Adipocytes promote breast tumorigenesis through TAZ-dependent secretion of Resistin

Significance Adipocytes are the most abundant and perhaps most active components of the tumor microenvironment in obese individuals that potentiate breast tumorigenesis through secretory mechanisms. The modulation of adipocytes can be novel therapy targets for breast cancer. Here, we revealed a specific upregulation of adipocytic TAZ through the FFA/PPARγ axis in diet-induced adiposity. Adipocytic TAZ knockdown or deficiency in mice inhibits adipocyte-induced breast cancer proliferation and stemness through impaired expression and secretion of Resistin. Immunostaining in triple-negative breast cancer samples showed that higher adipocytic TAZ/Resistin expression associates with higher clinical stages and poorer survival, demonstrating promising therapeutic targets.

For differentiation of human preadipocytes, cells were grown to confluence, and induced in medium containing 0.5 mM isobutyl-methylxanthine, 10 µM dexamethasone, 10 μM insulin, 0.2 nM triiodothyronine (T3) (#T-2752, Sigma-Aldrich) for four days and maintained in medium with 10 μM insulin for one day. The treatment was repeated three times, after which the cells were maintained in DMEM with 10 μM insulin until day 21 and subjected to oil red O staining to detect cytoplasmic triglyceride for mature adipocyte verification.

Dual luciferase reporter system
Mouse TAZ and Resistin promoter were cloned into pGL3-luciferase reporter vector (Promega, Madison, WI, USA). The luciferase reporter plasmid and pCMV-Renilla plasmid were transiently transfected into 293T cells, 6 hr after transfection, cells were treated with free fatty acids or vehicle for indicated concentration if needed, 12 or 16 hr after treatment, 293T cells were lysed and the fluorescence values were measured as the manufacturer suggested.

Free fatty acid preparation
As reported (2)(3)(4), free fatty acid stock solution was prepared by conjugating free fatty acid with fatty acid-free BSA. In detail, 100 mM free fatty acid stock solution was dissolved in 100 mM NaOH by heating at 70 °C (SA at 90°C). The stock solution was then appropriately diluted in prewarmed 20% BSA solution (final BSA concentration 0.5% (w/v)) when use. Vehicle (control medium) contained 100 mM NaOH and BSA without lipid.

Flow cytometry of ALDH + cells
E0771 cells were washed with phosphate-buffered saline (PBS) and then detached with trypsin/ (0.05% EDTA). Detached cells were washed twice with PBS containing 10% FBS (wash buffer) and resuspended in the wash buffer (10 6 cells/100 μL). For Aldefluor assay, E0771 cells were centrifuged and resuspended in Aldefluor assay buffer containing ALDH substrate (BODIPYaminoacetaldehyde) and incubated at 37°C for 45 min. A specific inhibitor of ALDH, diethylaminobenzaldehyde (DEAB), was used as a negative control (#01700, Stem Cell Technologies, Vancouver, BC, Canada). The labeled cells were washed once and resuspended in wash buffer, and then analyzed on a FACS Vantage (BD Biosciences, San Jose, CA, USA).

Cellular ATP determination
Breast cancer cells were plated into a 96-well plate at a low density of about 10 %, 24 hr later, the supernatant was aspirated and cellular ATP level was determined using a detection kit (#G9241, Promega) as the manufacturer suggested and recorded as the hour 0, the rest cells were then washed and cultured with Fibro-CM or TAZ knockdown-Adipo-CM, every 12 hr, the cellular ATP levels were measured and recorded.

Glucose tolerance tests and insulin tolerance tests
For glucose tolerance tests (GTT), animals were fasted for 16 hr overnight (17:00 pm-09:00 am) with free access to drinking water. The glucose level was assessed following glucose injection (2.0 g/kg) intraperitoneally. Serum glucose levels were determined immediately before and 15, 30, 60 and 120 min after glucose injection using a glucometer (OneTouch Ultra, Bayer, Berlin, Germany). For insulin tolerance tests (ITT), mice were fasted for 4 hr (9:00 am-13:00 pm) and then injected with human insulin (0.75 U/kg for C57BL/6J background mice) intraperitoneally. Blood glucose levels were determined immediately before and 15, 30, 60 and 120 min after insulin injection.

