Modeling human tumor angiogenesis in a three-dimensional culture system by Giorgio Seano, Giulia Chiaverina, Paolo Armando Gagliardi, Laura di Blasio, Roberto Sessa, Federico Bussolino, and Luca Primo Blood Volume 121(21):e129-e137 May 23, 2013 ©2013 by American Society of Hematology

Characterization of 3D hAR cultures. Characterization of 3D hAR cultures. (A) hARs sprout in the presence of EGM-2 but only after fibroadipose tissue cleaning. Representative micrographs of hARs with or without fibroadipose tissue cleaning, cultured with 5% FCS EBM-2 (Basal medium) or with EGM-2 (complete medium). The bottom micrograph is a magnification of the red dotted line square. Scale bar represents 200 μm. (B) 3D isosurface rendering of hAR sprouting of whole-mount immunofluorescence staining for VE-cadherin (red) and nuclei (blue) acquired through confocal microscopy. Tickmarks on axis represent 50 μm. (C) Confocal images of hAR sprouts treated with Ac-LDL or Dextran in green and stained with anti-VEGFR2 Ab in red and with DAPI in blue. Scale bar represents 10 μm. (D) Sprout lumen is shown by 3D rendering of magnification of (B) and z-stack section of yellow dotted line rectangle of hAR sprouting outgrowth stained for VE-cadherin (red) and nuclei (blue). The yellow arrow indicates the capillary lumen. Scale bar represents 5 μm. (E) Pericytes coverage. Immunostaining of whole-mount hARs with anti-VEcadherin (red), NG-2 (green), and nuclear staining with DAPI (blue). Scale bar represents 10 μm. Giorgio Seano et al. Blood 2013;121:e129-e137 ©2013 by American Society of Hematology

Modulation of angiogenic sprouts by growth factors. Modulation of angiogenic sprouts by growth factors. (A) Representative micrographs of hARs treated with basal medium (EBM-2), basal medium plus VEGF-A (10 ng/mL), or complete medium (EGM-2) for 15 days. Scale bar represents 200 μm. (B) The left panel shows representative output data of computer-assisted image analysis, as shown in supplemental Figure 3A. The right panel shows quantification of the angiogenic outgrowth at 15 days of hAR culture. 5% FCS EBM-2 plus heparin (basal medium) with the addition of indicated concentrations of specific GFs or complete EGM-2 (complete medium). Values shown are box-and-whisker plots, normalized to the median of complete medium hARs, of 3 independent experiments, each from 6 different umbilical cords (*P < .05 vs basal medium hARs; **P < .01). (C) Time course of angiogenic outgrowth lengths (normalized to day 16) and velocity (normalized to day 14) of hARs cultured with complete medium (EGM-2). Values shown are mean ± SD of 3 independent experiments, each from 6 different umbilical cords. Graphs represent the computer-assisted quantification shown in supplemental Figure 2A. Giorgio Seano et al. Blood 2013;121:e129-e137 ©2013 by American Society of Hematology

Validation of antiangiogenic drugs with hAR assays. Validation of antiangiogenic drugs with hAR assays. (A) hARs treated with antiangiogenic drugs. Quantification of angiogenic outgrowth at 15 days of culture in EGM-2. hARs treated with indicated concentrations of Sunitinib, SU5416, PI-103, Avastin, and anti-VEGF Ab. For Combretastatin, a vascular-disrupting agent, the treatment started at day 19 (shown in supplemental Figure 6B). Values shown are box-and-whisker plots, normalized to the median of DMSO or control IgG-treated hARs, of 3 independent experiments, each from 6 different umbilical cords (*P < .05 vs DMSO or control [ctr] IgG hARs; **P < .01; ***P < .001). (B) Evaluation of IC50 of antiangiogenic drugs on human and mAR assays. Nonlinear regression R2 > 0.7. Giorgio Seano et al. Blood 2013;121:e129-e137 ©2013 by American Society of Hematology

