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.

Dysregulation of microRNAs in colonic field carcinogenesis: implications for screening.

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.

Association of stem-like cells in gender-specific chemoprevention against intestinal neoplasia in MIN mouse.

This study was undertaken to examine the gender-sensitivity and chemopreventive responsiveness of celecoxib on intestinal stem-like cells as a biomarker of colon carcino-genesis, using the MIN mouse model. Male and female MIN mice (6-7-weeks old) were randomized to either control diet or to a diet supplemented with celecoxib (1,500 ppm). The animals were euthanized ten weeks later and the intestines were flushed and opened longitudinally to assess tumor count. Small intestinal segments were formalin-fixed and tissue sections were subjected to immunohistochemical evaluation of DCAMKL1, a known marker of stem-like cells. We found that in animals receiving control (AIN 76A diet) alone, female MIN mice had a higher polyp count than males (52.32 ± 13.89 vs. 35.43 ± 16.05; p<0.0005). However, compared to control diet groups, celecoxib supplementation caused a larger reduction in the number of polyps in females than their male cohorts (6.38 ± 1.43 vs. 12.83 ± 6.74; a reduction of 88% in females to 64% in males). Significant differences (p=0.013) were observed in the number of DCAMKL1-stained cells in the crypts of the wild-type (WT) (10.01 ± 1.07 stem cells per high powered field; HPF) compared to the MIN mice (24.15 ± 8.08 stem cells per HPF), illustrating increased stem-like cells in animals that are more prone to neoplasia. DCAMKL1 labeled stem-like cells were equal in number in the male and female groups receiving the control AIN 76A diet alone (females, 25.73 stem-like cells/HPF); males, 24.15 stem-like cells/HPF). However, females showed a greater reduction in the number of DCAMKL1-labeled stem-like cells with celecoxib supplementation than the respective males (16.63 ± 4.23 vs. 21.56 ± 9.06; a reduction of 35.4% in females to 10.7% in males). We conclude that a higher number of stem-like cells in the uninvolved mucosa paralleled tumorigenesis and mirrored greater chemopreventive responsiveness of female MIN mice compared to males.

Neo-angiogenesis and the premalignant micro-circulatory augmentation of early colon carcinogenesis.

Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 µm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.

Role of cytoskeleton in controlling the disorder strength of cellular nanoscale architecture.

Cytoskeleton is ubiquitous throughout the cell and is involved in important cellular processes such as cellular transport, signal transduction, gene transcription, cell-division, etc. Partial wave spectroscopic microscopy is a novel optical technique that measures the statistical properties of cell nanoscale organization in terms of the disorder strength. It has been found previously that the increase in the disorder strength of cell nanoarchitecture is one of the earliest events in carcinogenesis. In this study, we investigate the cellular components responsible for the differential disorder strength between two morphologically (and hence microscopically) similar but genetically altered human colon cancer cell lines, HT29 cells and Csk shRNA-transfected HT29 cells that exhibit different degrees of neoplastic aggressiveness. To understand the role of cytoskeleton in nanoarchitectural alterations, we performed selective drug treatment on the specific cytoskeletal components of these cell types and studied the effects of cytoskeletal organization on disorder strength differences. We report that altering the cell nanoarchitecture by disrupting cytoskeletal organization leads to the attenuation of the disorder strength differences between microscopically indistinguishable HT29 and CSK constructs. We therefore demonstrate that cytoskeleton plays a role in the control of cellular nanoscale disorder.