Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Gerhard Christofori ( gerhard.christofori@unibas.ch ).

The patient-derived xenograft transplantation (PDX) mouse model of metastatic human breast cancer, described and characterized as x-3078 in (), was kindly provided by M. Bentires-Alj, DBM, University of Basel, Switzerland.

2-3 months old female RAG2 -/- ;common γ receptor -/- (RSG), NMRI nu/nu, or NOD-Scid;common γ receptor -/- (NSG) mice were used for tumor transplantation experiments. No statistical methods were used to predetermine sample size. For all experiments presented in this study, the sample size was large enough to determine statistically significant effects. Once transplanted tumor cells formed palpable tumors, mice were randomized and allocated to different treatment groups. The investigators were not blinded to allocation during the experiments, yet they were blinded to allocation during the outcome assessment. The mouse colony was housed in a certified animal facility with a 12 hr light/dark cycle in a temperature-controlled room (22 ± 1°C) with free access to water and food, in accordance with Swiss guidelines.

All animal experiments have been performed under approval by the Kantonales Veterinäramt Basel-Stadt under permit numbers 1878, 1907 and 1908.

Py2T (), MTflECad, MTΔECad (), and ZsGreen 4T1 () were generated in the laboratory of G. Christofori (University of Basel) and previously described. MDA-MB-231 LM2 GFP () were obtained from J. Massague (Memorial Sloan Kettering Cancer Center, New York, NY, USA) and 3T3-L1 cells () were obtained from B. Deplancke (EPFL, Lausanne). Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Sigma-Aldrich) supplemented with fetal calf serum (FCS, 10%; Sigma-Aldrich), glutamine (2 mM; Sigma-Aldrich), penicillin (100 U; Sigma-Aldrich) and streptomycin (0.2 mg/l; Sigma-Aldrich). All cell lines were grown at 37°C, 5% CO, 95% humidity.

Method Details

In Vitro Adipogenesis Cells were seeded at density of 20,000 cells/cm2 and incubated overnight at 37°C in 5% CO 2 . To induce differentiation of 3T3-L1, the growth medium was replaced with medium containing 5 μg/ml insulin, 1 μM Dexamethasone (dissolved in ethanol), 2 μM Rosiglitazone (dissolved in DMSO) for 48 hr, then in medium containing 5μg/ml insulin for 48 hr, then in medium containing 1 μM Rosiglitazone. To induce differentiation in cancer cells, cells were seeded and incubated overnight and then treated with 200 ng/ml human recombinant BMP2 (Sigma, B3555) for 3 days, with 200 ng/ml BMP2 and 2μM Rosiglitazone (Rosi) for 4 days, and with medium containing 2 μM Rosiglitazone for another 3 days. The addition of other growth factors or inhibitors is indicated in the figures, from day 0 to day 7 such as human recombinant TGFβ1 (R&D Systems, 240-B) at 2 ng/ml, and the MEK inhibitors PD98059 at 40 μM and Trametinib as indicated, and the TGFβ receptor 1 inhibitor SB431542 at 10 μM. Control cells were treated with medium containing DMSO.

In Vitro Chondrogenesis Cells were seeded at a density of 60,000 cells/cm2 in normal growth medium (DMEM supplemented with glutamine, penicillin, streptomycin and 10% FBS) and incubated at 37°C in 5% CO 2 overnight. Medium was removed and cells were washed with serum free Opti-MEM media (to remove traces of serum). Differentiation medium: Opti-MEM serum free media (Gibco) containing 1% ITS (BD), 10 μM Dexamethasone (Sigma Aldrich), 100 μM Ascorbate (Sigma Aldrich) and 10 ng/ml human recombinant TGFβ1. One day later, medium was removed, and cells were washed. From day 2 to day 21, differentiation medium was replaced every 48 hr with medium containing 10 ng/ml human recombinant BMP2 and BMP4. For control cells, normal culture medium was used.

