Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study
- Lusik Cherkezyan†1,
- Yolanda Stypula-Cyrus†1,
- Hariharan Subramanian1,
- Craig White1,
- Mart Dela Cruz2,
- Ramesh K Wali2,
- Michael J Goldberg3,
- Laura K Bianchi3,
- Hemant K Roy2 and
- Vadim Backman1Email author
© Cherkezyan et al.; licensee BioMed Central Ltd. 2014
Received: 15 July 2013
Accepted: 5 March 2014
Published: 14 March 2014
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.
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.
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.
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.
KeywordsChromatin Colon cancer Field cancerization Field effect Transmission electron microcopy
Chromatin arrangement has been extensively studied as it defines the physical and biochemical forces that govern genome function. Alterations in higher-order chromatin structure are associated with changes in gene expression, observed in many complex human diseases [1, 2]. In particular, cells undergoing neoplastic transformation are characterized by the coarse, asymmetric aggregation of densely packed chromatin . The tumorigenic changes in chromatin texture have been shown to be independent of cell-cycle progression . Through chromatin remodeling mechanisms, genetic/epigenetic alterations of tumor suppressor genes or proto-oncogenes initiate and advance neoplastic progression. As a consequence, there is a large body of literature devoted to the structural differences between normal and cancerous cell nuclei [3, 5]. However, the process of malignant alterations a nucleus undergoes at the earliest stages of carcinogenesis remains unclear.
The phenomenon of field cancerization (also known as field carcinogenesis, field effect, or field defect) is the concept that the altered genetic/epigenetic environment that gives rise to a focal tumor is present throughout the organ. That is, the aberrant genetic and environmental modifications create a fertile background on which individual tumors and lesions originate. Therefore, the tissue in the neoplastic field can be used to identify and study the earliest events in cancer progression [6, 7]. This phenomenon has been examined in many cancers, including colon, lung, esophageal, ovarian, cervical, breast, prostate, and head and neck . Colorectal cancer (CRC) presents a well-studied example of field cancerization due to its characteristic continuous epithelium which shares environmental influences. Many of the epigenetic [8, 9], proteomic , and structural [11, 12] alterations associated with CRC have been reported in the normal colonic mucosa adjacent to the tumor. Specifically, profound changes in the nuclear structure of cells in the microscopically normal rectal mucosa from patients with an adenoma or adenocarcinoma were suggested [13–15]. Moreover, these changes were correlated with the risk of recurrence of a colonic lesion . These studies indicate that structural changes in the nuclear chromatin in the field of colon cancer represent a pre-neoplastic event. Therefore, understanding of the nuclear structure and how it reflects the process of malignant transformation is vital for the development of improved tools for cancer diagnosis and risk assessment.
One promising approach to quantify chromatin organization and function is through the spatial correlation of chromatin density distribution and, in particular, its fractal dimension [17–20]. The fractality of chromatin organization, validated by various methods [19–23], is the property of its self-similarity at different physical length scales. The fractal dimension of chromatin packing, in turn, is related to the amount of volume occupied by the surface of chromatin (a higher fractal dimension reflects a higher amount of exposed chromatin surface). Mathematically, a fractal medium is characterized by a power-law spatial correlation function, ~r (D-3), with D being the fractal dimension of the medium. Reports show that the fractal dimension is increased in tumor cell nuclei. Moreover, the more aggressive the tumor, the less it resembles a mathematically ideal fractal [24, 25]. Given the importance of chromatin structure for genome function, it is crucial to understand chromatin reorganization at the early stages of carcinogenesis. While nanoscale structural alterations in the field of CRC have been reported, these changes have not been visualized and identified until now due to the diffraction-limited resolution of optical techniques [13, 15]. In order to further investigate premalignant chromatin structure, a technique with higher resolution is required.
In the present study, we take advantage of the nanoscale resolution of transmission electron microscopy (TEM) to investigate pre-microscopically detectable chromatin rearrangements in histologically normal-appearing cell nuclei in two models of early-stage CRC. We study pre-neoplastic chromatin rearrangements in human rectal cell nuclei from the field of CRC, as well as in animal colonic nuclei at a pre-malignant time point of the established azoxymethane (AOM)-injected rat model of 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 chromatin density distribution is quantified via the spatial correlation function. We found significant and similar changes in the heterochromatin content, clumping and positioning in early and field carcinogenesis. Moreover, we show that these alterations correspond to the well-known hallmarks of cancer, but manifested at smaller, microscopically undetectable length scales. These results signify that the alterations in chromatin observed in the field of a tumor represent an early-stage event of carcinogenesis. We propose that the nanoscale nuclear abnormalities identified here can be employed as a biomarker for cancer prevention and diagnosis.
