Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations

Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis

Surface shaded renderings illustrating four nuclear shape categories of human mammary epithelial cells (scale bar = 5 microns).

<p>Surface shaded renderings illustrating four nuclear shape categories of human mammary epithelial cells (scale bar = 5 microns).</p

Shape classification of nuclei.

<p>Shape classification of nuclei.</p

Variations in the organization of nuclear content between normal (left), fibrocystic (middle) and metastatic (right) mammary epithelial cells (scale bar = 5 microns).

<p>Light gray haze depicts cytoplasm. Nuclear membrane is blue, low density regions of chromatin are green, moderate density regions are yellow, high density regions are orange, and nucleoli are magenta. Increased clumpiness is apparent in abnormal cells, and metastatic cell contains large number of smaller high-density clumps. Opacity of nuclear membrane (in blue) has been deliberately decreased to enable viewing of nuclear interior. Corresponding videos of these renderings are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029230#pone.0029230.s008" target="_blank">Movies S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029230#pone.0029230.s009" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029230#pone.0029230.s010" target="_blank">S5</a>.</p

Discriminatory power of features.

<p>Discriminatory power of features.</p

List of features.

<p>List of features.</p

Instances of irregular MDA-MB-231 nuclear morphologies (scale bar = 5 microns).

<p>Panel (A) depicts a multilobular nucleus and panel (B) illustrates a cell with micronuclei. Left images show nuclear surface in blue and cytoplasm in gray, middle images show surface-shaded renderings of the nuclear volume, and right images depict volume renderings through the nuclear volume. Increasing nuclear density is color coded from green to red.</p