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0008-5472.CAN-15-3490.Full.Pdf Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-15-3490 Cancer Tumor and Stem Cell Biology Research Cancer Stem Cells Regulate Cancer-Associated Fibroblasts via Activation of Hedgehog Signaling in Mammary Gland Tumors Giovanni Valenti1, Hazel M. Quinn1, Guus J.J.E. Heynen1, Linxiang Lan1, Jane D. Holland1, Regina Vogel1, Annika Wulf-Goldenberg2, and Walter Birchmeier1 Abstract Many tumors display intracellular heterogeneity with sub- activation of Hedgehog signaling. CAFs subsequently secrete sets of cancer stem cells (CSC) that sustain tumor growth, factors that promote expansion and self-renewal of CSCs. recurrence, and therapy resistance. Cancer-associated fibro- In vivo treatment of tumors with the Hedgehog inhibitor blasts (CAF) have been shown to support and regulate CSC vismodegib reduce CAF and CSC expansion, resulting in an function. Here, we investigate the interactions between CSCs overall delay of tumor formation. Our results identify a novel and CAFs in mammary gland tumors driven by combined intracellular signaling module that synergistically regulates activation of Wnt/b-catenin and Hgf/Met signaling in mouse CAFs and CSCs. Targeting CAFs with Hedgehog inhibitors mammary epithelial cells. In this setting, CSCs secrete the may offer a novel therapeutic strategy against breast cancer. Hedgehog ligand SHH, which regulate CAFs via paracrine Cancer Res; 77(8); 1–14. Ó2017 AACR. Introduction and skin (10, 11). Mesenchymal stem cells (MSC) and immune cells establish permissive growing conditions for CSCs in gastric Tumor heterogeneity is believed to be dependent on a distinct and intestinal tumors (12, 13). In breast cancer, a number of subset of tumor cells that possess the capacity to sustain tumor cellular components of the microenvironment have been growth, referred to as cancer stem cells (CSC) or tumor-initiating reported to regulate CSCs, such as MSCs, macrophages, and cells (1). CSCs have the ability to self-renew, retaining their cancer-associated fibroblasts (CAF; refs. 14–19). features over rounds of cell divisions and to differentiate into CAFs are major cellular components in the stroma of breast multiple cell types, placing them at the top of tumor cell hierarchy. cancers and are involved in many aspects of tumor progression, These properties appear to enable CSCs to resist chemotherapy, from the in situ growth of primary tumors to the metastatic spread thereby facilitating relapse (2). CSCs express selective markers at of cancer cells (17–19). The hypothesis has recently been put the cell surface and are identified on the basis of their ability to forward that CSCs and CAFs reside preferentially at the tumor– propagate tumors when serially transplanted into recipient mice stroma interface and that both cell types interact with and support (3). Tumor cells showing CSC properties have been reported in each other in a reciprocal fashion (20). CAFs can also cooperate human and mouse mammary gland tumors (4–7). with other stromal cells of the tumor microenvironment, includ- CSCs can be regulated by extrinsic signals provided by stromal ing immune and vascular cells, to establish favorable conditions cells of the tumor microenvironment, which establish favorable for tumor growth (21). Recent work has shown that CAFs sustain conditions for CSC growth (8, 9). Multiple types of stromal cells CSC expansion in colorectal and lung tumors, through the pro- have been reported to interact with CSCs and to influence their duction of secreted factors (22–24). However, the elucidation of behavior. For instance, endothelial cells provide factors that this interaction and the identification of supporting factors for support CSC proliferation and self-renewal in tumors of the brain CSCs are less well investigated in breast cancer. Here, we unra- veled the reciprocal interactions between CSCs and CAFs in our previously described Wnt-Met mouse model of basal-like mam- 1Cancer Research Program, Max Delbrueck Center for Molecular Medicine (MDC) mary gland tumors (25). in the Helmholtz Society, Berlin, Germany. 2Experimental Pharmacology & Oncology (EPO), Berlin, Germany. Materials and Methods Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Mouse strains and genotyping All animal experiments were conducted in accordance with G. Valenti, H.M. Quinn, and G.J.J.E. Heynen are co-first authors of this article. European, National, and MDC regulations. Wnt-Met mice were Current address for G. Valenti: Department of Digestive and Liver Diseases, described previously (25). For genotyping mice, ear punches were Columbia University Medical Center, New York, NY. digested in lysis buffer (100 mmol/L Tris pH 8.0, 10 mmol/L Corresponding Author: Walter Birchmeier, Max Delbrueck Center for Molecular EDTA, 0.2% SDS, 200 mmol/L NaCl, 300 mg/mL proteinase K) at Medicine, Robert-Roessle-Str. 10, Berlin 13125, Germany. Phone: 4930-9406- 55C overnight. Samples were diluted 1:20 and employed for PCR 3800; Fax: 4930-9406-2656; E-mail: [email protected] reactions to amplify transgenes using appropriate set of primers. doi: 10.1158/0008-5472.CAN-15-3490 Products of the amplification were analyzed by agarose (Invitro- Ó2017 American Association for Cancer Research. gen) gel electrophoresis. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-15-3490 Valenti et al. Histology Lourmat imaging system SL-3. a-Tubulin was used as a protein Mammary glands were fixed in 4% formaldehyde (Roth) at 4C loading control. overnight and dehydrated in ethanol (Roth). Dehydrated mam- Isolation of mammary gland tumor cells mary glands were embedded in paraffin and cut into 7-mm Tumors were minced in DMEM/F12 (Invitrogen) supple- sections. Tissue sections were dewaxed, rehydrated, and stained mented with 5% FBS (Invitrogen), 5 mg/mL insulin (Sigma- with Hematoxylin (Fluka) and Eosin (Merck). After staining, Aldrich), 0.5 mg/mL hydrocortisone (Sigma-Aldrich), 10 ng/mL tissue sections were dehydrated and mounted with Entellan EGF (Sigma-Aldrich) containing 300 U/mL Collagenase type III (Merck). Images were acquired using bright-field microscopy (Worthington), and 100 U/mL Hyaluronidase (Worthington) (Zeiss). at 37C for 1-hour shaking. Resulting organoids were resus- pended in 0.25% trypsin-EDTA (Invitrogen) at 37Cfor1 fl Immuno uorescence and confocal microscopy minute and digested in dissociation medium–containing 2 fi Paraf n-embedded tissue sections were dewaxed and rehy- mg/mL Dispase (Invitrogen), 0.1 mg/mL DNase I (Worthing- drated. Antigen retrieval was performed to unmask epitopes ton) at 37C for 1-hour shaking. Samples were filtered with 40- by boiling sections for 20 minutes in EDTA-Tween buffer mm cell strainers (BD Biosciences) and incubated with 0.8% (10 mmol/L Tris, 1 mmol/L EDTA, 0.05% Tween-20, pH 9.0). NH4Cl solution on ice for 3 minutes (RBC lysis). Cells were Cryosections were incubated at room temperature for 15 minutes. labeled with Lineage Cell Depletion Kit (Miltenyi Biotec) and fi Adherent cells were xed in 4% formaldehyde (Roth) at room subjected to magnetic-activated cell sorting (MACS). temperature for 15 minutes. Three-dimensional (3D) structures were fixed in 2% formaldehyde at room temperature for 20 FACS minutes and permeabilized with 0.5% Triton X-100 (Selva) at Single-cell suspensions were incubated with conjugated pri- 4C for 10 minutes. Samples were incubated with blocking buffer mary antibodies at room temperature for 15 minutes (see Sup- containing 10% goat serum at room temperature for 1 hour. For plementary Table S1 for the full antibody list). Cells were next staining, samples were incubated with primary antibodies at 4C incubated with 7AAD (BioLegend) at room temperature for 5 overnight (see Supplementary Table S1 for the full antibody list). minutes to stain the DNA of dead cells. Cells were sorted using Samples were incubated with secondary antibodies conjugated FACSAria II (BD Biosciences) or analyzed using LSRFortessa (BD with Alexa488, Cy3, or Alexa647 (1:400, Jackson ImmunoRe- Biosciences). Data were analyzed using the FlowJo Analysis search) at room temperature for 1 hour. Samples were counter- Software. stained with 1 mg/mL DAPI solution (1:2,000, Sigma- Aldrich) and mounted with Immu-mount (Thermo Scientific). Pictures Mammosphere and 3D Matrigel cultures  4 were acquired by confocal fluorescence microscopy using a Leica Single-cell suspensions were plated at 8 10 cells/mL in TCS SPE Microscope. serum-free DMEM/F12 medium, supplemented with 2% B27 (Invitrogen), 20 ng/mL FGF (Invitrogen), 20 ng/mL EGF, 4 mg/mL heparin (Sigma-Aldrich), 5 mg/mL insulin, and 0.5 mg/ qRT-PCR mL hydrocortisone (mam-medium) for 7 to 14 days on plates Diluted cDNAs were transferred to 96-well PCR optical plates coated with 1.2% PolyHEMA (Sigma-Aldrich). Mammosphere fi (Thermo Scienti c). Selected primers are listed in Supplementary cultures were supplemented with mam-medium every 3 days. For fi Table S1. SYBR GreenER qPCR mix (Thermo Scienti c) was used. coculture experiments, 0.4-mm membrane pore transwell inserts qRT-PCR was performed using the iCycler IQTM 5 multicolor real- (BD Biosciences) were seeded with CAFs or control fibroblasts time detection system (Bio-Rad). Relative mRNA levels were (COFs) at 4  104 cells/mL in DMEM/F12 containing 1% FBS. determined following normalization to the housekeeping genes Transwell inserts were placed in wells containing single-cell Actb or Rpl19 and analysis with the comparative threshold cycle suspensions in mammosphere-forming conditions. Mammo- ÀDDCt (2 ) method. spheres were allowed to grow for 7 or 14 days. Numbers and sizes of the mammospheres were determined by phase-contrast SDS-PAGE and Western blot analysis microscopy. In experiments involving the use of Wnt-Met reporter Protein samples (30 mg) were incubated at 95C for 5 minutes mice, fluorescence microscopy was used to detect yellow fluores- in sample buffer (125 mmol/L Tris pH 6.8, 4% SDS, 20% glycerol, cent protein (YFP).
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