Mammosphere formation assay. Mammosphere culture was performed as previously described (13,

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1 Supplemental Text Materials and Methods Mammosphere formation assay. Mammosphere culture was performed as previously described (13, 17). For co-culture with fibroblasts and treatment with CM or CCL2, fibroblasts were grown on top of 0.4-μmpore-size transwell filters (Nunc; Rochester, NY) and CM collected from differentially treated cells, or recombinant CCL2 were added to ultralow attachment 6-well plates (Corning; Corning, NY) containing freshly plated single BC cells at the beginning of the mammosphere formation assay. On day 10, numbers of mammospheres (diameter 70 μm) were counted, and SFE was calculated based on the numbers of initially seeded cells. For secondary sphere formation, first-passage mammospheres from day 10 cultures were collected by gentle centrifugation (320 g) and dissociated into single cells by incubation in trypsin-edta solution (Invitrogen; Carlsbad, CA), before plating into new wells. The PKH67 fluorescence labeling experiments were performed as previously described (19). In brief, BC cells were labeled with PKH67 (Sigma-Aldrich) following the manufacturer s procedures, before plating into ultralow attachment wells for mammosphere formation. On day 10, spheres were analyzed by confocal microscopy, or dissociated into single cells prior to fluorescenceactivated cell sorting on PKH67 intensity. Sorted PKH67 hi and PKH67 low cells were then used in the secondary mammosphere formation assay. RNA extraction, reverse transcription (RT) and real-time quantitative PCR (qpcr). RNA extraction, RT, and qpcr were performed as described previously (40). Primer sequences used are: 5 - AAGATCTCAGTGCAGAGGCTCG-3 and 5 -TTGCTTGTCCAGGTGGTCCAT-3 for human CCL2, 5 - GGTGAGACCTGCCTGAATG-3 and 5 -GTTGGGGTCCTGGCATC-3 for human NOTCH1, 5 - GGTTTTTGGCGGCTTCCAAG-3 and 5 -TCAGTTCCGCCACGGTCTC-3 for human HES1, and 5 - CTACCACATCCAAGGAAGCA-3 and 5 -TTTTTCGTCACTACCTCCCCG-3 for human 18S rrna (as internal control for all RT-qPCR). An annealing temperature of 55 C was used for all the primers. Cytokine antibody array and Western blot analyses. CM collected from 10 6 of cells were concentrated ~10-fold using Amicon Ultra-15 3K centrifugal filter devices (Millipore; Billerica, MA), and then assayed using RayBio human cytokine antibody arrays 6 8 (RayBiotech; Norcross, GA), following the

2 manufacturer s protocol. Preparation of cell lysates and Western blot were carried out as described previously (41). Primary antibodies included: Phospho(P)-Stat3 Y705, Stat3, NICD1, NOTCH1, P-p38 T180/Y182 and p38 (Cell Signaling). Cell transfection, reporter assays, and RNAi studies. DNA transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer s protocol, as described previously (42). The FlexiTube GeneSolution sirnas for NOTCH1 and AllStars negative control sirnas were purchased from Qiagen (Valencia, CA), and transfected using DharmaFECT Duo Transfection Reagent (Dharmacon; Lafayette, CO) according to the manufacturer s procedures. For luciferase reporter assays, cells growing in 12-well plates were transfected with 0.5 µg of CCL2 or NOTCH1 promoter reporters (kindly provided by Drs. Leonid S. Metelitsa and Warren S. Pear, respectively), along with 0.01 µg of pcmv-renilla plasmid as an internal control. Firefly and Renilla luciferase activities were measured using the dual luciferase assay system (Promega; Madison, WI), as described previously (40). In vitro secretase assays. Solubilized membrane extracts were prepared as described previously (43). Synthetic fluorescent resonance energy transfer (FRET) peptide substrates for α- or γ-secretase (Calbiochem; 10 μl of 100 μm stock) were incubated with the membrane extracts (100 μg in 100 μl), in the presence or absence of the α/γ-secretase inhibitors INCB3619 (5 μm) or DAPT (10 μm) at 37ºC overnight in dark. At the end of the incubation, the fluorescence in each sample was measured by a fluorescence spectrometer using excitation wavelength at 325 nm and emission wavelength at 393 nm for the α-secretase substrate, and excitation wavelength at 355 nm and emission wavelength at 440 nm for the γ-secretase substrate. Xenografts. All animal experiments were approved by the institutional animal care and use committee at City of Hope. Six-week-old female NOD/SCID/IL2Rγ-null (NSG) mice were injected in the No. 4 mammary fat pad with a mixture of 10 5 freshly dissociated, unmodified XP primary BC cells and CAF isolated from the same BC specimen but in vitro transduced with phiv7-tetr-ires-gfp lentivirus carrying Dox-inducible CCL2 shrna (for fibroblast-specific CCL2 depletion) or pbabe-gfp retrovirus (for CCL2 neutralizing antibody treatment). In the shrna study, mice were divided into 2 groups (n = 8 per group) for treatment with Dox or control. For the group with Dox treatment, mice were administered 1

