Isolated limb perfusion in the sarcoma-bearing rat: a novel preclinical gene delivery system

Reliable site-specific delivery of genetic constructs remains a challenging component of gene-based therapy of solid tumors. Isolated limb perfusion (ILP) continues to be evaluated for treatment of locally advanced soft tissue sarcomas because this approach uniquely directs therapeutic agents into the tumor-bearing extremity without significant systemic leak. In light of these considerations, we tested the hypothesis that ILP could be used to deliver genes carried in viral vectors to the sarcoma-bearing rat extremity, resulting in demonstrable gene transfer into the tumor. ILP was performed in rats by cannulating the femoral artery and vein, isolating the hind limb from systemic circulation by tourniquet, and cycling perfusate for 15 min at a rate of 2.4 ml/min. Leakage into the systemic circulation was 7.5% of the total perfusate concentrated in the isolated limb, as determined by perfusion with technetium 99m-tagged RBCs. We used the ILP technique to perfuse rat hind limbs bearing syngeneic fibrosarcoma tumor nodules with the replication-defective adenovirus Ad5LacZ, which expresses the bacterial beta-galactosidase. 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside staining of the perfused limb tissues confirmed gene transfer to the tumor and peritumoral tissue, demonstrating that the tumor was part of the perfusion circuit and that gene therapy delivered via this method was feasible. These results suggest that adaptation of this preclinical gene delivery model to administer genetic constructs aimed at controlling tumor growth may prove beneficial to patients with extremity sarcomas.