Regulating copper homeostasis of tumor cells to promote cuproptosis for enhancing breast cancer immunotherapy

Ethical statement

All animal experiments were approved by the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology (IACUC Number: S904).

Animals

Female Balb/c mice (6-8 weeks old, 18–20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed in an animal facility under constant environmental conditions (room temperature, 22 ± 1 °C; relative humidity, 40–70%; and 12 h light-dark cycle), and all mice had free access to food and water. Tumor volume was calculated as 0.5 × (length × width × width). To minimize animal discomfort, according to the Guideline of Assessment for Humane Endpoints in Animal Experiments (Certification and Accreditation Administration of the P. R. China, RB/T 173-2018), in general experiments, the tumor burden should not exceed 5% of the animal’s normal body weight; in therapeutic experiments, it should not exceed 10% of the animal’s body weight (10% indicated that the diameter of the subcutaneous tumor on the back of a 25 g mouse reached 17 mm). At the end of the mouse experiments, mice were euthanized according to animal welfare standards (euthanasia of all animals was performed using isoflurane in small animal anesthetics).

Materials

Polyvinylpyrrolidone (PVP), copper chloride dihydrate (CuCl₂), sodium sulfide nonahydrate (Na₂S·9H₂O), 2,2’-azinobis(3-ethylbenzothiazoline -6-sulfonic acid ammonium salt) (ABTS), α-(4-pyridine-N-oxide)-N-tert butylnitrone (POBN), 2,2’-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH), and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Sodium hydroxide (NaOH), hydrazine hydrate solution (NH₂NH₂ · xH₂O), ethanol absolute, dimethyl sulfoxide (DMSO) isopropanol, sodium chloride, sodium dodecyl sulfate, anhydrous methanol, concentrated nitric acid, and hydrochloric acid was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Calcein acetoxymethyl ester (Calcein AM), propidium iodide (PI), reactive oxygen species assay Kit, BCA protein assay Kit, SDS-PAGE sample loading buffer 5X, hydrophobic polyvinylidene fluoride, (PVDF), penicillin–streptomycin, RNase-free dd H₂O, 4% paraformaldehyde fix solution and tween-20 was purchased from Shanghai Biyuntian Biotechnology Co., Ltd (Shanghai, China). Polyacrylamide hydrochloride (PAH) was purchased from Anhui Zesheng Technology Co., Ltd (Anhui, China). Hyaluronic acid (HA) was purchased from Sigma-Aldrich (Shanghai, China). RNA isolater, HiScript III All-in-one RT SuperMix Perfect for qPCR Kit and Taq Pro Universal SYBR qPCR Master Mix Kit was purchased from Vazyme Biotech Co., Ltd (Nanjing, China). ECL Chemiluminescence Substrate Kit was purchased from Beijing Labgic Technology Co., Ltd (Beijing, China). Pancreatin was purchased from Gino Saber Biotechnology Co., Ltd (Zhejiang, China). Omni-Easy™ One-Step PAGE Gel Fast Preparation Kit was purchased from Shanghai Yaenzyme Biopharmaceutical Technology Co., Ltd (Shanghai, China). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd (Zhejiang, China). PBS and DMEM was purchased from Thermo Fisher Scientific Co., Ltd (Shanghai, China). Phenylmethanesulfonyl fluoride, Tris(hydroxymethyl)methyl aminomethane, and Glycine was purchased from Beijing Kehbio Technology Co., Ltd (Beijing, China). All solutions were prepared using ultrapure water from the Milli-Q system (≥18.20 M Ω).

Cell lines

Mouse breast cancer cells (4T1: CRL-2539), mouse embryonic fibroblasts (NIH-3T3: CRL-1658), and mouse monocyte macrophage leukemia cells (RAW264.7: TIB-71) cell line was obtained from Wuhan Saikangte Biotechnology Co., Ltd. Mouse breast cancer cells-luciferase labeled (4T1-LUC: CRL-2539-LUC2) cells was cell line obtained from Wuhan service Biotechnology Co., Ltd. All three cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines in this study get tested without mycoplasma contamination. Each cell line was morphologically confirmed according to the information provided by the cell-source center, and the main 4T1, NIH-3T3, and RAW264.7 cell lines were authenticated with short tandem repeat (STR) analysis (Supplementary Tables 2–4).

