C5a/C5aR pathway blocking promoted CuS-mediated cancer therapy effect by inhibiting cuproptosis resistance

Abstract

Background

Breast cancer is the most diagnosed malignancy and a leading cause of cancer-related deaths among women globally. Cuproptosis plays a significant role in tumor progression and therapeutic response. Increasing studies suggest that targeting cuproptosis presents a promising strategy for cancer therapy, such as through the development of copper nanoparticles as therapeutic agents. However, resistance to cuproptosis has emerged as a critical hallmark of cancer. Therefore, it is essential to further investigate the mechanisms underlying cuproptosis resistance to enhance its therapy effect.

Methods

The relationship between breast cancer progression and the C5a/C5aR pathway or cuproptosis was determined by single-cell RNA sequencing analyses, RNA-sequence analyses, bioinformatic analyses, survival analyses and immunohistochemistry. The antitumor effects of CuS nanoparticles and C5a receptor antagonists (C5aRA) were assessed by in vitro and in vivo strategies including cell counting kit-8, colony formation assay, relative reactive oxygen species level assay, western blots, real-time quantitative PCR, immunohistochemistry, immunofluorescence assay, flow cytometry and the xenograft mice models. Complement system activation by CuS nanoparticles was tested by ELISA.

Results

Our results indicated that activation of the C5a/C5aR pathway contributes to cuproptosis resistance by upregulating ATP7B expression via the Wnt/β-catenin pathway. Consequently, combining CuS nanoparticles with lazer treatment and C5aRA markedly enhanced the antitumor efficacy of CuS nanoparticles by overcoming cuproptosis resistance, leading to a synergistic effect in cancer therapy that included cuproptosis-targeting therapy, immunotherapy, and photothermal therapy.

Conclusions

This study reports, for the first time, proved C5a/C5aR pathway-mediated cuproptosis resistance in cancer cells, and combining CuS nanoparticles and C5aRA offers a superior and novel therapeutic strategy for cancer.

HY, BD and XH are joint first authors.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Targeting cuproptosis has emerged as a prominent focus in cancer therapy; however, cancer cells have developed various strategies to evade cuproptosis. The C5a/C5aR pathway has been shown to promote breast cancer progression and induce ferroptosis resistance in cancer cells, yet its role in cuproptosis remains unexplored.

WHAT THIS STUDY ADDS

• In the present study, we demonstrate that the C5a/C5aR pathway contributes to cuproptosis resistance in cancer cells. The combination of CuS nanoparticles with lazer treatment and C5a receptor antagonists (C5aRA) significantly enhances the antitumor efficacy of CuS nanoparticles by overcoming cuproptosis resistance, resulting in a synergistic effect that integrates cuproptosis-targeting therapy, immunotherapy, and photothermal therapy.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• This study, for the first time, establishes that the C5a/C5aR pathway mediates cuproptosis resistance in cancer cells and demonstrates that the combination of CuS nanoparticles and C5aRA presents a promising and novel therapeutic strategy for cancer.

Background

Breast cancer (BC) stands as the most frequently diagnosed cancer among women globally. Globally, 2.3 million new cases and 670,000 deaths from female BC occurred in 2022. Annual rates increased by 1–5% in half of examined countries. By 2050, new cases and deaths will have increased by 38% and 68%, respectively, disproportionately impacting low-Human Development Index (HDI) countries.¹ Despite significant improvements in survival rates over the past two decades, the global incidence of BC continues to escalate.² Consequently, there is a pressing need to develop innovative initiatives and strategies aimed at preventing and treating this pervasive disease.

Complement elements such as C3a or C5a appear to participate in some processes of tumor progression, including the regulation of tumor angiogenesis and immune cells recruitment and phenotype.^{3 4} Indeed, C5a was demonstrated to promote tumor metastasis of BC by altering T-cell responses in the metastatic niche.⁵ Besides, our previous research also provided evidence that BC development may rely on C5a/C5aR interaction, for which the MAPK/p38 pathway participated in downregulating the p21 expression. And C5a receptor antagonists (C5aRA) exhibited significant attenuation of BC cell growth in a mouse model.⁶ It was also reported that the C5a/C5aR pathway could promote chemotherapy resistance in glioblastoma and squamous carcinogenesis.^{7 8} Our previous work also demonstrated that the C5a/C5aR pathway could be activated by iron nanoparticles (NPs) and lead to ferroptosis resistance in BC.⁹

Cuproptosis presents promising applications in tumor therapy. Its mechanism of action is distinct from other known cell death pathways, such as apoptosis, ferroptosis, pyroptosis and necroptosis. It primarily relies on the accumulation of intracellular copper, which binds directly to lipoylated components of the tricarboxylic acid (TCA) cycle, leading to the aggregation of fatty acylated proteins and the loss of iron-sulfur (Fe-S) proteins. This process induces proteotoxic stress, ultimately resulting in cell death.¹⁰

Many tumor cells exhibit elevated levels of lipoylated TCA enzymes, particularly the pyruvate dehydrogenase complex, and show a greater dependence on mitochondrial metabolism rather than glycolysis. Recent studies have widely explored the use of copper ion-based metal carriers for targeting cancer cells. Additionally, cuproptosis offers an effective strategy to overcome chemotherapy and radiotherapy resistance, given its independence from apoptotic mechanisms.^10–12 However, cancer cells also develop many strategies to resist cuproptosis. The presence of copper efflux mechanisms and high levels of reducing agents, such as glutathione (GSH), in tumor cells may inhibit cuproptosis. ATP7A and ATP7B proteins are two homologous Cu-ATPases that facilitate copper transport into small vesicles within the cell membrane through ATPase hydrolysis. Subsequently, they expel copper via exocytosis following cell membrane fusion, thus preventing excessive accumulation of copper ions in the cell and promoting cellular self-protection.¹³ As a highly expressed reducing agent in tumor cells, GSH serves as a natural copper chaperone. Therefore, enhancing GSH consumption and inhibiting copper efflux are crucial strategies to increase the sensitivity of tumors to cuproptosis. Researchers have proved that buthionine sulfoximine, an intracellular GSH-depleting agent, can enhance tumor cell sensitivity to cuproptosis.¹⁴ Therefore, understanding the specific mechanism of resistance to cuproptosis in tumor cells is of great significance for developing therapeutic strategies for cuproptosis.

