Mitochondrial copper depletion suppresses triple-negative breast cancer in mice

Abstract

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

Extended Figure 1.. Cytotoxicity of CDN measured with trypan blue exclusion method.

Cell viability of (a) MDA-MB-231 and (b) MDA-MB-468 cells after treatment with various concentrations of CDN, TPA or ATN224 (mean ± s.e.m., n=3 independent samples). (c) Metal remedy experiment measuring the cell viability of MDA-MB-231 cells after 24 h treatment with CDN (CDM: 1 μM) with (n=3 independent samples) or without (n=6 independent samples) various metal ion supplement (mean ± s.e.m., P values from unpaired t test, two-tailed). (d) Titration study showing the remedy effect of various concentrations of copper ions on MDA-MB-231 cell viability measured by trypan blue method (mean ± s.e.m., n=6 independent samples for CDN group, n=3 independent samples for copper addition group, P values from unpaired t test, two-tailed).

Extended Figure 2.. Acute OCR response of MDA-MB-231 cells after 1 h treatment with CDN.

OCR was measured with a serial injection of oligomycin (1 μM), FCCP (1 μM) and rotenone and antimycin A (0.5 μM) (mean ± s.e.m., n=6 biologically independent samples).

Extended Figure 3.. Comparison on the cytotoxicity, OCR inhibition and mitochondria membrane potential damaging effect between CDN and established complex I inhibitors.

MDA-MB-231 or MDA-MB-468 cells were incubated with 1 μM of CDN, IACS-010759 or BAY 87–2243. (a) Cell viability after 24 h of treatment was measured by MTS assay (mean ± s.e.m., n=3 biologically independent samples, P values from unpaired t test, two-tailed). (b) OCR and (c) ECAR were determined via Seahorse assay at 1 h after incubation. The results were normalized by cell number (mean ± s.e.m., n=6 biologically independent samples, P values from unpaired t test, two-tailed)). (d) Representative confocal microscopy images of cells stained with MitoTracker Green (MT-G, green) and DAPI (blue) after 24 h treatment. (scale bar: 50 μm). Three experiments were repeated independently with similar results.

Extended Figure 4.. CDN-enabled copper depletion elevated cellular oxidative stress.

(a) SOD1 levels of MDA-MB-231 after treatment with TPA and CDN at concentrations indicated by western blot analysis. Two experiments were repeated independently with similar results. (b) SOD1 activity of TNBC cells treated with CDN (1 μM) or ATN224 (5 μM) (mean ± s.e.m., n=3 biologically independent samples, P>0.05 for MDA-MB-468, P value from unpaired t test, two-tailed). (c) Cellular superoxide level measured by the chemiluminescence signal of CLA after treatment of SPN (50 μg/ml), CDN treatment (CDM: 1 μM, SPN: 50 μg/ml), TM (1 μM) and TPA (1 μM), shown as a ratio of treated to non-treated control (mean ± s.e.m., n=3 biologically independent samples, P values from unpaired t test, two-tailed). (d) Immunofluorescence staining of γH2AX (left) and 4-hydroxynonenal (4-HNE, right) on MDA-MB 231 cells at 24 h after incubation with SPN (50 μg/ml), CDN (CDM: 1 μM, SPN: 50 μg/ml), TPA (1 μM) or ATN224 (1 μM). (Scale bar=50 μm). Three experiments were repeated independently with similar results.

Extended Figure 5.. Intensities of isotopologues of metabolites (glutamine, cysteinyl glycine, glutathione (reduced), 4-oxoproline, hydroxyproline) produced from ¹³C₅¹⁵N₂-labeled glutamine in vitro.

Illustration of the corresponding pathways through which these metabolites are produced is also shown. Metabolites with isotopologues shown are written in red. Other important intermediate metabolites are written in black. Non-labeled ¹²C and ¹⁴N are shown as black-filled circles while labeled ¹³C are shown as red-filled circles and labeled ¹⁵N are shown as green-filled circles. Raw intensities are normalized by the protein concentration. Data are shown as mean ± s.e.m. (n = 5 for biologically independent samples, P values from unpaired t test, two-tailed).

Extended Figure 6.. Intensities of isotopologues of metabolites (lactic acid, glutamate, asparagine, and taurine) produced from ¹³C515N₂-labeled glutamine in vitro and illustration of the corresponding pathways through which these metabolites are produced.

