Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [18F]FDG uptake and MRI measurement of hyperpolarized [1-13C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6-13C2]glucose and measuring 13C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1-13C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [18F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [18F]FDG uptake by infiltrating immune cells. Quantification of [18F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b+/CD45+ macrophages as the most [18F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [18F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1-13C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [18F]FDG uptake.

Significance:

These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1-13C]pyruvate metabolism versus PET measurement of 18F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death.

Tumors are increasingly categorized according to their biology (1), and a new generation of drugs are being developed that target the mutated pathways often found in tumors (2). Treatment response is typically assessed using morphologic imaging (CT or MRI; ref. 3) with response being categorized according to guidelines such as RECIST (4). However, changes in size are often slow to manifest after treatment (5–7). To complement the advances in therapy, new imaging techniques are required that can accurately assess early treatment responses. These could facilitate switching nonresponders to a more effective treatment, limit unnecessary side effects and costs of an ineffective therapy, and speed up clinical translation of new treatments.

Biochemical changes can occur before volumetric changes, and therefore targeted metabolic imaging could improve the timescale of response detection (8). PET using the glucose analogue, 2-([18F]fluoro)-2-deoxy-D-glucose ([18F]FDG), has been used to assess treatment response, notably in lymphomas (9, 10). However, concerns about the “flare” effect, a poorly understood phenomenon of increased tracer uptake soon after treatment attributed to agonistic effects of therapy on macrophage infiltration and activation, has often led to delayed [18F]FDG-PET imaging of treatment response (11–14).

13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized [1-13C]pyruvate metabolism has been shown to give an early indication of tumor treatment response (15, 16). However, for hyperpolarized [1-13C]pyruvate to enter routine clinical practice, it must demonstrate that it can provide information not available from [18F]FDG studies. In this study, we used a multivalent TRAIL agonist (MEDI3039) targeting the human death receptor five (DR5) to activate the extrinsic pathway of apoptosis in xenograft models of human colorectal and breast cancer (17) and compared the ability of hyperpolarized [1-13C]pyruvate and [18F]FDG-PET to detect early response to treatment in vivo. A reduction in label flux between hyperpolarized [1-13C]pyruvate and the endogenous lactate pool preceded changes in tumor volume and reliably detected treatment response, whereas PET measurements of [18F]FDG uptake largely failed to detect response.

Cell culture

Colo205 human colon adenocarcinoma cells (ATCC) and MDA-MB-231 triple-negative breast adenocarcinoma cells (ATCC) were transduced with a lentiviral vector expressing mStrawberry red fluorescent protein and luciferase (18). Colo205 cells were cultured in RPMI medium (Life Technologies), supplemented with 2 mmol/L l-glutamine and 10% heat-inactivated FBS (Life Technologies). MDA-MB-231 cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS. Both cell lines tested negative for Mycoplasma and were used within ten passages from the original stocks.

Animal preparation

Animal experiments were performed in compliance with a project license issued under the Animals (Scientific Procedures) Act of 1986. Protocols were approved by the Cancer Research UK, Cambridge Institute Animal Welfare and Ethical Review Body.

Colo205 or MDA-MB-231 cells (5 × 106) were resuspended in 0.2 mL PBS or a 50:50 mix of Matrigel (Corning) and complete DMEM, respectively, and implanted s.c. in the flanks of female BALB/c nu/nu mice (Charles River). Tumors were imaged when they reached approximately 0.8 cm3. For imaging, mice were fasted for 6 to 8 hours (19) and kept in a warmed chamber (32°C) for 1 hour prior to induction of anesthesia using 1% to 2.5% isoflurane (Isoflo, Abbotts Laboratories Ltd.) in a 50:50 mix of air (1 L/min) and oxygen (1 L/min). MEDI3039, a TRAIL agonist (Medimmune), was administered i.v. at 0.4 mg/kg (17, 20).

Hyperpolarization of [1-13C]pyruvate

A 44 mg sample of [1-13C]pyruvic acid (Cambridge Isotope Laboratories) containing 15 mmol/L of OX063 trityl radical (GE Healthcare) and 1.5 mmol/L of gadoterate meglumine (Dotarem, Guerbet) was hyperpolarized at approximately 1.2 K by microwave irradiation at 94.110 GHz and 100 mW in a 3.35 T Hypersense polarizer (Oxford Instruments) for approximately 1 hour (21). The frozen sample was rapidly dissolved in 6 mL buffer containing 40 mmol/L HEPES, 94 mmol/L NaOH, 30 mmol/L NaCl, and 100 mg/L EDTA heated to 180°C and pressurized to 10 bar to yield a final [1-13C]pyruvate concentration of approximately 75 mmol/L.

Imaging treatment response

Colo205 (n = 18, Supplementary Table S1) and MDA-MB231 (n = 22, Supplementary Table S2) tumor–bearing mice underwent bioluminescence (BLI), fluorescence (FLI), MR, and PET-CT imaging performed in the same 2-hour sessions before and 24 hours after treatment with MEDI3039 (Supplementary Fig. S1). FLI and BLI were performed using a Xenogen IVIS 200 (Perkin Elmer). Fluorescence images of mStrawberry expression were acquired using a DSRed filter (λex = 500–550 nm, λem = 575–650 nm) and corrected for background autofluorescence. Bioluminescence images were acquired 5 minutes after i.p. injection of 150 mg/kg of 15 mg/mL D-luciferin. Regions of interest (ROI) were analyzed using Living Image v4.5 software (Perkin Elmer).

After BLI, 12.9 ± 1.8 MBq [18F]FDG (in approximately 100 μL; Alliance Medical) was injected intravenously. MRI was performed in a 7.0 T horizontal bore magnet (Agilent) using an actively decoupled dual-tuned 13C/1H volume transmit coil (Rapid Biomedical) and a 20 mm diameter 13C receiver coil (Rapid Biomedical). For anatomical reference, eight axial T2-weighted 1H images were acquired using a fast-spin echo sequence with a slice thickness of 2.5 mm, field-of-view 40 × 40 mm, and matrix size of 256 × 256 points. 13C images were acquired using spectral spatial pulses and a 3D dual-spin echo acquisition (22). Flip angles were 7° for [1-13C]pyruvate and 45° for [1-13C]lactate. Five [1-13C]pyruvate images were acquired prior to the first [1-13C]lactate image, after which, each metabolite was excited with a temporal resolution of 2 seconds per metabolite. Injection of hyperpolarized [1-13C]pyruvate, which can cause transient hypoxia, was delayed for 1 hour post-[18F]FDG injection to minimize any effects on [18F]FDG uptake (23). Hyperpolarized [1-13C]pyruvate (15 mL/kg) was injected i.v. over 8 seconds, and the pulse sequence was started 2 seconds after the start of infusion. Images were acquired over 90 seconds and analyzed in Matlab (Mathworks). A 3D tumor ROI was defined on the T2-weighted image, and the rate of hyperpolarized 13C label exchange was assessed by measuring the ratio of the areas under the pyruvate and lactate labeling curves (AUC). This ratio is related directly to the apparent first-order rate constant describing label exchange between the injected pyruvate and the endogenous lactate pool (24).

