Targeting Vasculogenesis to Prevent Progression in Multiple Myeloma

Targeting vasculogenesis to prevent progression in multiple myeloma

Moschetta M, et al.

SUPPLEMENTARY INFORMATION CONTENT

Supplementary Data

Supplementary Methods

Supplementary Figure and Table legends

Supplementary Tables

SUPPLEMENTARY DATA

SCID-mu model.

SCID-mu model is based on the subcutaneous implantation of femurs from donor to recipient mice (Figure 3A). First, we examined whether the bone marrow of the transplanted femurs will be functional and lead to hematopoietic recovery, and we used isolated femurs from whole body GFP-SCID donor mice and implanted them in normal SCID-bg mice. Ten and 15 days after GFP-femur implantation these mice were bled and the presence of GFP+ circulating cells were assessed by flow-cytometry in isolated PBMCs; circulating GFP+ cells resembling the GFP+ circulating population of whole body SCID-GFP mice (Supplementary Figure 4A) were detectable in mice implanted with GFP+ femurs but not in naïve mice (Supplementary Figure 4B). In a subsequent experiment, mice were first implanted with 2 femurs from donor mice; 2 weeks after femur implantation these mice were injected i.v. with MM1.S-GFP-luc+ cells to assess whether the MM cells were able to home to both the host and the implanted BM; Supplementary Figure 4C shows that MM cells home to the implanted femurs as showed by BLI imaging (Supplementary Figure 4C, arrows show sites of femur implantation). IHC studies performed (Supplementary Figure 4D) in femurs harvested after sacrifice showed localization of hCD138+ MM1.S cells inside the BM of implanted femurs together with an increased vessel formation, mostly in the sites of MM localization.

SUPPLEMENTARY METHODS

Cells

MM cell lines (human MM1.S, MM1.S-GFP-luc+, MM1.S-RFP-luc+ and IM9, and murine 5TGM1 and Vk12598) were used in this study. The MM1.S cell line was purchased from ATCC (Manassas, VA); the MM1.S-GFP-luc+ cell line was previously obtained through multiple in vivo selection 1 while the MM1.S-RFP-luc+ cell line was generated directly by parental MM1.S cell line without in vivo selection. The 5TGM1 cell line was a kind gift of Dr. David G. Roodman (Indiana University, Indianapolis, Indiana, USA), whereas the IM9 cell line was obtained by Doctor Kiyohiko Hatake (Japanese Foundation for Cancer Research, Tokyo, Japan). These cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum FBS (Sigma Chemical, St Louis, MO), 2mM L-glutamine, 100 U/mL penicillin, and 100µg/mL streptomycin (GIBCO, Grand Island, NY). The Vk*MYC derived transplantable Vk12598 cell line was a kind gift of Doctor Marta Chesi (Mayo Clinic, Scottsdale, AZ) and was maintained and expanded by in vivo re-transplantation in immunocompetent C57BL/6 mice (Jackson Laboratories, Bar Harbor, Maine) 2.

The human umbilical vein endothelial cells (HUVECs) (Lonza, Walkersville, MD) were cultured in complete EGM-2 medium [EBM-2 medium (Lonza) supplemented with EGM-2 SingleQuot Kit Supplements and Growth Factors (Lonza); hereon called “endothelial cell specific medium”]. MM patient samples were obtained after approval from the Dana-Farber Cancer Institute Institutional Review Board (DFCI IRB). Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Peripheral mononuclear cells (PBMCs) from the peripheral blood (PB) of MM patients and healthy subjects were obtained after red blood cell lysis using specific buffer (BioLegend, San Diego, CA), according to the manufacture’s protocol.

Detection of EPCs in the peripheral blood (PB) of patients with MM

PBMCs were isolated from PB of MM patients at different stages of disease 3, and normal blood donor subjects (n=12 sMM, n=19 MM, and n=11 healthy donors, respectively); and washed twice in auto-MACS buffer (Miltenyi Biotec), then stained with Brilliant Violet 421-anti human CD34 (BD Pharmingen) and Alexa Fluor 647 anti-human VEGFR2 antibodies (BD Pharmingen) for 30 minutes on ice, and analyzed by flow cytometry for presence of EPCs (CD34+ VEGFR2+ cells) on a FACS ARIA II flow cytometry system (BD Biosciences, San Jose, CA). Number of EPC per μl of blood was counted by using this formula: (CD34+ cells)*(CD34 gated VEGFR2+ cells)*(white blood cell count/ μl)/10000 (Supplementary Figure 1A).

