The hollow fiber assay: Continued characterization with novel approaches

The hollow fiber assay, a unique in vivo model, permits the simultaneous ev aluation of compound efficacy against multiple cell lines in two physiologi cal compartments This assay has been used to characterize in vivo activity of cytotoxic compounds. The purpose of the present study was to characteriz e and optimize this assay for compounds with a defined mechanism of action, specifically cell cycle inhibition. Two human tumor cell lines and one nor mal human cell line were loaded into polyvinylidene fluoride hollow fibers at two or more cell concentrations and grown in mice for 3-10 days. The dat a demonstrate the importance of characterizing the initial loading density of various cell lines in the evaluation of compounds. All studies were perf ormed with cells in the linear part of the cell growth curves. Initial load ing densities of 1-2 x 10(4) cells/fiber gave the greatest opportunity for growth in the three human cell lines tested (HCT116 colon carcinoma, NCI-H4 60 non-small cell carcinoma, and AG 1523 normal fibroblast). Utilizing the MTT assay, standard curves were constructed to correlate the fnal number of cells with optical density (OD) readings at 540nm in. order to calculate c ell numbers in the fibers. Insights into the mechanism of action of cisplat in have been gained using Western blot analysis of the cell cycle markers P CNA (a protein present throughout the cell cycle) and Rb (a protein that ac ts as a tumor suppressor gene product) from the hollow fiber cells. In cisp latin-treated NCI-H460 cells both PCNA and Rb phosphorylation decreased, su ggesting the arrest of the cells prior to the S phase. Standard therapeutic agents, cisplatin, racemic flavopiridol, cyclophosphamide and mitomycin C, were evaluated independently in the hollow fiber assay and the xenograft m odel. The data demonstrate that compounds active in the hollow fiber assay are also active in the xenograft.