This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ABSTRACT Many esophageal diseases can arise during development or throughout life. Therefore, wel... more ABSTRACT Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.
Introduction: Accurate identification of neoplastic or pre-neoplastic lesions is traditionally de... more Introduction: Accurate identification of neoplastic or pre-neoplastic lesions is traditionally dependent on microscopic identification of nuclear abnormalities. It is both time-consuming and challenging in early-stage lesions and difficult to detect. Laser-emission microscopy (LEM) that uses laser emission (not fluorescence) is an emerging technology that takes advantage of the finding that highly-proliferating cells like those in pre-neoplastic lesions can be distinguished from less proliferative cells by a lower laser emission dot density (LDD) when stained with a nuclear dye. We have successfully used this technique to detect (early) nuclear changes in a high-fat induced colon cancer model. LEM has also been used successfully to differentiate healthy tissue from cancerous tissue in human patients. Methods: This LEM system is capable of rapidly scanning tissue as large as 2 cm×2 cm in ~10 min. Both laser emission and regular fluorescence are imaged simultaneously, thus providing information regarding the lasing spot density and location within the human colon tissues. In this study, we employed colon biopsies from 12 human subjects (at risk for colorectal cancer). These subjects participated in a 90-day intervention, placebo-controlled, randomized trial. 8 subjects ingested known chemopreventives and 4 subjects took a placebo. Colon biopsies were taken from the sigmoid colon at baseline and after 90 days. The formalin-fixed, paraffin-embedded tissues were subjected to LEM microscopic assessment. For this, the tissues were sandwiched between two mirrors to form a laser cavity and laser excitation was applied. By comparing the laser emission spectra, highly proliferative tissue could be differentiated from less proliferative tissue. Post-treatment biopsies were compared to their baseline status. These biopsies were also stained with Ki67 to obtain their proliferation index to compare with the LEM results. Results: On average, there was a 20% increase in the LDD among the 8 subjects on chemopreventives while no change was seen in Ki67 positivity. In placebo group, Ki67 was decreased by 14% and LDD was 12% increased. On individual comparison, LDD and Ki67 data were correlated positively in 6 subjects. Conclusion: The preliminary data suggest that in histologically-normal but “at-risk” human colon LEM may pick early nuclear changes that may not be detected by a proliferation marker. Table.A comparison of lasing dot density (LDD) data to Ki67 positivity (Whole slide analysis*)Mean LDDPercent Positive NucleiChange RatioSubject IDPrePostPrePostLDDKi67Data correlated?Placebo 1491336504.75.40.741.15NoPlacebo 2851078159.28.70.920.95YesPlacebo 3573183558.96.01.460.67NoPlacebo 4659288469.76.71.340.69NoChemopreventive#1 1296733436.513.61.132.10YesChemopreventive#1 2528261406.57.71.161.20YesChemopreventive#1 3468453429.67.01.140.73NoChemopreventive#1 4758173358.25.60.970.68YesChemopreventive#2 1323046255.34.91.430.91NoChemopreventive#2 2508078408.29.11.541.11YesChemopreventive#2 35587400110.46.80.720.66YesChemopreventive#2 4491373226.94.91.490.71NoPlacebo combined1.12±0.340.86±0.23Chemopreventive combined1.20±0.281.01±0.49*Both mucosa and submucosa were involved in the analysis Citation Format: Muhammad N. Aslam, Yun-Lu Sun, Ingrid Bergin, Mohamed Ali H. Jawad-Makki, Karsten Knuver, Danielle (Kim) Turgeon, James Varani, Xudong Fan. A novel laser emission microscopy for early detection of colon cancer: Analysis of at-risk human colon from an interventional trial (research in progress) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT113.
