Animal model
Ethical clearance was obtained from the Stellenbosch University animal research committee (no. SU-ACUM13-00,015 and no. ACU-2020–14,751). The experiments involving the use of laboratory animals were carried out in accordance with the Animal Welfare Act and recommendations of the Institutional Animal Care and Committee of Stellenbosch University. This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) to improve the reporting of research involving animals.
Three-week-old female C57BL6 mice (n = 74) were obtained from the Stellenbosch University Central Research Facility and were housed at the Stellenbosch University Animal Unit in individually ventilated cages (IVC) at temperature-controlled conditions (i.e., 22ºC) and underwent a 12-h light/dark cycle. Mice were allowed an acclimatization period of one week with ad libitum access to mouse pellets and tap water (Fig. 1). The general welfare of the animals was monitored daily. After one week of acclimatization, seventy-four mice were randomly divided into two main groups, namely the high fat diet (HFD, n = 36) and standard diet (SD, n = 38) groups (Fig. 1). To induce obesity, mice were fed a HFD containing 60% kcal fat (D12492, OpenSource Diets®, Research Diets Inc., New Jersey, USA), whereas a SD containing 10% kcal fat (D12450J, OpenSource Diets®, Research Diets Inc., New Jersey, USA) were used to generate lean control mice (Supplementary data, Table S1) [16]. According to literature, C57BL/6 mice are sensitive to diet-induced obesity (DIO) [17]. Body weight was monitored weekly over the study period and mice were on their respective diets up until the end point of the study.
After eight weeks on their respective diets, the DIO phenotype was established and the two groups were further randomly divided into two groups each, namely the tumour (T) and non-tumour (NT) groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells suspended in Hanks Balanced Salt Solution (HBSS) (Sigma Chemical Co., St Louis, MO, USA) in the fourth mammary pad, using a 23-guage needle syringe. The tumour groups were inoculated with E0771 triple negative breast cancer cells (HFD + T, n = 18; SD + T, n = 18) and the non-tumour groups were not inoculated with cancer cells (HFD + NT, n = 18; SD + NT, n = 20) (Fig. 1).
Once the tumours became palpable (200 – 300 mm2), DXR treatment was initiated. The mice were randomly divided into vehicle control (V, isovolumetric intra-peritoneal injection of HBSS) and DXR treatment (D5794, LKTR laboratories, Minnesota, USA) groups. Mice were restrained and three successive dosages of 4 mg/kg DXR were administered every three days (cumulative dosage of 12 mg/kg) via intraperitoneal injection. In humans, the dosage of 12 mg/kg DXR is equivalent to 36 mg/m2 and falls within the relevant dosage range of DXR treatment (15—90 mg/m2) administered to cancer patients in the clinical setting [18]. The eight experimental groups were assigned as follow: High fat diet + Tumour + Doxorubicin (HFD + T + DXR, n = 9); High fat diet + Non-Tumour + Doxorubicin (HFD + NT + DXR, n = 10); Standard diet + Tumour + Doxorubicin (SD + T + DXR, n = 10); Standard diet + Non-Tumour + Doxorubicin (SD + NT + DXR, n = 10), High fat diet + Tumour + Vehicle (HFD + T + V, n = 9); High fat diet + Non-Tumour + Vehicle (HFD + NT + V, n = 8); Standard diet + Tumour + Vehicle (SD + T + V, n = 8); Standard diet + Non-Tumour + Vehicle (SD + NT + V, n = 10) (Fig. 1).
The mice were weighed every second day and the last body weight was recorded on the day of euthanasia. Tumour growth was measured using a Harpenden caliper (mm) and individual tumour volumes were calculated according to the following equation [19]:
$$Tumour\;volume\;(mm^3)=\;\frac1{2\;\left(length\;x\;width^2\right)}$$
Fasting blood glucose, triglyceride and lactate levels were taken using the tail prick method where 5 μl of blood was collected on test strips using the Accu-Chek ® Performa Nano (Roche Diagnostics, Mannheim, Germany) and Accutrend Plus® (Roche Diagnostics, Mannheim, Germany) respectively. The mice were euthanized 3 days after the last DXR treatments were administered. The mice were anesthetized with 3% isoflurane (Isofor, Safeline, Pharmaceuticals, Florida, South Africa) and were euthanized by cervical dislocation. Hepatic tissues were excised, where half of the tissues were snap frozen in liquid nitrogen and stored at—80 °C for western blotting and Oil red O staining (n = 4). The other half of the tissues were preserved in 10% formalin for histological analysis (n = 4–5).
Blood analysis
Blood plasma samples were used to quantify TNF-a, IL-6, IL-10, leptin (PPX-04-MXCE327, Thermo Fisher Scientific, United States) using a custom ProcartaPlex panel and matched mouse Luminex kit. Insulin was quantified using a Milliplex mouse adipokine magnetic bead panel MAP kit (MADKMAG-71 K, Burlington, Massachusetts, United States). All analyses were performed according to the manufacturers’ protocols and specifications. Analytes were measured simultaneously using a MAGPIX system plate reader (APX1042, Bio-Rad, California, United States) and data (expressed in pg/ml) was processed on Bioplex Software 6.1 (Bio-Rad, California, United States).
