Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model

BMSCs were isolated from femur bone marrow of male BALB/c mouse. Mononuclear 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 containing 10% fetal bovine serum (Gibco BRL, Inc) at an initial seeding density of 1 × 10⁵ 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/cm^2. 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 × 10⁷cells/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 detection with flow cytometry.

AdenoVec-GFP-sFlt-1 and AdenoVec-GFP (InvivoGen, San Diego, CA) were transfected into HEK-293 cells using jetPEI (Qbiogene, Nottingham, UK) according to the manufacturer's protocol. Seven to 14 days later, the recombinant adenoviruses were harvested from the cultures and the viruses were further amplified in 293 cells, and then tittered by plaque assay. BMSCs were infected with adenoviruses at a multiplicity of infection (MOI) of 3000 for 2 hours followed by replacing the infection medium. Twenty-four hours later, the virus-infected BMSCs were harvested for subsequent experiments [11].

Western blot analysis was performed as described previously [13]. Briefly, the secreted proteins in supernatants of the culture were precipitated by TCA-DOC/acetone and the western blot assay was performed using standard method with primary monoclonal antibody of anti-sFlt-1 (1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a biotinylated secondary antibody, and the proteins on the membrane were visualized by Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).

The mouse endothelial cells proliferation inhibition assay was performed as described previously for examining suppression of sFlt-1 in the conditioned media on VEGF-driven proliferation of endothelial cells [14]. The conditioned media were obtained from BMSCs infected with Adv-GFP-sFlt-1, Adv-GFP, or PBS (mock infected), respectively. Endothelial cells were grown in 24-well plates at 37°C in DMEM containing 10% FBS. At 50% confluence, the cells were washed with PBS after removal of the DMEM media, and then 0.5 ml of conditioned media was added. Fifteen minutes later, VEGF was added to each well with a final concentration of 10 ng/ml and the cells were incubated at 37°C. After 72 h, the cells were trypsinized, and the number of viable cells was counted using a trypan blue assay.

Alginate-encapsulated tumor cell assays were performed as described previously [13]. Briefly, tumor cells, BMSCs expressing sFlt-1 or mixed cells were resuspended 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 subcutaneously into an incision made on the dorsal side. Incisions were closed with surgical clamps. After 21 days, mice were injected intravenously with 100 μl 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.

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 2×10⁵ LLC in leg or i.v. injection of 2×10⁵ CT26 cells from tail vein in 100 μl 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, 1×10⁶ of BMSCs infected with Adv-GFP-sFlt-1, BMSCs infected with Adv-GFP, unifected BMSCs or 100 μl 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, respectively, 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 handling and care of animals, all possible steps were taken to avoid animals' suffering and efforts were made to use the minimum number of animals.

The tissues were fixed in 10% neutral buffered formalin solution and embedded in paraffin. Sections of 3–5 μm 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 immunohistochemical staining were observed by two pathologists in a blinded manner.

Fluorescent in situ hybridization was performed according to the mouse Y chromosome probe manual (STAR*FISH; Cambio, Cambridge, England) with some modifications. Cryostat sections of 5 μm in thickness were fixed in Carnoy's fixative for three times, 10 min each, and the sections were incubated in pepsin solution (1% in 0.1N HCl) for 10 minutes and rinsed in PBS. Serial ethanol dehydration was done (1.5 min each), and the slides were air-dried at room temperature. Sections were denatured at 65°C for 2 min in preheated 70% formamide and 2×SSC buffer, pH 7.0, and were then 'quenched' with ice-cold 70% ethanol for 1.5 min. Serial ethanol dehydration was done again. The mouse Y chromosome probe labeled with Cy3 was denatured at 65°C for 10 min and applied to the sections. The sections were coverslipped and sealed with rubber cement for incubation overnight in a hydrated slide box at 37°C. The next day, the coverslips were carefully removed. The sections were washed twice in preheated 50% formamide in 2×SSC buffer for 5 min each at 45°C and were then gently washed twice in preheated 1×SSC buffer for 5 min each at 45°C.