Fluorescence endoscopy is emerging for guiding biopsy and early cancer detection in the gastroint... more Fluorescence endoscopy is emerging for guiding biopsy and early cancer detection in the gastrointestinal tract. A multimodal scanning fiber endoscope (mmSFE) with 2 fluorescence labeled peptides is designed to image overexpressed biomarkers associated with esophageal adenocarcinoma (EAC) and high-grade dysplasia (HGD), thus detecting early neoplasia. Quantification of multiplexed fluorescence images is critical, which ‘red-flags’ suspicious regions and supports diagnosis. The Target/Background (T/B) ratio is calculated to quantitatively evaluate fluorescence images. However T/B ratios are based on fluorescence intensities alone, so adding morphological features can be critical to providing evidence for diagnosis. Moreover, currently the T/B ratio is calculated for a single fluorescence channel. A protocol for multiplexed fluorescence quantification is needed. Materials and Methods: Peptides targeting EGFR and ErbB2 are labeled with NIR fluorescence Cy5 and IRdye800, respectively. A mmSFE with 2.4mm flexible shaft and wide-field forward-view imaging is designed to image these two near-infrared fluorophores with an additional reflectance channel for anatomical identification. In first-in-human clinical trials the two peptides are topically sprayed, briefly incubated, and then rinsed in subjects at high risk of EAC. Both fluorescence channels were captured simultaneously with the co-registered reflectance channel at 30Hz. After removing artifacts, frames were manually selected using morphological features. T/B ratios were then calculated for all fluorescence channels in selected frames. Results and Conclusions: T/B ratios for HGD subjects are greater than for healthy subjects in at least one of the fluorescence channels. Future work will design algorithms to automatically select suspicious regions based on morphological features. More analysis will be performed based on co-registered fluorescence channels.
Early detection of precursor lesions for colorectal cancer can greatly improve survival. Pre-neop... more Early detection of precursor lesions for colorectal cancer can greatly improve survival. Pre-neoplasia can appear flat with conventional white light endoscopy. Sessile serrated adenomas (SSA) are precursor lesions found primarily in the proximal colon and frequently appear flat and indistinct. We performed a clinical study of n=37 patients using a multimodal endoscopy with a FITC-labeled peptide specific for SSA. Lesions were imaged with white light, reflectance and fluorescence. White light images were acquired before the peptide was applied and were used to help localize regions of abnormal tissues rightly. Co-registered fluorescence and reflectance images were combined to get ratio images thus the distance was corrected. We calculated the target/background ratio (T/B ratio) to quantify the images and found 2.3-fold greater fluorescence intensity for SSA compared with normal tissues. We found the T/B ratio for SSA to be significantly greater than that for normal colonic mucosa with 89.47% sensitivity and 91.67% specificity at the threshold of 1.22. An ROC curve for SSA and normal mucosa was also plotted with area under curve (AUC) of 0.93. The result also shows that SSA and adenoma are statistically significant and can be identified with 78.95% sensitivity and 90.48% specificity at the threshold of 1.66. An ROC curve was plotted with AUC of 0.88. Therefore, our result shows that the application of a multimodal endoscope with fluorescently labeled peptide can quantify images and works especially good for the detection of SSA which is a premalignant flat lesion conferring a high risk of subsequently leading to a colon cancer.
Cellular and molecular gastroenterology and hepatology, 2022
Background & Aims TP53 mutations underlie Barrett’s esophagus (BE) progression to dysplasia a... more Background & Aims TP53 mutations underlie Barrett’s esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. Methods Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. Results The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2–E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate β-TrCP1–mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding–deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. Conclusions Loss of RNF128 Iso2–UBCH5C and persistence of the Iso1–UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.
Diaz and colleagues have carefully studied the effects of omeprazole on the expression of cytochr... more Diaz and colleagues have carefully studied the effects of omeprazole on the expression of cytochrome P-450 in primary cultures of human hepatocytes. When omeprazole was added to the culture medium in
Adenoma colonoid expansion. Individual colonoids from each of the three adenomas were examined un... more Adenoma colonoid expansion. Individual colonoids from each of the three adenomas were examined under phase-contrast microscopy after 8 and 13 days in culture (under each condition) and photographed. Photoshop CS6 image analysis tool was used to assess the surface area encompassed by individual colonoids and the difference in colonoid size between day-8 and day-13 was used to calculate the growth index (net change between the two time-points). Values shown are means and standard deviations. The number of individual colonoids assessed ranged from n=250 to n=424 for adenoma 282. For adenoma 590, colonoids ranged from n=53 to n=77. For adenoma 584, numbers ranged from n=15 to n=111. Statistical significance was assessed by ANOVA followed by student t-test (two-tailed) for unpaired data. Asterisks indicate significance at p<0.01 level. Insert: Phase-contrast images of the same fields on day-8 and day-13. Colonoids were more differentiated (thick walled, smooth surface and lack of small buds) at Day 13 with calcium 1.5mM as compared to colonoids exposed to calcium 0.15mM. Bar=200 μm.
Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal ref... more Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.
bioRxiv (Cold Spring Harbor Laboratory), May 20, 2021
European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when ex... more European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines. Keywords: esophagus, adult patient-derived cultures, biobank, disparity, ancestry Introduction The esophagus connects the upper pharynx with the stomach and is lined by a stratified squamous epithelium (Rosekrans et al., 2015). The esophagus is prone to many diseases including esophageal squamous cancer (Kim et al., 2017), eosinophilic esophagitis (Blevins et al., 2018), metaplasia (Barrett's esophagus) due to reflux and inflammation, and esophageal adenocarcinoma (Saraggi et al., 2016). Despite risk factors being equal across populations (Spechler et al., 2002; El-Serag et al., 2004), there is a higher incidence of esophageal adenocarcinoma in the Caucasian/European American (EA) population when compared to populations of African descent, including the Black/African American (AA) population (
Polyethylene glycol-electrolyte lavage solutions are widely used to prepare the colon for colonos... more Polyethylene glycol-electrolyte lavage solutions are widely used to prepare the colon for colonoscopy. Unfortunately, some patients find this preparation difficult to complete. Recent studies of a sodium phosphate-based laxative have shown both good patient tolerance and good bowel preparation. In these studies, the laxative has generally been prescribed in two doses, with the second dose taken early the morning of colonoscopy. Because the morning dose is inconvenient for many patients, we compared giving a common polyethylene glycol-based electrolyte lavage solution the day before colonoscopy with our method of giving both doses of sodium phosphate-based laxative the day before colonoscopy: one dose at 4 PM and the second dose at 8 PM. We judged efficacy by an assessment of residual liquid and fecal matter in the colon and judged tolerance by the results of a symptom questionnaire completed by each patient immediately before the procedure. Our results in more than 200 patients showed similar efficacy ratings and similar symptom scores for both preparations, but patients rated the sodium phosphate-based preparation as easier to tolerate. In conclusion, in selected patients this new dosing method for sodium phosphate is preferable to large-volume, whole-gut lavage solutions.
Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we... more Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomatous polyps. Methods: Fresh biopsy samples were transported in culture medium containing 1.5mM Ca2+ and antimicrobials. The mucosa/submucosa was surgically separated from the muscularis propria and incubated in 10mM dithiothreitol followed by 8mM ethylenediaminetetraacetic acid. Crypts were agitated free, washed with slow spins, and immediately embedded in a 3-dimensional environment at a density of 50 crypts/50μl Matrigel. After 3 hours, media was replaced to remove non-viable cellular products. Serum-free medium, containing low Ca2+ (0.15mM), was used to maintain crypt cultures. Cell proliferation and differentiation were assessed with markers Ki67 and E-cadherin by confocal microscopy. The fluorescence signal was quantified for the bottom 1/3 of the crypts (35 cells from base on one crypt edge) using morphometric imaging software. In parallel studies, abnormal crypt-like structures were isolated from adenomatous polyps/adenocarcinoma and maintained in a similar culture system. Results: At day-1 in culture, Ki67 expression was strong in the proliferating cells at the crypt base, with E-cadherin staining increasing towards the crypt apex. By day-3, E-cadherin expression became much more prominent and Ki67 decreased dramatically. This culture system preserved the in vivo phenotype of the crypts and allowed for the colonic epithelial cells to follow the physiological sequence of progression from proliferation to differentiation. Adenomatous tissue in culture at day-1 showed expression for Ki67 and E-cadherin but lacked spacial