Western blot
Hepatic tissue samples were placed on ice and allowed to thaw. Samples were suspended in 300 μl cold modified radio-immunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors. Surgical scissors cleaned with 100% ethanol was used to cut tissues into smaller pieces while on ice. Samples were homogenised (KineMatica PolytronTM PT2100, Fisher Scientific) while on ice. Samples were centrifuged (14, 000 RCF (g), 20 min, 4 °C) to yield distinct layers and the supernatant layer was removed and transferred into sterile Eppendorf tubes. Samples were then centrifuged again at 14, 000 RCF (g), 20 min, 4 °C. The process of removing the supernatant was repeated followed by protein determination using a Bradford assay. Protein samples were prepared with Laemmli’s sample buffer and were loaded onto 4–20% Criterion™ TGX Stain-Free™ Precast Gels (mini-PROTEAN® TGX™ Gels, Bio-Rad), following protein separation at 100 V for 10 min and 120 V for 60 min in Tris/Glycine/SDS running buffer (BioRad, CA, USA). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo RTA Midi PVDF transfer kit, BioRad, CA, USA) with the Trans-Blot Turbo Transfer System (BioRad, CA, USA) using mixed molecular weight. The membranes were blocked in 5% milk prepared in tris-buffered saline with tween 20 (TBS-T) for 2 h at room temperature (RT) and then incubated in primary antibody, at 4 °C overnight. On the following day, the membranes were incubated with secondary antibody for 1 h at RT. Primary and secondary antibody details are listed in the additional file (Supplementary data, Table S2). After incubation, the membranes were developed on the ChemiDoc™ MP System. Specific bands were visualized and detected using enhanced chemiluminescence (ECL) substrate detection (BioRad, CA, USA). Quantification of protein samples were normalized to total protein and expressed as a percentage of the control.
Histology
In all animals, a small portion of the right lateral lobe of the liver tissue was fixed in 10% neutral formalin buffered solution prior tissue processing (n = 4–5). Hepatic tissues were processed using an automated tissue processor (HistoCore PEARL, Leica Biosystems) on a 12-h cycle followed by infiltration with paraffin embedded wax (Leica EG 1150 H). Tissues were sectioned into 5 μm sections using a microtome (Leica RM 2125 RT) and tissue sections were placed onto positively charged histobond microscope slides. Two histochemical stains were carried out on the sectioned tissue; H&E stain for morphometric and pathological evaluation and Masson’s trichrome stain to evaluate the presence of fibrosis within hepatic tissue. The right median lobe of hepatic tissue was snap frozen in liquid nitrogen and sectioned into 7 μm sections using a cryostat (Leica CM 3050 S Research Cryostat, Leica Biosystems) to evaluate the presence of lipid accumulation using Oil red O staining (n = 4–5). Sections were placed onto positively charged histobond microscope slides and left to defrost. Sections were stained in Oil red O in dextrin staining solution (Sigma-Aldrich, 01,391, SA) for 25 min. Coverslips were mounted onto the slides using aqueous mounting media (Sigma-Aldrich, G 0918) prior to imaging under a microscope (Nikon ECLIPSE E400). The relative number of red pixels (lipid droplets) were quantified using Image J software v1.52a.
Histopathology
Three individuals, blinded to the treatment allocations, scored five images per tissue sample in each treatment group using the non-alcoholic fatty liver disease (NAFLD) activity score (NAS). This is an accredited and validated scoring system frequently used to evaluate hepatic steatosis, inflammation, and hepatocyte-specific pathology [20]. Each scorer was provided with a grading sheet to familiarize themselves with the scoring system one day prior to analysis. The scores were tabulated and used for inter- and intra-observer analysis. H&E-stained tissues were used to evaluate structural changes that occurs within hepatocytes and Masson’s trichrome-stained tissues were used to assist with the evaluation of liver fibrosis. Steatosis was scored according to its percentage per microscopic field, location (zone, 1, 2 or 3), and the presence of micro- and/or macrovesicular steatosis. With regards to inflammation, the location, lobular and/or portal, were evaluated per 100X microscopic field. Following H&E staining, microvesicular steatosis were indicated by black arrows and macrovesicular steatosis were indicated by red arrows (Fig. 5). Ten images per sample were taken and analysis was performed from right to left across the liver tissue sections. The H&E-stained and Masson’s trichrome-stained liver tissues were evaluated as follow: To assess the extent of steatosis, the presence of hydropic changes, micro- and macrovesicular steatosis were evaluated. To determine whether inflammation occurred, the presence of portal mononuclear cell infiltration, haematopoiesis, and Kupffer cell proliferation were evaluated. Characteristics of hepatocytes and the portal system, such as hepatocyte swelling, sinusoidal and central vein dilation, were also evaluated.
Statistical analysis
The western blot experiments were conducted with biological repeats of n = 4 and technical repeats of n = 1. Bio-Rad Image Lab™ software v6.0.1 was used for normalization of the protein specific intensities against total protein intensities. For the Oil Red O staining, ten images per sample were quantified in Image J software v1.52a and the relative number of red pixels were analysed among the different treatment groups. Statistical analysis was performed using GraphPad Prism v7.0. To determine whether the data was normally distributed, a normality test was performed using the Shapiro-Wilks test. To describe the differences between two groups, a Mann Whitney t-test was used. A three-way ANOVA (analysis of variance) followed by Fishers LSD post hoc test was used to describe the differences between three/or more groups and to determine the relationship between the three variables present in this study, namely, diet, disease, and treatment. The results were reported as mean ± standard error of the mean (SEM) and p < 0.05 was considered statistically significant. Histological experiments were conducted with biological repeats of n = 4–5 and technical repeats of n = 2.