High-intensity focused ultrasound (HIFU), as a non-invasive breast-conserving method, has led to revolutionary changes in the treatment of breast cancer [1,2]. However, HIFU faces challenges such as limitations in imaging and tumor localization, along with the attenuation of ultrasound energy at greater depths. These limitations could hinder the effective ablation of larger or deeper tumors, leading to residual tumor tissue [3]. Beyond tumor ablation, HIFU therapy may enhance the production of tumor antigens and bioactive substances, such as damage-associated molecular patterns (DAMPs), thereby stimulating innate antitumor immune responses to eliminate residual tumor cells [4,5]. However, postoperative tumor cells often exhibit low immunogenicity, and the hypoxic conditions following HIFU surgery further exacerbate the immunosuppressive tumor microenvironment (ITM). These factors result in a limited intensity of HIFU-induced innate immune response [6].

Immunogenic cell death (ICD) triggers T lymphocyte activation and the adaptive immune response through the release of DAMPs, such as calreticulin (CRT), high mobility group protein 1 (HMGB1), and adenosine triphosphate (ATP) [7,8]. As a major way for generating robust antitumor response, its significance is self-evident. However, HIFU therapy employs a current scanning strategy that results in focal domain temperatures of 60–90 °C [9], impeding the release of DAMPs [10]. Moreover, the cavitation and mechanical effects necessary for exposing cell surface antigens are insufficient [11]. Consequently, these factors lead to low immunogenicity in residual tissue, and prevent the body from eliciting robust and sustained innate immune response. Accumulating evidences have shown that enhancing ICD is an effective approach to increase the immune characteristics of tumor cells [12,13]. Among various ICD inducers, banoxantrone (AQ4N) distinguishes itself due to its specificity for the hypoxic postoperative microenvironment [14]. Owing to negligible cytotoxicity and immunotherapy effects under aerobic conditions, AQ4N would be activated to significantly induce ICD exclusively in hypoxic microenvironment [15].

Notably, the intricate interactions between tumor cells and tumor microenvironment (TME) serve as conducive “soil” for tumor cell proliferation and metastasis [16,17]. Particularly under severe hypoxic conditions after HIFU surgery, tumor cells exacerbate the ITM by recruiting immunosuppressive regulatory T cells through upregulation of the chemokine ligand CCL28, and promoting polarization of tumor-associated macrophages (TAMs) toward M2 immunosuppressive phenotype via hypoxia-inducible factor (HIF-1α) [18]. The ITM substantially impedes tumor antigen presentation, hampers dendritic cell maturation, and limits T-cell infiltration [19], thereby diminishing immune responses [20,21]. SR-717, as a non-nucleotide STING agonist [22,23], was recently shown to stimulate the STING signaling pathway to promote type I interferon (IFN-I) production, activate immune cells, and increase their intratumoral infiltration [24]. Therefore, SR-717 would effectively overcome the immunosuppression exacerbated by hypoxia following HIFU surgery.

On the other hand, in exacerbated hypoxic TME following HIFU treatment, tumor cells produce substantial amounts of lactic acid via the glycolytic pathway [25], which accumulates in tumor tissues. The elevated lactic acid suppresses immune cell functions, including effector T cells and natural killer (NK) cells, as well as promotes macrophage polarization toward the M2 phenotype, which are adverse for the antitumor immunotherapy [26]. Therefore, depleting lactic acid levels in tumors could effectively help to overcome ITM. Owing to their excellent lactate neutralization ability [27], calcium carbonate (CaCO3) nanoparticles can suppress the polarization of TAMs toward the immunosuppressive M2 phenotype, promote their polarization toward the immune-activating M1 phenotype [28]. Additionally, CaCO3 enhance activity and infiltration of T and NK cell by regulating the pH of the TME, thus providing new avenues for achieving long-term modulation of the ITM [29].

Guided by the aforementioned ideas, we developed a hypoxia-responsive albumin-crosslinked nanosystem for enhancing post-HIFU surgery immunotherapy through the co-delivery of AQ4N and SR-717. Specifically, this nanosystem utilizes hypoxia-responsive azobenzene linker to enable covalent cross-linking of HAS while encapsulating AQ4N, SR-717, and CaCO3, resulting in the synthesis of structurally stabilized HACS NPs. The HACS NPs dissociate within the hypoxic tumor microenvironment, enabling the controlled release of their therapeutic payloads. The results showed exogenous AQ4N could serve as PA contrast agent to guide precise HIFU treatment and contribute to more ablation of tumor tissue, thus creating severely hypoxic environment conducive to subsequent therapy. After surgery, AQ4N effectively induced the ICD in tumor cells. Meanwhile, SR-717 and CaCO3 worked synergistically to overcome immune resistance. As STING agonist, SR-717 amplified STING pathway activation, leading to the upregulation of cytokines and IFN-β. This, in turn, promoted DC maturation and CD8+ T-cell infiltration and ultimately established robust and long-term antitumor immunity. Consequently, high-efficacy suppression of both primary and metastatic tumor growth was achieved in breast tumor-bearing mouse models. Overall, this study provides a successful paradigm for advancing immunotherapy following various tumor surgical procedures, including HIFU.