In vitro EC-CFU and ECFC colony forming assays.

A 15-ml sample of venous blood was used for the EC-CFU or ECFC colony assays. Samples were processed within four hours after collection, and PBMCs were isolated after red-blood cell lysis. For EC-CFU assay, PBMCs from MM patients at different stages of disease (n=15 smoldering MM, n=15 active MM) and healthy donor controls (n=8) were washed once with PBS and once in endothelial cell specific medium supplemented with 20 percent fetal-calf bovine serum, penicillin (100 U per milliliter), and streptomycin (100 μg per milliliter) (here called “colony medium”). A total of 2.0x107 isolated cells were subsequently resuspended in colony medium and plated on 24 well dishes (1.5x106 cells per well) pre-coated with human fibronectin (BD BioCoat, BD, Bedford, MA), after a 48h pre-plating step in a fibronectin-coated 24-well plate, to remove adherent cells, as previously reported 4. Growth medium was changed every three days, and the numbers of colonies were counted 10 to 15 days after plating. Total colonies were counted manually by using an inverted light microscopy (Olympus CKX41, Olympus; Tokyo, Japan) at 10x magnification. EC-CFU phenotypic characterization was performed in PBMCs from 3 subjects after EC-CFU assay by flow-cytometry using the following Abs and the relative isotype control Abs: PE anti-human VEGFR2 Ab (BD Pharmingen), APC anti-human CD45, (BD Pharmingen), FITC anti-human CD31 (BD Pharmingen), APC anti-human CD133/1 (Milteny Biotec), Brilliant Violet 421 anti-human CD34 (BD Pharmingen).

Human ECFCs were derived from PBMCs from both MM patients at different stages of the disease (n=8 smoldering MM, n=14 active MM) or healthy donor controls (n=5). PBMCs were washed once with PBS and once in endothelial specific medium, and then resuspended in colony medium and seeded into 12-well plates (2x106 cells/well) previously coated with type 1 rat collagen, as reported 5. For collagen coating, rat tail type 1 collagen at 3.41 initial concentration (BD, Biosciences) was diluted with a 17 M acetic acid solution to the final concentration of 50 μg/ml and used to coat 12 well plates by adding 100 μl of this solution per well for 1 hour at room temperature, as previously reported 5.

After PBMC plating, the colony medium was changed every other day for 7 days and then, every three days. Individual ECFC colonies emerged 20 to 30 days after cell seeding and were re-plated separately. For each patient, colonies were counted manually by using an inverted light microscopy (Olympus CKX41, Olympus; city, at 10x magnification). After re-plating colony medium was changed every three days. Individual ECFC colonies from 2 patients at passage 3 or lower were analyzed by flow cytometry for the expression of endothelial and hematopoietic cell surface markers by using the following Abs and corresponding isotype control Abs: Alexa Fluor 647-anti-human VEGFR2 Ab (BD Pharmingen), APC anti-human CD45 Ab (BD Pharmingen), FITC anti-human CD31 Ab (BD Pharmingen), APC anti-human CD133/1 Ab (Miltenyi Biotec), Brilliant Violet 421 anti-human CD34 Ab (BD Pharmingen), Alexa 647 anti-human CD144 (VE-cadherin) Ab (BD Pharmingen), and PE anti human Tie2/Tek Ab (R&D systems, Minneapolis, MN).

MM cell proliferation assay, MM cell sorting, BRDU-7ADD cell-cycle analysis and apoptosis detection in the co-culture system.

MM1.S-GFP-luc+ and MM1.S-RFP-luc+ cells were plated at 1x104 cells/well in a BD Falcon 96-well plate, alone or in co-culture with HUVECs (3x103 cells) or primary ECFCs (3x103). Co-cultures were performed in RPMI-1640 containing 10% fetal bovine serum FBS (Sigma Chemical, St Louis, MO), 2mM L-glutamine, 100 U/mL penicillin, and 100µg/mL streptomycin (GIBCO, Grand Island, NY). After 24, 48 and 72h hours, 0.15 mg/ml D-luciferin was added and incubated for 1 hour. Luminescence was measured with a luminometer for cell growth quantification.

