What do the ultrastructural alterations tell us about early carcinogenesis? There are three potential facets of the alterations: intracellular—these include the compaction of higher-order chromatin structure and cytoskeleton-mediated alterations—and extracellular, putatively associated with an abnormal cross-linking of ECM. As discussed above, nanocytology data revealed that some of the most pronounced alterations occur in the cell nucleus (i.e., chromatin structure). There are several lines of evidence implicating chromatin remodeling leading to chromatin compaction as an early event in carcinogenesis. Transmission electron microscopy (TEM) confirmed that chromatin is condensed in histologically normal rectal colonocytes in field carcinogenesis (n=65) of patients with adenomas, colonocytes biopsied from the AOM-treated rats (n=150), and the CSK-knockdown cell lines (the tumor-suppressor gene CSK-knockdown is a more aggressive cell line derived from HT29 cells, n=40) (p<0.01). The length scale of the changes was ~50-400 nm, which is consistent with the range observed by the optical techniques. Chromatin de-compaction was experimentally induced by treating cells with valproic acid, which inhibits histone deacetylase (HDAC), a well-known chromatin compactor. ISOCT and PWS imaging of the valproic acid-treated cells showed a decrease in D and Ld.
Of note is that chromatin compaction at larger length scales (greater than 5 nm) is a well-known hallmark of dysplasia - focal lesions arising on the background of field carcinogenesis - that is widely used as a marker of neoplasia in histopathology of essentially all types of cancer. The emerging picture, therefore, is that of a progressive chromatin compaction from histologically normal cells in field carcinogenesis to dysplastic cells. Evidently, chromatin clumping that is so ubiquitously observed in dysplastic cells is not unique to this stage of carcinogenesis but begins as more subtle, histologically undetectable chromatin remodeling at smaller length scales.
Another facet of intracellular ultrastructural alterations are those related to the cytoskeleton51
. Biological implications of these changes were studied in two model systems: the AOM-treated rat model of colon cancer and HT29±CSK shRNA cell lines. Not only were twenty two out of 384 cytoskeleton-regulating genes altered by real-time PCR including an early overexpression of the EB1 proto-oncogene but also the LEBS-detectable alterations and Ld increase were mitigated by treatment with a cytoskeletal inhibitor colchicine and by EB1-knockdown, thus indicating cytoskeletal abnormalities in the histologically normal cells in early carcinogenesis.
Finally, ECM alterations in field carcinogenesis (e.g., increase in D and decrease in μ's
) were similar, albeit smaller in magnitude, to the ECM changes previously observed in tumor microenvironment53
. Scanning electron microscopy of rectal biopsies from patients with adenomas (n=16) showed increased cross-linking and local alignment of collagen fibrils/fibers, consistent with ISOCT and LEBS measurements. Furthermore, lysyl oxidase (LOX) was overexpressed in field carcinogenesis. Collagen cross-linking and the resulting increase in the mechanical stiffness of tumor stroma are well known hallmarks of tumor microenvironment with LOX being implicated as a potent collagen cross-linker27, 54
. These mechanical and structural processes have been implicated in promoting tumor microenvironment. Again, we conclude that matrix alterations such as collagen cross-linking are not unique to tumor microenvironment but instead develop early in carcinogenesis, at the stage of field carcinogenesis.
Physiologically, the microvascular and ultrastructural changes are not completely independent but instead interconnecting facets of early carcinogenesis feeding on each other. For example, the alterations of higher order chromatin structure have been implicated and may affect a myriad of genomic processes including gene transcription and post-transcriptional modifications (Fig. ). Indeed, a shift from a genomic homeostasis cannot occur without chromatin remodeling and thus alterations in chromatin nanoarchitecture. A greater number of hyperproliferative colonocytes that are found not only at the bottom of colonic crypts but also in what would normally be the maturation/differentiation zone of the crypts, leads to a hypoxic state, which drives neo-angiogenesis (EIBS)46
. A change in metabolism such as the Warburg effect is another EIBS stimulus. EIBS helps support what otherwise could be an unsustainable metabolic state. Lysyl oxidase (LOX) is also driven by tissue hypoxia and leads to collagen cross-linking and increased stiffness of the stroma. Some LOX-like enzymes deacetylate histones thus directly affecting chromatin structure and gene expression. Furthermore, increased ECM stiffness promotes tumor microenvironment and has been shown to increase FAK (focal adhesion kinase)/SRC phosphorylation with the consequent activation of the signaling pathway leading to a more proliferative and invasive phenotype55
, which, as early data indicates, manifests in cellular nanoarchitectural alterations.
Clearly, there might (and expected to) be other facets of microvascular and ultrastructural alterations in early and field carcinogenesis. Although little is still known about these changes and their role in tumor formation, overall the data indicates that field carcinogenesis alterations are, in essence, subtler forms of similar alterations in the tumor, including neo-angiogenesis, chromatin remodeling (e.g., chromatin clumping) and collagen matrix cross-linking. In other words, tumor phenotype does not develop de novo but appears to already be embedded in the predisposing field carcinogenesis while the alterations are amplified and become considerably more pronounced in frankly malignant cells and tumors.