A 15-minute static PET acquisition began at 90 minutes after injection of [18F]FDG using a nanoScan PET/CT (Mediso). A helical CT was acquired for anatomical reference and attenuation correction. PET images, with a nominal isotropic resolution of 0.3 mm, were reconstructed using a 3D ordered subset expectation maximization (OSEM) method in one to three coincidence modes, eight iterations, and six subsets. Images were normalized and corrected for decay, dead-time, random events, and attenuation. The images were analyzed using Vivoquant 3.0 software (InviCRO). A 3D tumor ROI was drawn manually, and Otsu thresholding was applied to better delineate the tumor. Mean and maximum standardized uptake values (SUV) were calculated using:

formula

where cimg is the activity concentration (MBq/mL) derived from the image ROI, ID is the injected dose, and BW is the body weight of the animal.

Dynamic [18F]FDG-PET

Three-hour PET scans were acquired in a separate cohort of Colo205 tumor–bearing mice (n = 9) following injection of 8.12 ± 1.13 MBq [18F]FDG before and 24 hours after treatment with MEDI3039. Scans were reconstructed with a nominal isotropic resolution of 0.6 mm using a 3D OSEM method with one to five coincidence modes, two iterations, and six subsets, with CT acquisition and PET corrections applied as for static image acquisitions. Scans were reconstructed into 50 time frames (5 seconds × 12, 0–1 minutes; 2 minutes × 30, 0–60 minutes; 15 minutes × 8, 60–180 minutes). A 3D ROI drawn manually over the inferior vena cava between the level of the kidneys and diaphragm was thresholded at 75% of the maximum activity and used as an image-derived input function. A 3D tumor ROI was Otsu thresholded on the 165- to 180-minute dataset. Patlak analysis was used to estimate the net influx rate of [18F]FDG (ki) from the linear portion of the graph between 20 and 60 minutes (25). The mean r2 for linear regression fitting was 0.97 ± 0.03.

Dynamic contrast-enhanced MRI

A separate cohort of Colo205 tumor–bearing mice (n = 3 per group, drug- and vehicle-treated) underwent dynamic contrast-enhanced (DCE)-MRI before and 24 hours after treatment with MEDI3039. Baseline spin-lattice relaxation rates (R1,pre = 1/T1) were measured using an inversion recovery fast low angle shot (FLASH) sequence (TR 5.5 ms, TE 2.5 ms, 10 s delay between acquisitions, 2 averages per inversion time). To obtain a precontrast R1 map, these data were fitted voxel-by-voxel to a monoexponential function:

formula

where TI is the inversion time. A T1-weighted gradient-echo pulse sequence was used with 4 × 1.5 mm thick axial slices with 0.25 mm gaps covering the tumor region, field-of-view 40 × 40 mm, data matrix 128 × 128, TR 110 ms, and TE 9 ms. Ten baseline images were collected prior to the injection, over 8 seconds, of 200 μmoles/kg i.v. Dotarem (Guerbet). Forty images were acquired immediately after injection, and a further nine timepoints generated from averaging blocks of nine images acquired every 10 minutes up to 90 minutes after injection. Images were converted to R1 relaxation rate maps using:

formula

where S0 is the relaxed signal (TR >> T1) and θ is the flip angle used in the gradient-echo sequence (27°). Changes in R1 were directly converted to gadolinium (Gd3+) concentration curves as described in ref. 26.

Whole-body [18F]FDG autoradiography and mStrawberry fluorescence imaging

A separate cohort of Colo205 tumor–bearing mice (n = 3 per group, drug- and vehicle-treated) were injected i.v. with [18F]FDG, culled after 90 minutes, and immediately frozen by submersion in liquid nitrogen–cooled isopentane. Axial cryosections (10 μm thick) were thaw-mounted onto glass slides (CM3050S Cryostat, Leica). The slides were apposed to a storage phosphor screen (GE Healthcare) overnight to produce autoradiographs with a pixel size of 10 μm using a Typhoon Biomolecular Imager (Amersham). Red fluorescence images of the cryosections were acquired using a 532 nm laser, long-pass 550 nm filter, and a pixel size of 10 μm. The slides were subsequently hematoxylin and eosin (H&E)–stained, and fluorescence, autoradiography, and H&E images were coregistered manually.

Determining the cellular fate of [18F]FDG using fluorescence-activated cell sorting

A separate cohort of Colo205 tumor–bearing mice (n = 3 per group, drug- and vehicle-treated) were injected i.v. with 140 ± 4.9 MBq [18F]FDG and after 90 minutes the tumors excised and single-cell suspensions were prepared by digestion in 1 mg/mL collagenase I (Sigma-Aldrich) and 0.1 mg/mL DNase I (Roche) at 37°C for 45 minutes with trituration at 15-minute intervals. The cells were washed in PBS/2 mmol/L EDTA and labeled by incubation with anti-CD45 (30-F11), anti-CD11b (M1/70; Biolegend), and live/dead fixable viability dye e780 (ThermoFisher Scientific) at 4°C for 1 hour. CD11b+/CD45+ phagocytes, CD45-/mStrawberry+ tumor cells, CD45-/mStrawberry- nonhematopoietic, nontumor cells, and e780+ dead cells were sorted on a BD Influx flow sorter (BD Biosciences). Flow cytometric data were analyzed using FlowJo V10.0 (FlowJo LLC). The radioactivity (cpm) of each cell population was determined using a well-counter (Nuklear-Medizintechnik) and converted to Bq using a calibration curve.

