Ultrasound-activated Cu2−XSe/TiO2 heterostructured nanosonosensitizer enables coordinated induction of ferroptosis and cuproptosis for tumor immunotherapy

Shibing Wang; Tingting Deng; Xue Yang; Yuan Chen; Yuli Zhou; Hairui Tian; Jiarui Li; Meili Li; Teng Lin; Cong Huang; Xiangmin Tong

doi:10.1186/s12951-026-05115-5

. 2026 Sep 27;24:915. doi: 10.1186/s12951-026-05115-5

Ultrasound-activated Cu_2−XSe/TiO₂ heterostructured nanosonosensitizer enables coordinated induction of ferroptosis and cuproptosis for tumor immunotherapy

Shibing Wang ^1,^2,^✉, Tingting Deng ³, Xue Yang ^2,⁴, Yuan Chen ⁵, Yuli Zhou ¹, Hairui Tian ³, Jiarui Li ³, Meili Li ³, Teng Lin ^3,^✉, Cong Huang ^3,^✉, Xiangmin Tong ^1,^2,^✉

PMCID: PMC13625417 PMID: 42816848

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subtype characterized by the absence of actionable molecular targets, high metastatic propensity, and limited responsiveness to current immunotherapies. Although immune checkpoint inhibitors show promise, their clinical efficacy is constrained by low tumor immunogenicity and a highly immunosuppressive tumor microenvironment (TME). This study reports a hyaluronic acid (HA)-functionalized TiO₂@Cu₂–_XSe@HA nanosystem that promotes the coordinated induction of ferroptosis and cuproptosis, thereby enhancing sonodynamic immunotherapy against TNBC. It features a rationally designed Cu₂–_XSe/TiO₂ heterostructured nanosonosensitizer and an integrated therapeutic strategy combining ultrasound-activated sonodynamic effects with the coordinated induction of ferroptosis and cuproptosis. Under ultrasound irradiation, the Cu₂−_XSe/TiO₂ heterointerface promotes efficient electron transfer and reactive oxygen species (ROS) generation, markedly amplifying sonodynamic efficacy. Meanwhile, released Cu²⁺ depletes intracellular glutathione, disrupts redox homeostasis, thereby promoting copper-dependent proteotoxic stress. In parallel, Cu²⁺ induces autophagic degradation of glutathione peroxidase 4, further enhancing ferroptotic signaling. This coordinated induction effectively suppresses tumor growth, remodels the immunosuppressive TME, and potentiates antitumor immunity, as further corroborated by single-cell RNA sequencing. Collectively, this study provides a rational strategy for integrating sonodynamic therapy with dual non-apoptotic cell death pathways, offering a promising strategy to sensitize TNBC to immunotherapy.

Graphical abstract

graphic file with name 12951_2026_5115_Figa_HTML.webp

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12951-026-05115-5.

Keywords: Triple-negative breast cancer, Nanosonosensitizers, Ferroptosis, Cuproptosis, Immunotherapy, cGAS–STING signaling

Introduction

Triple-negative breast cancer (TNBC), an aggressive subtype accounting for approximately 15–20% of all breast cancer cases, is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression [1–3]. Clinically, TNBC is associated with a higher incidence in younger women, rapid disease progression, and a strong propensity for distant metastasis, resulting in poorer prognosis and higher mortality compared to other breast cancer subtypes [4]. Current treatment strategies primarily rely on conventional modalities, including surgery, chemotherapy, and radiotherapy [5]. However, the lack of actionable molecular targets renders targeted therapies ineffective, while high genetic heterogeneity and intrinsic chemoresistance frequently lead to therapeutic failure. Moreover, postoperative recurrence and metastasis remain major clinical challenges [6]. Although immunotherapy, particularly immune checkpoint inhibitors (ICIs), has emerged as a promising approach, its clinical efficacy in TNBC remains limited due to low tumor immunogenicity and a profoundly immunosuppressive tumor microenvironment (TME) [7–11]. Therefore, the development of effective therapeutic strategies to enhance immunotherapy responsiveness in TNBC is urgently needed.

Reprogramming the immunosuppressive TME to improve immunotherapeutic efficacy has recently attracted considerable attention [12, 13]. In this context, emerging forms of programmed cell death (PCD), including pyroptosis [14], ferroptosis [15], and cuproptosis [16], have demonstrated the potential to activate tumor-specific immunity by modulating immune responses. Among these, ferroptosis and cuproptosis represent distinct non-apoptotic cell death pathways with particular advantages in overcoming drug resistance and promoting antitumor immunity [17]. Cuproptosis is triggered by intracellular copper accumulation and primarily targets mitochondrial metabolism, leading to oxidative damage and disruption of key enzymes in the tricarboxylic acid (TCA) cycle [18]. In contrast, ferroptosis is an iron-dependent process driven by reactive oxygen species (ROS)-mediated lipid peroxidation [19]. Despite their distinct initiation mechanisms, these pathways exhibit crosstalk through shared mitochondrial metabolic networks [20]. Notably, their coordinated activation can induce synergistic tumor cell death with superior therapeutic efficacy compared to single-pathway targeting [21]. However, insufficient intracellular copper, iron, or ROS levels, together with the immunosuppressive TME and intrinsic cellular defense mechanisms, severely limit their activation efficiency. Thus, developing strategies to efficiently induce ferroptosis and cuproptosis in a coordinated manner remains a critical challenge.

Sonodynamic therapy (SDT) utilizes ultrasound (US) to activate sonosensitizers for ROS generation and tumor growth inhibition. Compared with photodynamic therapy (PDT), SDT offers superior tissue penetration and enables effective treatment of deep-seated tumors, highlighting its translational potential [22]. Increasing evidence indicates that ROS generated by nanosonosensitizers during SDT can induce lipid peroxidation and trigger ferroptosis, while excessive ROS-mediated mitochondrial damage sensitizes tumor cells to cuproptosis [23, 24]. As the core component of SDT, sonosensitizers are broadly classified into organic and inorganic types [25]. Organic sonosensitizers (e.g., hematoporphyrin and 5-aminolevulinic acid) have shown clinical potential but are limited by poor stability, rapid metabolism, phototoxicity, and insufficient tumor specificity [26]. In contrast, inorganic titanium dioxide (TiO₂) nanoparticles (NPs) exhibit high stability, low phototoxicity, and favorable tumor accumulation via the enhanced permeability and retention (EPR) effect, making them attractive candidates for SDT [25, 27]. However, the wide band gap (≈ 3.2 eV) and low electron–hole separation efficiency of TiO₂ result in limited ROS generation. To overcome these limitations, various modification strategies have been explored, including defect engineering, heteroatom doping, metal incorporation, and heterostructure construction. Among these, heterostructure engineering is particularly effective in suppressing electron–hole recombination and enhancing charge separation efficiency. Copper selenide (Cu_2−XSe) has attracted increasing attention due to its unique electronic structure, excellent physicochemical properties, and ability to form heterojunctions with TiO₂, thereby modulating band alignment and enhancing charge transfer [28]. Importantly, Cu_2−XSe also serves as a functional copper reservoir within the TME. Released Cu²⁺ ions exert dual biological effects: they deplete intracellular glutathione (GSH), disrupt redox homeostasis and generate toxic Cu⁺ species to induce cuproptosis; concurrently, Cu²⁺ can promote the autophagic degradation of glutathione peroxidase 4 (GPX4), thereby amplifying ferroptotic signaling [29].

Therefore, this study develops a hyaluronic acid (HA)-functionalized Cu_2−XSe-integrated TiO₂ nanosystem for TNBC therapy through the coordinated induction of ferroptosis and cuproptosis. The platform incorporates advances in both material design and therapeutic strategy: the heterostructured TiO₂@Cu_2−XSe@HA nanosonosensitizer is rationally engineered to enhance charge separation, ROS generation, and tumor-targeting capacity while integrating ultrasound-driven sonodynamic effects with copper-mediated mechanisms that promote ferroptosis and cuproptosis, thereby remodeling the immunosuppressive TME. As shown in Scheme 1, this TiO₂@Cu_2−XSe@HA heterostructured nanoplatform enables the following functions in immunologically “cold” tumors such as TNBC: (i) the engineered Cu₂−_XSe/TiO₂ heterointerface promotes interfacial electron transfer and markedly enhances ROS generation under US irradiation, thereby amplifying the efficacy of SDT; (ii) excessive ROS accumulation induces lipid peroxidation and ferroptosis, while concomitant mitochondrial dysfunction sensitizes tumor cells to cuproptosis; (iii) in parallel, HA functionalization enables targeted delivery to TNBC cells and facilitates tumor microenvironment-responsive Cu²⁺ release, further disrupting redox homeostasis and promoting Cu-dependent proteotoxic stress; and (iv) Cu²⁺-mediated autophagic degradation of GPX4 further reinforces ferroptotic cell death. Through the coordinated activation of ferroptosis and cuproptosis, this nanosystem achieves potent tumor growth inhibition and remodels the immunosuppressive TME, thereby enhancing antitumor immune responses, as further supported by single-cell RNA sequencing (scRNA-seq). Thus, we established a mechanistically informed nanoplatform that integrates SDT with dual non-apoptotic cell death pathways, offering a viable strategy to potentiate immunotherapy in TNBC. This coordinated cell-death induction strategy may inform the design of other nanosonosensitizers to improve immunotherapy for solid tumors.

Scheme 1 — Schematic illustration of the design and therapeutic mechanism of TiO₂@Cu_2-XSe@HA NPs for ferroptosis-cuproptosis synergized tumor immunotherapy. (a) Synthetic route of TiO₂@Cu_2-XSe@HA NPs. (b) Upon ultrasound activation, the Cu_2-XSe/TiO₂ heterostructured nanosonosensitizer induces ferroptosis and cuproptosis in a coordinated manner, thereby enhancing antitumor immune responses

Results and discussion

Design and characterization of TCH NPs

Spindle-shaped anatase TiO₂ NPs were synthesized from tetrabutyl titanate (TBT) via a solvothermal process in acetic acid. Transmission electron microscopy (TEM) images revealed that the TiO₂ NPs were uniformly dispersed and exhibited well-defined morphology with an average particle size of ≈ 133 nm (Figs. 1a, b and S1). Subsequently, the obtained TiO₂ were ultrasonically dispersed in an aqueous CuCl₂ solution, followed by the addition of NaHSe to in situ generate Cu₂–_XSe nanodomains on the TiO₂ surface, yielding TiO₂@Cu₂–_XSe NPs (TC NPs). HA was further introduced for surface modification to obtain TiO₂@Cu₂–_XSe@HA NPs (TCH NPs). As shown in Figs. 1c and S2, Cu_2−XSe nanodomains were densely and uniformly anchored on the TiO₂ surface, forming a well-defined heterostructure. Elemental mapping analysis further confirmed the structural composition of TCH NPs. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images, corresponding energy-dispersive X-ray spectroscopy (EDS) maps, and line-scan profiles revealed homogeneous distributions of Ti and O throughout the nanoparticles, while Cu and Se were predominantly localized in the outer domains. High-resolution TEM (HRTEM) images displayed distinct lattice fringes with interplanar spacings of 0.35 and 0.20 nm, corresponding to the (101) and (220) plane of anatase TiO₂ and the Cu₂−_XSe phase, respectively (Fig. S3), further confirming the formation of the TiO₂/Cu₂−_XSe heterojunction.

