Oxygen‐driven cuproptosis synergizes with radiotherapy to potentiate tumor immunotherapy

The immunological implications of cuproptosis, a form of cell death highly sensitive to oxygen presence, remain largely unexplored in the context of tumor immunotherapy. Herein, we initially investigate the positive correlation between cuproptosis and tumor immunotherapy through bioinformatics analysis. Subsequently, an oxygen generator loaded with copper ions (Cu/APH‐M) has been constructed, which serves as an effective carrier of copper ions and crucially enhances the oxygenation of the tumor microenvironment. Importantly, Cu/APH‐M‐mediated dual strengthening of cuproptosis and radiotherapy could not only trigger a powerful antitumor immunity related to immunogenic cell death by RNA‐sequencing analysis, but also effectively inhibit the growth of both distal and in situ low rectal tumors after combined immunotherapy, creating a robust immune memory effect. Our work reveals the beneficial effects of enhanced cuproptosis in radio‐immunotherapy and elucidates its underlying mechanisms, which provides a novel approach for the synergistic integration of cuproptosis with immunotherapy and radiotherapy, broadening the scope of cuproptosis‐mediated tumor therapy.

environments. [1,2]From one perspective, copper serves as a cofactor for numerous enzymes integral to key cellular functions, encompassing mitochondrial respiration and biosynthesis among others. [3,4]From another perspective, copper dysregulation may result in toxicity to cells. [5]uproptosis, a type of regulated cell death induced through excess of Cu 2+ , has been recently discovered. [6]This form of copper-induced programmed cell death is distinct from other cell death pathways, including apoptosis, pyroptosis, necroptosis, and ferroptosis.Copper ions, once transported into the cell, directly bind to lipoamide acylated dihydrolipoamide S-acetyltransferase (DLAT) in the tricarboxylic acid cycle pathway, leading to abnormal aggregation of DLAT protein.
The increase in insoluble DLAT triggers cellular toxicity.Ferredoxin 1 (FDX1), serving as an upstream regulator of protein lipoamide acylation, participates in the regulation of the acylation of proteins, including DLAT.Concurrently, FDX1 reduces Cu 2+ to the more toxic Cu + , inhibiting the synthesis of iron-sulfur cluster proteins in the respiratory chain complex, which induces proteotoxic stress reactions, ultimately culminating in cell death. [7,8]Consequently, the judicious modulation of copper ion concentrations within cancer cells may offer a promising novel strategy for cancer treatment. [9,10]More importantly, it should be noted that cuproptosis is a form of cell death that relies on mitochondrial respiration, implying that the levels of oxygen (O 2 ) would significantly affect the sensitivity of tumor cells to cuproptosis. [11]However, with rapid proliferation of cancer cells often results in the hypoxic microenvironment in the solid tumors. [12,13]Theoretically, the relieving tumor hypoxic microenvironment may effectively enhance the cytotoxic effects of cuproptosis on cancer cells.[16] As a freshly recognized mode of cell death, cuproptosis induced the immunology and its interaction with tumor immunotherapy remain unclear.Therefore, whether cuproptosis could serve as a significant supplement to tumor immunotherapy with low clinical response to improve therapeutic efficiency needs to be further explored.
Similar to cuproptosis, the efficacy of radiotherapy, as one of the most commonly employed cancer treatment strategies in clinical, is also closely related to the tumor hypoxic microenvironment. [17,18]Although radiotherapy, as a local treatment, could induce certain immune effects, [19,20] it only produces abscopal effects in a very small number of individuals. [21,22][25] For instance, radiotherapy could induce angiogenesis, increase the activity of matrix metalloproteinases, [26] and enhance the expression of programmed cell death 1 ligand 1 (PD-L1) in cancer cells. [27,28][34] In the previous work, we utilized bimetallic nanozymes with high catalase (CAT) activity to effectively decompose the overexpressed hydrogen peroxide at the tumor site into oxygen, thereby alleviating the tumor hypoxic microenvironment and enhancing radio-immunotherapy of tumors. [35]Hence, the development of a copper ion carrier based on bimetallic nanozymes provides significant insights for the regulation of the TME and the enhancement of both cuproptosis and immunotherapy.Prussian blue nanoparticles (PB) are a detoxifying agent frequently employed in clinical practice with excellent biosafety. [36]The chemically etched hollow mesoporous Prussian blue nanoparticles (HPB) could not only be used as a drug carrier, but also doped with diversified ions (Mn 2+ , Ni 3+ , Co 3+ , etc.) to form multifunctional hybrid nanoparticles, whose doped metal ions could be released under mildly acidic conditions. [37,38]n this work, Au-Pt bimetallic nanozymes were in situ coated on HPB with a one-step reduction method, and then doped with Cu 2+ to prepare a copper-ion-loaded oxygen generator carrier ( Cu/AP H-M), which not only served as an effective carrier of copper ions but also efficiently relieved the tumor hypoxic microenvironment, thereby simultaneously enhancing the efficacy of both cuproptosis and radiotherapy.Interestingly, through RNA-sequencing (RNA-seq) analysis and in vivo assessment, we found that such dual-enhancement therapy mediated by Cu/AP H-M could also trigger a robust antitumor immunity (such as promoting the upregulation of Caspase-1, Calreticulin, heat shock protein (HSP70), HSP90, TNF-α, adenosine-triphosphate (ATP), etc., and reversing the upregulation of tumor PD-L1 induced by radiotherapy).When combined with immunotherapy, it effectively inhibited the growth of distant and in situ low rectal tumors, producing a long-term immune memory effect (Figure 1A).Our work, commencing with O 2 , reported the beneficial role of augmented cuproptosis in both radiotherapy and immunotherapy, elucidated its underlying mechanisms, and broadened the application spectrum of cuproptosis in tumor therapy, opening up new avenues for the integrative combination of cuproptosis, radiotherapy, and immunotherapy in clinical.