Subjects and samples
This study was conducted with the approval of the NorthShore University HealthSystem Institutional Review Board (IRB). Human biopsies were obtained from endoscopically normal rectal mucosa with an informed consent obtained from each subject prior to the procedure. Histopathologically all tissue samples appeared normal. Ten patient biopsies were used in this study, which included five normal and five from patients with adenomas (ranging in adenomatous polyp size from 2 to 10 mm). The biopsies were first placed in Karnovsky’s fixative for 2 weeks to preserve structure. The fixative consists of 0.1 M phosphate buffered solution containing 5% glutaraldehyde. Following standard protocol, the samples were stained with osmium tetraoxide (OsO4, commonly used to visualize DNA structure [26–28]), dehydrated, and then embedded in resin. Samples were then sectioned with an ultramicrotome to a thickness of 70 nm.
Animal procedures were performed at NorthShore University HealthSystem, with the approval of Institutional Animal Care and Use Committee (IACUC). Eighteen Fisher 344 rats (150–200 g; Harlan, Indianapolis, IN) were randomized to two weekly treatments of 15 mg/kg AOM (Midwest Research Institute, Kansas City, MO) or saline. Rats were euthanized at a premalignant time point, 10 weeks post injection, and necropsy was performed to confirm the absence of adenomas in the colon. To maintain good structural morphology, High Pressure Freezing (HPF) of animal colon samples was performed using a Leica EM-PACT2 high-pressure freezer at the Biological Imaging Facility (BIF) of Northwestern University. Automatic Freeze Substitution (AFS) was performed using a Leica AFS2 system. Samples were then embedded in Epon 812 resin (Electron Microscopy Sciences, Hatfield, PA) and thin-sectioned using Leica Ultracut S microtome into 90 nm sections onto copper grids.
Run length and heterochromatin percentage
Higher-order chromatin structure differentially regulates genomic loci through partitioning into active (euchromatin) and inactive (heterochromatin) domains (as reviewed in Ref. ). Changes in heterochromatin content and distribution have long been used as a marker for disease. Following standard TEM preparation and staining protocol, heterochromatic regions are heavily stained while euchromatic regions are lightly stained. Thus, nuclear TEM images were binarized to separate between these darkly-stained areas filled with the dense heterochromatin and the light euchromatin. For analysis, 1) the total amount of heterochromatin, and 2) the characteristic size (run length) of its clumps were measured. The first was calculated as the percentage of the nuclear area occupied by heterochromatin. The second was quantified via a parameter termed run length, which is defined as the average number of connected, consecutive pixels with values corresponding to heterochromatin. Each micrograph was scanned in horizontal and vertical directions to record the run lengths of all heterochromatin occurrences. The average of all recorded values was defined as the heterochromatin run length of the image.
Distribution with respect to periphery
Spatial correlation function
While A ρ and l c account for the variability in depth of staining and image size, the third parameter of the correlation function, D, uniquely quantifies the shape of the mass density spatial correlation function. By fitting the appropriate value of D, we are able to quantify 1) the relative length scale composition of the nucleus (increase in D implies a shallower decay of correlation function and, therefore, higher relative presence of larger length scales), 2) the functional forms of spatial correlation of mass distribution (e.g. Gaussian at D → ∞, decaying exponential at D = 4, stretched exponential at 3 < D < 4 and power law at D < 3). Thus, D delivers an excellent qualitative and quantitative measure of the experimentally obtained spatial correlation of chromatin density.
To fit the experimentally obtained B ρ (r), an array of Whittle-Matern family correlation functions was created with values of D ranging from 2 to 6 and l c ranging from 0.66r max to r max . The values of D and l c , which yield the best agreement (evaluated via r-squared values of the fit) between the experimental and fitted curves, were calculated for every image.
Binary quantification of chromatin compaction
Grey-scale quantification of chromatin compaction
We also quantified the shape of the chromatin correlation function by fitting the experimentally measured B ρ(r) from every micrograph to the family of Whittle-Matern correlation functions. This analysis revealed that 1) there is a significant increase in the width of correlation function, and, therefore, in the dominance of larger length scales in pre-cancerous nuclei (quantified via D); and 2) the type of spatial correlation function changes from fractal in the case of healthy nuclei (D < 3, in agreement with [19–23]) to a stretched exponential in precancerous cell nuclei (Figure 7). While the first finding supports a previously reported increase in run length, the second finding gives a different perspective on describing both nuclear structure and dynamics. The change in the type of spatial correlation has major implications in the physical forces and size-selectivity that regulate nuclear function. Interestingly, this transition in the type of spatial correlation function from fractal to a stretched exponential observed in cells was previously described in malignant ovarian tissue reorganization .
Proper higher-order three-dimensional organization of chromatin is crucial for normal cell function, influencing gene expression and DNA replication and repair. Abnormalities in chromatin organization have been described in a variety of diseases [1, 2]. While abnormal chromatin aggregation and clumping observed by an optical microscope is a well-studied hallmark of carcinogenesis, little is known about changes in nuclear organization that precede this stage of cancer progression. This is largely due to the diffraction limit of light microscopy used in conventional histology, which is unable to characterize intracellular structures smaller than 250 nm. Thus, to identify the pre-microscopically detectable abnormalities in higher-order chromatin structure at the earliest stages of carcinogenesis, imaging technology with a greater resolution is needed. Here, we use nanoscale-resolution TEM to study the chromatin organization in microscopically normal-appearing cell nuclei that may be undergoing early-stage tumorigenic alterations. We provide quantitative and qualitative descriptions of the pre-neoplastic nuclear ultrastructural (i.e. microscopically indiscernible) changes in the field of CRC in humans, as well as in early premalignant-stage CRC in AOM-treated rats.
