The influence of chromosome density variations on the increase in nuclear disorder strength in carcinogenesis

Microscopic structural changes have long been observed in cancer cells and used as a marker in cancer diagnosis. Recent development of an optical technique, partial-wave spectroscopy (PWS), enabled more sensitive detection of nanoscale structural changes in early carcinogenesis in terms of the disorder strength related to density variations. These nanoscale alterations precede the well-known microscopic morphological changes. We investigate the influence of nuclear density variations due to chromosome condensation on changes of disorder strength by computer simulations of model chromosomes. Nuclear configurations with different degrees of chromosome condensation are realized from simulations of decondensing chromosomes and the disorder strength is calculated for these nuclear configurations. We found that the disorder strength increases significantly for configurations with slightly more condensed chromosomes. Coupled with PWS measurements, the simulation results suggest that the chromosome condensation and the resulting spatial density inhomogeneity may represent one of the earliest events in carcinogenesis.     

Biological Mechanisms Underlying Structural Changes Induced by Colorectal Field Carcinogenesis Measured with Low-Coherence Enhanced Backscattering (LEBS) Spectroscopy

We previously reported the utility of Low-Coherence Enhanced Backscattering (LEBS) Spectroscopy in detecting optical changes in uninvolved rectal mucosa, changes that are indicative of the presence of advanced colorectal adenomas elsewhere in the colon (field carcinogenesis). We hypothesized that the alterations in optical signatures are due to structural changes in colonocytes. To elucidate those colonocyte changes, we used LEBS and an early time point in an animal model of colorectal field carcinogenesis – rats treated with azoxymethane (AOM). Changes in LEBS markers in intact mucosa from AOM-treated rats could be at least partially attributed to changes in colonocytes. To investigate the molecular mechanisms underlying the colonocyte abnormalities in premalignant colon, we took a candidate approach. We compared expression profiles of genes implicated directly or indirectly in cytoskeletal dysregulation in colorectal tissues from saline-treated versus AOM-treated rats. Our data suggest that a number of genes known to affect colon tumorigenesis are up-regulated in colonocytes, and genes previously reported to be tumor suppressors in metastatic cancer are down-regulated in colonocytes, despite the colonocytes being histologically normal. To further understand the role of the cytoskeleton in generating changes in optical markers of cells, we used pharmacological disruption (using colchicine) of the cytoskeleton. We found that differences in optical markers (between AOM- and control-treated rats) were negated by the disruption, suggesting cytoskeletal involvement in the optical changes. These studies provide significant insights into the micro-architectural alterations in early colon carcinogenesis, and may enable optimization of both bio-photonic and molecular risk stratification techniques to personalize colorectal cancer screening.     

Optical study of stress hormone‐induced nanoscale structural alteration in brain using partial wave spectroscopic microscopy

Chronic stress affects nano to microscale structures of the brain cells/tissues due to suppression of neural growths and reconnections, hence the neuronal activities. This results in depression, memory loss and even death of the brain cells. Our recently developed novel optical technique, partial wave spectroscopic microscopy has nanoscale sensitivity, and hence, can detect nanoscale changes in brain tissues due to stress. In this study, we applied this technique to quantify the stress related structural changes in the corticosterone‐treated mouse model of stress. Our results show that brains from corticosterone‐treated mice showed higher nanoscale structural disorder in the hippocampal region as compared to the brain from normal (vehicle) mice. The increase in structural alteration correlates with the duration of the stress. We further quantified the relative changes and the spatial localization of these changes in this mouse model and found out that the maximum changes occurred nearly symmetrically in both regions of the hippocampus. The mRNA for stress‐related genes, brain‐derived neurotrophic factor and tyrosine kinase‐coupled receptor were also significantly reduced in the hippocampus of corticosterone‐treated mice compared to that in control mice. These results indicate that chronic corticosterone treatment induces nanoscale structural alterations in mouse brain that corresponds to changes in stress‐related gene expression.     

Quantification of photonic localization properties of targeted nuclear mass density variations: Application in cancer‐stage detection

Light localization is a phenomenon which arises due to the interference effects of light waves inside a disordered optical medium. Quantification of degree light localization in optical media is widely used for characterizing degree of structural disorder in that media. Recently, this light localization approach was extended to analyze structural changes in biological cell like heterogeneous optical media, with potential application in cancer diagnostics. Confocal fluorescence microscopy was used to construct “optical lattices,” which represents 2‐dimensional refractive index map corresponding to the spatial mass density distribution of a selected molecule inside the cell. The structural disorder properties of the selected molecules were evaluated numerically using light localization strength in these optical lattices, in a single parameter called “disorder strength.” The method showed a promising potential in differentiating cancerous and non‐cancerous cells. In this paper, we show that by quantifying submicron scale disorder strength in the nuclear DNA mass density distribution, a wide range of control and cancerous breast and prostate cells at different hierarchy levels of tumorigenicity were correctly distinguished. We also discuss how this photonic technique can be used in examining tumorigenicity level in unknown prostate cancer cells, and potential to generalize the method to other cancer cells.      