organization. Conclusion: Intact colonic crypts from normal and abnormal human mucosa can be viably maintained in 3-D culture for 72 hours. This primary human in vitro 3-D model may be used to model efficacy of colonic cancer chemopreventives. Epithelial Cell Proliferation and Differentiation in Cultured Human Crypts (fold changes) Markers Zero-time 24 hours 48 hours 72 hours Ki67 1.00 0.36 0.18 0.00 E-cadherin 1.00 2.00 2.89 3.22 Citation Format: Michael K. Dame, Yan Jiang, Danielle Kim Turgeon, Henry Appelman, Muhammad N. Aslam, Kelly Copley, Durga Attili, Dean E. Brenner, James Varani. Human colon mucosal crypts in culture: a model to assess colon chemoprevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2013-3711
Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal ref... more Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for EAC development. GERD and esophagitis occur at similar rates among Blacks and Caucasians; yet, progression to EAC is significantly elevated among Caucasians. Unique protective factors in the epithelium of Blacks may contribute to this disparity. Our research team recently reported that the detoxification enzyme GSTT2 is higher in the esophageal squamous epithelium of Blacks compared to Caucasians with potential linkages to previously identified genomic variants in the GSTT2 locus (a 37 kb deletion and a 17 bp promoter duplication among Caucasians). Thus, the current study seeks to evaluate whether primary esophageal cell cultures isolated from Black or Caucasian cohorts can serve as discerning and relevant model systems to investigate risk factors linked to EAC progression, assess efficacy of mitigating agents and differential response...
Cancer Epidemiology and Prevention Biomarkers, 2007
CN21-04 Ornithine decarboxylase (ODC) is the first enzyme in polyamine synthesis and is a target ... more CN21-04 Ornithine decarboxylase (ODC) is the first enzyme in polyamine synthesis and is a target of the MYC gene. MYC is regulated by the adenomatous polyposis coli (APC) tumor suppressor gene in intestinal and colonic epithelial cells in humans and rodents (1). Non-steroidal anti-inflammatory drugs (NSAIDS) influence arachidonic acid metabolism in part by inhibiting the activities of cyclooxygenase 1 (COX1) and/or cyclooxygenase 2 (COX2), but NSAIDS also activate polyamine catabolism and export via COX-independent mechanisms (2, 3). Difluoromethylornithine (DFMO) is an irreversible inhibitor of ODC, which has been developed as a cancer chemopreventive agent (4). DFMO or any of several NSAIDS alone reduces colon carcinogenesis in rodent models, and the combination of DFMO and NSAIDS acts at least synergistically in both rodent and human cell models of colonic neoplasia (1). A polymorphism affecting ODC expression has been associated with risk of polyp recurrence in humans with prior...
Introduction: Epidemiologic and experimental data suggests that colon cancer should be largely pr... more Introduction: Epidemiologic and experimental data suggests that colon cancer should be largely preventable through dietary or chemical intervention. Polyamines are actively involved in proliferation and differentiation of many tissues. Lowering of intracellular levels via inhibition of the initial step (ornithine decarboxylase) in the polyamine synthesis pathway by the specific enzyme-activated inhibitor DFMO results in a decrease of adenomas and cancers in many preclinical organ-site cancer models, most notably in the colon. Methods: From 1989-1997 we conducted a series of pilot and Phase IIa (short term) and Phase IIb (long term) trials in which we determined the lowest dose of DFMO at which polyamine content of the colonic flat mucosa would be lowered, with the goal of finding a non-toxic dose. In 1997 we debated whether to do a definitive trial with DFMO alone or to combine it with another chemoprevention compound. We settled on combining DFMO with the NSAID sulindac at low dose...
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ABSTRACT Many esophageal diseases can arise during development or throughout life. Therefore, wel... more ABSTRACT Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.
