Role of HGF in Obesity-Associated Tumorigenesis: C3(1)-Tag Mice As a Model for Human Basal-Like

Role of HGF in obesity-associated tumorigenesis: C3(1)-TAg mice as a model for human basal-like breast cancer

METHODS

Reagents and antibodies

Basal-like breast cancer-like 4T1 epithelial cells (CRL-2539) were obtained within the past six months from the American Type Culture Collection (ATCC; Manassas, VA) and at time of purchase were characterized and authenticated by the ATCC using a fingerprinting process called short tandem repeat (STR) analysis of DNA, which is among the most informative polymorphic markers in the genome (ATTC.org). Cells were authenticated weekly using a morphology check as suggested by ATCC technical bulletin 8 (ATTC.org). Cell tracker dyes Green 5-chloromethylfluorescein diacetate (CMFDA; Excitation wavelength = 492/Emission wavelength = 517) were obtained from Invitrogen (Carlsbad, CA). Anti-mouse HGF antibody that detects total HGF (both pro and cleaved) and anti-mouse c-Met antibody that binds pro- and cleaved c-Met were obtained from R&D Systems (Minneapolis, MN). Anti-mouse SV40-TAg antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-mouse phospho-c-Met (phospho Y1003; pc-Met) antibody (Ab61024) and HGF ELISA kit (Ab100686) that recognizes all forms of HGF were obtained from Abcam (Cambridge, MA). Anti-mouse α-smooth muscle actin (# 04-1094) was obtained from Millipore (Billerica, MA). Estrogen and progesterone ELISA kits were obtained from Novatein Biosciences (Woburn, MA).

C3(1)-TAg Mouse Model

Diets:Diets obtained from Research Diets Inc. (New Brunswick, NJ) were matched for protein, vitamins, and minerals, and provided 10% kcal (“10%”); 45% kcal (“45%”); or 60% kcal (“60%”) from fat. Details of the diet components are provided in Suppl. Table 1. Corn oil replaced soybean oil to avoid potential complications of soy isoflavones in breast cancer oncogenesis, and cornstarch and maltodextrin replaced sucrose, as sucrose is highly lipogenic [1].

Tumor latency, number, growth and volume:Mice were anesthetized using 3% vaporized isofluorane/oxygen mixture in an induction chamber followed by removal of fur using a commercial depilatory to prevent interference in ultrasound measurement followed by serial ultrasound measurements carried out weekly from the time of tumor identification until sacrifice. Two dimensional ultrasound pictures of the tumors were acquired at 5 µm sections. From the ultrasound measurements post-imaging, the largest width (short diameter) and length (long diameter) in mm were determined for each tumor. The tumor volumes were calculated using the formula: length x width2 x 0.5. The percent change in volume over time was calculated: (End volume – start volume)/Start volume * 100. The total number of tumors per mouse was counted at sacrifice.

Tissue Harvest: Three weeks after detection of the first tumor, mice were anesthetized by an intraperitoneal (i.p.) injection of avertin (Fisher Scientific, Pittsburgh, PA). Blood was collected by cardiac puncture into a tube with 10 µl of 0.05 mM EDTA after mice were fasted 6 hours. Plasma was separated by centrifuging blood at 5000 x g for 5 min. Plasma was stored at –80°C. Mammary glands without palpable or visible tumors were collected as ‘normal’, although atypia of ductal epithelium could be present in C3(1)-TAg mice after 8 weeks of age [2]. Normal and tumor tissue was snap frozen in liquid nitrogen and saved for mRNA and protein isolation. Portions of the tissues were placed into a cassette and formalin fixed for immunohistochemical analysis.

Plasma and Tissue Cytokines Panel: Metabolically relevant hormones including leptin, insulin, IL-6, MCP-1 and TNF-α were measured in the plasma and tissues collected at sacrifice using the Milliplex MAP Mouse Metabolic Hormone Magnetic Bead Panel in the Luminex MAGPIX system (EMD Millipore, Billerica, MA). Normal mammary gland and tumors frozen in liquid nitrogen were pulverized under liquid nitrogen and were homogenized in RIPA buffer with protease and phosphatase inhibitors.

Immunohistochemistry of HGF, c-Met, pc-Met and SV40-TAg in Normal Mammary Glands and Tumors: Immunohistochemistry (IHC) was performed for HGF, c-Met, pc-Met and SV40-TAg following the protocol previously described in Sampey et. al. [1]. Anti-mouse HGF antibody and anti-mouse c-Met antibody were used at a dilution of 1:400 with secondary donkey anti-goat antibody (1:500; Jackson Immunoresearch; # 705-065-147). Anti-mouse SV40-TAg antibody was used at a dilution of 1:250 with secondary Ultravision Quanto Mouse on Mouse HRP kit using manufacturer’s recommendations (Thermo Scientific, Pittsburgh, PA; # TL-060-QHDM). Anti-mouse pc-Met was used at a dilution of 1:500 with secondary goat anti-rabbit (1:500; Jackson Immunoresearch; # 111-065-144) antibody. Following staining, slides were scanned into the Aperio Scanscope CS system (Aperio Technologies, Vista, CA) at a magnification of 40X and staining was quantified using the Aperio Imagescope software. The scanned slides were analyzed using the appropriate algorithms as described previously[3-5]. The Aperio Imagescope software and the positive pixel counts for diaminobenzidine (DAB) staining in the color deconvolution algorithm was utilized for HGF and pc-Met and membrane IHC algorithm for c-Met quantification[3,4]. N = 6 random areas from sections (n = 2 per mouse) were quantified and averaged per tumor per animal (n = 5 mice per diet exposure group).