ELISA for Human and mouse Resistin
Human Resistin ELISA kit was purchased from GenStar (#C630-02, Beijing, China), Mouse Resistin ELISA kit was from Invitrogen (# EMRETN). Samples were measured as the manufacturer suggested. Briefly, serum or supernatant samples were diluted with dilution buffer and added into the capture antibody precoated 96-well plates followed by incubated in 37°C or room temperature for 90 or 150 min. The plates were then washed 4 times with wash buffer for 2 min each, followed by adding biotinylated capture antibody and incubated in 37°C or room temperature for 60 min. After washing for 4 times, the plates were added with Horseradish Peroxidase working buffer in 37°C or room temperature for 30 or 45 min. Finally, each well was added with 100 µl tetramethylbenzidine substrate and incubated at room temperature for 10 min in the dark. The reaction was stopped by adding 100 µl of stop buffer to each well and the OD value was measured at 450 nm immediately by an ELISA reader (Magellan, Tecan Group AG, Männedorf, Switzerland).

RNA isolation and RT-qPCR
Cultured cells were lysed by TRIzol TM Reagent (#15596018, Invitrogen) (adipose tissues and other tissues were cut into pieces in TRIzol and immediately mashed together until homogenous with a homogenizer), total RNA was isolated according to the manufacturer's instructions. mRNA was converted to cDNA with the cDNA synthesis kit (AE311-03, TransGen Biotech, Beijing, China). RT-qPCR was performed with diluted cDNA (1:4) in three wells for each primer and SYBR green master mix (Bio-Rad) on Bio-Rad iCycler iQ Real Time PCR system. All RT-qPCR experiments were repeated at least three independent times. Primers used are listed on SI Appendix, Table S4.

Conditioned medium system and mass spectrometry
The medium of 3T3-L1 fibroblast or 3T3-L1 adipocyte were removed, the plates were washed two times with sterile PBS. 4 mL serum-free DME containing 2% fatty acid free-BSA per 10 cm dish was added to the plate and incubated for 12 hr. After incubation, the medium was collected and centrifuged at 500 × g and filtered through a 0.22 μm filter to remove cellular debris. The conditioned medium was then incubated with 4T1 or E0771 cells for 0.5 hr to detect the changes of intrinsic signaling pathways. For crystal violet staining, cells were cultured in conditioned medium supplemented with 0.2% FBS for 48-72 hr and then fixed with 4% paraformaldehyde solution and stained with 0.2% crystal violet solution.
For mass spectrometry assay, the confluent 3T3-L1 fibroblast or TAZ siRNA transfected adipocytes in 10 cm dish were washed twice with PBS and directly cultured in 4 mL serum-free DMEM for 12 hr. After centrifugation and filtration through a 0.22 μm filter, the medium was flash-frozen in liquid nitrogen and freeze-dried for 3 days using ALPHA 1-2 LD plus freeze dryer (Christ, Osterode, Germany). The dry power from 3T3-L1 fibroblast or adipocyte was dissolved in sterile PBS at 200 mg/mL. 10 mg of each sample was loaded to SDS-PAGE gel for silver staining, and the bands under 55 kDa were subjected to mass spectrometry sequencing and data analysis. Briefly, the gels were minced and destained with 50% acetonitrile in 50 mM ammonium bicarbonate and then added 10 mM DTT to reduce proteins at 56℃, followed by alkylation with 55 mM iodoacetamide at room temperature in the dark. After that, the samples were trypsin digested overnight at 37℃ with gentle shaking. Peptides were then extracted by using 1% trifluoroacetic acid in 50% acetonitrile followed by vacuum-dried and reconstituted in Immunoneutralization 3T3-L1 fibroblast or adipocyte conditioned medium was incubated with Resistin-neutralization antibody (PeproTech, 10 μg/mL) or the same amount of goat IgG control at 4°C overnight to make it neutralized thoroughly by inversions, after centrifugation at 500 × g, the supernatant was placed on 4T1 or E0771 cells for 0.5 hr to detect the phosphorylation level changes of growth signaling molecules.
The diameter of sphere was measured using Image J software (NIH, USA).

Isolation of adipocytes and immune cells
Primary adipocytes and immune cells were isolated according to the published methods (5,6).
In brief, Mammary adipose tissue was resected and digested in DMEM/F12 medium containing 0.2% (wt/vol) collagenase type I and 0.1% (wt/vol) BSA at 37 °C with shaking for 1 hr, after dispersion, mature adipocytes were floated on the surface. After three times further gentle dispersion, washes and centrifuge, the pure adipocytes were yielded. The debris containing immune cells, fibroblast and undigested adipose tissue was further separated, filtered through a 250-μm mesh filter and labeled with anti-CD45.1-FITC antibody (#110706, Biolegend, San Diego, CA, USA) for FACS sorting and analysis.

