Cell culture
Cells were obtained from the American Type Culture Collection and maintained in a humidified incubator at 5% CO2 and 37 °C. For hypoxic exposure, cells were grown in an INVIVO2 400 hypoxic workstation (Baker Ruskinn) using a continuous flow of a humidified mixture of 0.1% O2, 5% CO2, and 94.9% N2. Cells were maintained in Dulbecco’s modified Eagle’s medium (10 mM glucose) (Gibco) supplemented with 10% fetal bovine serum (FBS).
Gene silencing by RNA interference
The lentiviral transduction particles containing a FABP7 shRNA expression cassette (Mission® shRNA, TRCN0000059744) or a non-targeting shRNA sequence (SHC002U) were purchased from Sigma-Aldrich. Cells were transduced with a MOI of 3, in the presence of 6 μg/ml polybrene (Sigma Aldrich) for 24 h. Cells expressing the shRNA were selected in puromycin (Invitrogen)-containing medium (2 μg/ml). After the selection, cells were suspended in FBS containing 5% DMSO (v/v) and stored at − 80 °C. In a set of experiments, cells were refreshed in every 1 month.
Lipid peroxidation assay
Cellular lipid peroxidation levels were measured using Image-iT® Lipid Peroxidation Kit (Thermo Fisher) according to the manufacturer’s instructions. Cells were exposed to hypoxia for 24 h or 4 Gy of ionizing radiation. The fluorescence was measured with an Attune NxT Flow Cytometer (Thermo Fisher) using two different filter sets: the one at excitation/emission of 488/530 nm for detecting oxidized lipids and the other at excitation/emission of 561/620 nm for detecting reduced lipids. Lipid peroxidation levels were calculated as a ratio of the intensity of green (530 nm) fluorescence to that of red (620 nm) fluorescence.
Cell-cycle analysis
To evaluate cell-cycle distribution, cells were washed with ice-cold PBS, resuspended in 1 ml of PBS, and stored after the dropwise addition of 3 ml of ice-cold 70% ethanol at 4 °C until analysis. Cells were washed twice with ice-cold PBS and stained with a PI solution (100 mg/ml) (Sigma Aldrich) containing DNase-free RNase (12 mg/ml) and 1% of Triton X100. After overnight incubation at 4 °C, cells were analyzed with Attune NxT Flow Cytometer using a filter set at excitation/emission of 488/590 nm.
Quantitative PCR
RNA was isolated using TRIZOL® Reagent (Invitrogen), and complementary DNA was generated from the RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturers’ instructions. Real-time PCR was performed on a 7900HT Fast Real Time PCR System (Applied Biosystems) using the SensiMixTM SYBR No-Rox kit (Bioline). The comparative threshold cycle method was used to present the relative gene expressions. Expression data were normalized to the expression of the two control genes: ACTB and HPRT1. Primer sequences were as follows: ACTB forward: ATTGGCAATGAGCGGTTC; ACTB reverse: GGATGCCACAGGACTCCAT; HPRT1 forward: CCAGTCAACAGGGGACATAAA; HPRT1 reverse: CACAATCAAGACATTCTTTCCAGT; FABP7 forward: TGAAACCACTGCAGATGATAGAA; FABP7 reverse: TTTCTTTGCCATCCCATTTC; PRDM16 forward: ATGGGAGCAAATACTGACGG; PRDM16 reverse: CACGCAGAACTTCTCACTGC; PGC-1α forward: GCCAAACCAACAACTTTATCTCTTC; PGC-1α reverse: CACACTTAAGGTGCGTTCAATAGTC; UCP1 forward: TCTACGACACGGTCCAGG; UCP1 reverse: GTCTGACTTTCACGACCTCTG.
Western blots
Cell were lysed in RIPA buffer supplemented with cOmplete® Protease Inhibitor Cocktail (Roche) and PhosSTOP® Phosphatase Inhibitor Cocktail (Roche). The lysates were centrifuged at 20,000g at 4 °C for 15 min, and the supernatants were incubated with DTT (100 mM) and NuPAGE® LDS Sample Buffer (Invitrogen) at 70 °C for 10 min. Proteins were separated on Novex® 4–12% Tris-Glycine Mini Gels (Invitrogen) and transferred to a PVDF membrane. The membrane was incubated with 5% skim milk at room temperature for 1 hr and subsequently with primary antibodies at 4 °C for overnight. For the detection of FABP7, the step of centrifuging cell lysates was omitted, and 5% BSA was used as a blocking solution. Primary antibodies were as follows and used at 1:1000 dilution unless otherwise stated: rabbit anti-FABP7 (#13347, Cell Signaling Technology), rabbit anti-UCP1 (U6382, Sigma Aldrich), rabbit anti-PGC-1α (1:200 v/v) (sc-13067, Santa Cruz Biotechnology), rabbit anti-PRDM16 (1:500 v/v) (ab106410, Abcam), rabbit anti-CREB (#9197, Cell Signaling Technology), and rabbit anti-phospho-CREB (#9198, Cell Signaling Technology). Appropriate secondary horseradish peroxidase-linked antibodies were used (Dako, UK). Immunoreactivity was detected with ECL Prime Western Blotting Detection Reagent (Amersham) and visualized using ImageQuant LAS 4000 mini (GE Healthcare).
