Thermoradiotherapy-Driven Enhancement of Cuproptosis by Copper-Nitroimidazole Based Nanoparticles

Abstract

Introduction

Cuproptosis, a novel form of cell death tied to copper homeostasis and protein lipoylation, holds significant promise for breast cancer treatment. However, its efficacy is severely hindered by the tumor microenvironment (TME) heterogeneity, such as hypoxia and elevated glutathione (GSH) levels.

Methods

Herein, we synthesized CuNI nanoparticles via a facile hydrothermal method, which could serve as both a copper carrier and a photothermal agent, to enhance the accumulation of copper in tumor site. Following intravenous injection, CuNI accumulated and persisted in tumors via the enhanced permeability and retention effect (EPR) effect. Subsequent gradient 808 nm laser irradiation and radiotherapy (RT) were administered, CuNI could convert light energy to heat energy, which could alleviate hypoxia TME, while RT further depleted GSH and synergistically generates reactive oxygen species (ROS) with CuNI, synergistically amplifying CuNi-mediated cuproptosis.

Results

This co-treatment triggered immunogenic cell death (ICD), activating dendritic cells and T-cell responses to reverse the “cold” immune microenvironment. In vivo studies demonstrated complete tumor suppression with no overt toxicity.

Conclusion

The CuNI + NIR + RT strategy, leveraging “cuproptosis/ICD synergy”, offers a novel paradigm for the clinical translation of cuproptosis in breast cancer.

Introduction

Breast cancer remains a major clinical challenge, as existing therapies targeting canonical cell death mechanisms often have suboptimal outcomes due to the complex tumor microenvironment (TME).^1–4 Exploring new cell death pathways to enhance efficacy is a key focus in tumor treatment research. Copper, an essential micronutrient, acts as a critical catalytic cofactor in energy metabolism and signal transduction. The newly discovered cuproptosis, a non-apoptotic cell death modality mediated by copper homeostasis dysregulation, shows great potential in tumor therapy.^5,6 Its molecular mechanism is mainly regulated by ferredoxin 1 (FDX1)-driven protein lipoylation: excess copper ions, after FDX1-mediated modification, directly bind to dihydrolipoamide S-acetyltransferase (DLAT) in the tricarboxylic acid (TCA) cycle, inducing its oligomerization and triggering cytotoxicity.⁷ Meanwhile, FDX1 reduces Cu²⁺ to Cu⁺, which further destabilizes iron-sulfur clusters. These processes collectively provoke proteotoxic stress, ultimately leading to tumor cell death.^5,6,8–10 However, the unique pathological features of TME severely limit cuproptosis efficacy: hypoxic conditions upregulate the HIF-1α pathway, inhibiting DLAT expression and blocking cuproptosis; high glutathione (GSH) levels in tumors maintain redox homeostasis, scavenging reactive oxygen species (ROS) and chelating free copper ions to weaken cuproptosis.^11–13 Thus, overcoming TME limitations to efficiently enhance cuproptosis is a critical issue.

Multimodal synergistic therapeutic strategies, particularly those leveraging stimuli-responsive nanoplatforms to activate immunity, represent a transformative approach that may revolutionize precision oncology.¹⁴ Photothermal therapy (PTT), an emerging physical treatment, can directly improve the hypoxic microenvironment by dilating tumor blood vessels and increasing blood perfusion, while downregulating HIF-1α expression to relieve hypoxia-induced suppression of cuproptosis.^15–19 The hyperthermia generated by photothermal therapy can directly ablate tumor cells, thereby promoting immunogenic cell death (ICD).^1,20–22 Radiotherapy (RT) induces ROS bursts via ionizing radiation, depletes intracellular GSH in tumor cells, disrupts redox homeostasis, and further amplifies cuproptosis.^23–29 More importantly, massive ROS from PTT-RT synergy triggers immunogenic cell death (ICD), promotes dendritic cell (DC) maturation and cytotoxic T cell infiltration, effectively activates the “cold” tumor immune microenvironment, and forms long-term anti-tumor immune responses.^19,30,31

