Research and Exploration of an Improved HIFUa Method: “na-FUS+HIFUa”

Abstract

Background: High-intensity focused ultrasound ablation (HIFUa) has been shown to rapidly ablate tumour tissues. However, HIFUa alone often fails to stimulate sufficient and long-lasting antitumour immunity. To overcome this limitation, we investigated the use of non-ablation focused ultrasound (na-FUS), which has potent immunomodulatory effects, combined with HIFUa. In this study, we evaluated the potential of this improved HIFUa method, referred to as “na-FUS+HIFUa", along with the addition of gemcitabine (GEM), to enhance antitumour immunity, inhibit tumour development, and prevent metastasis in murine 4T1 tumours. Methods: This study was conducted in three stages. First, Ultrasonic images, TTC (2,3,5-triphenyltetrazolium chloride) and HE (hematoxylin-eosin) were used to prove na-FUS does not lead to coagulation necrosis. Then, we verify the efficacy and related immune mechanism of “na-FUS+HIFUa" in treating 4T1 breast cancer BALB/c mice. In unilateral tumor model mouse, the tumor was removed for ribonucleic acid sequencing (RNA-seq) transcriptome sequencing and immunofluorescence. Flow cytometry was performed use tumor and spleen. In bilateral tumor model mice, the treated tumor, untreated tumor, and spleen were removed for flow cytometry to detect infiltrating lymphocytes. Finally, GEM was combined to further elucidates the effect of “na-FUS+HIFUa" compared to HIFUa. Flow cytometry was performed use tumors and spleen. All mice underwent tumor growth monitoring and survival records, as well as counting the number of lung metastases. Results: Through RNA-seq analysis, we observed differential mRNA expression related to the immune microenvironment in the tumours. Gene Set Enrichment Analysis (GSEA) revealed a higher number of differentially expressed genes associated with adaptive immune response in the “na-FUS+HIFUa" group compared to the HIFUa group. Heatmap analysis demonstrated increased expression of marker genes associated with immune response following “na-FUS+HIFUa" treatment. Flow cytometry results further confirmed that “na-FUS+HIFUa" induced higher levels of local intratumoural lymphocyte infiltration, including CD4⁺ and CD8⁺ T cells, compared to HIFUa treatment alone (p < 0.05). Moreover, the combination of “na-FUS+HIFUa+GEM" significantly enhanced both local and systemic immune responses, leading to increased lymphocyte infiltration in the treated tumours, untreated tumours, and spleen. This combination approach also inhibited the development of lung metastases and improved overall survival. Conclusion: We have successfully developed a novel, non-invasive and feasible method for combining na-FUS with HIFUa. The pretreatment of na-FUS improved the efficacy of HIFUa and promoted the infiltration of CD4⁺T lymphocytes and CD8⁺T lymphocytes within the tumor. Combining GEM can further enhance the therapeutic effect of “na-FUS+HIFUa".