Metabolites with isotopologues shown are written in red. Other important intermediate metabolites are written in black. Non-labeled ¹²C and ¹⁴N are shown as black-filled circles while labeled ¹³C are shown as red-filled circles and labeled ¹⁵N are shown as green-filled circles. Raw intensities are normalized by both the protein concentration. Data are shown as mean ± s.e.m. (n = 5 for biologically independent samples, P values from unpaired t test, two-tailed).

Extended Figure 7.. Ex vivo imaging of MDA-MB-231 tumor bearing mice administered with fCDN.

Nude mice bearing orthotopic MDA-MB-231 tumors were injected i.v. with fCDN (CDM dose: 1.35 mg/kg, n=6 independent animals). At 24 h after injection, mice were sacrificed, and major organs were collected and imaged with IVIS. (a). Representative images of ex vivo imaging emitted at 540 nm and 740 nm (excited at 500 nm). The ratiometric fluorescence graphs were shown as Em540/Em740. Average fluorescence efficiency from all organs was quantified at the (b) 540 nm emission (from the SPN group) and (c) 740 nm emission (from the CDM group). (d) The ratio of fluorescence emission between 540 nm and 740 nm. For all the boxplots: center line, median; box limits, first and third quartiles; whiskers, min to max values.

Extended Figure 8.. Cumulative toxicity profile for CDN.

(a) Hematological (i-vii) and liver panel (viii-x) analysis of mice after receiving either saline or CDN (CDM dose: 1.35 mg/kg, intravenous administration weekly, 7 doses in total). (mean ± s.e.m., n=5 independent animals for blood test, n=3 independent animals for liver panel test). (b) Representative haematoxylin and eosin (H&E) staining of normal tissue slice from mice treated with saline or treatment strategy shown in Figure 6a for CDN (scale bar: 50 μm). Slides from 5 independent animals were imaged and showed similar results.

Extended Figure 9.. Acute toxicity profile for CDN.

(a) Body weight changes of mice after i.v. injection of saline or single large dose of CDN (CDM: 100 mg/kg, n=3 independent animals). (b) Blood test parameters of treated mice comparing to control mice (mean ± s.e.m., n=3 independent animals). (c) representative H&E stained tissue slices of indicated organs from CDN treated and control mice (scale bar: 20 μm). Slides from 3 independent animals were imaged and showed similar results.

Extended Figure 10.. Cytotoxicity of CDN to receptor-positive breast cancer and prostate cancer cells.

(a) viability of receptor-positive breast cancer cell lines (HCC1428, MCF7, T47D) and TNBC cells (MDA-MB-231, MDA-MB-468, BT-20) after 24 h of treatment with CDN measured by MTS assay (mean ± s.e.m., n=3 biologically independent samples). (b) Prostate cancer cell lines, PC3 and 22Rv1, also responded to CDN treatment. Viability after 24 h of CDN treatment was presented as percentage of cell control without treatment (mean ± s.e.m., n=3 biologically independent samples).

Figure 1.. Design and characterization of CDN.

(a) Molecular components of CDN and the illustrated nanoparticle formulation. When binding with Cu(I) or Cu(II), the fluorescence of CDM is quenched but the photoacoustic signal from the semiconducting polymer remains unchanged, allowing for quantitative analysis of the chelation process. (b) UV-Vis spectrum of CDN. CDM absorption peaks at 610 nm and the polymer peaks at 1100 nm. (c) Size and morphology of CDN measured by DLS (mean ± s.e.m., n=4 independent experiments) and TEM imaging (n=2 independent experiments, 2 images acquired per experiment). Inset: TEM image of CDN with uranyl acetate staining (scale bar: 100 nm). PDI: polydisperse index. (d) Zeta potential measurement of SPN with or without CDM loading (mean ± s.d., n=5 independent samples for SPN and n=4 independent samples for CDN). (e) Fluorescence signal changes of CDN (CDM: 1 μM, Em: 760 nm) upon binding with Cu(I) or Cu(II) in buffered solution (mean ± s.d., n=3 independent samples). (f) Percentage of fluorescence signal of CDN solution (CDM: 5 μM) after mixing with various metal ions to that without metal addition (Na⁺: 5 mM, Mg²⁺: 5 mM, K⁺: 5 mM, Ca²⁺: 5 mM, Zn²⁺: 100 μM, Fe²⁺: 20 μM, Mn²⁺: 1 μM, Ni²⁺: 0.5 μM, Cd²⁺: 0.5 μM, Co²⁺: 0.5 μM) (mean ± s.d., n=3 independent samples). Concentrations of the tested metal ions are selected based on the physiological abundancy. Blue: measurement after CDN mixing with indicated physiologically abundant or trace metal ions; red: measurement after addition of extra 5 μM of Cu(II) to previous mixture of CDM and metal ions. (g) Representative fluorescence imaging of CDN mixed with different equivalents of Cu(I) in the agar phantom (n=3 independent samples). Signal intensity is normalized based on the photoacoustic signal at 1100 nm for each well.