The X-ray photoelectron spectroscopy (XPS) survey spectrum of TCH NPs exhibited characteristic peaks corresponding to Cu 2p, O 1s, Se 3d, C 1s, and Ti 2p (Fig. 1d) [30]. High-resolution XPS spectra (Fig. 1e–g) further confirmed the chemical states of the constituent elements, with Cu present as both Cu²⁺ (933.44 eV) and Cu⁺ (931.69 eV), indicating mixed valence states favorable for redox cycling [31]. X-ray diffraction (XRD) patterns (Fig. 1h) matched well with standard diffraction data for TiO₂ and TiO₂@Cu_2−XSe, with no detectable impurity peaks, confirming the high crystallinity and phase purity of the synthesized NPs. As demonstrated by XPS results, compared with pristine TiO₂ and pure Cu_2−XSe, the characteristic binding energies of Ti in TiO₂@Cu_2−XSe shift to higher values, while the binding energies of Cu move toward lower positions (Fig. S4). This binding energy shift originates from chemical interaction and spontaneous electron transfer across the p-n heterojunction interface constructed between TiO₂ and Cu_2−XS, with previously reported findings [32]. Mott–Schottky plots showed positive slopes for TiO₂@Cu_2−XSe NPs, demonstrating that the TiO₂@Cu_2−XSe NPs display apparent n-type-dominant semiconducting behavior. Changes in the slope and flat-band potential after Cu_2−XSe modification indicated interfacial electronic interactions within the heterojunction (Fig. S5). To optimize Cu_2−XSe loading, TiO₂@Cu_2−XSe NPs were synthesized at Ti/Cu feeding molar ratios of 10:1, 10:2, and 10:3. ICP-OES analysis confirmed that Cu loading increased with the Cu precursor content (Table S1). Under identical conditions, the 10:2 formulation exhibited the highest ROS generation. Further increasing the Cu content to a 10:3 ratio moderately reduced sonodynamic activity, potentially because excessive Cu_2−XSe formed free PVP-stabilized NPs, promoted aggregation, overcovered the TiO₂ surface, or introduced additional charge-recombination sites. Accordingly, a Ti/Cu feeding ratio of 10:2 was selected for subsequent experiments.

UV–vis absorption spectra (Fig. 1i) exhibited a pronounced absorption peak around 808 nm, indicating successful integration of Cu₂−_XSe and enhanced optical absorption in the near-infrared region. As illustrated in Fig. 1j, Cu₂–_XSe incorporation induced a downward shift in the conduction band of TiO₂, which facilitated interfacial electron transfer and suppressed electron–hole recombination within the heterostructure. Dynamic light scattering (DLS) analysis (Fig. 1k–m) demonstrated that TiO₂, TC, and TCH NPs exhibit hydrodynamic diameters of 185.90 nm, 254.4 nm, and 297.6 nm, respectively, after incubation in phosphate-buffered saline (PBS) for 5 days, indicating good colloidal stability. The corresponding polydispersity index (PDI) values remained low (0.15 ± 0.02, 0.18 ± 0.03 and 0.17 ± 0.02), further confirming uniform size distribution. Zeta potential measurements (Fig. 1n) revealed a negative surface charge for TCH NPs, which is beneficial for colloidal stability and prolonged circulation. As schematically illustrated in Fig. 1o, the observed interfacial electronic interactions and proposed band alignment “may facilitate” interfacial charge transfer and may contribute to the enhanced ROS generation observed under US irradiation. Collectively, these results confirmed the successful construction, favorable dispersibility, and physicochemical stability of TCH NPs, providing a robust foundation for subsequent sonodynamic and immunotherapeutic applications.

CDT/SDT therapeutic effects triggered by TiO₂@Cu_2−XSe NPs

After confirming the successful construction and favorable physicochemical properties of TCH NPs, US-triggered sonodynamic and chemodynamic activities were systematically evaluated. As a classical inorganic sonosensitizer, TiO₂ is capable of generating ROS under US irradiation [27]; however, its therapeutic efficiency is often limited by rapid electron–hole recombination and insufficient charge utilization. By coupling Cu₂–_XSe with TiO₂, the interfacial electronic structure of the composite was expected to be effectively regulated, thereby facilitating charge carrier separation and enhancing sonodynamic processes. In addition, the Cu-containing component was anticipated to provide catalytic activity for Fenton-like reactions, endowing the nanosystem with chemodynamic functionality under US stimulation. This design rationale is consistent with previous reports demonstrating that heterostructure engineering of TiO₂ improves SDT efficiency via band structure modulation, while copper-based components contribute to catalytic oxidative activity.

The sonodynamic performance of TCH NPs was first investigated using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen probe (¹O₂) [11]. As shown in Fig. 2a, under US irradiation, the characteristic absorbance of DPBF at 418 nm progressively decreased with increasing TCH NP concentration, indicating a concentration-dependent enhancement in ¹O₂ generation. Similarly, prolonged irradiation resulted in a continuous decline in DPBF absorbance (Fig. 2b), demonstrating a clear time-dependent sonodynamic effect. Quantitative analysis further showed that the PBS-only group induced minimal DPBF consumption, whereas the TiO₂ + US group exhibited a more pronounced decrease, confirming the intrinsic sonodynamic activity of TiO₂ (Fig. 2c). Notably, the TCH NPs + US group showed the most substantial depletion of DPBF, indicating that Cu₂–_XSe incorporation markedly enhanced US-triggered ¹O₂ generation. We also directly compared DPBF depletion in the US, TiO₂ + US, and TiO₂@Cu_2−XSe + US groups under otherwise identical conditions (Fig. S6). The TiO₂@Cu_2−XSe + US group exhibited the greatest DPBF depletion, indicating enhanced ¹O₂ generation. This enhancement may be attributed to the formation of the heterostructure, which facilitates interfacial charge separation, suppresses charge-carrier recombination, and thereby improves the utilization of ultrasound-induced charge carriers for ROS generation.

EPR spectroscopy was performed to directly detect radical generation. As shown in Fig. 2d, negligible signals were observed in the PBS group. In contrast, the TiO₂ + US group exhibited discernible signals, further confirming the sonosensitizing role of TiO₂. Importantly, the TCH NPs + US group displayed the strongest signal intensity among all groups, demonstrating superior ROS generation capability upon US activation. These findings, together with the DPBF results, indicate that Cu_2-XSe/TiO₂ coupling effectively enhances SDT performance, most likely by promoting charge separation and suppressing electron–hole recombination at the heterointerface. Following validation of enhanced sonodynamic activity, the chemodynamic potential of the nanosystem was evaluated. Given that copper-based nanomaterials act as efficient Fenton-like catalysts and that CDT efficacy depends on the accessibility and redox activity of catalytic metal centers, Cu release behavior was first examined. More importantly, we performed additional EPR measurements to specifically examine hydroxyl radical (·OH) generation. The experimental groups included PBS, TiO₂ + US, and TiO₂@Cu_2-XSe + US, and the results were analyzed together with the original time-dependent EPR data (Fig. S7). Only weak ·OH signals were detected in the PBS and TiO₂ + US groups, whereas TiO₂@Cu_2-XSe + US produced a markedly stronger ·OH signal. As shown in Fig. 2e, TiO₂@Cu_2-XSe + US consistently exhibited higher Cu release than TiO₂@Cu_2-XSe alone. This observation suggests that US not only activates the TiO₂ sonosensitizer but also enhances access to catalytically active copper sites, thereby promoting Cu-mediated Fenton-like reactions.

Methylene blue (MB) degradation was subsequently used to evaluate catalytic oxidation capacity [33]. As shown in Fig. 2f, the characteristic absorption of MB gradually decreased over time, indicating continuous generation of oxidative species. Increasing TCH NP concentration further accelerated MB degradation (Fig. 2g), demonstrating concentration-dependent catalytic activity. These results are consistent with Cu release data and confirm effective Cu-associated catalytic activity in the nanosystem. Given that CDT relies on metal-mediated conversion of endogenous peroxides into highly cytotoxic radicals, the observed time- and concentration-dependent MB degradation supports the occurrence of Cu-driven oxidative catalysis. Further comparisons among treatment groups provided additional evidence for CDT enhancement. As shown in Fig. 2h, i, the TiO₂ + US group exhibited slight MB degradation ability, which was ascribed to the fact that TiO₂ could generate a small amount of •OH under US irradiation. In contrast, the TiO₂@Cu_2-XSe group showed obvious MB degradation, confirming the intrinsic chemodynamic therapy activity of the Cu₂₋ₓSe component. Notably, the TiO₂@Cu_2-XSe + US group exhibited the most significant MB depletion, which could be attributed to the combined effect of sonodynamic therapy and chemodynamic therapy, as well as the US-stimulated release of more copper ions.

The photoacoustic (PA) signal of TiO₂@Cu_2−XSe increased in a concentration-dependent manner and remained stable across different media and observation periods (Figs. 2j–l), supporting its suitability for in vivo PA imaging. Following administration, detectable PA signals persisted at the tumor site for up to 24 h, with TCH NPs exhibiting higher tumor-associated PA intensity than TiO₂@Cu_2−XSe (Fig. 2m). This enhancement indicates increased tumor accumulation, which may arise from the combined contributions of the EPR effect and HA–CD44-mediated tumor affinity. Collectively, these findings support the use of PA imaging to monitor tumor accumulation and provide a basis for the subsequent ultrasound-triggered therapeutic effects of TCH NPs.

Active targeting, internalization, and safety evaluation of TCH NPs in vitro and in vivo

CD44 is highly expressed on the surface of many malignant cells, including 4T1 breast cancer cells, whereas its expression is substantially lower in non-malignant cells such as human umbilical vein endothelial cells (HUVECs) [9]. Owing to this differential expression, HA-modified NPs were expected to exhibit selective affinity toward CD44-rich tumor cells. In this study, the relative expression levels of CD44 in 4T1 cells and HUVECs were verified by western blotting (Fig. 3a).

Fig. 3 — Active targeting, internalization, and in vivo tumor accumulation of TCH NPs. (a) Western blot analysis of CD44 expression in 4T1 cells and HUVECs. (**b, c**) Time-dependent internalization of RhB-labeled TCH NPs by 4T1 cells from 0.5 to 48 h measured by flow cytometry, with corresponding quantitative analysis (n = 3). (**d–f**) Cellular uptake of RhB-labeled TCH NPs visualized by CLSM and flow cytometry, including competitive inhibition with free HA, with corresponding quantitative fluorescence analysis (n = 3). Scale bar, 20 μm. (**g, h**) In vivo fluorescence imaging of 4T1 tumor-bearing mice at 0.5, 6, 12, 24, and 48 h after intravenous administration of ICG-labeled TiO₂@Cu_2−XSe (TC) or TiO₂@Cu_2−XSe@HA (TCH) NPs, with quantitative analysis of tumor-associated fluorescence intensity. * P < 0.05, ** P < 0.01, *** P < 0.001. NPs, nanoparticles; HA, hyaluronic acid; RhB, rhodamine B; bio-TEM, biological transmission electron microscopy; HUVECs, human umbilical vein endothelial cells; PBS, phosphate-buffered saline

To directly examine the intracellular localization of TCH NPs, 4T1 cells were incubated with the NPs, collected, and subsequently observed by biological transmission electron microscopy (Bio-TEM). As shown in Fig. S8, TCH NPs were clearly detected within the cytoplasm of 4T1 cells. TCH NPs were also labeled with rhodamine B (RhB), and intracellular uptake was quantified based on fluorescence intensity [34, 35]. Flow cytometry analysis showed a clear time-dependent increase in intracellular fluorescence from 0.5 to 48 h, indicating progressive accumulation of TCH NPs within 4T1 cells (Fig. 3b, c). Confocal laser scanning microscopy (CLSM) revealed a consistent uptake pattern, further supporting efficient and time-dependent internalization of TCH NPs by 4T1 cells (Fig. 3d). To investigate the role of HA-mediated targeting, competitive inhibition experiments were performed. As shown in Figs. 3e and f and 4T1 cells pretreated with excess free HA exhibited significantly reduced fluorescence after incubation with RhB-labeled TCH NPs (RhB-TCH), compared with cells without HA pretreatment. In contrast, minimal internalization of RhB-TCH NPs was observed in HUVECs, likely due to their low CD44 expression. These results indicate that TCH NPs were preferentially internalized by 4T1 cells via CD44-mediated recognition. Tumor-targeting capability was further evaluated in vivo using ICG-labeled NPs for near-infrared fluorescence imaging. Following intravenous administration, HA-mediated TCH NPs exhibited a markedly enhanced tumor-associated fluorescence compared with non-HA-coated TiO₂@Cu_2−XSe NPs (TC NPs), demonstrating improved tumor accumulation (Fig. 3g, h).Quantitative analysis showed that the fluorescence intensity of ICG-TCH NPs in tumors was 1.29-fold higher than that of ICG-TC NPs (Fig. 3h) at 12 h, further confirming the enhanced tumor accumulation conferred by HA functionalization [36].