Immunofluorescence (IF) co-staining of HGF and fibroblasts: To determine whether fibroblasts secrete HGF in vivo, normal mammary glands and tumors were stained for HGF and fibroblast marker – α – smooth muscle actin. Anti-mouse HGF antibody at 1:400 dilution with secondary Alexa-Fluor® 488 conjugated bovine anti-goat IgG at 1.500 dilution was co-stained with anti-mouse α-smooth muscle actin (# 04-1094) at 1:200 dilution with secondary Alexa-Fluor® 647 conjugated donkey anti-rabbit IgG at 1:500 dilution. DAPI was used for nuclear staining. The slides were observed under a Zeiss LSM 710 confocal microscope. Photomicrographs were obtained under a magnification of 63X using oil immersion lenses.

Mice for primary fibroblast isolation for co-culture studies:To study whether growth factors mediate the growth and progression of tumors, age-matched mice were fed either 10% or 60% fat diets starting at approximately 13 weeks of age (prior to latency) for a total of 4 weeks of diet exposure. Three separate age-matched diet groups were used: Group A (n = 5 mice each on 10% and 60% diets); Group B (n = 4 mice each on 10% and 60% diets); Group C (n = 2 mice on 10% and n = 7 mice on 60% diets). Mice were sacrificed as above and normal inguinal mammary glands or tumors were collected to isolate fibroblasts for ex vivo studies.

Isolation of fibroblasts:Fibroblasts were isolated as in Fleming et. al.[6] Briefly, lymph nodes from the mammary glands were removed prior to collection. Tissues were minced (~ 5 mm) in PBS (1X) buffer and placed in DMEM with 10% fetal bovine serum (FBS), 5mM glutamine and 10mM penicillin-streptomycin. All cells were incubated in a humidified incubator under 5% carbon dioxide. Fibroblasts migrated onto scratched 6 well plates over a 1 week period, after which NAFs and CAFs derived from mice in groups A, B and C (see above) were harvested and pooled for culture.

Co-culture studies of stromal-epithelial interactions

Co-Culture:4T1 cells were plated onto a 10 mm plate at a density of 1 million cells and grown to a confluency of 50-60% in RPMI-1640 media supplemented as above. The cells were then incubated for 45 min with a CellTrackerTM Green 5-chloromethylfluorescein diacetate (CMFDA) at 7.5 – 10 µM (Invitrogen, Carlsbad, CA) as previously described [7] to visualize co-cultures. Fibroblasts (NAF and CAF) from 10% and 60% high fat diet-fed mice were co-cultured with CellTrackerTM dye-stained 4T1 cells (CRL-2539) in a 1:1 ratio (50,000 fibroblasts: 50,000 4T1) in the presence or absence of 0.5 µg/ml anti-mouse HGF antibody (R&D Systems, Minneapolis, MN) per well in a 12 well plate in 2 ml of RPMI growth media. Mono-cultures of 4T1 cells and fibroblasts were plated as controls (total plated was 100,000 cells). Media was collected over a period of 24h at various time points as indicated.

Western immunoblot analysis for phospho- and total c-Met: Fibroblast conditioned media from the 60%-CAFs diluted 1:1 with fresh media was then used to treat 4T1 cells to induce phosphorylation c-Met. Untreated 4T1 cells and cells treated with recombinant mouse HGF (50 ng/ml) for 15 min were used as negative and positive controls, respectively.

Cell Proliferation: Live cell counts andBrdU assay: Following 8 h serum starvation, 4T1 cells were platedon a 96 well plate at a density of 25,000 cells/well for BrdU and 50,000 cells/10 cm plate for cell counts and assayed at the end of 48 h incubation with the treatment conditions. Fibroblast conditioned media from 10%-NAF, 60%-NAF, 10%-CAF, and 60%-CAF diluted 1:1 with fresh media was used to treat 4T1 cells to examine potential regulation of cell proliferation. Serum free media-treated cells were used as non-proliferating controls. Untreated cells and HGF (50 ng/ml) were also used as controls. Following 48 h incubation, cells were harvested by trypsinization and centrifuged at 3000 rpm for 3 min. The cells were resuspended in 400 µl PBS with 1 µl of propidium iodide (PI) for staining dead cells. The cells were counted on a flow cytometer and cells stained with PI were excluded from analysis (data not shown). In addition, wells without cells were used as blank controls for testing BrdU specificity. BrdUassay was performed as recommended by the manufacturer (Abcam, Cambridge, MA).

