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 209

suitable for the proliferation of bone marrow-derived stem cells [10]. Therefore,
we hypothesize that systemically delivered BMSCs would preferentially home to
tumor tissues and participate in the formation of tumor stroma. Therefore, it will
be useful to develop a new targeting system based on genetically modified BMSCs
for tumor gene therapy. The feasibility of this approach has been demonstrated by
Matus Studeny's experimemt [11].
Metastasis is the most common and fundamental characteristics of solid tu-
mors. Most deaths of cancerous patients result from the ruthless growth of me-
tastases that are resistant to conventional remedies. This urges us to develop a
novel therapeutic strategy for inhibiting tumor metastasis. The concept that tu-
mor growth and metastasis relies on angiogenesis has been widely proven and
accepted. The signal axis of vascular endothelial growth factor (VEGF) and its
receptors (VEGFR-1, VEGFR-2 and VEGFR-3) is involved in the formation and
progress of tumors. The soluble VEGF receptor-1 (sFlt-1) has been shown to be
effective in inhibition of cancerous angiogenesis [12].
In this report, we developed a novel strategy of tumor gene therapy in which
BMSCs loaded with recombinant adenoviruses expressing sFlt-1 could effectively
suppress tumor growth through inhibiting angiogenesis and metastases and pro-
long the lifespan in mouse model, indicating that BMSCs might be employed as
an effective carrier for tumor gene therapy.
Methods
bMscs isolation and culture
BMSCs were isolated from femur bone marrow of male BALB/c mouse. Mononu-
clear cells were separated by centrifugation over a Ficoll gradient (Sigma Chemical
Co., St. Louis, MO) of 1.077 g/ml. The cells were cultured in L-DMEM con-
taining 10% fetal bovine serum (Gibco BRL, Inc) at an initial seeding density
of 1 × 10 5 cells/cm2 at 37°C with 5% CO2 and 95% humidified atmosphere.
The nonadherent cells were removed and washed with PBS after 24 hours, and
the monolayer of adherent cells were cultured until they reached to confluence.
The cells were then trypsinized (0.25% trypsin with 0.1% EDTA), subcultured
at densities of 5000-6000 cells/cm2. and used for experiments during passages
five to eight. CD34, CD29 and CD44 expressions in BMSCs were determined
by flow cytometry. Briefly, the trypsinized BMSCs were adjusted to 1 × 10 7 cells/
ml in media (1% bovine serum albumin, 0.2% sodium azide in PBS), and then
stained with FITC-conjugated monoclonal antibodies (BD Biosciences, San Jose,
CA, USA) on ice according to the manufacturer's protocol, and followed by de-
tection with flow cytometry.