Human subjects

All protocols were reviewed and approved by the Pennington Biomedical Research Center Institutional Review Board and all human participants provided written informed consent (PBRC #23040). Human ASCs were obtained from 24 Caucasian females (4 groups, 6 donors per group) undergoing elective liposuction procedures, as previously described [18, 19]. Briefly, ASCs were isolated from processed lipoaspirates from subcutaneous abdominal adipose tissue of obese (Ob+Ab+) or non-obese (Ob-Ab+) subjects and from non-abdominal subcutaneous adipose depots of obese (Ob+Ab-) and non-obese (Ob-Ab-) subjects. Liposuction aspirates were incubated in 0.1% type I collagenase (Sigma) and 1% powered bovine serum albumin (BSA, fraction V; Sigma, St. Louis, MO, USA) dissolved in 100 ml of phosphate buffered saline (PBS) supplemented with 2 mM calcium chloride. This mixture was placed in a 37°C shaking water bath at 75 rpm for 60 minutes and then centrifuged to remove oil, fat, primary adipocytes and collagenase solution, leaving behind a pellet of cells. Cells were resuspended in complete culture media (CCM), which consisted of α-Minimal Essential Medium (αMEM; GIBCO; Grand Island, NY, USA), 20% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), 100 units per ml penicillin/100 μg/mL streptomycin (P/S; GIBCO), and 2 mM L-glutamine (GIBCO), and plated on 150 cm2 culture dishes. Fresh CCM was added every two to three days until cells achieved 80 to 90% confluence, at which time cells were harvested with 0.25% trypsin/1 mM EDTA (GIBCO) and cryopreserved prior to experimental use. Non-abdominal subcutaneous adipose tissue was isolated from the hip, knee, thigh, ankle, flank, upper toroso, scapula, forearm, arm and back. The mean BMI for each of the four donor groups was as follows: Ob+Ab+ (32.7 ± 3.7), Ob+Ab- (31.1 ± 0.6), Ob-Ab+ (22.7 ± 1.9) and Ob-Ab- (22.5 ± 1.2). The mean age of the subjects for each group of donors was as follows: Ob+Ab+ (42.5 ± 8.9), Ob+Ab- (44.0 ± 12.4), Ob-Ab+ (38.8 ± 7.0) and Ob-Ab- (52.4 ± 18.0). No statistical significance in age was observed between the donor groups.

Cell culture

ASCs

Frozen vials of ASCs were thawed and cultured on 150 cm2 culture dishes (Nunc, Rochester, NY, USA) in 25 ml CCM and incubated at 37°C with 5% humidified CO 2 . After 24 hours, viable cells were harvested with 0.25% trypsin/1 mM EDTA and replated at 100 cells/cm2 in CCM. Media was changed every two to three days. For all experiments, sub-confluent cells (≤70% confluent) between passages 2 to 6 were used.

To characterize the cells, ASCs were induced to undergo osteogenic and adipogenic differentiation. For osteogenic differentiation, ASCs were cultured in six-well plates in CCM until 70% confluent and media was replaced with fresh media containing osteogenic supplements, consisting of 50 μM ascorbate 2-phosphate (Sigma), 10 mM β-glycerol phosphate (Sigma) and 10 nM dexamethasone. After three weeks, cells were fixed in 10% formalin for 1 hour at 4°C and stained for 10 minutes with 40 mM Alizarin Red (pH 4.1) to visualize calcium deposition in the extracellular matrix. Images were acquired at 4× magnification on Nikon Eclipse TE200 (Melville, NY, USA) with Nikon Digital Camera DXM1200F using the Nikon ACT-1 software version 2.7. For adiogenic differentiation, ASCs were cultured in six-well plates in CCM until 70% confluent, and media was replaced with fresh media containing adipogenic supplements, consisting of 0.5 μM dexamethasone (Sigma), 0.5 mM isobuytlmethylxanthine (Sigma) and 50 μM indomethacin (Sigma). After three weeks, cells were fixed in 10% formalin for 1 hour at 4°C, stained for 10 to 15 minutes at room temperature with Oil Red O (Sigma) to detect neutral lipid vacuoles, and images were acquired at 10× magnification on Nikon Eclipse TE200 with Nikon Digital Camera DXM1200F using Nikon ACT-1 software version 2.7.

To determine the ability to form colony forming units (CFU), ASCs at passage 3 were plated at a density of 100 cells on a 10 cm2 plate in CCM and incubated for 14 days. Plates were rinsed three times with PBS, and 10 mL of 3% crystal violet (Sigma) was added for 30 minutes at room temperature. Plates were washed three times with PBS and once with tap water. Each experiment was performed in triplicate.

