Brown-fat-mediated tumour suppression by cold-altered global metabolism – Nature

Cell culture

The mouse MC-38 colon adenocarcinoma cell line was provided by R. Hernández. The mouse E0771 breast cancer cell line was purchased from CH3 BioSystems. The mouse pancreatic cancer cell line Panc02 was provided by M. Schnurr. The human colorectal carcinoma HCT 116 tumour cells, human pancreas duct epithelioid carcinoma PANC-1, mouse fibrosarcoma T241 and mouse melanoma B16-F10 were purchased from ATCC. T241, HCT 116, Panc02, PANC-1 and B16-F10 cells were cultured and maintained in a Dulbecco modified Eagle’s medium (DMEM, D6429, Merck) supplemented with 10% FBS, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (P4333, Merck). MC-38 cells were cultured and maintained in DMEM supplemented with 10% FBS, 10 mM HEPES, 0.1 mM MEM non-essential amino acid (M7145, Merck), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. E0771 cells were cultured and maintained in Roswell Park Memorial Institute (RPMI) 1640 (SH30027.01, GE Healthcare Life Science) medium supplemented with 10% FBS, 10 mM HEPES, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. All of the cell lines used in our study were negative for mycoplasma as detected by a microplasma kit (LT07-318, Lonza).

Animals

C57Bl/6 and SCID mice were obtained from the breeding unit at the Department of Microbiology, Tumour and Cell Biology, Karolinska Institute, Stockholm, Sweden and from the Model Animal Research Center of Nanjing University. Ucp1−/− mice in the C57BL/6 background were purchased from the Jackson Laboratory (003124). Female 6–8-week-old FVB/NJGpt-Tg(MMTV-PyMT)/Gpt mice were purchased from GemPharmatech (T004993). Male 9–10-week-old C57Bl/6J background ApcMin/+ mice (002020) were obtained from the Jackson Laboratory. Adult males and female mice aged 5–12 weeks were caged at 22 ± 3 °C under 12 h–12 h light–dark cycles before cold and warm exposure. Mice were randomly divided into groups for all of the experiments. All of the mouse studies were approved by the North Stockholm Animal Ethical Committee, Stockholm, Sweden (N3/17 and 6196-2019 with a maximal tumour volume of 2.5 cm3 and 2.0 cm3 for subcutaneous tumours and intraorgan tumours, respectively), or by the Animal Experimental Ethical Committee of the Fudan University, Shanghai, China (20190430 with a maximal tumour volume of 3.0 cm3). No statistical methods were used to predetermine sample size. The experimenter was not blinded to the assignment of the groups and the evaluation of the results. Tumour experiments, including subcutaneous, intraorgan implantation and genetic tumour models were performed at the Karolinska Institutet, Sweden and Fudan University, China. In both locations, subcutaneous tumour volumes did not exceed 2.5 cm3 and intraorgan tumour volumes did not exceed 2.0 cm3 in any of the experiments.

Cold exposure

For cold exposure, mice were housed and placed in an opened cage in the climate room at the Department of Microbiology, Tumour and Cell Biology, Karolinska Institute, Stockholm, Sweden, at the Department of Cellular and Genetic Medicine, School of Basic Medical Sciences, Fudan University, or in a climate chamber (HPP750, Memmert or HWS-350FT, Binglin Electronics). Male and female C57Bl/6 mice aged between 5 and 10 weeks were adapted at 18 °C for 1 week before exposing to a cold temperature of 4 °C in the climate room or 5 °C in the HPP750 climate chamber or exposed to a thermoneutral temperature of 30 °C as a control, followed by tumour implantation.

