Activation of cuproptosis amplifies immunogenic cell death for anti-tumor immunotherapy in gastric cancer

doi:10.21203/rs.3.rs-5276654/v1

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Activation of cuproptosis amplifies immunogenic cell death for anti-tumor immunotherapy in gastric cancer

https://doi.org/10.21203/rs.3.rs-5276654/v1

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Cuproptosis is a novel form of cell death mediated by protein lipid acylation and highly related to mitochondrial metabolism. Copper directly binds to the fatty acylation component of the tricarboxylic acid (TCA) cycle, resulting in toxic protein stress and ultimately leading to cell death. Immunogenic cell death (ICD) can effectively enhance tumor immunogenicity and induce systemic anti-tumor immunity. However, the relationship between copper-induced cell death and immunogenicity in gastric cancer remains unclear. We utilized the R package to conduct KEGG and GO analysis on differentially expressed genes (DEGs) associated with crucial copper-induced cell death genes in gastric cancer, confirming the potential of copper-induced cell death genes to enhance anti-tumor immunity. Examination of online databases revealed a significant reduction in the expression of key genes related to copper-induced cell death in gastric cancer tissues compared to normal gastric tissues. The expression of cuproptosis-related genes exhibited a negative correlation with the abundance of Treg and MDSC cells, while showing a positive correlation with the expression of ICD-related genes. Furthermore, we validated the impact of key copper-induced cell death genes on subcutaneous tumor growth and ICD in vivo. Tumor tissues with high FDX1 expression demonstrated increased levels of CD8⁺ T cells, perforin 1 (PRF1), high mobility group box 1 (HMGB1), and Cu²⁺. In conclusion, the activated copper-dependent death-key genes ultimately facilitate immunogenic cell death in gastric cancer, thereby augmenting the anti-tumor immune response in gastric cancer.

cuproptosis

gastric adenocarcinoma

immunogenic cell death

FDX1

HMGB1

Gastric cancer, which ranks third in terms of cancer-related deaths, is one of the most malignant tumors worldwide ¹. Patients with advanced gastric cancer are frequently diagnosed at a late stage, and the majority of these patients undergo treatment with immunotherapy or chemotherapy ². However, the effectiveness of a single treatment is limited, leading to an increasing focus among clinicians and researchers on the use of combination therapy.

Copper, functioning as an essential trace element within the human body under normal circumstances, assumes a pivotal role in maintaining intracellular and extracellular copper homeostasis ³. Any disruption to the delicate balance of copper homeostasis can profoundly impact diverse biological processes ³. In recent years, copper-induced cell death has gained significant attention as a novel form of cell death. Ferredoxin1(FDX1), as an upstream regulator of copper-induced cell death, mediates the reduction of bivalent copper ions to monovalent copper ions and facilitates the fatty acylation of dihydrolipoamide S-acetyltransferase (DLAT) ⁴. Abnormal aggregation of fatty acylated DLAT and the loss of Fe-S cluster proteins lead to protein toxic stress and ultimately induce cell death ^4,5. Recent researches have demonstrated that the synergistic utilization of copper ion carriers in conjunction with immunotherapy may potentially enhance the efficacy of cancer treatment in clinical settings ^6–9. Consequently, there exists a burgeoning interest in comprehending the intricate mechanisms underlying copper-mediated cell death for its potential application in cancer treatment.

When tumor cells are stimulated by external factors and undergo cell death, they undergo a transition from being non-immunogenic to becoming immunogenic ¹⁰. This subsequently triggers the body’s immune response against the tumor, known as immunogenic cell death (ICD), which was conceptualized in 2005 ¹¹. Damage-associated molecular patterns (DAMPs) released during ICD can engage pattern recognition receptors (PRRs) located on the surface of dendritic cells (DCs), triggering a cascade of cellular responses and ultimately activating both innate and adaptive immune reactions ^12,13. The primary effects of ICD encompass the induction of calreticulin (CALR) exposure, secretion of high mobility group box-1 (HMGB1), release of ATP, and engagement with pattern recognition receptors on dendritic cells’ surface to activate them and subsequently initiate an immune response ¹⁴. There may also be release of CXCL10 ¹⁵. CALR plays a pivotal role in maintaining calcium homeostasis, regulating gene expression, facilitating molecular chaperoning, and promoting cell adhesion ¹⁶. HMGB1 exhibits its functionality by binding to nucleosomes to induce DNA bending, participating in crucial processes such as DNA transcription and replication, and mediating inflammatory responses ¹⁷. Existing research has demonstrated that the activation of ICD can significantly enhance anti-tumor immunity ^18–21. Following ICD induction, specific immune responses against tumors are initiated, leading to improved long-term efficacy of anticancer medications through direct targeting and elimination of cancer cells or by stimulating anti-tumor immune reactions ¹⁵.