The systemic biosafety of TCH NPs was subsequently evaluated through blood biochemistry, hematological analysis, and organ histopathological examination. As shown in Fig. S9a-f, no significant changes were observed in leukocyte or erythrocyte counts, renal function markers (blood urea nitrogen and creatinine), or hepatic enzymes (aspartate aminotransferase and alanine aminotransferase) following treatment. These results indicate the absence of detectable liver or kidney toxicity. Consistently, hematoxylin and eosin (H&E) staining revealed no obvious histopathological abnormalities in major organs, including the heart, liver, spleen, lung, and kidney (Fig. S9g). Collectively, these findings demonstrate the favorable biocompatibility and biosafety profile of TCH NPs, supporting their potential for in vivo antitumor applications.

In vitro anticancer efficacy and ROS-induced mitochondrial dysfunction under combined CDT and SDT

The anticancer activity of TCH NPs was evaluated in vitro. As shown in Fig. 4a, the CCK-8 assay demonstrated that TCH + US induced pronounced concentration-dependent cytotoxicity in 4T1 cells, consistent with the combined effects of US-triggered SDT and Cu-mediated CDT. The Cell Counting Kit-8 (CCK-8) assay yielded consistent results (Fig. 4b). In contrast, HUVECs exhibited higher tolerance toward TCH NPs, with cell viability remaining above 80% even at 200 µg/mL after 24 h of incubation (Fig. 4c). These results indicate favorable selectivity toward tumor cells, likely associated with CD44 overexpression.

To further evaluate cytotoxicity, live/dead staining was performed following different treatments. Inverted fluorescence microscopy revealed modest cell death in the TCH and TH + US groups, whereas 4T1 cells treated with TCH + US exhibited the highest level of cell death (Fig. 4d, e). Apoptosis analysis was subsequently conducted using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide staining. Flow cytometry showed that the proportion of Annexin V-positive 4T1 cells in the TCH + US group reached 63.54%, which was markedly higher than that in the TH + US group (23.76%) and the TCH-alone group (17.57%), corresponding to 2.67-fold and 3.62-fold increases, respectively (Fig. S10). These findings demonstrate the pronounced cytotoxicity associated with combined CDT and SDT. Together with the cellular uptake data, they further suggest that enhanced internalization of TCH NPs contributes to the observed cytotoxic effect [3, 11]. As shown in Fig. 4f-h, TCH + US treatment induced significant and concentration-dependent increases in ROS levels, whereas negligible changes were observed in control group. Flow cytometry and confocal microscopy analyses yielded consistent results. Collectively, these findings indicate that TCH + US effectively promotes intracellular ROS accumulation, which is a key mediator of their anticancer activity.

Excessive ROS generation is known to disrupt mitochondrial redox homeostasis, ultimately leading to mitochondrial dysfunction [37]. Once ROS accumulation surpasses the cellular defense capacity, mitochondrial injury is further amplified, and antioxidant systems become progressively exhausted, forming a self-reinforcing damaging cycle [38, 39]. To evaluate mitochondrial damage, mitochondrial membrane potential (MMP) was assessed using the JC-1 probe. As shown in Fig. 4i, j, the TCH + US group exhibited a more pronounced decrease in MMP compared with control group, indicating severe mitochondrial depolarization. Among all groups, TCH + US induced the most substantial loss of membrane potential. Consistently, mitochondrial permeability transition pore (mPTP) opening increased in parallel with MMP decline (Fig. 4k–m), further confirming mitochondrial dysfunction. Mitochondrial superoxide production was evaluated using the MitoSOX probe. A strong red fluorescence signal was observed in the TCH + US group (Fig. S11), indicating excessive mitochondrial ROS generation. These results suggest that TCH combined with US markedly aggravated mitochondrial damage, likely through disruption of the electron transport chain. Indeed, analysis of mitochondrial respiratory chain complexes showed that TCH + US treatment significantly suppressed the activities of complexes I, III, IV, and V (Fig. S12). Taken together, these findings demonstrate that TCH NPs combined with US irradiation exert potent anticancer effects by promoting excessive ROS accumulation and severe mitochondrial dysfunction through the combined effects of CDT and SDT.

TCH-induced ferroptosis through copper-dependent autophagic degradation of GPX4

Ferroptosis is an iron-dependent, caspase-independent form of regulated cell death characterized by lipid peroxidation [9, 40, 41]. Increasing evidence has shown that the combination of CDT and SDT can promote lipid peroxide (LPO) accumulation and trigger ferroptosis [42, 43]. Consistent with ferroptotic features, TEM revealed pronounced mitochondrial ultrastructural abnormalities in the TCH + US group, including reduced cristae density and shortened mitochondrial morphology, indicative of impaired oxidative phosphorylation (Fig. 5a). Recent studies have demonstrated that Cu²⁺ plays a pro-ferroptotic role by binding to cysteine residues on GPX4, thereby promoting Tax1-binding protein 1 (TAX1BP1)-dependent autophagic degradation of this key antioxidant enzyme. This mechanism suggests that exogenous Cu²⁺ may amplify ferroptotic cell death (Fig. 5b). To further investigate this process, 4T1 cells were transfected with a green fluorescence protein (GFP)-tagged GPX4 construct. Confocal imaging revealed weakened GPX4 fluorescence and elevated expression of the autophagosome marker MAP1LC3A/B-II in the TCH + US group (Fig. 5c), which was further confirmed by high-resolution confocal microscopy (Fig. 5d). These observations indicate that GPX4 was recruited as a substrate for autophagic degradation. To further determine whether GPX4 degradation was mediated by autophagy, we performed additional autophagy inhibition experiments using chloroquine (CQ), a widely used pharmacological inhibitor of autophagic flux. As shown in Fig. S13a, 4T1 cells were transfected with a GFP-tagged GPX4 construct to directly monitor the intracellular distribution and abundance of GPX4. Confocal imaging showed that GPX4 fluorescence was markedly weakened in the TCH + US group, accompanied by increased expression of the autophagosome marker MAP1LC3A/B-II, suggesting that TCH + US treatment promoted autophagosome formation and GPX4 degradation. In contrast, in the TCH + US + CQ group, the autophagic signal was reduced, and GPX4 fluorescence was partially restored compared with the TCH + US group. These results indicate that inhibition of autophagy prevented GPX4 degradation, supporting the conclusion that GPX4 was recruited as a substrate for autophagic degradation under TCH + US treatment. To further determine whether the inhibition of GPX4 degradation affected ferroptosis-related lipid peroxidation, we performed lipid ROS staining. As shown in Fig. S13b, control cells exhibited only weak green fluorescence, whereas cells in the TCH + US group displayed markedly enhanced fluorescence intensity, indicating elevated lipid peroxidation. However, the TCH + US + CQ group exhibited restored green fluorescence intensity compared with the TCH + US group, suggesting that autophagy inhibition reduced lipid peroxidation. Consistently, western blot analysis further demonstrated that TCH + US treatment downregulated the ferroptosis-suppressing proteins GPX4 and FSP1, while upregulating ACSL4 (Fig. S13c–d). By contrast, CQ treatment restored the expression of GPX4 and FSP1 and decreased ACSL4 expression, indicating that ferroptosis was attenuated when autophagy was inhibited. Taken together, these results provide direct pharmacological evidence that TCH + US-induced GPX4 degradation occurs through a autophagic process and that this autophagy-mediated GPX4 degradation contributes to ferroptosis activation.

Fig. 5 — TCH-induced ferroptosis via copper-dependent autophagic degradation of GPX4. (a) Bio-TEM images showing mitochondrial ultrastructural changes in 4T1 cells after treatment. Mitochondria are indicated by red circles. (b) Schematic illustration of copper-dependent autophagic degradation of GPX4 leading to ferroptosis. (c) Confocal images showing colocalization of GFP-tagged GPX4 with the autophagosome marker MAP1LC3A/B in 4T1 cell treatment. (d) High-resolution fluorescence images confirming GPX4–MAP1LC3A/B colocalization. (e) Lipid peroxidation in 4T1 cells detected using the BODIPY 581/591-C11 probe. Scale bar, 20 μm. (**f–i**) Flow cytometry analysis and quantitative evaluation of lipid peroxidation. (j) Intracellular MDA levels in 4T1 cells. (k) Intracellular GPX4 enzymatic activity. (**l, m**) Western blot analysis of GPX4, FSP1, and ACSL4 expression, with corresponding quantitative analysis. * P < 0.05, ** P < 0.01, *** P < 0.001. CDT, chemodynamic therapy; SDT, sonodynamic therapy; US, ultrasound; ROS, reactive oxygen species; GPX4, glutathione peroxidase 4; FSP1, ferroptosis suppressor protein 1; ACSL4, acyl-CoA synthetase long-chain family member 4; TEM, transmission electron microscopy; MDA, malondialdehyde; NPs, nanoparticles

Given that TCH induced ROS accumulation and mitochondrial dysfunction while removing key inhibitory factors of ferroptosis, lipid peroxidation was further evaluated using the BODIPY 581/591-C11 probe under hypoxic conditions (1% O₂). As shown in Fig. 5e, control cells exhibited weak green fluorescence, whereas TCH + US group displayed markedly enhanced fluorescence intensity, indicating elevated lipid peroxidation. Flow cytometry analysis yielded consistent results (Fig. 5f–i). Subsequently, intracellular malondialdehyde (MDA) levels were quantified. TCH + US treatment significantly increased MDA production, further supporting enhanced lipid peroxidation (Fig. 5j). In parallel, GPX4 enzymatic activity was markedly reduced (Fig. 5k). Western blot analysis further demonstrated downregulation of ferroptosis-suppressing proteins GPX4 and FSP1, along with upregulation of ACSL4 (Fig. 5l, m). Collectively, these results demonstrate that TCH NPs induce ferroptosis through ROS overproduction and copper-dependent autophagic degradation of GPX4, with ferroptotic signaling further enhanced by US irradiation through the combined effects of CDT and SDT.

TCH-induced cuproptosis and cGAS–STING pathway activation under combined SDT/CDT treatment

Depletion of intracellular GSH enhances the susceptibility of tumor cells to ROS-mediated damage and may also facilitate cuproptosis [44]. To quantify intracellular GSH levels, a commercial glutathione assay kit based on the reaction between GSH and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was employed. This reaction generates 2-nitro-5-thiobenzoate (TNB), which exhibits characteristic absorbance at 412 nm, allowing quantitative determination of GSH levels (Fig. S14). As shown in Fig. 6a, treatment with TCH NPs significantly reduced intracellular GSH levels, and this depletion was further enhanced under US irradiation. This reduction was attributed to the redox activity of Cu²⁺ released from TCH NPs.

