Sharon et al. Supplementary Data
Supplementary Data
Table 2: Summary of proteomics analysis: highest intensity extracellular proteins in tumor cell CM.
Rank / Gene name / Protein name / Protein ID(uniprot) / Average intensity
(Log2)
1 / Fn1 / Fibronectin; Anastellin / G5E8M2 / 36.1
2 / Spp1 / Osteopontin / F8WIP8 / 33.7
3 / Timp2 / Metalloproteinase inhibitor 2 / Q6PI17 / 33.4
4 / Ppia;Gm9234 / Peptidyl-prolyl cis-trans isomerase / Q5SVY2 / 33.3
5 / Psap / Sulfated glycoprotein 1 / E9PZ00 / 33.3
6 / Cp / Ceruloplasmin / G3X9T8 / 33.3
7 / Thbs1 / Thrombospondin-1 / P35441 / 32.9
8 / Hspa8 / Heat shock cognate 71 kDa protein / P63017 / 32.4
9 / Ctsb / Cathepsin B / P10605 / 32.4
10 / B2m / Beta-2-microglobulin / P01887 / 32.1
11 / Lgals3bp / Galectin-3-binding protein / Q07797 / 32.1
12 / Wfdc2 / WAP four-disulfide core domain protein 2 / Q9DAU7 / 31.8
13 / Lgals1 / Galectin-1 / P16045 / 31.7
14 / Cfl1;Gm6180 / Cofilin-1 / P18760 / 31.5
15 / Clu / Clusterin / Q549A5 / 31.4
Supplementary Figure legends
Figure S1: OPN does not affect NMFs proliferation: NMFs were incubated in SFM for 24 hours, followed by treatment with 3µg/ml rOPN, Met-1 CM or CM+neOPN for 24 or 48 hours. Con=SFM. Cell viability was assessed by XTT assay. (*P<0.05, n=3).
Figure S2: CD44 expression is up regulated in CAFs and in activated NMFs: A. CD44 is expressed in NMFs:NMFs were stained with anti-mouse CD44-FITC (green), nuclei are stained blue with DAPI (original magnification X40). B. CD44 expression is up regulated in mammary CAFs:qRT-PCR analysis of CD44 gene expression in NMFs and CAFs isolated by FACS sorting from normal mammary glands or from PyMT tumors. Results were normalized to the housekeeping gene GAPDH. Error bars represent SD of triplicates. (*P<0.05, n=3) C.Tumor cell CM induces up regulation in cell surface CD44 on NMFs: FACS analysis of cell surface expression of CD44 receptor in NMFs and in NMFs treated with either NMFs CM (Con) or with MET-1 CM (CM). Cells were stained with anti-CD44-FITC (*P<0.05, n=2). D. CD44 is expressed in mouse mammary carcinoma: Immunohistochemistry of CD44 in tissue sections of tumors from orthotropic tumors of MET-1 cells. Representative picture of 10 fields analyzed from 2 mice. Scale bar=100µM.
Figure S3: Knockdown of OPN does not affect tumor cell growth in vitro: A. XTT assay. Equal numbers of Met-1 or shOPN Met-1 cells were plated for 24 hours and viability was measured with XTT kit. B. Equal numbers of Met-1 or shOPN Met-1 cells were plated as in A. Cells were counted after 24 hours. C. Representative images of cultured Met-1 and shOPN Met-1 cells.
Supplementary Methods
Preparation of conditioned media
Growth media was removed from cells at about 75% confluency in 10cm plates, cells were washed thoroughly, and incubated in 10 mL of serum-free DMEM for 48 hours at 37°C and 5% CO2. Conditioned media was collected from cells and filtered through 45-μm filters (Millipore).
Fibroblast assays with CM
Tissue culture plates were prepared by adding collagen mix [50µg rat tail collagen I (BD Biosciences) with 1ml 0.02N Acetic acid] to each well and allowing it to set at 37°C for at least 30 min. plates were washed X2 with PBS and fibroblasts were plated on the collagen at a density of 2.5 × 105 cells per well (6 well) or 3 × 104 cells per well (24 well) in normal growth media and allowed to settle overnight. Growth media was removed from the fibroblasts and replaced with serum-free media (SFM) for additional 24 hours. SFM was then replaced by fresh SFM, left unchanged (NMF CM),or replaced with conditioned media from MET-1 breast cancer cells. For OPN signaling blockade assays, cells were incubated in conditioned media that were pretreated with blocking antibody to OPN (Abcam, Cat# ab11503, 3µg/ml) for 24hrs or with Arg-Gly-Asp peptide (Sigma, 10µg/ml), or with Rat anti-mouse CD44 (Biolegend, Cat# 103014, 5µg/ml) for 1 hour.
