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.
Methodology
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.
Principal Findings
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.
Conclusions
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.
Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.
Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells. We investigated changes in the cellular respiration and gene expression diversity resulting from adaptation to repeated episodes of acute hypoxia in a premalignant progression model. We find differential, progression stage-specific alterations in phenotypic heterogeneity and identify cells with aberrant phenotypes. To our knowledge, this study is the first demonstration of an integrated approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells in the context of disease progression.
Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers.
In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett’s esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF). Functional assays revealed both cell types showed repressed proliferation and significant changes in motility (speed, displacement and directionality) as a result of interactions between the two cell types. Cellular interactions appear to be mediated through both direct cell-cell contact and secreted ligands. The findings of this study are important in that they reveal, for the first time, the effects of cellular communication on gene expression and cellular function between premalignant (dysplastic) epithelial cells and their normal counterparts.
The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an ‘epigenetic’ drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue. Using a combination of novel optical computed tomography (CT)-based quantitative 3D absorption microscopy and conventional confocal fluorescence microscopy, we show that subjecting malignant cells to vorinostat preferentially alters their 3D nuclear architecture relative to non-cancerous cells. Optical CT (cell CT) imaging of fixed single cells showed that drug-treated cancer cells exhibit significant alterations in nuclear morphometry. Confocal microscopy revealed that vorinostat caused changes in the distribution of H3K9ac-marked euchromatin and H3K9me3-marked constitutive heterochromatin. Additionally, 3D immuno-FISH showed that drug-induced expression of the DNA repair gene MGMT was accompanied by spatial relocation toward the center of the nucleus in the nuclei of metaplastic but not in non-neoplastic cells. Our data suggest that vorinostat’s differential modulation of 3D nuclear architecture in normal and abnormal cells could play a functional role in its anti-cancer action.
Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett’s esophagus (BE) as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous) stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.