Analysis by flow cytometry of the cell surface marker profile was conducted by harvesting ASCs with 0.25% trypsin/1 mM EDTA for three to four minutes at 37°C. A total of 3×105 cells were concentrated by centrifugation at 500 x g for five minutes, suspended in 50 μl PBS and labeled with the primary antibodies. The following primary antibodies were used: Anti-CD45-PeCy7, anti-CD11b-PeCy5, anti-CD166-PE, anti-CD105-PE, anti-CD90-PeCy5, anti-CD34-PE, isotype-control FITC human IgG1 and isotype-control PE human IgG2a were purchased from Beckman Coulter (Indianapolis, IN, USA). Anti-CD44-APC was purchased from BD Biosciences (San Jose, CA, USA). The samples were incubated for 30 minutes at room temperature and washed three times with PBS. The samples were then analyzed with Galios Flow Cytometer (Beckman Coulter, Brea, CA, USA) running Kaluza software (Beckman Coulter). To assay cells by forward and side scatter of light, FACScan was standardized with microbeads (Dynosphere Uniform Microspheres; Bangs Laboratories Inc.; Thermo Scientific, Waltham, MA, USA). At least 10,000 events were analyzed and compared with isotype controls.

Breast cancer cell lines

MCF7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO), supplemented with 10% FBS and P/S. Cells were grown at 37°C with 5% humidified CO 2 , fed every two to three days, and split 1:4 to 1:6 when they reached 90% confluency.

Synthesis of GFP breast cancer cells

To produce retroviruses carrying green fluorescent protein (GFP) and neomycin resistance (neo), 293T cells were transfected by means of a modified calcium chloride transfection protocol when cells reached 90 to 95% confluency. The following amount of DNA was used to transfect cells on a 10 cm plate: 10 μg pMSCVneo-GFP vector, 10 μg pVPACK-Gp-dl packaging plasmid, and 10 μg pCI-VSV-G envelope-encoding plasmid. Twenty-four hours after transfection, cells were washed with PBS, replaced with fresh media, and collected after 48 hours. To transduce MCF7 or MDA-MB-231 cells, conditioned media containing retrovirus was added to MCF7 or MDA-MB-231 cells at 70% confluency. MCF7 cells were selected with 1 mg/ml of Genticin (Invitrogen, Carlsbad, CA, USA), while MDA-MB-231 cells were selected with 500 μg/ml of Genticin for two week and GFP expression was verified with flow cytometry.

Breast cancer cell and ASC co-culture

MCF7 cells or MDA-MB-231 cells were co-cultured with Ob-Ab- (n = 6), Ob-Ab+ (n = 6), Ob+Ab-(n = 6), or Ob+Ab+ (n = 6) ASCs (1:1 ratio) at 200 cells/cm2 in DMEM supplemented with 10% FBS and P/S. After seven days, cells were harvested, washed and analyzed by flow cytometry. The percentage of GFP positive cells (MCF7 cells or MDA-MB-231) was determined with Gallios Flow Cytometer running Kaluza software (Beckman Coulter, Brea, CA, USA) (Figure 1A). Where indicated, MCF7 cells were co-cultured with Ob-Ab-, Ob-Ab+, Ob+Ab-, or Ob+Ab+ ASCs (1:1 ratio) grown in CCM made with charcoal dextran stripped-FBS (CDS-FBS) with or without supplemental estrogen (E 2 ; 10 nM), leptin neutralizing antibody (R&D Systems; Minneapolis, MN, USA), or letrozole (Sigma). For RNA isolation, MCF7 cells were sorted after co-culture with the Becton-Dickinson FACSVantage SE Cell Sorter with DiVa option (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with the DiVa software v5.02 (BD).

Figure 1 Direct co-culture of breast cancer cells with ASCs result in increased proliferation in vitro . (A) After seven days of co-culture (CC) with six adipose stem cells (ASC) donors per group, quantification of MCF7 cells and MDA-MB-231 (231) cells was based on the percentage of GFP+ cells in the population multiplied by the total number of cells in each condition. (B) To determine the influence of ASCs on MCF7 cells indirectly, conditioned media (CM) from pooled ASCs (n = 6 donors per group) were collected and added to MCF7 cells. After seven days, the total number of MCF7 cells was counted. Values reported are the mean of three independent experiments, each performed in triplicate. Bars, ± SD. *, P <0.05; #, P <0.01. Full size image

ASC conditioned media

ASCs, pooled from six donors per group, were plated on at 150 cm2 culture dish at 100 cells/cm2. After overnight culture, media was replaced with serum free αMEM medium. After seven days, conditioned media was collected and filtered to remove debris. ASC conditioned media from each group was plated on top of MCF7 cells set up in triplicates. After seven days, the total number of MCF7 cells were counted with a hemocytometer. Three independent experiments were conducted, each in triplicate.

