In vivo experiments
Animals
We utilized virgin Sprague Dawley female rats that were bred in our laboratory and weighed between 180–200 g. The rats were housed in a temperature-controlled room (22–24 ºC) with a 12-hour light-dark cycle (lights on 06.00–18.00 h). They had free access to rat chow (Gepsa Feeds, Córdoba, Argentina) and tap water, or were administered 6-N-Propyl-2-thiouracyl (PTU) solution ad libitum.
All procedures were conducted in accordance with the ARRIVE guidelines and Guiding Principles in the Care and Use of Animals of the US National Institute of Health, and were approved by the Institutional Animal Care and Use Committee of the School of Medical Science, Universidad Nacional de Cuyo (Protocol approval # 85/2016).
Experimental protocols
To study the effect of HypoT on mammary carcinogenesis, the rats were treated per os with a single dose of 7,12-Dimethylbenz[a]anthracen (DMBA; 15 mg/rat) at 55 days of age to induce mammary tumors. The day of DMBA administration was considered day 0 of the experiment. Animals were divided at random into two experimental groups: HypoT (6-N-propyl-2-thiouracil –PTU- in drinking water, n = 30) and untreated (tap water, EUT, n = 30). Afterward, each group was subdivided into animals that developed tumors (tumor EUT and tumor HypoT) and those that did not develop them (non-tumor EUT and non-tumor HypoT). The non-tumor EUT and HypoT groups were used to assess the effects of HypoT on the ability of adipocytes to modify the biological behavior of tumorigenic and non-tumorigenic mammary epithelial cells, and then to compare them with those produced by tumor-associated adipocytes (tumor EUT and tumor HypoT).
Tumor appearance was monitored every 72 hours in all animals. The incidence, latency, and progression of tumors were recorded. Rats were euthanized via decapitation when tumors grew beyond 1000 mm3 or at the end of the experiment on day 250 for rats that did not develop mammary tumors. The time of euthanasia was standardized to 10:00 h on the day of diestrus. Before sacrifice, all animals were weighed and their vaginal smears were examined. Blood samples were collected and allowed to clot at room temperature. Serum was separated for hormone determinations. Histopathological and immunohistochemical analysis were performed on a piece of each tumor and MAT. Additionally, fragments of MAT were collected to obtain CMs for in vitro experiments.
Induction of hypothyroidism
The administration of PTU (P3755, Sigma-Aldrich, St Louis, Missouri, USA) at a concentration of 0.1 g l− 1 in the drinking water was used to induce HypoT, starting on day 3 until euthanasia. The respective control group (EUT) was maintained in standard conditions throughout the study period15.
Induction of mammary tumors
Mammary tumors were generated by DMBA (D3254, Sigma-Aldrich) dissolved in sunflower oil at a concentration of 5 mg ml− 1. The dose of 15 mg/rat was administered on day 0 using an intragastric probe, 3 h after food and water deprivation to ensure a complete absorption of the drug. Food and water were replaced 2 h after DMBA administration. DMBA is widely used as a model carcinogen in cancer research47–49 and is known to act as a tumor initiator that transforms normal breast tissue into a tumor 15,50.
Hormone determinations
Thyroid-stimulating hormone (TSH) was measured by double antibody radioimmunoassay 15. The hormone was radio-iodinated using the Chloramine T method and purified by passage through Sephadex G75 (G7550, Sigma-Aldrich). The results were expressed in terms of the rat TSH RP-3 standard preparation. Assay sensitivity was 0.5 µg l− 1 serum, and the inter- and intra-assay coefficients of variation were < 10%.
Triiodothyronine (T3) and thyroxine (T4) concentrations in sera were measured by radioimmunoassay using commercial kits for total hormones (RK-6CT1 and RK-1CT1 double antibody radioimmunoassay from the Institute of Isotopes Ltd., Budapest; respectively) 15. Inter- and intra-assay coefficients of variation were < 10%.
Latency, incidence, and progression of tumors
Latency was defined as the interval between DMBA administration and the appearance of first breast tumor. Incidence was considered as the percentage of rats that developed tumors throughout the research period with respect to the total number of rats per group. The major (DM) and minor (dm) diameters, which were measured every 72 hours using a caliber, were used to estimate the tumor volume (TV = dm2xDM / 2). Tumor growth rate was calculated as tumor volume / [day of sacrifice – day of appearance of the first tumor]. Tumor-free survival was assessed by measuring the time elapsed from DMBA administration until either the day of sacrifice when the tumor volume exceeded 1000 mm3 or day 250, whichever occurred first.
Tumors and MAT histology
A small piece of tumor and MAT surrounding the tumor or inguinal mammary gland from each rat were processed for histopathologic studies by fixing in buffered formalin, dehydrated in ethanol (Biopack, Argentina), and embedded in paraffin wax. Sections of 3–5 µm thickness were cut with a Hyrax M 25 microtome and stained with hematoxylin–eosin (H&E) to define the histopathological changes in the mammary glands and to classify tumors according to published criteria 51. Images were taken with a Nikon Eclipse E200 Microscope (Nikon Corp., Japan) fitted with a Micrometric SE Premium (Nikon Corp., Japan) digital still camera under 100x, 400x, and 600x magnifications. To note, only ductal tumors were included in the study. Papillary and lobular tumors were excluded.