Correlation of cuproptosis-related genes with cancer immunotherapy
Lipoylation is a post-translational modification based on mitochondrial lipids that is critical for cuproptosis.FDX1 serves as an upstream regulator of protein lipoylation modification.On the one hand, it is involved in regulating lipoylation of proteins (including DLAT).On the other hand, FDX1 reduces Cu 2+ to the more toxic Cu + , leading to the inhibition of Fe-S cluster protein synthesis and inducing cell death.LIAS is a member of the lipoic acid synthase family and catalyzes the synthesis of lipoic acid.DLAT is the substrate protein for lipoylation.Cu + directly binds to lipoylated DLAT, causing the oligomerization of DLAT and triggering cell death.Therefore, these three genes are the key genes that promote the occurrence of cuproptosis.To investigate the immunological significance of cuproptosis and its potential as a crucial enhancer for tumor immunotherapy, high-throughput sequencing data of mRNA gene expression profiles in colorectal cancer were downloaded from the public database, the cancer genome atlas (TCGA) and a total of 183 colorectal cancer samples, categorized as MSI-H and MSI-L, were selected for further analysis.Initially, 34 immunogenic cell death (ICD)-related genes and 13 key cuproptosisrelated genes, including genes promoting cuproptosis: LIAS, DLD, DLAT, FDX1, LIPT1, SLC31A1, PDHA1, PDHB, and genes inhibiting cuproptosis: ATP7B, ATP7A, MTF1, GLS, CDKN2A were obtained and three key cuproptosis promoting genes (LIAS, DLAT, and FDX1) were selected to conduct a Mantel test analysis with ICD-related genes, aiming to validate the correlation between cuproptosis and ICD.Presented in Figure 1B, it was observed that genes promoting cuproptosis correlated positively with the majority of ICD-related genes.Furthermore, an analysis on the level of expression of DLAT, FDX1, and LIAS in relation to immune cells infiltration was performed in the tumor immune microenvironment.The bubble demonstrated a close correlation between the infiltration of immune cells and the expression of DLAT, a key protein in cuproptosis (Figure 1C).Moreover, through scatter plots analysis, the expression of DLAT was positively correlated with the expression of macrophages, dendritic cells (DCs), CD8 + T cells, and natural killer cells in tumor tissue (Figure 1D).Meanwhile, by conducting a consensus clustering analysis on the chip expression profile data of cuproptosis-related genes in the aforementioned 183 colorectal cancer samples, two distinct patterns, cluster C1 and C2, were identified.The heatmap of the consensus matrix presented clear boundaries, demonstrating the accuracy and robustness of the clustering results (Figure S1a).Moreover, the gene set enrichment analysis (GSVA) algorithm was also employed to assess the cuproptosis activity levels in each patient.As depicted in Figure S1b, the cuproptosis level in the C1 group was markedly higher in comparison to the C2 group, further indicating a higher activity level of cuproptosis in the C1 subgroup.Further, the Mcp counter algorithm was utilized to calculate the level of immune cell infiltration in each sample.The box plot results revealed that immune cells infiltration, such as B cells and T cells, was higher in the C1 group in the presence of elevated incidence of cuproptosis (Figure S1c), suggesting that cuproptosis might facilitate the infiltration of immune cells in tumor.In brief, the aforementioned bioinformatics analysis indicated a robust correlation between the relevant genes promoting cuproptosis and the antitumor immune response.Therefore, theoretically, by augmenting cuproptosis within tumors could potentially provide a significant assistance for tumor immunotherapy.