However, despite numerous studies documenting the correlation between copper-induced cellular death and immunotherapy, there is currently a dearth of research investigating the association between genes linked to copper-triggered cell death and markers indicative of ICD. In this study, we initially conducted an analysis of the correlation between the copper death key gene FDX1 and immunotherapy through public database. Subsequently, we examined the clinical characteristics and prognosis of FDX1 in gastric cancer. Additionally, we confirmed that high expression of FDX1 can suppress the proliferation of subcutaneous tumors of gastric cancer in vivo. In addition, the immunogenic death-related molecule HMGB1 was increased in tumors with high FDX1 expression compared to the control group. Our study was intended to investigate whether activation of cuproptosis amplifies immunogenic cell death for anti-tumor immunotherapy, providing new insights for immunotherapy in gastric cancer.

The activation model of cuproptosis amplifies immunogenic cell death in gastric cancer (Fig. 1).

Copper-death key gene promotes antitumor immunity.

To investigate the role of copper-death key genes in gastric cancer, KEGG enrichment analysis was conducted to investigate the differentially expressed genes associated with FDX1 in gastric cancer. Results revealed significant enrichments in pathways such as citrate cycle, 2-oxocarboxylic acid metabolism, and antigen processing and presentation (Fig. 2A). Furthermore, the GO analysis revealed that the differentially expressed FDX1 genes in gastric cancer were significantly enriched in the integrin and C-X-C chemokine pathways (Fig. 2B). The integrin consists of two distinct subunits, α and β. In mammals, there are 18 α subunits and 8 β subunits, which can combine to form 24 different pairs of αβ integrins ²². The previous study ²³ has shown that integrin β7 (ITGB7) is expressed on the surfaces of some immune cells and plays a critical role in facilitating the homing of immune cells to the intestinal tract. The decreased expression of ITGB7 on tumor-derived CD8⁺ T cells may be linked to CD8⁺ T cells anti-tumor effectiveness. Furthermore, ITGB7 can impede colorectal tumor progression by preserving anti-tumor immunity. The chemokine family exhibits diverse biological functions, including chemotaxis, tumor growth, and angiogenesis ²⁴. CXCLs orchestrate inflammation and immune responses by mediating the migration of key white blood cell populations such as neutrophils, monocytes, and macrophages ²⁵. Furthermore, the Gene Set Enrichment Analysis (GSEA) showed significant enrichment of IL-2 STAT5 signaling and epithelial-mesenchymal transition in the group with low FDX1 expression (Fig. 2C, D). In summary, FDX1 may enhance the anti-tumor immune response in gastric cancer.

Reduced expression of cuproptosis-related genes in gastric cancer.

Analysis of the HPA database revealed that the expression levels of copper-death related genes were higher in normal tissues compared to tumor tissues, suggesting a potential association between high expression of these genes and improved prognosis (Fig. 3A). Univariate hazard regression analysis revealed that elevated expression of copper-related death genes and immunogenic death genes were associated with a lower risk in the overall survival analysis of gastric cancer patients (Fig. 3B). In summary, elevated FDX1 expression in patients may indicate a more favorable prognosis.

The expression of cuproptosis related genes is positively associated with ICD markers in GC.