[1,6-13C2]glucose infusion and 13C and 1H NMR spectroscopy of tumor extracts

A separate cohort of Colo205 tumor–bearing mice (n = 3 per group, drug- and vehicle-treated) were infused with [1,6-13C2]glucose as described previously (27). Tumors were freeze-clamped and blood obtained by cardiac puncture. Tissue extracts were prepared by addition of 5 μL/mg of 2 mol/L perchloric acid (PCA) and homogenization in a Precellys 24 homogenizer (Stretton Scientific). Extracts were pH corrected to 7.0, lyophilized, and dissolved in deuterium oxide. Two μmoles trimethylsilylpropanoic acid were added as a chemical shift standard (0 p.p.m.). High-resolution 1H and 1H-decoupled 13C NMR spectra were acquired at 294 K using a 5 mm probe and a 600 MHz NMR spectrometer (Bruker). The acquisition conditions for 13C spectroscopy were 30° flip angle, 15,000 transients, spectral width of 36.8 kHz, acquisition time of 1.8 seconds, and a relaxation delay of 1.2 seconds. The acquisition conditions for 1H spectroscopy were 90° flip angle, 1,024 transients, spectral width of 10 kHz, acquisition time of 3.3 seconds, and relaxation delay of 2 seconds. Data were phased and baseline corrected and peak integrals calculated using Topspin 4.0 (Bruker).

Analysis of [18F]FDG and radiolabeled metabolites in tumor extracts

A separate cohort of Colo205 tumor–bearing mice (n = 3 per group, drug- and vehicle-treated) were anesthetized and injected i.v. with 146.2 ± 7.8 MBq [18F]FDG (∼200 μL). After 90 minutes, tumors were excised and homogenized in ice-cold 4 mol/L PCA using a Precellys 24 homogenizer (Stretton Scientific). The extracts were neutralized with 8 mol/L KOH and passed through a 0.2 μm syringe filter (Whatman). Radio-high performance liquid chromatography (radioHPLC) was performed using a Dionex UltiMate 3000 HPLC system (ThermoFisher Scientific; ref. 28). Samples (100 μL) were injected into the system, separated using a Partasil SAX 10 μm column (250 mm × 4.6 mm, Sigma-Aldrich), and eluted with a linear gradient of 300 mmol/L sodium dihydrogen phosphate containing 2% methanol (A) and 2% methanol in water (B). The gradient profile was 0–15 min 5% A, 15–25 min 50% A (isocratic), with a flow rate of 1.5 mL/min. Radioactivity was detected using a fLumo HPLC NaI detector (Berthold Technologies) connected to the column outflow. Metabolite retention times for [18F]FDG and [18F]FDG-6-P were assigned using standards, whereas 2-([18F]fluoro)-2-deoxy-6-phospho-D-gluconolactone ([18F]FD-PGL) and 2-([18F]fluoro)-2-deoxy-D-glucose-1,6-bisphosphate ([18F]FDG-1,6-P2) were assigned using data from ref. 28.

Western blotting

Freeze-clamped tumor samples (n = 7 per group, drug- and vehicle-treated) were homogenized in 10 μL/mg modified RIPA buffer (50 mmol/L HEPES, 1 mmol/L EDTA, 0.7% sodium deoxycholate, 1% Nonidet P-40, 0.5 mol/L lithium chloride, pH 7.6, 1 cOmplete mini EDTA-free protease inhibitor; Sigma-Aldrich) using a Precellys 24 homogenizer. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (iBlot 2, ThermoFisher Scientific). Membranes were blocked with 1:1 Odyssey blocking buffer and TBS and incubated with antibody solutions (Supplementary Table S3) at 4°C overnight. Antibodies were detected using multiplexed IRDye secondary antibodies and images acquired using an Odyssey CLx (LI-COR Biosciences).

Enzyme activity and ATP assays

Lactate dehydrogenase (LDH) activity was determined spectrophotometrically in tumor extracts (n = 7 per group, drug- and vehicle-treated) prepared in RIPA buffer (29). Colorimetric kits were used to determine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity (ab204732, Abcam) and ATP concentration (ab83355, Abcam), whereas a fluorometric kit was used to determine pyruvate kinase (PK) activity (ab83432, Abcam). A PHERAstar FS microplate reader (BMG Labtech) was used for all spectrophotometric measurements.

Immunohistochemistry

Sections of formalin-fixed paraffin-embedded tumors were stained for cleaved caspase-3 (CC3) and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL; ref. 20). Percentage positivity for each stain was calculated using positive pixel count algorithms in Aperio ImageScope 12.3.3 (Leica).

Analysis

Statistical and graphical analysis was performed using Prism v6.0 (GraphPad). Statistical tests performed were paired or unpaired t tests with errors representing SD, unless stated otherwise. P values are summarized in figures tables as: *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P = 0.0001–0.001; ****, P < 0.0001.

MEDI3039 induces cell death and tumor regression

Colo205 tumor–bearing mice (n = 3) were treated with 0.4 mg/kg MEDI3039 i.v. weekly for 4 weeks and fortnightly thereafter (a total of 14 doses over 6 months). There was a decrease in tumor volume from 0.95 ± 0.1 cm3 to 0.03 ± 0.02 cm3 12 days after initial treatment, after which, tumor volumes decreased to below the detection limit on T2-weighted MRI (Fig. 1A–D). Bioluminescence and red fluorescence intensity decreased over the first month, after which, one tumor started to regrow, whereas the other two continued to decrease in intensity (Fig. 1E–L). At 6 months, the tumor that regrew had reached its size limit, whereas in the other 2 mice the tumors remained undetectable.

Figure 1.

MEDI3039 treatment induces long-term regression of Colo205 tumors. A–D, Tumor volumes (n = 3) were determined by T2-weighted MRI measurements. E–H, mStrawberry fluorescence. I–L, Bioluminescence. The bioluminescence scale was decreased by a factor of 10 for the images acquired on day 29.

Figure 1.

MEDI3039 treatment induces long-term regression of Colo205 tumors. A–D, Tumor volumes (n = 3) were determined by T2-weighted MRI measurements. E–H, mStrawberry fluorescence. I–L, Bioluminescence. The bioluminescence scale was decreased by a factor of 10 for the images acquired on day 29.

