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Blood, Vol. 107, Issue 7, 2774-2776, April 1, 2006
Evidence for incorporation of bone marrow–derived endothelial cells into perfused blood vessels in tumors
Blood Duda et al.
107: 2774
Supplemental materials for: Duda et al
Methods Transgenic mouse and tumor models
One transgenic strain, developed on a C57BL6 background, harbors GFP driven by the chick  -actin promoter and the CMV intermediate early enhancer (Actb-GFP; C57BL/6-Tg(ACTB-EGFP)1Osb/J). Another, developed on a FVB background, homozygously expresses GFP under the endothelial Tie2 promoter (Tie2-GFP, stock Tg(TIE2GFP)287Sato/J). These mice were acquired from Jackson Laboratories, Bar Harbor, ME, USA and subsequently bred in our animal facility. Bone marrow transplantation
Bone marrow transplants were performed after characterizing the sensitivity of the two strains to whole body irradiation through longitudinal studies. Whole body irradiation, in a single dose of 9 Gy for FVB mice and 12 Gy for C57BL6 mice, was administered to recipient mice. The recipient mice were salvaged by injection of 5 × 106 bone marrow cells harvested from donor mice, following a standard protocol. In brief, the donor mice were sacrificed by pentobarbital overdose (200 mg/kg body weight, i.p. injection) prior to marrow extraction. Bone marrow cells were harvested from donors by flushing tibias with cold saline. The level of bone marrow reconstitution was verified by flow cytometry analyses. Wild type mice receiving GFP-cell transplants are referred to as WT/Tie2-GFP and WT/Actb-GFP, respectively. Tie2-GFP mice that received wild-type marrow cells are referred to as Tie2-GFP/WT-BMT. Tumor cell lines
Isogenic tumor implantation was carried out both in wild type and mutant mice, using mammary carcinoma lines (TG1-1 and MCa8 for FVB), lung adenocarcinoma lines (LA-P0297 for FVB, and Lewis Lung carcinoma, Lewis lung carcinoma for C57BL6 mice) and B16 melanoma for C57BL6 mice. Removal of primary lung adenocarcinoma isografts resulted in multiple distant tumors particularly in the lungs, in both strains of mice. Finally, a group of WT/Tie2-GFP-BMT mice was maintained in the colony and monitored for the development of spontaneous tumors in aging mice (two tumors developed in mice at around 12 months of age, the other two in 18 months old mice).
For tumor implantation, 1 × 106 neoplastic cells were suspended in 50 µl of Hanks’ Balanced Salt Solution (Invitrogen, Grand Islands, NY, USA) and injected into the mammary fat pads or subcutaneously in mice. For intravital multiphoton microscopy1,2, 2 × 105 neoplastic cells were superficially injected under the pial surface of the brain (for the cranial window model3; n = 4). Prior to all surgeries, recipient mice were anesthetized using intramuscular injections of ketamine HCl (9 mg, Ketaset, Aveco, Fort Dodge, IA, USA) and xylazine HCl (0.9 mg, Anased, Lloyd Laboratories, Shenandoah, IO, USA) at 1 ml per 100 g body weight. Tumor implantation was performed at least 3 months after the irradiation and transplantation procedures described. Tissue preparation and histology
BMD-EC incorporation in functional (blood-perfused) vessels was achieved by i.v. perfusion staining with biotinylated lectin2 (0.1 ml of 1 mg/ml biotinylated Lycopersicon esculentum (Tomato) lectin (Vector Labs, Burlingame, CA, USA). Mice were sacrificed after 5 minutes of perfusion staining and whole body perfusion fixation was performed using 4% paraformaldehyde. Tumors were excised, dehydrated overnight at 4°C in 30% sucrose in PBS, embedded in OCT compound and stored at —80°C. Fluorescence tissue counterstaining was performed using the nuclear dye DAPI (Molecular Probes, Eugene, OR, USA). Tumor sections (20-30 µm-thick) were mounted on slides using Vectashield mounting media for fluorescence (Vector Labs). The method allowed identification of functional vascular structures in tumors while preserving the morphology. Tissue sections