To examine the relationship of cuproptosis related genes and anti-tumor immunity in gastric cancer, the expression of copper-related death genes was examined for its correlation with immune infiltration by using the online database TISIDB (Fig. 4A). The database analysis uncovered a notable negative correlation between the levels of FDX1, PDHA1 and DLAT expressions with Treg cells (Fig. 4B). Additionally, a negative correlation was observed between FDX1, PDHA1, DLAT and MDSC cells (Fig. 4C). GEPIA dataset revealed the significant positive correlation between the expression levels of cuproptosis related genes and HMGB1, which serves as one of the markers for ICD. In addition, the expression levels of copper-related death-associated genes and CALR are also have a positive association (Fig. 4D). The correlation between HMGB1 and immune cells was significantly demonstrated in Fig. 4E, revealing a striking negative association between the expression level of HMGB1 and the abundance of Treg and MDSC cells. The above findings indicate a favorable association between genes linked to copper-induced cell death and genes associated with immunogenic cell death in gastric cancer.

Single cell analysis of cuproptosis related genes and ICD genes in gastric cancer.

The TISCH (Tumor Immune Single-cell Hub) database was utilized to investigate the expressions of FDX1, LIAS, DLD, DLAT, PDHA1, PDHB, HMGB1 and CALR in gastric cancer tissues and their intricate relationship with the tumor microenvironment (TME). The single-cell analysis of both datasets (STAD_GSE134520 and STAD_GSE167297) revealed that the distribution patterns of FDX1, LIAS, DLD, DLAT, PDHA1 and PDHB in individual cells were consistent with those observed for ICD markers HMGB1 and CALR (as shown in Fig. 5A and B), demonstrating a remarkable alignment between these molecular entities. This alignment suggests a potential interplay between genes related to copper-induced cell death and markers indicative of ICD within the complex tumor microenvironment of gastric cancer.

High expression of cuproptosis-related genes is associated with a positive prognosis within the TME in gastric cancer.

Kaplan Meier Plotter database was utilized to analyze the survival curves of the copper death gene in gastric cancer. Patients with gastric cancer who exhibit high expression levels of FDX1 and LIAS tend to have a longer overall survival, suggesting that increased expression of genes related to copper-induced cell death may serve as a predictive marker for a better prognosis (Fig. 6A). Similarly, in patients characterized by Tregs enrichment, higher levels of FDX1 and LIAS expression were associated with improved outcomes but were less significant compared to those without Tregs enrichment (Fig. 6B). Additionally, patients with high expression of copper death genes showed a favorable prognosis in the presence of enriched CD8⁺ T cells, but this association was not observed with decreased CD8⁺ T cells. This suggests that the positive impact of copper-related death-related genes on gastric cancer patients’ prognosis may depend on the tumor immune cells infiltration (Fig. 6C, D).

FDX1 inhibits tumor growth and enhances ICD in vivo.

To investigate the impact of FDX1 on tumor proliferation and markers associated with ICD in vivo, we subcutaneously implant MFC cells into 615 mice and allow for periodic assessment of tumor volume at three-day intervals (Fig. 7A). Intriguingly, in vivo investigations revealed that FDX1 exerted a potent inhibitory effect on gastric cancer cell proliferation. Notably, heightened expression levels of FDX1 resulted in a significant reduction in both xenograft weight and volume (Fig. 7B, C). Quantification analysis conducted on tumor tissues generated by overexpressed FDX1 cells unveiled elevated values for PRF1, HMGB1 and Cu²⁺ content when compared to those observed within the control group (Fig. 7D). Moreover, Figures E showed augmented accounts for CD8⁺ T cell within tumor tissue samples originating from FDX1-overexpressing cells. The findings from the in vivo experiments provide evidence that cuproptosis could promote ICD in gastric cancer.

Somatic mutation analysis

To further explore the FDX1 mutation in human cancer, we employed the online tool cBioPortal to analyze a total of 1590 samples. The frequency of FDX1 alterations across gastric cancer is visually represented in Fig. 8A. In addition, cBioPortal offers comprehensive data on structural variants, mutations, and CAN data. Our findings indicate that amplification exhibits the highest mutation frequency among all TCGA tumors (2.04%), with deep deletion accounting for the majority of this occurrence (0.68%), as depicted in Fig. 8A. Figure 8B illustrates the use of cBioPortal to visualize high levels of FDX1 associated with amplified FDX1. According to Fig. 8C, missense mutation of FDX1 is identified as the primary mutation type in gastric cancer.