Close modal

Histologic and reporter gene evaluation of early response to therapy

Colo205 and MDA-MB-231 tumor–bearing mice underwent combined BLI, FLI, [1-13C]pyruvate- MRI, and [18F]FDG-PET imaging before and 24 hours after treatment with MEDI3039 (Colo205, n = 10; MDA-MB-231, n = 12) or drug vehicle (saline; Colo205 n = 8; MDA-MB-231, n = 9; Table 1; Supplementary Tables S1 and S2). CC3 staining increased from 21.8 ± 11.6% to 58.51 ± 14.4% (P = 0.0002, n = 7 per group, drug- and vehicle-treated) in Colo205 tumors (Fig. 2A–C) and from 19 ± 5.1% to 57.7 ± 19.3% (P = 0.006, n = 5 drug-treated and n = 4 vehicle-treated) in MDA-MB-231 tumors (Fig. 2D–F). TUNEL staining increased from 8.0 ± 6.7% to 19.4 ± 6.3% (P = 0.007, n = 7 per group, drug- and vehicle-treated) in Colo205 tumors (Fig. 2G–I) and 6.6 ± 2.0% to 21.1 ± 6.1% (P = 0.003, n = 5 drug-treated and n = 4 vehicle-treated) in MDA-MB-231 tumors (Fig. 2J–L). There was no significant decrease in tumor volumes after 24 hours (Supplementary Fig. S2A–S2F). mStrawberry fluorescence decreased in MDA-MB-231 tumors by 45.3 ± 28.6% (P = 0.0002, n = 12), but there was no change in Colo205 tumors (P = 0.30, n = 8). However, fluorescence in Colo205 tumors had decreased by 48 hours after treatment (P = 0.04; n = 3). With the exception of one animal, tumor bioluminescence decreased after treatment, with 57.3 ± 53.3% (P = 0.0053; n = 10) and 68.5 ± 24.8% (P = <0.0001; n = 12) decreases in Colo205 and MDA-MB-231 tumors, respectively. Measurements of ATP concentration in Colo205 tumor extracts showed a decrease from 0.36 ± 0.1 to 0.14 ± 0.05 μmol/g w.w. (P = 0.0008; n = 11) at 24 hours after treatment.

Table 1.

Imaging, ex vivo, and histologic detection of treatment response 24 hours after MEDI3039 treatment

T2 MRIBioluminescenceFluorescence[1-13C]pyruvate MRSI[18F]FDG-PET[18F]FDG autoradiography[18F]FDG excised tumorsHistology
Tumor typeVolume (cm3)Mean radiance (p/s/cm2/sr)Mean radiant efficiency [(p/s/cm2/sr)/(μW/cm2)]Lactate/pyruvate ratioSUVmaxSUVmeanki (min−1)Tumor/muscle ratio%ID/gCC3 (% positivity)TUNEL (% positivity)
Colo205 
 Pretreatment 0.879 ± 0.425 2.455 × 109 ± 3.445 × 109 1.465 × 109 ± 9.009 × 108 2.525 ± 0.549 2.103 ± 0.250 1.131 ± 0.160 0.048 ± 0.01     
 (n = 10) (n = 10) (n = 8) (n = 7) (n = 7) (n = 7) (n = 9)     
 Posttreatment 0.829 ± 0.491 4.723 × 108 ± 5.678 × 108 2.068 × 109 ± 1.981 × 109 1.414 ± 0.391 2.134 ± 0.362 1.087 ± 0.190 0.037 ± 0.012 4.096 ± 0.268 7.613 ± 0.697 58.510 ± 14.440 19.360 ± 6.311 
 (n = 10) ** (n = 10) (n = 8) ** (n = 7) (n = 7) (n = 7) (n = 9) (n = 3) (n = 6) *** (n = 7) ** (n = 7) 
 Precontrol 0.705 ± 0.252 6.961 × 108 ± 9.705 × 108 1.373 × 109 ± 1.494 × 109 1.975 ± 0.548 2.261 ± 0.580 1.230 ± 0.223      
 (n = 7) (n = 8) (n = 7) (n = 5) (n = 7) (n = 7)      
 Postcontrol 0.721 ± 0.289 1.092 × 109 ± 1.406 × 109 1.288 × 109 ± 1.191 × 109 2.106 ± 0.630 2.219 ± 0.161 1.182 ± 0.206  4.011 ± 0.224 8.846 ± 0.828 21.800 ± 11.580 8.005 ± 6.728 
 (n = 7) (n = 8) (n = 7) (n = 5) (n = 7) (n = 7)  (n = 3) (n = 6) (n = 7) (n = 7) 
MDA-MB-231 
 Pretreatment 0.939 ± 0.488 1.288 × 109 ± 4.629 × 108 3.002 × 109 ± 1.180 × 109 2.236 ± 0.386 2.219 ± 0.399 1.045 ± 0.172      
 (n = 8) (n = 12) (n = 12) (n = 7) (n = 10) (n = 10)      
 Posttreatment 0.862 ± 0.450 4.148 × 108 ± 4.340 × 108 1.634 × 109 ± 1.015 × 109 1.382 ± 0.331 1.977 ± 0.346 0.877 ± 0.158    57.650 ± 19.300 21.120 ± 6.061 
 (n = 8) **** (n = 12) *** (n = 12) ** (n = 7) (n = 10) * (n = 10)    ** (n = 5) ** (n = 5) 
 Precontrol 0.664 ± 0.281 1.541 × 109 ± 1.503 × 109 2.349 × 109 ± 1.067 × 109 2.126 ± 0.588 2.125 ± 0.558 0.998 ± 0.214      
 (n = 9) (n = 9) (n = 9) (n = 9) (n = 9) (n = 9)      
 Postcontrol 0.686 ± 0.304 1.067 × 109 ± 6.385 × 108 2.174 × 109 ± 1.110 × 109 2.442 ± 0.607 2.039 ± 0.685 0.913 ± 0.275    18.980 ± 5.092 6.644 ± 1.944 
 (n = 9) (n = 9) (n = 9) (n = 9) (n = 9) (n = 9)    (n = 4) (n = 4) 
T2 MRIBioluminescenceFluorescence[1-13C]pyruvate MRSI[18F]FDG-PET[18F]FDG autoradiography[18F]FDG excised tumorsHistology
Tumor typeVolume (cm3)Mean radiance (p/s/cm2/sr)Mean radiant efficiency [(p/s/cm2/sr)/(μW/cm2)]Lactate/pyruvate ratioSUVmaxSUVmeanki (min−1)Tumor/muscle ratio%ID/gCC3 (% positivity)TUNEL (% positivity)
Colo205 
 Pretreatment 0.879 ± 0.425 2.455 × 109 ± 3.445 × 109 1.465 × 109 ± 9.009 × 108 2.525 ± 0.549 2.103 ± 0.250 1.131 ± 0.160 0.048 ± 0.01     
 (n = 10) (n = 10) (n = 8) (n = 7) (n = 7) (n = 7) (n = 9)     
 Posttreatment 0.829 ± 0.491 4.723 × 108 ± 5.678 × 108 2.068 × 109 ± 1.981 × 109 1.414 ± 0.391 2.134 ± 0.362 1.087 ± 0.190 0.037 ± 0.012 4.096 ± 0.268 7.613 ± 0.697 58.510 ± 14.440 19.360 ± 6.311 
 (n = 10) ** (n = 10) (n = 8) ** (n = 7) (n = 7) (n = 7) (n = 9) (n = 3) (n = 6) *** (n = 7) ** (n = 7) 
 Precontrol 0.705 ± 0.252 6.961 × 108 ± 9.705 × 108 1.373 × 109 ± 1.494 × 109 1.975 ± 0.548 2.261 ± 0.580 1.230 ± 0.223      
 (n = 7) (n = 8) (n = 7) (n = 5) (n = 7) (n = 7)      
 Postcontrol 0.721 ± 0.289 1.092 × 109 ± 1.406 × 109 1.288 × 109 ± 1.191 × 109 2.106 ± 0.630 2.219 ± 0.161 1.182 ± 0.206  4.011 ± 0.224 8.846 ± 0.828 21.800 ± 11.580 8.005 ± 6.728 
 (n = 7) (n = 8) (n = 7) (n = 5) (n = 7) (n = 7)  (n = 3) (n = 6) (n = 7) (n = 7) 
MDA-MB-231 
 Pretreatment 0.939 ± 0.488 1.288 × 109 ± 4.629 × 108 3.002 × 109 ± 1.180 × 109 2.236 ± 0.386 2.219 ± 0.399 1.045 ± 0.172      
 (n = 8) (n = 12) (n = 12) (n = 7) (n = 10) (n = 10)      
 Posttreatment 0.862 ± 0.450 4.148 × 108 ± 4.340 × 108 1.634 × 109 ± 1.015 × 109 1.382 ± 0.331 1.977 ± 0.346 0.877 ± 0.158    57.650 ± 19.300 21.120 ± 6.061 
 (n = 8) **** (n = 12) *** (n = 12) ** (n = 7) (n = 10) * (n = 10)    ** (n = 5) ** (n = 5) 
 Precontrol 0.664 ± 0.281 1.541 × 109 ± 1.503 × 109 2.349 × 109 ± 1.067 × 109 2.126 ± 0.588 2.125 ± 0.558 0.998 ± 0.214      
 (n = 9) (n = 9) (n = 9) (n = 9) (n = 9) (n = 9)      
 Postcontrol 0.686 ± 0.304 1.067 × 109 ± 6.385 × 108 2.174 × 109 ± 1.110 × 109 2.442 ± 0.607 2.039 ± 0.685 0.913 ± 0.275    18.980 ± 5.092 6.644 ± 1.944 
 (n = 9) (n = 9) (n = 9) (n = 9) (n = 9) (n = 9)    (n = 4) (n = 4) 