Considering this, we first engineered a copper ion delivery system, CuNI nanoparticles, and employed gradient 808 nm laser irradiation and RT to directly potentiate cuproptosis, while further triggering immunogenic cell death (ICD) and immune activation (Scheme 1). Administered via tail vein injection, CuNI reached the tumor site and persisted long-term. Initially, 808 nm NIR-I laser irradiation triggers PTT: it alleviates tumor hypoxia to relieve cuproptosis inhibition, and generates ROS through photothermal conversion property of CuNI, laying the foundation for subsequent therapy. RT further depleted GSH, elevated ROS levels, and synergized with PTT to enhance CuNI-mediated cuproptosis. This multimodal strategy integrated advantages of PTT, RT and cuproptosis, and activated systemic anti-tumor immunity via ICD, achieving durable tumor eradication. In vivo experiments confirmed that CuNI combined with NIR + RT completely inhibited tumor growth without obvious toxicity via the “synergistic cuproptosis/ICD activation” mode. In summary, this novel synergistic therapeutic system provided new ideas to break through clinical application bottlenecks of cuproptosis, promising to be a new paradigm for breast cancer treatment.

Scheme 1.

Schematic illustration of photothermal-radio therapy-driven enhancement of cuproptosis by copper-nitroimidazole based nanoparticles.

Results and Discussion

Initially, through regulating the molar ratio of Cu²⁺ to dinitroimidazole (2-NI), CuNI nanoparticles with tunable particle sizes were successfully synthesized via a facile hydrothermal route under ambient temperature conditions. As illustrated in Figure 1A, transmission electron microscopy (TEM) images reveal that CuNI nanoparticles exhibit a square morphology, with an average particle size of approximately 50–60 nm. Previous studies have corroborated that nanomaterials within the particle size range of 30–200 nm possess superior stability and enhanced surface activity.³² Figure 1B confirmed the homogeneous distribution of Cu and N elements throughout the CuNI nanoparticles. To further evaluate their dispersibility, CuNI nanoparticles were co-incubated with PBS buffer. As presented in Figure 1C, dynamic light scattering (DLS) measurements were performed continuously for three days to monitor the particle size of CuNI, and the results demonstrated that CuNI nanoparticles exhibit excellent dispersibility and stability. Furthermore, it has been well-documented that nanoparticles within this specific size range can accumulate in tumor tissues via the enhanced permeability and retention (EPR) effect, while evading premature renal filtration.³³ Subsequently, CuNI nanoparticles were subjected to further characterization using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), with a focus on analyzing the crystal structure and surface chemical composition/valence states of the material. The XRD results revealed that, in contrast to NI, CuNI exhibits a distinct metal-organic framework (MOF)-like crystal structure, which is ascribed to the chelation between Cu²⁺ and NI, along with the deprotonation of triethylamine and its regulatory effect on particle size (Figure 1D). Figure 1E–F displayed the binding energy of Cu. In addition, Figure 1F depicted the Cu 2p spectra showing two peaks at 955.1 and 935.1 eV, which presumably are the 2p_1/2 and 2p_3/2 peaks of Cu, respectively. XPS also revealed the presence of its oscillatory satellite peaks. Additionally, a good photothermal agent can respond to an 808 nm laser to generate thermal energy. Figure 1G presented the temperature change curves of CuNI with different concentrations after being irradiated by the 808 nm laser. Taking PBS as a control group, after 5 minutes of irradiation, the temperature of the PBS solution shows almost no obvious change, while the temperature of 100 μg/mL CuNI can rise to 42.1°C under the same conditions (Figure 1G). Figures S1-2 showed the on/off cycle of the laser for 3 times of CuNI. After 3 consecutive irradiations, the photothermal performance of CuNI remains unchanged, confirming its photothermal stability. CuNI can catalyze H₂O₂ to generate reactive oxygen species (ROS), thereby directly killing tumor tissues. To verify this property, we conducted a methylene blue (MB) degradation experiment. The core principle is to utilize peroxidase (POD)-like activity to catalyze H₂O₂ to oxidize methylene blue (a redox indicator), causing methylene blue to change from blue to colorless, and the characteristic peaks were verified by UV-Vis absorption spectroscopy. With the increase of CuNI concentration, its ability to degrade MB gradually becomes stronger (Figure 1H). CuNI can respond to glutathione (GSH) degradation and release Cu²⁺. As shown in Figure 1I, CuNI can exist stably without GSH, while after co-incubation with GSH, the nanostructure of CuNI is destroyed, thereby releasing Cu²⁺. This result indicates that CuNI is expected to release Cu²⁺ in the TME with high GSH, thereby triggering cuproptosis.