CuS is a commonly used inorganic material with significant potential as a cuproptosis agent for cancer therapy.¹⁵ CuS NPs exert anticancer effects through dual mechanisms: cuproptosis and photothermal therapy (PTT). The cuproptosis mechanism mediated by CuS NPs involves the following detailed steps: CuS NPs undergo pH-dependent degradation in the acidic tumor microenvironment (TME) (pH~6.5), releasing Cu⁺ ions. This process is accelerated by the overexpression of GSH in cancer cells, which reduces Cu²⁺ to Cu⁺. These ions accumulate in mitochondria, where they directly bind to lipid-acylated enzymes, inducing toxic protein oligomerization and loss of Fe-S cluster proteins. This disrupts mitochondrial respiration, leading to proteotoxic stress and cell death via cuproptosis.¹⁶ PTT: CuS NPs induce PTT through the following mechanisms: CuS NPs exhibit strong absorption in the near-infrared (NIR) region due to their localized surface plasmon resonance and narrow bandgap (~1.7 eV). On NIR lazer irradiation, absorbed photons excite electrons, which relax non-radiatively, converting light energy into heat. The generated heat elevates the local temperature (typically >42°C), inducing irreversible damage to tumor cells via protein denaturation, membrane disruption, and DNA damage.¹⁷ However, using CuS NP directly as a therapeutic agent poses challenges due to issues such as tissue compatibility, which may result in suboptimal treatment outcomes.

In the present study, we revealed that the C5a/C5aR pathway promoted ATP7B expression by activating the Wnt/β-catenin pathway and subsequently led to cuproptosis resistance in BC cells. Besides, we also demonstrated that CuS NPs could inhibit BC progression by inducing cancer cell cuproptosis, remodeling the tumor immune environment and photothermal therapy function led by 808 nm lazer. However, C5a/C5aR pathway activation induced by CuS NPs attenuated the therapeutic effect by inhibiting cuproptosis. Therefore, combining CuS NPs with C5aRA could enhance its antitumor effect by increasing cancer cells sensitivity to cuproptosis. This study provided a novel insight into the development of a novel strategy that synergized various therapeutic mechanisms for BC treatment.

Methods

Single-cell RNA sequencing analyses

Single-cell RNA-sequencing data analysis was performed by NovelBio Bio-Pharm Technology with NovelBrain Cloud Analysis Platform. We selected 20 single-cell samples, which included 10 cases of breast epithelial tissues from healthy individuals and 10 cases of untreated triple-negative breast cancer (TNBC) primary tumor tissues from Gene Expression Omnibus (GEO) datasets. The sources of this data are presented in online supplemental table 1. We applied fastp with default parameters, filtering the adapter sequence and removing the low-quality reads to achieve the clean data. Unique Molecular Identifiers (UMI)-tools were applied for single-cell transcriptome analysis to identify the cell barcode whitelist. The UMI-based clean data was mapped to the mouse genome (Ensemble V.100) using STAR mapping with customized parameter from UMI-tools standard pipeline to obtain the UMI counts of each sample. Cells contained over 200 expressed genes and mitochondria UMI rate below 20% passed the cell quality filtering and mitochondria genes were removed in the expression table. After excluding low-quality cells and contaminating lineage cells, we finally obtained 83,052 cells (normal: 46,225 cells, tumor: 36,827 cells). Seurat package (V.3.1.4, https://satijalab.org/seurat/) was used for cell normalization and regression based on the expression table according to the UMI counts of each sample and per cent of mitochondria rate to obtain the scaled data. Principal Component Analysis (PCA) was constructed based on the scaled data with the top 2,000 high variable genes and the top 10 principals were used for Uniform Manifold Approximation and Projection (UMAP) construction. Using the graph-based cluster method (resolution=0.8), we acquired the unsupervised cell cluster result based on the PCA top 10 principal components, and we calculated the marker genes by the FindAllMarkers function with the Wilcoxon rank-sum test algorithm under the following criteria: (1) lnFC>0.25; (2) p value<0.05; (3) min.pct>0.1. To identify the cell type in detail, the clusters of the same cell type were selected for UMAP analysis, graph-based clustering and marker analysis. The cell type identity was based on SingleR (https://github.com/dviraran/SingleR). Pathway analysis was used to find out the significant pathway of the differential genes according to the Gene Ontology (GO) database. We turned to Fisher’s exact test to select the significant pathway, and the threshold of significance was defined by p value and False Discovery Rate (FDR). The SCENIC analysis was performed by using the pySCENIC (V.0.11.2) and hg19-tss-centered 10 kb-10 species databases for RcisTarget, GRNboost, and AUCell. The input matrix was the normalized expression matrix that was from Seurat. Cuproptosis gene set was obtained from literature.¹⁸ We applied the single-cell trajectories analysis using Monocle2 (http://cole-trapnell-lab.github.io/monocle-release) using DDR-Tree and default parameters. Before Monocle analysis, we select marker genes of the Seurat clustering result and raw expression counts of the cells that passed filtering. Based on the pseudo-time analysis, branch expression analysis modeling (analysis) was applied for branch fate-determined gene analysis. For copy number variation (CNV) estimation, cells defined as endothelia, fibroblast and macrophage were used as reference to identify somatic copy number variations with the R package InferCNV (V.0.8.2). We scored each cell for the extent of CNV signal, defined as the meaning of squares of CNV values across the genome. Putative malignant cells were then defined as those with CNV signal above 0.05 and CNV correlations above 0.5.

Patients and clinical specimens

Primary BC tissues and paired tumor-adjacent normal tissues (collected 5 cm from the tumor margin) were obtained from 44 treatment-naïve patients with TNBC who underwent surgical resection with lymph node dissection at Chongqing Southwest Hospital, China between 2008 and 2010. None of the patients underwent preoperative chemotherapy or radiation therapy. Patients detail of tissue origin of BC was shown in online supplemental table 2.

Cells, antibodies and chemicals

Mouse BC cell line 4T1, E0771, mouse melanoma cell line B16F10 and mouse colon cancer cell line CT26, human BC line MDA-MB-231 and MCF7 were all purchased from Procell Life Science and Technology. 4T1, E0771 and MDA-MB-231 cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (high glucose) (Gibco, Life Technologies, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA) and maintained at 37°C in a humidified incubator with 5% CO₂. MCF7 cells were routinely cultured in Modified Eagle’s Medium (Gibco, Life Technologies, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA) and maintained at 37°C in a humidified incubator with 5% CO₂. CT26 and B16F10 were cultured in Roswell Park Memorial Institute (RPMI)-1640 Medium (Gibco, Life Technologies, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, USA) and maintained at 37°C in a humidified incubator with 5% CO₂. All mediums were supplemented with 10% Fetal Bovine Serum (FBS) and 100 U/mL penicillin-streptomycin.

