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Supplementary Materials and Methods Cell purification and cultures BM stromal cells (BMSCs) were obtained after the adhesion of mononuclear cells (BMMCs) to polystyrene flasks and cultured in DMEM medium (Euroclone, Pero, Milan, Italy) with the addition of 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA). Primary MM plasma cells were isolated from non-adherent BMMCs using magnetic cell sorting with anti-CD138-conjugated microbeads (MACS; Miltenyi, Auburn, CA, USA) and cultured in RPMI-1640 medium (Euroclone) with the addition of 10% FBS. Fibroblasts were purified from BMSCs through D7-FIB-conjugated (anti-fibroblasts) microbeads1 (Miltenyi) and cultured in DMEM medium containing 20% FBS. CD146+ cells (mesenchymal stem cells) were obtained from BMSCs through anti-CD146 microbeads (Miltenyi) and cultured in DMEM medium with 10% FBS. MM endothelial cells (MM ECs) were isolated from BMSCs using Ulex Europaeus-conjugated microbeads2 and cultured in DMEM medium with 10% FBS. CD133+ hematopoietic stem and progenitor cells (HSPCs) were mobilized in 7 MM patients with cyclophosphamide and granulocyte-colony stimulating factor prior to autologous stem cell transplantation and harvested from the peripheral blood using a COBE Spectra cell separator (Gambro Inc., Stockholm, Sweden). A CliniMACS device (Miltenyi) selected CD133+ cells using anti-CD133 microbeads (Miltenyi). Cells (1×106 cells/mL) were cultured for 14 days in fibronectin (FN)-coated 24-well plates (Becton Dickinson-BD, San Jose, CA, USA) in expansion medium (Iscove’s modified Dulbecco medium (IMDM), Euroclone) with the addition of 10% FBS, 10% horse serum, 10-6M hydrocortisone (all from Sigma), vascular endothelial growth factor (VEGF; 60 ng/mL), fibroblast growth factor-2 (FGF-2; 10 ng/mL), hepatocyte growth factor (HGF; 10 ng/mL), stem cell growth factor (SCGF; 100 ng/mL), epidermal growth factor (EGF; 10 ng/mL), and insulin-like growth factor-1 (IGF1; 10 ng/mL, all from Peprotech, Rocky Hill, NJ, USA)3. Cells were then grown for 7 days in differentiation culture medium (IMDM medium) supplemented with VEGF (60 ng/mL) and SCGF (100 ng/mL)4. 1 The purity (>95%) of the cell populations was determined using the FACScanto II cytofluorimetry system (Becton Dickinson-BD, San Jose, CA, USA). To study the endothelial to mesenchymal transition (EndMT) and mesenchymal transition (MT) processes, ECs, HSPCs, and MSCs were cultured in the presence of conditioned medium (CM) from paired CAFs or RPMI8266 cells either with or without TGFβ (10 ng/ml, Peprotech) for 3, 7, or 14 days as specified in the text. Cells were then used for phenotypic and functional assays. Experiments involving TGFβ inhibition were performed by adding the competitive TGFβ-receptor 1 (TGFβ-R1) inhibitor SD208 (100-200 ng/ml; Tocris-bioscience, R&D Systems, Minneapolis, MN, USA) to the CM. Chemotaxis, adhesion, and angiogenesis assays in vitro For the adhesion assay, RPMI8266 cells and CAFs were pre-incubated with blocking mAbs against VLA-4 (Chemicon, Temecula, CA, USA), VLA-5 (Abcam, Cambridge, UK), β1 (Beckman Coulter, Miami, FL, USA), β3 (Chemicon, Billerica, MA, USA), αVβ3 (Millipore, Billerica, MA, USA), β7 (BioLegend, San Diego, CA, USA), and FN (HFN7.1 clone, Thermo-scientific, Fremont, CA, USA) for 1 h. The involvement of the SDF1α/CXCR4 receptor in CAF/MM plasma cell interactions was studied by treating CAFs with the CXCR4 antagonist AMD3100 (50 μM, SigmaAldrich, St. Louis, MO, USA) for 4 h during the chemotaxis and adhesion assays. CM from CAFs and MM cells and the measurement of cytokine secretion CAFs were grown to 80% confluence and incubated in serum-free DMEM medium (SFM) for 48 h. MM cells (1×106 cells/mL) were plated in SFM RPMI-1640 for 48 h. Culture supernatants were centrifuged (200×g for 10 min) and stored at -80°C as CM. SDF1α, TGFβ, IGF1, IL-6, VEGF, and FGF2 were measured using an enzyme-linked immuno-sorbent assay (ELISA, R&D Systems). Real-time RT-PCR Total RNA was isolated from 5×105 CAFs using the RNeasy Mini kit (Qiagen, Milano, Italy) and reverse-transcribed into total cDNA with the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). Real-time RT-PCR was performed using the StepOne system (Applied Biosystems, Foster 2 City, CA, USA). Specific primers for the αSMA, FSP1, FAP, and GAPGH genes were obtained from the Assay-On-Demand Product of Applied Biosystems (Hs00243201_m1; Hs00990806_m1; Hs00559595_m1; 4326317E, respectively). Relative gene expression levels were normalized to GAPDH expression, and fold changes were calculated using the 2-∆∆Ct method. Immunohistochemistry and immunofluorescence Formalin-fixed, 4-µm BM sections from the iliac crest of MM and MGUS patients and the femurs of 5T33MM and naïve mice were stained with anti-plasma cell (DAKO, Golstrup, Denmark), antiFSP1 (Millipore), and anti-αSMA (Abcam) mAbs. Four-micron sections of frozen xenografted MM plasmocytomas were stained with anti-CD31 mAb (Abcam) using a biotin-streptavidin method, and vessels were counted in 3 to 4 fields (at 250X magnification) spanning the entirety of three sections per sample and expressed as an average5. For double-labeling immunofluorescence, cells adherent to 4-well chamber slides were fixed and permeabilized with cold methanol (Sigma-Aldrich). The slides were stained with anti-αSMA-FITC (Abcam) and anti-FSP1 (Sigma-Aldrich) mAbs followed by anti-rabbit PE-conjugated (R&D Systems) antiserum. Cell nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Sections were examined using an Olympus fluorescence microscope (Olympus Italia, Rozzano, Italy). REFERENCES 1. Jones EA, Kinsey SE, English A, Jones RA, Straszynski L, Meredith DM, Markham AF, Jack A, Emery P, McGonagle D. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum. 2002 Dec;46(12):3349-60. 2. Vacca A, Ria R, Semeraro F, Merchionne F, Coluccia M, Boccarelli A, Scavelli C, Nico B, Gernone A, Battelli F, Tabilio A, Guidolin D, Petrucci MT, Ribatti D, Dammacco F. Endothelial cells in the bone marrow of patients with multiple myeloma. Blood. 2003 Nov 1;102(9):3340-8. Epub 2003 Jul 10. 3. Ria R, Piccoli C, Cirulli T, Falzetti F, Mangialardi G, Guidolin D, Tabilio A, Di Renzo N, Guarini A, Ribatti D, Dammacco F, Vacca A. Endothelial differentiation of hematopoietic stem and progenitor cells from patients with multiple myeloma. Clin Cancer Res. 2008 Mar 15;14(6):1678-85. doi: 10.1158/1078-0432.CCR-07-4071 4. 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