Construction and evaluation of Cu/AP H-M oxygen generator
Cuproptosis is a type of cell death that relies on mitochondrial respiration, thus the degree of oxygen could notably influence the sensitivity of cancer cells to cuproptosis.Solid tumors are often hypoxic, so optimizing the hypoxic microenvironment of the tumor could enhance the cytotoxicity of cuproptosis.To this end, we designed a copper-ion-loaded oxygen generator carrier ( Cu/AP H-M), hoping that it could effectively deliver Cu 2+ and relieve the tumor hypoxic microenvironment, thereby enhancing both the efficacy of cuproptosis and radiotherapy.First, PB were prepared by hydrothermal synthesis and then etched to obtain HPB.Subsequently, a one-step reduction method was employed to in situ coat nanozymes on the surface of HPB (Figure S2) and then doped with Cu 2+ to obtain Cu/AP H (Figure 2A).As illustrated in transmission electron microscopy (TEM) image (Figure 2B), Au-Pt nanozymes were densely coated on HPB, and the energy dispersive X-ray spectroscopy mapping showed successful doping of Cu 2+ into the oxygen generator (Figure 2C).To further increase the biocompatibility and tumor-targeting capacity of the copper-ion-loaded oxygen generator carrier, they were modified with cancer cell membrane.As shown in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figure 2D), CT26 cancer cell membrane was successfully modified onto Cu/AP H to obtain Cu/AP H-M.The dynamic light scattering results (Figure 2E) displayed that the size of Cu/AP H-M was ∼115 nm, consistent with the TEM results (Figure 2B).Moreover, after cell membrane modification, the Zeta potential of Cu/AP H decreased to around −34 mV (Figure 2F), which would be beneficial for its long circulation in vivo.After successfully constructing the copper-ionloaded oxygen generator carrier, the CAT-like activity of Cu/AP H-M initially evaluated using the CAT Kit.The results indicated that Cu/AP H-M possessed 50.7 ± 4.39% CAT relative activity just at 50 μg/mL (Figure 2G).Subsequently, the property of Cu/AP H-M to generate O 2 by decomposing hydrogen peroxide (H 2 O 2 ) at different pH (7.4 and 5.6) was further evaluated.Here, Ti(SO 4 ) 2 , that could react with H 2 O 2 to display a yellow color and characteristic ultraviolet-visible absorbance peaks at 405 nm, was employed as a chromogenic agent to evaluate the capability of Cu/AP H-M in decomposing H 2 O 2 (Figure S3a,b).As depicted in Figure 2H, 50 μg/mL of Cu/AP H-M could completely decompose 10 mM H 2 O 2 within 60 min at 37 • C, which could also be observed by the gradual fading of the H 2 O 2 -Ti(SO 4 ) 2 solution as shown in the photographs (Figure S3c).Concurrently, to explore the persistent catalytic capacity of Cu/AP H-M in decomposing H 2 O 2 , H 2 O 2 (10 mM) was repeatedly added to the Cu/AP H-M (50 μg/mL) solution every 60 min under pH 5.6.The results from Figure S4 indicated that Cu/AP H-M could continuously and effectively decompose H 2 O 2 without affecting its own enzymatic activity.Afterwards, a portable dissolved oxygen meter was used to monitor the O 2 concentration in the aforementioned solution.As demonstrated in Figure 2I, compared with HPB-M and Cu H-M, Cu/AP H-M could rapidly elevate the O 2 content in the solution to 19.344 mg/L within 20 min, and such effect could be sustained through the repeated addition of H 2 O 2 (Figure 2J).Finally, under the slightly acidic conditions of tumors (pH 5.6), the delivery and release of Cu 2+ from the copper-ion-loaded oxygen generator carrier were simulated.As depicted in Figure 2K, Cu/AP H-M could cumulatively release 85.16 ± 0.61% of loaded Cu 2+ within 48 h.The above results indicated that through defined chemical and physical methods, the copper-ion-loaded oxygen generator carrier could be successfully constructed.Cu/AP H-M not only exhibited high CAT activity but also could continuously and effectively decompose H 2 O 2 to produce O 2 and release Cu 2+ in a simulated in vivo TME.This provides a solid preliminary foundation for the subsequent effective induction of enhanced cuproptosis and radiotherapy at the cellular and animal levels.