Synthesis of Cu₉S₈

Weighing 480 mg of polyvinylpyrrolidone was dissolved in 50 mL of ultrapure water, 200 μL of CuCl₂ solution (0.5 M) was added and stirred for 5 min, then 50 mL of NaOH solution at pH = 9, and 16 μL of hydrazine hydrate (85%) were added to form a bright yellow Cu₂O suspension. After stirring for 5 min, 400 μL of Na₂S solution (1.33 M) was added, and the reaction was carried out at 60 °C for 2 h. At the end of the reaction, the solution was washed by centrifugation with a water/ethanol solution with a volume ratio of 2:1 at 10,000×g three times. The copper ions content was determined by ICP-MS and the mass of Cu₉S₈ was calculated to be 3.5 mg.

Synthesis of CPH, CAP, CAPH, CAPSH

To 1 mL of Cu₉S₈ (1 mg/mL) solution, 0.1 mg of PAH was added and stirred for 12 h. The solution was washed by centrifugation three times. Adding 1 mg of HA and stirring for 8 h. Centrifuging and washing three times to obtain CPH. To 1 mL of Cu₉S₈ (1 mg/mL), adding 4 mg of AIPH and stirring for 12 h. Adding 0.1 mg of PAH and stir for 12 h. Centrifuging and washing three times to obtain CAP. About 1 mg of CAP is stirred with 1 mg of HA for 8 h. Centrifuging and washing three times to obtain CAPH. CAPS was obtained by incubating 0.12 mg of CAP with 3 μg of siRNA at 4 °C for 2 h. About 0.12 mg of HA was added and stirred for 8 h. The sample was washed by centrifugation three times to obtain CAPSH. The sequence of siATP7A is as follows: 5′-CCCGAGUGAUAGCAGAGUUUAdTdT-3′, The siATP7A was synthesized by Jinkairui Biotechnology Co., Ltd.

Photothermal properties of Cu₉S₈

About 200 µL of Cu₉S₈ at different concentrations (12.5, 25, 50, and 100 μg/mL) were taken in the wells of removable enzyme labeling plates, and the samples were irradiated with a 1064 nm laser at 0.75 W/cm² for 5 min, and the temperature changes of the samples with the laser irradiation were recorded with an infrared thermography camera. The same method was used to determine the temperature change of Cu₉S₈ at a concentration of 100 μg/mL at different power densities (0.25, 0.50, and 0.75 W/cm²), and three parallels were set up for each sample. To test the photothermal stability of the probes, 200 µL of Cu₉S₈ at a concentration of 100 μg/mL was placed in the wells of a removable enzyme plate. The samples were then irradiated using a 1064 nm laser at 0.75 W/cm² for 5 min. After irradiation, the samples were cooled for 5 min, and the temperature change was recorded using an infrared thermography camera. The photothermal conversion efficiency of Cu₉S₈ was calculated using Eq. (1):

hs = mc/τ; Q_Dis = hs × (T_{max of water} - T_surr), T_max represents the peak temperature at which Cu₉S₈ undergoes heating, T_surr denotes the surrounding ambient temperature, T_max of the water signifies the maximum temperature reached during the water heating process. The mass of the solution is denoted by “m”, the specific heat capacity of water is represented as “c”, the laser power is expressed as “I”, and the absorption value at 1064 nm is indicated as A₁₀₆₄.

Detection of free radicals

AIPH underwent thermal decomposition, resulting in the production of alkyl radical groups that reacted with ABTS to form ABTS^+•. The solution containing a mixture of CAPH and ABTS was exposed to a 1064 nm laser with a power density of 0.75 W/cm² for 5, 10, and 15 min. After centrifugation, the UV absorption spectrum of the solution was measured using UV-vis absorption spectroscopy in the range of 400–1000 nm. To determine the type of free radicals generated, we mixed 150 μL of AIPH, Cu₉S₈, and CAPH with 100 mM POBN. The illuminated group was then irradiated with a NIR laser with a power density of 0.75 W/cm² for 5 min. On the other hand, the non-illuminated group was mixed for 5 min and then directly detected. After the different treatments, we measured the ESR signals of the samples at 25 °C using an ESR spectrometer.

