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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.
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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
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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. Loges S, Fehse B, Brockmann MA, Lamszus K, Butzal M, Guckenbiehl M, Schuch G, Ergün
S, Fischer U, Zander AR, Hossfeld DK, Fiedler W, Gehling UM. Identification of the adult
human hemangioblast. Stem Cells Dev. 2004 Jun;13(3):229-42.
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5. Berardi S, Caivano A, Ria R, Nico B, Savino R, Terracciano R, De Tullio G, Ferrucci A, De
Luisi A, Moschetta M, Mangialardi G, Catacchio I, Basile A,Guarini A, Zito A, Ditonno
P, Musto P, Dammacco F, Ribatti D, Vacca A. Four proteins governing overangiogenic
endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets.
Oncogene. 2012 May 3;31(18):2258-69. doi: 10.1038/onc.2011.412. Epub 2011 Oct 3.
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