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Primary Anti-cancer Drug Screening Activities

Hollow Fiber Assay

Process

Advancement of potential anticancer agents from identification in the in vitro screen to preclinical development is enhanced with demonstration of in vivo efficacy in one or more animal models of neoplastic disease. Most such models require considerable materials in terms of laboratory animals and test compound as well as substantial amounts of time and cost to determine whether a given experimental agent or series of agents have even minimal anti-tumor activity. The hollow fiber assay described below has demonstrated the ability to provide quantitative indices of drug efficacy with minimum expenditures of time and materials and is currently being utilized as the initial in vivo experience for agents found to have reproducible activity in the in vitro anticancer drug screen.

Methodology of the Hollow Fiber Assay

A standard panel of 12 tumor cell lines are used for the routine hollow fiber screening of the in vitro actives. These include NCI-H23, NCI-H522, MDA-MB-231, MDA-MB-435, SW-620, COLO 205, LOX, UACC-62, OVCAR-3, OVCAR-5, U251 and SF-295. In addition, alternate lines can be used for specialized testing of compounds on a nonroutine basis. The cell lines are cultivated in RPMI-1640 containing 10% FBS and 2 mM glutamine. On the day preceeding hollow fiber preparation, the cells are given a supplementation of fresh medium to maintain log phase growth. For fiber preparation, the cells are harvested by standard trypsinization technique and resuspended at the desired cell density ((2-10 X 106 cells/ml). The cell suspension is flushed into 1 mm (internal diameter) polyvinylidene fluoride hollow fibers with a molecular weight exclusion of 500,000 Da. The hollow fibers are heat-sealed at 2 cm intervals and the samples generated from these seals are placed into tissue culture medium and incubated at 370 in 5% CO2 for 24 to 48 hours prior to implantation. A total of 3 different tumor lines are prepared for each experiment so that each mouse receives 3 intraperitoneal implants (1 of each tumor line) and 3 subcutaneous implants (1 of each tumor line). On the day of implantation, samples of each tumor cell line preparation are quantitated for viable cell mass by a stable endpoint MTT assay so that the time zero cell mass is known. Mice are treated with experimental agents starting on day 3 or 4 following fiber implantation and continuing daily for 4 days. Each agent is administered by intraperitoneal injection at 2 dose levels. The doses are based on the maximum tolerated dose (MTD) determined during prior acute toxicity testing. The fibers are collected from the mice on the day following the fourth compound treatment and subjected to the stable endpoint MTT assay. The optical density of each sample is determined spectrophotometrically at 540 nm and the mean of each treatment group is calculated. The percent net growth for each cell line in each treatment group is calculated and compared to the percent net growth in the vehicle treated controls. A 50% or greater reduction in percent net growth in the treated samples compared to the vehicle control samples is considered a positive result. Each positive result is given a score of 2 and all of the scores are totaled for a given compound. The maximum possible score for an agent is 96 (12 cell lines X 2 sites X 2 dose levels X 2 [score]). A compound is considered for xenograft testing if it has a combined ip + sc score of 20 or greater, a sc score of 8 or greater, or produces cell kill of any cell line at either dose level evaluated. This scoring system has been validated by DCTDC statisticians in CTEP to represent a level of detection expected to score current "standard" agents as active.