In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer

Abstract

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 xenografts^2,3. 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.

Extended data Figure 1.. Mitochondrial markers in KL mouse lung tumors.

Whole cell lysates from lung tumors isolated from KL mice were immunoblotted with indicated antibodies. Tumors with high levels of surfactant protein C (SP-C:actin ratio > 0.5) were defined as adenocarcinomas (blue box), while tumors with low SP-C (SP-C:actin ratio < 0.5) were defined as squamous cell carcinomas (red box). Each lane represents individual tumor isolated from KL mice. This Western blot was done on 20 individual tumors isolated from KL mice from three independent experiments.

Extended data Figure 2.. Measuring mitochondrial membrane potential in vitro in A549 and L3161C cells.

a, Gating strategy used for quantification of TMRE signal. R2 – region representing single cells was used for quantification of the TMRE signal. b, Overlay histogram showing shifts in TMRE staining in L3161C cells strained with Vehicle, 8 μM Oligomycin, and 8 μM Oligomycin + 4 μM FCCP. c, TMRE measurements in A549 cells treated with indicated concentrations of Phenformin or FCCP for 3 hr (n = 3 biological replicates). d, TMRE measurements in mouse cell line L3161C treated with indicated concentrations of Phenformin or FCCP for 3 hr (n = 3 biological replicates). e, Viability of A549 cells treated with indicated concentrations of Phenformin for 3 hr (n = 3 biological replicates). f, Uptake of ¹⁸FBnTP probe measured by gamma counter in A549 cells treated with 1 mM Phenformin for 3 hr (n = 5 biological replicates). g, Oxygen consumption rate (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 hr (n = 3 biological replicates). j, Uptake of ¹⁸FBnTP probe measured by gamma counter in mouse cell line L3161C treated with Vehicle, 8 μM Oligomycin, or 8 μM Oligomycin with 4 μM FCCP for 3 hr (n = 6 biological replicates). k, Viability of L3161C cells treated as in j (n = 6 biological replicates). Data are shown as mean +/− SD. Experiments in c-i, were repeated twice with similar results. Experiments in j and k were done once.

Extended Data Figure 3.. Short-term treatment with phenformin does not lead to changes in proliferation or apoptosis.

a, Transverse ¹⁸FBnTP/CT overlay (left panel) of mouse lung (middle panel) after treatment with phenformin. H&E of a lung lobe with associated adenocarcinoma (ADC) tumor (right panel). b, Representative slides stained with H&E (left panel), Cleaved Caspase 3 (middle panel), and Ki67 (right panel) from tumors from KL mice treated with Vehicle (top panel) or Phenformin (bottom panel). This experiment was done once on slides from n = 5 mouse lungs (Vehicle) and n = 6 mouse lungs (Phenformin). c, Quantification of staining for Ki67 and d, Cleaved Caspase 3 for tumors from KL mice treated with Vehicle (n = 5 mice) or Phenformin (n = 6 mice). This experiment was done once. e, Phenformin in lung tumors isolated form KL mice was quantified using liquid chromatography/mass spectroscopy. Tumors were isolated from mice treated with Vehicle (n = 6) or 100 mg/kg (n = 2) or 200 mg/kg Phenformin (n = 2) for 5 days. This experiment was done once. f, Representative ¹⁸FBnTP PET/CT overlay of a tumor formed by transthoracically implanted L3161C cell line. H – heart, T – tumor. This image is representative of at least 20 PET/CT images. g, H&E slide of a tumor formed by transthoracically implanted L3161C cell line imaged in f. h, H&E staining of tumor formed by L3161C mouse cell line. j, Representative slides stained with Ki67 (top panel), and Cleaved Caspase 3 (bottom panel) from tumors formed by transthoracically transplanted L3161C cells that were treated with Vehicle, Metformin, Phenformin. This experiment was done once on slides from n = 8 tumors (Vehicle), n = 5 tumors (Metformin), n = 6 tumors (Phenformin). The data are represented as the mean +/− SD. Statistical significance was calculated using unpaired two-tailed t-test.

