VEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization

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1 Cell Reports, Volume 9 Supplemental Information VEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization Sharon Lim, Yin Zhang, Danfang Zhang, Fang Chen, Kayoko Hosaka, Ninghan Feng, Takahiro Seki, Patrik Andersson, Jingrong Li, Jingwu Zang, Baocun Sun, and Yihai Cao

2 SUPPLEMENTAL INFORMATION Supplemental Experimental procedures Mouse tumor model. In tumor removal experiments, primary tumors were surgically removed at the size cm 3, and the surgical incisions were sutured. Treatment with antibodies started at day 2 post surgery. All mice were sacrificed by inhalation with a lethal dose of CO 2, blood samples were collected through cardiac puncture, and plasma was prepared using EDTA-coated tubes, followed by centrifugation at g for 20 min. Unless freshly used, all samples were stored at 80 C for further analysis. Vascular permeability. Lysinated rhodamine dextran at the molecular weight of kda was intravenously injected into vector or VEGF tumor-bearing mice. Fresh bone samples were prepared from sacrificed mice and was immediately frozen on dry ice. The compact bones were carefully peeled off and the bone marrow were fixed with 4% PFA at 4 C overnight, followed by staining with a rat anti-mouse endomucin antibody (1:200 dilution in PBST) at 4 C overnight. A secondary fluorescentconjugated antibody (Goat anti-rat Alexa 555, Invitrogen, A21434; 1:400 dilution in PBST) was incubated at RT for 2 h. Bone marrow samples were mounted with Vectashield (Vector Labs, H-1000) and positive signals were detected with a confocal microscope. Bone marrow irradiation. C57Bl6 mice were irradiated with a lethal dose of 900- gamma-rad. Irradiated mice were sacrificed at day 7 and fresh bone samples were fixed with 4% PFA at 4 C overnight, followed by washing with PBS prior to paraffin- 1

3 embedding. Paraffin-embedded samples of 5 µm thickness were stained with Hematoxylin-Eosin or were subjected to immunohistochemistry using the same protocol as described below. FACS Experiment. Blood samples were collected by cardiac puncturing, and BMCs collected from femur and tibia bones were centrifuged at rpm for 4 min. Samples were blocked with 10% healthy mouse serum in PBS at RT for 15 min. Anti- VEGFR1 and anti-vegfr2 antibodies were incubated at the concentration of 64 µg/ml in PBS on ice for 1 h, followed by washing with cold PBS. Samples were further incubated with a goat anti-rat Cy5-labeled antibody (1:400 in PBS) on ice for 30 min, followed by washing twice with PBS. For triple staining, a mouse hematopoietic lineage FITC cocktail (1:12.5, ebioscience, ) containing an anti-mouse Ly-6A/E PerCP-Cyanine 5.5 antibody (1:200, ebioscience, ) and anti-mouse CD117 PE antibody (1.100, ebioscience, ) in combination with an anti-mouse CD31 PE antibody (1:100, BD Pharmingen, ), or an anti-mouse CD45 FITC antibody (1.150,BioLegend, ) were used after VEGFR1 and VEGFR2 antibody staining. Antibodies were incubated on ice for 45 min followed by washing with cold PBS. Samples was fixed in 1% PFA and analyzed with BD FACS scanner. For each sample, events were analyzed. MACS-based cell separation. BMCs collected from fresh femur and tibia bones were centrifuged at rpm for 10 min. Samples were incubated with MASC endothelial cell microbeads at 4 C for 15 min (Miltenyi Biotec, ), followed by washing with cold washing buffer. Samples were further added to LS MACS separators and the magnetically labeled endothelial cells were incubated with 2

