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Last Updated: 06/21/23

Milestone (1995)

Hollow fiber assay adopted as prerequisite for xenograft studies

The assay has demonstrated the ability to provide quantitative initial indices of in vivo drug efficacy and is currently being used as the initial in vivo experience for agents found to have reproducible activity in the in vitro anticancer drug screen. Drugs showing activity with unique mechanisms of action are being further developed as potential clinical candidates or have already made the transition from bench to bedside. Some examples are Velcade, 17-AAG, and dimethanesulfonate.

In 1995, DTP implemented a solid tumor efficacy model based on cell growth inside biocompatible hollow fibers.1,2 The hollow fiber assay, which was developed by Dr. Melinda Hollingshead, chief, Biological Testing Branch, has the ability to provide quantitative indices of drug efficacy in heterogeneous tumors with minimal expenditures of time and materials. It is currently being used as the initial in vivo experience for agents found to have reproducible activity in the in vitro anticancer drug screen.

The hollow fiber assay has several advantages over standard animal efficacy models. First, demonstrating that potential anticancer agents have in vivo efficacy in one or more animal models of neoplastic disease can require considerable investments in terms of laboratory animals and quantity of test compound. Second, conducting studies in animal models requires substantial amounts of time and resources for the testing procedure itself. Even when such studies can be conducted, it is possible that the experimental agent or series of agents will exhibit only minimal antitumor activity. Third, cancer treatments that appear promising in tissue culture are often less effective in solid tumors, in part because of the proliferative and microenvironmental heterogeneity that develops in these tumors as they grow.3

The hollow fiber assay at full capacity allows screening of 50 or more compounds per week in a 10-day assay. In addition to requiring less than two weeks to complete, it requires at most only 450 mg of material, as opposed to the multigram quantities required for xenograft studies. Compounds that retard the growth of the selected tumor cell lines are recommended for the next level of testing.


Small hollow fibers (1 mm in diameter, 2 cm long, molecular weight exclusion of 500,000 Da), made of polyvinylidene fluoride and containing cells from human tumors, are inserted underneath the skin and in the body cavity of the mouse. A standard panel of 12 tumor cell lines, including lung, breast, colon, melanoma, ovarian, and central nervous system, is used for the routine hollow fiber screening of in vitro actives. In addition, alternative lines can be used for specialized testing of compounds.

Each mouse receives three tumor cell lines as intraperitoneal (ip) implants and three subcutaneous (sc) implants. Mice are treated with experimental agents starting on day 3 or 4 following fiber implantation and continuing daily for four days. Each agent is administered by intraperitoneal injection at two dose levels.

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 × 2 sites × 2 dose levels × 2 [score]). A compound is considered for possible xenograft testing if it has a combined ip + sc score of 20 or greater or an sc score of 8 or greater, or if it produces cell kill of any cell line at either dose level evaluated.

1 Biological Testing Branch, Developmental Therapeutics Program. Primary anti-cancer drug screening activities.

2 National Cancer Institute. Drug Discovery at the National Cancer Institute: Questions and Answers.

3 Hollingshead MG, Alley MC, Camalier RF, Abbott BJ, Mayo JG, Malspeis L, Grever MR. In vivo cultivation of tumor cells in hollow fibers. Life Sci 1995;57:131–141.


In vivo cultivation of tumor cells in hollow fibers.

Growth and chemotherapeutic response of cells in a hollow-fiber in vitro solid tumor model.