The following commercially available antibodies were used for western blotting and immunohistochemistry (IHC) assay: α-tubulin (Affinity, AF4651), FDX1 (Proteintech, 12592–1-AP), β-actin (Beyotime, AF5003), DLAT (Proteintech, 13426–1-AP), LIAS (Proteintech, 11577–1-AP), β-catenin (Proteintech, 51067–2-AP), ATP7B (Proteintech, 19786–1-AP), C5b-9 (Beyotime, AF6360), C5aR (Proteintech, 11577–1-AP), HRP-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime, Cat#A0208) and HRP-labeled Goat Anti-Mouse IgG (H+L) (Beyotime, Cat#A0216) as secondary antibody, Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) (Beyotime, Cat#A0423).

The chemical reagents used in this study included elesclomol (MCE, HY-12040), CuCl₂ (Aladdin, 7447-39-4), DKK1 (MCE, HY-15763), crystal violet (Beyotime, C0121), DCFH-DA (Beyotime, S0033S), CuS NPs (Nanjing Jike Biotechnology), Mito-Tracker Red CMXRos (Beyotime, C1035), peptine C5aRA (GL Biochem, China).

RNA extraction and quantitative real-time PCR

RNAeasy Animal RNA Extraction Kit (Spin Column) (Beyotime, R0026) was applied to extract total RNA according to the manufacturer’s instructions, and the NanoDrop 2000c instrument (Thermo Fisher, USA) was applied for quantifying RNA concentration. High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) was used for the reverse transcription of 2 µg total RNA. The quantitative real-time PCR was performed using the SYBR Green Kit (Takara, Japanese) and Light Cycler 480 II system (Bio-Rad, China). β-actin was adopted for normalization with the 2−ΔΔct method.

Primers used were listed as follows:

Mouse-ATP7B-F: 5-GGGGACGATGCCTGAACAG-3,

Mouse-ATP7B-R: 5-GCCGGGCAAAGCAAGTTTAG-3;

Human-ATP7B-F:5-GCCAGCATTGCAGAAGGAAAG-3;

Human-ATP7B-R:5-TGATAAGTGATGACGGCCTCT-3;

Mouse-β-actin-F:5-AAGGTCGGAGTCAACGGATTTG-3;

Mouse-β-actin-R:5-CCATGGGTGGAATCATATTGGAA-3.

Human-β-actin -F:5-CATGTACGTTGCTATCCAGGC-3;

Human-β-actin -R:5-CTCCTTAATGTCACGCACGAT-3;

Cell viability assay

Cell viability assay was applied with the cell counting kit-8 (CCK-8) kit (Servicebio, G4103). Briefly, cells were seeded into a 96-well plate overnight and treated as indicated, and CCK-8 reagents were added to each well, after which the plates were placed in a humidity incubator (37°C, 5% CO₂). The absorbance measurements were carried out at 450 nm using a Countstar Spectrophotometer (151000712). Samples were prepared at least in triplicates.

Mice xenograft model

Female Balb/c mice, female C57BL/6 mice (6–8 weeks of age) were obtained from the Animal Institute of Academy of Medical Science (Beijing, China) and housed in individual ventilated cages at the Institute of Immunology of the Army Medical University (Chongqing, China). 4T1, CT26 or B16F10 cells were resuspended with phosphate-buffered saline (PBS), and 3×10⁵ cells were subcutaneously injected into each mouse. When implanted tumors reached 100–300 mm³, mice were randomized and divided into indicated groups and then intraperitoneally injected with 0.9% NaCl, CuS NPs (10 mg/kg), C5aRA (1 mg/kg), CuS NPs (10 mg/kg) + C5 aRA (1 mg/kg) two times and monitored using Vernier calipers. Tumor volume (mm³)=0.5×tumor length×tumor width². At the endpoint, the xenograft tumors from the euthanized mice were photographed and weighed. Animal care and experiments were conducted in compliance with Institutional Animal Care and Use Committee and National Institutes of Health (NIH) guidelines.

Immunofluorescence and microscopy

Cells were seeded on coverslips in a 6-well plate overnight and treated as indicated. Briefly, the cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in 0.25% Triton X-100 for 10 min and blotted with 3% Bovine Serum Albumin (BSA) for 1 hour. The cells were incubated with DLAT antibody (1:500) at 4°C overnight. Then, the cells were washed three times with 0.1% PBS, incubated with the appropriate secondary antibodies for 1 hour at 37°C, washed and sealed with mounting medium including DAPI (Servicebio, G1012-10ML). Images were captured on confocal. For mitochondrial staining, cells were incubated with 200 nM MitoTracker Red CMXRos (Servicebio, C1035-50 μg) for 30 min prior to paraformaldehyde fixation.

Cell viability assay

4T1 cells were seeded overnight in 96-well plates (5×10³ cells/well). Cells were treated with elesclomol-Cu (ES-Cu) and/or other reagents for 48 hours. Viability was assessed by CCK-8 kit (Beyotime, Cat#C0037). The absorbance (OD) was measured at 450 nm using a BioTek plate reader (BioTek).

Mouse C5a quantification

The concentrations of C5a in the mouse plasma samples were measured by ELISA using Mouse C5a ELISA kit (Cloud-Clone, USA) according to the manufacturer’s instructions. After proper dilution, the corresponding reagents were added and samples were measured by a 96-well microplate reader (Bio-Rad, Hercules, California, USA).

Assessment of intracellular ROS generation

The DCFH-DA assay was used to measure intracellular reactive oxygen species (ROS) generation. Briefly, 4T1 cells were seeded at a density of 1×10⁴ cells per well in 96-well culture plates and cultured for 12 hours to allow cell adhesion. Subsequently, the original culture medium was exchanged with 200 µL of Dulbecco's Modified Eagle Medium (DMEM) medium containing PBS, CuS NPs (100 µg/mL), CuS NPs (100 µg/mL) +C5 a (100 nM), CuS NPs (100 µg/mL) +C5 a (100 nM) +C5aRA (100 nM). Following incubation for 48 hours at 37°C, the cells were washed with PBS and then incubated with the culture medium containing 10 µM DCFH-DA for 20 min at 37°C. To calculate intracellular ROS, the cells were imaged by an automatic microplate reader (Olympus, Japan).

Protein extraction and western blotting

Cells or tumor tissues were lysed in T-PER Tissue Protein Extraction Reagent (Thermo, Cat#78510) containing protease inhibitors cocktail (CWBIO, Cat#CW2200S). Lysates were centrifuged at 12,000 rpm for 15 min. Protein extracts were solubilized in loading buffer (Bioground, Cat#BG0022). Equal amounts of lysate were separated on SDS-PAGE (ExpressPLUS, Cat#M42015C) and transferred onto a 0.45 µm polyvinyl difluoride membrane (Beyotime, Cat#FFP39). The protein was identified by incubating the membrane with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies.

