subsequent experimental protocol is"na-FUS+HIFUa".2."na-FUS+HIFUa"induced more immune gene expression than"HIFUa"The gene pool enrichment analysis showed that"na-FUS+HIFUa"could induce more adaptive immune genes than HIFUa treatment.The heat map also showed that"na-FUS+HIFUa"therapy had a stronger ability to induce the expression of related immune response marker genes than HIFUa.Moreover,the expression of tumor progression-related marker genes decreased after"na-FUS+HIFUa"treatment.3."na-FUS+HIFUa"promoted stronger CD4~+and CD8~+T lymphocyte infiltrationFor the unilateral tumor model,by immunofluorescence staining we observed that the infiltration of CD8~+T cells in the tumors treated with"na-FUS+HIFUa"was higher than that treated with HIFUa(P<0.05).Flow cytometry also showed similar results.Compared with HIFUa group and control group,CD4~+and CD8~+T cells in"na-FUS+HIFUa"tumors were significantly increased(P<0.05).In spleen,CD4~+T cells and B cells were significantly increased in"na-FUS+HIFUa"group(P<0.05).The survival of"na-FUS+HIFUa"also benefited significantly(P<0.05).For bilateral tumor models,CD4~+T cells,CD8~+T cells cells were also significantly increased in"na-FUS+HIFUa"on the treatment side(P<0.05).However,no significant elevation of related cells was observed in the untreated side and spleen(P>0.05).4.GEM combined with"na-FUS+HIFUa"could enhance anti-tumor effectsIn the bilateral tumor model,CD4~+T cells and CD8~+T cells of the treated side of the mice were significantly increased in"na-FUS+HIFUa+GEM"group(P<0.05).In the untreated side of the tumor,CD8~+T cells also was significantly increased in"na-FUS+HIFUa+GEM"group(P<0.05).In addition,CD3~+T lymphocyte in spleen of"na-FUS+HIFUa+GEM"group were significantly increased(P<0.05).It showed that the combination of GEM could enhance the systemic anti-tumor immunity induced by"na-FUS+HIFUa".Moreover,in"na-FUS+HIFUa+GEM"group,tumor growth were significantly inhibited both in the treated and untreated sides of mice and lung metastasis were significantly reduced(P<0.05).Both in unilateral tumor model and bilateral tumor model,"na-FUS+HIFUa+GEM"group had significant survival advantages(P<0.05).ConclusionsThis study proved that"na-FUS+HIFUa"is a new,non-invasive and effective tumor treatment method."na-FUS+HIFUa"not only improves local tumor control,but also prolongs survival and induces distant effects.The pretreatment of na-FUS improves the curative effect of HIFUa,promotes the CD4~+and CD8~+T lymphocyte infiltration.The combination of GEM can further enhance the efficacy of"na-FUS+HIFUa".