After harvest of monolayer cells with spindle-like morphology from mouse bone marrow, three CD markers (CD34, CD29 and CD44) were used for characterizing BMSCs. The cells were positive for CD29, CD44, and negative for CD34, indicating that they didn't belong to hematopoietic progenitor cells, but rather bone marrow-derived stromal cells (BMSCs) or mesenchymal stem cells (MSCs). BMSCs were cultured to reach to 90% confluence and incubated with adenoviruses at a MOI of 3000 for 2 hours. Twenty four hours later, all of the cells were GFP-positive checked by fluorescence microscopy and the cells were ready for tail vein infusion after PBS washing two times (Figure 1A). The secreted sFlt-1 in the supernatants of conditioned media was verified by Western blot (Figure 1B) and the function of sFlt-1 was validated by endothelial cell growth inhibition assay in vitro. The concentrated conditioned media obtained from BMSCs infected with Adv-GFP-sFlt-1 or with Adv-GFP were applied to the mouse endothelial cells grown in 24-well plates, followed by stimulation with a 10 ng/ml of VEGF 15 minutes later. Conditioned media from BMSCs infected with Adv-GFP-sFlt-1 resulted in inhibition of endothelial cells proliferation by about 50% compared with that from BMSCs infected with Adv-GFP or uninfected BMSCs (Figure 1C, P < 0.05), indicating that the BMSCs infected with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1.

The mouse CT26 and LLC metastasis models were used to determine whether BMSCs expressing sFlt-1 suppress the growth of metastatic neoplasm. The metastatic nodules > 3 mm were assessed as an indicator of angiogenesis because the growth of tumors > 3 mm is thought to be vasculature sensititive [19]. Most of the mice systemically administered with control BMSCs or NaCl solution developed macroscopic lung metastases, whereas mice treated with BMSCs expressing sFlt-1 decreased lung metastases. The tumor nodules (> 3 mm) of lung surface were significantly decreased in the mice treated with BMSCs expressing sFlt-1 (P < 0.05) (Figure 2A, B). Lung weight, which correlates with total tumor burden, was also reduced significantly in the team of mice administered with BMSCs expressing sFlt-1 (Figure 2C). Booming proliferation of tumor cells and severe destruction of lung tissues were observed in the H.E. staining for control mice, whereas in the mice treated with BMSCs expressing sFlt-1, cancerous cells were less nourished and lung tissues were relatively normal (Figure 2D). Benefited from the decrease of tumor burden in the lung metastases, the lifespan of the tumor-bearing mice was prolonged significantly (Figure 2E, P < 0.05). The similar results were also observed from mouse LLC metastasis model (Figure 3).

Systemically delivered BMSCs which express sFlt-1 were shown to induce apparent inhibition of angiogenesis in vivo compared with control mice by immunohistochemistry. Angiogenesis within tumor tissue was estimated by counting the number of microvessels on the section staining with an anti-CD31 antibody. Very few newborn microvessels were found around the tumor cells in the BMSCs-sFlt-1 group, whereas abundant microvessels existed in three controls (Figure 4A, B, P < 0.05). The inhibition of angiogenesis in mice treated with BMSCs expressing sFlt-1 was further confirmed in alginate encapsulation assay. The surface vessel densities and the FITC-dextran uptake were apparently reduced in beads which contain relatively high ratio of sFlt-1-bearing BMSCs to LLC cells (Figure 4C, D, P < 0.05). Furthermore, an another alginate assay showed that the anti-angiogenesis effect of local production of sFlt-1 (came from the alginate beads being filled with sFlt-1-bearing BMSCs and tumor cells, or the BMSCs intravenous injected) was stronger than systemic production of sFlt-1 (came from the alginate beads being filled with sFlt-1-bearing BMSCs and implanted in the distant site) (Figure 4-E and 4F). Being starved of the nourishment by the inhibition of angiogenesis in neoplasm, augmentation of apoptosis of cancerous cells was detected by the TUNEL assay (Figure 5, P < 0.05). The inhibition of angiogenesis and the consequent induction of apoptosis underlie the mechanism of the metastases decrease and the life-span prolongation in tumor-bearing mice.

It has been reported that ubiquitous tissue distribution of adipose-derived stem cells in normal mouse was observed when the cells were systematically administrated [20]. We are concerned about the existence of BMSCs in the metastases and the surrounding tissues. To detect the tumor tropism of BMSCs after systemically administration, fluorescent in situ hybridization of Y chromosome was performed on frozen section of CT26 lung metastasis after BMSCs administration one week. Positive signals were observed in the frozen section of lung metastases exclusively, whereas the relatively normal area of lung tissue was totally negative for the fluorescent signals (Figure 6). The tropism of BMSCs to the sites undergoing active cell growth and differentiation makes them a smart delivery system of therapeutic agents, especially in gene therapy of cancer and severe destruction of tissues.