Cu/AP H-M-mediated dual strengthening of cuproptosis and radiotherapy
After thoroughly evaluating the performance of the Cu 2+loaded O 2 generator carrier, dual strengthening of cuproptosis and radiotherapy mediated by Cu/AP H-M was further verified at the cellular level.As shown in Figure 3A, it was firstly confirmed through flow cytometry that Cu/AP H-M could be taken up by CT26 tumor cells, indicating its ability to deliver Cu 2+ into the cells.Subsequently, different concentrations of Cu/AP H-M (0, 50, 100, 200 μg/mL) were jointly incubated with CT26 tumor cells for a period of 12 h, the intracellular copper ion content was quantified by inductively coupled plasma-optical emission spectrometer (ICP-OES).The findings indicated that when the concentration of Cu/AP H-M reached 100 μg/mL, the proportion of internalized Cu/AP H-M in the cells was 9.7 ± 0.11% (Figure 3B).Afterwards, western blot was used to further detect whether Cu 2+ transported into cells by Cu/AP H-M could induce cuproptosis.As depicted in Figure 3C, following treatment with different concentrations of Cu/AP H-M (0, 10, 20, 40 μg/mL), there was a clear oligomerization of DLAT in CT26 cells, which was consistent with the results in semi-quantitative measurements (Figure S5).Moreover, compared to the blank control group, key proteins in the cuproptosis process such as ACO2, LIAS, and FDX1 were all downregulated, while HSP70 was upregulated, the trend of which aligned with the positive control group (Elesclomol-Cu), suggesting that Cu/AP H-M could indeed induce cuproptosis in tumor cells.Meanwhile, under hypoxic culture conditions, the enhancing effect of Cu/AP H-M on cuproptosis was further investigated.Compared to Cu H-M, 20 μg/mL of Cu/AP H-M was more effective in inducing DLAT oligomerization and significantly reduced the expression of ACO2 and FDX1, revealing that Cu/AP H-M could strongly enhance the occurrence of cuproptosis (Figure 3D).In order to intuitively display the behavior of Cu/AP H-M intracellular, CT26 cells treated with 40 μg/mL Cu/AP H-M for 12 h were photographed using a bio-TEM.As depicted in Figure 3E, following treated Cu/AP H-M, the morphology of the mitochondria was noticeably swelled and vacuolated, and the cristae within the mitochondrial inner membrane disappeared, indicating that the mitochondrial function might be damaged or destroyed after cuproptosis.Importantly, Cu/AP H-M could also be clearly observed near the mitochondria intracellular.This clearly suggested that Cu/AP H-M were not only internalized by CT26 cells, but also accumulated nearby the mitochondria, where Cu 2+ was released to induce cuproptosis.After confirming that Cu/AP H-M could effectively induce and augment cuproptosis, the amplification effects of Cu/AP H-M on radiotherapy was further investigated at the cellular level, as well as the cytotoxic effects on tumor cells when combined with cuproptosis.As depicted in Figure 3F, at a concentration of 50 μg/mL, Cu/AP H-M significantly enhanced the combination of cuproptosis and radiotherapy, reducing cell survival rate to 52.67 ± 3.68%.Meanwhile, upon elevating the concentration to 100 μg/mL, the cell survival rate plummeted to just 21.24 ± 2.2%.It is worth noting that during the process of cuproptosis, FDX1 converts Cu 2+ into the more harmful Cu + , resulting in the suppression of Fe-S cluster protein production, which serves a pivotal part in the process of DNA repair and protection, thereby synergistically sensitizing cells to radiotherapy, consistent with the aforementioned cytotoxicity results.To further validate this, CT26 cells treated with Cu/AP H-M, 6 Gy, Cu H-M+6 Gy, AP H-M+6 Gy, and Cu/AP H-M+6 Gy, respectively, were stained with γH2AX.The data revealed that both Cu H-M and Cu/AP H-M-induced cuproptosis could effectively promote DNA damage caused by radiotherapy, thereby sensitizing cells to radiotherapy (Figure 3G,H).The abovementioned findings were further substantiated by cell cloning assay (Figure 3I,J).Hence, it has been initially confirmed at the cellular level that Cu/AP H-M could effectively transport Cu 2+ into cells, leading to mitochondrial dysfunction and damage, which in turn triggered enhanced cuproptosis.Concurrently, it was discovered that the occurrence of cuproptosis, due to its inhibition of Fe-S cluster protein synthesis, synergistically enhanced the cytotoxic effects of radiotherapy on tumor cells.This combination will provide a robust assurance for the dual enhancement of Cu/AP H-M-mediated cuproptosis and radiotherapy against tumors in vivo.