In Vitro Osteogenesis Cells were seeded at density of 20,000 cells/cm2 in normal growth medium (DMEM supplemented with glutamine, penicillin, streptomycin and 10% FBS) and incubated at 37°C in 5% CO 2 for 48 hr. From day 2 until day 21 differentiation medium was used: Supplemented DMEM containing 1mg/ml β-Glycerophosphate (Calbiochem), 100 nM Dexamethasone and 100 μM L-Ascorbate, 1mM Sodium Pyruvate (Sigma) and 10ng/ml human recombinant BMP4 was added at each time point. Control cells were incubated with culture medium.

RNA Isolation and RT-PCR Total RNA was prepared using TriReagent (Sigma-Aldrich) for cells or RNeasy mini kit (74104, Qiagen) for tissues. For RNA sequencing, total RNA was isolated using the miRNeasy mini kit (217004, Qiagen). RNA was reverse transcribed with M-MLV reverse transcriptase (Promega), and transcripts were quantified by PCR using SYBR-green PCR MasterMix (Invitrogen). Riboprotein L19 primers were used for normalization. PCR assays were performed in triplicate, and fold induction was calculated using the comparative Ct method (ΔΔ Ct). Primers used for quantitative RT-PCR are listed in the Key Resources Table

Immunoblotting Cells were lysed in boiling lysis buffer (0.29 M Tris-HCl pH 6.8, 4.7% SDS, 23% glycerol). Protein concentration was determined using the BCA assay kit (Pierce). Equal amounts of protein were diluted in SDS-PAGE loading buffer (10% glycerol, 2% SDS, 65 mM Tris, 1 mg/100 ml bromophenol blue, 1% β-mercaptoethanol) and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose, 0.45 μM pore size membranes by wet transfer, blocked with 5% skim milk powder in TBS/0.05% Tween 20 and incubated with the antibodies indicated. HRP-conjugated secondary antibodies were detected by chemiluminescence using a Fusion Fx7 chemiluminescence reader (Vilber Lourmat, France).

siRNA Transfection 20 nM siRNAs (Ambion) against Zeb1 (s74841), Zeb2 (s76954, s76956 10nM each), Klf4 (s68835, s68836, s68837 6.66nM each), Snail1 (s74062) or a negative control (4390846) were prepared using Lipofectamine RNAiMax (Invitrogen) for the transfection according to the manufacturer’s instructions. Cells were seeded and incubated with siRNA. After 24 hr (day 1) medium was changed to medium containing BMP2 (treated) or normal medium (no treatment) according to in vitro adipogenesis protocol. 48 hr later (day 3) medium was changed to adipogenesis or control medium and siRNA transfection was repeated. Cells were harvested at day 5.

Adiponectin Secretion Supernatants of untreated cells (day 0) as well as control-treated cells and cells treated with the adipogenesis protocol for 10 days were harvested. Adiponectin concentrations were determined using the Adiponectin Mouse ELISA Kit (ab108785, Abcam) according to the manufacturer’s protocol.

Lipolysis Analysis Lipolysis was induced using Isoproterenol (final concentration 100 nM) in untreated cells (day 0) as well as in control-treated cells and cells treated with the adipogenesis protocol for 10 days. Glycerol release was measured using a Lipolysis Assay Kit (Colorimetric) (ab185433, Abcam) according to the manufacturer’s protocol.

Extracellular Metabolic Flux Analysis Gubser et al., 2013 Gubser P.M.

Bantug G.R.

Razik L.

Fischer M.

Dimeloe S.

Hoenger G.

Durovic B.

Jauch A.

Hess C. Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch. For analysis of metabolic signatures, a Seahorse XFe-96 metabolic extracellular flux analyzer was used (Seahorse Bioscience, North Billerica, MA, USA). Adipogenesis-treated (10 days) and undifferentiated control cells were used for metabolic analysis. Prior to performing the metabolic assays, differentiation media was exchanged with serum-free, unbuffered basal medium supplemented with 25 mM glucose and 2 mM glutamine (Seahorse, MA, USA). Mitochondrial perturbation profiling was used to determine mitochondrial respiratory and glycolytic parameters by sequential addition of oligomycin (1 μM), Carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone (FCCP) (0.6 μM), and rotenone/antimycin A (1 and 2 μM, respectively). Metabolic parameters were calculated as previously described ().

