Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5–10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045–6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.

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