The quantification of the percentages of stroma, mostly composed of adipocytes, and epithelial tissue in the mammary gland was performed by measuring the area occupied by the epithelium or adipose tissue in 8–10 fields per sample from the animals of each group using the ImageJ 1.42q software available at the NIH site (http://rsb.info.nih.gov/ij). The area was expressed as a percentage of the whole field as previously published15,45.
The size of the adipocytes in the preparations was measured, taking the greater and smaller diameters of individual cells at a magnification of 400x, using ImageJ 1.42 software. Approximately 100 cells were taken at random per section, and the average diameters were calculated for each preparation 19. Six to eight sections from each sample were evaluated.
Also, adipocytes were identified as unilocular (UL, containing a single large vacuole), paucilocular (PL, exhibiting a large vacuole surrounded by at least five small lipid droplets), or multilocular (ML, containing more than five small homogeneous lipid droplets) 18. Both, UL, PL, and ML adipocytes were evaluated in 10 randomly selected fields per animal using the same Eclipse E200 microscope under a magnification of 400x.
Apoptotic and mitotic indexes
The microscopic analysis was carried out by two independent observers (CLF and LEZ). The apoptotic and mitotic indexes were calculated by counting the total number of apoptotic bodies and mitotic figures, respectively, in histological sections stained with H&E in ten fields from each animal under a magnification of 400x. The mitotic/apoptotic ratio (M/A ratio) was calculated by dividing the mitotic index by the apoptotic index 15.
MCT1 and MCT4 expression
Mammary adipose tissue was homogenized in radio-immunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% w/v sodium dodecylsulfate, 1% w/v Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate and protease inhibitors). Homogenates were centrifugated at 12000 x g for 30 min, the supernatant collected, and protein concentration were quantified by Micro BCA (Protein Assay Kit, Thermo Scientific Inc.). The expression of MCT-1 (ab90582, 1:1000 dilution, Abcam plc) and MCT-4 (sc-50329, 1:500 dilution, Santa Cruz Biotechnology Inc) was measured in the homogenates by SDS-PAGE followed by immunoblotting, as indicated in the Western blot section below. Since the expression of commonly used proteins in the adipose tissue for quantitation are affected by HypoT induction, we used densitometric analysis of the intensity of the Ponceau staining as loading control 20. Finally, all protein levels in EUT conditions were set as 1.0 and the expressions for the same proteins in HypoT were referred to EUT.
In vitro experiments
Reagents
Reagents were purchased from Sigma (Buenos Aires, Argentina). While culture media and additives were obtained from Gibco BRL (Carlsbad, CA, USA), tissue culture flasks, dishes, and multi-well plates were acquired from Falcon Orange Scientific (Graignette Business Park, Belgium).
Sample collection and handling
Fragments of MAT (0.5 g) from both HypoT and EUT rats were removed from the surroundings of the tumor or from the inguinal mammary gland if there was no tumor. They were transferred to a Petri dish under a sterile laminar flow hood and washed with 50 ml phosphate buffer saline solution (PBS) supplemented with gentamicin (50 µg ml− 1). The tissues were transferred to a centrifuge tube containing PBS and spun slowly (2000 x g) for 5 min to remove red blood cells and debris. MAT was weighed and incubated with M199 culture medium (Invitrogen™, Thermo Scientific Inc., Massachusetts, USA) (1 g tissue per 10 ml M199) supplemented with gentamicin (50 µg ml− 1) for 1 h at 37°C in 5% CO2. Following this, M199 was removed, and fresh medium was added. After 24 h of incubation, MAT-CM was collected, centrifuged at 5000 x g for 5 min at room temperature, filtered and stored at -80°C until its use. M199 medium was used as Control-CM. Only MAT-CMs of animals that generated ductal breast tumors were made. These MAT-CMs were used in triplicate to conduct all the biological assays and each experiment was repeated three times. T4 and estradiol (E2) concentrations in MAT-CMs were determined by chemiluminescent immunoassays. In these same CMs, the expression of adiponectin (ab22554, 1:1000 dilution, Abcam plc) was measured by SDS-PAGE followed by immunoblotting, as indicated in the Western blot analysis section below. The assay for the MAT-CMs was done by loading equal volumes of each CM 17 and by using the densitometric analysis of the intensity of the Ponceau staining as loading control 20.
