Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.
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We thank C. Zamilpa, D. Abeydeera, W. Ladno, O. Sergeeva, D. Williams and T. Olafsen for assistance with PET/CT imaging of the mice. We thank M. Cilluffo, R. McMickle, V. Muhunthan, M. Han and E. Assali for laboratory assistance. We thank the Translational Pathology Core Laboratory at UCLA’s DGSOM for assistance with tumour sample preparation and processing. This research was supported by the NIH National Center for Advancing Translational Science (NCATS) UCLA CTSI Grant number UL1TR001881. D.B.S. was supported by the UCLA CTSI KL2 Translational Science Award grant numbers KL2TR001882 at the UCLA David Geffen School of Medicine, the UCLA Jonsson Comprehensive Cancer Center grant P30 CA016042, the Department of Defense LCRP grant number W81XWH-13-1-0459 and the NIH/NCI R01 CA208642-01. S.T.B. was supported by an NIH T32 training grant HL072752. A.J. was supported by a USHHS Ruth L. Kirschstein Institutional National Research Service Award T32 CA009056. G.A. and A.G. were supported by an NIH/NCI R01 CA208642-01 diversity supplement. J.T.L. was supported by NIH/NCI P30 CA016042. This research was supported by the Joyce and Saul Brandman Fund for Medical Research. We thank the Scott family and the Carrie Strong Foundation as well as B. and D. Goldfarb for their support.
S.M.D. is an advisory board member for EarlyDx Inc., T-Cure Bioscience Inc., Cynvenio Biosystems Inc. and the Johnson and Johnson Lung Cancer Initiative. D.B.S., M.M. and S.S. have filed a provisional patent US 62/901,947 and are listed as inventors for the use of 18F-BnTP to guide use of complex I inhibitors for the treatment of lung cancer.
Peer review information Nature thanks Kevin Brindle, Ralph Deberardinis and Jared Rutter for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Mitochondrial markers in KL mouse lung tumours.
Whole-cell lysates from lung tumours isolated from KL mice were immunoblotted with the indicated antibodies. Tumours with high levels of the ratio of SP-C to actin (>0.5) were defined as ADCs (blue box), whereas tumours with low SP-C to actin ratios (<0.5) were defined as SCCs (red box). Each lane represents an individual tumour isolated from KL mice. Western blot was done on 20 individual tumours isolated from KL mice from three independent experiments.
Extended Data Fig. 2 Measuring mitochondrial membrane potential in vitro in A549 and L3161C cells.
a, Gating strategy used for the quantification of TMRE signal. The R2 region representing single cells was used for quantification of the TMRE signal. b, Overlay histogram showing shifts in TMRE staining in L3161C cells treated with vehicle, 8 μM oligomycin or 8 μM oligomycin plus 4 μM FCCP. c, TMRE measurements in A549 cells treated with the indicated concentrations of phenformin or FCCP for 3 h (n = 3 biological replicates). d, TMRE measurements in mouse cell line L3161C treated with the indicated concentrations of phenformin or FCCP for 3 h (n = 3 biological replicates). e, Viability of A549 cells treated with the indicated concentrations of phenformin for 3 h (n = 3 biological replicates). f, Uptake of 18F-BnTP probe measured by gamma counter in A549 cells treated with 1 mM phenformin for 3 h (n = 5 biological replicates). g, OCR per cell measured in A549 cells treated acutely with 1 mM phenformin (n = 25 technical replicates). h, OCR per cell measured in mouse cell line L3161C treated acutely with 1 mM phenformin (n = 25 technical replicates). i, TMRE measurements in mouse cell line L3161C treated with vehicle, 8 μM oligomycin, or 8 μM oligomycin with 4 μM FCCP for 3 h (n = 3 biological replicates). j, Uptake of 18F-BnTP probe measured by gamma counter in mouse L3161C cells treated with vehicle, 8 μM oligomycin, or 8 μM oligomycin with 4 μM FCCP for 3 h (n = 6 biological replicates). k, Viability of L3161C cells treated as in j (n = 6 biological replicates). Data are mean ± s.d. Experiments in c–i, were repeated twice with similar results. Experiments in j and k were done once.
Extended Data Fig. 3 Short-term treatment with phenformin does not lead to changes in proliferation or apoptosis.
a, Transverse 18F-BnTP PET–CT overlay (left) of mouse lung (middle) after treatment with phenformin. H&E staining of a lung lobe with an ADC tumour (right). b, Representative slides stained with H&E (left), CC3 (middle) and Ki67 (right), from tumours from KL mice treated with vehicle (top) or phenformin (bottom). Experiment was performed once on slides from n = 5 (vehicle) and n = 6 (phenformin) mouse lungs. c, d, Quantification of staining for Ki67 (c) and CC3 (d) for tumours from KL mice treated with vehicle (n = 5 mice) or phenformin (n = 6 mice). Experiment was performed once. e, Phenformin in lung tumours isolated form KL mice was quantified using liquid chromatography–mass spectroscopy. Tumours were isolated from mice treated with vehicle (n = 6) or 100 mg kg−1 (n = 2) or 200 mg kg−1 phenformin (n = 2) for 5 days. Experiment was performed once. f, Representative 18F-BnTP PET–CT overlay of a tumour formed by transthoracically implanted L3161C lung cells into syngeneic recipient mice. This image is representative of at least 20 PET–CT images. g, H&E slide of a tumour formed as in f. h, Higher magnification image of H&E staining of tumour formed by L3161C mouse cell line as in f. j, Representative slides stained with Ki67 (top), and CC3 (bottom) from tumours formed by transthoracically transplanted L3161C cells that were treated with vehicle, metformin or phenformin. Experiment was performed once on slides from n = 8 (vehicle), n = 5 (metformin) or n = 6 (phenformin) tumours. Data are mean ± s.d. P values determined by unpaired two-tailed t-test.
