Anti-Angiogenesis Therapy Based on the Bone Marrow- Derived Stromal Cells Genetically Engineered to Express Sflt-1 in Mouse Tumor Model - Genetic Engineering: Recent Developments in Applications - page 211

in a 1.5% solution of sodium alginate and added dropwise into a swirling 37°C
solution of 250 mM calcium chloride. Alginate beads were formed with about 1
× 105 cells per bead. After mice were anesthetized, 4 beads were implanted sub-
cutaneously into an incision made on the dorsal side. Incisions were closed with
surgical clamps. After 21 days, mice were injected intravenously with 100 µ l of a
100 mg/kg FITC-dextran solution (Sigma). Beads were surgically removed, and
FITC-dextran was quantified against a standard curve of FITC-dextran.
Murine tumor Metastases Models and treatment
The colon carcinoma CT26, Lewis lung cancer LLC and fibrosarcoma MethA cell
lines were used in this study. Metastasis models of LLC and CT26 were generated
as described previously [15]. Briefly, female C57BL/6 and BALB/c mice were
received i.m. injections of 2×10 5 LLC in leg or i.v. injection of 2×10 5 CT26 cells
from tail vein in 100 µ l of PBS, respectively and then the animals were randomly
divided into four groups (10 mice/group). After 4 days for CT26 model and
10 days for LLC model of tumor inoculation, 1×10 6 of BMSCs infected with
Adv-GFP-sFlt-1, BMSCs infected with Adv-GFP, unifected BMSCs or 100 µ l
of 0.9% NaCl solution alone were administered via tail vein for four doses at 3
days intervals for CT26 model and 5 days intervals for LLC model. Mice received
LLC or CT26 injection were sacrificed on day 32 or day 16 after inoculation, re-
spectively, when control mice became moribund, and the lungs from each mouse
were removed and the surface lung metastases (>3 mm) were measured and scored
[15]. The lungs of animals were also fixed in 10% buffered formalin followed by
histological analysis. All of the mice used in this study were approved by the West
China Hospital Cancer Center's Animal Care and Use Committee. In the han-
dling and care of animals, all possible steps were taken to avoid animals' suffering
and efforts were made to use the minimum number of animals.
Histological Analysis
The tissues were fixed in 10% neutral buffered formalin solution and embedded
in paraffin. Sections of 3-5 µ m were stained with hematoxylin and eosin (H.E.).
Frozen sections were fixed in acetone, incubated, and stained with anti-CD31
antibody, then visualized with DAKO LSAB kit (DAKO, Carpinteria, CA), as
described previously. Vessel density was determined by counting the number of
microvessels per high-power field in the sections, as described [16]. Apoptosis in
tumor tissues was determined by terminal dUTP nick-end labeling (TUNEL)
method using an in situ cell death detection kit (Roche Molecular Biochemicals)
following the manufacturer's protocol [17,18]. Sections in H&E staining and