Mouse tumour models

Approximately 1 × 106 cells of T241 fibrosarcoma, E0771 breast cancer and B16 melanoma, 1–3 × 106 MC-38 tumour cells, 1.5 × 106 Panc02 cells and 5 × 106 PANC-1 cells in 100 μl phosphate-buffered saline (PBS) were subcutaneously implanted into each of C57BL/6 mouse, or immune-deficient SCID mice. For the intraliver injection model, in isoflurane-anaesthetized 7-week-old female C57BL/6 and 10-week-old male SCID mice, a subcutaneous incision was performed and the mice were injected with approximately 0.5 × 106 MC-38 or 1 × 106 HCT 116 cells in 30 μl PBS into the liver, followed by closure of the incisions with the sterile surgical suture (CR436, Jinhuan Medical). All surgically operated mice were subcutaneously treated with buprenorphine before and after surgical operation. Tumour sizes were measured with callipers and tumour volumes were calculated according to the standard formula (length × width2 × 0.52). For the subcutaneous tumour model, tumour-bearing mice were euthanized when the tumour size reached the indicated size and tissues including tumours, WAT depots, iBAT and liver were dissected for further assessments. Fresh tumour, WAT and BAT were immediately collected and kept on dry ice or liquid nitrogen for metabolomics and RNA-seq multi-omics analyses. Mice in the intraliver tumour models were euthanized 2–3 weeks or 6 weeks after tumour implantation. For RNA and protein extraction, fresh tissues were immediately frozen on dry ice and stored at –80 °C until further use. A portion of fresh tissues was immediately dissected, fixed with 4% paraformaldehyde (PFA) (MA0192, Meilunbio) overnight and subsequently used for histological and immunohistochemical analyses. Metabolic rates were measured when tumours reached an average size of approximately 1.0 cm3. Mice were analysed for metabolic rates, followed by euthanasia and tissue collection. Vehicle-treated mice using the same therapeutic regimens served as a control group.

Female 10-week-old MMTV-PyMT mice were adapted at 18 °C for 24 h before exposing to 4 °C or 30 °C conditions. MMTV-PyMT mice started to develop tumours in the mammary glands at the age of 12 weeks. For BAT-removal experiments, BAT of each animal was removed at the age of 9 weeks, cold exposure started at the age of 10 weeks and continued until the termination of the experiments. At week 4 after cold exposure, PET–CT analysis was performed and tumour nodules of the same size from the 4 °C- and 30 °C-exposed groups were analysed for 18F-FDG uptake. C57Bl/6J background ApcMin/+ male mice (aged 9 weeks) were acclimatized to 4 °C and 30 °C for 6 weeks. ApcMin/+ mice usually developed multiple intestinal adenomas between weeks 10 and 14. On week 6 after cold exposure, PET–CT analysis was performed and tumour nodules of the same size from the 4 °C- and 30 °C-exposed groups were analysed for 18F-FDG uptake.

Body and intratumour temperature

To measure mouse CBT, a temperature probe (TW2 and RET-3, Agntho’s AB) was placed in the rectum of each mouse. For measuring subcutaneous body temperature, a small incision on the interscapular region of each mouse under mild isoflurane anaesthesia was surgically created and the probe was subsequently inserted into the incision for measurements. Accurate temperature was read using the MicroTherma 2T thermometer. For measuring tumour temperature, the probe was put into the tumour of the mice. All measurements were performed at room temperature.

Metabolic analyses

Whole-body energy metabolism and non-shivering thermogenesis were quantitatively measured by oxygen consumption using the Oxymax CLAMS-HC comprehensive laboratory animal monitoring system with environmental enclosure (Columbus Instruments). The metabolic measurements were carried out at thermoneutral and cold temperatures. The oxygen sensor was warmed up for at least 6 h before calibration with the reference gases, that is, 100% nitrogen gas and a mixture of 20.5% O2 and 0.5% CO2. The environmental enclosure was warmed and cooled before placing the mice inside. For the whole-body energy metabolism, CO2 and O2 were measured and the data were collected every 12 min for each experimental mouse.

Glucose- and insulin-tolerance tests

Mice were starved for 4–6 h during the light phase with free access to water on the day of assessment. Glucose levels in the blood samples collected from the tail vein by a 25G needle were measured using a glucometer (Accu-Chek, Active, Roche Diagnostics) immediately before and at 15, 30, 60 and 120 min after oral feeding with 1.5 mg glucose with 10 μl per g body weight and intraperitoneal injection of insulin (0.5 U per kg body weight), respectively. Mice were kept at ambient temperature throughout these assays.

