Single cell and collective cell invasive aggregates demonstrated different metastatic potentials. (A) H & E sections of in ovo tumors revealed overall tumor histology. (B) Evidence of strand filing (top left panel, arrows) and single cells (bottom left panel) were seen in H & E sections of transforming growth factor-beta receptor II control (TβRIIfl/fl) tumors. Collective clusters were seen in transforming growth factor-beta receptor II knockout (TβRII KO) tumors. Images are representative of the tumor periphery and tumor-stromal boundaries. (C) Results from murine-specific Alu quantitative PCR found that collective aggregates of TβRII KO tumors achieved greater metastasis than single cells of TβRIIfl/fl tumors in ovo. CAM, chorioallantoic membrane; fib, fibroblasts. (D) TβRII KO epithelial cells possess a greater ability than TβRIIfl/fl cells to extravasate and survive post extravasation, quantified via an experimental metastasis assay and subsequent murine-specific Alu PCR (top graph). All timepoints and samples were compared with the 6-hour timepoint of TβRIIfl/fl cells and fibroblasts (dashed line). Representative images of epithelial cells (green) in relation to the lectin-labeled vasculature (red) were taken at all timepoints to confirm extravasation quantification and are shown beneath the graph (fibroblasts were unlabeled and therefore not shown). The 6-hour timepoint represented cells that arrested in the vasculature. Presence of carcinoma cells in the capillary bed, which is porous, was seen. At the 18-hour and 24-hour timepoints, proliferative capability of disseminated tumor cells was seen. This was evident in cells extravasating from the capillary bed, invading into areas of the CAM in close proximity to the vasculature, and exhibiting protrusive cellular processes. At the 72-hour timepoint, cohesive groups of cells with protrusive cellular processes were observed near vessels.