Animals and experimental set-up
Male Fischer F344-rats (6-week-old; n = 64) were purchased from Charles River GmbH (Sulzfeld, Germany) and kept under controlled conditions at 23 ± 2 °C and 55 ± 5% relative humidity. Rats were maintained on a 12:12 h light dark cycle with lights on from 6 am to 6 pm. Rodent chow and water were available ad libitum. Half of the group (n = 32) was randomly selected to receive a high-fat diet (diet-induced obesity: DIO; 29% carbohydrate, 21% crude protein, 35% crude fat and 5.2 kcal/g metabolizable energy; C1090–60 Altromin, Lage, Germany) for 6 (short-term tumor challenge) or 10 weeks (long-term tumor challenge) to induce obesity. Control animals (n = 32) received the corresponding control diet (58% carbohydrate, 21% crude protein, 4% crude fat and 3.5 kcal/g metabolizable energy; C1090–10, Altromin). Animals were weighed and handled by the scientists weekly. All research and animal care procedures were approved by the local Animal Care Committee of the “Landesverwaltungsamt Halle” (reference number 42502–2-1116MLU).
After feeding the high-fat diet, DIO animals started the experiments with a significant higher body weight as compared to their age-matched lean littermates.
Subsequently animals were i.v. inoculated with either 1 × 106 cells of an adenocarcinoma syngeneic tumor (MADB106) dissolved in 1 ml isotonic saline or only 1 ml of isotonic saline (NaCl) via the tail vein.
Subsequently half of the animals were killed at an early time point, 15 min after tumor cell challenge (short-term experiment). The other half of the group of rats was admitted to develop lung metastases over a period of 21 days and therefore represent the late time point (long-term experiment). Rats of the long-term experiment received the diet according to their group (control or DIO) for the 21 days of lung metastases development until the end of the experiment.
Culture and CFSE labeling of MADB106 tumor cells
Cell culture and CFSE (fluorescein derivate 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester)-labeling of cells were conducted as described elsewhere [21]. In brief, the MADB106 mammary adenocarcinoma syngenic tumor is a selected variant cell line obtained from a pulmonary metastasis produced by the intravenous injection of the 9–10 dimethyl-1-2-benzanthracene-induced MADB106 parental adenocarcinoma in F344-rats. Injecting 1 × 106 MADB106-cells via the tail vein of animals leads to pulmonary metastasis after 21 days. For in situ quantification 15 min after injection, tumor cells were vitally dye stained using CFSE (Cell Trace CFSE Cell Proliferation Kit, Molecular Probes, Eugene, USA) before injection [22].
Blood and organ sampling
Rats were killed under general isoflurane anesthesia either after 15 min (short-term tumor challenge; short-term experiment) or 21 days (long-term tumor challenge; long-term experiment) by puncture of the abdominal aorta. Blood was withdrawn and spleen and liver were removed. Organs were immediately frozen in liquid nitrogen and stored at −80 °C for RNA isolation and lipid analysis. Heparinized blood samples were stored on crushed ice. Subsequently, erythrocytes in the blood were destroyed by a lysis buffer (155 mM NH4Cl, 10 mM KHCO3 and 0.01% EDTA) to obtain leukocytes for following cytometric analysis.
Lung preparation
Lungs were processed as described by von Hörsten et al. [22]. Briefly, a cannula was inserted into the trachea in situ. Lungs and the heart were dissected from the chest and rinsed with 10 ml 0.9% NaCl. Afterwards, lungs were transfused with 8 ml of O.C.T. embedding medium (Sakura, Tokyo, Japan; diluted 1/5 in PBS) for short-term experiment or with Bouin’s fixative (Sigma-Aldrich, Taufkirchen, Germany) for long-term experiment. Complete lungs were dissected at the hilum from the pulmonary trunk of the heart, frozen in liquid nitrogen and stored at −80 °C for consecutive immunohistochemically staining for the short term experiment or immediately immersion fixed in Bouin’s fixative for 24 h for long-term experiment.
Immunohistochemically analysis of the lungs (short-term experiment)
Frozen tissue samples of the right lobe were immersed in O.C.T. embedding medium and 5 μm thick sections were cut and placed on glass slides. Every 15th section was selected with a random start, yielding 15–21 sections per animal. Selected sections were mounted on coated glass slides (Starfrost, Knittel, Braunschweig, Germany) and air dried.