Colony formation assay

4T1 cells (800 cells well⁻¹) were seeded in 12-well plates for 72 hours in an incubator (37°C, 5% CO₂). 4T1 cells were subsequently treated with PBS, CuS NPs (100 µg/mL), CuS NPs (100 µg/mL) +C5 a (100 nM), CuS NPs (100 µg/mL) +C5 a (100 nM) +C5aRA (100 nM) and then incubated for another 48 hours. After treatment, the cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.2% crystal violet for 10 min. Images were captured by camera after washing with ultrapure water several times.

Biological safety assay

The tumor-bearing mice were intravenously injected with CuS NPs (10 mg/kg), after injection for 48 hours, mice serum was obtained to test the ALT (alanine aminotransferase), AST (aspartate aminotransferase), CREA (serum creatinine), TBIL (total bilirubin), DBIL (direct bilirubin), UREA (urine creatinine) level.

Histopathological analysis

After different treatments, mice from different groups were euthanized by CO₂ inhalation. The main organs of the mice, such as their hearts, livers, spleens, kidneys, lungs and tumors, were collected and fixed in 10% neutral buffered formalin for 48 hours. Then, these tissues were embedded in paraffin after being processed by alcohol and xylene and sectioned into 5 µm thickness.

Single-cell suspension of tissue and flow cytometry analysis

Tumor tissues were digested with Tumor Dissociation Kit (Miltenyi Biotec, Cat#130-096-730) using gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) and then crushed through mesh for single-cell suspension. To determine macrophage polarization, cells from tumor tissues were stained with Zombie.NIR Dye (BioLegend, Cat#B323327), anti-mouse CD45 (BioLegend, Cat#103132), CD11b (BioLegend, Cat#101263), I-A/I-E (BioLegend, Cat#107605), and CD11c (BioLegend, Cat#117307). After the staining process by following the manufacturer’s instructions, samples were analyzed by flow cytometry (CytoFLEX).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism (V.8.0) software. The statistical significance for comparisons of two groups was evaluated using the Student’s t-test. Statistical significance for comparisons of three or four groups was determined by one-way analysis of variance. All data were presented as the mean±SEM. P value<0.05 was considered statistically significant. Statistical significance was denoted (no significance, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.) in the figures and figure legends. All experiments were independently repeated at least three times.

Results

C5a/C5aR pathway was activated in breast cancer and closely related to the malignant degree of breast cancer cells

We selected 20 single-cell samples which included 10 cases of breast epithelial tissues from healthy individuals and 10 cases of untreated TNBC primary tumor tissues. After integrating the transcriptional data from all acquired cells, we first performed low-resolution UMAP clustering, generating two-dimensional graphs with nine distinct clusters from 20 samples. To classify the primary cell types within this atlas, we annotated each cluster according to their marker genes (figure 1A–C). We conducted a differential analysis comparing epithelial tissue from tumor patients with normal breast epithelial tissue. GO enrichment analysis of the differentially expressed genes revealed a significant association with the complement activation pathway (figure 1D), indicating notable activation of the complement system in tumor tissues. Specifically, we observed significant differences in the expression of proteins related to the complement C5-C5aR pathway between normal and tumor tissues, which were validated using single-cell data and IHC staining (figure 1E,F). To further assess the activation status of the C5a/C5aR pathway in tumor cells, we analyzed transcription factors and found that those regulating the key protein C5aR within the complement pathway were significantly activated in tumor tissues (figure 1G). These differences were also reflected in relevant proteins associated with complement C5a, supporting the notion that the complement C5a-C5aR pathway may play a crucial role in the progression of TNBC.

Given that BC cells originate from epithelial cells, we then performed high-resolution UMAP analysis and re-clustered the epithelial cells into 18 clusters (figure 1H). To further investigate the impact of the complement C5a-C5aR pathway on TNBC, we scored the epithelial cells based on complement activation (figure 1I). Next, we divided epithelial cells into two groups: high complement expression and low complement expression (figure 1K). To detect whether complement activation is related to the malignancy of BC cells, CNV analysis was conducted. CNV analysis has been widely used in Single-Cell RNA Sequencing (scRNA-seq) to investigate disease evolution and development. Then, we performed the CNV level of each epithelial cell cluster. Among all epithelial cell clusters, C16 exhibited remarkably lower CNV levels than other clusters, we set it as normal epithelial cells (figure 1J). Malignant cells displayed heterogeneous CNV score and higher CNV score correlated with higher complement activation score (figure 1L). These results indicate that complement activation is aberrantly heightened in BC and is closely associated with its malignant progression. Therefore, it is essential to investigate the specific mechanisms underlying the promotion of BC development, offering new insights and potential targets for its treatment.

C5a/C5aR pathway was related to cancer cell cuproptosis

To further explore the mechanism of the complement pathway affecting the development of BC, GO enrichment analysis of Differentially Expressed Genes (DEGs) of two clusters (high complement expression vs low complement expression) was conducted. The results revealed a significant correlation of the complement pathway with copper ion metabolism (figure 2A). Consequently, cuproptosis scoring was performed on the epithelial cell clusters (figure 2B) and we conducted a Pearson correlation analysis to elucidate the relationship between complement activation and the cuproptosis pathway (p<2.2e−16, R=0.27) (figure 2C). Furthermore, our Single-Sample Gene Set Enrichment Analysis (ssGSEA) analysis revealed that the cuproptosis pathway was activated in tumor epithelial cells (figure 2D). Notably, the cell population with a high cuproptosis score has a high degree of complement activation, which also indicates a high degree of cellular malignancy (figure 2E).

To dissect the evolutionary dynamics of breast epithelial lineages, we also constructed a pseudo-time cell trajectory analysis of the 18 epithelial cell clusters and generated a three-branch trajectory depicting the development from non-malignant to malignant cells (figure 2F). Due to its low CNV level, C16 was located at the bottom-right corner of the trajectory curve, suggesting the clear starting point of this evolving trajectory curve. After confirmation of this starting point, developmental routes were determined to begin with the initial state and then bifurcating into either Cell fate 1 or Cell fate 2 branches with high-CNV-levels endpoints (figure 2F). Hence, C4 and C14 were identified as malignant cell clusters since they were at the end of the trajectory curve with relatively high CNV levels (figure 2F). In a word, we identified that C16 represented the normal mammary ductal epithelial cells, while C4 and C14 were the malignant cell clusters of BC.