Fig. 6 — TCH-Induced Cuproptosis and cGAS–STING Pathway Activation under Combined SDT/CDT Treatment. (a) Intracellular GSH levels in 4T1 cells after indicated treatments (n = 3). (b) Confocal fluorescence images of intracellular Cu levels detected using a Cu-specific probe. Scale bar, 20 μm. (c) Confocal images showing DLAT aggregation in 4T1 cells following different treatments. Scale bar, 20 μm. (d) Western blot analysis of FDX1, LIAS, and ATP7B expression in 4T1 cells after treatment. (e) Quantitative colocalization analysis corresponding to panel (c). (f) High-resolution fluorescence images showing oxidative damage of mtDNA detected by 8-OHdG staining. Scale bar, 10 μm. (g) Confocal images showing colocalization of TOMM20 (mitochondria) and TFAM (mtDNA) in 4T1 cells after treatment. Scale bar, 10 μm. (h) Western blot analysis of cGAS, STING, and phosphorylated IRF3 (p-IRF3), with corresponding quantitative analysis. (i) Schematic illustration of cGAS–STING pathway activation. * P < 0.05, ** P < 0.01, *** P < 0.001. CDT, chemodynamic therapy; SDT, sonodynamic therapy; US, ultrasound; GSH, glutathione; ROS, reactive oxygen species; DLAT, dihydrolipoamide S-acetyltransferase; FDX1, ferredoxin 1; LIAS, lipoic acid synthase; ATP7B, ATPase copper transporting beta; mtDNA, mitochondrial DNA; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; cGAS, cyclic GMP–AMP synthase; STING, stimulator of interferon genes; IRF3, interferon regulatory factor 3

Recent studies have identified cuproptosis as a copper-dependent form of regulated cell death with emerging relevance in cancer therapy [45]. In this process, intracellular Cu²⁺ is reduced to Cu⁺ by ferredoxin 1 (FDX1) within mitochondria. The resulting Cu⁺ interacts with lipoylated dihydrolipoamide S-acetyltransferase (DLAT), inducing abnormal protein oligomerization and destabilization of iron–sulfur cluster proteins, thereby triggering proteotoxic stress and cell death. Consistent with this mechanism, intracellular copper levels increased following TCH treatment, and this effect was further enhanced in the TCH + US group, indicating that US activation promoted Cu accumulation in tumor cells (Figs. 6b and S15). To evaluate activation of the cuproptosis pathway, DLAT aggregation was assessed by immunofluorescence staining. As shown in Fig. 6c, e, pronounced punctate DLAT signals were observed, particularly in the TCH + US group, indicating enhanced protein aggregation. Western blot analysis further revealed downregulation of FDX1 and lipoic acid synthase (LIAS), both of which are closely associated with mitochondrial copper metabolism and iron–sulfur cluster homeostasis (Fig. 6d). In addition, ATP7B, a copper-exporting ATPase responsible for maintaining intracellular copper balance, was significantly downregulated following TCH + US treatment. Collectively, these findings indicate that TCH combined with US disrupted intracellular copper homeostasis and activated cuproptosis. To further investigate the relationship between ROS generation and the observed cuproptosis-associated alterations, we performed additional experiments using N-acetylcysteine (NAC), a widely used ROS scavenger. Intracellular copper levels were increased following TCH treatment and were further elevated after TCH + US treatment, indicating that US activation enhanced intracellular copper accumulation (Fig. S16a). In parallel, immunofluorescence staining revealed pronounced punctate DLAT signals in the TCH + US group, consistent with increased aggregation of the lipoylated mitochondrial protein DLAT (Fig. S16b). Importantly, NAC treatment reduced the punctate DLAT signals induced by TCH + US, indicating that excessive ROS generation contributed to DLAT aggregation and mitochondrial proteotoxic stress. However, DLAT aggregation remained higher than that in the PBS group after NAC treatment. These findings suggest that ROS plays an important role in promoting the cuproptosis-associated phenotype but does not fully account for the observed DLAT aggregation. ROS-independent factors, particularly intracellular copper accumulation and disruption of mitochondrial copper homeostasis, may also contribute to this process. Western blot analysis further showed that TCH + US treatment decreased the expression of ATP7B, FDX1, and lipoic acid synthase (LIAS) (Fig. S17). The downregulation of ATP7B, a major copper-exporting ATPase, may impair copper efflux and thereby contribute to intracellular copper accumulation. Meanwhile, the reduced expression of FDX1 and LIAS indicated disruption of mitochondrial protein lipoylation and copper-dependent mitochondrial metabolism. Following NAC treatment, the expression levels of ATP7B, FDX1, and LIAS were partially restored compared with those in the TCH + US group, although they remained lower than those in the PBS group. These results indicate that excessive ROS contributes substantially to the dysregulation of copper homeostasis and mitochondrial lipoylation-associated proteins. However, the incomplete reversal by NAC suggests that additional ROS-independent mechanisms, including impaired copper export and persistent intracellular copper accumulation, may also contribute to the observed cuproptosis-associated changes. Taken together, the intracellular copper accumulation, DLAT aggregation, and alterations in ATP7B, FDX1, and LIAS collectively support the involvement of a cuproptosis-associated process in TCH + US-induced tumor cell death. The NAC rescue experiments further demonstrate that ROS-mediated mitochondrial stress facilitates these changes but is not their sole cause.

Mitochondrial DNA (mtDNA) is highly susceptible to oxidative damage due to the absence of protective histones. To assess mtDNA oxidation under combined SDT and CDT conditions, 8-hydroxy-2′-deoxyguanosine (8-OHdG) staining was performed [44]. As shown in Fig. 6f, the TCH + US group exhibited strong Cy5 fluorescence corresponding to oxidized mtDNA. Moreover, the colocalization of TOMM20-positive mitochondria with 8-OhdG signals indicated that oxidative damage was predominantly localized within mitochondria. To further determine whether mtDNA was released into the cytosol, mitochondria and mtDNA were co-stained using TOMM20 (mitochondria) and TFAM (mitochondrial transcription factor A), respectively. In the control group, strong colocalization of TOMM20 and TFAM signals indicated intact mitochondrial DNA localization. In contrast, partial release of mitochondrial DNA was observed in the TCH + US group (Fig. 6g).

The cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS–STING) pathway is a central mediator of innate immune activation [11, 46]. Upon sensing cytosolic double-stranded DNA, cGAS catalyzes the synthesis of cyclic GMP–AMP (cGAMP), which activates STING and triggers downstream signaling, including phosphorylation of interferon regulatory factor 3 (IRF3) and induction of type I interferons [47]. To determine whether mtDNA release activated this pathway, key signaling proteins were analyzed by western blotting. As shown in Fig. 6h, TCH + US treatment significantly enhanced STING expression and IRF3 phosphorylation, consistent with mtDNA release. This conclusion was further supported by increased expression of interferon-β (IFN-β) (Fig. S18). Collectively, these results demonstrate that, under US irradiation, TCH NPs induced cuproptosis while promoting mitochondrial damage and mtDNA release, leading to cGAS–STING pathway activation and potentially contributing to the enhanced antitumor immune response observed with combined SDT/CDT treatment.

Ferroptosis- and cuproptosis-mediated immunogenic cell death and M2-to-M1-like macrophage repolarization under combined CDT/SDT treatment

Immunogenic cell death (ICD) is characterized by the release of damage-associated molecular patterns (DAMPs), which can activate antitumor immune responses [48]. In this study, TCH-induced ICD was evaluated by measuring canonical DAMPs, including calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP). CLSM showed that TCH + US treatment markedly promoted the translocation of CRT to the cell membrane (Fig. 7a) and reduced intracellular HMGB1 levels (Fig. 7b). Western blot analysis further demonstrated upregulation of CRT and downregulation of HMGB1 in 4T1 cells following TCH + US treatment (Fig. 7c, d). Enzyme-linked immunosorbent assay (ELISA) revealed that extracellular HMGB1 release was significantly elevated in the TCH + US group, reaching levels 1.85-fold and 1.48-fold higher than those in the TCH-alone and TH + US groups, respectively (Fig. 7e). Similarly, extracellular ATP levels were highest in the TCH + US group (Fig. 7f). Flow cytometry analysis yielded consistent results, further confirming enhanced DAMP release under these treatment conditions (Fig. 7g, h). These findings demonstrate that TCH combined with US effectively induced ICD and promoted immunogenic signaling.

Fig. 7 — Ferroptosis- and Cuproptosis-Mediated Immunogenic Cell Death and M2-to-M1-Like Macrophage Repolarization under Combined CDT/SDT Treatment. (**a, b**) CLSM images showing calreticulin (CRT) exposure (a) and HMGB1 localization (b) in treated 4T1 cells. Scale bar, 20 μm. (**c, d**) Western blot analysis of CRT and HMGB1 expression, with corresponding quantitative analysis. (**e, f**) Release of HMGB1 and ATP in the supernatant from 4T1 cells after different treatments (n = 3) (**g, h**) Flow cytometric analysis of CRT exposure and corresponding quantification. (i) Confocal images showing EdU-labeled tumor cell-derived DNA by Raw264.7 macrophages. Scale bar, 10 μm. (j) Flow cytometric analysis of CD86 expression in macrophages exposed to conditioned media (n = 3). (**k–m**) Confocal images and quantitative analysis of CD86 and CD206 expression in macrophages. Scale bar, 10 μm. (**n, o**) ELISA quantification of IL-6 and IL-1β secretion in Raw264.7 macrophages. * P < 0.05, ** P < 0.01, *** P < 0.001. ICD, immunogenic cell death; DAMPs, damage-associated molecular patterns; CRT, calreticulin; HMGB1, high-mobility group box 1; ATP, adenosine triphosphate; CLSM, confocal laser scanning microscopy; ELISA, enzyme-linked immunosorbent assay; US, ultrasound; CDT, chemodynamic therapy; SDT, sonodynamic therapy; dsDNA, double-stranded DNA; TLR9, Toll-like receptor 9; MyD88, myeloid differentiation primary response protein 88; NF-κB, nuclear factor kappa B; IL-1β, interleukin-1 beta; IL-6, interleukin-6

To investigate tumor–macrophage crosstalk, EdU-labeled 4T1 cells were treated with TCH + US and subsequently co-cultured with Raw264.7 macrophages. Distinct red EdU-positive signals were observed in the cytoplasm of macrophages, indicating efficient internalization of tumor cell-derived double-stranded DNA (dsDNA) (Fig. 7i). Furthermore, Raw264.7 macrophages exposed to conditioned medium from TCH + US-treated 4T1 cells exhibited significantly increased expression of the M1-associated marker CD86 compared with control groups (Fig. 7j). Confocal imaging further showed that CD86 levels were markedly upregulated following TCH + US treatment (Fig. 7k–m), indicating polarization toward an M1-like phenotype. Emerging evidence suggests that oxidized mitochondrial DNA (ox-mtDNA), as a type of DAMP, can activate Toll-like receptor 9 (TLR9)-mediated signaling in macrophages, leading to recruitment of myeloid differentiation primary response protein 88 (MyD88) and activations of nuclear factor κB (NF-κB) [49, 50]. Given that TCH + US treatment promoted the release of ox-mtDNA into both cytosolic and extracellular compartments, we further examined macrophage activation. ELISA results showed significantly increased secretion of pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6) in Raw264.7 macrophages exposed to conditioned medium from TCH + US-treated 4T1 cells (Fig. 7n, o). These findings indicate that DAMPs released from tumor cells following TCH + US treatment promoted macrophage repolarization toward an antitumor M1-like phenotype. Taken together, these results demonstrate that TCH combined with US not only induced ICD but also reprogrammed tumor-associated macrophages, thereby enhancing antitumor immune responses.

In vivo antitumor efficacy of TCH NPs combined with US irradiation

A schematic illustration of the treatment protocol is shown in Fig. 8a. The in vivo antitumor efficacy of TCH NPs was evaluated using a 4T1 tumor-bearing mouse model. Notably, the TCH + US group exhibited the most pronounced tumor growth inhibition compared with all other groups. Bioluminescence imaging was performed to monitor tumor progression. As shown in Fig. 8b, c, the TCH + US group displayed markedly reduced bioluminescence signals compared with the saline control, indicating effective suppression of tumor growth. The TCH and TH + US groups also showed reduced signals relative to the PBS and US-only groups, reflecting partial therapeutic effects attributable to CDT or SDT alone. Tumor growth curves and quantitative analysis further supported these findings (Fig. 8d, e). Tumors in the saline group exhibited rapid progression, whereas moderate inhibition was observed in the TCH and TH + US groups. In contrast, the TCH + US group showed the most significant suppression of tumor growth. Consistently, tumor weights measured at the endpoint were lowest in the TCH + US group (Fig. 8f), confirming its superior therapeutic efficacy. Importantly, no significant changes in body weight were observed across all treatment groups throughout the experimental period (Fig. 8g), indicating that TCH + US treatment did not induce appreciable systemic toxicity. Histological analysis of tumor tissues provided further evidence of therapeutic efficacy. H&E and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays revealed extensive tumor cell damage and apoptosis in the TCH + US group. In addition, immunohistochemical analysis showed reduced expression of HMGB1 and LIAS, indicating activation of ICD and cuproptosis, respectively (Fig. 8h). Collectively, these results demonstrate that TCH NPs, when combined with US irradiation, achieve potent in vivo antitumor efficacy through synergistic CDT and SDT effects, while maintaining favorable systemic safety.

