Mice, Tumor Growth, and Drug Administration

CB-17 SCID mice were obtained from Charles River Laboratories, Wilmington, MA, and were allowed to acclimate to the animal facility for one week prior to use in the study. The mice were kept on a 12-hour light and dark cycle and had free access to food and water. Mice were handled in accordance with the Guide for Care and Use of Laboratory Animals under the approval of the Institutional Animal Care and Use Committee (IACUC). Approximately 7.5 × 10⁶ - 1 × 10⁷ Igrov1 parental cells or Igrov1/T8 cells suspended in 200 μL matrigel (BD Biosciences, Two Oak Park, Bedford, MA) was injected into each hindlimb region of the mice. Both hindlimbs were injected in some mice, while others had only one hindlimb injected with cells. Tumors formed between 2 and 4 weeks following injection. At approximately 4 weeks when the tumors reached our established detectable size range, 50 mm³ – 250 mm³, the tumor xenografts were injected with TPT at 0 and 24 hours by three different administration routes: intra-tumoral (IT), retro-orbital (RO), and intra-peritoneal (IP). 177, 724, and 505 were also injected at 48 and 72 hours by one of the three administration routes to verify MDR and non-toxicity of the tumor xenografts. Subsequently, TPT combined with the reversal agent was administered every 24 hours for 4-5 days or until tumors regressed below our limit of detection (~15 mm³). The dosage of drug for each injection was determined based on analysis of in vitro EC₅₀, TD₅₀, and IC₅₀ results. Tumor measurements were acquired using a Scienceware® Digi-Max™ slide caliper (Sigma-Aldrich) and tumor volume was calculated by the equation: L*(W²/2), where L is the length in the rostral-caudal direction and W is the width measured transversely.(19,28) Statistical significance was evaluated using a two-tailed t-test and reported as a p-value. Mice were then euthanized and any remaining tumor was cut into histological sections, stained with H&E, and verified by light microscopy. Fibrosis and/or necrosis of remaining tumor sections was quantifiably measured by two investigators, initially blinded to each other's results, using a Lovin Field Finder Micro Slide grid (Electron Microscopy Sciences, Hatfield, PA). An average of both investigators’ measurements was calculated for each drug therapy group and incorporated into the size reductions of the tumors.