The heatmap showed how the expression of genes changed from the initial state to Cell fate 1 or 2 and identified three different transformation patterns (figure 2G). Marker genes in cuproptosis (ATP7B and LIAS) along with complement-related genes (C5 and C5AR2) exhibited an up-then-down expression pattern (figure 2G,H), which indicates that the increase of C5a in BC may be accompanied by the increase of ATP7B, thus leading to the occurrence of cuproptosis resistance. In a word, pseudo-time cell trajectory analysis indicated that complement-related proteins and cuproptosis-related proteins exhibited similar expression patterns, further suggesting an interplay between these two pathways. Interaction networks also illustrated the relationships between the proteins involved in C5a/C5aR pathways and cuproptosis (figure 2I). To further investigate the influence of the C5a/C5aR pathway on BC cells, we treated 4T1 cells and MDA-MB-231 cells with C5a and conducted RNA-sequence analysis. Gene Set Enrichment Analysis revealed that among various forms of programmed cell death (PCD)—including apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis–cuproptosis (labeled by red box) exhibited a high enrichment score, suggesting a potential relationship between the C5a/C5aR pathway and cuproptosis in BC cells (figure 2J,O). Figure 2P,Q presents a Volcano Plot and heat map that demonstrated the effective upregulation of the cuproptosis-related gene ATP7B in the C5a-treated group compared with the control group. The western blot and real-time quantitative PCR (RT-qPCR) results displayed in figure 2M,N and R,S further corroborated that ATP7B expression was enhanced by the C5a/C5aR pathway activation. ATP7B primarily functions by transporting copper into the trans-Golgi network lumen to facilitate the biosynthesis of copper-dependent enzymes and to enable the efflux of excess copper from cells by incorporating it into extracellular vesicles.¹⁹ Moreover, previous studies have indicated that the transcriptional activation of ATP7B could confer cuproptosis resistance in gastric cancer.²⁰ Therefore, we hypothesized that the upregulation of ATP7B by the C5a/C5aR pathway may contribute to cuproptosis resistance in cancer cells.

C5a/C5aR pathway drove cuproptosis resistance in cancer cells by upregulating ATP7B expression through activation of the Wnt/β-catenin pathway

To investigate whether the C5a/C5aR pathway could mediate cuproptosis resistance, we induced cuproptosis in 4T1 cells using ES-Cu, a known cuproptosis inducer.^{21 22} As illustrated in figure 3A, 4T1 cells treated with ES-Cu for 48 hours displayed marked disruption of cell membrane integrity. Notably, treatment with C5a reversed the disruption caused by ES-Cu. Furthermore, results from the CCK-8 assay depicted in figure 3B indicated that the ES-Cu induced cell death in 4T1 cells could be mitigated by C5a, while this reversal was abrogated by C5aRA. Subsequently, we aimed to determine whether the protective effect of C5a was attributed to the inhibition of cuproptosis. As a newly defined form of regulated cell death, cuproptosis is characterized by the aggregation of lipoylated proteins, such as dihydrolipoamide S-acetyltransferase (DLAT).¹⁰ Therefore, immunofluorescence (IF) experiments were conducted to visualize the distribution of DLAT in 4T1 cancer cells. Indeed, treatment with ES-Cu resulted in the formation of DLAT puncta (denoted by white arrows in figure 3C), indicating DLAT aggregation. In addition, co-treatment with C5a significantly inhibited DLAT aggregation in 4T1 cancer cells compared with ES-Cu treatment alone, and this inhibition was reversed by C5aRA (figure 3C). Collectively, these observations suggested that the C5a/C5aR pathway suppresses ES-Cu-induced cuproptosis. As demonstrated in figure 2N, the C5a/C5aR pathway could upregulate ATP7B, which plays a critical role in cuproptosis resistance. We further assessed the ATP7B expression in the ES-Cu-induced cuproptosis model. As shown in figure 3D, treatment with ES-Cu decreased ATP7B expression, while C5a increased it. This finding confirmed that the C5a/C5aR pathway contributed to cuproptosis resistance by upregulating the expression of ATP7B.

The Wnt/β-catenin signaling plays a crucial role as cancer cells adapt to therapeutic perturbations and changing biological stresses, developing resistance to cell death.^{23 24} Besides, it plays a critical role in BC initiation, metastasis, and chemotherapy resistance.²⁵ Interestingly, the heatmap presented in figure 3E,F illustrated the expression distribution of proteins involved in Wnt/β-catenin pathway activity, suggesting that the C5a/C5aR pathway can activate this pathway. Besides, prior research has established that ATP7B is regulated by the Wnt/β-catenin pathway.²⁰ Additionally, the C5a/C5aR pathway has been shown to activate the Wnt/β-catenin pathway in conditions such as diabetic kidney disease and colorectal cancer.^{26 27} Thus, we hypothesized that the C5a/C5aR pathway upregulated ATP7B through the activation of the Wnt/β-catenin pathway. To further validate this, we employed Dickkopf-1 (DDK1), a Wnt/β-catenin pathway inhibitor,²⁸ in C5a-treated cells (4T-1, E0071, MDA-MB-231, MCF7). RT-qPCR and western blot analysis in figure 3G,H revealed that the C5a/C5aR pathway induced ATP7B expression; however, this upregulation was reversed by Wnt/β-catenin pathway inhibitor DDK1. In summary, these results proposed that the C5a/C5aR pathway induced cuproptosis resistance in cancer cells by upregulating ATP7B expression through the activation of the Wnt/β-catenin pathway.

Cuproptosis-related genes expression was significantly different in BC and normal breast tissues and BC tissues displayed high ATP7B expression