Morphogenesis of GFP lentiviral-transduced hARs Morphogenesis of GFP lentiviral-transduced hARs. hARs stably transduced with lentiviruses carrying pLKO.1-GFP before gel embedding. Morphogenesis of GFP lentiviral-transduced hARs. hARs stably transduced with lentiviruses carrying pLKO.1-GFP before gel embedding. Three-dimensional rendering reconstruction of high-resolution confocal stacks of representative hARs at indicated days of culture. Magnification is shown in the right column. Scale bar represents 200 µm. Giorgio Seano et al. Blood 2013;121:e129-e137 ©2013 by American Society of Hematology

Lentiviral-mediated stable knockdown of VEGFR2 in hAR and mAR models. Lentiviral-mediated stable knockdown of VEGFR2 in hAR and mAR models. (A) Graph reports VEGFR2 expression in hAR-sprouted cells extracted from BME. hARs are transduced with lentiviruses carrying VEGFR2-targeted shRNA (shVEGFR2_87 and shVEGFR2_88) and scramble shRNA (shSRCL), or not infected (notINF). VEGFR2 mean fluorescence signals of VEGFR2-positive cells are shown and expressed as relative percentage of VEGFR2 levels of shSCRL hARs (*P < .05 vs shSCRL hARs; **P < .01). (B-C) Quantifications and representative micrographs of hARs transduced with lentiviruses carrying VEGFR2-targeted shRNA (shVEGFR2_87 and shVEGFR2_88) and scramble shRNA (shSRCL) or not infected (notINF). hARs were observed after 15 days of EGM-2 culture and are representative of 3 experiments, each from 6 umbilical cords. Values shown are box-and-whisker plots of sprout lengths normalized to the median of shSCRL hARs. Scale bar represents 200 µm (*P < .05 vs shSCRL hARs). (D) Graph showing VEGFR2 expression in mAR-sprouted cells extracted from BME. VEGFR2 mean fluorescence signals of VEGFR2-positive cells are shown and expressed as relative percentage of VEGFR2 levels of shSCRL mARs (*P < .05 vs shSCRL mARs). (E-F) Quantifications and representative micrograph of mARs transduced with lentiviruses carrying VEGFR2-targeted shRNA (shVEGFR2_69 and shVEGFR2_70) and scramble shRNA (shSCRL). mARs were observed after 5 days of EGM-2 culture and are representative of 3 experiments, each from 5 different mice. Values shown are box-and-whiskers plots of sprout lengths, normalized to the median of shSCRL mARs. Scale bar = 200 µm (*P < .05 vs shSCRL mARs). Giorgio Seano et al. Blood 2013;121:e129-e137 ©2013 by American Society of Hematology

Use of the hAR model to test the angiogenic potential of tumor cells. Use of the hAR model to test the angiogenic potential of tumor cells. (A) Representative photograph of hARs cocultured with LNCaP STC for 30 days in basal medium (EBM-2 plus 10% FCS). The inset shows higher magnification of the same photograph. Scale bar represents 400 µm. (B) Representative micrographs of angiogenic outgrowth of hARs with or without LNCaP STCs for 25 days. Scale bar represents 400 µm. (C) Time course of angiogenic outgrowth length (normalized to day 25 of hARs alone) of hARs cultured with complete medium (EGM-2) with or without LNCaP STCs (shown in B). Values shown are mean ± SD of 3 independent experiments, each from 6 different umbilical cords (*P < .05 vs hARs alone; **P < .01; ***P < .001). (D) Branching density analysis in tumor hARs. Representative micrographs and quantifications of angiogenic outgrowth of hARs with or without LNCaP STCs for 25 days. Values shown are box-and-whisker plots of branching points number divided by sprout lengths and normalized to the median of hARs alone (*P < .05 vs hARs alone). (E) Dose-response normalized inhibitor curve fittings for evaluation of IC50 of Sunitinib treatment on hARs alone or with LNCaP STCs. Equation “log(inhibitor) vs normalized response” Y = 100/(1+10^((X-LogIC50))). Nonlinear regression R2 > 0.7. Giorgio Seano et al. Blood 2013;121:e129-e137 ©2013 by American Society of Hematology