Oil Red O staining
The Oil Red O stock was prepared by 0.5 g Oil Red O (Sigma-aldrich, # 0-0625) in 100 mL isopropanol. Mature adipocytes were rinsed with PBS and then fixed by 4% formaldehyde for 15 min at room temperature. Fresh Oil Red O working solution was prepared by adding 6 mL stock to 4 mL distilled water and then filtered through a 0.22-μm filter. The mature adipocytes were rinsed with PBS for 3 times, and then added Oil Red O working solution for 30 min at 37 °C. The cells were rinse several times carefully with distilled water to remove excess stain and any precipitate that forms and dried to be scanned.

Immunohistochemical staining and evaluation
Tissues were formalin-fixed, paraffin-embedded and then sectioned. After de-paraffinization and rehydration, antigen retrieval was performed with 1 mM EDTA solution (pH 9.0) in a pressurized decloaking chamber for 3 min at 120°C. After peroxidase blocking, the slides were treated with 10% normal goat serum for 1 hr at room temperature followed by the primary For Ki67 staining analysis, periadipocyte areas were selected as 1 mm radially inward in TNBC samples or 500 µm radially inward in mice tumors along the cancer-adipocyte interface, periphery (no adipocyte) areas were selected as 1 mm radially inward in TNBC samples or 500 µm radially inward in mice tumors from the edge of tumor without adipocytes, interior areas were selected as in tumor interior and 1 mm or 500 µm distant from periadipocyte areas and periphery (no adipocyte) areas in TNBC or mice tumors. 5-10 20-fold fields were randomly selected to quantify the proportion of Ki67+ cells according to the size of the area, the average of all fields in an area was shown in the results.

EdU incorporation assay
The EdU incorporation assay was performed using a Cell-Light EdU Apollo 488 In Vitro Imaging Kit (Ribobio, Guangzhou, China) as previously described (7). Briefly, treated 4T1 and E0771 cells were cultured with 20 μM EdU for 2 hr and then fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were then washed with 2 mg/mL glycine for 5 min followed by washed twice with PBS contained 0.5% Triton X-100. 100 µL Apollo 567 stain reaction buffer was added into the cells and incubated for 30 min in dark. The cells were then washed three times with PBS contained 0.5% Triton X-100 and stained with 100 µL Hoechst 33342 (5 mg/mL) for 30 min at room temperature. EdU-labeled cells were counted in 8 randomly selected fields under a fluorescent microscope.

Chromatin Immunoprecipitation
Totally differentiated 3T3-L1 adipocytes in 15 cm dish were crosslinked with 1% formaldehyde for 15 min at room temperature. Cells were rinsed with ice-cold PBS and lysed in cell lysis buffer

Allograft experiments
Fresh E0771 cells were resuspended in PBS at a density of 5*10 6 /mL. 100 µL cell suspension were inoculated into the mammary fat pad of the thoracic (glands # 2) mammary glands in CD/HFD female mice through a 22-G needle. 6 days after injection, the length and width of the mammary tumors were measured with sliding calipers every day. Tumor volume in cm 3 was calculated by the formula: Volume = (width) 2 × length/2. Mice were sacrificed at day 13 when the length of the tumor was less than 1 cm and tumors were resected. Tumor weight was then determined and fixed in 4% paraformaldehyde solution for immunohistochemistry. For Resistin antibody therapy assay, the neutralizing antibody was purified from Resistin peptide (EAIDKKIKQDF-BSA) immunized New Zealand White rabbits by antigen affinity purification.
After tumor injection, the antibody in PBS was injected into the mammary fat pad on days 2, 4, 8 and 12 at 0.75 mg/kg body weight. The control group was injected the same volume and amount of rabbit IgG in PBS. In all assays, p < 0.05 was considered statistically significant and was annotated throughout as *p < 0.05, **p < 0.01, ***p < 0.001.  Heatmap of differentially expressed genes that encode secreted proteins in CD-and HFD-MAT.

SI Appendix, Figure Legends
(K) RT-qPCR analysis of differentially expressed genes that encode secreted proteins in CDand HFD-MAT after 12 weeks feeding. (L) RT-qPCR analysis of TAZ in CD-and HFD-MAT after 12 weeks feeding. n = 12 in each group. Data shown are mean ± SEM. Data were analyzed using Student's t test (A, B, F-tumor-weight, H, K, L) and two-way ANOVA (C, F-tumor-volume).
Mammosphere numbers were counted and analyzed (right). Data shown are mean ± SEM.