Extended Data Figure 4.. Expressing ND1 in mouse L3161C lung adenocarcinoma cell line reduces sensitivity of mitochondrial membrane potential to Phenformin in vitro and in vivo.

a, Basal OCR rate per cell for L3161C-pBabe (black) (n = 12 technical replicates) and L3161C-ND1 cells (red) (n = 12 technical replicates) treated with 50 μM Phenformin for 24 hr. b, Basal OCR rate per cell for L3161C-pBabe (black) (n = 12 technical replicates for all conditions, except n = 6 for 250 μM Phen and n = 9 for 500 μM Phen) and L3161C-ND1 cells (red) (n = 12 technical replicates) treated with indicated concentrations of Phenformin for 24 hr. This experiment was repeated once. The data are represented as the mean +/− SD. c, Waterfall plot for % change in maximum uptake of ¹⁸FBnTP post-treatment to pre-treatment for mice transthoracically implanted with L3161C cells expressing empty vector (L3161C+pBabe) (n = 5 mice) or expressing ND1 (L3161C+ND1) (n = 5 mice) and treated with 125 mg/kg Phenformin for 5 days. e, Waterfall plot of % change in maximum uptake of ¹⁸FBnTP in tumors formed by transthoracically implanted L3161C-pBabe (n = 3 mice) or L3161C-ND1 cells (n = 5 mice) that were treated with Vehicle for 5 days. Experiments in a-d were preformed once. Statistical significance was calculated using unpaired two-tailed t-test.

Extended Data Figure 5.. Multi-tracer imaging and IHC markers in lung tumors from KL mice.

a, Representative PET images of KL mice imaged with ¹⁸FBnTP and ¹⁸F-FDG on sequential days. Top panel shows CT and ¹⁸FBnTP image and bottom panel shows CT and ¹⁸F-FDG image. H - heart, T - tumor; tumors are indicated by arrows and circled. b, Whole lung slides stained with H&E, Tom20, Glut1, and CK5/TTF1 from Mouse 1, Mouse 2 and Mouse 3. Scale bar = 5 mm. c, Higher magnification of representative images from tumor circled in b form mouse 1, mouse 2, mouse 3 and that were stained with H&E, Tom20, Glut1, CK5 and TTF1. Scale bar = 25 μm. This data is representative of three independent mouse experiments.

Extended Data Figure 6.. PET/CT and biochemical analysis of KL tumors.

a, Crystal structure of complex I (PDB: 5lc5) is shown with NDUFS1 and NDUFV1 subunits is red and FeS clusters in yellow. b-c, PET/CT images from three KL mice that were imaged on sequential days with ¹⁸FBnTP (top panel) and ¹⁸F-FDG (bottom panel). Tumors are circled, H – heart. Maximum uptake value for each tumor 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 tumors from mouse 5372 (imaged in b) are shown – T1 in blue (low ¹⁸F-FDG and Glut1 levels; high ¹⁸FBnTP and high Ndufs1 and Ndufv1 levels); and T2 in red (high ¹⁸F-FDG and Glut1 levels; low ¹⁸FBnTP and low Ndufs1 and Ndufv1 levels). Experiments in b-d are representative of three independent mouse experiments. Experiment in e was done once.

Extended Data Figure 7.. Levels of Ndufs1 and Ndufv1 in KL tumors.

Whole cell lysates from lung tumors isolated from KL mice were immunoblotted with indicated antibodies. This Western blot was done on 20 individual tumors isolated from KL mice from three independent experiments.

Extended Data Figure 8.. Sensitivity of mouse and human lung cancer cell lines to complex I inhibitors phenformin and IACS-010759.

a, TMRE measurement comparing mouse adenocarcinoma cell line (n = 3 biological replicates) and mouse squamous cell carcinoma (n = 3 biological replicates) cell lines. b, Cell viability of mouse adenocarcinoma cell line (n = 3 biological replicates) and mouse squamous cell carcinoma cell line (n = 3 biological replicates) cells was measured in the presence of indicated concentrations of Phenformin for 48 hr. c, Cell viability of human ADC cell line (A549) (n = 3 biological replicates) and human SCC cell line (RH2) (n = 3 biological replicates) cells was measured in the presence of indicated concentrations of Phenformin for 48 hr. d, Cell viability of human ADC cell line (A549) (n = 3 biological replicates) and human SCC cell line (RH2) (n = 3 biological replicates) cells was measured in the presence of indicated concentrations of IACS-010759 for 48 hr. The data are represented as the mean +/− SD. Statistical significance was calculated using unpaired two-tailed t-test or one-way ANOVA (for b-d). Experiments were repeated twice with similar results.

