Novel Non-thermal Histotripsy Tumor Ablation Stimulates Anti-tumor Immunity And Enhances The Efficacy Of Immune Checkpoint Inhibitor

【Background】Immunotherapy has opened a new revolution in tumor therapy,and immune checkpoint inhibitors are an important part of cancer immunotherapy.At present,great progress has been made in the clinical treatment of immune checkpoint inhibitors,but the clinical objective response rate is not high,about 20%.Therefore,there is an urgent need for combination therapy to expand the beneficiaries of immune checkpoint inhibitors and improve the efficacy of immune checkpoints.Tumor physical therapy(such as radiotherapy and radiofrequency ablation treatment commonly used in clinical)combined with immune checkpoint inhibitor research is an important direction of combined immunotherapy.In theory,by stimulating inflammation or releasing tumor antigens in the tumor microenvironment,radiation therapy and tumor thermal ablation therapy can enhance the role of immune checkpoint inhibitors and even make previously resistant cancers sensitive to immunotherapy.However,although immunostimulatory effects have been observed in tumor thermal ablation and radiation therapy,the magnitude of these effects has not yet proven capable of consistently and stably augmenting the effect of immunotherapy.At the same time,radiation therapy and thermal ablation treatment have side effects such as radiation and thermal side injury.In addition,one potential immunostimulatory limitation of radiation therapy and tumor thermal ablation may be their inability to induce sufficient tumorous release of immunogenic or inflammatory subcellular components,such as neoantigens or damage-associated molecular patterns like calreticulin(CRT)or high mobility group box protein 1(HMGB1)that are capable of triggering strong tumor-directed adaptive immune responses.Therefore,it is also necessary to develop a new type of tumor physical therapy to open up new ways for tumor physical therapy.At the same time,it can expand the clinical efficacy of immune checkpoint inhibitors.Histotripsy is a new type of non-invasive,non-thermal,and non-ionizing tumor ablation method that uses overlapping high-pressure ultrasound pulses to destroy cellular architecture.At the focal point,focused ultrasound will cause acoustic cavitation to produce microbubbles,and produce precise extreme pressure change regions,causing the microbubbles to rapidly expand and collapse,thereby mechanically fractionating cells and homogenizing the target tissue into liquefied acellular homogenate.By extending the intervals between these pulses to milliseconds or longer,heat generation at the focal point can be avoided,thereby avoiding the denaturing effects of heat or ionizing radiation.However,whether Histotripsy can effectively ablate tumors and inhibit tumor growth is unknown,and whether Histotripsy can induce tumor immunogenic cell death and release tumor immunogenic antigens and damage-associated molecule patterns such as CRT and HMGB1 to stimulate anti-tumor immune response remains to be explored.Besides,the clinical effectiveness of immune checkpoint suppression is still not high.Whether Histotripsy can enhance the efficacy of immune checkpoint inhibitors remains to be evaluated.We hypothesized that Histotripsy will be able to effectively ablate tumors,promote tumor immunegenic cell death,stimulate the body’s anti-tumor immunity,and enhance the efficacy of immune checkpoint inhibitors.【Objective】1.To evaluate the safety and effectiveness of Histotripsy as a new type of tumor ablation.2.To evaluate whether Histotripsy can induce tumor immunogenic cell death,release immunogenic tumor neoantigens and DAMPs such as CRT and HMGB1,and then stimulate anti-tumor immune response and induce distant effects of tumor treatment.3.To determine whether Histotripsy can synergistically enhance the efficacy of immune checkpoint suppression.【Methods】We used B16GP33 or Hepa1-6 cells to establish C57BL/6 mouse subcutaneous and lung metastasis models.Tumors from each group were treated with Histotripsy ablation,radiotherapy,tumor radiofrequency ablation,or cytotoxic T lymphocyte-associated protein4(CTLA-4)blockade.The safety and effectiveness of Histotripsy ablation tumors were evaluated by body weight,tumor size monitoring,and H&E staining of major organs.The effect of Histotripsy on the antitumor immune response in intratumor,tumor draining lymph nodes and spleen was evaluated by flow cytometry.Immunohistochemistry and immunofluorescence were used to further confirm Histotripsy’s ability to induce T cell infiltration in tumors.The ability of Histotripsy to release immunogenic tumor antige n was confirmed by GP33 in vitro stimulation experiment.By establishing bilateral subcutaneous tumors and lung metastatic tumor models,the ability of Histotripsy to stimulate abscopal effects was evaluated.The ability of Histotripsy to induce immunogenic cell death and release DAMPs was analyzed by immunofluorescence and ELISA.We used tumor monitoring and flow cytometry analyze whether combination with Histotripsy therapy can improve the efficacy of immune checkpoint inhibitors.【Results】1.Histotripsy ablation did not significantly affect the body weight changes of mice.H&E staining showed no significant pathological changes in heart,liver,lung,kidney,and bowel after Histotripsy ablation,nor did it promote tumor micrometastasis.Moreover,Histotripsy ablation tumors can significantly inhibit tumor growth.2.Histotripsy ablation can significantly enhance the infiltratio n of immune cells including CD8~+T cells,natural killer cells,dendritic cells,neutrophils,macrophages and B cells,especially tumor specific CD8~+T cells Infiltration;at the same time Histotripsy is significantly stronger than radiation therapy and radiofreq uency ablation in promoting CD8~+T cell infiltration.3.Through GP33 in vitro stimulation experiments,we found that Histotripsy can effectively release tumor-associated antigens,preserve the integrity and immunogenicity of the antigen,and stimulate the activation of GP33-specific CD8~+effector T cells.4.Histotripsy ablation can significantly induce tumor-specific CD8~+T cell responses in tumor draining lymph nodes and spleen,and the ratio of GP33-specific CD8~+T cells to Tregs cells in the peripheral circulation changes favorably,and NK cells significantly increase and neutrophils rise.5.Histotripsy had a unique ability to inhibit the growth of contralateral distant tumors and promote the infiltration of CD8~+T cells in distant tumors,while no such effects have been observed with radiotherapy and radiofrequency treatment;meanwhile,Histotripsy can prolong survival time of bilateral tumor-bearing mice.In addition,the model of lung metastases showed that Histotripsy ablation of subcutaneous tumors can significantly promote the infiltration of C D8~+T cells in lung metastases and decrease the number of lung metastases.6.Immunofluorescence and ELISA experiments showed that Histotripsy ablation significantly promoted C RT translocation to the cell membrane on the endoplasmic reticulum,and promoted the release of HMGB1 from the nucleus to extranuclear and extracel ular,and gradually entered the peripheral blood circulation.7.Immune checkpoint inhibitors have a stronger ability to control the growth of distant tumors and promote the infiltration of CD8~+T cells in tumors than Histotripsy.When combined with immune checkpoint inhibitors and Histotripsy ablation,Histotripsy significantly enhanced the therapeutic efficacy of immune checkpoint inhibitors.When using the weakly immunogenic Hepa1-6 liver cancer model,Histotripsy combined with immune checkpoint inhibitors achieved similar results.【Conclusion】Histotripsy is a safe and effective new tumor ablation modality.Histotripsy induced tumor immune cell death,released immunogenic neoantigens,DAMPs such as CRT and HMGB1,and stimulated antitumor immune responses in the tumor,regional tumor draining lymph nodes and spleen,as well as systemic inflammatory responses.At the same time,Histotripsy promoted the tumour immune abscopal effect and enhanced the efficacy of immune checkpoint inhibitors.Histotripsy is a promising type of tumor ablation and may expand the impact and promise of cancer immunotherapy.