Figure 1.

Morphology and characterization of CuNI. (A) TEM image of CuNI nanoparticles. (B) HAADF-STEM image of CuNI. Scale bar: 100 nm. (C) hydrodynamic diameter of CuNI in PBS. (D) The XRD patterns of CuNI. (E) XPS spectra of CuNI. (F) Cu 2p high-resolution XPS spectra of CuPy-Au. (G) Temperature curves of CuNI (0, 25, 50 and 100 μg/mL) after 808 nm laser irradiation. (H) UV−vis absorbance of MB solution reacted with H₂O₂ + CuNI (0, 25, 50, 100 and 200 μg/mL). (I) Release curves of Cu²⁺ in CuNI with and without GSH.

After completing the characterization experiments of CuNI, to gain deeper insights into the anti-tumor effects of CuNI, we further expanded our investigations to cellular experiments. Initially, to investigate the anti-tumor proliferation effect of the combined treatment regimen, cell colony formation assays were conducted under both normoxic and hypoxic conditions. As shown in Figure 2A and B, the survival fraction of the CuNI + NIR + RT group was significantly lower than that of the other groups, directly proving that this combined strategy can greatly inhibit cell survival, and achieve the optimal killing effect. Inspired by its in vitro antitumor efficacy, we further explored its mechanism of action. Glutathione (GSH), as an endogenous antioxidant in tumor cells, can prevent cell damage by scavenging reactive oxygen species (ROS) generated by RT. As shown in Figure 2C, CuNI can enhance the efficacy of RT by depleting intracellular GSH. However, hypoxia (Figure S3) can attenuate this effect to a certain extent. The γ-H₂AX fluorescence assay was performed on the cells of each experimental group. γ-H₂AX, as a phosphorylated form of histone H₂AX, is a classic biomarker for DNA damage (such as double-strand breaks), and the red fluorescent foci correspond to DNA damage sites.³⁴ Under normoxic conditions, red γ-H₂AX fluorescence appeared in the RT group, indicating that RT can induce DNA damage under normoxia, while there was almost no red fluorescence in the CuNI group, and the red fluorescence was significantly enhanced in the CuNI + NIR + RT group. This observation indicated that the CuNI material alone exhibited a limited capacity to induce DNA damage. In contrast, the combined treatment regimen of CuNI with NIR irradiation and RT can further augment DNA damage, thereby exerting a synergistic therapeutic effect. Figure 2D demonstrated that the hypoxic environment inhibited RT-induced DNA damage. Nonetheless, the combined treatment of CuNI + NIR + RT still achieves a moderate level of DNA-damaging effect. The focus density of γ-H₂AX is positively correlated with the degree of DNA double-strand breaks and the risk of cell death, the quantitative results in Figures S4 and S5 showed that the focus density of γ-H₂AX in the CuNI + NIR + RT group was significantly higher than that in the other groups. We quantitatively detected the distribution map of CuNI entering cells in different time periods by ICP, and the results showed that CuNI would gradually accumulate in tumor cells, which was expected to cause the destruction of redox homeostasis (Figure S6). CuNI could trigger cuproptosis through the responsive release of Cu²⁺, while GSH depletion by RT and the photothermal effect induced by NIR irradiation can further promote the occurrence of cuproptosis. Oligomerization of dihydrolipoamide S-acetyltransferase (DLAT) is recognized as one of the canonical hallmarks of cuproptosis. Therefore, the fluorescent expression of DLAT in different treatment groups was monitored to verify the cuproptosis-inducing effect of CuNI. As clearly illustrated in Figure 2E, CuNI in combination with NIR irradiation and RT effectively triggered DLAT oligomerization and thereby induced cuproptosis. However, this cuproptosis-inducing effect was compromised under hypoxic conditions. As illustrated in Figure 2F and G, the Western blot results showed that the expression of FDX1, a key protein involved in cuproptosis, was significantly downregulated in the CuNI + NIR + RT group, which corresponded to the DNA damage and DLAT fluorescence results under both normoxic and hypoxic conditions.