In parallel, MM1.S-GFP-luc+ or MM1.S-RFP-luc+ cells were cultured in presence or absence of HUVECs for 24 hours; and subsequently sorted (gating on GFP+ or RFP+ cells) by using a FACS ARIA II flow-sorter. RNA was extracted by sorted cells by using RNeasy kit (Quiagen).

For the study of cell-cycle modulation and apoptosis of MM1.S-GFP-luc+ or MM1.S-RFP-luc+ after co-culturing with ECs, MM cells (1x106) were co-culture on a layer of HUVECs (80% confluency) for 24h then harvested and used for flow-cytometry analysis. Specifically, cell-cycle profiles of MM1.S-GFP-Luc+ and MM1.S-RFP-Luc+ cells co-cultured with or without HUVECs were analyzed by staining with bromodeoxyuridine (BrdU) and 7-aminoactinomycin D (7-AAD) (BrdU Flow Kit; BD Pharmingen) according to the manufacturer's instruction. Apoptosis in MM1.S-GFP-Luc+ and MM1.S-RFP-Luc+ cells was quantified by staining with Annexin V (BD Biosciences) and 7-AAD (BD Biosciences). The experiment was performed in triplicate. The samples were analyzed by flow cytometry (FACSCanto II; BD Biosciences).

Live confocal microscopy

Five x 105 HUVECs or ECFCs were seeded in 12-well glass bottom plate (MatTek) and cultured in colony medium. After reaching 80% confluence, cell medium was removed and an Ab staining solution containing MACS buffer, FcR blocking Reagent (Miltenyi Biotec) and Brilliant Violet 421 anti-human CD34 Ab (BD Pharmingen), or Alexa 647 anti-human CD144 (VE-cadherin) Ab (BD Pharmingen), or APC anti-human CD45 Ab (BD Pharmingen), or Alexa Fluor 488 anti-human CD31 Ab (BD Pharmingen) (100 μl/well) was added; plates were then transferred in the cell incubator for 45 minute to allow staining; after, Ab solution was removed and plates washed gently 2 times with sterile MACS buffer. Finally colony medium was added and cells were immediately observed on a Leica SP5X laser scanning confocal microscope (Leica, Buffalo Grove, IL, USA) and pictures analyzed by using Leica LAS AF lite software (Leica, Microsystems). MM1.S and IM9 cells (5x105) were transferred in a 12-well glass bottom plate, and the same Abs were added directly into the cell suspension medium. After 45-minute incubation, cells were collected and washed two times with MACS buffer. Finally resuspended in cell growth medium and reseeded in a 12-well glass bottom plate and immediately observed through confocal microscopy.

Transgenic mice experiments

ID3-/- mice and ID1-/- mice in a mixed C57Bl6/129Sv background were kindly provided by Doctor Benezra R. (Memorial Sloan–Kettering Institute, NY, USA). Mice were backcrossed onto C57BL/6 background (Jackson Laboratories) for 4-5 generation, and then crossed to obtain ID1+/+ ID3 +/+ mice (wild type littermates) and ID1+/-ID3-/- mice. Genotyping was performed by using Transnetyx automated genotype service (Transnetyx Inc.).

Wild type littermates (n=8) and ID1+/-ID3-/- mice (n=6) were injected i.v. with Vk12598 cells (4 x 106 total BM or spleen cells) obtained by C57BL/6 mice previously transplanted with Vk12598 cells 2.

Transgenic Vk*MYC mice (t-Vk*MYC) 6, and wild type C57BL/6 mice (healthy C57BL/6) (Charles River Breeding Laboratories, Calco, Italy) were housed in a pathogen-free animal facility, and treated in accordance with the European Community Guidelines. Vk*MYC transgenic mice were genotyped by Real Time-PCR in order to identify experimental Vk*MYC+/- animals 6. All in vivo experiments were approved by the Ethical Committee of Fondazione Centro San Raffaele (Milan, Italy). Mouse blood was collected in Eppendorf tubes by retro-orbital sampling for serum electrophoresis analysis (SPEP), and for EPC quantification.