Immunofluorescence
Cells were grown on cover slips and fixed with 4% paraformaldehyde at room temperature for 10 min. For the visualization of polarized mitochondria, cells were incubated with 150 nM of Mito Tracker® Red CMXRos (Molecular Probes) at 37 °C for 30 min prior to the fixation. Cells were permeabilized with 0.1% Triton X-100 for 5 min and then blocked in 5% normal horse serum for 30 min. They were incubated with rabbit anti-UCP1 diluted in blocking solution (1:500 v/v) (U6382, Sigma Aldrich) overnight at 4 °C, labeled with a secondary antibody conjugated with Alexa® 488 (Invitrogen) at room temperature for 30 min, and mounted with ProLong® Diamond Antifade Mountant with DAPI (Molecular Probes). For calculating UCP1-positive cell proportion, at least 6 images were acquired in each condition with a Delta Vision Elite High Resolution Microscope (GE Healthcare Life Science). For analyzing the colocalization of UCP1 and Mito Tracker, images were acquired with a Zeiss LSM 780 confocal microscope (Carl Zeiss) and reconstituted with ImageJ 1.51 g (National Institutes of Health).
Assessment of mitochondrial membrane potential
Mitochondrial membrane potential was analyzed with BDTM MitoScreen Kit (BD Bioscience) according to the manufacturer’s instructions. Cells were stained with JC-1 solution for 30 min and analyzed with an Attune NxT Flow Cytometer using two different filter sets: the one at excitation/emission of 488/530 nm for detecting polarized mitochondria and the other at excitation/emission of 561/585 nm for detecting depolarized mitochondria.
Assessment of mitochondrial respiration and cellular glycolytic function
Seahorse Cell Mito Stress Test Kit (Agilent) and Seahorse Glycolysis Stress Test Kit (Agilent) were used to assess mitochondrial respiration and cellular glycolytic function, respectively. Cells were plated in a 96-well Seahorse XF Cell Culture Microplate (40,000 cells/well) 1 day prior to the assay with normal growth media. For Mito Stress Test, the growth media was replaced to Seahorse Base Media (Agilent) supplemented with 10 mM glucose, 4 mM glutamine, and 1 mM sodium pyruvate (pH 7.4 at 37 °C), and the cells were transferred to non-CO2 incubator (37 °C) 1 hr prior to the assay. In the assay, 0.5 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin A were sequentially injected, and oxygen consumption rate (OCR) was monitored using Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience). For Glycolysis Stress Test, the growth media was replaced to Seahorse Base Media (Agilent) supplemented with 4 mM glutamine and 1 mM sodium pyruvate (pH 7.35 at 37 °C) for the preconditioning. In the assay, 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxy-glucose were sequentially injected, and extra cellular acidification rate (ECAR) was monitored. After the assays, the OCR and ECAR were normalized with the relative fluorescent intensities from CyQUANT® Cell Proliferation Assay Kit. All the parameters were generated and analyzed on a Seahorse XF report generator.
Measurement of cellular temperature
For the measurement of cellular temperature, the temperature-sensitive fluorescent nanoprobe (T probe) was used as previously described [5]. Cells were grown in μ-Slide 8 Well Glass Bottom (ibidi) and then incubated with the growth media containing 1.5 μg/ml of T probe for 16 h. After washing twice with growth media, fluorescence lifetime imaging microscopy (FLIM) was performed on Leica SP8X Inverted Confocal/Gated STED microscope (Leica microsystems) equipped with a thermal control chamber and an objective lens (HC PL APO 63 × /1.20 W with Motorized Correction Collar, Leica microsystems) under the continuous flow of 5% CO2. T probe was excited with a tunable pulsed white laser (561 nm, 40 Hz), and its emission was collected at 570–620 nm with 50 times of repetitions. Fluorescence lifetimes were calculated by monoexponential decay fitting (2.5–15 ns). Calibration curve of T probe was generated by collecting fluorescence lifetimes of stained cells at 3 different incubator temperatures: 32 °C, 37 °C, and 42 °C. After generating the calibration curve, fluorescence lifetimes in cells with/without the specific knockdown were collected at 37 °C of incubator temperature. Cells were equilibrated for at least 30 min at desired temperatures prior to measurements.