Figure 2.

In vitro anti-tumor effect of CuNI. (A) Colony formation assay results for 4T1 cells treated with different groups under normoxic or (B) hypoxic conditions. (C) GSH level of different groups. (D) DAPI and γ-H₂AX staining were used to visualize nuclear condensation and DNA fragmentation in cells treated as indicated under normoxic or hypoxic conditions. Scale bar: 20 μm. (E) DLAT fluorescence images of cancer cells after the indicated treatment. Scale bar: 20 μm. (F) Fe−S cluster protein expression under normoxic or hypoxic conditions. (G) gray analysis of FDX1 in tumor cells after the indicated treatments. ***P < 0.005; Student’s t-test.

PTT and RT can induce the release of damage-associated molecular patterns (DAMPs) and, in synergy with cuproptosis, enhance immunogenic cell death (ICD), thereby further triggering an immune activation effect.³⁵ Upon the induction of ICD in tumor cells, calreticulin (CRT) is exposed on the surface of dying tumor cells, while high-mobility group box 1 (HMGB1) is released from the cell nucleus. These DAMPs can promote the maturation of dendritic cells and the infiltration of tumor-specific T cells, thereby ameliorating the tumor microenvironment.³⁶ As shown in Figure 3A, the control group exhibits no detectable CRT expression. In contrast, the CuNI + NIR group achieved a certain degree of CRT translocation and exposure on the cell surface, while the CuNI + NIR + RT group demonstrated the strongest CRT expression. Following treatment with the CuNI + NIR + RT regimen, the intracellular HMGB1 green fluorescence was nearly completely diminished, and the extent of HMGB1 from the cells to the extracellular environment was most pronounced (Figure 3B), indicating that this combination therapy can robustly induce ICD. The combination of CuNI with external stimuli, including RT and NIR laser, is anticipated to enhance immunogenic cell death (Figure 3C). In Figure 3D, the double-positive scatter plot of the CuNI + NIR + RT group was denser and had a higher proportion, suggesting that this combined treatment significantly enhances the co-expression of CD80 and CD86, promoting the maturation of DCs, creating favorable conditions for the activation of T cells, and is beneficial to the anti-tumor immune response. Figure 3E demonstrated that CuNI + NIR + RT group could induce a remarkable ATP release. Figure 3F showed the quantization result of DC maturity. Figure 3G and H present the effects of different treatments on the cellular immune response from different immune-related indicators. Among them, the ATP release, IL-6 secretion, and TNF-α secretion of the CuNI + NIR + RT group were all significantly higher than those of other groups. This indicated that this combined treatment can most effectively induce ATP release to initiate the first step of the immune response, strongly promote DC maturation to provide a key prerequisite for T cell activation, amplify the anti-tumor immune signal, significantly induce the killing of tumor cells, and have the strongest effect in activating the immune microenvironment. Through the synergistic effect of multiple links, by means of molecular events related to ICD and changes in immune indicators, it remodels the tumor immune microenvironment and promotes the efficient initiation and enhancement of the anti-tumor immune response.

Figure 3.