RT2profiler™ PCR arrays

Breast cancer PCR arrays

Total cellular RNA was extracted from FACS purified MCF7 cells after co-culture with a pool of ASCs (n = 6 per group) or MCF7 control cells (not co-cultured) using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with DNase I digestion (Qiagen) according to manufacturer’s instructions. One μg of RNA was converted to cDNA with the RT2 First Strand Kit (SABiosciences, Frederick, MD, USA) according to the manufacturer’s protocol. Gene expression profiling was performed using the Breast Cancer RT2 Profiler PCR Array (SABiosciences) and RT2 qPCR Master Mix (SABiosciences). PCR amplification was performed in a Bio-Rad CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows: 95°C for 10 minutes, 40 cycles of 95°C for 15 sec and 60°C for 1 minute, followed by a dissociation curve. At the completion of the reaction, C t values were determined, and ΔΔ C t and fold change were determined using the RT2 Profiler PCR Array Data Analysis web portal (SABiosciences). Genes whose mRNA levels increased or decreased more than two-fold in MCF7 cells after co-culture with ASCs relative to MCF7 cells without co-culture were considered differentially expressed (P <0.05).

Obesity PCR arrays

Ob-Ab- (n = 6), Ob-Ab+ (n = 6), Ob+Ab- (n = 6) or Ob+Ab+ (n = 6) ASCs were expanded in CCM and collected for RNA extraction using the RNeasy Mini Kit, and the total cellular RNA was treated with DNase I per the manufacturer’s instructions. One μg of RNA was converted to cDNA with the RT2 First Strand Kit according to the manufacturer’s protocol. Gene expression profiling was performed using the Obesity RT2 Profiler PCR Array (SABiosciences) and RT2 qPCR Master Mix. Reaction settings and analysis was conducted as described above. Genes whose mRNA levels increased or decreased more than two-fold (P <0.05) in MCF7 cells after co-culture with ASCs relative to MCF7 cells without co-culture were considered differentially expressed.

Western blot

MCF7 cells and FACS sorted MCF7 cells after co-cultured with ASC donors (n = 6 per group) were incubated in phosphatase and protease inhibitors (Pierce, Thermo Scientific, Rockford, IL, USA), lysed with RIPA buffer (Pierce), and centrifuged. Cell lysate was also obtained from Ob-Ab- (n = 6), Ob-Ab+ (n = 6), Ob+Ab- (n = 6), or Ob+Ab+ (n = 6) ASCs cultured in CCM made with charcoal dextrose stripped FBS (Atlanta Biologicals, Lawrenceville, GA, USA). Where indicated, ASCs were treated with 10 nM 17β-estradiol (E 2 , Sigma, St. Louis, MO, USA) and/or 100 nM ICI182,780 (Sigma), and cell lysate was obtained. A total of 20 μg of protein was fractionated on 4 to 12% SDS-polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). The blots were blocked with blØk Noise Canceling Reagents (Millipore, Billerica, MA, USA) and probed using primary antibodies incubated overnight at 4°C, washed with phosphate-buffered solution with 0.01% TWEEN-20 (PBST), followed by a secondary antibody conjugated to horseradish peroxidase (HRP), washed with PBST and visualized with chemiluminescence reagent (Invitrogen) on an ImageQuant LAS 4000 (GE Healthcare Life Science; Piscataway, NJ, USA).

Antibodies against cyclin dependent kinase inhibitor 2A (CDKN2A), estrogen receptor-alpha (ESR1), and progesterone receptor (PGR) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-secreted frizzled-related protein 1 (anti-SFRP1), anti-mouse-HRP, anti-rabbit-HRP antibodies were purchased from Abcam (Cambrige, MA). Anti-glutathione S-transferase P (anti-GSTP1) was purchased from Cell Signaling Technologies (Danvers, MA, USA), anti-actin was purchased from Sigma, and anti-leptin was purchased from R&D Systems.

In vivo tumorigenicity assay

All procedures involving animals were conducted in compliance with State and Federal law, standards of the US Department of Health and Human Services, and guidelines established by Tulane University Institutional Animal Care and Use Committee (IACUC). All protocols were approved by the Tulane IACUC.

SCID/beige (CB17.Cg-PrkdcscidLystbg-J/Crl) immunocompromised female ovariectomized mice (five weeks old) were obtained from Charles River Laboratories (Wilmington, MA, USA). Mice were divided into treatment groups of five animals, with or without estrogen: MCF7 only, MCF7 plus Ob-Ab- ASCs (n = 6 donors), MCF7 plus Ob-Ab+ ASCs (n = 6 donors), MCF7 plus Ob+Ab- ASCs (n = 6 donors), and MCF7 plus Ob+Ab+ ASCs (n = 6 donors). Where indicated, estradiol pellets (0.72 mg, 60-day release, Innovative Research of America, Sarasota, FL, USA) were implanted subcutaneously in the lateral area of the neck.