NOTE: *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P = 0.0001–0.001; ****, P < 0.0001.

Figure 2.

Histologic assessment of tumor cell death following treatment with MEDI3039. Tumor sections were stained for CC3 and TUNEL (n = 7 per group, drug- and vehicle-treated). CC3 (A–C) and TUNEL (G–I) staining of Colo205 tumor sections taken 24 hours after treatment of the animals with MEDI3039 (treatment) or drug vehicle (control). CC3 (D–F) and TUNEL (J–L) staining of MDA-MB-231 tumor sections taken 24 hours after treatment of the animals with MEDI3039 (treatment) or drug vehicle (control). **, P = 0.001–0.01; ***, P = 0.0001–0.001.

Figure 2.

Histologic assessment of tumor cell death following treatment with MEDI3039. Tumor sections were stained for CC3 and TUNEL (n = 7 per group, drug- and vehicle-treated). CC3 (A–C) and TUNEL (G–I) staining of Colo205 tumor sections taken 24 hours after treatment of the animals with MEDI3039 (treatment) or drug vehicle (control). CC3 (D–F) and TUNEL (J–L) staining of MDA-MB-231 tumor sections taken 24 hours after treatment of the animals with MEDI3039 (treatment) or drug vehicle (control). **, P = 0.001–0.01; ***, P = 0.0001–0.001.

Close modal

Imaging early response to MEDI3039 treatment using 13C MRI and PET

3D 13C MRS images showed a posttreatment decrease of the [1-13C]lactate/[1-13C]pyruvate ratio (the ratio of the areas under the lactate and pyruvate labeling curves) in all animals, with a mean reduction of 42.2 ± 15.9% (P = 0.004; n = 7) and 36.3 ± 18.6% (P = 0.007; n = 7) in Colo205 (Fig. 3A–C) and MDA-MB-231 (Fig. 3D and E) tumors, respectively (Table 1). There was no change in [18F]FDG-PET SUVmax (2.1 ± 0.3 to 2.1 ± 0.4, P = 0.82, n = 7) or SUVmean (1.1 ± 0.2 to 1.1 ± 0.2, P = 0.6, n = 7) in Colo205 tumors. In MDA-MB-231 tumors, SUVmax did not change significantly (2.2 ± 0.4 to 2.0 ± 0.3, P = 0.2, n = 10), but there was a decrease in SUVmean (1.0 ± 0.2 to 0.9 ± 0.2; P = 0.048, n = 10). However, this was a decrease of 14 ± 21%, compared to a 36 ± 19% decrease in [1-13C]lactate/[1-13C]pyruvate ratio, indicating that changes in [1-13C]pyruvate metabolism were more sensitive for detecting response to treatment in these tumors.

Figure 3.

Comparison of hyperpolarized [1-13C]lactate/[1-13C]pyruvate ratios and [18F]FDG-PET SUVmax values in Colo205 and MDA-MB-231 tumors before and 24 hours after treatment with MEDI3039. A, A Colo205 tumor–bearing mouse before (top two rows of images) and after (bottom two rows of images) treatment. Maximum intensity projections overlaid on bone reconstructions (top and bottom rows) and an axial slice through the tumor (middle two rows). The tumor is indicated by white arrows and a dashed outline. B and D, [1-13C]lactate/[1-13C]pyruvate ratio before and after treatment in Colo205 (n = 7 drug-treated; n = 5 vehicle-treated; B) and MDA-MB-231 (n = 7 drug-treated; n = 9 vehicle-treated; D) tumors. C and E, [18F]FDG SUVmax before and after treatment in Colo205 (C) and MDA-MB-231 (E) tumors. Pre- and posttreatment images are identically scaled, and PET images are scaled from an SUV of 0–1.5. **, P = 0.001–0.01.

Figure 3.

