Three-dimensional culture of single cells from normal prelactating mammary gland, MINO precancer, and invasive carcinoma. (a, d, g) Inverted microscope phase contrast images as well as histologic images generated from paraffin-embedded 4 μm sections of the three-dimensional cultures stained by immunohistochemistry with (b, e, h) CK8–18 or (c, f, i) CK14 are shown. The magnification scale (lower right panel i) is identical for all histology panels and approximate for the inverted microscopy photographs. CK8–18 confirms that the major cell population is a luminal phenotype, but CK14 shows that there is also myoepithelial differentiation of single cells, documenting bipotential of the individual cells giving rise to these three-dimensional structures. A single MINOsphere from line 4w4 was transplanted from the three dimensional culture into the gland cleared fat pad of a 3-week-old female FVB/n mouse. The mammary gland was removed 10 weeks after transplant and a whole mount mammary gland preparation was made with hematoxylin stain to visualize cell density (j). After photography, the same gland was processed for histologic sectioning, and the resulting 4 μm hematoxylin and eosin stained tissue section is shown (k). At least three foci of tumor are seen in the differentiation zone of the MINO. One is indicated by the arrow. CK, cytokeratin; MINO, mammary intraepithelial neoplasia outgrowth.