To investigate the relationship between BC and cuproptosis, we selected five genes (ATP7B, DLAT, FDX1, SLC31A1, LIAS) closely associated with cuproptosis and subsequently performed an expression analysis in BC.¹⁰ As illustrated in figure 4A, we examined the expression patterns of cuproptosis-related genes (CRGs) in BC tissues versus non-tumor tissues using The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets, revealing that ATP7B, DLAT, FDX1 and SLC31A1 were significantly overexpressed in BC compared with normal tissues. In contrast, lipoic acid synthase (LIAS) exhibited higher expression levels in normal breast tissues. Following this, we conducted Kaplan-Meier analysis to assess the correlation between CRGs expression and survival outcomes in BC. The results of the overall survival (OS) analysis indicated that high expression levels of DLAT, FDX1, SLC31A1 and LIAS were associated with poorer prognoses in patients with BC, whereas the expression level of ATP7B is positively correlated with patient survival (figure 4B). To enhance the diversity of our database, we collected gene sequencing data of TNBC from two additional sources of the GEO database, alongside TCGA, for our analysis (in online supplemental figure 1). Consistent with the TCGA database analysis presented in the revised figure 4A,B, we examined the differences in cuproptosis-related genes between normal and tumor tissues and analyzed the survival duration disparities between high-expression and low-expression genes. These results provide further support for the role of cuproptosis in BC progression, suggesting that ATP7B may play a pivotal role. Additionally, we used IHC analysis to examine the expression levels of DLAT, FDX1 and ATP7B in tumorous tissues from patients with BC in comparison to adjacent non-tumorous tissues. As shown in figure 4C,D, DLAT, FDX1 and ATP7B expressions were significantly greater in tumorous tissues than in adjacent non-tumorous tissues. Numerous studies have consistently reported elevated copper concentrations in tumor tissues and serum across various cancer types, including BC,²⁹ gastric cancer and lung cancer.³⁰ In patients with BC, high serum copper levels closely correlate with tumor stage and disease progression.³¹ Increased serum copper can promote tumor cell growth by activating the PI3K-AKT signaling pathway.³² Conversely, copper overload may induce cuproptosis in BC cells, leading them to develop strategies to evade this process. Previous research has indicated that transcriptional activation of ATP7B can confer resistance to cuproptosis in gastric cancer.²⁰ Hence, we hypothesize that BC elevates ATP7B expression to counteract cuproptosis. These findings underscore the significant role of copper in the pathogenesis of BC and highlight the potential for targeted copper-based therapies in its treatment.

CuS nanoparticles displayed antitumor efficacy by inducing cancer cell cuproptosis in vitro

As copper plays a pivotal role in cellular metabolic processes, targeting copper metabolism, specifically through cuproptosis, has emerged as a promising strategy for cancer therapy. However, effectively inducing cuproptosis in tumor cells remains challenging due to cuproptosis resistance and the inconsistent efficacy of copper ionophores.^{20 33} As we have demonstrated, activation of the C5a/C5aR pathway could mediate cuproptosis resistance in cancer cells. We hypothesized that combining a cuproptosis inducer with the C5a/C5aR pathway blocking (C5aRA) could represent a novel and effective therapeutic strategy for cancer. First, we chose CuS NPs as the cuproptosis inducer for it was a copper-based NPs and has been proved to be able to inhibit melanoma growth by the PTT.³⁴ Initially, we assessed the cytotoxicity of CuS NPs on 4T1 cancer cells in vitro. The in vitro antitumor activity of CuS NPs against 4T1 tumor cells was evaluated using a CCK-8 assay. As depicted in figure 4E, CuS NPs exhibited promising antitumor effects, 100 µg/mL treatment resulted in a 47% decrease in cell viability of 4T1 cells. As illustrated in figure 4F, 4T1 cells incubated with CuS NPs (100 µg/mL) for 48 hours displayed significant disruption of membrane integrity. Furthermore, we confirmed the antitumor effects of CuS NPs through a plate colony formation assay, which demonstrated that the number of colonies in the CuS NPs group was approximately 10% lower than that in the control group (figure 4G,H). To investigate the mechanism by which CuS NPs induce cuproptosis, we visualized DLAT oligomerization via IF imaging. As shown in figure 4I, cells treated with CuS NPs exhibited a marked increase in DLAT aggregation. Additionally, the Fe-S cluster protein ferredoxin 1 (FDX1), a crucial regulator of copper-mediated cell death, can reduce Cu²⁺ to Cu⁺. The generated Cu⁺ could react with intracellular H₂O₂ to generate toxic ⋅OH via a Fenton-like reaction.^{10 35} Therefore, we assessed intracellular ROS generation following CuS NPs treatment. As shown in figure 4J, CuS NPs treatment elevated intracellular ROS levels. Moreover, the expression of the cuproptosis marker gene LIAS was significantly reduced in 4T1 cells after CuS NPs treatment (figure 4K). Overall, these findings indicated that CuS NPs can effectively induce cuproptosis in tumor cells.

Antitumor ability of CuS nanoparticles in vivo

Given the significant antitumor activity of CuS NPs observed in vitro, we proceeded to evaluate their therapeutic effects in vivo. Initially, we assessed the potential antitumor effects of CuS NPs using the 4T1 xenograft mouse model. Female Balb/c mice received subcutaneous injections of 3×10⁵ 4T1 cells. When the tumor volume reached approximately 80 mm³, the mice were randomly divided into two groups: one receiving 0.9% NaCl and the other receiving CuS NPs/lazer. Mice were intravenously injected with the respective drugs on day 5 and 8 (figure 4L). CuS NPs are reported to exhibit strong near-infrared light absorption and high photothermal conversion efficiency;^{34 36} therefore, an 808 nm lazer (2 W cm⁻²) was applied for 3 min, 6 hours post-injection, to enhance its antitumor effect. As shown in figure 4M–O, the tumor volume and weight of the 4T1 xenografts were significantly reduced following intravenous injection of CuS NPs combined with lazer treatment, compared with the control group. Consistent with in vitro findings, a significantly decreased expression level of LIAS was observed in the tumor tissues treated with CuS NPs (figure 4P). Furthermore, other copper-based NPs like NP@ESCu have been reported to remodel TME by inducing the maturation of dendritic cells (DCs) and promoting CD8⁺ T-cell infiltration within tumor tissues. Besides, it also stimulated the transition from the immunosuppressive M2 phenotype to the immunostimulatory M1 phenotype in tumor-associated macrophages (TAMs), while inhibiting the aggregation of myeloid-derived suppressor cells (MDSCs).³⁷ So, we also explore the effects of CuS NPs on the TME, tumors were collected from the mice for the analysis of immune parameters. The results indicated that the percentage of DCs in the tumors of mice treated with CuS NPs was higher than that in the control group treated with 0.9% NaCl. Conversely, the percentage of MDSCs decreased in the CuS NP-treated mice (figure 4Q,R). Therefore, CuS NPs effectively reprogrammed the TME, thereby activating a systemic antitumor immune response. In conclusion, CuS NPs represented a promising antitumor NP, suitable for applications in photothermal therapy, cuproptosis-related therapy, and immunotherapy.