PET–CT imaging

Mice that were exposed to 4 °C or 30 °C were fasted for 6 h before PET–CT scanning. 18F-fluoro-2-deoxy-d-glucose (18F-FDG) with a radiochemical purity of more than 95% was produced by a cyclotron (Siemens CTI RDS Eclips ST) using the Explora FDG4 module. In vivo PET–CT imaging scans and image analysis were performed using an Inveon Animal-PET-CT system (Siemens Preclinical Solution). Mice exposed to 4 °C or 30 °C were scanned 1 h after intravenous injection of 3.7 MBq (100 μCi) of 18F-FDG. Experimental animals were maintained with anaesthetization using 2.5% isoflurane/oxygen before and during the scanning. Three-dimensional ordered-subset expectation maximization (3D-OSEM)/maximum algorithm was used for image reconstruction. The maximal percentage-injected dose per gram was calculated and the SUV-BW of iBAT and tumour in either the subcutaneous region or liver was measured in a manually drawn region of interest. Inveon Acquisition Workplace software (Siemens Medical Solutions) was used for further analysis.

For human studies, CT scans were performed using the following protocols: 120 kv tube voltage, 120 mA tube current and 3.75 mm thickness of layers. For PET collection, 6–7-bed cycles were used for the whole-body scanning and each bed cycle was 2 min. Data were reconstructed using the viewpoint method.

Surgical removal of iBAT

For removal of iBAT, a small incision was surgically created in each mouse under isoflurane-inhalation anaesthetization. Blood vessels in the iBAT were crushed by a pinch using sterilized forceps to prevent excess bleeding after cutting out the tissues. iBAT tissues were carefully dissected using autoclaved surgical scissors, followed by the closure of the incision with the sterile surgical suture. Sham-operated mice were used as controls. All of the mice were allowed to recover for a week at ambient temperature before further analyses.

Survival assay

Survival studies of tumour-bearing mice were performed at Fudan University, Shanghai, China according to the ethical permit in which the humane end point (tumour size ≤ 3 cm3) was the criterium to euthanize each mouse. This end point and body condition score BCS-1 (moribund state) were not exceeded in any of the experiments.

RNA-seq analysis

For RNA-seq, isolated total RNA was collected from isolated tumour tissues and was measured using the NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific). The quality of RNA samples was assessed by agarose gel electrophoresis and using the Agilent 2100 Bioanalyzer (Agilent Technologies). All of the samples displayed a 260/280 ratio of around 2.0. cDNA libraries were constructed and sequenced by Majorbio Biotech. In brief, 200 ng RNA for each group was used for the library construction using the Truseq RNA sample prep Kit (Illumina). The constructed DNA was enriched by PCR amplification and then purified by Certified Low Range Ultra Agarose (Bio-Rad) gel electrophoresis. Clone clusters were generated on the Illumina cBot, using the Truseq PE Cluster Kit v3-cBot-HS, and high-throughput sequencing was performed on an Illumina Miseq sequencer, using Truseq SBS Kit v3-HS (200 cycles). Aligned reads were summarized using featureCounts (v.2.0.0) based on the Gencode vM19 annotation. Differential expression analysis between three 30 °C and two 4 °C samples was performed using the R (v.4.0.3) package DESeq2 (v.1.30.0). GSEA was performed with GSEA (v.4.1.0) using the GSEAPreranked tool, whereby genes were preranked on the basis of their P values and fold changes. Mouse gene sets for Gene Ontology analysis were downloaded from http://ge-lab.org/gskb/.