GLUT4 Translocation Control cells and cells treated with the adipogenesis protocol were incubated for 25 min with 100 nM insulin or normal growth medium and subsequently visualized for the expression and localization of GLUT4 by confocal microscopy (Leica SP5).

EdU Incorporation to Detect Proliferating Cells Cells before and after a full ten-day adipogenesis protocol were incubated for 24 hr with 1 μM EdU (Invitrogen) or 72 hr with 0.1μM EdU. Cells were fixed with 4% PFA for 20 min, washed with PBS, permeabilized with 0.5% NP40 and washed with PBS-T (PBS with 0,01% TX-100). ClickIT reaction was performed according to the manufacturer’s protocol (BaseClick), cells were washed with PBS-T and used for immunostaining. Images were acquired with a fluorescence Leica DMI 4000 microscope. For each replica, at least 7 fields were quantified using intensity quantification function in ImageJ software.

Immunofluorescence Microscopy Analysis Cells Cells were fixed with 4% PFA and permeabilized with 0.5% NP40. Cells were blocked with 3% BSA and incubated with primary antibody for 1.5 hr at room temperature or overnight at 4°C. After washing, cells were incubated with secondary antibodies for 1 hr at room temperature, washed and incubated with DAPI for 10 min, and washed and mounted with fluorescent mounting medium (Dako). Samples were imaged with a confocal microscope (LSM 510 Meta, Zeiss or Leica SP5) or with a fluorescence microscope (Leica DMI 4000). Cells expressing C/EBPα (Cat# 8178P) were manually quantified with ImageJ software for at least 5 fields per replica.

RNA Sequencing Analysis Total RNA was isolated from cells of two independent experiments using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction. RNA quality control was performed with a fragment analyzer using the standard or high sensitivity RNA analysis kit (DNF-471-0500 or DNF-472-0500) from Labgene and RNA concentration was measured by using the Quanti-iT™ RiboGreen RNA assay kit (Life Technologies/Thermo Fisher Scientific). 200 ng of RNA was utilized for library preparation with the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina). Poly-A+ RNA was sequenced by HiSeq SBS kit v4 (Illumina) on an Illumina HiSeq 2500 using protocols defined by the manufacturer. Dobin et al., 2013 Dobin A.

Davis C.A.

Schlesinger F.

Drenkow J.

Zaleski C.

Jha S.

Batut P.

Chaisson M.

Gingeras T.R. STAR: ultrafast universal RNA-seq aligner. Obtained single-end RNA-seq reads (63-mers) were mapped to the mouse genome assembly, version mm10, with RNA-STAR (), with default parameters except for allowing only unique hits to genome (outFilterMultimapNmax=1) and filtering reads without evidence in spliced junction table (outFilterType="BySJout"). Using RefSeq mRNA coordinates from UCSC ( genome.ucsc.edu , downloaded in December 2015) and the qCount function from QuasR package (version 3.12.1), we quantified gene expression as the number of reads that started within any annotated exon of a gene. The differentially expressed genes were identified using the edgeR package (version 1.10.1). Genes with FDR smaller than 0.05 and minimum log2 fold change of +/- 1 were used for downstream analysis.

Comparative Analysis Al Adhami et al., 2015 Al Adhami H.

Evano B.

Le Digarcher A.

Gueydan C.

Dubois E.

Parrinello H.

Dantec C.

Bouschet T.

Varrault A.

Journot L. A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation. We compared our RNA-sequencing data with already published RNA-sequencing data obtained from 3T3-L1 cells (). The batch effects correction was performed on the gene expression data using ComBat (PMID: 16632515 ). We then computed spearman rank correlation coefficients between the data sets and the heatmap was generated using hierarchical clustering (hclust function) in heatmap2. All the above-mentioned computations were performed in R (R-3.3.1) version.

Regulatory Motif Analysis Integrated System for Motif Activity Response Analysis (ISMARA) (PMID: 24515121 ) prediction tool was employed to identify regulatory motifs enriched in MTΔECad undergoing adipogenesis at different time points (0 hr, 6 hr, 3 days, 5 days, 7 days and 10 days) RNA-seq data. The data provided for ISMARA analysis was in FASTQ format and selected mm10 version as reference genome.

