In vivo animal studies

All research involving animals was performed at the IDIBELL animal facility in compliance with protocols approved by the IDIBELL Committee on Animal Care and following national and European Union regulations. Mouse models used in this study have been previously described: MMTV-Rank+/tg (Rank+/tg) [2, 5], MMTV-Neu (Neu+/−) [14], MMTV-PyMT (PyMT+/−) [13] and double transgenic PyMT+/−Rank+/tg and Neu+/−Rank+/tg [18]. Mice were monitored for tumor formation three times per week and animals bearing tumors bigger than 1 cm in diameter were considered as endpoint criteria for sacrifice.

MECs and mammary tumor cell isolation

Single cells were isolated from mammary glands or tumors as previously described [18, 34]. Briefly, fresh tissues were mechanically dissected with McIlwain tissue chopper and enzymatically digested with DMEM/F12 (Gibco), 0.3% collagenase A (Sigma), 2.5 U/ml dispase (Sigma), 20 mM HEPES and Penicillin/Streptomycin (ThermoFisher Scientific) for 45 min at 37 °C. For MEC isolation, fibroblasts were excluded by incubation with DMEM high glucose containing 10% FBS (Gibco) for 1 h at 37 °C. Single MECs were then isolated by trypsin (Gibco) incubation for 2 min at 37 °C followed by an incubation with 2.5 U/ml dispase I, 20 U/ml DNase I (Roche) for 5 min at 37 °C. Cell aggregates were removed by filtering the cell suspensions with 40 μm cell strainers (BD Falcon). For tumor cell isolation, samples were treated with trypsin for 2 min at 37°C and cell aggregates were removed by filtering the cell suspension with 70 μm strainers (BD Falcon).

Orthotransplantation assays

For orthotopic transplants, basal (Lin− CD24lo CD49fhi) and luminal (Lin− CD24hi CD49flo) MECs isolated from mammary glands as detailed above were diluted 1:1 in matrigel matrix (Culteck) and injected in a final volume of 40 µL in the abdominal mammary fat pad of immunocompromised female mice (50,000 cells/gland from Neu+/− and Neu+/−Rank+/tg mice) or syngeneic females (2,000 cells/gland from PyMT+/− and PyMT+/−Rank+/tg). Mice were monitored for tumor incidence and latency and sacrificed when tumors reached a volume of 1 cm3.

Tumor limiting dilution assay

Mammary tumor cells from PyMT+/− and PyMT+/−Rank+/tg primary tumors were isolated as described above, pooled, diluted 1:1 in matrigel matrix (Culteck) and injected in a final volume of 40 µL. 1.000,000, 10,000, 1,000, 100 and 10 cells were injected in the abdominal mammary fat pads of 8-week-old syngeneic mice (FVB background) and tumor incidence was monitored along time.

Flow cytometry

Single cells were labeled with antibodies against CD24-PE or CD24-FITC (5 μg/mL, M1/69 BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com), CD29-FITC (1.25 μg/mL, HMb1-1, BD Pharmingen), CD49f-a647 (2.5 μg/mL, GoH3, BD Pharmingen), CD61-PE or CD61-FITC (2.5 μg/mL, 2C9.G2, BD Pharmingen), Sca1-APC or Sca1-PE (0.5 μg/mL, Ly-6A/E, BD Pharmingen), and CD49b-a647 (1.25 μg/mL, HMa2 Biolegend, San Diego, CA, http://www.biolegend.com). Lymphocytes and endothelial cells were excluded in flow cytometry using CD45-PECy7 (0,125 μg/mL, 30-F11 Biolegend) and CD31-PECy7 (0,5 μg/mL, 390 Biolegend) antibodies, respectively. Flow cytometry analysis was performed using FACS Canto (Becton Dickinson, San Jose, CA) and Diva software package. Cell sorting was performed using MoFlo (Beckman Coulter) at 25 psi and using a 100 mm tip.

Tissue histology and immunostaining

Tissue samples were fixed in formalin and embedded in paraffin and 3–5 μm sections were cut for histological analyses and stained with hematoxylin and eosin. Lung metastases were detected and counted based on nuclear morphology and similarity with primary tumors. 15–16 sections per lung were quantified in primary Neu+/−Rank+/tg and Neu+/− tumors. Immunostaining was performed on 3 μm tumor sections. After citrate antigen heat retrieval, slides were stained with CK8 (TROMA, dshl, Developmental Studies Hybridoma Bank, Iowa City, Iowa), CK5 (AF-138, Covance, Princeton, NJ) and CK14 (AF-64, Covance) antibodies followed by incubation with corresponding fluorochrome conjugated secondary antibodies (Life Technlogies) and DAPI (Sigma). Slides were mounted with Prolong ® Gold Antifade (Life Technologies) and analyses were carried out in a Leica TCS SP5 confocal microscope. Images were captured using Las AF Lite software (Leica).

Gene expression analysis

Total RNA of frozen tumor pieces was prepared with Tripure Isolation Reagent (Roche) following the manufacturer’s instructions. Frozen tumors tissues were fractionated using glass beads (Sigma) and PreCellys® tissue homogenizer (Berting Technologies). cDNA was produced by reverse transcription using 1 μg of RNA following kit instructions (Applied Biosystems). Quantitative PCR was performed using LightCycler® 480 SYBR green MasterMix (Roche) and the primers used were already published [18].

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Analysis of the differences between mouse cohorts or conditions was performed with a two-tailed Student’s t-test, one-way ANOVA or Chi-Square test. p values are annotated; n.s. not significant. Estimation of tumor-initiating cells in limiting dilutions was calculated using the extreme limiting dilution analysis (ELDA) [35].