Cuproptosis, a form of cell death mediated through protein lipid acylation, is intricately linked with mitochondrial metabolism ⁴. The regulatory network governing copper-induced cell death involves a multitude of genes ^26–29, including FDX1, LIAS, DLAT, PDHA1, PDHB among others. As a crucial regulatory gene, FDX1 plays a vital role in the process of promote copper-induced cell death ⁴. Furthermore, numerous studies have demonstrated the association between FDX1 and immune cell infiltration. For instance, Zhang et al. demonstrated a positive correlation between copper metabolism-related risk scores and the recruitment of diverse immune cell populations, while also noting a negative correlation with the cytotoxicity of T cells against cancer cells ³⁰. Moreover, patients with higher copper metabolism-related risk scores displayed increased immune cell infiltration and prolonged survival following immunotherapy ³⁰. In Uterine Corpus Endometrial Carcinoma (UCEC), FDX1 was found to enhance the infiltration of CD8⁺ T cells while concurrently attenuating the proliferation and metabolic activity of UCEC cells ³¹. Additionally, FDX1 may have potential implications for prognostic prediction in thyroid cancer through its association with immune infiltration ³². Conversely, low expression of FDX1 in renal clear cell carcinoma is linked to shorter survival time and heightened immune activation, characterized by changes in tumor mutation burden, the tumor microenvironment, increased immune cell infiltration, and upregulated expression of immunosuppressive markers ³³. Therefore, the relationship between copper-induced cell death genes and anti-tumor immunity needs further investigation.

The TME is a complex network composed of various cell types, including tumor cells, immune cells, fibroblasts, and related cytokines ³⁴. The cells within the TME interact dynamically with cancer cells through numerous signaling mechanisms and pathways, participating in the onset, progression, metastasis, and response to treatment of tumors ³⁵. Within the TME, there are several types of immune-suppressive cells, such as MDSCs, tumor-associated macrophages, Tregs, and tumor-associated fibroblasts, alongside anti-tumor immune cells like natural killer cells and infiltrating CD8⁺ T cells ³⁵. The dysfunction of immune effector cells and the generation of numerous immunosuppressive cells in the tumor microenvironment are key factors contributing to tumor immune evasion ³⁶. ICD refers to a programmed cell death process that occurs within tumor cells, characterized by the release of various intracellular components that activate the immune system to generate an immune response against the tumor. This form of cell death plays a significant role in shaping the tumor immune microenvironment ³⁷. By releasing antigens and pro-inflammatory factors, immunogenic cell death can recruit and activate various immune cells, including T cells, natural killer cells, and antigen-presenting cells, thereby enhancing the immune cell’s ability to attack the tumor ³⁸. Therefore, there is a close relationship between immunogenic cell death and the tumor immune microenvironment, helping to promote immune surveillance of tumors and anti-tumor immune responses. Previous studies have indicated that copper death has an impact on reshaping the tumor microenvironment ³⁹, but the mechanism of its effect on ICD remains unclear.

In this study, the KEGG analysis revealed that the differentially expressed gene FDX1 was significantly enriched in the antigen presentation pathway. Previous studies have demonstrated that DAMPs released during ICD can effectively enhance the maturation and migration of APCs ^40,41. Therefore, it is hypothesized whether the differential expression of FDX1 may be involved in DAMP-mediated promotion of APC maturation. GO analysis showed that the differentially expressed genes of FDX1 were significantly enriched in the integrin pathway and chemokine pathway. CXCL10 is a member of the chemokine family, and there is CXCL10 release during ICD ¹⁵. Whether there is a high level of CXCL10 in gastric cancer, as previously reported ^42,43, that can enhance the T cell response in the tumor immune microenvironment of ovarian cancer. GSEA analysis indicated a significant enrichment of IL-2 STAT5 signaling and epithelial mesenchymal transition in the low FDX1 expression group. IL-2 is a cytokine with diverse effects, activating both regulatory T cells and effector T cells through three distinct signaling pathways (JAK-STAT, RAS-MAPK, and PI3K-AKT pathways) ⁴⁴. However, the crucial difference between Treg cells and Teff cells lies in the fact that Mst1-Mst2 enhances STAT5 signaling in Treg cells, while PTEN, PD-1 and CTLA-4 suppress PI3K-AKT signaling ⁴⁵. The differential gene function of the copper-dependent death key regulatory gene FDX1 was found to be enriched in immune regulation. Clinical case analysis revealed that genes related to copper-induced cell death were associated with a favorable prognosis in patients with gastric cancer.