Will knowledge of human genome variation result in changing cancer paradigms?

Our incomplete understanding of carcinogenesis may be a significant reason why some cancer mortality rates are still increasing. This lack of understanding is likely due to a research approach that relies heavily on genetic comparison between cancerous and non-cancerous tissues and cells, which has led to the identification of genes of cancer proliferation rather than differentiation. Recent observations showing that a tremendous degree of natural human genetic variation occurs are likely to lead to a shift in the basic paradigms of cancer genetics, in that there is a need to consider both the nature of the genes involved, and the idea that not every genetic variation identified in these genes may be associated with carcinogenesis. Based on studies using LCM and micro-genetic analyses, we propose that significant cancer initiating events may take place during the very early stages of development of cancer-susceptible tissues and that using such techniques might greatly help us in our understanding of carcinogenesis.     

Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection

Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. Sensitive techniques are required for measuring cellular levels of DNA damage. We describe in detail a novel immunoassay that makes use of the resolving power of capillary electrophoresis and the sensitivity of laser-induced fluorescence detection. An example is given of the detection of thymine glycol in DNA produced by irradiation of human cells with a clinical dose of 2 Gy. A detection limit of approximately 10(-21) mol allowed us to monitor the repair of the lesion and to suggest that the cellular repair response may be inducible.     

Diverse proteomic alterations in gastric adenocarcinoma

Gastric adenocarcinoma is one of the most common cancers in Asian countries including China. Although its incidence rates in the West are lower than that in Asia, gastric cancer is still a major health problem worldwide, being second only to lung cancers in the number of deaths it causes. Helicobacter pylori infection has been identified as the major pathogen, but the detailed pathogenesis of gastric carcinoma remains elusive. Due to the lack of suitable and specific biomarkers for early detection, most cases of the disease are diagnosed at late stages and the survival rate is low. In this study, we used a proteomic approach to globally analyze the protein profiles of paired surgical specimens of primary gastric adenocarcinoma and nontumor mucosa aiming at identifying specific disease-associated proteins as potential clinical biomarkers and for carcinogenetic study. Compared to nontumor tissues, multiple protein alterations were found in tumor tissues. Some of these alterations involve variations in the expression of cytoskeleton proteins, including an increase in cytokeratin 8 and tropomyosin isoform and a decrease in cytokeratin 20. Co-up-regulations of heat-shock proteins and glycolytic enzymes were observed in tumor tissues, indicating self-protective efforts of cells and the growing energy requirement during malignant transformation. Diverse regulations also occurred with proteins involved in cell proliferation and differentiation, such as GMP reductase 2 and creatine kinase B, and proteins bearing potential tumor suppressor activities, including prohibitin and selenium binding protein 1. More interestingly, a human stomach-specific protein, 18 kDa antrum mucosa protein, was found to be dramatically under-expressed in cancer tissues, implicating a possible special pathological role for this protein in gastric carcinogenesis. Further comprehensive evaluation by globally considering the altered factors may result in the discovery of a biomarker index for effective assessment of the disease and may provide in-depth information for better understanding the pathogenesis of gastric cancer.     

Dynamic changes of exhaustion features in T cells during oral carcinogenesis

ObjectivesThis study aimed to clarify the dynamic changes of exhaustion features in T cells during oral carcinogenesis.Materials and MethodsMice were randomly divided into 4NQO group and control group. The exhaustion features of CD4⁺ and CD8⁺ T cells of both groups were detected by flow cytometry. Furthermore, multiplex immunohistochemistry was used to evaluate the expression of inhibitory receptors in human normal, dysplastic, and carcinogenesis tissues. Finally, anti‐PD‐1 antibody treatment was performed at the early premalignant phase of oral carcinogenesis.ResultsThe proportion of naive T cells in 4NQO group was lower than those in control group, while the proportion of effector memory T cells was higher in 4NQO group. The expression of inhibitory receptors on CD4⁺ and CD8⁺ T cells increased gradually during carcinogenesis. In contrast, the secretion of cytokines by CD4⁺ and CD8⁺ T cells decreased gradually with the progression stage. Strikingly, those changes occurred before the onset of oral carcinogenesis. The expression of inhibitory receptors on T cells increased gradually as the human tissues progressed from normal, dysplasia to carcinoma. Interestingly, PD‐1 blockade at the early premalignant phase could reverse carcinogenesis progression by restoring T cell function.ConclusionsT‐cell dysfunction was established at the early premalignant phase of oral carcinogenesis; PD‐1 blockade at the early premalignant phase can effectively reverse T‐cell exhaustion features and then prevent carcinogenesis progression.     

Acquisition of mitochondrial dysregulation and resistance to mitochondrial-mediated apoptosis after genotoxic insult in normal human fibroblasts: a possible model for early stage carcinogenesis

Acquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5 μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24 h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis.     