Immunostaining of NK cells and CFSE-labeled MADB106 tumor cells was performed using monoclonal antibodies (mAbs) directed against the NK-RP1 receptor (CD 161 rat/ clone 10/78, BioRad AbD Serotec, Puchheim, Germany) on the NK cell surface and the intracellular CFSE antigen (anti-Fluorescein from mouse IgG1 clone B13-DE1, Roche Applied Science, Mannheim, Germany), respectively. Thereafter, APAAP (alkaline-phosphatase-anti-alkaline-phosphatase-complex) staining was performed. Sections were fixed in acetone for 10 min and washed with TBS–Tween (0.05% Tween 20, Serva, Heidelberg, Germany) followed by the incubation with the primary anti-CFSE-mAb overnight at room temperature in humid chambers. Sections were washed with TBS–Tween followed by incubation for 30 min with the bridging antibody (Dako Z 0259, 1/50, rabbit anti-mouse, Dako, Hamburg, Germany) diluted in 5% rat serum. After another rinse the APAAP complex (100 ml Dako D 0651, 1/50, mouse; in TBS–Tween) was added and the sections incubated for 30 min at room temperature. The incubations with bridging antibody and the addition of the APAAP complex were repeated once for 15 min followed by addition of the substrate Fast Blue (Sigma-Aldrich). Next the incubation with the primary antibody against CD161 was performed for 1 h followed by an identical secondary staining procedure except that Fast Red (Sigma) was used as substrate. Finally, sections were counterstained with hematoxylin and covered with glycergel mounting medium (Dako). The immunohistological investigations were carried out strictly under blind conditions. For counting of NK cells and NK cell-tumor cell contacts an area of 40 mm2 was evaluated by using the Software Image J software (US National Institutes of Health, Bethesda, MD, USA).
Visualization of lung metastasis (long-term experiment)
Due to the fixation of lungs in Bouin’s solution 21 days after inoculation of the MADB106 cells, subpleural lung surface metastases were identified by a light, white appearance. Surface metastases are 1–8 mm3, mushroom-shaped, distinctly separated, and raised above the lung surface. All visible surface metastases were quantified by masked counting.
Cytometric analysis
FACS (Fluorescence Activated Cell Sorting) analysis was performed using the following mouse anti-rat mAbs: CD3 conjugated with allophycocyanin (T cell receptor/CD3 APC), and CD161a conjugated with phycoerythrin (NK cells/NKRP1A+/CD161abright PE, BD Biosciences, San Diego, USA). Protected from light, cells were incubated for 30 min at 4 °C. Thereafter, PBMCs were washed twice with washing buffer (PBS supplemented with 1% BSA and 0.1% sodium azide), resuspended in measuring buffer (PBS supplemented with 0.1% BSA and 0.1% sodium azide) and samples were analyzed by flow cytometry using LSR Fortessa with BD FACSDiva Flow Cytometry Software Version 6.2 (BD Biosciences, San Diego, USA; Fig. 1). NK cells are represented by the CD161abright/CD3− population.
Lipid analysis
Lipids were extracted from liver with a mixture of n-hexane and isopropanol (3:2, v/v) as already described [23]. For determination of the concentrations of cholesterol and triacylglycerols (TAG) in the liver, aliquots of the lipid extracts were dried and the lipids dissolved using Triton X-100 (Sigma-Aldrich) [24]. Concentrations of cholesterol and TAG in liver were determined using an enzymatic reagent kit (Ecoline S+, DiaSys GmbH, Holzheim, Germany).
RT-PCR analysis
Total RNA was isolated by Precellys 24 (Pequlab, Erlangen, Germany) from frozen spleen samples using TRIZOL™ (Sigma Aldrich) according to the manufacture’s protocol. cDNA synthesis was carried out as described by Koenig & Eder [25]. The mRNA concentrations of genes were measured by realtime detection PCR (iQ5, BioRad, München, Germany) using SYBR® Green MIX (BioRad) and the specific primers (KiCqStart™ Primers, Sigma Aldrich, Hamburg, Germany, Additional file 1: Table S1) following the manufacture’s protocol. For determination of mRNA concentration a threshold cycle (Ct) was obtained from each amplification curve using the software Bio-Rad iQ5 (BioRad). Calculation of the relative mRNA concentration was made using the ΔΔCt method [26] with individual amplification efficiency for each primer, determined by a standard curve with different dilutions of primers. The housekeeping gene Cyp18 was used for normalization (Additional file 1: Table S1).
Statistics
Data analysis was performed using the Graph Pad Prism Software V5 (GraphPad, Inc., La Jolla, CA, USA). For one-way ANOVA Tukey-test and for two-way ANOVA Bonferroni-test with the main factors “diet” and “tumor” were used as post-hoc tests. Means were considered significantly different at p ≤ 0.05. Results are presented as means ± standard error of the mean (SEM).
* indicates significant differences of means (p ≤ 0.05) between all groups analyzed by one-way ANOVA.
# indicates significant differences of means (p ≤ 0.05) between rats receiving NaCl compared to rats receiving MADB106 cells analyzed by two-way ANOVA.
§ indicates significant differences between rats receiving control diet compared to DIO animals (p ≤ 0.05) analyzed by two-way ANOVA.