Encapsulation rate and drug release studies of AIPH

In a 1 mL solution of Cu₉S₈ (1 mg/mL), 4 mg of AIPH was introduced and stirred for 12 h. Subsequently, 0.1 mg of PAH was added, and the mixture was stirred for an additional 12 h. The absorption value of the supernatant at 365 nm was determined through centrifugation, and the mass of AIPH was calculated based on the standard curve. The encapsulation rate was calculated using Eq. (2):

The CAPH was dialyzed using a 500 Da dialysis bag, and the pH of the dialysate was adjusted to 5.4, 6.5, and 7.4. At regular intervals, the external fluid of the dialysis bag was sampled, and the UV absorption value was measured. The sample was then returned to the dialysis bag, and this process was repeated until the absorption value remained constant. The content of AIPH in the external fluid of the dialysis bag was calculated using the standard curve of AIPH.

Release of copper ions

A solution of Cu₉S₈ (1 mg/mL) was placed in a dialysis bag. The pH of the dialysate was then adjusted to 5.4, 6.5, and 7.4. Samples were collected at specific time intervals of 5, 10, 30 min and 1, 2, 6, 12, 24, 36 h. The amount of copper ions presented in the solution was measured using ICP-MS.

ICP-MS testing conditions

Sample pretreatment: After weighing the tumor tissues, homogenize them using a tissue homogenizer and digest them in concentrated nitric acid until the solution is clear and transparent. Standard curve: Prepare a standard curve using a standard solution of known concentration (the concentration of the standard curve is 10, 20, 40, 60, 80 μg/L). Instrument setting: set the element to be detected as copper, select the standard mode, and optimize the parameters. The plasma power was between 1000–1600 W. Set the argon flow rate at 0.8–1.2 L/min. Testing: The sample was fed into the plasma through the feeding system, and the signal intensity of the characteristic ions was recorded. Data analysis: The standard curve was plotted according to the counts per second (cps) of the standard solution versus concentration. The concentration of the elements in the sample is calculated by comparing the signal intensity of the sample with the standard curve.

Cytotoxicity assay

4T1 cells were inoculated in 96-well plates, with ~5 × 10³ cells per well and 5 parallels per group. Once the cells were fully attached to the wall, different concentrations of CPH (0, 10, 20, 40, 60, 80, and 100 µg/mL) were added. After 12 h, the medium was removed, and the cells were washed with PBS three times. Then, 100 µL of 1 mg/mL MTT solution was added to each well. After 4 h, the medium and MTT solution were aspirated out, and 100 µL of DMSO was added to each well. The plate was shaken for 20 min to ensure even distribution. Subsequently, the absorption at 490 nm of each well was measured using an enzyme marker to determine cell viability.

In vitro combination therapy experiments

The 4T1 cells were uniformly inoculated in 96-well plates with ~5 × 10³ cells per well. There were five parallel groups in each group, and they were cultured for 24 h. The cells were then treated with AIPH, CPH, and CAPH, respectively, for 12 h. After that, the medium was removed, and the cells were washed three times with PBS. In the illumination group, the cells were exposed to a 1064 nm laser at a power density of 0.75 W/cm² for 5 min. Then, the medium was added back, and the incubation continued for 6 h. Subsequently, 100 µL of 1 mg/mL MTT solution was added to each well. After 4 h, the MTT solution was aspirated out, and 100 µL of DMSO was added to each well. The 96-well plate was then placed on a shaking table and shaken uniformly for 20 min. Finally, the absorption of each well at 490 nm was measured with an enzyme marker to calculate the cell viability.

The 4T1 cells were inoculated homogeneously in 24-well plates with ~1 × 10⁵ cells per well and cultured for 24 h. The old medium was discarded, and the cells were treated with AIPH, CPH, and CAPH, respectively, for 12 h. After being washed three times with PBS, the light group was exposed to a laser with a power density of 0.75 W/cm² at 1064 nm for 5 min and then cultured for 4 h. The cells were co-stained using the Calcein AM/PI assay and observed under inverted fluorescence microscopy.