Optical quantitative pathology of cervical intraepithelial neoplasia in human tissues using spatial frequency analysis

An optical quantitative histological method in human tissues using spatial frequencies is demonstrated. Optical spatial frequency spectra from different stages of human Cervical Intraepithelial Neoplasia (CIN) tissue are evaluated as a potential quantitative pathological tool. The degree of randomness of tissue structures from normal to different stages of CIN tissue can be recognized by spatial frequency analysis. The standard deviation, σ of human normal and CIN tissue, is obtained by assuming the spatial frequency spectra as a Gaussian distribution. A support vector machine classifier (SVM) is trained in the subspace of σ. Twenty‐eight normal and CIN samples of varying grades are examined and compared with current diagnostic outcomes. Our results suggest that an excellent accuracy for diagnostic purposes can be achieved. This approach offers a simple, efficient and objective way to supplement histopathology in recognizing alterations from normal to different stages of cervical pre‐cancer, which are reflected by spatial information contained within the aperiodic and random structures of the different types of tissue. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Interleukin-4 stimulates collagen synthesis by normal and scleroderma fibroblasts in dermal equivalents.

Interleukin-4 (IL-4) is one of the products of T-lymphocytes and mast cells, inflammatory cells which accumulate in connective tissues at early stages of fibrosis. We tested the effects of IL-4 on human fibroblasts from normal and scleroderma skin seeded in three dimensional collagen lattices ("dermal equivalents"). IL-4 (10 and 100 U/ml) stimulated collagen synthesis in a dose-dependent manner. No significant alteration of lattice retraction and cell proliferation was observed. At the concentration 100 U/ml, Il-4 was approximately twice more efficient on collagen synthesis than Transforming Growth Factor beta (10 ng/ml). IL-4 secretion in connective tissues might be an important factor for the development of fibrotic processes.     

Ferritin is increased in diethylnitrosamine-altered rat hepatocytes

Ferritin was localized by immunoperoxidase in rat liver during the early stages of experimental carcinogenesis induced by diethylnitrosamine. Carcinogen - altered hepatocytes, indentified by their reduction in membrane-bound ATPase activity, showed a highly elevated content in ferritin (or ferritin subunits), compared to normal hepatocytes. This could correspond to an accumulation of free subunits in the cytoplasm or to a “non-specific” increase in ferritin synthesis related to the carcinogenic process. Our results suggest that some cytoplasmic proteins other than enzymes can be modified during the early stages of carcinogenesis and that ferritin accumulation detected by immunolocalization can be used as a valuable marker to identify foci of cellular alterations.     

Non-Invasive Detection of Early Retinal Neuronal Degeneration by Ultrahigh Resolution Optical Coherence Tomography

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.     

Monitoring of viral cancer progression using FTIR microscopy: a comparative study of intact cells and tissues

Fourier transform infrared microspectroscopy (FTIR-MSP) is an analytical method with a promising potential for detecting the spectral changes due to cancerous changes in cells. The purpose of the present study is monitoring biochemical spectral changes accompanying viral cancer progression in cells and tissues using FTIR-MSP. As a model system, we used cells in culture which were transformed to malignant cells by infection with murine sarcoma virus (MuSV) and cervical tissues at different neoplastic stages. In order to devise a systematic follow-up of the cancer progression, it was essential first to determine and validate consistent and significant spectral biomarkers, which can evidently discriminate between normal and cancerous cells/tissues. Then these biomarkers were used for the characterization and classification of early stages of malignant transformation utilizing discriminant classification function techniques. Our study points out that malignancy progression can be eminently graded for both cell lines and tissues. For example, using the array of four biomarkers: A(2958)A(2852)+A(2923),A(1121)/A(1015),A(1171)/A(1152)and|A(1082)-A(1056)|A(1028), we attained that the classification accuracies of different premalignant stages of cell lines and tissues were varied between 89.5 and 97.4%. These results strongly support the potential of developing FTIR microspectroscopy as a simple, reagent free method for early detection and accurate differentiation of premalignant stages.     

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.     

Glomerular Basement Membrane Selective Permeability in Short-term Streptozotocin-induced Diabetic Rats

In diabetes, the glomerular basement membrane undergoes thickening and structural alterations with loss of glomerular permselectivity properties. However, the onset of the alterations at early phases of diabetes is unclear. Aiming to determine the functional and structural alterations of the glomerular wall in the early stages of diabetes, we have studied the distribution of endogenous circulating albumin and type IV collagen in the glomerular basement membrane, using the immunocytochemical approach. The streptozotocin-injected hyperglycemic rat was our animal model. Renal tissues were examined after 10 days, 2, 4 and 6 months of hyperglycemia. Upon immunogold labelings, changes in the glomerular permeability to endogenous albumin were found altered as early as upon ten days of hyperglycemia. In contrast, no structural modifications were detected at this time point. Indeed, glomerular basement membrane thickening and an altered type IV collagen labeling distribution were only observed after four months of hyperglycemia, suggesting that functional alterations take place early in diabetes prior to any structural modification. In order to evaluate the reversibility of the glomerular alterations, two-month-old diabetic animals were treated with insulin. These animals showed a significant restoring of their glomerular permselectivity. Our results suggest a link between glycemic levels and alteration of glomerular permeability in early stages of diabetes, probably through high levels of glycated serum proteins.