Figure 2.. CDN induces TNBC cell death via intracellular copper depletion.

(a) Representative optical images of MDA-MB-231^luc cells after 24 h incubation with CDN with or without different concentrations of copper supplements or excessive EDTA for copper deprivation (CDM: 1 μM). CDM fluorescence depicts copper binding to CDN and CCL-1 bioluminescence imaging depicts intracellular labile copper. (b) MDA-MB-231 and (c) MDA-MB-468 cell viability (percentage of cell control without treatment) after 24 h incubation with different concentrations of CDN, CDM, TPA, TM or ATN224 in complete medium with 10% FBS measured by MTS assay (mean ± s.e.m., n=3 biologically independent samples). (d) Viability of MDA-MB-231 cells after 24 h treatment with SPN or CDN with or without copper, zinc, iron or manganese addition (CDM: 1 μM, SPN: 50 μg/ml) measured by MTS assay (mean ± s.d., n=3 biologically independent samples, P value from unpaired t test, two-tailed). (e) Viability of TNBC cells (MDA-MB-231 and MDA-MB-468) and normal human cells (MCF-10A, WI-38 and RWPE-1) after 24 h treatment with CDN (CDM: 1 μM) in serum-free media (mean ± s.e.m., n=3 biologically independent samples).

Figure 3.. CDN inhibits mitochondria complex IV.

(a) Mitochondria membrane potential of MDA-MB-231 cells after 24 h incubation with CDN (CDM: 1 μM) with or without copper supplements (10 μM) or other control agents (chelator concentration: 1 μM, SPN concentration: 50 μg/ml), indicated by JC-1 staining (10 μg/ml). Red fluorescence that is emitted by aggregated JC-1 characterizes a high membrane potential while green fluorescence emitted by JC-1 monomer characterizes low membrane potential (scale bar: 50 μm, n=3 independent experiments). (b) Cytochrome c oxidase activities of MDA-MB-231 and MDA-MB-468 cells after 24 h incubation with CDN (CDM: 1 μM), TPA (1 μM) or ATN224 (5 μM) (mean ± s.e.m., n=3 biologically independent samples, P value from unpaired t test, two-tailed). Results are expressed as percentage of cell control without treatment. (c, d) CDN inhibits cellular oxygen consumption in (c) MDA-MB-231 and (d) MDA-MB-468 cells. 24 h after incubation with CDN (CDM: 1 μM) or control agents (TPA: 1 μM, ATN224: 1 μM or 5 μM, SPN: 50 μg/ml), the cellular oxygen consumption rate was measured by Seahorse analyzer. Oligomycin (1 μM) was introduced after 28 min, FCCP (1 μM) was introduced after 54 min and rotenone/antimycin A (0.5 μM) was introduced after 80 min (mean ± s.e.m., n=8 biologically independent samples). (e) Inhibition of OCR can be mitigated with the addition of copper ion. MDA-MB-231 and MDA-MB-468 cells were incubated with 1 μM of CDN for 1 h with or without copper supplement (10 μM) (mean ± s.e.m., n=6 biologically independent samples; P value from unpaired t test, two-tailed). (f) Cellular ATP level (as percentage of control with no treatment) after 24 h incubation with CDN, TPA or ATN224 (chelator concentration: 1 μM) in FBS supplemented culture medium (mean ± s.e.m., n=3 biologically independent samples; P value from unpaired t test, two-tailed).

Figure 4.. CDN induced inhibition of mitochondrial OXPHOS changes metabolisms of TNBC cells.