Comparison of hyperpolarized [1-13C]lactate/[1-13C]pyruvate ratios and [18F]FDG-PET SUVmax values in Colo205 and MDA-MB-231 tumors before and 24 hours after treatment with MEDI3039. A, A Colo205 tumor–bearing mouse before (top two rows of images) and after (bottom two rows of images) treatment. Maximum intensity projections overlaid on bone reconstructions (top and bottom rows) and an axial slice through the tumor (middle two rows). The tumor is indicated by white arrows and a dashed outline. B and D, [1-13C]lactate/[1-13C]pyruvate ratio before and after treatment in Colo205 (n = 7 drug-treated; n = 5 vehicle-treated; B) and MDA-MB-231 (n = 7 drug-treated; n = 9 vehicle-treated; D) tumors. C and E, [18F]FDG SUVmax before and after treatment in Colo205 (C) and MDA-MB-231 (E) tumors. Pre- and posttreatment images are identically scaled, and PET images are scaled from an SUV of 0–1.5. **, P = 0.001–0.01.

Close modal

Confirmation that [18F]FDG-PET fails to detect response to MEDI3039

[18F]FDG tumor/muscle ratios derived from autoradiography (Supplementary Fig. S3A–S3G) and mean percentage injected dose per gram (%ID/g) from well-counting of excised Colo205 tumors (Supplementary Fig. S3H) also did not change significantly following treatment (Table 1). However, in a separate cohort of Colo205 tumor–bearing mice (n = 9), MEDI3039 treatment resulted in the net [18F]FDG influx rate (ki) decreasing from 0.048 ± 0.01 to 0.037 ± 0.012 min−1 (P = 0.051).

Colo205 tumor–bearing mice (n = 3) were further imaged 24, 48, and 72 hours after MEDI3039 treatment. Mean SUVmax showed no significant change, with a value of 1.67 ± 0.1 before treatment and 1.79 ± 0.5 (P = 0.77) 72 hours after treatment, despite significant decreases in tumor volume.

DCE-MRI showed an increase in perfusion after MEDI3039 treatment

In a separate cohort of Colo205 tumor–bearing mice (n = 3), treatment response was assessed using DCE-MRI (Supplementary Fig. S4). Tumor gadolinium (Gd3+) concentration increased in all mice after treatment from 0.05 ± 0.009 to 0.09 ± 0.02 mmol/L at 10 minutes after injection, although the small sample size meant this was not statistically significant. The rate of contrast agent clearance was similar in pretreatment and posttreatment tumors.

Determination of the cellular fate of [18F]FDG using fluorescence-activated cell sorting of disaggregated tumors

Tumor cells (mStrawberry+, CD45-) comprised 78.3 ± 5.2% of the sorted cells in untreated tumors (n = 3; Fig. 4A and C) and 71.6 ± 9.4% in MEDI3039-treated tumors (n = 3; Fig. 4B and C) at 24 hours after treatment (P = 0.3). Phagocytes (CD45+, CD11b+) comprised 1.6 ± 0.4% of cells in untreated tumors and 2.3 ± 1.7% after treatment (P = 0.5; Fig. 4A–C). Treatment resulted in a decrease in tumor cell [18F]FDG uptake (%ID/cell) from 2.1 × 10−9 ± 5.0 × 10−10% to 7.5 × 10−10 ± 2.7 × 10−10% (P = 0.01). Phagocytes were the most [18F]FDG-avid cell type, taking up approximately 5 × the uptake of tumor cells (Fig. 4D). Treatment also decreased [18F]FDG uptake per phagocyte, from 1.2 × 10−8 ± 2.5 × 10−9% to 6.5 × 10−9 ± 1.9 × 10−9 (P = 0.04). Due to there being approximately 50 × and 30 × more tumor cells than phagocytes in untreated and treated tumors, respectively, the contribution of phagocytes to [18F]FDG uptake in the tumor as a whole remained small despite their greater [18F]FDG uptake per cell. Correction for % cell type, represented as %ID per million total cells, showed that in untreated tumors, uptake in the tumor cell population was 2 × 10−3 ± 5 × 10−4% per million total cells in the tumor, whereas in phagocytes, it was 2 × 10−4 ± 8.7 × 10−5% per million total cells. After treatment uptake in the tumor cell population was 6 × 10−4 ± 5.5 × 10−4%, whereas in the phagocyte population, it was 1 × 10−4 ± 5.6 × 10−5% (Fig. 4E).

Figure 4.

[18F]FDG uptake in different cell populations in Colo205 tumors (n = 3 per group, drug- and vehicle-treated). Tumors were excised 24 hours after treatment of the mice with MEDI3039 or drug vehicle (control). Disaggregated tumors were flow sorted, and radioactivity in the different cell fractions was counted. Example sort profiles from a control tumor (A) and a tumor treated with MEDI3039 (B). C, The percentage of different cell types in the tumors. The percentage injected dose per cell for each cell type (D) and the percentage injected dose for each population of cells (i.e., the %ID per cell corrected for the percentage of each cell type) displayed as %ID per million sorted cells (E). *, P = 0.01–0.05.

Figure 4.

[18F]FDG uptake in different cell populations in Colo205 tumors (n = 3 per group, drug- and vehicle-treated). Tumors were excised 24 hours after treatment of the mice with MEDI3039 or drug vehicle (control). Disaggregated tumors were flow sorted, and radioactivity in the different cell fractions was counted. Example sort profiles from a control tumor (A) and a tumor treated with MEDI3039 (B). C, The percentage of different cell types in the tumors. The percentage injected dose per cell for each cell type (D) and the percentage injected dose for each population of cells (i.e., the %ID per cell corrected for the percentage of each cell type) displayed as %ID per million sorted cells (E). *, P = 0.01–0.05.

Close modal

MEDI3039-induced changes in the expression of enzymes and transporters involved in [18F]FDG and [1-13C]pyruvate metabolism in Colo205 tumors

Treatment decreased the expression of MCT1 and to a lesser extent MCT4 (P = 0.005 and P = 0.02, respectively, n = 7; Fig. 5A and B). LDH activity and expression were unchanged (P = 0.07 and P = 0.9, respectively, n = 7; Supplementary Table S4; Fig. 5C and H). Expression of GLUT1 and GLUT3 decreased (P = 0.01 and P = 0.0001, respectively, n = 7; Fig. 5D and E), whereas HK2 expression was unchanged (P = 0.1, n = 7; Fig. 5F). PARP cleavage by caspase-3, a feature of apoptosis, increased significantly after MEDI3039 treatment (P = 0.005, n = 7; Fig. 5G). There were no significant changes in the activities of GAPDH (Fig. 5I) or PK (Fig. 5J).

Figure 5.

Changes in membrane transporter and enzyme expression and enzyme activity changes following treatment with MEDI3039. Arrows above the Western blots indicate samples from posttreatment tumors. Expression of MCT-1 (A), MCT-4 (B), LDH-A (C), GLUT1 (D), GLUT3 (E), HK2 (F), and PARP (G); and enzyme activity of LDH (H), GAPDH (I), and PK (J). cPARP, cleaved PARP; uPARP, uncleaved PARP. *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P = 0.0001-0.001.

