GoKawiilAnimal studies
All animal studies were performed according to protocols approved by the University Committee on Animal Resources at the University of Rochester Medical Center. Gclcf/f mice27 were crossed with the MMTV-PYMT (The Jackson Laboratory, 022974)61 and Rosa26creERT2 (The Jackson Laboratory, 008463)62 mouse strains. For the tumour allograft model, Gclcf/fRosa26creERT2 MMTV-PYMT mice and control C57BL/6 mice (The Jackson Laboratory, 000664) were used (female, at least 12 weeks old). For tumour xenograft experiments, athymic nude NU/J mice (The Jackson Laboratory, 002019) were used (female, at least 8 weeks old).
For the tumour allograft model, Gclcf/fRosa26creERT2 MMTV-PYMT mice were euthanized and autochthonous tumours were collected, cut into 8 mm3 fragments and cryopreserved in liquid nitrogen. These fragments were later thawed on ice and implanted into the right fourth mammary fat pad of immunocompetent C57BL/6 mice. Once the grafted fragments had formed fully established tumours, they were collected, cut into 8 mm3 fragments and cryopreserved using the same procedure. Finally, these tumour fragments were thawed and grafted into recipient immunocompetent C57BL/6 mice and used for the designated experimental treatments. To activate Cre recombinase and induce Gclc deletion in tumour pieces from Gclcf/fRosa26creERT2 MMTV-PYMT mice, female mice were intraperitoneally injected with tamoxifen (Sigma-Aldrich, T5648) at 50–160 mg kg–1 once daily for 5 consecutive days. For tumour xenograft models, 1 × 106 HCC-1806 cells or 5 × 106 PC3 cells were injected into the right fourth mammary fat pad or hind flank of athymic nude NU/J mice (The Jackson Laboratory, 002019) in 100 µl sterile PBS or a 1:1 mixture of cell suspension in PBS and Matrigel (Corning, 356237) using a 29-gauge 1 ml insulin syringe. In all cases, animals received only one cell injection at a single location per experiment.
Tumour volume was estimated using the formula for an oblate spheroid \(v=\frac{\pi }{6}\times ({l}^{2})\times w\), where volume (v) is calculated using caliper measurements of the longest (l) and shortest (w) sides of the tumour. For pharmacological interventions, treatment of mice was initiated once the average tumour volume in the groups reached approximately 300 mm3. Mice were treated with pharmacological agents by either intraperitoneal (i.p.) injection or inclusion in the drinking water. For treatment via i.p. injection, GGsTop (Tocris, 4452; WuXi, 926281-37-0; or MCE, HY-108467) was diluted in sterile saline and injected at the indicated concentrations. For treatment via drinking water, GGsTop (WuXi, 926281-37-0) was diluted to 62.5 µg ml–1 (189 µM) and provided to mice ad libitum. Based on an average daily water consumption of 4 ml, the estimated daily dose of GGsTop for a 25 g mouse was 10 mg kg−1 per day (equivalent to 5 mg kg−1 twice a day i.p. injections). NAC (Sigma-Aldrich, A7250) was diluted in drinking water at a concentration of 30 mM, the pH was adjusted to 7.00 and it was provided to mice ad libitum.
Mice of desired strains were age-matched and assigned randomly to their treatment groups. For xenograft studies, animals were allocated into groups ensuring the mean, median and standard error of tumor size was similar across all groups. Investigators were not blinded to group allocation during experiments owing to technical limitations. Statistical methods were not used to chose sample sizes. The number of animals assigned per condition was selected to account for the variability of the examined phenotypes based on pilot experiment and past experience with the animal models (≥6 animals per experimental condition). Mice were euthanized at humane end points (when the tumour diameter exceeded a total of 20 mm in length, when ulceration occurred or when weight loss exceeded 20%). Tumours and tissues were snap-frozen on dry ice and stored at –80 °C or placed in 10% neutral buffered formalin (Fisher Scientific, 22-110-761) for 24 h and then stored in 100% methanol (Fisher Scientific, A412P-4).
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue sections (5 µm) were used for haematoxylin and eosin staining and immunohistochemical analyses. The tissues were dewaxed and rehydrated through a series of xylene and ethanol changes. For antibody staining, antigen retrieval was performed on the slides by incubating them in a steamer for 40 min in citrate buffer (Vector Labs, H-3300-250). Tissues were permeabilized with PBS supplemented with 0.1% Tween-20 for 10 min, followed by a peroxidase block with 3% H 2 O 2 (Sigma-Aldrich, 216763) for 20 min. The slides were washed in PBS and then blocked using 10% goat serum in PBS for 1 h at room temperature before adding primary antibodies diluted in the blocking buffer. Primary antibody (anti-GCSc 1:100 (SantaCruz, sc390811), anti-CD45 1:100 (Santa Cruz, sc1178), anti-F4/80 1:100 (Cell Signaling Technology, 70076), anti-DNA/RNA Damage 1:200 (Abcam, 62623), anti-NRF2 1:100 (Abcam, 31163) and anti-GGT1 1:200 (donated from M. H. Hannigan)63) incubation was carried out overnight at 4 °C. Biotinylated goat anti-mouse IgG or goat anti-rabbit IgG (Vector Labs, BA-9200 1:200 and BA-1000 1:200, respectively) secondary antibodies were added in blocking buffer and incubated for 1 h at room temperature. Stains were developed using VectaStain Elite ABC-HRP Peroxidase and DAB Substrate kits (Vector Labs, PK-7100 and SK-4100, respectively). Tissues were counterstained with haematoxylin, mounted with Permount mounting medium (Fisher Scientific, SP15500) and coverslipped for imaging. Images were taken using an Olympus VS120 virtual slide microscope and Visiopharm image analysis system.
Immunofluorescence
Formalin-fixed, paraffin-embedded tissue sections (5 µm) were used for immunofluorescence. The tissues were dewaxed and rehydrated through a series of xylene and ethanol changes. For antibody staining, antigen retrieval was performed on the slides by incubating them in a steamer for 40 min in citrate buffer (Vector Labs, H-3300-250). The slides were washed in water and then blocked using 10% goat serum in PBS for 1 h at room temperature before adding primary antibodies diluted in the blocking buffer. Primary antibody (anti-GCSc 1:10 (SantaCruz, sc390811) and anti-CD45 1:200 (Proteintech, 31243-1-AP)) incubation was carried out overnight at 4 °C. Secondary antibodies (AlexaFluor 594 anti-mouse 1:500 (Invitrogen, A11005) and AlexaFluor 488 anti-rabbit 1:500 (Invitrogen, A11034)) were added to blocking buffer and incubated at room temperature for 1 h. For immunofluorescence, the second-to-last wash included 1 µg ml–1 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich D9542) in PBS to stain cell nuclei. Immunofluorescence tissues were mounted with Immuno-Mount (Thermo Fisher, 9990402) and coverslipped for imaging. Immunofluorescence-stained slides were imaged using a Zeiss Axioimager.M2m with an AxioCam MRm camera and the AxioVision program for imaging.
Cell culture
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