Use of the membrane invasion culture system (mics) as a screen for anti‐invasive agents

Abstract

The Membrane Invasion Culture System (MICS) assay was adapted for relatively rapid screening of compounds and used to identify anti‐invasive drugs that inhibit human and murine tumor cell migration through a reconstituted basement membrane in vitro. Cell lines demonstrating low and high invasive and metastatic potentials were tested with all compounds for tumoricidal effects prior to evaluation in MICS at non‐cytotoxic doses. The effect on invasive potential in the MICS assay was determined in 3 categories: (1) 48 hr drug pre‐treatment prior to seeding in the MICS (exceptions: 90 min pre‐treatment with pertussis toxin and, for some studies, continuous exposure for 2‐7 days); (2) peptide or prostaglandins 2 hr after seeding and attachment to the membranes in MICS followed by continuous exposure; and (3) cells receiving neither drug nor peptide treatment and serving as controls in each MICS chamber. Since invasion involves cellular motility and deformability, some cytoskeleton disrupting agents were selected. Of these, vincristine, colcemid and colchicine inhibited invasion but taxol did not. Pre‐treatment with cAMP agonists produced conflicting results: dibutyryl cAMP and 8‐(4‐chloro‐phenylthio) cAMP resulted in 50% and 38% reduction in invasion, respectively, whereas 8‐bromo cAMP stimulated invasive potential by 30%. Forskolin and cholera toxin both significantly reduced invasiveness. Pre‐treatment with 5‐azacytidine and araC, to consider the role of methylation and proliferations decreased invasive ability. Anti‐metastatic drugs such as γ‐interferon and razoxane inhibited invasive potential but to varying degrees. Treatment of cells with prostaglandins E₂, F_2α, A₂, and D₂ were ineffectual; however, indomethacin mildly inhibits invasion (< 30%). With respect to the cell surface and metastatic potential, pre‐treatment with tunicamycin did not alter invasiveness; whereas pre‐treatment with swainsonine blocked invasion by approximately 50% at 0.3 μg/ml. The inhibition caused by swainsonine was enhanced (to 92%) if cells were continuously exposed to drug and was reversible. Several RGD‐containing peptides which inhibit attachment to various substrates did not modify invasion in the MICS if added after cell attachment was completed. The peptide which inhibits laminin attachment, CDPGYIGSR, inhibited invasion by 48%. Two peptides from the fibrinogen gamma chain were mildly inhibitory and peptides from TGF‐α inhibited invasion >60%. Collectively, these results demonstrate the potential utility of the MICS for prescreening compounds for anti‐invasive potential, and ultimately anti‐metastatic therapy.