Efficient induced immunogenic cell death of the 4T1 tumor cells by CuNI. (A) Schematic illustration of CuNI + NIR + RT-based enhancing immunogenic cell death. (B) Immunofluorescence image of CRT in different groups. (C) Immunofluorescence image of HMGB1 in different groups. (D) Representative flow cytometry plots in response to different treatments. (E) Adenosine triphosphate (ATP) secretion in the cell medium after various treatments. (F) Quantitative analysis of matured DCs. (G) Levels of the cytokine IL-6 and (H) TNF-α as determined by ELISA. ***P < 0.005; Student’s t-test.

Buoyed by the aforementioned findings, we proceeded to assess the in vivo therapeutic performance of CuNI. We utilized BALB/c mice bearing 4T1 tumor as animal model to verify the anti-tumor efficacy and mechanism of action of the CuNI + NIR + RT combined therapy. First, after intravenous injection of CuNI, we sacrificed the mice and harvested their primary organs for analysis (Figure S7). The results confirm that CuNI exhibits significant tumor accumulation via the enhanced permeability and retention effect, with the highest concentration detected in tumor tissues at 12 hours post-intraperitoneal administration. Then, the mice were randomly divided into the following five groups: (1) PBS + NIR; (2) RT; (3) CuNI; (4) CuNI + NIR and (5) CuNI + NIR + RT. Figure 4A presented the standardized experimental procedure for tumor treatment in animals. Through the standardized process of modeling, treatment and monitoring, the reproducibility of the experiment is ensured, so as to verify the anti-tumor efficacy of this combined therapy. As shown in Figure 4B, the tumor volumes of mice in the PBS + NIR group increased rapidly. In comparison, the RT group exerted a slight inhibitory effect on tumor volume, while the CuNI + NIR group achieved a certain degree of tumor-killing effect through photothermal effect and cuproptosis. Notably, the tumor volume in the CuNI + NIR + RT group decreased, demonstrating a relatively strong inhibitory effect on tumor growth. The tumor weight of the CuNI + NIR + RT group was significantly lighter than that of the other four groups, directly confirming that its tumor-inhibiting ability is optimal (Figure 4C). Figure 4D shows the body weight changes of the mice in each group at different time points. A stable body weight reflects good safety, and the CuNI + NIR + RT group showed no obvious body weight loss, indicating that the combined therapy did not cause severe toxicity and had controllable safety. To further investigate in vivo cytotoxic mechanism of CuNI, tumor tissues were collected from the mice following the completion of treatment and subjected to various histological staining assays. In the immunofluorescence staining of Figure 4E, when staining for HIF-1α, the weak green fluorescence in the CuNI + NIR + RT group indicated that the combined therapy improved the hypoxic tumor microenvironment; when staining for γ-H₂AX, the strong red fluorescence in this group proved that it efficiently induced tumor DNA damage; when staining for DLAT, the intensified green fluorescence indicated that DLAT was largely enhanced, triggering and promoting cuproptosis to induce tumor cell death; under H&E staining, the tumor tissue of the CuNI + NIR + RT group showed a disordered structure and large necrotic areas, intuitively demonstrating the strong killing effect of the combined therapy on tumors. Overall, it demonstrates the anti-tumor advantages and mechanism of action of the CuNI + NIR + RT combined therapy from multiple dimensions.

Figure 4.

In vivo antitumor effects of CuNI-based therapy. (A) Schematic of the in vivo treatment timeline. (B) Tumor growth curves. (C) Final tumor weights. (D) Mice body weight changes. (E) Tumor tissue staining for HIF-1α, γ-H₂AX, DLAT, and H&E to assess efficacy. Scale bar: 100 μm. ***P < 0.005; Student’s t-test.