4 100 µl Tris-HCl lysis buffer (ph 7.5) in the presence of a phosphatase inhibitor cocktail (5470S, Cell Signaling) on ice for 10 min and centrifuged at rpm for 15 min at 4 C. The supernatant was collected for immunoblotting. Supplemental Figure Legends Figure S1. Plasma levels of circulating VEGF and peripheral blood counts, related to Figure 1. (A) Plasma VEGF levels were measured from 7 8 mice of tumor-free, vector tumorbearing and VEGF tumor-bearing mice. (B) RBC, HGB and HCT from peripheral blood of tumor-free, vector tumor-bearing and VEGF tumor-bearing of wt C57Bl6 mice were measured (n = 7 8). (C) RBC, HGB, HCT, and WBC from peripheral blood of tumor-free, vector tumorbearing and VEGF tumor-bearing of Vegfr1 TK-/- mice were measured (n = 3 5). All data are represented as mean ± s.e.m. **P<0.01, ***P<0.001 (Student s t-test, twotailed). Figure S2. Effects of tumor-derived VEGF isoforms, and VEGFR1-binding ligands PlGF and VEGF-B in mobilization of BMCs, related to Figure 3. (A) H&E staining of BM of tumor-free, vector, VEGF 121, VEGF 165, and VEGF 189 tumor-bearing mice (n = 6). Arrows point to BMCs. (B) Endomucin staining of BM microvessels of tumor-free, vector, VEGF 121, VEGF 165, and VEGF 189 tumor-bearing mice. Endomucin (red) and DAPI (blue) double immunostaining. Arrows indicate microvessels. (C E) Quantification of BMC numbers (C), microvessel numbers (D) and sinusoidal areas of microvessels (E) in each group (n = 6 8). Scale bars of each panels, 50 µm. 3

5 All data are represented as mean ± s.e.m. **P<0.01, ***P<0.001 (Student s t-test, two-tailed). (F) H&E staining of BM of tumor-free, vector, PlGF, and VEGFB tumor-bearing mice (n = 6). Arrows point to BMCs. (G) Double immunostaining of endomucin (red) and DAPI (blue) in BM of tumorfree, vector, PlGF, and VEGFB tumor-bearing mice (n = 6). Arrows indicate microvessels. (H J) Quantification of BMC numbers (H), BM microvessel numbers (I), and sinusoidal areas of BM microvessels (J). (n = 6 8). Scale bars of each panels, 50 µm. All data are represented as mean ± s.e.m. Figure S3. BMC mobilization and sinusoidal vascular dilation after removal of primary tumors, related to Figure 4. (A) Effects of VEGFR1 and VEGFR2 blockades on BMC mobilization and microvascular dilation in tumor-free healthy mice (n = 6 8). Black arrows point to BMCs and yellow arrows indicate sinusoidal blood vessels. Numbers of BMCs and microvessels as well as sinusoidal vascular areas were quantified (n = 6 8). Not treated (NT). (B) BMC mobilization and microvascular dilation of VEGF-tumor-bearing mice and mice after removal of primary tumors. Black arrows point to BMCs and yellow arrows indicate sinusoidal blood vessels. Numbers of BMCs and microvessels as well as sinusoidal vascular areas were quantified (n = 6 8). (C) Effects of VEGF, VEGFR1 and VEGFR2 blockades on BMC mobilization and microvascular dilation in mice after removal of primary VEGF tumors. Black arrows point to BMCs and yellow arrows indicate sinusoidal blood vessels. Numbers of 4

6 BMCs and microvessels as well as sinusoidal vascular areas were quantified (n = 6 8). Scale bars of each panels, 50 µm. All data are represented as mean ± s.e.m. *P<0.05, ***P<0.001 (Student s t-test, two-tailed). Figure S4. FACS analysis of VEGFR1, VEGFR2 and total cell populations in peripheral blood and BM, related to Figure 5. (A) FACS analysis of VEGFR1 + or VEGFR2 + signals in CD31 + fraction in wt tumors. Human ovarian cancer cells were used as a negative control. (B) Analysis of peripheral blood VEGFR1, VEGFR2 and total cell populations in tumor-free, vector tumor- and VEGF tumor-bearing mice (n = 6). (C D) Percentages (C) and absolute numbers (D) of VEGFR1 and VEGFR2 in peripheral blood are presented (n = 6). (E) Analysis of BM VEGFR1, VEGFR2 and total cell populations in tumor-free, vector tumor- and VEGF tumor-bearing mice. (F G) Percentages (F) and absolute numbers (G) of VEGFR1 and VEGFR2 in BM are presented (n = 6). Figure S5. FACS analysis of VEGFR1, VEGFR2 in LSK and myeloid cell populations in BM, related to Figure 6. (A) FACS analysis of BM lineage + and lineage - cell populations (n = 6). (B C) Analysis of BM VEGFR1 + (B) and VEGFR2 + (C) subpopulations of LSK populations of tumor-free healthy, vector and VEGF tumor-bearing mice. (n = 6). (D E) Analysis of BM VEGFR1 + (D) and VEGFR2 + (E) subpopulations of CD45 + populations of tumor-free healthy, vector and VEGF tumor-bearing mice. (n = 6). 5

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