Biosafety evaluation of CuS NPs

Next, the biosafety of CuS NPs was evaluated in vivo. To this end, healthy mice received injections of 0.9% NaCl or CuS NPs through the tail vein. Blood was collected for further analysis after 48 hours. The physiological and biochemical parameters of blood from mice treated with different agents were thoroughly examined. The results indicated that ALT, AST, CREA, TBIL, and DBIL levels in mice treated with CuS NPs did not significantly differ from those in mice treated with 0.9% NaCl (figure 5A). Collectively, these findings suggested that CuS NPs exhibited low toxicity and sufficient safety in vivo. Under pathological conditions, major organs of the 4T1 xenograft mice were observed through H&E staining. No significant morphological changes were detected in the principal tissues and organs of mice treated with CuS NPs compared with those treated with 0.9% NaCl (figure 5B). Nevertheless, a significant limitation of metal NPs is their limited biocompatibility with blood components and the immune system.^{9 38} Studies have reported that other metal-based NPs like PEG-Fe₃O₄ iron oxide NPs can activate the complement system and provoke an inflammatory response.^{9 38} To investigate whether CuS NPs could activate the C5a/C5aR pathway, female Balb/c mice were administered CuS NPs. Following exposure, an increase in C5a levels was observed (figure 5C). Under pathological conditions, after treating 4T1 xenograft mice with CuS NPs, IHC staining of xenograft tumor tissues revealed a significant increase in the levels of C5b-9 and C5aR in these tissues (figure 5D). These results collectively demonstrated that CuS NPs could induce activation of the complement system under both normal physiological and pathological conditions.

C5a/C5aR pathway blocking enhanced the antitumor effect of CuS NPs on breast cancer cells in vitro

As CuS NPs can activate the C5a/C5aR pathway, which negatively affects tumor therapy by inducing cuproptosis resistance, we hypothesized that treatment targeting the C5a/C5aR pathway could enhance the antitumor efficacy of CuS NPs. To investigate this potential combined therapy efficiency, we initially treated 4T1 cells with CuS NPs, CuS NPs+C5 a, and CuS NPs+C5 a + C5 aRA. As illustrated in figure 5E, 4T1 cells incubated with CuS NPs for 48 hours displayed significant disruption of cell membrane integrity, which was inhibited by C5a treatment. Furthermore, colony formation assays confirmed that blocking the C5a/C5aR pathway could augment the antitumor effects of CuS NPs (figure 5F,G). Subsequently, the results of the CCK-8 assay, shown in figure 5H, indicated that while CuS NPs exhibited promising antitumor effects, the presence of C5a increased 4T1 cell viability, suggesting that C5a compromised the therapeutic efficacy of CuS NPs. However, when the C5a/C5aR pathway was inhibited by C5aRA, this attenuation was reversed. To ascertain whether the decreased anti-tumor effect induced by C5a/C5aR was due to inhibited cuproptosis, IF experiments were conducted to visualize the distribution of DLAT in 4T1 cancer cells. Indeed, CuS NPs induced the formation of DLAT puncta (indicated by white arrows), indicating DLAT aggregation. Notably, co-treatment with C5a and CuS NPs significantly inhibited DLAT aggregation in 4T1 cancer cells compared with treatment with CuS NPs alone, while C5aRA treatment enhanced DLAT aggregation (figure 5I). Additionally, C5a reduced the generation of ROS induced by CuS NPs treatment, and this effect was reversed by C5aRA treatment (figure 5J). In 4T1 cells, LIAS expression decreased after CuS NPs treatment but was subsequently increased by C5a (figure 5K). Collectively, these results suggest that the combination of CuS NPs with C5a/C5aR pathway blockade enhanced cuproptosis in 4T1 cells, thereby improving the efficacy of CuS NPs in tumor therapy.

C5a/C5aR pathway blocking promoted the antitumor effect of CuS NPs in multiple tumor models

Next, we evaluated the in vivo anticancer efficacy of CuS NPs in combination with C5aRA under lazer irradiation in Balb/c mice bearing 4T1 tumors. The mice were randomly assigned to four groups (n=5 mice per group) and received intravenous injections on day 5 and 8 as follows: (1) 0.9% NaCl, (2) CuS NPs/lazer, (3) C5aRA, and (4) CuS NPs/lazer in combination with C5aRA. For the irradiation groups, tumor sites were exposed to an 808 nm lazer (2 W cm⁻²) for 3 min 6 hours after injection. Consistent with our in vitro findings, the 4T1 xenograft model demonstrated that the combination of CuS NPs/lazer and C5aRA significantly inhibited in vivo growth of 4T1 cells compared with treatments with either agent alone (figure 6A–D). Furthermore, IHC results indicated that LIAS expression was reduced in the combination group compared with single-agent treatment group (figure 6E). Additionally, the combination therapy remodeled the TME by decreasing MDSCs and increasing DCs, thus activating a systemic antitumor immune response (figure 6F,G). Subsequently, the major organs of the mice were examined using H&E staining. No significant morphological changes were observed in the major tissues and organs of mice treated with CuS NPs and C5aRA (figure 6H), indicating that the combination of CuS NPs with C5aRA exhibited low toxicity and sufficient safety in vivo. We also assessed the therapeutic effects of this combination in other xenograft tumor models, and the results shown in figure 7A–H demonstrated that the combination exhibited a superior therapeutic effect in both B16-F10 and CT-26 xenograft tumors. From all these experiments, we concluded that the CuS NPs combined with C5aRA display exceptional antitumor efficacy. This synergy enhances the antitumor effect by promoting cancer cell cuproptosis, remodeling the TME, and exhibiting photothermal therapeutic effects.

Discussion

represents one of the most prevalent malignant cancers among women globally, characterized by a high incidence and recurrence rate.^{39 40} According to the latest statistics from 2022, BC alone accounts for nearly one-third of all new cancer diagnoses in women in the USA, totaling 287,850 new cases.⁴¹ Despite the development of diagnostic and therapeutic strategies that acknowledge the heterogeneity of BC, effective approaches to enhance prognosis for recurrence-free survival and OS remain insufficient. Furthermore, the resistance of patients to chemotherapy, radiotherapy, or endocrine therapy poses a significant challenge in achieving long-term survival.⁴²

Growing evidence indicates that the complement system plays a crucial role in modulating cancer immunity, contributing to tumor initiation and development to varying degrees.^{4 43} The C5a/C5aR pathway has been linked to tumor progression and poor prognosis in patients with BC.^{5 6} Tumor-promoting effects of C5a have also been documented in various murine cancer models, including breast, cervical, lung, ovarian, colorectal, and melanoma types.^44–47 Prior research has investigated the role of the C5a/C5aR pathway in the progression of BC. Maciej M Markiewski demonstrated that ribosomal protein S19 interacts with C5aR1, thereby promoting BC growth by facilitating the recruitment of MDSCs to tumors.⁴⁸ Additionally, Ou et al revealed that C5aR1+neutrophils enhance the glycolytic capacity of BC cells through the ERK1/2-WTAP-ENO1 signaling pathway.⁴⁹ Xi Li provided evidence that inhibiting C5aR1 reprograms TAMs in two murine BC models: one intrinsically sensitive to PARP inhibitors (T22) and the other resistant (T127). In these models, Rps19/C5aR1 signaling is selectively elevated in TAMs_C3, which predominantly express genes related to an anti-inflammatory/protumor state.⁵⁰