Cell uptake

ICP-MS detection

Adding CAPSH to the culture medium of 3T3 and 4T1 cells and co-culture. Collect the cells at 2, 4, and 8 h. After counting, nitric acid was added for digestion, and ICP-MS was used to measure the copper content within the cells.

Flow cytometry detection

Loading Cy5.5 onto CAPSH and adding it to the culture medium of 3T3 and 4T1 cells. Co-culture and collecting the cells at 2, 4, and 8 h. Using flow cytometry to detect the fluorescence intensity of Cy5.5.

Detection of free radicals in cells

The 4T1 cells were uniformly inoculated in 24-well plates with ~1 × 10⁵ cells per well and cultured for 24 h. The old medium was then discarded, and the cells were treated with AIPH, CPH, and CAPH, respectively, for a duration of 12 h. The intracellular content of reactive oxygen species was assessed using the DCFH-DA reactive oxygen species detection kit.

In vitro detection of ICD activation

Sterile coverslips were placed in a 12-well culture plate, and 4T1 cells were seeded and cultured for 24 h. After different treatments, the cells were incubated for an additional 6 h. Subsequently, the medium was removed, and the cells were washed twice with PBS. The 4T1 cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Next, the cells were blocked with 5% BSA at room temperature for 20 min. Following blocking, the cells were incubated overnight at 4 °C with anti-HMGB1, anti-HSP70, and anti-CRT antibodies. After overnight incubation, the cells were washed three times with PBS and incubated with secondary antibodies at room temperature for 1 h. Finally, the cells were washed three times with PBS, and the coverslips were carefully mounted on slides with DAPI antifade mounting medium for confocal fluorescence imaging and flow cytometry detection. 4T1 cells were seeded in a 12-well plate and cultured for 24 h. After different treatments, the cells were incubated for an additional 6 h. Subsequently, the ATP content in the culture medium was detected using an ATP assay kit.

Copper distribution in tumors and organs

To study the distribution of copper in tumors and organs, 30 tumor-bearing mice were selected. Each mouse was administered an intravenous injection of 150 μL of CAPSH (2 mg/mL) through the tail vein. At the following time points: 0.5, 1, 2, 4, 6, 8, 12, and 24 h, as well as 5 and 15 days post-injection, three mice were euthanized at each time point. The heart, liver, spleen, lungs, kidneys, and tumor tissues were then harvested. After weighing the tissues, each sample was homogenized in a tissue grinder with 400 μL of PBS. Subsequently, 5 mL of concentrated nitric acid was added to the samples, which were then stored in the dark for several hours. Once the samples were completely digested at 120 °C in a digestion apparatus until the solution was clear and transparent, the copper content was measured using ICP-MS.

Blood half-life study

For the study of blood half-life, 150 μL of CAPSH (2 mg/mL) was administered intravenously to three BALB/c mice via the tail vein. Blood samples were collected at the following time points: 10 min, 30 min, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h post-injection. Each blood sample was then digested with concentrated nitric acid until the solution was completely clear. The concentration of copper in the blood was determined using ICP-MS.

RNA extraction

The adherent cells were washed three times with PBS after different treatments, 1 mL of RNA isolator was added, and the cells were blown down and collected in a centrifuge tube. Next, 200 µL of chloroform was added to the solution. The mixture was vigorously shaken for 15 s to form an emulsion and then left to stand on ice for 5 min. After that, the solution was centrifuged at 12,000×g for 15 min at 4 °C. The upper aqueous phase was aspirated into a new centrifuge tube. To this tube, an equal volume of pre-cooled isopropanol was added. The mixture was mixed upside down and left to stand on ice for 10 min. Subsequently, the tube was centrifuged at 12,000×g for 10 min at 4 °C. Usually, a white precipitate is visible at this stage. The supernatant was removed, and 1 mL of 75% ethanol was added. The tube was inverted several times and left at room temperature for 3–5 min. Then, it was centrifuged at 12,000×g for 5 min at 4 °C, and the supernatant was discarded. The precipitate was dried for 2–5 min, and an appropriate amount of RNase-free dd H₂O was added to dissolve it. Finally, the ratios of A₂₀₆/A₂₃₀ and A₂₆₀/A₂₈₀ were measured using a microspectrophotometer. The primer sequences used in quantitative polymerase chain reaction experiments are provided in Supplementary Table 5.