Figure 5.

Changes in membrane transporter and enzyme expression and enzyme activity changes following treatment with MEDI3039. Arrows above the Western blots indicate samples from posttreatment tumors. Expression of MCT-1 (A), MCT-4 (B), LDH-A (C), GLUT1 (D), GLUT3 (E), HK2 (F), and PARP (G); and enzyme activity of LDH (H), GAPDH (I), and PK (J). cPARP, cleaved PARP; uPARP, uncleaved PARP. *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P = 0.0001-0.001.

Close modal

Measurement of glycolytic flux in MEDI3039-treated Colo205 tumors using [1,6-13C2]glucose

Colo205 tumor–bearing mice (n = 3 per group, drug- and vehicle-treated) were infused with [1,6-13C2]glucose for 150 minutes. 13C enrichment of blood glucose was 50.0 ± 7.2% and was unaffected by treatment. The expected tumor metabolite labeling patterns are shown in Fig. 6C. There was a decrease in tumor [3-13C]lactate concentration from 1,259 ± 297 μmoles/g w.w. to 363 ± 148 μmoles/g w.w. 24 hours after treatment (P = 0.01; Fig. 6A, B, and D), a decrease in [3-13C]alanine concentration, from 233 ± 59 to 37.6 ± 19 μmoles/g w.w. (P = 0.03), and a decrease in TCA cycle metabolite and [4-13C]glutamate labeling (Fig. 6A and B). There was no significant change in [1,6-13C2]glucose concentration in the tumors, which decreased from 425 ± 169 to 279 ± 135 μmoles/g w.w. (P = 0.3).

Figure 6.

13C NMR measurements of Colo205 tumor extracts following [1,6 13C2]glucose infusions into tumor-bearing mice 24 hours after drug vehicle (n = 3) or MEDI3039 (n = 3) treatment. Example 13C NMR spectra from a control tumor (A) and a treated tumor (B). Chemical shift assignments: 1, β-glucose C1; 2, α-glucose C1; 3, β-glucose C6; 4, α-glucose C6; 5, lactate C3; 6, alanine C3. Insets, spectra between 30 and 37 ppm. showing the difference in C3 and C4 glutamate labeling before treatment and after treatment. C, Diagram showing the 13C labeling pattern (blue) following [1,6 13C2]glucose infusion. D, Comparison of 13C-labeled glucose and lactate concentrations in control- and MEDI3039-treated tumors.**, P = 0.001–0.01. D, doublet; GLU, glutamate; S, singlet.

Figure 6.

13C NMR measurements of Colo205 tumor extracts following [1,6 13C2]glucose infusions into tumor-bearing mice 24 hours after drug vehicle (n = 3) or MEDI3039 (n = 3) treatment. Example 13C NMR spectra from a control tumor (A) and a treated tumor (B). Chemical shift assignments: 1, β-glucose C1; 2, α-glucose C1; 3, β-glucose C6; 4, α-glucose C6; 5, lactate C3; 6, alanine C3. Insets, spectra between 30 and 37 ppm. showing the difference in C3 and C4 glutamate labeling before treatment and after treatment. C, Diagram showing the 13C labeling pattern (blue) following [1,6 13C2]glucose infusion. D, Comparison of 13C-labeled glucose and lactate concentrations in control- and MEDI3039-treated tumors.**, P = 0.001–0.01. D, doublet; GLU, glutamate; S, singlet.

Close modal

Measurement of [18F]FDG and its metabolites in tumor extracts

HPLC peaks at 2 and 6 minutes corresponded to [18F]FDG and [18F]FDG-6-P, respectively (Supplementary Fig. S5A and S5B). The ratio of [18F]FDG/[18F]FDG-6-P was 6.2 ± 3% in untreated tumors and 7.0 ± 3.7% after treatment (n = 3 per group, drug- and vehicle-treated, P = 0.8; Supplementary Fig. S5C). HPLC peaks were also observed at 8.5, 9, 20, and 22.5 minutes. The peak at 20 minutes, assigned to [18F]FD-6-PGL (28), was decreased in treated tumors, suggesting that MEDI3039 treatment reduced flux into the pentose phosphate pathway.

Imaging with hyperpolarized [1-13C]pyruvate has the potential to be used clinically to detect early tumor responses to treatment (15, 30). Preclinical studies in breast cancer and glioma models demonstrated reductions in label flux from [1-13C]pyruvate to lactate prior to a reduction in tumor volume (31, 32), whereas in other studies, hyperpolarized [1-13C]pyruvate was no more sensitive than measurements of tumor volume or [18F]FDG uptake in detecting treatment response (33–35).

Drug treatment can have direct effects on metabolic pathways, for example, etoposide can inhibit oxidative phosphorylation (36, 37), and PI3K inhibitors can decrease LDH expression (38). Therefore, any effects observed with metabolic imaging may be drug-specific rather than a generic measure of treatment response (36). Here, we used a targeted agent to induce apoptosis that avoided potential direct effects of the drug on glycolytic enzyme expression. This allowed study of the specific effects that cell death has on the tumor metabolism of hyperpolarized [1-13C]pyruvate and [18F]FDG. Engineering the tumor cells to express mStrawberry and luciferase gave independent markers that could be used to confirm treatment response in vivo. Although MEDI3039 activates the extrinsic pathway of apoptosis, other mechanisms of cell death, particularly secondary necrosis, undoubtedly coexist following treatment and are not discriminated between in these experiments (39).

Survival increased from under 2 weeks in control animals to a minimum of 6 months in treated mice and there was complete response in two of three tumors. Treatment-induced cell death was demonstrated histologically by marked increases in CC3 and TUNEL staining at 24 hours, and functionally by a decrease in mStrawberry fluorescence at 24 and 48 hours in MDA-MB-231 and Colo205 tumors, respectively. The delayed decrease in Colo205 tumors was attributed to delayed clearance of the mStrawberry protein, possibly indicating a higher proportion of macrophage uptake and apoptosis as compared with necrotic cell death (40). Cell death resulted in a decrease in ATP concentration in both tumor types and consequent reduction in bioluminescence. In both tumor types, label flux from hyperpolarized [1-13C]pyruvate to lactate was decreased, and this preceded changes in tumor volume.