Introduction: Accurate identification of neoplastic or pre-neoplastic lesions is traditionally de... more Introduction: Accurate identification of neoplastic or pre-neoplastic lesions is traditionally dependent on microscopic identification of nuclear abnormalities. It is both time-consuming and challenging in early-stage lesions and difficult to detect. Laser-emission microscopy (LEM) that uses laser emission (not fluorescence) is an emerging technology that takes advantage of the finding that highly-proliferating cells like those in pre-neoplastic lesions can be distinguished from less proliferative cells by a lower laser emission dot density (LDD) when stained with a nuclear dye. We have successfully used this technique to detect (early) nuclear changes in a high-fat induced colon cancer model. LEM has also been used successfully to differentiate healthy tissue from cancerous tissue in human patients. Methods: This LEM system is capable of rapidly scanning tissue as large as 2 cm×2 cm in ~10 min. Both laser emission and regular fluorescence are imaged simultaneously, thus providing information regarding the lasing spot density and location within the human colon tissues. In this study, we employed colon biopsies from 12 human subjects (at risk for colorectal cancer). These subjects participated in a 90-day intervention, placebo-controlled, randomized trial. 8 subjects ingested known chemopreventives and 4 subjects took a placebo. Colon biopsies were taken from the sigmoid colon at baseline and after 90 days. The formalin-fixed, paraffin-embedded tissues were subjected to LEM microscopic assessment. For this, the tissues were sandwiched between two mirrors to form a laser cavity and laser excitation was applied. By comparing the laser emission spectra, highly proliferative tissue could be differentiated from less proliferative tissue. Post-treatment biopsies were compared to their baseline status. These biopsies were also stained with Ki67 to obtain their proliferation index to compare with the LEM results. Results: On average, there was a 20% increase in the LDD among the 8 subjects on chemopreventives while no change was seen in Ki67 positivity. In placebo group, Ki67 was decreased by 14% and LDD was 12% increased. On individual comparison, LDD and Ki67 data were correlated positively in 6 subjects. Conclusion: The preliminary data suggest that in histologically-normal but “at-risk” human colon LEM may pick early nuclear changes that may not be detected by a proliferation marker. Table.A comparison of lasing dot density (LDD) data to Ki67 positivity (Whole slide analysis*)Mean LDDPercent Positive NucleiChange RatioSubject IDPrePostPrePostLDDKi67Data correlated?Placebo 1491336504.75.40.741.15NoPlacebo 2851078159.28.70.920.95YesPlacebo 3573183558.96.01.460.67NoPlacebo 4659288469.76.71.340.69NoChemopreventive#1 1296733436.513.61.132.10YesChemopreventive#1 2528261406.57.71.161.20YesChemopreventive#1 3468453429.67.01.140.73NoChemopreventive#1 4758173358.25.60.970.68YesChemopreventive#2 1323046255.34.91.430.91NoChemopreventive#2 2508078408.29.11.541.11YesChemopreventive#2 35587400110.46.80.720.66YesChemopreventive#2 4491373226.94.91.490.71NoPlacebo combined1.12±0.340.86±0.23Chemopreventive combined1.20±0.281.01±0.49*Both mucosa and submucosa were involved in the analysis Citation Format: Muhammad N. Aslam, Yun-Lu Sun, Ingrid Bergin, Mohamed Ali H. Jawad-Makki, Karsten Knuver, Danielle (Kim) Turgeon, James Varani, Xudong Fan. A novel laser emission microscopy for early detection of colon cancer: Analysis of at-risk human colon from an interventional trial (research in progress) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT113.