After completion of the initial treatment, the residual primary tumors were surgically removed, and the mice remained without detectable tumors for at least 30 days before being rechallenged with 4T1 cells. This experiment was performed to assess whether treatment-associated immune activation persisted beyond the initial therapeutic period. Following rechallenge, flow cytometric analysis of the rechallenged tumors revealed increased proportions of tumor-infiltrating CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) and granzyme B-positive cytotoxic CD8⁺ T cells (CD45⁺CD3⁺CD8⁺GZMB⁺) in the TCH + US group, reaching 36.2% and 41.4%, respectively (Fig. S19a–d). These findings support persistent cytotoxic T-cell activation following a prolonged interval without detectable tumors. Furthermore, the proportion of splenic CD8⁺CD44⁺CD62L⁻ effector memory T cells (T_EM) reached 35.0% in the TCH + US group (Fig. S19e, f), indicating the presence of a memory-associated T-cell phenotype. Collectively, the increased infiltration of cytotoxic T cells in the rechallenged tumors and the expansion of the splenic T_EM population are consistent with sustained treatment-associated immune activation. Separately, Kaplan–Meier analysis of overall survival in the initial six-group therapeutic experiment, measured from treatment initiation, showed prolonged observed survival in the TCH + US group compared with the control group (Fig. S20).

To strengthen the mechanistic basis of our conclusions, we performed additional pharmacological rescue experiments using ferrostatin-1 (Fer-1), a canonical ferroptosis inhibitor, and tetrathiomolybdate (TTM), a copper chelator commonly used to suppress copper-dependent cell death. The contribution of ferroptosis and cuproptosis to the therapeutic efficacy of TCH NPs was further evaluated in a 4T1 tumor-bearing mouse model. The experimental groups included TCH + US alone, TCH + US combined with Fer-1, TCH + US combined with TTM, and TCH + US combined with both Fer-1 and TTM. TCH + US produced the most pronounced inhibition of tumor growth. Administration of either Fer-1 or TTM partially attenuated this antitumor effect, whereas simultaneous administration of Fer-1 and TTM resulted in a more substantial reversal of tumor growth inhibition (Fig. S21a-d). These findings indicate that both ferroptosis and copper-dependent cell death contribute to the therapeutic activity of TCH + US and that simultaneous inhibition of both pathways produces a stronger rescue effect than inhibition of either pathway alone.

No significant changes in body weight were observed among the treatment groups throughout the experimental period, suggesting acceptable systemic tolerability (Fig. S21e). Histological examination further supported the therapeutic findings. H&E staining, TUNEL and Ki-67-stained analysis revealed extensive tumor tissue damage and increased tumor cell death following TCH + US treatment. These effects were partially reduced by either Fer-1 or TTM and were further attenuated when the two inhibitors were administered together (Fig. S21f-h). Immunohistochemical analysis showed decreased GPX4 and LIAS expression in tumors treated with TCH + US, consistent with the involvement of ferroptosis- and cuproptosis-associated molecular alterations, respectively (Fig. S21i-j). Importantly, these changes were alleviated following treatment with the corresponding inhibitors. Thus, the molecular findings, together with the pharmacological rescue experiments, provide stronger evidence that ferroptosis and copper-dependent cell death are functionally involved in the antitumor activity of TCH + US. We also evaluated whether inhibition of these cell-death pathways affected treatment-induced antitumor immunity. TCH + US markedly increased CRT exposure, indicating enhanced immunogenic cell death. CRT expression was reduced by either Fer-1 or TTM (Fig. S21k). Consistent with these findings, the proportion of mature bone marrow-derived dendritic cells increased from 13.8% in the saline group to 27.8% in the TCH + US group, whereas combined Fer-1 and TTM treatment reduced this proportion to 16.4% (Figs. S22a and S22c). Similarly, the proportion of CD8 + T cells increased from 21.3% in the saline group to 34.8% following TCH + US treatment but decreased to 24.8% when Fer-1 and TTM were administered together (Figs. S22b and S22d). The elevated levels of IFN-γ and IL-6 induced by TCH + US were also attenuated by Fer-1 or TTM (Fig. S22e-f). Collectively, these results suggest that ferroptosis and copper-dependent cell death contribute not only to direct tumor suppression but also to the subsequent induction of immunogenic cell death and antitumor immune activation.

To determine the functional contribution of cGAS-STING signaling to TCH + US-mediated therapy, additional in vivo experiments were performed using H-151, a pharmacological STING inhibitor, in 4T1 tumor-bearing mice. TCH + US markedly suppressed tumor growth, whereas coadministration of H-151 partially diminished this effect (Fig. S23a-d), indicating that STING signaling contributes to the therapeutic efficacy of TCH + US. No significant changes in body weight were observed during treatment, suggesting acceptable systemic tolerability (Fig. S23e). Consistently, H&E, TUNEL, and Ki-67 staining revealed extensive tumor damage, increased tumor cell death, and reduced tumor cell proliferation after TCH + US treatment, all of which were partially reversed by H-151 (Fig. S23f-h). Immunohistochemical analysis further showed increased STING expression following TCH + US treatment, whereas this increase was attenuated by H-151 (Fig. S23i-j). Together, these molecular and pharmacological findings support the functional involvement of cGAS–STING signaling in TCH + US-mediated tumor suppression. We next investigated whether STING inhibition affected the antitumor immune response induced by TCH + US. The proportion of CD8 + T cells increased from 13.5% in the saline group to 26.0% after TCH + US treatment but decreased to 16.9% following H-151 coadministration (Fig. S24a-b). CD8 + IFN-γ + T cells exhibited a similar trend (Fig. S24c-d), demonstrating that STING inhibition weakened TCH + US-induced cytotoxic T-cell activation. These results suggest that cGAS-STING signaling contributes to both tumor control and the subsequent antitumor immune response. Nevertheless, because the causal relationship between mtDNA release and STING activation was not directly established, mtDNA release is considered to be associated with, and potentially contribute to, cGAS–STING activation rather than serving as its sole upstream trigger.

TCH-US treatment reshapes the tumor immune microenvironment

To systematically evaluate the impact of TCH-US treatment on the TME, we performed scRNA-seq analysis on mice tumor samples from PBS-treated (control) and TCH-US-treated groups [51]. Uniform Manifold Approximation and Projection visualization (Fig. 9a) revealed distinct clustering of diverse cell populations, including plasma cells, migrating conventional dendritic cells, T cells, smooth muscle cells, endothelial cells, tumor cells, neutrophils, fibroblasts, NK cells, and macrophages. The overall major cell type was largely comparable between the PBS and TCH-US groups (Fig. 9b). We next focused on the dynamics of T cell subset dynamics, considering their critical role in antitumor immunity. To characterize the molecular profiles of these T cell states, we examined the expression of canonical signature markers (Fig. 9c). Consistent with established markers, Foxp3, Il2ra, and Ctla4 were enriched in regulatory T cells, whereas Gzmb, Prf1, and Klrd1 characterized the effector T cell cluster. Notably, exhaustion markers such as Havcr2 and Pdcd1 were upregulated in the exhausted T cell cluster. Quantitative analysis of cell proportions (Fig. 9d) demonstrated a significant increase in the proportion of effector T cells in the TCH-US group compared to that of the PBS group. Conversely, the proportion of Tregs was markedly reduced in the TCH-US group, accompanied by a decreasing trend in naive T cells. This shift suggests that TCH-US treatment promotes an effector-dominant T cell landscape while suppressing immunosuppressive Treg populations. Considering the functional plasticity of macrophages in the TME, we further evaluated their polarization states. Gene set scoring analysis (Fig. 9e) revealed that the M1 Score (pro-inflammatory phenotype) was significantly elevated in the TCH-US group, whereas the M2 Score was significantly decreased. Collectively, TCH-US treatment perturbs tumor cell metabolism and survival as well as reprograms the immune microenvironment by promoting M1 macrophage polarization and enhancing cytotoxic T cell responses.

To further elucidate the molecular mechanisms underlying these phenotypic changes, we performed KEGG pathway enrichment analysis (Fig. 9f). The results indicated that genes upregulated in the TCH-US group were significantly enriched in pathways associated with HIF-1 signaling, ferroptosis, cysteine and methionine metabolism, and central carbon metabolism in cancer. Thus, TCH-US treatment induces pronounced metabolic stress and rewires energy metabolism within the TME.

Consistent with the enrichment of ferroptosis-related pathways, we further quantified specific cell death-associated signatures. Violin plot analysis (Fig. 9g) demonstrated that both the cuproptosis and ferroptosis scores were significantly elevated in the TCH-US group as compared to the PBS group, confirming effective activation of these regulated cell death pathways. Furthermore, the expression profiles of key metabolic and cell cycle-associated genes were examined via heatmap analysis (Fig. 9h). Genes including Hsp90ab1, Pgk1, Ldha, and Ccnd1 exhibited distinct expression patterns between the groups, further corroborating the metabolic reprogramming identified in the pathway analysis.

In vivo tumor immune microenvironment remodeling mediated by TCH NPs

As shown in Fig. 10a, b, treatment with TCH + US markedly increased the infiltration of cytotoxic CD8⁺ T cells within tumor tissues. The proportion of CD8⁺ T cells reached 35.0% in the TCH + US group, compared with 22.3% in the saline group. In parallel, the proportion of regulatory T cells (Tregs) was reduced following treatment, with decreases of 11.5%, 15.6%, and 7.49% observed in the TH + US, TCH, and TCH + US groups, respectively, relative to the control group (Fig. 10c, d). These changes indicate effective remodeling of the tumor immune microenvironment, characterized by enhanced effector T-cell responses and reduced immunosuppressive Treg infiltration, thereby contributing to immune reactivation and tumor suppression. Collectively, the simultaneous increase in cytotoxic T-cell infiltration and reduction in Treg-mediated immunosuppression suggest that TCH + US treatment remodels the tumor immune microenvironment, which may contribute to its antitumor efficacy.

To further evaluate innate immune remodeling, the polarization states of tumor-associated macrophages were analyzed. Flow cytometry revealed a marked decrease in M2-like macrophages and a significant increase in M1-like macrophages in the TCH + US group compared with the control group (Fig. 10e, f,h). The TCH + US group exhibited the highest M1/M2 macrophage ratio, indicating a pronounced shift toward an antitumor macrophage phenotype. Myeloid-derived suppressor cells (MDSCs), identified as CD45⁺CD11b⁺Gr-1⁺ cells, are key contributors to the immunosuppressive TME. As shown in Fig. 10g and i, the proportion of MDSCs decreased to 6.67%, 7.75%, and 4.79% in the TH + US, TCH, and TCH + US groups, respectively, compared with 9.94% and 10.5% in the control and US-only groups. These findings indicate that TCH + US effectively alleviated immunosuppression within the TME. Consistent with these observations, ELISA analysis showed significantly increased levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-6, and IFN-γ, particularly in the TCH + US group (Fig. 10j–l). In addition, immunofluorescence staining further confirmed that the TCH + US group exhibited the lowest infiltration of M2-like tumor-associated macrophages (Fig. 10m). Collectively, these results demonstrate that TCH NPs, especially under US irradiation, effectively remodel the tumor immune microenvironment by enhancing cytotoxic T-cell responses, reducing immunosuppressive cell populations, and promoting macrophage polarization toward an M1-like phenotype, thereby contributing to robust antitumor immunity.

In vivo antitumor efficacy and immune activation following combination therapy with anti–PD-L1

Cancer immunotherapy is widely regarded as a promising strategy for the treatment of malignant tumors; however, its clinical efficacy in TNBC remains limited [52]. Based on the observed ability of TCH NPs to remodel the immunosuppressive tumor microenvironment through synergistic CDT and SDT, we further hypothesized that TCH NPs could potentiate immune checkpoint blockade. To test this hypothesis, the therapeutic efficacy of TCH NPs in combination with anti–PD-L1 (aPD-L1) was evaluated in 4T1 tumor-bearing mice according to the regimen shown in Fig. 11a. As shown in Fig. 11b–e, aPD-L1 monotherapy resulted in limited tumor suppression, whereas the TCH + US+aPD-L1 combination produced significantly greater tumor growth inhibition than either aPD-L1 alone or TCH + US treatment. Notably, no significant body weight loss was observed across all treatment groups (Fig. S25), indicating that the combination of TCH NPs and aPD-L1 did not induce additional systemic toxicity. This was further supported by histological analysis, which showed no obvious damage to major organs (Fig. S26). Biodistribution analysis showed that, following intravenous administration, TCH NPs predominantly accumulated in the liver, kidneys, and tumor tissues. ICP analysis of Cu biodistribution showed that Cu associated with the administered TCH formulation was detected mainly in the liver, kidney, and tumor and decreased markedly in major organs over 48 h (Fig. S27). These findings suggest that TCH NPs did not disrupt systemic metal homeostasis. Histopathological evaluation demonstrated that the TCH NPs + aPD-L1 + US group exhibited the most extensive tumor cell damage, as evidenced by H&E and TUNEL staining (Fig. S28). In addition, Ki-67 staining showed, the lowest proliferative activity in this group. Consistent with ferroptotic activation, GPX4 expression was markedly reduced. Furthermore, increased CRT and decreased intracellular HMGB1 levels indicated enhanced ICD, likely associated with HMGB1 release during tumor cell death.

