Abstract Colorectal cancer (CRC) is a commonly diagnosed cancer and the third leading cause of cancer-related deaths in the United States. About 50,000 people will die from this disease in 2011. CRCs develop through an accumulation of genetic and epigenetic aberrations and have been broadly classified into three molecular subtypes - chromosomal instability (CIN), microsatellite instability (MSI), and CpG Island Methylator Phenotype (CIMP). Research studies on CRC have predominantly focused on its molecular biological and genetic basis, but very little is known about the three-dimensional cell morphological variations between these subclasses. We investigated alterations in the three-dimensional (3D) nuclear architecture of the three molecular subtypes of CRC, comparing them to a benign adenoma, using representative epithelial cell line models: AAC1 (benign), AAC1-SB10 (CIN), HCT-116 (MSI) and RKO (CIMP). We applied micro-optical computed tomography (Cell-CT) to image one hundred individual hematoxylin-stained cells of each of the four types with submicron, isotropic spatial resolution. We performed automated 3D image analysis on the volumetric cell images, quantifying fifty descriptors (features) to characterize the nuclear morphology and coarse chromatin organization. We evaluated the statistical efficacy of our 3D features in distinguishing among the cell types. Our results demonstrated previously unreported morphological variations among the controls and three types of CRC. Relative to the benign adenoma cells, in contrast to CIN and MSI, the CIMP subtype exhibited markedly smaller cell and nuclear sizes, smaller nucleus-to-cytoplasm (NC) ratio and a predominantly smooth nuclear interior, lacking in clumpy chromatin. However, the CIMP cell nuclei exhibited the highest optical density among the four cell classes. MSI cells showed the clumpiest nuclear texture, followed by the benign adenoma cell nuclei. These results offer new perspectives on nuclear structure variations in benign colon adenoma and colorectal cancer cells, warranting further investigation. They suggest that the hypermethylation seen in the CIMP phenotype may result in demonstrable chromatin compaction with a concomitant reduction in nuclear size. The chromatin clumpiness observed in the MSI cells may reflect increased mutations at nuclear scaffold binding sites, which tend to be characterized by single and di-nucleotide repeats. We intend to pursue studies on human biopsy cells with ultimate goal of testing the efficacy of Cell-CT for CRC diagnosis, subtyping and staging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4057. doi:1538-7445.AM2012-4057