Differential Effects of Iressa on growth and morphology. MAM-1 co-cultures were treated for 24–48 h by replacing conditioned media with fresh media in the absence (Control) or presence of 1 μM Iressa. A. Phase contrast photomicrographs of MAM-1 co-cultures following treatment with 1 μM Iressa were taken using a 10× objective. Tumor cell nests (T) decrease in cellularity over time and stromal cells (S) thicken and increase in cellularity. By 48 h tumor cell nests erode, cells flatten, and show evidence of apoptosis while stromal cells develop into multi-cell layer nests recapitulating the morphology of a fibrotic response in vitro. B. Immunofluorescence of MAM-1 that were dual-labeled with HER2/neu-TRITC and α-SMA-FITC to identify the tumor (red) and stromal (green) subpopulations, respectively. Photographs taken under low power (10×) show decreased cellularity of HER2+ nests (red) and increased cellularity and density in the α-SMA+ stroma (green). These shifts in the cellularity of the separate subpopulations are emphasized by the nuclear counterstain with DAPI (blue). Under higher magnification (40×) HER2+ nests of tumor cells (red) have decreased in cellularity and flattened into a monolayer while the cellularity and α-SMA reactivity has increased in the stromal elements surrounding the tumor cells generating the thickened appearance that is associated with fibrosis. Note in particular the membrane accentuation of HER2/neu in the tumor cells treated with Iressa and the dense bundles of a-SMA reactive fibers in Iressa treated stroma.