Dual strengthening of cuproptosis and radiotherapy eliminate primary tumor and trigger robust antitumor immunity
Given that Cu/AP H-M could effectively dual-strengthen cuproptosis and radiotherapy, we further evaluated its in vivo behavior and antitumor effects to explore the potentially antitumor immune response triggered by augmented cuproptosis.First, through pharmacokinetic, in vivo imaging system (IVIS) imaging and biodistribution studies, after tumor cell membrane modification, Cu/AP H exhibited a significantly prolonged circulation time in vivo, along with excellent tumor targeting properties.In CT26 subcutaneous tumor-bearing mice, it was observed that Cu/AP H-M could effectively accumulate at the tumor site 12 h post-injection and retain in the tumor site for an extended period (Figures 4A,B, S6, and S7).Furthermore, minimal residual Cu/AP H-M was found in normal tissues, which might be ascribed to the modification of the tumor cell membrane and homotypic targeting (Figure S8a).The efficient targeting and enrichment of Cu/AP H-M could further alleviate the hypoxic TME.Tumor slices of mice 3 days after Cu/AP H-M injection revealed a significant improvement in the hypoxic microenvironment at the tumor site (Figure 4C), and a prominent downregulation of hypoxia inducible factor-1α (HIF-1α) (Figures 4D and S8b).Furthermore, the expression of key cuproptosis proteins, ACO2 and FDX1, in tumor tissues were also examined.As shown in Figure 4E,F, compared to Cu H-M, Cu/AP H-M not only improved the hypoxic microenvironment but also effectively promoted the occurrence of cuproptosis (Figure 4G), which aligned with the results in vitro.Subsequently, in vivo antitumor efficiency of Cu/AP H-M combined with radiotherapy was evaluated.Demonstrated in Figure 4H, compared to the single augmented cuproptosis group ( Cu/AP H-M) and augmented radiotherapy group ( AP H-M+6 Gy), the combination of cuproptosis and radiotherapy ( Cu H-M+6 Gy) significantly strengthened the inhibition of tumors.Such effect might be attributed to the synergistic sensitization effect of cuproptosis on radiotherapy.Surprisingly, the combination of augmented cuproptosis and augmented radiotherapy ( Cu/AP H-M+6 Gy) nearly eliminated the tumors completely and effectively increased the survival rate (100%) and lifetime of the mice (Figure 4I).Hematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay of the tumor on the day 7 post-treatment also demonstrated that the therapeutic effect of Cu/AP H-M+6 Gy was markedly stronger than other groups (Figure 4J,K).Importantly, such treatment strategy also exhibited good biocompatibility, with no detrimental effect on the mice's health (Figure S9) or body weight (Figure S10).
To investigate the immune effects and mechanism resulting from augmented cuproptosis and radiotherapy, RNAseq analysis was conducted on CT26 cells treated with 6 Gy, Elesclomol-Cu, Cu/AP H-M, and Cu/AP H-M+6 Gy.As shown in Figure S11a,b, treatments such as Cu/AP H-M and Cu/AP H-M+6 Gy caused significant changes in the overall gene expression of tumor cells such as the triggering of numerous classical inflammatory signaling pathways, indicating the potential influence of cuproptosis on other therapeutic strategies.Whereafter, the relevant genes associated with immune effects (TNF-α, HSP90AA1, P2RX7, CASP1, CALR, CXCL-10, BAX, IFN-β, and CD274) were further extracted (Figure 5A) for enrichment analysis (Figure 5B-I).Compared to the control group, Cu/AP H-Mmediated augmented cuproptosis exhibited the upregulation of TNF-α, HSP90AA1, P2RX7, CASP1, CXCL-10, BAX, and IFN-β genes (mostly related to ICD or inflammatory factors), which was consistent with the trend observed in the Elesclomol-Cu group.Meanwhile, Cu/AP H-M+6 Gy, which mediated the dual strengthening of cuproptosis and radiotherapy, also generated similar effects as mentioned above.Additionally, surprisingly, both Elesclomol-Cu and Cu/AP H-M-induced cuproptosis could reverse the upregulation of CD274 gene (encoding PD-L1 expression) caused by radiotherapy (Figure 5J).Therefore, the results from RNAseq analysis indicated that Cu/AP H-M-mediated augmented cuproptosis not only effectively promoted the upregulation of genes related to ICD and inflammation in tumor cells, but also reversed the upregulation of the PD-L1 gene caused by radiotherapy.Such reversal could inhibit tumor cells evade immune cell attacks and promote antitumor immune response.To investigate the downstream effects of the aforementioned gene regulation mediated by augmented cuproptosis on protein expression and cytokine secretion, the ATP levels in medium were measured in vitro after Cu/AP H-M and Cu/AP H-M+6 Gy treatments.Presented in Figure 5K, the ATP levels were upregulated to 1.03 ± 0.45 μM and 1.4 ± 0.24 μM, respectively, which was consistent with the upregulation of the P2RX7 gene (P2RX7: an ATP channel protein involved in regulating the release of intracellular ATP).At the animal level, tissue sections of CT26 tumors revealed a conspicuous upregulation in the expression levels of Caspase-1 (Figure 5L,M), TNF-α (Figure 5N,O), Calreticulin (Figure S12a), HSP70 (Figure S12b), and HSP90 (Figure S12c) following treatment with Cu/AP H-M and Cu/AP H-M+6 Gy.Moreover, the overexpression of PD-L1 induced by radiotherapy was also surprisingly reversed (Figure 5P,Q).All of those findings collectively demonstrated that the dual strengthening of cuproptosis and radiotherapy could effectively eliminate subcutaneous solid tumors.Additionally, the augmented cuproptosis could successfully trigger a robust antitumor immune response, thereby paving the way for the combination of cuproptosis with immunotherapy.