Exchange of hyperpolarized 13C label between pyruvate and lactate depends on tumor perfusion, MCT and LDH expression, and lactate pool size. Because DCE-MRI measurements showed a small increase in tumor perfusion and there was no change in LDH expression, the decrease in exchange must have been due to the large decrease in lactate pool size, which through the accompanying decrease in NADH concentration will decrease LDH activity, and by decreased MCT1 and MCT4 transporter expression, which will decrease pyruvate transport (41, 42). We have shown previously, albeit in another tumor cell type, that LDH and the MCTs have comparable flux control coefficients for the exchange and that the exchange is linearly dependent on lactate concentration (41, 42). Given the large decrease in lactate concentration and more modest decrease in MCT expression, it seems likely that the decreased exchange is largely the result of the decrease in lactate concentration, resulting from a decrease in glycolytic flux, and consequent decrease in LDH activity. Furthermore, we observed significant PARP cleavage, which has previously been correlated with depletion of the NAD(H) pool and consequent decrease of LDH activity (33, 34).

The lack of a significant decrease in [18F]FDG uptake in treated tumors was surprising given the degree of cell death observed histologically. [18F]FDG accumulation in tissues is multifactorial, with no consistent relationship between [18F]FDG uptake and GLUT and hexokinase expression (43, 44). In dynamic PET measurements [18F]FDG uptake remained irreversible 3 hours after injection demonstrating that glucose-6-phosphatase activity, previously implicated in the failure of response detection with [18F]FDG-PET, was minimal in these tumors (45). Although we observed significant decreases in GLUT1 and GLUT3 expression, HK2 expression did not change. Therefore, in these tumors, HK2 activity appears to dominate [18F]FDG trapping, which would explain the persistence of [18F]FDG uptake following treatment. Although the levels of ATP were decreased in the tumor, these were evidently sufficient to maintain [18F]FDG phosphorylation. In Colo205 tumors, the trend toward a decrease in net [18F]FDG influx rate (ki) after treatment indicates that a kinetic analysis may detect changes that are not apparent from the SUV. However, the hyperpolarized [1-13C]pyruvate/[1-13C]lactate ratio, in which pyruvate delivery is accounted for, may be a more practical clinical approach than kinetic analysis of [18F]FDG uptake, which requires prolonged dynamic image acquisition and arterial blood sampling.

13C NMR measurements of [1,6-13C2]glucose metabolism and radioHPLC measurements of [18F]FDG metabolism showed that treatment of Colo205 tumors reduced glycolytic flux to lactate and flux into the TCA cycle but that phosphorylation of [18F]FDG was maintained. This suggests that, contrary to the generally accepted view, [18F]FDG-6-P accumulation in tumors is not necessarily reflective of glycolytic flux and that the differential response seen with [18F]FDG-PET and [1-13C]pyruvate is not due to a switch to oxidative metabolism after treatment. That hyperpolarized [1-13C]pyruvate can detect changes in glycolytic flux, but not [18F]FDG, is a reflection of the fact that the rate of exchange between pyruvate and lactate is partly dependent on the lactate concentration, which is determined primarily by glycolytic flux, whereas the accumulation of [18F]FDG is only dependent on transport and phosphorylation (41).

The failure of [18F]FDG to detect treatment response has frequently been attributed to activation of phagocytic cells and to a lesser extent other immune cells, resulting in a paradoxical increase in tumor [18F]FDG uptake (12, 14, 46, 47). Here, [18F]FDG uptake per cell was quantified by sorting labeled cell populations using FACS. We defined a phagocytic population as CD45+/CD11b+, presumed to be mostly macrophages (48). The majority of this population was also positive for mStrawberry+, both before and after treatment, indicating tumor cell phagocytosis. Although macrophages were the most glycolytically active cell type, their low numbers meant that the majority of [18F]FDG] in the tumor accumulated in tumor cells. Using microautoradiography, Kubota and colleagues reported that high [18F]FDG uptake corresponded to areas infiltrated with macrophages, which is frequently cited as the basis of the metabolic flare effect (49). Although our findings corroborate this previous work, we show that although macrophages are the most [18F]FDG-avid cell type, they represent such a small component of the tumors that their overall contribution to [18F]FDG uptake is relatively insignificant. This excludes inflammatory cell infiltration as a cause of the failure of [18F]FDG to detect treatment response in this study.

The general implications of our findings are limited by the use of subcutaneous human xenografts in immunocompromised mice, which have greatly reduced numbers of T cells. However, the proportion of macrophages in Colo205 tumors was similar to the proportion reported in most early human colorectal tumors (<5%; ref. 50). In tumors with a greater degree of immune cell infiltration, [18F]FDG uptake may have a more significant impact on [18F]FDG-PET results. A further limitation of the experiments performed to elicit mechanisms responsible for changes in [18F]FDG and [1-13C]pyruvate metabolism was the small cohort sizes in some experiments (n = 3 in each group for several of the experiments). Nevertheless, the majority of results were statistically significant.

We have shown that hyperpolarized [1-13C]pyruvate can be used to detect treatment-induced tumor cell death, with decreases in lactate labeling preceding a reduction in tumor volume, whereas there was no significant change in [18F]FDG or glucose uptake, despite a large decrease in glycolytic flux. Tumor [18F]FDG-6-P accumulation was not significantly affected by inflammatory cell infiltration.

No potential conflicts of interest were disclosed.

Conception and design: R.L. Hesketh, D.Y. Lewis, S. Ros, K.M. Brindle

Development of methodology: R.L. Hesketh, A.J. Wright, D.Y. Lewis, A.E. Denton, R. Grenfell, J.L. Miller, R. Bielik

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.L. Hesketh, J. Wang, A.J. Wright, A.E. Denton, R. Grenfell, J.L. Miller, R. Bielik, M. Fala, B. Xie, D.-e. Hu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.L. Hesketh, J. Wang, A.J. Wright, D.Y. Lewis, A.E. Denton, R. Bielik, M. Gehrung

Writing, review, and/or revision of the manuscript: R.L. Hesketh, J. Wang, A.J. Wright, D.Y. Lewis, J.L. Miller, R. Bielik, S. Ros, K.M. Brindle

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.L. Hesketh, J. Wang, S. Ros, B. Xie

Study supervision: D.Y. Lewis, K.M. Brindle

We would like to thank Matt Clayton, Mike Mitchell, and Ryan Asby for their help with animal experiments and Jane Gray and Ian Hall for their expertise in Western blotting and ex vivo fluorescence imaging. We would also like to thank the members of the PET-CT department at Addenbrookes' Hospital for their generous provision of [18F]FDG.

The work was supported by a Cancer Research UK Programme grant (17242) and by the CRUK-EPSRC Imaging Centre in Cambridge and Manchester (16465) awarded to K.M. Brindle.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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