Detection of intracellular protein expression

The cell precipitate was collected, washed three times with PBS, and the liquid was aspirated. Protein lysate (RIPA: PMSF = 1000:1) was added to the cell precipitate and thoroughly mixed. It was then placed on an ice bath and lysed for 1 h. Finally, it was centrifuged at 12,000×g, 4 °C for 5 min. The protein concentration in the supernatant was determined by the BCA Protein Concentration Assay Kit and mixed with loading buffer solution thoroughly, placed in heating denaturation at 98 °C for 10 min, and placed in the refrigerator at −20 °C for storage. The protein samples were separated by SDS-PAGE gel electrophoresis and transferred to the PVDF membrane. The membrane was blocked with 5% BSA in TBST for 1 h at room temperature and immunoblotted with primary antibody overnight at 4 °C. The membrane was then washed three times with TBST and incubated with HRP-labeled secondary antibody at room temperature for 1 h. Finally, the protein bands were visualized using the Enhanced Chemiluminescence (ECL) Substrate Kit.

In vivo safety evaluation

To assess the biosafety of CAPSH, blood samples (400 µL per mouse) were obtained from the heart after the completion of treatment. A portion of 300 µL was placed in a procoagulant tube containing separating gel and centrifuged for 10 min. The resulting supernatant was used for liver and renal function analysis, including AST, ALT and UREA. An additional 100 µL was collected in an anticoagulant tube for routine blood tests, such as RBC, WBC, PLT, and HGB measurements. Furthermore, the heart, liver, spleen, lung, and kidney of the mice were collected, weighed, fixed in 4% paraformaldehyde, dehydrated, embedded, and sectioned for H&E staining and histopathological examination using a light microscope.

In vivo antitumor efficiency

Two tumor models were utilized to evaluate the effectiveness of antitumor treatment in vivo. The subcutaneous tumor model was established by injecting ~1 × 10⁶ 4T1 cells per mouse into the lower right back of Balb/c mice. The in situ tumor model was created by injecting ~1 × 10⁶ 4T1-LUC cells per mouse into the first pair of mammary glands in the chest of Balb/c mice. Once the tumor volume reached 80–100 mm³, the mice in the subcutaneous tumor model were randomly divided into six groups (seven mice per group): PBS, AIPH + L, Cu₉S₈ + L, CAPH, CAPH + L, CAPSH + L. Similarly, the mice in the in situ tumor model were randomly divided into six groups (four mice per group). Each group was administered 150 μL of different samples through the tail vein, with a dosage of 15 mg/kg for Cu₉S₈ in the Cu₉S₈ + L, CAPH, CAPH + L, CAPSH + L group, and 12 mg/kg for AIPH in the AIPH + L, CAPH, CAPH + L, CAPSH + L group. After 24 h of sample injection, the light group irradiated the tumor with a laser having a wavelength of 1064 nm and a power density of 0.75 W/cm² for 5 min. The changes in body weight and tumor volume were recorded every other day. Tumor growth monitoring in mice with in situ tumor models using an animal fluorescence imager.

Statistics and reproducibility

Data were expressed as mean ± SD. Significance between the two groups was assessed by unpaired two-tailed Student’s t-test, and between each of the multiple groups was calculated using one-way ANOVA. Values with P < 0.05 were considered significant. Exact p values were provided accordingly in the figures. All the statistical analyses were performed using GraphPad Prism (9.5.0). In this study, the sample size was determined based on prior experimental experience and standard practices. We did not use statistical methods to predetermine the sample size. During data analysis, no data were excluded. We have provided a detailed description of the experimental methods to ensure that other researchers can replicate the experiment and verify the reproducibility of the results. Flow-cytometry data were analyzed with FlowJo (ver. 10.8.1). ImageJ (ver. 1.4.3.67) were used to analyze fluorescent grayscale. Alpha EaseFC 4.0 was used to analyze the WB. Confocal images were analyzed with FluoView31S (ver.2.3.1.163).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54469-7