Studies On Therapeutic Efficacy Of Sonodynamic Therapy Combined With Immune Checkpoint Blockade On Breast Cancer

Breast cancer,as a malignant tumor,is highly prevalent in the global female population.Traditional chemoradiotherapy can’t achieve desired effect,and psychological effects of surgical treatment existed widely in patients.Moreover,it is also prone to metastasis and recurrence after surgery.In recent years,immunotherapy,which has been applied to breast cancer,achieved gratifying outcomes.However,only a few proportions of patients（10-20%）were benefited from it,limited effects were exhibited in most patients.To enhance the therapeutic effect of immunotherapy,more attention has been paid for exploring a variety of approaches of combination therapy.As a relatively non-invasive treatment,sonodynamic therapy can penetrate tissue deeper with little damage to surrounding normal tissues,which is becoming an ideal local treatment.Calcium are involved in various physiological processes of organisms,and the maintenance of its homeostasis is essential for normal activities of cells.Calcium overload is considered to be a major cause of cell death.In this study,the combination of sonodynamic therapy,calcium overload and immune checkpoint blockade was used to improve the therapeutic efficacy for breast cancer.The main works and results of are as follows:1.TiO₂ nanoparticles coated with calcium phosphate（CaP）were prepared（designed as TiO₂@CaP）.The structure of TiO₂@CaP was observed by TEM.The elements analysis was carried out by X-ray diffraction（XRD）and FSEM.Atomic absorption spectroscopy（ICP-MS）showed that compared with neutral condition（p H7.4）,more free Ca²⁺was released by CaP under acidic conditions（p H6.5 and p H5.0）.The reactive oxygen species（ROS）production of TiO₂@CaP was detected by DPBF probe.The result revealed that ROS was produced from TiO₂@CaP under ultrasound（US）（2.1W/cm²,10min）.2.In vitro cell experiments verified TiO₂@CaP had good biocompatibility.Under US treatment,the cell-killing effect of TiO₂ and TiO₂@CaP was elevated along with the increase of nanoparticles concentration.However,owing to existence of Ca²⁺,TiO₂@CaP has a more excellent antitumor ability.The confocal laser scanning microscope（CLSM）and flow cytometry（FCM）were used to detect concentration variation of intracellular Ca²⁺,which was significantly increased in TiO₂@CaP compared to TiO₂.The change of calreticulin（CRT）,high mobility group protein B1（HMGB1）and ATP was respectively detected by FCM,ELISA.The results proved that after treated with TiO₂@CaP and ultrasound,the expression of CRT,HMGB1 and ATP were all up-regulated,indicating more immunogenic cell death（ICD）has been induced by TiO₂@CaP-SDT.3.We established 4T1 tumor-bearing mice model and demonstrated the immune system was activated by TiO₂@CaP-SDT.After TiO₂@CaP-SDT,the proportion of mature dendritic cells in tumor-draining lymph nodes was significantly increased,indicating the dendritic cells were activated by tumor-associated antigen.4.We validated the therapeutic effect of TiO₂@CaP-SDT combined with immune checkpoint inhibitor PD-1 on breast cancer.Compared with other groups,the tumor growth rate in TiO₂@CaP-SDT+anti-PD-1 group was significantly slowed down.FCM analysis suggested the infiltration of specific T cells in tumor microenvironment was increased.Besides,ELISA confirmed that the level of cytokines IFN-γand TNF-αin serum were elevated significantly.Furthermore,the significant suppression of TiO₂@CaP-SDT+anti-PD-1 on distal tumors was also verified in 4T1 bilateral tumor model,which suggested the system immune response induced by TiO₂@CaP-SDT+anti-PD-1 combination therapy may inhibit the metastasis.Taken together,the sonosensitizer-TiO₂@CaP used in this study produced ROS with US.The presence of CaP increased Ca²⁺levels within tumor cells by generating free Ca²⁺in acid environment,causing calcium overload.Both of ROS and calcium overload had a mutual synergistic effect and induced ICD.In addition,PD-1 antibody ameliorated tumor immunosuppressive microenvironment,TiO₂@CaP-SDT+anti-PD-1 combination therapy significantly improved the antitumor effect.At the same time,the activation of immune response exhibited killing effect on the distal tumors.In summary,TiO₂@CaP-SDT combined with immunotherapy exhibited an ideal effect in animal models,thus was promising to provide a feasible and effective therapeutic strategy for clinical treatment of breast cancer.