Recently, metal metabolism-associated cell death mechanisms such as ferroptosis and cuproptosis have garnered increasing attention in cancer therapy research.^{51 52} Cuproptosis is induced by the accumulation of intracellular copper ions (Cu²⁺), which subsequently bind to lipoylated components in the mitochondrial TCA cycle. This binding aggregates Cu-bound lipoylated mitochondrial proteins and downregulates Fe-S cluster proteins, leading to proteotoxic stress and ultimately PCD.¹⁰ Since its discovery, cuproptosis, like ferroptosis, has attracted considerable interest for its potential as a target for novel anticancer therapeutic strategies.^{33 37 53–55} Many studies have focused on inducing cuproptosis in cancer cells for therapeutic purposes. Tsvetkov et al observed that ES-Cu induces cellular damage through cuproptosis.¹⁰ Furthermore, copper NPs, such as CuO and CuS, have been designed for antitumor therapy.^{33 56} Boda Guo developed an ROS-sensitive polymer (PHPM) to co-encapsulate ES and Cu, forming NPs (NP@ESCu). NP@ESCu was rapidly taken up by tumor cells, resulting in a 4.2-fold increase in intracellular Cu and downregulation of the Fe-S cluster protein LIAS, facilitating cuproptosis. And current research on nanomedicine is focusing on cuproptosis-based synergistic cancer therapies. There is a strong desire to combine cuproptosis with other anticancer modalities, such as photodynamic therapy, photothermal therapy, ferroptosis, autophagy, and immunotherapy.³⁷ CuS NPs, which can induce cuproptosis while exhibiting strong near-infrared light absorption and high photothermal conversion efficiency, represent a promising material for cancer therapy.^{34 36}

However, effectively inducing cuproptosis in tumor cells remains challenging due to variations in copper ion metabolism, tumor resistance to cuproptosis, and the inconsistent efficacy of copper ionophores. Reports indicated that gastric cancer cells can activate the Wnt/β-catenin signaling pathway, conferring resistance of cancer stem cells to cuproptosis.²⁰ Therefore, further analysis of the mechanisms underlying cuproptosis resistance is crucial for developing therapies based on copper particle-induced cuproptosis. Our previous study demonstrated that the C5a/C5aR pathway can lead to ferroptosis resistance in BC cells.⁹ However, the relationship between the C5a/C5aR pathway and cuproptosis resistance remains elusive. Additionally, a significant drawback of metal NPs is their lack of biocompatibility with blood components and the immune system. It has been reported that PEG-Fe₃O₄ iron oxide NPs can activate the complement system and induce inflammatory responses.^{9 38} Further investigation into the mechanisms of cuproptosis resistance and the properties of copper NPs can enhance the refinement of therapeutic approaches targeting cuproptosis.

In addition to targeting cancer cells, the TME plays a crucial role in tumor progression and is increasingly recognized as a promising therapeutic target in cancer treatment. This approach focuses on reprogramming the immune microenvironment to enhance the identification and eradication of tumors. Numerous studies have explored the relationship between the cuproptosis pathway and immune responses. It has been reported that cuproptosis-related signatures are associated with immune cell infiltration in colorectal cancer, including DCs, CD8⁺ and CD4⁺ T cells, regulatory T cells, and macrophages.⁵⁷ Similarly, in BC, it has been shown that the expression of the cuproptosis-related gene PDHA1 correlates closely with the infiltration levels of CD4⁺ memory T cells, M0 and M1 macrophages, and mast cells. These findings are significant for understanding the link between cuproptosis and immunotherapy.⁵⁵ Furthermore, copper-based NPs NP@ESCu not only induce the maturation of DCs and promote CD8⁺ T-cell infiltration within tumor tissues but also stimulate the transition from the immunosuppressive M2 phenotype to the immunostimulatory M1 phenotype in TAMs, while inhibiting the aggregation of MDSCs.³⁷

In our study, we demonstrated the activation of the C5a/C5aR pathway in BC and its correlation with disease progression. Furthermore, this pathway contributes to cuproptosis resistance in cancer cells by upregulating ATP7B expression through the activation of the Wnt/β-catenin pathway. Consequently, we hypothesized that inhibiting the C5a/C5aR pathway could enhance cuproptosis-targeted therapy. Based on these findings, we proposed a novel combination strategy that integrates CuS NPs (CuS) with lazer therapy and C5aRA to inhibit the C5a/C5aR pathway. Our results showed that the synergy between CuS NPs and C5aRA produced a remarkable antitumor effect across multiple xenograft cancer models. This combination therapy led to increased cancer cell cuproptosis, enhanced immunotherapeutic effects through the proliferation of DCs and the reduction of MDSCs in the tumor immune microenvironment, as well as significant photothermal therapy outcomes. Furthermore, C5aRA mitigated the adverse effects of complement activation induced by CuS NPs. The rational and most compelling aspect of the synergy between CuS NPs and C5aRA lies in their ability not only to eliminate the detrimental effects of complement activation from CuS NP treatment but also to enhance cuproptosis in cancer cells. This strategic combination exhibited excellent antitumor efficacy through the promotion of cancer cell cuproptosis, remodeling of the TME, and effective photothermal therapy.

Conclusion

Our results indicated that the activation of the C5a/C5aR pathway plays a significant role in BC cuproptosis resistance by upregulating ATP7B expression through the Wnt/β-catenin signaling pathway. Consequently, the combination of CuS NPs with lazer treatment and C5aRA significantly enhances the antitumor efficacy of CuS NPs, effectively overcoming cuproptosis resistance. This approach results in a synergistic effect in cancer therapy, integrating cuproptosis-targeting therapy, immunotherapy, and photothermal therapy and paving the way for the development of advanced copper NP-based therapies for tumors. The complete workflow of this study is depicted in figure 7I.

Data availability statement

Data are available upon reasonable request.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

The animal study was approved by the Army Medical University ethics committee. The study was conducted in accordance with the local legislation and institutional requirements. The maximal tumor size/burden permitted by the ethics committee is 2000 mm³ and the maximal tumor size/burden in the study was not exceeded. The studies involving humans were approved by the Army Medical University ethics committee. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

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