Focused ultrasound thermal ablation and CD40 agonism reprograms breast tumor immunity to drive regression and memory

Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we investigated whether subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across immunologically and molecularly distinct murine BC models.

Subtotal ultrasound-guided T-FUS was applied to E0771, BRPKP110, EMT6, and 4T1 tumors in mice treated with systemic agonistic αCD40. Histology, ATP bioluminescence imaging, and multispectral flow cytometry were used to define acute tumor injury and immune remodeling. Therapeutic efficacy was evaluated by tumor growth, survival, T-cell depletion, and contralateral tumor rechallenge.

Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4+ and CD8+ T cells. When layered onto this immunogenic milieu, αCD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 30% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Combination treatment also extended survival across three of four models. Efficacy required both CD4+ and CD8+ T cells, as depletion of either compartment abrogated tumor control. Complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T-cell activation.

Subtotal T-FUS cooperates with CD40 agonism to drive durable, T cell-dependent BC regression and immunologic memory. These findings establish T-FUS as an immune-potentiating partner for CD40 agonism and position this combination as a clinically scalable, in situ vaccination-like strategy with potential to broaden immunotherapy benefit across breast cancer subtypes, including luminal tumors that remain largely refractory to immune checkpoint blockade.