The analysis of heterochromatin distribution revealed an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in both models of early cancer (Figures 4, and 5). Note that these heterochromatin rearrangements, also identified as chromatin coarsening, are a well-known feature of cancer when resolved by an optical microscope (for review, see ). We have also observed profound changes in the spatial correlation of chromatin density at length scales smaller than 250 nm (Figure 7a,b), which remarkably coincides with the diffraction limit of optical microscopes. Thus, we established that in the series of sequential events involved in malignant transformation, nanoscale chromatin compaction and rearrangement precedes microscale chromatin compaction and rearrangement.
Additionally, we found a change in the type of chromatin density correlation from fractal in normal nuclei to a stretched exponential in the pre-cancerous nuclei (Figure 7). This important finding has implications beyond the purely structural. The fractal nature of chromatin distribution in normal cells, or self-similarity at different length scales, ensures that the diffusion rates for transcription factors are independent of their size. Meanwhile, the stretched exponential distribution is described by a characteristic length scale, implying preferred diffusion of particular-size proteins. Thus, the change in the type of chromatin spatial correlation may alter the diffusion of macromolecules inside the nucleoplasm and hence the chromatin’s accessibility to transcription factors. We conclude that the extensive structural chromatin alterations reported here represent the earliest known events in carcinogenesis which likely drive the changes in gene expression during neoplastic transformation.
The novel characterization of premalignant nuclear architecture reported here establishes new biomarkers of early tumorigenesis. This opens the door for a set of new technologies to develop and perform tests for the diagnosis and risk stratification of cancer. From a clinical perspective, TEM-based methods are impractical, as they are time consuming, costly, and rely on heavy processing of the sample. Meanwhile, optical techniques present distinct advantages of time- and cost-effectiveness, and can be performed by primary care physicians, complementing the pathological examination and enhancing diagnosis through implementation of an automated analysis. The development of ultrastructural biomarkers can aid in cancer diagnosis when detected by scattering-based techniques, such as optical coherence microscopy, light-scattering spectroscopy, confocal light absorption and scattering spectroscopy, and partial wave spectroscopic microscopy [43–45].
Several potentially important measures of nuclear structure could not be quantified in the present study due to its technological limitations, as follows. The precise shape of the nucleus, known to be altered in cancer, could not be characterized due to the dehydration involved in sample fixation protocol that is likely to distort the native shape of nuclei. Nuclear size and total number of nucleoli were not measured as they are highly dependent on the sample sectioning. The absolute values of chromatin mass density variations could not be determined due to the employed staining. Finally, the imaging resolution of the employed TEM instrumentation did not allow us to resolve structural changes at length scales smaller than the chromatin fiber. Future studies involving advanced electron microscopy techniques with improved spatial resolution and using protocols that better preserve native chromatin structure will provide new insights into pre-neoplastic transformation of the nuclear structure.
Furthermore, to fully understand the changes in genomic activity resulting from the established structural alterations, it is important to note that the transcriptional status of an individual gene is not always determined by its nuclear location and the degree of local chromatin condensation. Despite the correlation between gene density, transcriptional activity, and nuclear positioning, there are also many reports demonstrating deviations from this rule. For example, heterochromatin is not always transcriptionally silent [46–48]; active genes can be present in the periphery of the nucleus; and inactive genes can be located in the interior [49–51]. Therefore, the effect of early tumorigenic structural changes in 3D chromatin structure on genome function needs to be further investigated. This may be achieved by integrating the subdiffractional sensitivity of the interferometric spectroscopy of scattered light  and the targeted enhancement of optical contrast  by careful selection of transcription activity-related protein markers .
In this manuscript we identify significant quantitative and qualitative changes in chromatin distribution in field and early carcinogenesis. We confirm that the ultrastructural field effect changes of nuclear organization represent the initial steps that lead to the development of well-known, microscopically detectable hallmarks of cancer. We conclude that the established alterations in higher-order chromatin structure is a crucial early event in tumorigenesis. Identifying pre-neoplastic changes in the tumorigenic field is a promising area of research in order to develop novel tools for cancer prediction and diagnosis.
Transmission electron microcopy
This work was funded by NIH Grants No. R01CA128641, No. R01CA165309, No. R01 EB003682, No. U54CA143869, and NSF Grant No. CBET-1240416. The authors would like to thank the staff at the Biological Imaging Facility at Northwestern University for their assistance in TEM imaging, and especially thank Ms. Charlene Wilke. The authors also thank Dhwanil Damania, Vladimir Turzhitsky, Nikhil N. Mutyal, and Andrew J. Radosevich for their assistance in TEM sample preparation.
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