3 mg/ml Dox in 5% sucrose through the drinking water starting at 2 days before transplantation. In the study with CCL2 antibody, mice were divided into 3 groups (n = 8 per group). CCL2 neutralizing antibody (Catalog #AB-279-NA) or goat IgG control (Catalog #AB-108-C) purchased from R&D Systems (Minneapolis, MN), or equal volume of PBS, were administered through intraperitoneal (i.p.) injection three times a week at 5 mg/kg, starting at the next day after transplantation. The same CCL2 neutralizing antibody and IgG control were used in sphere formation assay (Fig. 1F) and CM preparation (Fig. 4G) at a final concentration of 30 ng/ml. When tumor became palpable, tumor volume was assessed every other day by caliper measurements using the formula (width 2 length)/2 (mm 3 ). At the end of each experiment, harvested tumors were mechanically and enzymatically dissociated, and the epithelial tumor cells and CAFs were purified by flow cytometry using human ESA (for tumor cells) and the GFP label on CAFs as markers.

4 Supplemental Figures Figure S1. Marker expression in primary fibroblasts and BC cells. (A) Western blot analysis of Vimentin and GAPDH in NAF2 and CAF after treatment with the CM from BT474 or XP BC cells for 3 or 10 days. (B) Immunofluorescence assay indicating SMA expression in a fraction of Vimentin-expressing primary CAF Bar equals 100 μm. (C) Flow cytometry indicating ESA expression in the majority of primary XP epithelial tumor cells and lack of ESA in primary CAF

5 Figure S2. Serial confocal images of representative mammospheres formed by PKH67-labeled BT474 cells in the presence or absence of CCL2. Scale bar equals 20 μm.

6 Figure S3. CCL2 expression in CAF3 cells upon CM treatments. Total RNA isolated from CAF3 that had been treated with CM from indicated BC cells for 4 or 24 h in the presence or absence of Stattic was analyzed for CCL2 mrna level by RT-qPCR. Data were normalized to 18S in each sample. * p<0.001 compared to the control (the first column).

7 Figure S4. Sustained CCL2 induction in CAFs co-cultured with tumor cells. RNA was isolated from CAF that had been co-cultured with XP tumor cells for indicated time in the presence or absence of Stattic. Expression of CCL2 was assessed by RT-qPCR. * p<0.001 compared to the control (the first column).

8 Figure S5. Expression of CCR2 and CCR4 in BC cells. RNA was extracted from indicated BC cells and subjected to RT-qPCR for the expression of CCR2 and CCR4. Data were normalized to 18S in each sample.

9 Figure S6. HES1 and HEY1 reporter assays in BT474 cells. Luciferase activity was analyzed in reportertransfected BT474 cells at 24 h post CCL2 treatment +/- inhibitors of γ-secretase (DAPT; 10 nm) or α-secretase (INCB3619; 5 nm). Each bar represents the mean ± S.D. of 3 independently transfected wells. * p<0.001 compared to the control (the first column).

10 Figure S7. In vitro secretase cleavage assay. The solubilized plasma membrane extracts prepared from BT474 cells treated with CCL2 or vehicle were incubated with synthetic fluorescent resonance energy transfer (FRET) substrates for α- or γ-secretase. As controls for the specificity of FRET substrates, DAPT or INCB3619 was added to the untreated BT474 membrane extracts. At the end of the incubation, the fluorescence absorbance (ABS) in each sample was measured, as indicated in the graph. Each bar represents the mean ± S.D. of 3 independent wells. * p<0.001 compared to the control (the first column).

11 Figure S8. Representative IHC images of XP/CAF xenograft tumors treated with PBS, IgG, or anti-ccl2. Serial sections of tumors were stained for human CCL2, NOTCH1, and SMA. Bar equals 100 μm.

12 Figure S9. Representative IHC images of CCL2 and NOTCH1 in primary BC specimens. Images from two specimens were shown. Bar equals 100 μm.