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

Abstract

BackgroundNuclear 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.MethodsFor 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.ResultsWe 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.ConclusionsWe 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.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

“…This relation is derived from the properties of fractal media with conserved mass and volume-as compaction increases locally, the variations in mass density (heterogeneity) must also increase. Previous molecular dynamics simulations have further confirmed that increases in δn*L c correspond to an increase in macromolecular compaction, and experimental results have shown that this increase within the nucleus quantitatively describes an increase in chromatin heterogeneity (21,25,26).…”

“…One of the primary features of tumorigenesis is a shift in the fractal physical organization of chromatin, correlating both with the formation of tumors and their invasiveness. In early carcinogenesis, we have previously applied a fixed-cell-imaging technique, partial-wave spectroscopic (PWS) microscopy and transmission electron microscopy (TEM), to detect physical changes in the chromatin nanoarchitecture, indicating that the topology of chromatin is a critical component in cellular function at the earliest stages of tumor formation (21). PWS microscopy allows examination of the intracellular organization concealed by the diffraction limit with length scale sensitivity in the range of 20-200 nm, the range at which existing label-free live-cell imaging techniques are deficient, due to the relationship between the nanoscale spatial variations of macromolecular density and the resulting variance in the spectrum of backscattered light (22,23).…”

Reconstruction of explicit structural properties at the nanoscale via spectroscopic microscopy

“…We believe that all of the above factors must have contributed to the observed fourfold difference in the RI correlation lengths within cancer cell lines and histologically normal tissue. Lastly, based on previous studies focused on the quantification of the internal organization of biomaterials via light or electron microscopy, 20,23 we believe that data acquired from 10 to 30 fields of view (30 to 150 biological cells depending on cell type) will be sufficient to account for biological variability and determine σ n Δ and l c values typical for a given biological sample. In the future, automated image acquisition can be implemented to acquire and analyze whole-slide images of biological samples (up to 1500 images per slide as has already been implemented elsewhere).…”

Nanoscale refractive index fluctuations detected via sparse spectral microscopy

“…Thus, PWS is a promising tool for detecting early-stage microscopically undetectable tumorigenic alterations in biological cells and tissues such as changes in chromatin organization [9].…”

“…PWS microscopy achieves sensitivity to nanoscale structures within biological cells by using the spectroscopic content of microscope images, and quantitatively measures nanoarchitectural changes in cells associated with carcinogenesis [6,7]. These intracellular, macromolecular alterations are recognizable as some of the earliest indicators of carcinogenesis and are detectable throughout an affected organ, not just at the tumor site, via a phenomenon known as the field effect of carcinogenesis [8,9]. Because PWS is sensitive to nanoscale structure below the diffraction limit of traditional microscopy systems, including those used for wholeslide imaging, it can be used for minimally invasive cancer screening via the field effect.…”