Dual strengthening of cuproptosis and radiotherapy boost immunotherapy to suppress distant tumors
Based on the effective antitumor effects of augmented cuproptosis and robust antitumor immunity, the CT26 subcutaneous bilateral tumor model was established to explore the potential of immunotherapy for distant tumors mediated by Cu/AP H-M+6 Gy and αPD-L1 (Figure 6A).As shown in Figure 6B,C, treatment with Cu/AP H-M+6 Gy plus αPD-L1 resulted in almost complete inhibition of both the growth of the primary tumors and the distant tumors.To further evaluate the immune effects of the aforementioned antitumor efficacy, immunological analysis was performed by flow cytometry.First, single-cell suspensions were prepared from the left inguinal lymph nodes of mice on the 5th day after treatments, which were stained with CD11c, CD80, and CD86 flow antibodies (Figure S13).The observation was made that the percentage of mature DC cells in the lymph nodes of mice subjected to treatment with Cu/AP H-M+6 Gy plus αPD-L1 was upregulated to 25.5 ± 2% (Figure 6D,E).This suggested that the antitumor immune response induced by augmented cuproptosis and radiotherapy could enhance the activation of antigen-presenting cells, providing support for subsequent activation of the antitumor immune cycle.Subsequently, T cell (Figure S14) and macrophage (Figure S15) populations in the primary and distant tumors were analyzed using flow cytometry with CD3, CD8, Granzyme B, CD11b, F4/80, CD86, and CD206 flow antibodies.As displayed in Figure 6F-H, both in the primary (Figure S16a-c) and distant tumors, there was a significant increase in the proportion of infiltrating CD3 + CD8 + Granzyme B + cells after the abovementioned treatments and the trend was generally consistent, which would further boost the killing of tumor cells.Furthermore, the tumor tissue exhibited an increase in the proportion of M1-polarized macrophages (Figure S17a,b), while a downward trend was observed in the proportion of M2-polarized macrophages (Figures S18 and 6I).The aforementioned therapeutic approach did not notably impact the body weight of the mice during the entire course of treatment, indicating its biosafety (Figure S19).Therefore, the robust antitumor immunity mediated by the dual strengthening of cuproptosis and radiotherapy was expected to further boost the effectiveness of immunotherapy and promote the activation of the antitumor immune cycle.

Dual strengthening of cuproptosis and radiotherapy synergize immunotherapy prevent tumor recurrence and inhibit low rectal cancer
The triggering of immune memory empowers the immune system to avert the reoccurrence of cancer, which is of significant importance in tumor immunotherapy. [39]Based on the aforementioned research, it could be concluded that Cu/AP H-M-mediated augmented cuproptosis, in combination with radio-immunotherapy, not only eliminated the primary solid tumor but also enhanced immunotherapy to suppress distant tumors.Therefore, to investigate whether our strategy could promote the development of immune memory, on the 60th day after removal of subcutaneous tumors by Cu/AP H-M+6 Gy and Cu/AP H-M+6 Gy plus αPD-L1, spleens were harvested from the mice to prepare single-cell suspensions, which were then stained with CD3, CD8, CD44, and CD62L flow antibodies for analysis (Figure S20a).As shown in Figures 7A and S20b, the proportion of effector memory T cells (TEM: CD3 + CD8 + CD62L − CD44 + ) in the spleens of mice treated with Cu/AP H-M+6 Gy increased to 39.14 ± 1.86%, while the Cu/AP H-M+6 Gy plus αPD-L1 group exhibited a more significant increase in the proportion of T EM cells, reaching 48.6 ± 3.56%.Moreover, the mice that had developed immune memory were rechallenged with CT26 tumor cells, as presented in Figure 7B,C.It was observed that the tumors in the mice with immune memory almost did not grow, and the survival time of the mice was extended to 90 days with a survival rate of 100%.Throughout such duration, no notable reduction in the mice's body weight was observed (Figure S21).The above results indicated that Cu/AP H-M-mediated augmented cuproptosis and radiotherapy not only promoted tumor immunotherapy but also generated lasting immune memory responses, hence thwarting tumor recurrence.
In rapid sequence, an in situ low rectal cancer model was established in BALB/c mice to simulate the occurrence and treatment of clinical colorectal cancer (Figure 7D).As shown in the IVIS imaging images in Figure 7E, Cu/AP H-M+6 Gy plus αPD-L1 treatment significantly inhibited the growth of luciferase-expressing CT26 cells.Furthermore, 83.3% of the mice treated with our treatment strategy exhibited a survival period exceeding 35 days (Figure 7F), which was further supported by the tumor size images obtained from mice on day 20 after treatment (Figure S22).Therefore, our strategy is anticipated to offer novel tactics and procedures for addressing clinical colorectal cancer.