Culture of tumorigenic and non-tumorigenic breast epithelial cell lines
The cell lines (MCF-7, MDA-MB-231, and MCF10-A) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). MCF-10-A cells are non-tumorigenic and ER + mammary epithelial cells. MCF-7 and MDA-MB-231 are tumorigenic mammary epithelial cell lines. MCF-7 cells are ER+, while the MDA-MB-231 are not, which is why they have attributed a more aggressive role in breast cancer. MCF-7 and MDA-MB-231 cells were cultured in DMEM-F12 medium with 10% fetal bovine serum (FBS. Gibco, Fisher Scientific, USA. Internegocios, Argentina). MCF-10A cells were cultured in DMEM-F12 medium supplemented with 10% FBS, 2 µg/ml insulin, 0.5 µg/ml cortisol, and 0.02 ng/ml EGF. Cell lines were kept at 37°C in a 5% CO2 atmosphere.
Cell proliferation assay
Cell proliferation assay was conducted following a protocol already reported 52,53. Non-tumorigenic (5x103 MCF-10A cells/well) and tumorigenic (2x103 MCF-7 or MDA-MB-231 cells/well) breast epithelial cell lines were seed into 96-well plates with DMEM-F12 supplemented with 10% of FBS for 24 h. Then, after treatment with 50% tumor or non-tumor MAT-CMs of HypoT or EUT or M199 and 50% DMEM-F12 medium with 2% of FBS for another 24 h, the cell proliferation was detected using a commercial colorimetric kit (Cell titer 96 AQueous One Solution Cell Proliferation Assay, MTS). Results are expressed as percentage of color intensity and normalized to cells grown in M199 (control-CM).
Trypan blue viability test
The three cell lines (5x104 cells/well) were incubated on 12-well plates with DMEM-F12 supplemented with 10% FBS for 24 h. Then, they were incubated with 50% MAT-CMs and 50% DMEM-F12 with 2% of FBS for 24 h more. Cell viability was calculated as the number of viable cells divided by the total number of cells within the grids on the hemacytometer. Trypan blue solution (0.1%) was added to the cell suspension (Azul Tripan Biopack, Argentina). The number of non-viable cells (stained with blue) and living cells (without blue color) in total cells (counting 1000 cells per group) were then counted. Cell viability was calculated as the number of viable cells divided by the total number of cells within the grids on the hemacytometer.
Cellular adhesion assay
Cell adhesion assays were carried out in accordance with a previously published procedure 54. Briefly, 96-well plates were coated overnight in 5% CO2 at 37°C with 100 µl MAT-CMs or M199. The plates were then blocked for 1 h at 37°C with 100 µl FBS. After washing with PBS 1X, the three cell lines (5x104 cells/well) were suspended in a serum-free DMEM-F12 medium, seeded, and allowed to adhere to the CMs factor-coated wells for 1 h at 37°C in 5% CO2. Non-adherent cells were aspirated. MTS assay was used to evaluate residual cells. Cell adhesion to MAT-CMs of HypoT and EUT factors was expressed as a percentage of M199.
Wound-healing assay
The effect of MAT-CMs on the migration of non-tumorigenic and tumorigenic mammary cell lines was evaluated by wound-healing according to procedures that have been previously reported 55. Wounds were produced by scratching the cell layer using sterile 200 µl plastic pipette tips on 100% confluent wells of MCF10-A, MCF-7, and MDA-MB-231 cells cultured in a 96-well plate with DMEM-F12 supplemented with 10% FBS. Then, they were rinsed with PBS to remove cell debris. Cells were further cultured with 50% MAT-CMs and 50% DMEM F12 with 2% of FBS and allowed to migrate into the denuded area for 6 h. Images at time zero (0 h) and 6 h later were acquired by an inverted phase-contrast microscope (Olympus CKX-41; 4x objective). A quantitative analysis of the wound closure was measured by Java Image J (NIH, Bethesda, MD, USA) computer program utilizing the freehand selection mode and determined the relative wound closure regarding to control of the wounded area after 6 h.
Western blot analysis
RIPA buffer was used to lyse cells and Micro BCA (Protein Assay Kit, Thermo Scientific Inc.) to quantify the total proteins in samples. Proteins were separated in an SDS-PAGE 13% gel, and electrotransferred to a PVDF membrane. After that, the membrane was blocked with bovine serum albumin (Sigma-Aldrich, 0055 K) and then incubated with the corresponding primary antibodies against Caspase 3 (ab4051, 1:300 dilution, Abcam plc), PARP (ab32138, 1:500 dilution, Abcam plc) and β-actin (sc-47778, 1:1000 dilution, Santa Cruz Biotechnology Inc.) overnight at 4°C. Afterward, membranes were incubated for 90 min at room temperature in the presence of the horseradish peroxidase-conjugated secondary antibody (1:3000 polyclonal goat anti-rabbit biotinylated, Dako Cytomation, Denmark). After five washes in TBS-T, specific bands were detected by chemiluminescence (ECLTM, Amersham, Sigma Aldrich, UK) using a ChemiDoc XRS + System with Image Lab Software from Bio-Rad and then quantified by densitometry using digital image processing by the FIJI Image processing package. In the cell extracts, β-actin level was used as loading control to determine that equal quantities of proteins were loaded in the gel.