Modeling lymphangiogenesis in a three-dimensional culture system

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1 Modeling lymphangiogenesis in a three-dimensional culture system Françoise Bruyère, Laurence Melen-Lamalle, Silvia Blacher, Guy Roland, Marc Thiry, Lieve Moons, Francis Frankenne, Peter Carmeliet, Kari Alitalo, Claude Libert, Jonathan P Sleeman, Jean-Michel Foidart & Agnès Noël Supplementary figures and text: Supplementary Figure 1: Characterization of three-dimensional lymphatic ring cultures. Supplementary Figure 2: Effect of VEGF-C pathway on lymphatic endothelial cell outgrowth. Supplementary Figure 3: Effect of growth factors on lymphatic endothelial cell outgrowth. Supplementary Figure 4: Effect of medium conditioned by aortic smooth muscle cells. Supplementary Figure 5: Effect of proteolytic pathway on lymphatic endothelial cell outgrowth. Supplementary Figure 6: Induction of lymphangioma in MMP-2-deficient and wild type mice. Supplementary Figure 7: Visualization of lymphatic thoracic duct excision. Supplementary Figure 8: Illustration of the method of quantification. Supplementary Methods Note: Supplementary Videos 1 4 are available on the Nature Methods website. 1

2 a Supplementary Figure 1 b Von Wil CD34 c d Thy1.1 CD45 e f αsma Alpha SMA Negative control Supplementary Figure 1: Characterization of three-dimensional lymphatic ring cultures. Outgrowing lymphatic endothelial cells were immunopositive (in green) for Von Willebrand factor (Von Wil) (a) and negative for CD34 (b), Thy1.1 (c) and CD45 (d). Red staining corresponds to nuclear staining with propidium iodide (a-d). A few alpha smooth muscle (asma) positive cells were detected close to the lymphatic rings, but they never covered lymphatic endothelial cells (e). Blue staining corresponds to nuclear staining with DAPI (d). A negative control (f) using the secondary antibody reveals the non-specific staining of the lymphatic ring. Scale bar : 4 µm.

3 Supplementary Figure 2 VEGF-C Control (4% ultroser) 5ng/ml 1ng/ml svegfr-3 Control (4% ultroser +1ng/ml VEGF-C) 1µg/ml 5µg/ml MAZ51 Control (1% FCS) 3µg/ml 16µg/ml Clt (4% Ultroser) 5 ng/ml 1 ng/ml NS Clt (4% Ultroser + VEGF-C) 1 µg/ml 5 µg/ml Clt (1% FCS) 3 µg/ml 9.5 µg/ml 16 µg/ml Supplementary Figure 2: Effect of VEGF-C pathway on lymphatic endothelial cell outgrowth. Representative micrographs of lymphatic rings cultured in the presence of increasing concentrations of VEGF-C, svegfr-3 in the presence of 1 ng/ml VEGF-C and MAZ51 to rings cultured with 1% FCS. The graph represents the computerized quantification shown in Fig. 4.

4 Supplementary Figure 3 PlGF Control (4% ultroser) 5ng/ml 1ng/ml FGF2 Control (4% ultroser) 5ng/ml 2ng/ml Suramin Control (1% FCS) 3mg/ml 6mg/ml PlGF FGF-2 Clt (4% Ultroser) 5 ng/ml 1 ng/ml 2 ng/ml Suramin Clt (4% Ultroser) 5 ng/ml 1 ng/ml 2 ng/ml Clt (1% FCS) 3 mg/ml 6 mg/ml PDGF Control (4% ultroser) 5mg/ml 2mg/ml PDGF Clt (4% Ultroser) 1 ng/ml 5 ng/ml 2 ng/ml 1.4

5 PDGF inhibitor Control (4% ultroser+pdgf).35µg/ml 3.5µg/ml PDGF inhibitor + serum Control (1% FCS) 1.7µg/ml 3.5µg/ml PDGF-R inhibitor Clt (4% Ultroser +PDGF).35 µg/ml 3.5 µg/ml 1.4 PDGF-R inhibitor + serum Clt (1% FCS) 1.7 µg/ml 3.5 µg/ml 7 µg/ml Supplementary Figure 3: Effect of growth factors on lymphatic endothelial cell outgrowth. Representative micrographs of lymphatic rings cultured in the presence of increasing concentrations of PlGF, FGF-2, suramin (FGF trapper), PDGF, and a PDGF receptor (PDGF-R) inhibitor in the presence of 5ng/ml PDGF or with 1% FCS. The graph represents the computerized quantification shown in Fig. 4.

