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Scanning Microscopy

Abstract

Retinoic acid (RA) and butyric acid (BA) were investigated for their effect on in vitro migration of highly metastatic murine B16a melanoma cells. These potential antitumor agents are known to alter the cytoskeleton. Our initial studies determined the 72 h cytostatic /cytotoxic concentration of RA (1 X 10-6 M / > 1 X 10-5M) and BA (1.5 mM) / > 2.0 mM). Cytostasis by RA and BA was confirmed by autoradiography and radioisotope incorporation. For migration assays, cells were plated on 3 and 5 μm diameter pore polycarbonate membranes. Complete media was added containing RA or BA at time of plating. For BA pretreatment studies, BA was added to cells for 72 h prior to plating cells in fresh BA on the membranes. Top and bottom surfaces of the membranes were examined after 72 h of incubation by scanning electron microscopy. Al though RA and BA induced cells on top of the membrane to change morphology as shown by phase, transmission and scanning electron microscopy, only BA enhanced the deformability of cells to allow for passage through the 3 μm diameter pores. Butyric acid enhanced migration through 3 μm diameter pore membranes by 511%. For 5 μm diameter pore membranes, 55.2% of the plated number of untreated early passage cells migrated to the bottom surface as compared to 57.3% for BA-treated cells and 14.9% for RA-treated cells. However, if cellular proliferation over the 72 h period was factored in, BA increased migration by 456% over the controls and pretreatment of cells with BA for 72 h prior to plating increased migration by 893%. Without considering proliferation, RA inhibited migration by 75% over controls. The decrease in migration observed in RA-treated cells was due to an inhibitory effect on cellular migration and a decrease in proliferation.

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