During the correlation analysis, a negative association was observed between genes associated with copper-induced cell death and immune suppressor cells Tregs and MDSC which are known to have the potential to facilitate tumor evasion of the immune system ⁴⁶. Furthermore, correlation analysis demonstrated a positive correlation between regulatory genes involved in copper-induced cell death and immunogenic markers. We considered whether the activation of copper-induced death would additionally enhance immunogenic cell death. In vivo experiments have validated that the upregulation of the pivotal regulatory gene responsible for copper-induced cell death results in an augmentation of both perforin release and HMGB1 content, a death-associated immunogenic molecule. In addition, the concentration of copper ions in tumor tissues increased after FDX1 enhancement. Based on the findings from flow cytometry analysis, elevated FDX1 levels are associated with an increase in the percentage of CD8⁺ T cells within subcutaneous tumor tissues. These findings indicated that cuproptosis related genes could amplify immunogenic cell death and enhance anti-tumor immunity in gastric cancer. The importance of this study lies in providing a clear pathway for the development of drugs tailored for clinical treatment of gastric cancer, with the goal of improving therapeutic outcomes for the gastric cancer patients.

Data Recourses

The RNA sequencing (RNA-seq) data, corresponding clinicopathological data, somatic cell mutation, and copy number variation (CNV) files of gastric cancer (GC) were acquired from the Cancer Genome Atlas (TCGA) database (https://xena.ucsc.edu/).

R package

The KEGG analysis of FDX1 were determined using R software (version:4.3.2), where the “org.Hs.eg.db” package was used to print curve plots. p value < 0.05 was considered statistically significant. The GO analysis of FDX1 were determined using R software, where the “clusterprofiler” package was used to print curve plots. p value < 0.05 was considered statistically significant.

The Human Protein Atlas

The HPA database (https://www.proteinatlas.org/) offers protein expression data through three analysis modules: cell, tissue, and pathology. It also provides support for generating survival curves for cancer patients. The proteome analysis is based on 27,520 antibodies targeting 17,288 unique proteins and consists of 12 independent parts. In this study, we primarily utilized the tissue and pathology sections of the database. The tissue module was employed to retrieve the expression of copper death-related genes in normal gastric tissue, while the pathology module was used to assess the expression of copper death-related genes in gastric cancer tissue. Statistical analysis was conducted using a staining intensity scoring method.

GEPIA2

GEPIA2 (http://GEPIA 2 (cancer-pku.cn)) is an incredibly user-friendly and easily accessible database that effortlessly facilitates the analysis of RNA-seq expression data derived from a staggering 9,736 tumor samples and 8,587 normal samples obtained from the prestigious TCGA and GTEx projects. We have effectively utilized this remarkable website to acquire correlation analysis results between genes associated with copper-induced cell death and markers indicative of immunogenic cell death.

TISIDB

TISIDB (http://TISIDB (hku.hk)) serves as an online platform that focuses on the interaction between tumors and the immune system, bringing together various diverse forms of data. For each cancer type, the relative abundance of TILs were inferred by using gene set variation analysis (GSVA) based on gene expression profile.

TISCH

The Single Cell Tumor Immune Center (TISCH) (http://TISCH (comp-genomics.org)) database provides comprehensive information at the single-cell level regarding tumor microenvironments, encompassing gene expression and cell types such as immune cells, fibroblasts, and endothelial cells. Users can conveniently conduct targeted searches for specific genes or tumor locations.

Kaplan Meier-plotter analysis

Kaplan Meier Plotter (http://Kaplan-Meier plotter (kmplot.com)) is a powerful tool utilized for investigating the impact of multiple genes on tumor survival rates in gastric cancer patients. It leverages comprehensive databases such as EGA, GEO and TCGA to conduct survival analysis, which involves tracking cohorts and events over time to evaluate prognosis. The log-rank test is employed to compare survival rates among different groups. Our study specifically focused on assessing the prognostic significance of copper death-related genes in gastric cancer patients.