MCF7 cells (106) alone or MCF7 cells (106) in combination with ASCs (106) suspended in a total volume of 50 μl of sterile PBS were mixed with 100 μl of reduced growth factor Matrigel (BD Biosciences, Bedford, MA, USA). Cells were injected subcutaneously into the fifth mammary fat pad on both sides. All procedures in animals were carried out under anesthesia using a mixture of isoflurane and oxygen delivered by mask.

Tumor size was measured every three days using digital calipers and calculated as previously described [20]. At necropsy, animals were euthanized by cervical dislocation after exposure to CO 2 . Tumors were removed and frozen in liquid nitrogen or fixed in 10% neutral buffered formalin and paraffin embedded for further analysis.

Immunohistochemistry

Formalin-fixed, paraffin-embedded (FFPE) tumor sections were deparaffinized, rehydrated in a graded solution of Sub-X solutions, stained with hematoxylin and eosin or quenched with 0.3% H 2 O 2 (Sigma), rinsed with PBST, blocked with 1% BSA and stained with primary antibodies against Ki-67 (Abcam) or human progesterone receptor (PGR; DAKO North America, Inc., Carpinteria, CA, USA) overnight at 4°C. Each tumor section was subsequently washed in PBST, incubated with appropriate HRP-conjugated secondary antibody for one hour at room temperature, and washed with PBST. For colorimetric staining, slides were then incubated in 3,3'-Diaminobenzidine (DAB; Vector Laboratories; Burlingame, CA, USA), washed with PBST, counterstained with hematoxylin, and rinsed with deionized water. Slides were sealed with Permount Mounting Medium (Sigma). For apoptosis analysis, the TACS-XL in situ Apopotosis Detection Kit (R&D Systems) was used according to the manufacturer’s instructions. After staining, tumor sections were counterstained and sealed as mentioned above. Images were acquired at 10× and 40×. Quantification of the percentage of positivity was assessed using ImageScope (Aperio, Vista, CA, USA) and determined by the percentage of positive pixels divided by the total number of pixels in a given section.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Ob-Ab- (n = 6 donors), Ob-Ab+ (n = 6 donors), Ob+Ab- (n = 6 donors), or Ob+Ab+ (n = 6 donors) ASCs cultured in CCM were collected for total cellular RNA extraction using a RNeasy Mini Kit. Where indicated, ASCs were cultured in CCM containing charcoal dextrose-stripped FBS, with or without supplementation with 10 nM E 2 and/or 100 nM ICI182,780. RNA was then purified with DNase I digestion (Invitrogen), and reverse transcribed using the SuperScript VILO cDNA synthesis kit (Invitrogen). Quantitative real-time PCR was performed using the EXPRESS SYBR GreenER qPCR SuperMix Kit (Invitrogen) according to the manufacturer’s instructions. The following primer set sequence for leptin (forward 5′-gaagaccacatccacacacg-3′, reverse 5′-agctcagccagacccatcta-3′) and aromatase (forward 5′-cagaggccaagagtttgagg-3′, reverse 5′-acactagcaggtccctttgg-3′) were used. β-actin (forward 5′-caccttctacaatgagctgc-3′ and reverse 3′-tcttctcgatgctcgacgga-5′) was used as an internal reference point. At the completion of the reaction, ΔΔC t was calculated to quantify mRNA expression.

Oncomine analysis

A set of 440 normal breast tissues and invasive ductal carcinomas (IDC) deposited by The Cancer Genome Atlas (TCGA) was analyzed using the Oncomine Research Edition to assess leptin expression. Details of the standardized normalization techniques and statistical calculations can be found on the Oncomine website (http://www.oncomine.com).

KM plot analysis

To determine the five-year relapse-free survival of patients diagnosed with breast cancer based on leptin expression, an online survival analysis tool was utilized and can be found on the Kaplan-Meier Plotter website (http://www.kmplot.com). Details of the standardized normalization techniques and characterization of high or low expression have been previously described [21].

Statistical analysis

All values are presented as means ± standard deviation (SD). The statistical differences among two or more groups were determined by ANOVA, followed by post-hoc Dunnet multiple comparison tests versus the respective control group. The statistical differences between two groups were performed by Student’s t-test. Statistical significant was set at P <0.05. Analysis was performed using Prism (Graphpad Software, San Diego, CA, USA).