Fig. 11 — In Vivo Antitumor Efficacy and Immune Activation Following Combination Therapy with Anti–PD-L1. (a) Schematic illustration of the treatment protocol for TCH NPs combined with anti–PD-L1 (aPD-L1) therapy in 4T1 tumor-bearing mice. (b, c) Quantitative analysis of bioluminescence intensity of tumor and tumor volume after the indicated treatments (n = 3). (d) Representative in vivo bioluminescence images showing tumor progression after different treatments (n = 3). (e) Relative tumor volume after the indicated treatments (n = 3). (f, g) Flow cytometric analysis of intratumoral CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) with corresponding quantitative analysis. (h, i) Flow cytometric evaluation and quantification of IFN-γ expression in CD8⁺ T cells. (j) Immunofluorescence images of tumor sections stained for CD3 and CD8. Scale bar, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. CDT, chemodynamic therapy; SDT, sonodynamic therapy; US, ultrasound; aPD-L1, anti–programmed death-ligand 1; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; GPX4, glutathione peroxidase 4; CRT, calreticulin; HMGB1, high-mobility group box 1; ICD, immunogenic cell death; IFN-γ, interferon gamma; TNF-α, tumor necrosis factor alpha; IL-6, interleukin-6

To further assess immune activation, tumor-infiltrating lymphocytes were analyzed by flow cytometry. The TCH NPs + aPD-L1 + US group exhibited a significant increase in CD8⁺ T-cell infiltration (Fig. 11f, g). Moreover, these CD8⁺ T cells displayed elevated IFN-γ expression (Fig. 11h, i), indicating enhanced cytotoxic function. This immune activation was accompanied by increased levels of pro-inflammatory cytokines, including TNF-α, IL-6, and IFN-γ (Fig. S29). Consistently, immunofluorescence staining of tumor sections revealed the highest CD8⁺ T-cell infiltration in the TCH NPs + aPD-L1 + US group (Fig. 11j). Collectively, these findings demonstrate that, the combination of TCH NPs with immune checkpoint blockade effectively enhances ferroptosis, ICD, and antitumor immune responses, thereby achieving superior therapeutic efficacy in TNBC.

Conclusion

In conclusion, this study presents a hyaluronic acid–functionalized Cu_2−XSe-loaded TiO₂ nanosystem as a promising therapeutic strategy for TNBC. By integrating tumor-targeted delivery, microenvironment-responsive Cu²⁺ release, and enhanced sonodynamic therapy, this nanoplatform effectively amplified ROS generation, disrupted intracellular redox homeostasis, and triggered ferroptosis and cuproptosis in TNBC cells. Importantly, the coordinated induction of these two non-apoptotic cell death pathways contributed to tumor cell killing and remodeling of the immunosuppressive TME, thereby improving tumor immunogenicity and enhancing the responsiveness of TNBC to immune checkpoint blockade. Furthermore, this system activated the cGAS–STING pathway and promoted immunogenic cell death, leading to robust antitumor immune responses. This generalizable paradigm of “sonodynamic activation + dual non-apoptotic cell death + immune activation” may provide a promising therapeutic strategy for other immunologically “cold” tumors. Collectively, this work provides a rational design strategy for integrating sonodynamic therapy with metal ion–mediated programmed cell death and immunomodulation, offering a potentially translatable approach to overcome therapeutic resistance and enhance immunotherapy efficacy in TNBC.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1.^{(12.5MB, docx)}

Author contributions

Shibing Wang, Tingting Deng, and Xue Yang contributed equally to this work. Shibing Wang: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing-original draft. Tingting Deng: Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing – review & editing. Xue Yang: Investigation, Validation, Formal analysis. Yuan Chen: Investigation, Validation. Yuli Zhou: Investigation, Resources, Software, Data curation, Visualization. Hairui Tian: Validation, Formal analysis. Jiarui Li: Resources, Methodology, Writing – review & editing. Meili Li: Supervision, Project administration, Writing-review & editing. Teng Lin: Supervision, Project administration, Writing-review & editing. Cong Huang: Conceptualization, Supervision, Funding acquisition, Project administration, Writing-review & editing. Xiangmin Tong: Conceptualization, Supervision, Funding acquisition, Project administration, Writing-review & editing. All authors discussed the results and approved the final version of the manuscript.

Funding

The project is funded by the Foundation of Science Technology Department of Zhejiang Province (No. LKLZ26H080001, Owner: Shibing Wang), Guangdong Basic and Applied Basic Research Foundation (No. 2025A1515011491, Owner: Cong Huang), Construction Fund of Key Medical Disciplines of Hangzhou-Laboratory Diagnostics (No. 2025HZZD01, Owner: Shibing Wang), the Key R&D Program of Hangzhou Science and Technology Bureau (20241203A11, Owner: Xiangmin Tong), Hangzhou Natural Science Foundation of China (2024SZRYBC080002, Owner: Shibing Wang), Science and Technology Project of Zhejiang Provincial Health Commission (No. 2021KY517, Owner: Yuan Chen), Medical Scientific Research Foundation of Guangdong Province (No. A2024042, Owner: Cong Huang), Guangdong Province Medical Research Fund (No. A2024042, Owner: Cong Huang).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The approval of the study was obtained from Institution Animal Ethics Committee of the Ethics Committee of Guangzhou Miles Biosciences (IACUC Issue No. MIS2026012).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

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Contributor Information

Shibing Wang, Email: wangshibing@hmc.edu.cn.

Teng Lin, Email: 13502952420@163.com.

Cong Huang, Email: chuang200628@163.com.

Xiangmin Tong, Email: tongxiangmin@163.com.