Metabolomics analysis

Metabolomics analysis was performed in house or with the assistance from Shanghai Biotree Biotech. Metabolites in MC-38 tumours and adipose tissues were measured by capillary electrophoresis time of flight mass spectrometry (CE–TOFMS) using the Agilent 7100 CE Capillary Electrophoresis system equipped with an Agilent 6230 Time-of-Flight mass spectrometer (Agilent Technologies) according to previously reported methods. In brief, snap-frozen tumour and adipose tissue samples (approximately 100 mg for tumour and 30 mg for adipose tissues) were homogenized with 500 μl of methanol containing internal standards (H3304-1002, Human Metabolome Technology) and 500 μl of chloroform and 200 μl of ultrapure water (50% v/v) were subsequently added into the sample solution. The solution was centrifuged at 2,300g for 5 min at 4 °C to remove debris. Subsequently, 300 μl of the aqueous fraction was centrifugally filtered through a 5 kDa cut-off filter using an Ultrafree MC-PLHCC filter (UFC3LCCNB_HMT, Human Metabolome Technology). The filtrate was dried by a dryer for 180 min and dissolved in 25 μl of both Milli-Q water-containing reference compounds (H3304-1004, Human Metabolome Technology). The amount of all anion- and cation-charged metabolites in the samples was measured by CE−TOFMS. For analysis of cationic metabolites, a fused silica capillary (H3305-1002, 50 μm internal diameter × 80 cm total length, Human Metabolome Technologies) with a cation buffer solution (H3301-1001, Human Metabolome Technologies) as the electrolyte was used. The sample was injected at a pressure of 50 mbar for 10s. To determine anionic metabolites, a chemically coated cationic polymer capillary (50 µm internal diameter × 80 cm total length, H3305-1002, Human Metabolome Technologies) was used with an anion buffer solution (H3302-1021, Human Metabolome Technology) as the electrolyte. The sample was injected at a pressure of 50 mbar for 25 s. For measuring cation- and anion-charged metabolites, the CE capillary was preconditioned by flushing the running electrolyte for 3 or 4 min before sample injection. Each sample was inserted with a pressure injection of 50 mbar for 10 s under 20 °C of the capillary temperature and 27 kV and 30 kV applied voltage for measurement of cation- and anion-charged metabolites, respectively. Sheath liquid (H3301-1020, Human Metabolome Technology) was delivered at 10 µl min−1 to the CE interface by a pump using the Agilent 1200 series pump equipped with a sheath flow splitter. Electrospray ionization–TOFMS was conducted in positive-ion mode with 4 kV capillary voltages for cationic metabolites and was set in the negative-ion mode with 3.5 kV for anionic metabolites.

The spectrometer was scanned from m/z 50 to 1,000 at 1.5 cycles per second during separation and detection. An automatic recalibration of each acquired spectrum was performed using the masses of reference standards. For the CE–TOFMS system control and data acquisition, we used an Agilent MassHunter software for TOFMS (Agilent Technologies). All target metabolites were identified by matching their m/z values and migration times with the normalized m/z values and migration times of corresponding authentic standard compounds. Data were quantified by comparing the ratio of analyte peak area/internal standard peak area of sample solution to that of the reference standard mixture. The quantitative formula was as follows: X nmol mg−1 = (sample relative area)/(standard relative area) × (concentration of internal standard in methanol: 50 µM) × (amount of methanol for metabolite isolation (500 µl)) × 1/(sample weight) × (concentration of metabolite in the reference standard mixture (50 µM))/(concentration of internal standard in the reference standard mixture (100 µM)). The sample relative area = (metabolite peak area in the sample solution)/(internal standard area in the sample solution). The standard relative area = (metabolite peak area in the reference standard mixture)/(internal standard area in the reference standard mixture). Chromatograms of each metabolite were extracted and quantified using the Agilent MassHunter Qualitative Analysis software (Agilent Technologies). For lipidomics analysis, 50 μl mouse serum sample was diluted into extraction buffer (isopropyl alcohol (CAEQ-4-013493-4000, CNW Technologies): n-hexane (CAEQ-4-011518-4000, CNW Technologies) = 2:3 (v:v) with internal standards (CDAA252795, ANPEL). Samples were sonicated in an ice-water bath for 5 min, then centrifuged at 4 °C for 15 min at 12,000 rpm. Then, 400 μl of supernatant was dried in a speed vac, stored under nitrogen and reconstituted in 160 μl of n-hexane. After centrifugation at 12,000 rpm for 5 min, the supernatant was projected for gas chromatography–mass spectrometry (5977B, Agilent) for further analysis.