were evaluated for content of BMD-ECs and vascular parameters. Co-localization of GFP-positive, bone marrow-originating cells with lectin-stained capillaries was verified and quantified by confocal microscopy. Tie2 promoter-driven GFP expression was distinguished from autofluorescence by using a multispectral imaging system (CRI Inc., Cambridge, MA, USA). The number of GFP+ vessels in sections was counted in 6-10 regions of interest and normalized by the total number of lectin-stained vessels. Vessel diameters were measured as the smallest diameter for each vessel lumen. Flow cytometric analyses
Four-color flow cytometry was used to analyze GFP positive cells after obtaining single-cell suspensions from tumor tissues using enzymatic digestion (using 30 minutes digestion at 37°C with collagenase type 2, Worthington Biochemical Corporation Lakewood, NJ, USA). For analyses of cell phenotype, we used fluorecently labeled, monoclonal antibodies against mouse CD31, Sca-1, Mac-1 (CD11b), Tie2, CD45 (all BD Pharmingen, San Diego, CA, USA) and CD133 (Chemicon, Temecula, CA, USA). Flow cytometry was performed on FACSVantage Instruments (Becton Dickinson, San Jose, CA, USA). Analysis was performed using FloJo software (Tree Star, Inc., San Carlos, CA, USA) after collection of 50,000-100,000 mononuclear cell events. Supplemental Material References 1. Brown EB, Campbell RB, Tsuzuki Y, et al. In vivo measurement of gene expression, angiogenesis, and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med. 2001;7:864-868.
2. Duda DG, Fukumura D, Munn LL, et al. Differential transplantability of tumor-associated stromal cells. Cancer Res. 2004;64:5920-5924.
3. Hobbs SK, Monsky WL, Yuan F, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proceedings of the National Academy of Sciences of the United States of America. 1998;95:4607-4612.
Files in this Data Supplement:
- Figure S1. Detection of BMDCs in tumors grown in WT/Actb-GFP-BMT mice (JPG, 81.9 KB)
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(A) Flow cytometry plots from dissociated lung tumor tissue: The small population of GFP+CD31—CD45— BMDCs was positive for Sca1, but negative for CD133. (B, C) Representative images of lectin perfusion-staining (shown in red) of Lewis lung carcinoma (B) and B16 melanoma (C) vessels: despite the massive GFP-BMDC infiltration, particularly in LLC tissues, there was little or no direct incorporation of BMD-ECs in functional tumor vessels. Insert in B shows blood vessels surrounded by GFP cells that are not directly involved in the endothelial lining Images are 1.72 mm across in B (423 µm across in the insert) and 826 µm across in C. (DAPI nuclear staining shown in blue.)
- Figure S2. Detection of GFP in brain tissue in Tie2-GFP mice (JPG, 126 KB)
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(A-D) GFP was readily detectable in endothelial cells in normal brain tissue and in mammary carcinoma tissues isografted in the brain and imaged by confocal microscopy. (A, C) Representative images showing only GFP expression under Tie2 promoter in brain endothelial cells (B, D) Same images as in A and C after merging the endogenous GFP (shown in green) with the Texas Red-streptavidin staining of biotinylated-lectin perfused vessels (shown in red) and DAPI nuclear counterstaining (in blue). Images are 1.72 mm across.
- Figure S3. Mammary tumor tissue in a Tie2-GFP mouse (A) and in WT/Tie2-GFP-BMT mice (B-D) imaged in frozen sections by confocal microscopy (JPG, 110 KB)
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Note the absence of GFP in perfused vessels of tumors isografted in the fat pad (B), and the expression of GFP in the majority of mammary tumor vessels when isografted in the brain (C, D). Vessels were perfused with biotinylated-lectin and stained with streptavidin Texas Red (shown in red), while the nuclei were stained with DAPI (in blue). Images are 1.72 mm across.
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