CONCLUSION
Cuproptosis is a form of cell death that has been recently identified, therefore, elucidating its antitumor immune effect is of great significant implications for clinical cancer treatment.In our research, the positive correlation between cuproptosis and tumor immunotherapy was initially explored by bioinformatics analysis.Subsequently, Cu 2+ -loaded O 2 generator carrier were successfully constructed and prepared to regulate the tumor hypoxic microenvironment and deliver Cu 2+ .After evaluating the superior performance of Cu/AP H-M, we further elucidated in vitro that it could effectively transport Cu 2+ and induce augmented cuproptosis.Notably, augmented cuproptosis could also improve the effectiveness of radiotherapy by impeding Fe-S cluster protein production.In vivo study, the dual strengthening of cuproptosis and radiotherapy mediated by Cu/AP H-M not only successfully eradicated subcutaneous tumors but also promoted the upregulation and release of Caspase-1, TNF-α, Calreticulin, HSP70, HSP90, ATP, etc., triggering a powerful antitumor immune response.Simultaneously, the augmented cuproptosis mediated by Cu/AP H-M could reverse the increased expression of PD-L1 in tumor cells induced by radiotherapy, reducing the immunological escape of tumor cells.After combined immunotherapy, the dual strengthening of cuproptosis and radiotherapy not only effectively activated the antitumor immune cycle, inhibiting the growth of distal tumors and in situ low rectal tumors, but also generated a potent immune memory response to thwart future tumor invasions (Figure 7G).In summary, our work introduced for the first time the beneficial role of augmented cuproptosis in radiotherapy and immunotherapy, elucidating its potential mechanism.Our strategy presented a novel approach for the synergistic integration of cuproptosis with immunotherapy and radiotherapy and elucidated the specific mechanisms of the interaction between cuproptosis and radiotherapy, as well as immunotherapy, expanding the application range of cuproptosis in cancer treatment.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper.

F I G U R E 1
Schematic diagram of dual strengthening of cuproptosis and radiotherapy potentiating radio-immunotherapy and bioinformatics analysis of the beneficial correlation between cuproptosis and immunotherapy.(A) Schematic diagram of Cu/AP H-M oxygen generator dual strengthening both cuproptosis and radiotherapy, synergistic boosting antitumor immunotherapy.(B) The correlation coefficients between immunogenic cell death (ICD)-related genes and cuproptosis-related genes were displayed.(C) The bubble plot displays the correlation between expression levels of dihydrolipoamide S-acetyltransferase (DLAT), Ferredoxin 1 (FDX1), and LIAS and immune cells.(D) Positive correlation at the cellular level between the expression of DLAT and macrophages, dendritic cells, CD8 + T cells, and natural killer (NK) cells in tumor tissues.p-Values in (D) were calculated using the DESeq2 package in R for differential analysis.

F I G U R E 2
The fabrication, characterization, and functional evaluation of Cu/AP H-M.(A) Synthetic procedure of Cu/AP H-M.(B) Transmission electron microscopy (TEM) images of copper-doped gold-platinum bimetallic nanozyme-coated hollow mesoporous Prussian blue nanoparticles ( Cu/AP H). (C) Energy dispersive X-ray spectroscopy (EDX) mapping of Cu/AP H. (D) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of cancer cell membrane modified Cu/AP H ( Cu/AP H-M).(E) Dynamic light scattering (DLS) of Prussian blue (PB), hollow mesoporous Prussian blue (HPB), AP H, Cu/AP H, and Cu/AP H-M.(F) Zeta potential of PB, HPB, AP H, Cu/AP H, and Cu/AP H-M.(G) Catalase activity of HPB-M, Cu H-M, and Cu/AP H-M detected by CAT Kit.(H) H 2 O 2 (10 mM) degradation after treated with PBS, HPB-M, Cu H-M, and Cu/AP H-M (50 μg/mL) at different pH (7.4 and 6.5) and time points.(I) O 2 generation curve post-H 2 O 2 treated with PBS, HPB-M, Cu H-M, and Cu/AP H-M (50 μg/mL) at predesigned time points.(J) Repetitive O 2 generation after treated with Cu/AP H-M (50 μg/mL) by repeated addition of H 2 O 2 .(K) Cumulative release of Cu 2+ from Cu/AP H-M at different pH (7.4 and 6.5).Error bar represent mean ± standard deviation (SD) (n = 3).p-Values in (G) were calculated by one-way analysis of variance (ANOVA) test (*p < 0.05; ***p < 0.001).