Extended Data Figure 9.. Characteristics of tumors from KL mice treated with Vehicle or IACS-010759.

a, ¹⁸FBnTP uptake in tumors in KL mice before the start of the treatment with Vehicle or 15 mg/kg IACS-010759. Each dot represents a tumor; n = 44 (Vehicle), n = 66 (IACS). b and c, Images from H&E stained lung sections from KL mice treated with Vehicle (b) or 15 mg/kg IACS-010759 (c) for 12 days with tumors delineated with red lines. Quantification of this data is shown in Figure 4l. This experiment was done once.

Extended Data Figure 10.. Intra-tumoral heterogeneity in KL mice.

Higher magnification of tumor shown in Figure 4n, and Figure 4o with Glut1 staining (left panel) and CK5/TTF1 staining (right panel). Areas corresponding to ADC and SCC are indicated, with rectangular boxes corresponding to zoomed in images shown in Figure 4n. This data is representative of three independent mouse experiments.

Figure 1.. ¹⁸FBnTP PET imaging and biodistribution analysis of KL lung tumors identified differential uptake between lung adenocarcinomas (ADC) and squamous cell carcinomas (SCC).

a, PET/CT overlay of a KL mouse with lung tumors, imaged with ¹⁸FBnTP. Right panel is rotated 90° compared to left panel. Heart (H) and tumor (T) are indicated by arrows. L – liver; GI – gastrointestinal tract; K – Kidney; B – Bladder. b, Biodistribution of ¹⁸FBnTP probe in tissue from wild type FVB mice measured by gamma counter ex vivo after 1 hr uptake (n = 5 mice). c, Biodistribution of the ¹⁸FBnTP probe in normal tissue of KL mice measured by % injected dose/gram after 1 hr uptake (n = 12 mice). d, ¹⁸FBnTP uptake in lung ADC and SCC from KL mice (n = 5 mice, n = 10 ADC tumors, n = 7 SCC tumors). e, Representative transverse image of the heart and lungs of a KL mouse imaged with CT (left panel) and ¹⁸FBnTP (right panel). H – heart, T1 – adenocarcinoma (ADC), T2 – squamous cell carcinoma (SCC). f, IHC staining of T1 and T2 tumors from panel e. TTF1 – thyroid transcription factor 1; CK5 – keratin 5; Tom20 – translocase of outer membrane 20. Scale bar = 100 μm. The data are represented as the mean +/− SD. Statistical significance was calculated using unpaired two-tailed t-test. Experiments in b, c were done once. Data in a, d, e, f are representative of experiments repeated thrice, with similar results obtained.

Figure 2.. Treatment of KL mice with the complex I inhibitor phenformin suppresses ¹⁸FBnTP uptake in lung tumors.

a, Schematic drawing representing voltage dependent uptake of ¹⁸FBnTP into the mitochondria. Bioenergetics driven by electron transport chain (ETC), and oxidative phosphorylation (OXPHOS) are shown. b, Schematic drawing of imaging and treatment regiments for KL mice treated with 125 mg/kg/day Phenformin. c, Representative images from a KL mouse before (top panel) and after (bottom panel) treatment for 5 days with 125 mg/kg/day Phenformin. Values for maximum percent injected dose (%ID/g) for the heart and tumor are indicated. H – heart. c, Quantification of maximum percent injected dose (%ID/g) for tumors in Vehicle (Veh) and Phenformin (Phen) groups before treatment (Pre-treatment). Each dot represents an individual tumor; n = 11 tumors for Vehicle and Phenformin groups. d, Quantification of maximum ¹⁸FBnTP uptake for tumors in Vehicle and Phenformin groups after treatment (Post-treatment) with 125 mg/kg/day Phenformin for 5 days. Each dot represents an individual tumor; n = 11 tumors for Vehicle and Phenformin groups. e, Average ¹⁸FBnTP uptake in KL mice after 5 day treatment with Vehicle (n = 48 tumors; 10 mice) or Phenformin (n = 23 tumors; 10 mice). Each dot represents an individual tumor. f, Waterfall plot for % change in probe uptake post-treatment to pre-treatment for mice treated with Vehicle or Phenformin for 5 days. Each bar represents an individual tumor. The data are represented as the mean +/− SD. Statistical significance was calculated using unpaired two-tailed t-test. Data in d, e, f are from two separate mouse experiments. Data in g are from three individual mouse experiments.