Cell culture

The mouse gastric cancer cell lines MFC was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MFC cell was cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 ng/mL streptomycin) at 37°C in a humidified atmosphere of 5% CO₂.

ELISA

Intratissue HMGB1 and PRF1 content were determined with Mouse PRF1(Perforin 1) ELISA Kit (Elabscience, E-EL-M0890c) and HMGB1 analysis kit (mlbio, MM-44107M2). After grinding and homogenizing fresh tumor tissue with zirconia, centrifuge the supernatant at 4°C for detection. Remove the well-coated plate from the kit and add the sample to be tested and standard substance, then incubate at 37°C for 90 minutes. Discard the liquid in the plate, then immediately add biotinylated antibody and incubate at 37°C for 60 minutes. Wash three times, add HRP enzyme conjugate, incubate at 37°C for 30 minutes, discard the liquid, wash five times. Add substrate solution and incubate at 37°C for 15 minutes. Finally, add termination solution, then measure the OD value at a wavelength of 450nm.

The contents of Cu²⁺ analysis

Intratissue Cu²⁺ was determined with Tissue Copper Content Assay Kit (solarbio, BC5565). After homogenizing fresh tumor tissue with zirconium beads, the supernatant should be centrifuged at 4°C for analysis. Utilize a 96-well plate and follow the instructions provided in the kit. Subsequently, measure the OD value at a wavelength of 580nm and use it in the formula to calculate the Cu²⁺ content in the sample being studied.

Flow cytometry analysis

To examine the tumor infiltrating lymphocytes, tumor tissue was digested in a specific medium consisting of 100 mL of DMEM supplemented with 2 mL of FBS, 50 µl of hyaluronidase (Solarbio, H8030), 0.1 g of collagenase I (Solarbio, C8140), and DNase I (Solarbio, D8071) at 37 ℃ for 30 min. Cells were stained with respective antibodies (CD45, Thermo Fisher, 11-0451-82; CD8a, Thermo Fisher, 46-0081-82) at 4 ℃ for 30 min. The stained cells were measured using FACS Calibur (BD Biosciences). The data were analyzed by utilizing the FlowJo software (Treestar Inc.).

The cBioPortal Website

The online tool cBioPortal is designed to explore, visualize, and interpret multimodal cancer genomes data (http://cbioportal.org). The site's intuitive web interface allows users with no background in bioinformatics to understand the complex cancer genomics landscape, and enables researchers to explore genetic alterations across genes, pathways and samples using a query interface combined with customized data storage.

Statistical analysis

The statistical analysis for the images was conducted using R software (version 4.3.2), SPSS 27.0, and GraphPad Prism 9.5. Unpaired t tests were used to compare normally distributed variables between two groups, with significance levels set at *< 0.05 and **<0.01.

CRediT authorship contribution statement

D. P. and C. S. developed the study concept and were responsible for the study design. Q. S. and Y. Z. wrote the manuscript and analyzed data. M. L. revised the manuscript. All authors contributed to the article and approved the submitted version.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data provided in the present study can be obtained by contacting the first author.

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No competing interests reported.

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You are reading this latest preprint version

Activation of cuproptosis amplifies immunogenic cell death for anti-tumor immunotherapy in gastric cancer

Status:

Version 1

Abstract

Figures

Introduction

Results

Somatic mutation analysis

Discussion

Materials and Methods

Data Recourses

R package

The Human Protein Atlas

GEPIA2

TISIDB

TISCH

Kaplan Meier-plotter analysis

Cell culture

ELISA

The contents of Cu²⁺ analysis

Flow cytometry analysis

The cBioPortal Website

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1

Activation of cuproptosis amplifies immunogenic cell death for anti-tumor immunotherapy in gastric cancer

Status:

Version 1

Abstract

Figures

Introduction

Results

Somatic mutation analysis

Discussion

Materials and Methods

Data Recourses

R package

The Human Protein Atlas

GEPIA2

TISIDB

TISCH

Kaplan Meier-plotter analysis

Cell culture

ELISA

The contents of Cu2+ analysis

Flow cytometry analysis

The cBioPortal Website

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1

The contents of Cu²⁺ analysis