6 Supplementary Figure Clt (SMC medium) SMC medium+ PlGF Distance to the ring (mm) 1 Clt (SMC medium) SMC medium + FGF Distance to the ring (mm) Supplementary Figure 4: Effect of medium conditioned by aortic smooth muscle cells Lymphatic rings were cultured in the presence of medium conditioned by aortic smooth muscle cells (SMC) untreated (Clt, SMC medium) or treated with PlGF (1ng/ml) (SMC + PlgF) (a) or FGF (1ng/ml) (SMC + FGF) (b).

7 Supplementary Figure 5 Aprotinin Control (1% FCS) 5µg/ml 25µg/ml Clt (1% FCS) 5 µg/ml 1 µg/ml 25 µg/ml PAI-1 KO WT TIMP2 KO PAI-1 WT PAI-1 -/- 2. Control (1% FCS) 7µg/ml 14µg/ml Clt (1% FCS) 1 µg/ml 7 µg/ml 14 µg/ml RO Control (1% FCS).5mg/ml 5mg/ml Clt (1% FC.5 mg/ml mg/ml 5 mg/ml

8 MMP-2 KO WT KO MMP-2 WT MMP-2 -/ Supplementary Figure 5: Effect of proteolytic pathway on lymphatic endothelial cell outgrowth. Representative micrographs of lymphatic rings cultured in the presence of increasing concentrations of aprotinin, TIMP-2, or synthetic MMP inhibitor (RO ). Lymphatic rings cultured in the presence of autologous mouse serum derived from PAI-1-deficient mice (PAI -/- ), Mmp-2-deficient mice (Mmp-2 -/- ), and the corresponding wild type mice (WT). The graph represents the computerized quantification shown in Fig. 5.

9 Supplementary Figure 6 a. c. b. Lymphangioma surface (cm2) KO liver d. diaphragm MMP2 KO MMP2 WT WT KO WT Lymphangioma surface (%) MMP2 KO MMP2 WT Supplementary Figure 6: Induction of lymphangioma in MMP-2-deficient and wild type mice. Injection of incomplete Freund s adjuvant led to the formation of lymphangiomas at the surface of liver (a, b) and diaphragm (c, d) in MMP-2- deficient mice (KO) and their corresponding wild type mice (WT). a, c: Representative macroscopic lesions delineated by black borders. b,d: Quantification. Results are expressed as the surface of liver covered by lymphangioma (b) or the percentage of diaphragm surface covered by lymphangioma (d). :P<.5; : P<.5

10 a Supplementary Figure 7 b c Step 1 Aorta Aorta d e Step 2 Aorta Aorta f g Step 3 Supplementary Figure 7: Visualization of lymphatic thoracic duct excision. The lymphatic thoracic duct is dissected out of the mouse thoracic cavity filled with sterile PBS (a). Fat tissue above the aorta is first ressected (b, c) (Step 1) before the careful removal of fat tissue surrounding the lymphatic thoracic duct (d, e) (Step 2). Once the duct is completely separated from fat, both ends are cut with scissors (Step 3) and immediately transferred into an ice-cold DMEMcontaining culture dish (f). Lymphatic thoracic duct is then cut into about 1mm long pieces and maintained in serum-free medium prior embedding into a collagen gel (g). Arrow heads delineate the lymphatic duct. Scale bars (a): 5mm, (b,d,f,g): 2mm, (c,e): 1mm

11 Supplementary Figure Distance to the ring (mm) Distance to the ring (mm) Supplementary Figure 8: Illustration of the method of quantification. Two images corresponding to different growing rates were first binarized and a grid of concentric rings was drawn by making successive dilatations at similar intervals (one pixel) of the thoracic duct boundary. The number of intersections between microvessels and this grid was determined for each image. Microvessel distribution is defined as the number of intersections plotted as a function of the distance from the ring.

