Blood Outgrowth Endothelial Cells (BOEC) Contribute to Tumor Vascularization and Can Be Used for Delivery of Cancer Gene Therapy.

Continuous and adequate levels of angiogenesis inhibitors at the site of tumor vasculature may be necessary to obtain optimal antitumor effect. Following recent observations that circulating in blood endothelial cells enhance tumor vasculogenesis we have designed grown from peripheral blood endothelial cells to deliver anti-angiogenic antitumor therapy. BOEC, after several-fold expansion, expressed endothelial markers: VE-cadherin, CD31, von Willebrand factor, and P1H12 and responded to functional endothelial assays: uptake of acetylated low-density lipoprotein, and vascular tube formation. BOEC were also found in functional vessels in vivo, when NOD-SCID mice implanted subcutaneously with Lewis lung carcinoma or H226 lung cancer, were injected via tail vein with 5x10⁵ BOEC. Staining for CD31 on tumor sections of BOEC injected mice demonstrated increased blood vessel density (BOEC 1982±1281, sham 328±229 (P<0.001)). In FsaII tumor model, systemic BOEC injection resulted in increased tumor volumes and higher tumor oxygenation when compared with sham injected animals. Injected via tail vein BOEC distributed initially throughout organs with high vascularization, but in 72 hours remained only in tumor, liver, spleen, and bone marrow. Given the ability of BOEC to survive within tumor vasculature, we designed BOEC to deliver endostatin and soluble VEGFR-1 (sFlt1) genes to the tumor’s endothelium. A retroviral MSCV vector was constructed to contain green fluorescent protein (GFP) and human endostatin gene (hES) or sFlt1. The hES or sFlt1 containing BOEC were evaluated for vWF, CD31, and VE-cadherin, and found positive. Transduced with endostatin or sFlt1 gene BOEC had a slower growth rate in culture, produced detectable endostatin (1.84 ng/ml) or sFlt-1 (665 pg/ml), and were able to inhibit MVEC growth in co-culture condition. When endostatin transduced BOEC were injected to mice with tumor tumor vascularization was decreased (EBOEC 290±172 versus BOEC 1982±1281(p<0.001)). This resulted in tumor volume reduction in mice injected with EBOEC (0.364±0.227cm³ versus sham 0.802±0.431cm³ (p=0.102)). This early data suggest that BOEC have the potential for tumor-specific delivery of cancer gene therapy.