Functional Enrichment Analysis and Heatmaps We performed functional enrichment analysis of differentially expressed genes for biological processes or pathways in R (3.3.1) using several publically available Bioconductor resources including GO.db (version 3.4.0), GOstats (version 2.40.0) (PMID: 17098774 ), KEGG.db (version 3.2.3) and ReactomePA (version 1.18.1) (PMID: 26661513 ). The significance of each biological processes or pathways identified was calculated using the hypergeometric test (equivalent to Fisher’s exact test) and those with p values ≤0.05 were considered significant. Heatmaps were generated in R (3.3.1) using packages RColorBrewer (1.1-2) and gplots (3.0.1) using normalized CPM (counts per million) data obtained for RNA-sequencing experiments.

In Vivo Adipogenesis For primary tumor transplantation experiments, 0.5x106 GFP-expressing Py2T cells were injected into the mammary fat pad of female RAG2-/-;common γ receptor-/- (RSG) mice. 2 weeks after injection, mice were treated daily with i.p injections of drugs or vehicle for two weeks. High dose group, 5 mg/kg PD98059 and 16 mg/kg Rosiglitazone dissolved in vehicle; low dose group, 2 mg/kg PD98059 and 16 mg/kg Rosiglitazone dissolved in vehicle; control group, vehicle alone (4% DMSO, 30% PEG300, 5% Tween 80). For tumor invasion and metastasis experiments, female NMRI nu/nu mice were injected into the mammary fat pad with 0.5x106 ZsGreen-expressing 4T1 cells and female NOD-Scid;common γ receptor-/- (NSG) mice were injected into the mammary fat pad with 1x106 GFP-expressing MDA-MB-231 LM2 cells. MDA-MB-231 LM2 cells were obtained from J. Massague (Memorial Sloan Kettering Cancer Center, New York, NY, USA) and prepared separately with PBS 1:1 mix, and suspended with Cultrex (R) BME 2 RGF (ORGANOID MATRIX) PathClear (R)∗ (TRVIGEN/AMSBIO) before implantation. After initial tumor growth, tumor volume was measured, and mice were divided into four treatment groups as follows: Group 1: Control treated with vehicle (0.5% hydroxypropyl methylcellulose and 0.2% Tween 80 in distilled water (pH 8.0) and 0.7% DMSO). Group 2: Rosiglitazone (20 mg/kg) dissolved in vehicle. Group 3: Trametinib (0.3 mg/kg) dissolved in vehicle. Group4: Rosiglitazone (20 mg/kg) and Trametinib (0.3 mg/kg) dissolved in vehicle. Mice were treated daily p.o. for three weeks. Animals were sacrificed, tumor volumes and weights were measured, and tumors and lungs were embedded in paraffin for H&E staining and OCT for immunofluorescence staining and frozen for protein isolation. All studies involving mice have been approved by the Swiss Federal Veterinary Office (SFVO) and the Cantonal Veterinary Office of Basel Stadt (licenses 1878, 1907, and 1908).

PDX Human Breast Cancer Model Gao et al., 2015 Gao H.

Korn J.M.

Ferretti S.

Monahan J.E.

Wang Y.

Singh M.

Zhang C.

Schnell C.

Yang G.

Zhang Y.