Drug treatment

CL-316,243 disodium salt (1499, Tocris Biosciences) was administered systemically by daily intraperitoneal injection at a dose of 1 mg kg−1 in PBS. After 21 days of treatment, the mice were euthanized for further analysis.

Infrared thermal imaging

Mice in thermoneutral and cold environments were anaesthetized and kept at room temperature for 5 min for imaging. Thermal images on the back of mice were captured using an infrared thermal imager (Fortric 285, Fortric). Setting environmental temperature as the baseline, thermal images of mice were acquired and were further analysed using Fotric AnalyzIR.

Histology and immunohistochemistry

The paraffin-embedded tissue sections of 5 μm thickness were incubated at 60 °C for approximately 2 h to melt the covered paraffin. After the incubation, the warm slides were put in Tissue-Clear (1466, Sakura) for deparaffinization and dehydrated in serial steps using 99%, 95% and 70% ethanol. For haematoxylin and eosin (H&E) staining, the dehydrated slides were stained with H&E. For immunohistochemical staining, tissue slides were boiled by microwave for 20 min in an unmasking solution (H3300, Vector Laboratories) and subsequently blocked with 4% either goat or donkey serum. To visualize cells in the adipose tissues and TME, we stained the WAT, BAT and tumours with antibodies listed below. For the TME, the slices of tumour tissue were stained with rabbit anti-mouse cleaved-caspase-3 (1:200, 9661, Cell Signaling), rabbit anti-mouse Ki-67 (1:100, PA5-19462, Thermo Fisher Scientific), rabbit anti-mouse FSP1 (1:300, 07–2274, Merck), rabbit anti-mouse IBA1 (1:200, 019–19741, FUJIFILM Wako) or rabbit anti-mouse CD45 (1:200, ab10558, Abcam) antibodies, followed by staining with species-matched secondary antibodies as follows: Alexa Fluor 555-labelled goat anti-rabbit (1:300, A21482, Thermo Fisher Scientific) or Alexa Fluor 488-labelled donkey anti-rabbit (1:300, A21206, Thermo Fisher Scientific) antibodies. For detection of hypoxia in the tumour, the slides were stained with a rabbit anti-CA9 antibodies (1:300, NB100–417, Novus) for primary staining and Alexa Fluor 488-labelled donkey anti-rabbit or Alexa Fluor 555-labelled goat anti-rabbit antibodies for the secondary staining. For adipose tissues, the slide with the samples was stained with rabbit anti-mouse UCP1 (1:200, ab 10983, Abcam), rabbit anti-mouse COX4 (1:300, GTX114330, GeneTex), and guinea pig anti-mouse perilipin (1:300, 20R-PP004, Fitzgerald Industries) antibodies, followed by dyeing with Alexa Fluor 555 goat anti-rabbit and Alexa Fluor 647 goat anti-guinea pig (1:200, A-21450, Thermo Fisher Scientific) secondary antibodies. Positive signals were detected using a fluorescence microscope equipped with a camera (Nikon, DS-QilMC) using NIS-Elements D3.2 (Nikon). For H&E staining, the image was captured using a light microscope (Nikon Eclipse TS100) with the camera (DS-Fil, Nikon) using NIS-Elements F v.3.0 software (Nikon). Images were analysed using Adobe Photoshop CS5 extended software and ImageJ.

Whole-mount staining

Paraformaldehyde-fixed tumours, sWAT and iBAT tissue samples were digested with 20 μg ml−1 proteinase K (EO0491, Thermo Fisher Scientific) in a 10 mM Tris-HCl buffer (pH 7.4) for 5 min and blocked with 3% skim milk, followed by staining overnight at 4 °C with goat anti-mouse CD31 antibodies (1:200, AF3628, R&D systems). After rigorous rinsing with PBS, blood vessels were detected with donkey anti-goat Alexa Fluor 555-labelled secondary antibodies (1:300, A21432, Thermo Fisher Scientific), mounted in Vectashield mounting medium (Vector Laboratories), and stored at −20 °C in the dark before microscopy examination using the Nikon C1 confocal microscope (Nikon). The images were recorded using the camera of a Nikon C1 confocal microscope using the EZ-C1 v.3.91 software (Nikon). Captured images were further analysed using Adobe Photoshop CS5 extended software.