Figure 3.. ¹⁸FBnTP detects mitochondrial complex I inhibition in vivo.

a, Schematic drawing of the transthoracic (TT) implantation of KPL lung adenocarcinoma (ADC) cells into syngeneic recipient mouse, imaging and treatment regiments. b, Waterfall plot for % change in ¹⁸FBnTP uptake post-treatment to pre-treatment for mice treated with a single dose of Vehicle (n = 7 mice) or 0.25 mg/kg Oligomycin (n = 9 mice) or 0.5 mg/kg Rotenone (n = 7 mice). Statistical significance was calculated using one way ANOVA. c, Schematic drawing of the treatment and imaging regiment for syngeneic mice implanted transthoracically with KPL lung ADC cells and treated with Vehicle or Complex I inhibitor for the indicated time. d, Waterfall plot for % change in ¹⁸FBnTP uptake post-treatment to pre-treatment for mice treated with Vehicle (n = 4 mice) or 500 mg/kg Metformin (n = 5 mice) for 5 days. e, Waterfall plot for % change in ¹⁸FBnTP uptake post-treatment to pre-treatment for mice treated with Vehicle (n = 4 mice) or 125 mg/kg Phenformin (n = 6 mice) for 5 days. Experiments in b, d, and e were performed once. Statistical significance for d and e was calculated using unpaired two-tailed t-test.

Figure 4.. Multi-tracer PET imaging of KL lung tumors with ¹⁸FBnTP and ¹⁸F-FDG.

a, PET/CT images of a KL mouse with two tumors, T1 and T2. Top panel – ¹⁸FBnTP/CT overlay, bottom panel – ¹⁸F-FDG/CT overlay. H – heart. b, Western blot of T1 and T2 isolated from mouse imaged in a and probed with indicated antibodies. c, Western blot of tumors isolated from KL or KP mice and probed with indicated antibodies. d-i, Experiments performed on A549 (ADC) and RH2 (SCC) cell lines. d, Western blot probed with indicated antibodies. Loading control (β-actin) is shown for each blot; intensity of bands relative to actin is indicated. e, TMRE measurement (n = 3 biological replicates). f, Basal oxygen consumption rate (OCR) (n = technical 15 replicates). g, Extracellular acidification rate (ECAR) (n = technical 15 replicates). h, Coomassie staining of mitochondria isolated from A549 and RH2 cells separated on blue native gel. SC – supercomplex, I – complex I, V – complex V, III – complex III. Densitometry of the CI band corresponding to CV and CIII is indicated. i, Complex I activity in A549 and RH2 cells (n = technical 25 replicates). j, Schematic of the treatment regiment for KL mice. k, Percent of lung area occupied by tumors in KL mice treated with Vehicle or 15 mg/kg IACS-010759. N = 9 mice (Vehicle), n = 13 mice (IACS). l, Percent of Ki67 positive cells per tumor area quantified for TTF1 positive tumors (TTF1^POS) for KL mice treated as indicated in j (left panel; n=861 [Vehicle]; n=1068 [IACS]) and CK5 positive tumors (CK5^POS) tumors (right panel; n=31 [Vehicle]; n=26 [IACS]). The data are shown as the mean +/− SEM. m, Transverse view of a KL mouse imaged with ¹⁸FBnTP PET/CT (top panel) and ¹⁸F-FDG PET/CT (bottom panel). H – heart; tumor is outlined. n, H&E stain of the PET imaged tumor. Tumor histology is indicated. Scale bar = 1.0 mm. o, Representative images of Glut1 (left panel) and CK5/TTF1 (right panel) stained tumor. Scale bar = 25 μm. The data are represented as the mean +/− SD. Statistical significance was calculated using unpaired two-tailed t-test. Experiments in a, b, c, d-h were done twice with similar results. Experiments in i, k, l were done once. Data in m-o are representative of three independent mouse experiments.