References

1.Xiong W, Cheng Z, Chen H, Liang H, Wang M, Chen Y, et al. Biomimetic tumor cell membrane-encapsulated nanoparticles combine NIR-II photothermal therapy and chemotherapy for enhanced immunotherapy in triple-negative breast cancer. Adv Funct Mater. 2024;34(52):2410841. [Google Scholar]
2.Chen D, Chen X, Yang M, Li Q, Weng S, Kou S, et al. H3K36me2 methyltransferase NSD2/WHSC1 promotes triple-negative breast cancer metastasis via activation of ULK1-dependent autophagy. Autophagy. 2025;21(8):1824–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Wang H, Ma K, Li Z, Lou S, Yang D, Zhang H, et al. Augmenting cuproptosis and anti-metastatic immunity in breast cancer by copper-based nanoplatform for synergistic immunotherapy via lactate metabolic reprogramming and hypoxia alleviation. J Control Release. 2026;392:114716. [DOI] [PubMed] [Google Scholar]
4.Lynce F, Mainor C, Donahue RN, Geng X, Jones G, Schlam I, et al. Adjuvant nivolumab, capecitabine or the combination in patients with residual triple-negative breast cancer: the OXEL randomized phase II study. Nat Commun. 2024;15(1):2691. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Leon-Ferre RA, Goetz MP. Advances in systemic therapies for triple negative breast cancer. BMJ. 2023;381:e071674. [DOI] [PubMed] [Google Scholar]
6.Ou Y, Zhang K, Shuai Q, Wang C, Hu H, Cao L, et al. USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells. Acta Pharm Sin B. 2025;15(5):2593–611. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Meng M, Wu J, Feng Y, Lin L, Chen J, Pang X, et al. A comprehensive strategy based on high clinical translational nanosystem for programmable immunotherapy of triple negative breast cancer. Adv Mater. 2024;36(27):2314309. [DOI] [PubMed] [Google Scholar]
8.Chang D, Yang J, Li Y, Min S, Ma Y, Xu T, et al. Spatiotemporally controlled nanomicelles for synergistic phototherapy and immune reprogramming in triple-negative breast cancer. ACS Nano. 2025;19(41):36323–41. [DOI] [PubMed] [Google Scholar]
9.Li Z, He S, Xie L, Zeng G, Huang J, Wang H, et al. Boosting ferroptosis and immunotherapy for colorectal cancer by lactate-related metabolic reprogramming. Adv Funct Mater. 2024;34(52):2411247. [Google Scholar]
10.Li Z, Jiang J, Zhou Y, Mao X, Wen H, Chen Z, et al. Inulin-based oral chemotherapy modulates gut microbiota and immune microenvironment through inhibition of neutrophil extracellular trap formation for improving cancer therapy. Adv Sci (Weinh). 2026;13(20):e14318. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Li Z, Chen H, Wang C, Deng T, Huang J, Ye H, et al. Augmenting cGAS/STING-mediated innate immunity by copper-based nanoplatform for synergistic cancer immunotherapy via inducing irreversible DNA damage. J Control Release. 2025;386:114075. [DOI] [PubMed] [Google Scholar]
12.Mao C, Deng F, Zhu W, Xie L, Wang Y, Li G, et al. In situ editing of tumour cell membranes induces aggregation and capture of PD-L1 membrane proteins for enhanced cancer immunotherapy. Nat Commun. 2024;15(1):9723. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Cheng N, Zhou Q, Jia Z, Mu Y, Zhang S, Wang L, et al. Functionalized biomimetic nanoparticles loaded with salvianolic acid B for synergistic targeted triple-negative breast cancer treatment. Mater Today Bio. 2025;30:101441. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Sun W, Wang H, Qi Y, Li M, Zhang R, Gao Z, et al. Metal-phenolic vehicles potentiate cycle-cascade activation of pyroptosis and cGAS-STING pathway for tumor immunotherapy. ACS Nano. 2024;18(34):23727–40. [DOI] [PubMed] [Google Scholar]
15.Lin L, Fang Z, Liu G, Liu Y, Li Z, Pan D, et al. Prodrug-based combinational nanomedicine remodels lipid metabolism for reinforced ferroptosis and immune activation. Acta Pharm Sin B. 2025;15(5):2746–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Guan M, Cheng K, Xie X, Li Y, Ma M, Zhang B, et al. Regulating copper homeostasis of tumor cells to promote cuproptosis for enhancing breast cancer immunotherapy. Nat Commun. 2024;15(1):10060. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Deng K, Gao W, Wen Y, Huang J, Li X, Yang X, et al. A functional nanocomposite tri-activates cuproptosis, ferroptosis, and mitophagy death pathway to oppose malignancies. Adv Healthc Mater. 2026;15(6):e03530. [DOI] [PubMed] [Google Scholar]
18.Mao L, Lu J, Wen X, Song Z, Sun C, Zhao Y, et al. Cuproptosis: mechanisms and nanotherapeutic strategies in cancer and beyond. Chem Soc Rev. 2025;54(13):6282–334. [DOI] [PubMed] [Google Scholar]
19.Zhang H, Ma J, Hou C, Luo X, Zhu S, Peng Y, et al. A ROS-mediated oxidation-O-GlcNAcylation cascade governs ferroptosis. Nat Cell Biol. 2025;27(8):1288–300. [DOI] [PubMed] [Google Scholar]
20.Huang L, Zhu J, Wu G, Xiong W, Feng J, Yan C, et al. A strategy of adding fuel to the flames enables a self-accelerating cycle of ferroptosis-cuproptosis for potent antitumor therapy. Biomaterials. 2024;311:122701. [DOI] [PubMed] [Google Scholar]
21.Zhi H, Yin W, Chen S, Zhang X, Yang Z, Man F, et al. Lactate metabolism regulating nanosystem synergizes cuproptosis and ferroptosis to enhance cancer immunotherapy. Biomaterials. 2026;325:123538. [DOI] [PubMed] [Google Scholar]
22.Cheng X, Yin Y, Fang Y, Zhou X, Guo H, Fan D, et al. Breaking the depth barrier: dual-activated radical storm from a natural product-based nanoplatform for spatiotemporally controlled deep tumor sonodynamic therapy. Adv Funct Mater. 2026;36(5):e14995. [Google Scholar]
23.Zhu Y, Niu X, Ding C, Lin Y, Fang W, Yan L, et al. Carrier-free self-assembly nano-sonosensitizers for sonodynamic-amplified cuproptosis-ferroptosis in glioblastoma therapy. Adv Sci (Weinh). 2024;11(23):2402516. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ye L, Yu C, Xia J, Ni K, Zhang Y, Ying X, et al. Multifunctional nanomaterials via cell cuproptosis and oxidative stress for treating osteosarcoma and OS-induced bone destruction. Mater Today Bio. 2024;25:100996. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Zhu W, Sun Y, Xu K, Xie C, Yang M, Ye Y, et al. Ultrasmall manganese-doped TiO2–x overcomes the efficacy-safety dilemma of sonodynamic therapy via tumor microenvironment-responsive reactive oxygen species amplification and clearance. ACS Nano. 2025;19(44):38440–56. [DOI] [PubMed] [Google Scholar]
26.Collins VG, Hutton D, Hossain-Ibrahim K, Joseph J, Banerjee S. The abscopal effects of sonodynamic therapy in cancer. Br J Cancer. 2025;132(5):409–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Sun S, Huang X, Yang N, Lei H, Pei Z, Han Z, et al. Fluorinated titanium oxide (TiO₂–xFx) nanospindles as ultrasound-triggered pyroptosis inducers to boost sonodynamic immunotherapy. ACS Nano. 2024;18(30):19756–70. [DOI] [PubMed] [Google Scholar]
28.Li N, Li L, Qiu Y, Liu X, Zhang J, Gao Y, et al. Hollow cubic CuSe@CdS with tunable size for plasmon-induced Vis-NIR driven photocatalytic properties. Nanoscale. 2024;16(16):8151–61. [DOI] [PubMed] [Google Scholar]
29.Xue Q, Yan D, Chen X, Li X, Kang R, Klionsky DJ, et al. Copper-dependent autophagic degradation of GPX4 drives ferroptosis. Autophagy. 2023;19(7):1982–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Qiao X, Liang J, Qiu L, Feng W, Cheng G, Chen Y, et al. Ultrasound-activated nanosonosensitizer for oxygen/sulfate dual-radical nanotherapy. Biomaterials. 2023;301:122252. [DOI] [PubMed] [Google Scholar]
31.Feng X, Lin T, Chen D, Li Z, Yang Q, Tian H, et al. Mitochondria-associated ER stress evokes immunogenic cell death through the ROS-PERK-eIF2α pathway under PTT/CDT combined therapy. Acta Biomater. 2023;160:211–24. [DOI] [PubMed] [Google Scholar]
32.Chen Z, Zhu X, Xiong J, Wen Z, Cheng G. A p-n junction by coupling amine-enriched brookite-TiO₂ nanorods with CuxS nanoparticles for improved photocatalytic CO₂ reduction. Mater (Basel). 2023;16(3):960. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Chen Z, Li Z, Huang H, Shen G, Ren Y, Mao X, et al. Cancer immunotherapy based on cell membrane-coated nanocomposites augmenting cGAS/STING activation by efferocytosis blockade. Small. 2023;19(43):e2302758. [DOI] [PubMed] [Google Scholar]
34.Guo W, Chen Z, Li Z, Huang H, Ren Y, Zhao B, et al. Improved immunotherapy for gastric cancer by nanocomposites with capability of triggering dual-damage of nuclear/mitochondrial DNA and cGAS/STING-mediated innate immunity. Chem Eng J. 2022;443:136428. [Google Scholar]
35.Guo W, Chen Z, Li Z, Huang H, Ren Y, Li Z, et al. Cancer cell membrane biomimetic mesoporous silica nanotheranostics for enhanced ferroptosis-mediated immuogenic cell death on gastric cancer. Chem Eng J. 2023;455:140868. [Google Scholar]
36.Xiao T, Hu W, Fan Y, Shen M, Shi X. Macrophage-mediated tumor homing of hyaluronic acid nanogels loaded with polypyrrole and anticancer drug for targeted combinational photothermo-chemotherapy. Theranostics. 2021;11(14):7057–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Xu X, Pang Y, Fan X. Mitochondria in oxidative stress, inflammation and aging: from mechanisms to therapeutic advances. Signal Transduct Target Ther. 2025;10(1):190. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Drilon A, Jenkins C, Iyer S, Schoenfeld A, Keddy C, Davare MA. ROS1-dependent cancers: biology, diagnostics and therapeutics. Nat Rev Clin Oncol. 2021;18(1):35–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Glorieux C, Liu S, Trachootham D, Huang P. Targeting ROS in cancer: rationale and strategies. Nat Rev Drug Discov. 2024;23(8):583–606. [DOI] [PubMed] [Google Scholar]
40.Friedmann Angeli JP, Krysko DV, Conrad M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405–14. [DOI] [PubMed] [Google Scholar]
41.Yu C, Chen D, Feng L, Zang P, He G, Liu J, et al. Piezocatalytic nanoplatform drives Mg2+-release-amplified ferroptosis-immune synergy for tumor microenvironment reprogramming. ACS Nano. 2026;20(12):10024–42. [DOI] [PubMed] [Google Scholar]
42.Wei X, Jiang Y, Chenwu F, Li Z, Wan J, Li Z, et al. Synergistic ferroptosis-immunotherapy nanoplatforms: multidimensional engineering for tumor microenvironment remodeling and therapeutic optimization. Nano Micro Lett. 2026;18(1):56. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Zhang M, Yang D, Dong C, Huang H, Feng G, Chen Q, et al. Two-dimensional MXene-originated in situ nanosonosensitizer generation for augmented and synergistic sonodynamic tumor nanotherapy. ACS Nano. 2022;16(6):9938–52. [DOI] [PubMed] [Google Scholar]
44.Cheng R, Li Z, Luo W, Chen H, Deng T, Gong Z, et al. A copper-based photothermal-responsive nanoplatform reprograms tumor immunogenicity via self-amplified cuproptosis for synergistic cancer therapy. Adv Sci (Weinh). 2025;12(19):e2500652. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Chen L, Min J, Wang F. Copper homeostasis and cuproptosis in health and disease. Signal Transduct Target Ther. 2022;7(1):378. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Wang Y, Luo J, Alu A, Han X, Wei Y, Wei X. cGAS-STING pathway in cancer biotherapy. Mol Cancer. 2020;19(1):136. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Samson N, Ablasser A. The cGAS-STING pathway and cancer. Nat Cancer. 2022;3(12):1452–63. [DOI] [PubMed] [Google Scholar]
48.Meier P, Legrand AJ, Adam D, Silke J. Immunogenic cell death in cancer: targeting necroptosis to induce antitumour immunity. Nat Rev Cancer. 2024;24(5):299–315. [DOI] [PubMed] [Google Scholar]
49.Yao H, He Q, Wei S, Xiang L, Luo Y, Huang C, et al. Targeted inhibition of macrophage STING signaling alleviates inflammatory injury and ventricular remodeling in acute myocardial infarction. Acta Pharm Sin B. 2025;15(8):4030–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Fan X, Han J, Zhong L, Zheng W, Shao R, Zhang Y, et al. Macrophage-derived GSDMD plays an essential role in atherosclerosis and cross talk between macrophages via the mitochondria-STING-IRF3/NF-κB axis. Arterioscler Thromb Vasc Biol. 2024;44(6):1365–78. [DOI] [PubMed] [Google Scholar]
51.Li Z, Li M, Yang L, Chen J, Ye Q, Qian W, et al. Oncolytic vaccinia virus armed with anti-CD47 nanobody elicit potent antitumor effects on multiple tumor models via enhancing innate and adoptive immunity. J Immunother Cancer. 2024;12(12):e009473. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Wan M, Pan S, Shan B, Diao H, Jin H, Wang Z, et al. Lipid metabolic reprograming: the unsung hero in breast cancer progression and tumor microenvironment. Mol Cancer. 2025;24(1):61. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(12.5MB, docx)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

[CR1] 1.Xiong W, Cheng Z, Chen H, Liang H, Wang M, Chen Y, et al. Biomimetic tumor cell membrane-encapsulated nanoparticles combine NIR-II photothermal therapy and chemotherapy for enhanced immunotherapy in triple-negative breast cancer. Adv Funct Mater. 2024;34(52):2410841. [Google Scholar]

[CR2] 2.Chen D, Chen X, Yang M, Li Q, Weng S, Kou S, et al. H3K36me2 methyltransferase NSD2/WHSC1 promotes triple-negative breast cancer metastasis via activation of ULK1-dependent autophagy. Autophagy. 2025;21(8):1824–42. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Wang H, Ma K, Li Z, Lou S, Yang D, Zhang H, et al. Augmenting cuproptosis and anti-metastatic immunity in breast cancer by copper-based nanoplatform for synergistic immunotherapy via lactate metabolic reprogramming and hypoxia alleviation. J Control Release. 2026;392:114716. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Lynce F, Mainor C, Donahue RN, Geng X, Jones G, Schlam I, et al. Adjuvant nivolumab, capecitabine or the combination in patients with residual triple-negative breast cancer: the OXEL randomized phase II study. Nat Commun. 2024;15(1):2691. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Leon-Ferre RA, Goetz MP. Advances in systemic therapies for triple negative breast cancer. BMJ. 2023;381:e071674. [DOI] [PubMed] [Google Scholar]

[CR6] 6.Ou Y, Zhang K, Shuai Q, Wang C, Hu H, Cao L, et al. USP51/GRP78/ABCB1 axis confers chemoresistance through decreasing doxorubicin accumulation in triple-negative breast cancer cells. Acta Pharm Sin B. 2025;15(5):2593–611. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Meng M, Wu J, Feng Y, Lin L, Chen J, Pang X, et al. A comprehensive strategy based on high clinical translational nanosystem for programmable immunotherapy of triple negative breast cancer. Adv Mater. 2024;36(27):2314309. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Chang D, Yang J, Li Y, Min S, Ma Y, Xu T, et al. Spatiotemporally controlled nanomicelles for synergistic phototherapy and immune reprogramming in triple-negative breast cancer. ACS Nano. 2025;19(41):36323–41. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Li Z, He S, Xie L, Zeng G, Huang J, Wang H, et al. Boosting ferroptosis and immunotherapy for colorectal cancer by lactate-related metabolic reprogramming. Adv Funct Mater. 2024;34(52):2411247. [Google Scholar]

[CR10] 10.Li Z, Jiang J, Zhou Y, Mao X, Wen H, Chen Z, et al. Inulin-based oral chemotherapy modulates gut microbiota and immune microenvironment through inhibition of neutrophil extracellular trap formation for improving cancer therapy. Adv Sci (Weinh). 2026;13(20):e14318. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Li Z, Chen H, Wang C, Deng T, Huang J, Ye H, et al. Augmenting cGAS/STING-mediated innate immunity by copper-based nanoplatform for synergistic cancer immunotherapy via inducing irreversible DNA damage. J Control Release. 2025;386:114075. [DOI] [PubMed] [Google Scholar]

[CR12] 12.Mao C, Deng F, Zhu W, Xie L, Wang Y, Li G, et al. In situ editing of tumour cell membranes induces aggregation and capture of PD-L1 membrane proteins for enhanced cancer immunotherapy. Nat Commun. 2024;15(1):9723. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Cheng N, Zhou Q, Jia Z, Mu Y, Zhang S, Wang L, et al. Functionalized biomimetic nanoparticles loaded with salvianolic acid B for synergistic targeted triple-negative breast cancer treatment. Mater Today Bio. 2025;30:101441. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Sun W, Wang H, Qi Y, Li M, Zhang R, Gao Z, et al. Metal-phenolic vehicles potentiate cycle-cascade activation of pyroptosis and cGAS-STING pathway for tumor immunotherapy. ACS Nano. 2024;18(34):23727–40. [DOI] [PubMed] [Google Scholar]