To investigate the activation effects of different treatment regimens on immune cells in the tumor immune microenvironment, we verified the immune synergistic effects of the combined treatment of CuNI + NIR + RT from the functional dimensions of DC maturation and T cell infiltration. DC cells, as the core for initiating and regulating ICD, play a crucial role by activating T cells. In the CuNI + NIR + RT group, DCs showed the optimal maturation status: the representative flow cytometry results showed that the peak maturation degree reached about 28.2%, which was 2.7 times that of the RT group and 1.6 times that of the CuNI + NIR group (Figure 5A, C), fully confirming that this combined treatment could effectively promote DC maturation and lay a solid foundation for T cell activation. Further detection revealed that the number of CD8⁺T cells in the CuNI + NIR + RT group increased significantly (Figure 5B and D), which was consistent with the phenomenon of the highest degree of T cell activation in the tumor, indicating that this combined regimen can efficiently recruit and activate T cells. Meanwhile, as shown in Figure 5E and F, the levels of TNF-α and interferon-γ (IFN-γ) in the tumor microenvironment of the CuNI + NIR + RT group were significantly higher than those in other groups, and the tumor killing effect was enhanced by the higher secretion of cytokines. After the completion of treatment, organ sections were collected and subjected to hematoxylin and eosin (H&E) staining in order to evaluate CuNI + NIR + RT treatment-related histopathological changes in major organs (heart, liver, spleen, lung and kidney). As shown in Figure S8, no inflammatory responses or tissue damage were observed in either the PBS + NIR group or the CuNI + NIR + RT group, indicating that the synergistic therapeutic strategy involving CuNI combined with dual external irradiation (RT and NIR) not only exerts favorable in vitro and in vivo therapeutic efficacy, but also exhibits excellent biosafety and biocompatibility.

Figure 5.

Activation of anti-tumor immunity by CuNI-based therapy. (A) Flow plots of CD80+ and CD86+ DCs. (B) Flow plots of CD4+ and CD8+ T cells. (C) Quantified CD80+ and CD86+ DCs. (D) Quantified CD8 T cells. (E) ELISA measurements of secretions of TNF-α. (F) ELISA measurements of secretions of IFN-γ. ***P < 0.005; Student’s t-test.

Experimental Section

Materials and Reagents

All the reagent kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. The other solvents and chemical reagents used in this work were purchased from Sinopharm Chemical Reagent (China) and Aladdin-Reagent (China). All antibodies were purchased from Beijing Biosynthesis Biotechnology (China).

Characterization of CuNI NPs

The morphology and structure of CuNI were observed by transmission electron microscopy (TEM) (JEM-F200, Japan). X-ray diffraction patterns were recorded using a Bruker D8 Advance X-ray diffractometer with desorption measurement. The particle size was measured by DLS (Malvern Zeta sizer Nano ZS90). The release experiment of Cu²⁺, Photothermal conversion ability and detection of ·OH were carried out according to the previous work.^5,6,18

Cell Culture and Relative Experiments

4T1 cell line were obtained from the Cell Bank of the Chinese Academy of Sciences and incubated in RPMI-1640 medium supplemented with 10% FBS in a humidified atmosphere. Cells were incubated with 5 different groups for 12 h under hypoxia or normoxia condition: (1) PBS + NIR; (2) RT (4Gy); (3) CuNI; (4) CuNI + RT; (5) CuNI + NIR + RT. The CuNI concentration was 50 μg/mL in group 3, 4 and 5. Then, cells in group 1 and 5 were irradiated with the 808 nm laser at a power density of 0.5 W/cm² for 5 min. Subsequently, cells in groups 2, 4 and 5 were exposed to 4 Gy X-ray irradiation. Subsequently, relevant commercial detection kits (following their corresponding experimental protocols), fluorescent staining kits, and antibodies were used for staining experiments.^17,37,38 For the clonogenic proliferation assay, crystal violet staining was performed.