12 SUPPLEMENTARY METHODS Gel whole-mount immunostaining On day 11 of culture, lymphatic rings were harvested from the agar cylinder, washed for 3 min in PBS, fixed for 3 min with 8 % methanol, and kept at 4 C in 7 % ethanol until used or fixed for 3 min with 4% paraformaldehyde and kept in an azide/pbs solution until used. For immunostaining, gels were washed three times with PBS and blocked in 1.5% BSA-3% Gloria milk for 1 h at room temperature. The gels were then incubated overnight with appropriate primary antibodies: rabbit anti-lyve-1 (1/6), rat anti-vegfr-3 (1/5) (both provided by K. Alitalo, Finland), rabbit anti-von Willebrand Factor (1/2) (DAKO, Glostrup, Denmark), rat anti-cd45 (1/4) (BD Pharmingen, Franklin Lakes, NJ), rat anti-cd34 (1/5) (BD Pharmingen, Franklin Lakes, NJ), anti-alpha Smooth Muscle Actin (asma) (1/1) (Sigma-Aldrich, St Louis, MO) and mouse anti-thy1.1 (1/1) (Chemicon, Billerica, MA). After four washes with PBS, gels were incubated with the appropriate FITC-coupled secondary antibodies for 6 min at room temperature (rabbit anti-rat Ig (1/1; DAKO, Glostrup, Denmark); swine anti-rabbit Ig (1/4; DAKO, Glostrup, Denmark)). After washing with PBS, gels were mounted on a microscope slide with Vectashieldpropidium iodide mounting medium or DAPI (Vector Laboratories, Burlingame, CA). Transmission electron microscopy Lymphatic rings cultures were fixed after 11 days of culture for 1 h at 4 C with % glutaraldehyde in a Sörensen.1 M phosphate buffer (ph 7.4) and postfixed for 3 min with 1% osmium tetroxide. After dehydration, samples were embedded in Epon. Ultrathin sections obtained with a Reichert Ultracut S ultramicrotome were contrasted with uranyl acetate and lead citrate. Observations were made with a Jeol 1 CX II transmission electron microscope at 6 kv. Gelatinolytic zymography Media conditioned by thoracic duct rings derived from wild type or Mmp-2-deficient mice were collected after 11 days of culture. Protein samples (2 µg) were separated in 1% SDSpolyacrylamide gels containing 1 mg/ml gelatin (Sigma-Aldrich, St Louis, MO). After electrophoresis, SDS was removed from the gels by two incubations in 2% Triton X-1 for 3 min. Gels were incubated overnight at 37 C in 5 mm Tris-HCl (ph 7.4), M NaCl and 5 mm CaCl 2. Gels were then stained for 2 min with Coomassie Blue and destained for 2 h. Medium conditioned by HT18 fibrosarcoma cells in presence or absence of Concanavaline A (ConA) was used as controls. Aortic smooth muscle cell culture Aortic smooth muscle cell purchased from Lonza (Switzerland) were routinely grown according to manufacturer s instructions in SmGM medium complemented with the singlequots Kits (Lonza, Switzerland). Cells were seeded in 6-well plates (Falcon) (3, cells/well) and incubated in serum-containing medium for 24h. Cells were then washed twice with PBS and subsequently incubated with 3ml of serum-free SmGM not supplemented (untreated control) or supplemented with 1 ng/ml PlGF or FGF-2. Induction of lymphangioma Lymphatic endothelial tumor (lymphangioma) formation was induced by two intraperitoneal injections of incomplete Freund s adjuvant as described 5,6. After 4 weeks, the surface of lymphangioma masses appearing at the surface of diaphragme and liver was measured by using the Image J software. The lymphatic origin of tumors was assessed by positivity for LYVE-1.