Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study

BACKGROUND: Nuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention. METHODS: For this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function. RESULTS: We established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential. CONCLUSIONS: We characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the development of well-documented, microscopically detectable hallmarks of cancer

Transmission electron microscopy imaging to analyze chromatin density distribution at the nanoscale level

Transmission electron microscopy (TEM) is used to study the fine ultrastructural organization of cells. Delicate specimen preparation is required for results to reflect the "native" ultrastructural organization of subcellular features such as the nucleus. Despite the advent of high-resolution, fluorescent imaging of chromatin components, TEM still provides a unique and complementary level of resolution capturing chromatin organization at the nanoscale level. Here, we describe the workflow, from tissue preparation, TEM image acquisition and image processing, for obtaining a quantitative description of chromatin density distribution in plant cells, informing on local fluctuations and periodicity. Comparative analyses then allow to elucidate the structural changes induced by developmental or environmental cues, or by mutations affecting specific chromatin modifiers at the nanoscale level. We argue that this approach remains affordable and merits a renewed interest by the plant chromatin community

Preservation of cellular nano-architecture by the process of chemical fixation for nanopathology.

Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy and have not been examined with nanoscale sensitive imaging techniques. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two live cell models: a drug-based model of Hela cells differentially treated with daunorubicin and a cell line comparison model of two cells lines with inherently distinct chromatin organizations. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology

Linker histones are fine-scale chromatin architects modulating developmental decisions in Arabidopsis

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