Fluorescence endoscopy is emerging for guiding biopsy and early cancer detection in the gastroint... more Fluorescence endoscopy is emerging for guiding biopsy and early cancer detection in the gastrointestinal tract. A multimodal scanning fiber endoscope (mmSFE) with 2 fluorescence labeled peptides is designed to image overexpressed biomarkers associated with esophageal adenocarcinoma (EAC) and high-grade dysplasia (HGD), thus detecting early neoplasia. Quantification of multiplexed fluorescence images is critical, which ‘red-flags’ suspicious regions and supports diagnosis. The Target/Background (T/B) ratio is calculated to quantitatively evaluate fluorescence images. However T/B ratios are based on fluorescence intensities alone, so adding morphological features can be critical to providing evidence for diagnosis. Moreover, currently the T/B ratio is calculated for a single fluorescence channel. A protocol for multiplexed fluorescence quantification is needed. Materials and Methods: Peptides targeting EGFR and ErbB2 are labeled with NIR fluorescence Cy5 and IRdye800, respectively. A mmSFE with 2.4mm flexible shaft and wide-field forward-view imaging is designed to image these two near-infrared fluorophores with an additional reflectance channel for anatomical identification. In first-in-human clinical trials the two peptides are topically sprayed, briefly incubated, and then rinsed in subjects at high risk of EAC. Both fluorescence channels were captured simultaneously with the co-registered reflectance channel at 30Hz. After removing artifacts, frames were manually selected using morphological features. T/B ratios were then calculated for all fluorescence channels in selected frames. Results and Conclusions: T/B ratios for HGD subjects are greater than for healthy subjects in at least one of the fluorescence channels. Future work will design algorithms to automatically select suspicious regions based on morphological features. More analysis will be performed based on co-registered fluorescence channels.
Early detection of precursor lesions for colorectal cancer can greatly improve survival. Pre-neop... more Early detection of precursor lesions for colorectal cancer can greatly improve survival. Pre-neoplasia can appear flat with conventional white light endoscopy. Sessile serrated adenomas (SSA) are precursor lesions found primarily in the proximal colon and frequently appear flat and indistinct. We performed a clinical study of n=37 patients using a multimodal endoscopy with a FITC-labeled peptide specific for SSA. Lesions were imaged with white light, reflectance and fluorescence. White light images were acquired before the peptide was applied and were used to help localize regions of abnormal tissues rightly. Co-registered fluorescence and reflectance images were combined to get ratio images thus the distance was corrected. We calculated the target/background ratio (T/B ratio) to quantify the images and found 2.3-fold greater fluorescence intensity for SSA compared with normal tissues. We found the T/B ratio for SSA to be significantly greater than that for normal colonic mucosa with 89.47% sensitivity and 91.67% specificity at the threshold of 1.22. An ROC curve for SSA and normal mucosa was also plotted with area under curve (AUC) of 0.93. The result also shows that SSA and adenoma are statistically significant and can be identified with 78.95% sensitivity and 90.48% specificity at the threshold of 1.66. An ROC curve was plotted with AUC of 0.88. Therefore, our result shows that the application of a multimodal endoscope with fluorescently labeled peptide can quantify images and works especially good for the detection of SSA which is a premalignant flat lesion conferring a high risk of subsequently leading to a colon cancer.
Cellular and molecular gastroenterology and hepatology, 2022
Background & Aims TP53 mutations underlie Barrett’s esophagus (BE) progression to dysplasia a... more Background & Aims TP53 mutations underlie Barrett’s esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown. Methods Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability. Results The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2–E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate β-TrCP1–mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding–deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells. Conclusions Loss of RNF128 Iso2–UBCH5C and persistence of the Iso1–UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.
Diaz and colleagues have carefully studied the effects of omeprazole on the expression of cytochr... more Diaz and colleagues have carefully studied the effects of omeprazole on the expression of cytochrome P-450 in primary cultures of human hepatocytes. When omeprazole was added to the culture medium in
Adenoma colonoid expansion. Individual colonoids from each of the three adenomas were examined un... more Adenoma colonoid expansion. Individual colonoids from each of the three adenomas were examined under phase-contrast microscopy after 8 and 13 days in culture (under each condition) and photographed. Photoshop CS6 image analysis tool was used to assess the surface area encompassed by individual colonoids and the difference in colonoid size between day-8 and day-13 was used to calculate the growth index (net change between the two time-points). Values shown are means and standard deviations. The number of individual colonoids assessed ranged from n=250 to n=424 for adenoma 282. For adenoma 590, colonoids ranged from n=53 to n=77. For adenoma 584, numbers ranged from n=15 to n=111. Statistical significance was assessed by ANOVA followed by student t-test (two-tailed) for unpaired data. Asterisks indicate significance at p<0.01 level. Insert: Phase-contrast images of the same fields on day-8 and day-13. Colonoids were more differentiated (thick walled, smooth surface and lack of small buds) at Day 13 with calcium 1.5mM as compared to colonoids exposed to calcium 0.15mM. Bar=200 μm.
Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal ref... more Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.
bioRxiv (Cold Spring Harbor Laboratory), May 20, 2021
European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when ex... more European Americans (EA) are more susceptible to esophageal tissue damage and inflammation when exposed to gastric acid and bile acid reflux and have a higher incidence of esophageal adenocarcinoma when compared to African Americans (AA). Population studies have implicated specific genes for these differences; however, the underlying cause for these differences is not well understood. We describe a robust long-term culture system to grow primary human esophagus in vitro, use single cell RNA sequencing to compare primary human biopsies to their in vitro counterparts, identify known and new molecular markers of basal cell types, and demonstrate that in vivo cellular heterogeneity is maintained in vitro. We further developed an ancestrally diverse biobank and a high-content, image based, screening assay to interrogate bile-acid injury response. These results demonstrated that AA esophageal cells responded significantly differently than EA-derived cells, mirroring clinical findings, having important implications for addressing disparities in early drug development pipelines. Keywords: esophagus, adult patient-derived cultures, biobank, disparity, ancestry Introduction The esophagus connects the upper pharynx with the stomach and is lined by a stratified squamous epithelium (Rosekrans et al., 2015). The esophagus is prone to many diseases including esophageal squamous cancer (Kim et al., 2017), eosinophilic esophagitis (Blevins et al., 2018), metaplasia (Barrett's esophagus) due to reflux and inflammation, and esophageal adenocarcinoma (Saraggi et al., 2016). Despite risk factors being equal across populations (Spechler et al., 2002; El-Serag et al., 2004), there is a higher incidence of esophageal adenocarcinoma in the Caucasian/European American (EA) population when compared to populations of African descent, including the Black/African American (AA) population (
Polyethylene glycol-electrolyte lavage solutions are widely used to prepare the colon for colonos... more Polyethylene glycol-electrolyte lavage solutions are widely used to prepare the colon for colonoscopy. Unfortunately, some patients find this preparation difficult to complete. Recent studies of a sodium phosphate-based laxative have shown both good patient tolerance and good bowel preparation. In these studies, the laxative has generally been prescribed in two doses, with the second dose taken early the morning of colonoscopy. Because the morning dose is inconvenient for many patients, we compared giving a common polyethylene glycol-based electrolyte lavage solution the day before colonoscopy with our method of giving both doses of sodium phosphate-based laxative the day before colonoscopy: one dose at 4 PM and the second dose at 8 PM. We judged efficacy by an assessment of residual liquid and fecal matter in the colon and judged tolerance by the results of a symptom questionnaire completed by each patient immediately before the procedure. Our results in more than 200 patients showed similar efficacy ratings and similar symptom scores for both preparations, but patients rated the sodium phosphate-based preparation as easier to tolerate. In conclusion, in selected patients this new dosing method for sodium phosphate is preferable to large-volume, whole-gut lavage solutions.
Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we... more Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomatous polyps. Methods: Fresh biopsy samples were transported in culture medium containing 1.5mM Ca2+ and antimicrobials. The mucosa/submucosa was surgically separated from the muscularis propria and incubated in 10mM dithiothreitol followed by 8mM ethylenediaminetetraacetic acid. Crypts were agitated free, washed with slow spins, and immediately embedded in a 3-dimensional environment at a density of 50 crypts/50μl Matrigel. After 3 hours, media was replaced to remove non-viable cellular products. Serum-free medium, containing low Ca2+ (0.15mM), was used to maintain crypt cultures. Cell proliferation and differentiation were assessed with markers Ki67 and E-cadherin by confocal microscopy. The fluorescence signal was quantified for the bottom 1/3 of the crypts (35 cells from base on one crypt edge) using morphometric imaging software. In parallel studies, abnormal crypt-like structures were isolated from adenomatous polyps/adenocarcinoma and maintained in a similar culture system. Results: At day-1 in culture, Ki67 expression was strong in the proliferating cells at the crypt base, with E-cadherin staining increasing towards the crypt apex. By day-3, E-cadherin expression became much more prominent and Ki67 decreased dramatically. This culture system preserved the in vivo phenotype of the crypts and allowed for the colonic epithelial cells to follow the physiological sequence of progression from proliferation to differentiation. Adenomatous tissue in culture at day-1 showed expression for Ki67 and E-cadherin but lacked spacial organization. Conclusion: Intact colonic crypts from normal and abnormal human mucosa can be viably maintained in 3-D culture for 72 hours. This primary human in vitro 3-D model may be used to model efficacy of colonic cancer chemopreventives. Epithelial Cell Proliferation and Differentiation in Cultured Human Crypts (fold changes) Markers Zero-time 24 hours 48 hours 72 hours Ki67 1.00 0.36 0.18 0.00 E-cadherin 1.00 2.00 2.89 3.22 Citation Format: Michael K. Dame, Yan Jiang, Danielle Kim Turgeon, Henry Appelman, Muhammad N. Aslam, Kelly Copley, Durga Attili, Dean E. Brenner, James Varani. Human colon mucosal crypts in culture: a model to assess colon chemoprevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2013-3711
Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal ref... more Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for EAC development. GERD and esophagitis occur at similar rates among Blacks and Caucasians; yet, progression to EAC is significantly elevated among Caucasians. Unique protective factors in the epithelium of Blacks may contribute to this disparity. Our research team recently reported that the detoxification enzyme GSTT2 is higher in the esophageal squamous epithelium of Blacks compared to Caucasians with potential linkages to previously identified genomic variants in the GSTT2 locus (a 37 kb deletion and a 17 bp promoter duplication among Caucasians). Thus, the current study seeks to evaluate whether primary esophageal cell cultures isolated from Black or Caucasian cohorts can serve as discerning and relevant model systems to investigate risk factors linked to EAC progression, assess efficacy of mitigating agents and differential response...
Cancer Epidemiology and Prevention Biomarkers, 2007
CN21-04 Ornithine decarboxylase (ODC) is the first enzyme in polyamine synthesis and is a target ... more CN21-04 Ornithine decarboxylase (ODC) is the first enzyme in polyamine synthesis and is a target of the MYC gene. MYC is regulated by the adenomatous polyposis coli (APC) tumor suppressor gene in intestinal and colonic epithelial cells in humans and rodents (1). Non-steroidal anti-inflammatory drugs (NSAIDS) influence arachidonic acid metabolism in part by inhibiting the activities of cyclooxygenase 1 (COX1) and/or cyclooxygenase 2 (COX2), but NSAIDS also activate polyamine catabolism and export via COX-independent mechanisms (2, 3). Difluoromethylornithine (DFMO) is an irreversible inhibitor of ODC, which has been developed as a cancer chemopreventive agent (4). DFMO or any of several NSAIDS alone reduces colon carcinogenesis in rodent models, and the combination of DFMO and NSAIDS acts at least synergistically in both rodent and human cell models of colonic neoplasia (1). A polymorphism affecting ODC expression has been associated with risk of polyp recurrence in humans with prior...
Introduction: Epidemiologic and experimental data suggests that colon cancer should be largely pr... more Introduction: Epidemiologic and experimental data suggests that colon cancer should be largely preventable through dietary or chemical intervention. Polyamines are actively involved in proliferation and differentiation of many tissues. Lowering of intracellular levels via inhibition of the initial step (ornithine decarboxylase) in the polyamine synthesis pathway by the specific enzyme-activated inhibitor DFMO results in a decrease of adenomas and cancers in many preclinical organ-site cancer models, most notably in the colon. Methods: From 1989-1997 we conducted a series of pilot and Phase IIa (short term) and Phase IIb (long term) trials in which we determined the lowest dose of DFMO at which polyamine content of the colonic flat mucosa would be lowered, with the goal of finding a non-toxic dose. In 1997 we debated whether to do a definitive trial with DFMO alone or to combine it with another chemoprevention compound. We settled on combining DFMO with the NSAID sulindac at low dose...
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