Cell proliferation assay
Cells were seeded in 96-well plate at a density of 1000 cells/well and exposed to hypoxia or ionizing radiation. Cell number was determined using CyQUANT® Cell Proliferation Assay Kit (Molecular Probes) according to the manufacturer's instructions. Fluorescence was measured with a SpectraMax microplate reader (Molecular Devices) with excitation at 485 nm and emission detection at 530 nm.
Clonogenic assay
Cells were seeded in 100-mm dish at a density of 125 cells/dish, and, 24 h later, they were exposed to hypoxia or ionizing radiation. For hypoxia experiments, cells were cultured under hypoxia for 72 h and then placed back to normoxia. Cells were allowed to grow for 14–15 days until colonies became visible and clear. Colonies were fixed with acetic acid/methanol solution (1:7 v/v) for 5 min, stained with 0.5% crystal violet solution for 2 h, and rinsed with tap water. Size and number of colonies were measured using a ColCount automated colony counter (Optronix). Plating efficacy (PE) and surviving fraction (SF) were calculated from the following equations: (PE) = (number of colonies formed/number of cells seeded) × 100 (%) and (SF) = (number of colonies formed after irradiation)/(number of cells seeded × PE) [21]. For radiation experiments, cells were irradiated 24 h after seeding with the IBL 637 Cesium-137 γ-ray machine (The dose rate was 0.0485 Gy/s).
Immunohistochemistry
Breast cancer primary tumor and the paired normal mammary glands were collected through partial or total mastectomy at the Department of Breast Surgery, Kyoto University Hospital. Written informed consent was obtained from all patients prior to sample collection. The study protocol was approved by the Ethics Committee for Clinical Research, Kyoto University Hospital (authorization number G424). The sections were incubated with citrate buffer at 120 °C for 5 min and with 3% hydrogen peroxide/methanol solution for 30 min and then blocked in PBS containing 5% normal goat serum and 1% bovine serum albumin for 10 min. They were incubated with rabbit anti-UCP1 diluted in blocking solution (1:500 v/v) (U6382, Sigma Aldrich) overnight at 4 °C. Staining was performed using ENVISION+HRP (DAKO) and DAB+ (DAKO) according to the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin solution and imaged using an optical microscope (BZ-9000, Keyence, Osaka, Japan). UCP1 expression was scored as “negative/weak,” “moderate,” and “strong” by 2 independent evaluators. The association between UCP1 expression and clinicopathological features was assessed using χ-square test.
Gene expression analysis and survival analysis using breast cancer cohorts
Gene expression and clinical data for both Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) [22] and The Cancer Genome Atlas (TCGA) [23] breast studies were downloaded from the cBioPortal for cancer genomics at https://www.cbioportal.org. We considered microarray data for METABRIC and RNA Seq V2 RSEM normalized gene expression data for TCGA. Genes with NA values in more than half of the samples were filtered out as a pre-processing step. Likewise, we removed samples with NA values in more than half of the genes. Sequencing data was then managed by the transformation log2 (x + 1), where x stands for the original expression value. The gene signature as defined previously was used to investigate the extent of hypoxia of METABRIC and TCGA samples [24]. The analysis was performed by using the R software (https://www.r-project.org/). In particular, we used the sigQC R package [25] to understand if the properties of such previously identified gene signature were conserved on the above-mentioned datasets. After confirming that we could use the signature, we focused on two measures of signature summary provided by sigQC (i.e., the median score and the gene set enrichment score computed via the single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm) for further elaboration. The correlation between the hypoxia scores and the genes of interest (UCP1 and FABP7) was computed as Spearman’s rank correlation and reported on scatterplots (ggpubr package). To investigate the association between the patients’ survival time and different covariates (i.e., hypoxia, UCP1, and FABP7 expressions), we used the Cox proportional-hazards model (survival package [26]). We then took advantage of the Kaplan-Meier (KM) method to estimate the survival probability from observed survival times (survival package). Since we wanted to display how estimated survival depends upon the UCP1 and FABP7 values (low/high), we divided the samples in groups by using two different methods, i.e., the median and then k-means, k = 2 (stats package). In this paper, we show the result using k-means as a representative since we found that both methods showed similar results.
Statistical analyses
Statistical analysis of numerical data and generation of graphs was carried out on Prism 6.0 (GraphPad) using unpaired Student’s t tests. All results are represented with means ± SD unless otherwise stated. Significance of difference is represented by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001.