FACS analysis and sorting
Data were collected on FACS Calibur (Becton Dickinson) equipped with 488, 637 and 407 lasers. For analysis, a typical forward- and side-scatter gate was set to exclude dead cells and aggregates. FlowJo software was used to quantify 1x104 cells per sample. CD44-FITC (BioLegend cat# 103015, 5ug/ml) signal was collected in the FL1 channel through a 585/42 bandpass filter. For each experiment, cells were stained with 7AAD (Invitrogen, Cat# D1306, 5ug/ml) to establish background staining and to set quadrants before calculating the percentage of positive cells.
CAF isolation was performed as we previously described (1). In all fibroblasts isolation and analysis tumor cells and immune cells were excluded with antibodies EpCAM and CD45, respectively. (CD326 (EpCAM)-APC, Miltenyi Biotech, cat #130-096-417 and CD45-FITC, eBioScience cat #11-0451-81).
Oncomine data set analysis
Data was pooled from six publicly available microarray data sets of breast tumors and their corresponding normal tissue: For each data set, OPN expression level is presented as the median expression in multiple breast cancer tissues from human invasive ductal carcinomas (IDC) or in normal breast tissue from the same patients (Normal).
The data sets are from the following studies: TCGA (No Associated paper) composed of 76 patients with IDC. Richardson Breast 2 (Richardson et al., 2006 PMID: 16473279) composed of 47 patients with IDC treated at Brigham and Women`s Hospital. Karnoub Breast (Karnoub et al., 2007 PMID: 17914389) composed of 22 patients with IDC treated at Whitehead Institute for Biomedical Research. Zhao Breast (Zhao et al., 2004 PMID: 15034139) composed of 40 patients with IDC treated at Department of Surgery, Stanford University School of Medicine. Radvanyi Breast (Radvanyi et al., 2005 PMID: 16043716) composed of 29 patients with IDC treated at Sanofi Pasteur, Toronto. Gluck Breast (Gluck et al., 2011 PMID: 21373875) composed of 154 patients with IDC treated at Sylvester Comprehensive Cancer Center.
Collagen contraction assays
A total of 2×105 fibroblasts were suspended in a collagen mixture [100 μL of cells in normal growth media mixed with 100 μL of collagen mix (68.75µl DMEM, 0.72µl 1N NaOH, 31.25µl Rat Tail Collagen High concentration, Type 1 (BD bioscience)] per well of 24-well plates and allowed to set at 37°C for 45 min. Fibroblast-containing gels were incubated in SFM, MET-1 conditioned media, or MET-1 shOPN conditioned media for 24 hours. Gels were photographed at various time points. ImageJ software was used to measure gel area and assess contraction.
Scratch assays
6 well tissue culture plates were collagen coated and rinsed with PBS. NMFs were seeded in each well to a final density of 300,000 cells per well and maintained at 37°C and 5% CO2 for 24h to permit cell adhesion and the formation of a 70% confluent monolayer. Culture medium was then immediately removed (along with any dislodged cells). The removed medium was replaced with SFM or SFM supplemented with different substances (as indicated), or Met-1 CM or Met-1 CM supplemented with inhibitors as described above for 24 hours. Confluent monolayers were then scratched with a sterile pipette tip to leave a scratch of approximately 0.4–0.5 mm in width and cells were incubated for additional 17 hours. All scratch assays were performed in quadruplicate. ImageJ software was used to quantify and analyze scratch area.
Quantitative real-time PCR
Total RNA was isolated from cell pellets using Nucleuspin RNA II Kits (MACHERY-NAGEL). DNase treatment and cDNA synthesis were conducted using iScript cDNA Syntesis (BIORAD). Quantitative real-time PCRs (qRT-PCR) for mouse mGUS, GAPDH, CXCL1, CXCL2, COX-2, IL6 and OPN were conducted using iTaq Univarsal SYBR Green Supermix (BIORAD).
Western blotting
Lysates were prepared from cell pellets in RIPA lysis buffer (Sigma). Protein determination was performed with Pierce BCA Protein Assay Kit. Gels were transferred to nitrocellulose membranes (BioRad) and probed with antibodies for OPN (Santa Cruz, sc-21742), ERK (#9108), AKT (#9272), p-ERK (#4370) p-Akt (#4060); Cell Signaling Technology.
Immunocytoflourescence
NMFs were grown in 24 wells on glass coverslips coated with collagen for 24hrs. Cells were fixed with 100% methanol for 30 min, followed by washing 3 times with PBS. Cells were stained with anti-mouse CD44 conjugated to Alexa Flour 488 (BioLegend 103022,5ug/ml) for 1hour. Coverslips were mounted with DAPI.
In vitroviability assays
For the XTT viability assay, 5×103cells were seeded per well in a 96-well flat-bottomed tissue culture plate. After 24 hr of incubation, cell viability was assessed with an XTT-based assay according to the manufacturer instructions (Biological Industries). Absorbance at 450 nm (OD450) was determined for each well using an automated microplate reader (SpectraMax 190; Molecular Devices, Sunnyvale, CA).
Supplementary References:
1.Sharon Y, Alon L, Glanz S, Servais C, Erez N. Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS). J Vis Exp 2013(71).
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