MM1.S-GFP-luc+ xenograft and in vivo anti-VEGFR2 therapy with DC101.

MM1.S-GFP-luc+ (5x106 cells) cell line was xenotransplanted in SCID-bg mice through i.v. injection; Tumor growth was assessed by using in vivo bioluminescence imaging (BLI). Mice were injected with 75 mg/kg of Luciferin (Xenogen, Hopkington, MA), and tumor growth was detected by bioluminescence 3 min after the injection, using Xenogen In Vivo Imaging System (Caliper Life Sciences, Hopkinton, MA), as previously reported 1. For survival analysis, mice were sacrificed after hind-limb paralysis development, or after development of any sign of distress (moribund state, weight loss, or respiratory distress).

Therapeutic activity of DC-101 anti-murine VEGFR2 Ab (ImClone Systems LLC, Eli Lilly & Co.) was evaluated in vivo in the MM1.S-GFP-luc+ orthotopic xenograft model. SCID-bg mice were injected i.v. with 5x106 MM1.S-GFP-luc+ cells. A group of mice was treated with rat untargeted IgG (eBioscience, 40 mg/Kg body weight (BW) at 3 day intervals, n=10), starting 4 days after tumor cell injection, another with DC101 Ab (40 mg/Kg body weight (BW) i.p. at 3 day intervals, n=10) 4 days after tumor cell injection (early treatment), and another with DC101 Ab 3 weeks after tumor cell injection (late treatment, n=10). In another experiment SCID-bg mice were injected i.v. with 5x106 MM1.S-GFP-luc+, and then treated with PBS (200 μl i.p., n=7) or bortezomib (Selleck Chemicals, 0.5 mg/kg, twice/week; i.p., n=7) 3 weeks after tumor cell injection. Tumor growth was assessed by using in vivo bioluminescence imaging (BLI). Mice were injected with 75 mg/kg of Luciferin (Xenogen, Hopkington, MA), and tumor growth was detected by bioluminescence 3 min after the injection, using Xenogen In Vivo Imaging System (Caliper Life Sciences, Hopkinton, MA), as previously reported 1. For survival analysis, mice were sacrificed after hind-limb paralysis development, or after development of any sign of distress (moribund state, weight loss, or respiratory distress).

Serum electrophoresis, serum total and gamma protein quantification

Whole blood (100 ml) was obtained from mice by submandibular bleeding. To obtain serum, blood was left coagulating for 30-45 minutes at room temperature, and then centrifuged for 15 minutes at 4.000 rpm to allow serum-cell separation. Serum was then collected, and used for total protein quantification through Bradford method (Bio-Rad Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories), then diluted 1:2 in normal saline buffer for serum protein electrophoresis (SPEP) analysis. SPEP was performed using precasted agarose gels (Helena Laboratories, Beaumont, TX), and a SPIFE 3000 semi-automated gel electrophoresis system (Helena Laboratories, Beaumont, TX), as previously reported 2. Gels were scanned by an Epson Perfection V700 Photo Scanner at 300 dots per inch, and band intensity was quantified by clinically certified QuickScan 2000 software (Helena Laboratories, Beaumont, TX) to estimate serum gamma protein concentration.

SCID-mu model characterization and recruitment model.

For the in vivo recruitment model, femurs were isolated from healthy donor SCID-bg mice or healthy NOD.Cg-PrkdcscidTg(CAG-EGFP)1Osb/KupwJ (from hereon called SCID-GFP mice, Jackson Laboratories), immersed in growth factor reduced Matrigel and immediately implanted subcutaneously by using a trocar needle in healthy recipient SCID-bg mice (2 femurs/mouse).

For the SCID-mu characterization studies, 2 femurs were implanted in the ventral position of SCID-bg mice (n=3); 10 days and 15 days after femur implantation, mice were bled, and the PBMCs obtained after red blood cell lysis were studied for the presence of circulating GFP+ cells through flow-cytometry. In a similar study, SCID-bg mice (n=3) were injected with MM1.S-GFP-luc+ cells (5x106) 15 days after femur implantation. Mice were then followed for tumor growth with in vivo BLI weekly until the development of paralysis. At this point, mice were euthanized and the implanted femurs were harvested for histological analysis.