Extended Data Fig. 4 Expressing ND1 in mouse L3161C lung ADC cell line reduces sensitivity of mitochondrial membrane potential to phenformin in vitro and in vivo.
a, Basal OCR rate per cell for L3161C cells expressing empty vector (pBabe; black) (n = 12 technical replicates) or L3161C cells expressing ND1 (L3161C-ND1; red) (n = 12 technical replicates) treated with 50 μM phenformin for 24 h. b, Basal OCR rate per cell for L3161C-pBabe (black) (n = 12 technical replicates for all conditions, except n = 6 for 250 μM and n = 9 for 500 μM phenformin) and L3161C-ND1 cells (red) (n = 12 technical replicates) treated with the indicated concentrations of phenformin for 24 h. Data are mean ± s.d. c, Waterfall plot of the percentage change in maximum uptake of 18F-BnTP after treatment relative to before treatment for mice transthoracically implanted with L3161C cells expressing empty vector (pBabe; n = 5 mice) or ND1 (n = 5 mice) and treated with 125 mg kg−1 phenformin for 5 days. d, Waterfall plot of the percentage change in maximum uptake of 18F-BnTP in tumours formed by transthoracically implanted L3161C-pBabe (n = 3 mice) or L3161C-ND1 cells (n = 5 mice) treated with vehicle for 5 days. Experiments in a–d were performed once. P values determined by unpaired two-tailed t-test.
Extended Data Fig. 5 Multi-tracer imaging and immunohistochemistry markers in lung tumours from KL mice.
a, Representative PET and computed tomography (CT) images of three KL mice imaged with 18F-BnTP (top) and 18F-FDG (bottom) on sequential days. H, heart; T, tumour. Arrows and circles denote tumours. b, Whole lung slides stained with H&E, TOM20, GLUT1, or CK5 plus TTF1 from three mice. Scale bars, 5 mm. c, Representative higher magnification images of the tumours circled in b stained with H&E, TOM20, GLUT1, CK5 plus TTF1 as indicated. Scale bars, 25 μm. Data are representative of three independent mouse experiments.
Extended Data Fig. 6 PET–CT and biochemical analysis of KL tumours.
a, Crystal structure of complex I (PDB accession 5lc5), with NDUFS1 and NDUFV1 subunits in red, and FeS clusters in yellow.b–d, PET–CT images from three KL mice that were imaged on sequential days with 18F-BnTP (top) and 18F-FDG (bottom). Tumours are circled. Maximum uptake value for each tumour after normalization to maximum uptake of the heart is indicated. e, Western blot analysis from lung nodules that were isolated from mice imaged in b–d. Two lung tumours from mouse 5372 (imaged in b) are shown—T1 in blue (low 18F-FDG and GLUT1 levels; high 18F-BnTP, NDUFS1 and NDUFV1 levels); and T2 in red (high 18F-FDG and GLUT1 levels; low 18F-BnTP, NDUFS1 and NDUFV1 levels). Experiments in b–d are representative of three independent mouse experiments. Experiment in e was performed once.
Extended Data Fig. 7 Levels of NDUFS1 and NDUFV1 in KL tumours.
Whole-cell lysates from lung tumours isolated from KL mice were immunoblotted with the indicated antibodies. This western blot was done on 20 individual tumours isolated from KL mice from three independent experiments.
Extended Data Fig. 8 Sensitivity of mouse and human lung cancer cell lines to complex I inhibitors phenformin and IACS-010759.
a, TMRE measurement as determined by flow cytometry comparing mouse ADC (n = 3 biological replicates) and mouse SCC (n = 3 biological replicates) cell lines. b, Cell viability of mouse ADC (n = 3 biological replicates) and mouse SCC (n = 3 biological replicates) cells was measured in the presence of indicated concentrations of phenformin for 48 h. c, Cell viability of human ADC (A549; n = 3 biological replicates) and human SCC (RH2; n = 3 biological replicates) cells was measured in the presence of indicated concentrations of phenformin for 48 h. d, Cell viability of human ADC (A549; n = 3 biological replicates) and human SCC (RH2; n = 3 biological replicates) cells was measured in the presence of indicated concentrations of IACS-010759 for 48 h. Data are mean ± s.d. P values determined by unpaired two-tailed t-test or one-way ANOVA (for b–d). Experiments were repeated twice with similar results.
Extended Data Fig. 9 Characteristics of tumours from KL mice treated with vehicle or IACS-010759.
a, Uptake of 18F-BnTP in tumours from KL mice before the start of treatment with vehicle or 15 mg kg−1 IACS-010759. Each dot represents a tumour; n = 44 (vehicle), n = 66 (IACS) tumours. b, c, H&E staining images from lung sections from KL mice treated with vehicle (b) or 15 mg kg−1 IACS-010759 (c) for 12 days, with tumours delineated by red lines. Quantification of these data is shown in Fig. 4l. Experiment was performed once.
Extended Data Fig. 10 Intra-tumoral heterogeneity in KL mice.
Higher magnification images of tumour shown in Fig. 4n, o with GLUT1 staining (left) and CK5 and TTF1 staining (right). Areas corresponding to ADC and SCC are indicated, with rectangular boxes corresponding to magnified images shown in Fig. 4n. Data are representative of three independent mouse experiments.
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Momcilovic, M., Jones, A., Bailey, S.T. et al. In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer. Nature 575, 380–384 (2019). https://doi.org/10.1038/s41586-019-1715-0
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