et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. 3 pieces. Tumor pieces were maintained in PBS until transplantation. 8-13 weeks-old NSG (female) mice were anesthetized with Isofluoran, and tumors were transplanted into the 9th mammary fat pad. The wounds were closed with metal clips, and the animals were placed under infrared light until full recovery. For pain relief, Meloxicam was administered (5 mg/kg body weight per day) for two days. Four weeks post-transplantation, the mice were divided into four cohorts and treatment was initiated as described (control, Rosiglitazone, Trametinib, and a combination of Rosiglitazone and Trametinib). Eight weeks post-transplantation, mice were anesthetized with Isofluoran, and tumors were surgically removed, measured and embedded in paraffin and OCT. Mice recovered for five days prior to treatment continuation. Animal health was monitored daily. Four months post-transplantation, mice were euthanized, and lungs isolated and embedded in paraffin for H&E staining and OCT for immunofluorescence staining. The patient-derived xenograft transplantation (PDX) mouse model of metastatic human breast cancer, described and characterized as x-3078 in (), was kindly provided by M. Bentires-Alj, DBM, University of Basel, Switzerland. A tumor propagated in NSG female mice was dissected and cut in app. 1 mmpieces. Tumor pieces were maintained in PBS until transplantation. 8-13 weeks-old NSG (female) mice were anesthetized with Isofluoran, and tumors were transplanted into the 9th mammary fat pad. The wounds were closed with metal clips, and the animals were placed under infrared light until full recovery. For pain relief, Meloxicam was administered (5 mg/kg body weight per day) for two days. Four weeks post-transplantation, the mice were divided into four cohorts and treatment was initiated as described (control, Rosiglitazone, Trametinib, and a combination of Rosiglitazone and Trametinib). Eight weeks post-transplantation, mice were anesthetized with Isofluoran, and tumors were surgically removed, measured and embedded in paraffin and OCT. Mice recovered for five days prior to treatment continuation. Animal health was monitored daily. Four months post-transplantation, mice were euthanized, and lungs isolated and embedded in paraffin for H&E staining and OCT for immunofluorescence staining.

Immunofluorescence Microscopy Analysis Tissues Tumors were fixed in 4% PFA for 2 hr followed by overnight incubation in 20% sucrose to cryopreserve the tissue, both at 4°C. Then, tumors were snap frozen in Tissue-Tek OCT compound (Thermo Scientific) and stored at -80°C. 7 μm thick tumor sections were cut, dried for 30 min, rehydrated with PBS, permeabilized with 0.2% Triton X-100 for 20 min and blocked with 5% normal goat serum (NGS; Sigma-Aldrich) for 1 hr. As an exception, when performing stainings with anti-cCasp3 antibodies, blocking was performed using 20% NGS. Subsequently, primary and secondary antibodies were diluted in blocking solution and incubated overnight at 4°C and 1 hr at room temperature, respectively. Nuclei were stained with 4′,6-Diamidin-2-phenylindol (DAPI; Sigma-Aldrich) followed by mounting the slides with Dako mounting medium (Dako). For adipocyte images, 80 μm thick tumor sections were cut and treated as indicated previously with the exception of 2 hr blocking duration and 6 hr of secondary antibody staining. For CD31, cleaved Caspase-3, PH3 and E-cadherin staining, images were acquired with a Leica DMI 4000 microscope. For Perilipin, H2Kd, HLA staining, images were taken with a confocal spinning disc microscope with CFI Plan Apo 20x, 0.75 NA objective lens (Nikon) with VisiView software (Visitrion). 3D reconstruction images were generated using Imaris’ Surface function (Imaris x64 9.1.2).

Blood Vessel Density To determine blood vessel density, CD31+ vessels were quantified in 8-15 microscopic fields per tumor (depending on the tumor size) normalized to the respective tumor area.

Cell Proliferation and Apoptosis Areas of cells positive for phospho-histone 3 (PH3; proliferating cells) or cleaved caspase-3 (cC3; cells undergoing apoptosis) were quantified in 8-20 microscopic fields per tumor (depending on the tumor size) and were normalized to the respective area of Zs-Green positive tumor cells.

Lung Metastasis Quantification Histological lung sections were stained with hematoxylin and eosin (H&E) and imaged using an Axio Imager scanning microscope (Zeiss), and the number of lung metastasis per section was quantified using VIS software (Visiopharm https://www.visiopharm.com ).

BODIPY Staining on Frozen Sections Cryosections (7 μm or 80 μm thick) were rehydrated in PBS and incubated with BODIPY in the dark at room temperature for 30 min. Sections were washed and incubated with DAPI for 10 min, then washed and mounted with fluorescent mounting medium (Dako). Images were taken by laser scanning confocal microscopy (Leica SP5). Representative images for adipocytes expressing GFP were prepared with IMARIS 8.2.1 software.