Extracellular acidification rate (ECAR) elevated in both (a) MDA-MB-231 and (b) MDA-MB-468 cells at 24 h after incubation with CDN (CDM: 1 μM). After CDN treatment, MDA-MB-468 showed minimal cellular response to the subsequent injections of oligomycin (1 μM, introduced after 28 min), FCCP (1 μM, introduced after 54 min) and rotenone/antimycin A (0.5 μM, introduced after 80 min). MDA-MB-231 exhibited no response to oligomycin and rotenone/antimycin A. Unlike CDN, other control agent treatment (TPA: 1 μM, ATN224: 1 μM or 5 μM, SPN: 50 μg/ml) slightly decreased or did not impact the ECAR levels (mean ± s.e.m., n=8 biologically independent samples). (c) (i) glucose uptake and (ii) lactate secreted into the medium over 24 h incubation with ATN224 (5 μM) or CDN (CDM: 1 μM) (mean ± s.e.m., n=3 biologically independent samples, P value from unpaired t test, two-tailed). Results are expressed as percentage of cell control without treatment. (d) Illustration of glucose metabolism pathways affected by CDN treatment. (e) Percentage of M+3 lactic acid over the total pool. Intensities of isotopologues of metabolites, including, (f) glutamine, (g) glutamate, (h) UDP-N-acetylglucosamine, produced from ¹³C₆-labeled glucose in vitro. Metabolites with isotopologues shown are written in red. Other important intermediate metabolites are written in black. Non-labeled ¹²C are shown as black-filled circles while labeled ¹³C are shown as red-filled circles. Raw intensities are normalized by protein concentration. Data are shown as mean ± s.e.m. (n=5 biologically independent samples, P value from unpaired t test, two-tailed).

Figure 5.. CDN depletes copper efficiently in vivo.

(a) Labile copper concentration detected by CCL-1 bioluminescence imaging after i.v. injection of CDN (1.35 mg/kg) in MDA-MB-231^luc tumor bearing mice (mean ± s.e.m., n=3 independent animals). (b) Longitudinal monitoring of nanocomplex delivery and copper binding via optical imaging of CDN after i.v. injection of CDN. Photoacoustic signal in the tumor region (blue) reflects the accumulation and retention of CDN while fluorescence signal reports the copper binding to CDM (red). (mean ± s.d., n=3 independent animals) (c) D-luciferin bioluminescence imaging and (d) CCL-1 imaging of mice receiving SPN, TPA or CDN treatment (chelator dose: 1.35 mg/kg). MDA-MB-231^luc tumor bearing mice were i.v. administered with the indicated agents every three days with a total of 5 doses. Images were acquired at day 25 after the first injection (mean ± s.e.m., n=5 independent animals, P value from unpaired t test, two-tailed). (e) The labile copper levels in the tumor region for different treatment groups were quantified as CCL-1 to D-luciferin flux ratio (mean ± s.e.m., n=5 independent animals; P value from unpaired t test, two-tailed). (f) In vivo mitochondrial membrane potential measured by MAL3 bioluminescence imaging. MDA-MB-231^luc tumor bearing mice were imaged before and after injection with PBS, SPN or CDN. The total photon flux of MAL3 bioluminescence signals was normalized by D-luciferin bioluminescence signal (mean ± s.e.m., n=5 independent animals, P value from unpaired t test, two-tailed).

Figure 6.. CDN inhibits TNBC tumor growth.

(a) Treatment strategy for long-term survival study with MDA-MB-231 tumor bearing mice. Six control and treatment groups were included in the study: control, SPN, TPA, ATN224, CDM and CDN. Mice received SPN, TPA, CDM or CDN (chelator dose: 1.35 mg/kg) via i.v. injection weekly and ATN224 via oral gavage daily (0.7 mg/kg/day). (b) Tumor growth curve of each individual mouse in different treatment groups (n=12 independent animals). In the CDM treatment group, * indicates individuals that received early euthanasia under the instruction of veterinarian due to skin rashes. (c) Survival curve of different treatment groups up to 70 days after the initial treatment. (d) Tumor growth curve of MDA-MB-468 tumor bearing mice during CDN and control agent treatment (mean ± s.e.m., n=5 independent animals). At day 40 after initial treatment, tumor burden in CDN treatment group was significantly smaller than that in the non-treated control (P=0.016), SPN group (P=0.003), TPA group (P=0.004) and ATN224 group (P=0.004) (P value from unpaired t test, two-tailed). Three days after the 7^th dose, mice were sacrificed, and tumors were subject to metabolite analysis. (e) Glucose, (f) lactate and (g) alanine levels were determined (mean ± s.d., n=5 independent animals, P value from unpaired t test, two-tailed).