FACS analysis

Approximately 3 × 106 MC-38 GFP tumour cells in 100 μl PBS were subcutaneously implanted into each C57BL/6 mouse. Tumour tissues were collected around 0.5 cm3 in size, and the fresh tumour tissues were cut into small pieces, followed by collagenase digestion. In brief, the minced tumours were enzymatically digested with 0.15% collagenase I (17100-017, Gibco) and 0.15% collagenase II (17101015, Gibco) in PBS for 40 min at 37 °C, and centrifuged at 1,500 rpm at 4 °C for 10 min. The pellets were resuspended with PBS containing 1% FBS. Single-cell pellets were obtained by filtration using 70 μm cell strainers followed by centrifugation at 1,300 rpm at 4 °C for 10 min. The pellets were fixed using 1% PFA in PBS for 15 min at room temperature and washed in PBS. The cell pellets were further resuspended in PBS, and cold 70% ethanol was added dropwise to the pellets while vortexing. The samples were stored at 4 °C for further analysis. To the DNA content, fixed single-cell suspensions in PBS were incubated with 20 mg ml−1 propidium iodide (PI) (P3566, Invitrogen) and 100 mg ml−1 RNase (EN0531, Thermo Scientific) final on ice for 30 min. PI signals in GFP-positive cells were immediately analysed on the BD FACSCalibur Flow Cytometer (BD Bioscience), and data analysis was performed using the CellQuest Pro software (v.6.0, BD Bioscience). Gating strategies are shown in Supplementary Fig. 1. GFP-positive MC-38 cells and PI unstained samples were used to set each gate.

RNA isolation and PCR analysis

Total RNA was extracted from cells and tissues using the TRIzol (15596026, Invitrogen) and GeneJET RNA Purification Kits (K0732, Thermo Fisher Scientific) according to the manufacturer’s instructions. The total RNA was reverse-transcribed and cDNAs were used for PCR and qPCR analyses using the primers listed in Supplementary Table 1. Samples were stored at −20 °C and processed for qPCR using the ABI Prism 7500 System (Applied Biosystems). Each qPCR sample was performed in triplicate and 20 µl reactions contained the Power SYBR Green PCR Master Mix (4367659, Thermo Fisher Scientific) or Hieff qPCR SYBR Green Master Mix (11203ES03, Yeasen), 150 nM forward and reverse primers and 1 µl cDNA. The qPCR protocol was performed for 40 cycles and each cycle consisted of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min.

LDH activity

Freshly isolated mouse CRC tumours were washed and immersed with the assay buffer, and then homogenized using a Dounce homogenizer. Tissue lysates were applied to the LDH activity assay using the LDH assay kit (ab102526, Abcam) according to the manufacturer’s instructions.