Supplemental Figures:

Supplemental Figure 1: Study design for C3(1)-Tag mice.

Supplemental Figure 2:Obesity does not increase SV40 TAg tissue expression. A) Representative photomicrographs (20X) of the IHC analysis of negative control and SV40 - TAg staining(arrows) in 10%, 45%, and 60% diet groups. B) Total SV40-TAg protein levels in normal mammary glands were quantified using the Aperio ImageScope color deconvolution algorithm for n = 2 sections from 5 mice per diet group. * 45% vs. 60% (P = 0.022). C) Representative photomicrographs (20X) of the IHC analysis of negative control and SV40 - TAg staining in tumors isolated from 10%, 45%, and 60% diet groups. D) Total SV40-TAg protein levels in tumors were quantified using the Aperio ImageScope color deconvolution algorithm and quantified.

Supplemental Table 1: Custom purified diet components (Research Diets Inc.)

Custom Product # / D11012202
10% diet / D11012203
45% diet / D11012204
60% diet
% / gm / kcal / gm / kcal / gm / kcal
Protein / 19 / 20 / 24 / 20 / 26 / 20
Carbohydrate / 67 / 70 / 41 / 35 / 26 / 20
Fat / 4 / 10 / 24 / 45 / 35 / 60
Total / 100 / 100 / 100
kcal/gm / 3.8 / 4.7 / 5.2
Ingredient / gm / kcal / gm / kcal / gm / kcal
Casein / 200 / 800 / 200 / 800 / 200 / 800
L-Cystine / 3 / 12 / 3 / 12 / 3 / 12
Corn Starch / 575 / 2300 / 220.6 / 882 / 68.8 / 275
Maltodextrin 10 / 125 / 500 / 125 / 500 / 125 / 500
Cellulose, BW200 / 50 / 0 / 50 / 0 / 50 / 0
Corn / 25 / 225 / 25 / 225 / 25 / 225
Lard / 20 / 180 / 177.5 / 1598 / 245 / 2205
Mineral Mix S10026 / 10 / 0 / 10 / 0 / 10 / 0
DiCalcium Phosphate / 13 / 0 / 13 / 0 / 13 / 0
Calcium Carbonate / 5.5 / 0 / 5.5 / 0 / 5.5 / 0
Potassium Citrate, 1 H20 / 16.5 / 0 / 16.5 / 0 / 16.5 / 0
Vitamin Mix V10001 / 10 / 40 / 10 / 40 / 10 / 40
Choline Bitartrate / 2 / 0 / 2 / 0 / 2 / 0
FD&C Yellow Dye #5 / 0.05 / 0 / 0 / 0 / 0 / 0
FD&C Red Dye #40 / 0 / 0 / 0.05 / 0 / 0 / 0
FD&C Blue Dye #1 / 0 / 0 / 0 / 0 / 0.05 / 0
Total / 1055 / 4057 / 858.2 / 4057 / 773.9 / 4057

1. Sampey BP, Vanhoose AM, Winfield HM, Freemerman AJ, Muehlbauer MJ, Fueger PT, Newgard CB, Makowski L (2011) Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose inflammation: comparison to high-fat diet. Obesity (Silver Spring) 19 (6):1109-1117. doi:10.1038/oby.2011.18

2. Green JE, Shibata MA, Yoshidome K, Liu ML, Jorcyk C, Anver MR, Wigginton J, Wiltrout R, Shibata E, Kaczmarczyk S, Wang W, Liu ZY, Calvo A, Couldrey C (2000) The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma. Oncogene 19 (8):1020-1027. doi:10.1038/sj.onc.1203280

3. Polcz ME, Adamson LA, Lu X, Chang MN, Fowler LJ, Hobbs JA Increased IL-6 detection in adult and pediatric lymphoid tissue harboring Parvovirus B19. Journal of Clinical Virology (0). doi:

4. Ruifrok A, Johnston D (2001) Quantification of histochemical staining by color deconvolution. Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology 23:291 - 299

5. Sampey BP, Freemerman AJ, Zhang J, Kuan PF, Galanko JA, O'Connell TM, Ilkayeva OR, Muehlbauer MJ, Stevens RD, Newgard CB, Brauer HA, Troester MA, Makowski L (2012) Metabolomic profiling reveals mitochondrial-derived lipid biomarkers that drive obesity-associated inflammation. PloS one 7 (6):e38812. doi:10.1371/journal.pone.0038812

6. Fleming JM, Miller TC, Kidacki M, Ginsburg E, Stuelten CH, Stewart DA, Troester MA, Vonderhaar BK (2012) Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo. Breast cancer research : BCR 14 (3):R97. doi:10.1186/bcr3215

7. Camp JT, Elloumi F, Roman-Perez E, Rein J, Stewart DA, Harrell JC, Perou CM, Troester MA (2011) Interactions with fibroblasts are distinct in Basal-like and luminal breast cancers. Molecular cancer research : MCR 9 (1):3-13. doi:10.1158/1541-7786.MCR-10-0372