In vitro Marrow-Derived Dendritic Cells Maturation Study

BMDCs were isolated from 8-week-old Balb/c mice bone marrow. For BMDCs maturation assay, 8×10⁴ 4T1 cells were treated with 5 different groups: (1) PBS + NIR; (2) RT (4Gy); (3) CuNI; (4) CuNI + RT; (5) CuNI + NIR + RT. Then, the treated 4T1 cells were cocultured with 1×10⁶ BMDCs. Then BMDCs were stained with anti-CD11c, anti-CD80, and anti-CD86. Finally, the cells were sorted using flow cytometer (Beckman-Coulter, USA). The secretion levels of cytokines including TNF-α and IL-6 in the samples were tested with ELISA kits.

Animal Tumor Models

Female Balb/c mice aged 4–6 week were purchased from Vital River Company (Beijing, China). 100 μL of 4T1 cell suspension (1×10⁶ cells) were subcutaneously injected into each mouse to establish the tumor models. The animal experiments were carried out according to the protocol approved by the Ministry of Health in People’s Republic of PR China and were approved by the Administrative Committee on Animal Research of Guangxi Medical University (Approval number: 2025-KYL (022)).

All anesthesia procedures for vertebrate subjects in this study were designed and implemented in strict accordance with internationally recognized ethical guidelines for animal experiments (including relevant standards of the American Veterinary Medical Association, AVMA).

In vivo Biodistribution Study

In the quantitative biodistribution analysis, mice were intravenously injected with 200 μL CuNI. At 6 h, 12 h, and 24 h post-administration, the mice were sacrificed, and the main organs, including the heart, liver, spleen, lung, kidney and stomach, were excised and weighed. Then, the Cu content in the samples was analyzed using ICP-AES.

In vivo Antitumor Study

The 4T1 tumor model was used. When tumors reached about 200 mm³, tumor bearing mice were divided randomly into 5 different groups (n=5): (1) PBS + NIR; (2) RT (4Gy); (3) CuNI; (4) CuNI + RT; (5) CuNI + NIR + RT. The CuNI dose was 5 mg/kg. The NIR and RT (4 Gy) performed 12 h after intravenous injection. Mice body weight was monitored every 3 days. After treatment, all the mice were sacrificed. The blood samples from these mice were collected for blood biochemistry analysis. Five main organs (heart, liver, spleen, lung and kidney) and tumors of all mice were harvested, washed with PBS, and fixed with paraformaldehyde for histological analysis. And the tumor tissues were weighed, and fixed in 4% neutral buffered formalin, processed routinely into paraffin, and sectioned at 4 μm. Then the sections were stained with DLAT, γ-H₂AX, HIF-1α and H&E finally examined by using a confocal laser scanning microscope (CLSM; IX81, Olympus, Japan). Detection of immune activation in vivo were carried out according to the previous work.^39–42

Statistical Analysis

Data analyses were conducted using the GraphPad Prism 5.0 software. Significance between every two groups was calculated by the Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.005.

Conclusions

In conclusion, this study developed a multimodal synergistic therapeutic strategy combining CuNI nanoparticles with NIR and RT. CuNI NPs exhibit distinct physicochemical properties and excellent dispersibility and stability. Under 808 nm laser irradiation, CuNI NPs generate stable photothermal effects to alleviate tumor hypoxia. When combined with RT, this multimodal therapy could disrupt redox homeostasis and works synergistically with Cu²⁺ to boost ROS production, enhancing cuproptosis induction. Experimental results verified the efficacy and favorable biological safety of this strategy from both in vitro and in vivo perspevtives. Furthermore, the combinatorial effects of RT and cuproptosis further promote ICD, which in turn enhances DCs maturation and T-cell infiltration, ultimately activating the immunosuppressive tumor immune microenvironment. This study provides a promising new paradigm for cuproptosis-based anti-tumor treatment.

Funding Statement

This work was supported by grants from the (Guangxi Young Elite Scientist Sponsorship Program (2025YESSGX006), Beijing MDK Public Welfare Foundation Research Fund (No. MDK 2022-1001), Pandeng Fund of Harbin Medical University Cancer Hospital (No. PDTS 2024A-03) and Postdoctoral Scientific Research Developmental Fund of Heilongjiang (No. LBH-Q22).

Disclosure

The authors declare no competing interests in this work.