[CR15] 15.Lin L, Fang Z, Liu G, Liu Y, Li Z, Pan D, et al. Prodrug-based combinational nanomedicine remodels lipid metabolism for reinforced ferroptosis and immune activation. Acta Pharm Sin B. 2025;15(5):2746–63. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Guan M, Cheng K, Xie X, Li Y, Ma M, Zhang B, et al. Regulating copper homeostasis of tumor cells to promote cuproptosis for enhancing breast cancer immunotherapy. Nat Commun. 2024;15(1):10060. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Deng K, Gao W, Wen Y, Huang J, Li X, Yang X, et al. A functional nanocomposite tri-activates cuproptosis, ferroptosis, and mitophagy death pathway to oppose malignancies. Adv Healthc Mater. 2026;15(6):e03530. [DOI] [PubMed] [Google Scholar]

[CR18] 18.Mao L, Lu J, Wen X, Song Z, Sun C, Zhao Y, et al. Cuproptosis: mechanisms and nanotherapeutic strategies in cancer and beyond. Chem Soc Rev. 2025;54(13):6282–334. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Zhang H, Ma J, Hou C, Luo X, Zhu S, Peng Y, et al. A ROS-mediated oxidation-O-GlcNAcylation cascade governs ferroptosis. Nat Cell Biol. 2025;27(8):1288–300. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Huang L, Zhu J, Wu G, Xiong W, Feng J, Yan C, et al. A strategy of adding fuel to the flames enables a self-accelerating cycle of ferroptosis-cuproptosis for potent antitumor therapy. Biomaterials. 2024;311:122701. [DOI] [PubMed] [Google Scholar]

[CR21] 21.Zhi H, Yin W, Chen S, Zhang X, Yang Z, Man F, et al. Lactate metabolism regulating nanosystem synergizes cuproptosis and ferroptosis to enhance cancer immunotherapy. Biomaterials. 2026;325:123538. [DOI] [PubMed] [Google Scholar]

[CR22] 22.Cheng X, Yin Y, Fang Y, Zhou X, Guo H, Fan D, et al. Breaking the depth barrier: dual-activated radical storm from a natural product-based nanoplatform for spatiotemporally controlled deep tumor sonodynamic therapy. Adv Funct Mater. 2026;36(5):e14995. [Google Scholar]

[CR23] 23.Zhu Y, Niu X, Ding C, Lin Y, Fang W, Yan L, et al. Carrier-free self-assembly nano-sonosensitizers for sonodynamic-amplified cuproptosis-ferroptosis in glioblastoma therapy. Adv Sci (Weinh). 2024;11(23):2402516. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Ye L, Yu C, Xia J, Ni K, Zhang Y, Ying X, et al. Multifunctional nanomaterials via cell cuproptosis and oxidative stress for treating osteosarcoma and OS-induced bone destruction. Mater Today Bio. 2024;25:100996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Zhu W, Sun Y, Xu K, Xie C, Yang M, Ye Y, et al. Ultrasmall manganese-doped TiO2–x overcomes the efficacy-safety dilemma of sonodynamic therapy via tumor microenvironment-responsive reactive oxygen species amplification and clearance. ACS Nano. 2025;19(44):38440–56. [DOI] [PubMed] [Google Scholar]

[CR26] 26.Collins VG, Hutton D, Hossain-Ibrahim K, Joseph J, Banerjee S. The abscopal effects of sonodynamic therapy in cancer. Br J Cancer. 2025;132(5):409–20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Sun S, Huang X, Yang N, Lei H, Pei Z, Han Z, et al. Fluorinated titanium oxide (TiO₂–xFx) nanospindles as ultrasound-triggered pyroptosis inducers to boost sonodynamic immunotherapy. ACS Nano. 2024;18(30):19756–70. [DOI] [PubMed] [Google Scholar]

[CR28] 28.Li N, Li L, Qiu Y, Liu X, Zhang J, Gao Y, et al. Hollow cubic CuSe@CdS with tunable size for plasmon-induced Vis-NIR driven photocatalytic properties. Nanoscale. 2024;16(16):8151–61. [DOI] [PubMed] [Google Scholar]

[CR29] 29.Xue Q, Yan D, Chen X, Li X, Kang R, Klionsky DJ, et al. Copper-dependent autophagic degradation of GPX4 drives ferroptosis. Autophagy. 2023;19(7):1982–96. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Qiao X, Liang J, Qiu L, Feng W, Cheng G, Chen Y, et al. Ultrasound-activated nanosonosensitizer for oxygen/sulfate dual-radical nanotherapy. Biomaterials. 2023;301:122252. [DOI] [PubMed] [Google Scholar]

[CR31] 31.Feng X, Lin T, Chen D, Li Z, Yang Q, Tian H, et al. Mitochondria-associated ER stress evokes immunogenic cell death through the ROS-PERK-eIF2α pathway under PTT/CDT combined therapy. Acta Biomater. 2023;160:211–24. [DOI] [PubMed] [Google Scholar]

[CR32] 32.Chen Z, Zhu X, Xiong J, Wen Z, Cheng G. A p-n junction by coupling amine-enriched brookite-TiO₂ nanorods with CuxS nanoparticles for improved photocatalytic CO₂ reduction. Mater (Basel). 2023;16(3):960. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Chen Z, Li Z, Huang H, Shen G, Ren Y, Mao X, et al. Cancer immunotherapy based on cell membrane-coated nanocomposites augmenting cGAS/STING activation by efferocytosis blockade. Small. 2023;19(43):e2302758. [DOI] [PubMed] [Google Scholar]

[CR34] 34.Guo W, Chen Z, Li Z, Huang H, Ren Y, Zhao B, et al. Improved immunotherapy for gastric cancer by nanocomposites with capability of triggering dual-damage of nuclear/mitochondrial DNA and cGAS/STING-mediated innate immunity. Chem Eng J. 2022;443:136428. [Google Scholar]

[CR35] 35.Guo W, Chen Z, Li Z, Huang H, Ren Y, Li Z, et al. Cancer cell membrane biomimetic mesoporous silica nanotheranostics for enhanced ferroptosis-mediated immuogenic cell death on gastric cancer. Chem Eng J. 2023;455:140868. [Google Scholar]

[CR36] 36.Xiao T, Hu W, Fan Y, Shen M, Shi X. Macrophage-mediated tumor homing of hyaluronic acid nanogels loaded with polypyrrole and anticancer drug for targeted combinational photothermo-chemotherapy. Theranostics. 2021;11(14):7057–71. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Xu X, Pang Y, Fan X. Mitochondria in oxidative stress, inflammation and aging: from mechanisms to therapeutic advances. Signal Transduct Target Ther. 2025;10(1):190. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Drilon A, Jenkins C, Iyer S, Schoenfeld A, Keddy C, Davare MA. ROS1-dependent cancers: biology, diagnostics and therapeutics. Nat Rev Clin Oncol. 2021;18(1):35–55. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Glorieux C, Liu S, Trachootham D, Huang P. Targeting ROS in cancer: rationale and strategies. Nat Rev Drug Discov. 2024;23(8):583–606. [DOI] [PubMed] [Google Scholar]

[CR40] 40.Friedmann Angeli JP, Krysko DV, Conrad M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405–14. [DOI] [PubMed] [Google Scholar]

[CR41] 41.Yu C, Chen D, Feng L, Zang P, He G, Liu J, et al. Piezocatalytic nanoplatform drives Mg2+-release-amplified ferroptosis-immune synergy for tumor microenvironment reprogramming. ACS Nano. 2026;20(12):10024–42. [DOI] [PubMed] [Google Scholar]

[CR42] 42.Wei X, Jiang Y, Chenwu F, Li Z, Wan J, Li Z, et al. Synergistic ferroptosis-immunotherapy nanoplatforms: multidimensional engineering for tumor microenvironment remodeling and therapeutic optimization. Nano Micro Lett. 2026;18(1):56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Zhang M, Yang D, Dong C, Huang H, Feng G, Chen Q, et al. Two-dimensional MXene-originated in situ nanosonosensitizer generation for augmented and synergistic sonodynamic tumor nanotherapy. ACS Nano. 2022;16(6):9938–52. [DOI] [PubMed] [Google Scholar]

[CR44] 44.Cheng R, Li Z, Luo W, Chen H, Deng T, Gong Z, et al. A copper-based photothermal-responsive nanoplatform reprograms tumor immunogenicity via self-amplified cuproptosis for synergistic cancer therapy. Adv Sci (Weinh). 2025;12(19):e2500652. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Chen L, Min J, Wang F. Copper homeostasis and cuproptosis in health and disease. Signal Transduct Target Ther. 2022;7(1):378. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Wang Y, Luo J, Alu A, Han X, Wei Y, Wei X. cGAS-STING pathway in cancer biotherapy. Mol Cancer. 2020;19(1):136. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR47] 47.Samson N, Ablasser A. The cGAS-STING pathway and cancer. Nat Cancer. 2022;3(12):1452–63. [DOI] [PubMed] [Google Scholar]

[CR48] 48.Meier P, Legrand AJ, Adam D, Silke J. Immunogenic cell death in cancer: targeting necroptosis to induce antitumour immunity. Nat Rev Cancer. 2024;24(5):299–315. [DOI] [PubMed] [Google Scholar]

[CR49] 49.Yao H, He Q, Wei S, Xiang L, Luo Y, Huang C, et al. Targeted inhibition of macrophage STING signaling alleviates inflammatory injury and ventricular remodeling in acute myocardial infarction. Acta Pharm Sin B. 2025;15(8):4030–46. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR50] 50.Fan X, Han J, Zhong L, Zheng W, Shao R, Zhang Y, et al. Macrophage-derived GSDMD plays an essential role in atherosclerosis and cross talk between macrophages via the mitochondria-STING-IRF3/NF-κB axis. Arterioscler Thromb Vasc Biol. 2024;44(6):1365–78. [DOI] [PubMed] [Google Scholar]

[CR51] 51.Li Z, Li M, Yang L, Chen J, Ye Q, Qian W, et al. Oncolytic vaccinia virus armed with anti-CD47 nanobody elicit potent antitumor effects on multiple tumor models via enhancing innate and adoptive immunity. J Immunother Cancer. 2024;12(12):e009473. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR52] 52.Wan M, Pan S, Shan B, Diao H, Jin H, Wang Z, et al. Lipid metabolic reprograming: the unsung hero in breast cancer progression and tumor microenvironment. Mol Cancer. 2025;24(1):61. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Ultrasound-activated Cu2−XSe/TiO2 heterostructured nanosonosensitizer enables coordinated induction of ferroptosis and cuproptosis for tumor immunotherapy

Shibing Wang

Tingting Deng

Xue Yang

Yuan Chen

Yuli Zhou

Hairui Tian

Jiarui Li

Meili Li

Teng Lin

Cong Huang

Xiangmin Tong

Abstract

Graphical abstract

Supplementary Information

Introduction

Scheme 1.

Results and discussion

Design and characterization of TCH NPs

Fig. 1.

CDT/SDT therapeutic effects triggered by TiO2@Cu2−XSe NPs

Fig. 2.

Active targeting, internalization, and safety evaluation of TCH NPs in vitro and in vivo

Fig. 3.

In vitro anticancer efficacy and ROS-induced mitochondrial dysfunction under combined CDT and SDT

Fig. 4.

TCH-induced ferroptosis through copper-dependent autophagic degradation of GPX4

Fig. 5.

TCH-induced cuproptosis and cGAS–STING pathway activation under combined SDT/CDT treatment

Fig. 6.

Ferroptosis- and cuproptosis-mediated immunogenic cell death and M2-to-M1-like macrophage repolarization under combined CDT/SDT treatment

Fig. 7.

In vivo antitumor efficacy of TCH NPs combined with US irradiation

Fig. 8.

TCH-US treatment reshapes the tumor immune microenvironment

Fig. 9.

In vivo tumor immune microenvironment remodeling mediated by TCH NPs

Fig. 10.

In vivo antitumor efficacy and immune activation following combination therapy with anti–PD-L1

Fig. 11.

Conclusion

Supplementary Information

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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Ultrasound-activated Cu_2−XSe/TiO₂ heterostructured nanosonosensitizer enables coordinated induction of ferroptosis and cuproptosis for tumor immunotherapy

CDT/SDT therapeutic effects triggered by TiO₂@Cu_2−XSe NPs