Simulation-guided non-thermal low-intensity ultrasound reprograms the tumor immune microenvironment and engages systemic antitumor immunity in a syngeneic orthotopic mouse model of breast cancer

<jats:p>Introduction: Therapeutic ultrasound has been extensively studied in ablative and sonodynamic contexts, leaving the intrinsic bioactivity of continuous non-thermal low-intensity ultrasound (LIU) largely uncharacterized. Objectives: To characterize the tumor biological and immunomodulatory effects of non-thermal continuous LIU in complementary in vitro and in vivo breast cancer models, underpinned by a standardized exposure platform characterized through finite element simulations and experimental validation. Methods: Acoustic and thermal fields were characterized and optimized using in silico simulations and validated against hydrophone and temperature measurements to ensure homogeneous, non-thermal exposure (1MHz, 1W/cm2, 100% duty cycle). 4T07 murine mammary carcinoma spheroids received 20min LIU treatment, and metabolic activity, apoptosis, and intracellular stress-associated markers were assessed. In a syngeneic orthotopic 4T07 mammary carcinoma model in BALB/c mice, up to six LIU treatment cycles were administered; tumor growth, survival, histopathology, immunohistochemistry, bulk tumor RNA sequencing, spleen volume, and plasma cytokine profiles were assessed. Results: In vitro and intratumoral temperatures remained within the physiological range (39 degrees C or lower) throughout exposure. In spheroids, LIU reduced ATP content by more than 40% and significantly increased apoptotic, Hsp70⁺ and Hsp90⁺ cell fractions. In vivo, cyclic LIU slowed tumor growth, increased intratumoral necrosis, and significantly prolonged time to humane endpoint compared to untreated controls. LIU promoted early intratumoral myeloid cell infiltration and shifted the tumor transcriptome (2,573 differentially expressed genes), with enrichment in gene sets associated with immunogenic cell death, pattern-recognition, inflammatory, and innate and adaptive immune programs and downregulation of pro-tumorigenic pathways. LIU enriched the transcriptional signatures of M1 macrophage polarization and, notably, B-cell compartment engagement, which has not previously been reported for standalone continuous mechanical ultrasound. LIU significantly attenuated tumor-associated splenomegaly and elevated plasma IL-1alpha, TNF-alpha, and IL-10. Conclusion: These results establish a reproducible preclinical platform and provide a hypothesis-generating mechanistic basis for evaluating LIU as an adjunct to immune checkpoint blockade.</jats:p>