Immunoblotting

Tissue lysates from tumours in different mouse models were extracted using a lysis buffer (C3228, Sigma-Aldrich) with a mixture of proteinase inhibitors (1:100, 8340, Sigma-Aldrich) and a phosphatase inhibitor cocktail (1:100, 5870, Cell Signaling) by a homogenizer (15555819, Thermo Scientific). Protein concentrations in tissue lysates were measured using the BCA assay. An equal amount of protein from each experimental group and a protein ladder (26616/26620, Thermo Scientific) was placed onto an SDS–PAGE gel (4561086/4561083, Bio-Rad) or a 10% SDS–PAGE gel (PG112, EpiZyme). The separated protein on the gel was transferred onto a polyvinylidene difluoride membrane (IPFL00010, Millipore), followed by blocking using 5% bovine serum albumin (BSA, 11413164, Fisher Scientific) in Tris borate EDTA (TBE) and staining with primary antibodies at 4 °C overnight. The membranes were subsequently stained with specific species-matched secondary antibodies conjugated with IRDye dissolved in 3% BSA in TBE for 1 h at room temperature. For visualization of positive signals, the Odyssey CLx system (LI-COR) was used. The positive signals were quantified using an Image Studio v.3.1 (LI-COR). β-Actin was used as the loading control for all blots. Primary and secondary antibodies were as follows: PI3K p85 (1:1,000, 4257, Cell Signaling Technology), phosphorylated PI3K p85 (1:1,000, 4228, Cell Signaling Technology), AKT (1:1,000, 9272, Cell Signaling Technology), phosphorylated AKT (1:1,000, 4051, Cell Signaling Technology), mTOR (1:1,000, 2972, Cell Signaling Technology), phosphorylated mTOR (1:1,000, 2971, Cell Signaling Technology), β-actin (1:1,000, 3700, Cell Signaling Technology), rabbit anti-mouse beta-actin polyclonal (1:1,000, 20536-1-AP, Proteintech), donkey anti-rabbit IRDye 680RD (1:15,000, 926–68073, LI-COR Biosciences) and donkey anti-mouse IRDye 800CW (1:15,000, 926–32212, LI-COR Biosciences) antibodies. For some of the samples, primary antibodies including rabbit anti-PI3K p85 alpha (1:1,000, ab191606, Abcam), rabbit anti-phosphorylated PI3K p85 alpha (1:1,000, ab182651, Abcam), rabbit anti-AKT (1:1,000, ab8805, Abcam), rabbit anti-phosphorylated AKT (1:1,000, ab38449, Abcam), rabbit anti-GLUT1 (1:1,000, ab115730, Abcam) and mouse anti-GAPDH (1:1,000, A01020, Abbkine) antibodies were used. Secondary antibodies including goat anti-mouse HRP-conjugated IgG (1:5,000, AS003, ABclonal) and goat anti-rabbit HRP-conjugated IgG (1:5,000, AS014, ABclonal) antibodies were used. Target proteins were visualized using the EZ ECL pico luminescence reagent (AP34L025, Life-iLab) with the Molecular Imager ChemiDoc XRS System (Bio-Rad). Full blot gels are provided in Supplementary Fig. 2.

Human study

All of the human studies were approved by the Ethical Review Committee in the Qilu Hospital, Shandong University, Shandong Province, China (KYLL-202011-011-02), and informed consent for human research and the publication of the PET–CT images was obtained from all of the participants. Healthy volunteers including 3 men (body mass index, 23.0 ± 0.4 kg m−2) and 3 women (body mass index, 23.1 ± 0.5 kg m−2) aged between 22 and 25 years were recruited for the cold-exposure studies. Before cold exposure, all of the volunteers were fasted overnight and kept at 28 °C for 1 h. After 1 h, 18F-FDG at a dose of 0.1 mCi kg−1 was intravenously injected into each of the volunteers, followed by a PET scan (GE Discovery STE16, GE Healthcare). The healthy male and female individuals with T-shirts and shorts were exposed to a mildly cold ambient temperature of 16 °C for 2–6 h per day for consecutive 14 days. On day 13, all of the volunteers were fasted overnight and continued cold exposure for 2 h the next morning. PET scans were performed while the individuals intermittently soaked their feet in ice water. PET scan images were collected.

For patients with cancer, an 18-year-old female patient with Hodgkin’s lymphoma participated in a pilot study during the intervals of the fifth cycle of combination chemotherapy of adriamycin, bleomycin, vinblastine and dacarbazine for 5 cycles. The cold ambient temperature was approximately 22 °C regulated by air-conditioning for 1 week and the patient wore light clothing. The patient fasted overnight before PET–CT examination. The warm temperature exposure was about 28–30 °C, regulated by air-conditioning for 4 days. On day 4 after warm exposure, the patient was examined using PET–CT after overnight fasting. An experienced nuclear-medicine physician examined and analysed the PET and PET–CT images, and BAT activation and tumour 18F-FDG absorption were quantified.

Statistical analysis

Sample numbers, the number of biological replicates and statistical analysis methods are provided in the figure legends. Statistical analysis was performed using Microsoft 365 Excel, GraphPad Prism v.9.2.0, and R (v.4.0.3). Data are presented as the means of determinants ± s.e.m. For RNA-seq, P values were calculated using the Wald test. P < 0.05 was deemed to be statistically significant.

Reporting summary

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

Leave a Comment

Your email address will not be published.