F I G U R E 4
Cu/AP H-M oxygen generator regulate the tumor hypoxic microenvironment and promote cuproptosis to eliminate solid tumors.(A) Biodistribution after intravenous injection of DiD-Cu/AP H-M at different time points.(B) Mean fluorescence intensity statistics of DiD-Cu/AP H-M in major organs and tumor at 48 h post-intravenous injection.(C) Hypoxia immune-fluorescence images of CT26 tumor slices collected from mice 3 days post-treatment of Cu/AP H-M (blue: nucleus; green: hypoxia).(D) The immunofluorescence slices of HIF-1α expression in CT26 tumors treated by Cu/AP H-M for 3 days (blue: nucleus; red: HIF-1α).(E) The immunofluorescence slices of ACO2 expression in CT26 tumors treated by Cu/AP H-M for 3 days (blue: nucleus; red: ACO2).(F) The immunofluorescence slices of Ferredoxin 1 (FDX1) expression in CT26 tumors treated by Cu/AP H-M for 3 days (blue: nucleus; green: FDX1).(G) Mean fluorescence intensity statistics of FDX-1 and ACO2 in CT26 tumor slices collected from mice after 3 days treated with Cu/AP H-M.Error bars represent mean ± standard deviation (SD) (n = 3).(H) CT26 tumors growth curves after treatment with Cu/AP H-M, AP H-M+6 Gy, Cu H-M+6 Gy, and Cu/AP H-M+6 Gy. (I) Survivorship curves of CT26 tumor-bearing mice after treatments.Error bars represent mean ± SD (n = 5).(J) Hematoxylin-eosin staining (H&E) full slices of tumor tissue at day 7 post-treatments.(K) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stained tumor slices from mice collected at 3 day post-treatment.p-Values were calculated by one-way analysis of variance (ANOVA) test in (G), and two-way ANOVA test in (H) and (I) (*p < 0.05; ***p < 0.001).F I G U R E 5 Cu/AP H-M-mediated augmented cuproptosis promote antitumor immune responses.(A) Heatmap of gene expressions associated with immunogenic death in CT26 cells treated with above-mentioned treatments.(B-J) Differential analysis of TNF-α (B), HSP90AA1 (C), P2RX7 (D), CASP1 (E), CALR (F), CXCL-10 (G), BAX (H), IFN-β (I), and CD274 (J) gene expression in (B).(K) The concentration of ATP released in the medium from CT26 cells at 8 h post-treated with 6 Gy, Cu/AP H-M, and Cu/AP H-M+6 Gy. (L and M) Immunofluorescence slices of Capase-1 (green) expression (L) and mean fluorescence intensity statistics (M) in CT26 tumors at 3 day post-treatment.(N and O) Immunofluorescence slices of TNF-α (red) expression (N) and mean fluorescence intensity statistics (O) in CT26 tumors at 3 day post-treatment.(P and Q) Immunofluorescence slices of PD-L1 (red) expression (P) and mean fluorescence intensity statistics (Q) in CT26 tumors at 3 day post-treatment.Error bars represent mean ± standard deviation (SD) (n = 3).p-Values in (B-K), (M), (O), and (Q) were calculated by one-way analysis of variance (ANOVA) test (*p < 0.05; **p < 0.01; ***p < 0.001).

F I G U R E 7
Cu/AP H-M-mediated therapeutic strategy induce memory immune response and suppress low rectal cancer.(A) Representative flow cytometry plots of effector memory T cells (transmission electron microscopy [TEM]: CD3 + CD8 + CD44 + CD62L − ) in spleen of mice 60 days after treated with Cu/AP H-M+6 Gy and Cu/AP H-M+6 Gy+αPD-L1.(B and C) Tumor growth (B) and survivorship curves (C) of mice in rechallenged tumor experiments conducted after 60 days of the initial tumors being eliminated.Error bars represent mean ± standard deviation (SD) (n = 5).(D) Schematic diagram of Cu/AP H-M+6 Gy+αPD-L1 to inhibit the growth of in situ low rectal cancer.(E) In vivo bioluminescence images used to monitor the growth and metastases of luciferase-expressing CT26 colorectal cancer cells after treatment with Cu/AP H-M+6 Gy+αPD-L1.(F) Survivorship curves of low rectal cancer mice after treatments.(G) Mechanistic diagram of Cu/AP H-M oxygen generator dual strengthening both cuproptosis and radiotherapy potentiating radio-immunotherapy efficacy.Error bars represent mean ± SD (n = 6).p-Values were calculated by two-way analysis of variance (ANOVA) test in (B), (C), and (F) (***p < 0.001).
Pei Pei, Yuhong Wang, and Wenhao Shen contributed equally to this work.This work was partially supported by the National Natural Science Foundation of China (U1932208, 32171382, 12275003, 32301169), the Key Research and Development Program of Social Development of Jiangsu Province (BE2022725), the Natural Science Foundation of Jiangsu Province (BK20190946), the Key Scientific Research Project of Colleges and Universities of Anhui Province of China (2022AH050683), the Anhui Provincial Natural Science Foundation (2308085QH281), the Gusu Leading Talent Program for Innovation and Entrepreneurship (grant no.ZXL2021441), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Suzhou Health Youth Backbone Talent "National Tutorial System" Training Project (Qngg2023005), and the Beijing Sisko Clinical Oncology Research Foundation (Y-tongshu2021/qn-0366).