The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 () and SPARC-related modular calcium binding 2 (). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.
The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, while less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5.LGR5-positive cells were isolated from 4 adenoma organoid lines and were subjected to RNA-sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA-seq data from adenoma and normal organoids, we found correlations between LGR5 and CRCspecific genes, including DKK4 (dickkopf WNT signaling pathway inhibitor 4) and SMOC2 (SPARC related modular calcium binding 2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.
Vitiligo is a disease of the skin characterized by the appearance of white spots. Significant progress has been made in understanding vitiligo pathogenesis over the past 30 years, but only through perseverance, collaboration, and open-minded discussion. Early hypotheses considered roles for innervation, microvascular anomalies, oxidative stress, defects in melanocyte adhesion, autoimmunity, somatic mosaicism, and genetics. Because theories about pathogenesis drive experimental design, focus, and even therapeutic approach, it is important to consider their impact on our current understanding about vitiligo. Animal models allow researchers to perform mechanistic studies, and the development of improved patient sample collection methods provides a platform for translational studies in vitiligo that can also be applied to understand other autoimmune diseases that are more difficult to study in human samples. Here we discuss the history of vitiligo translational research, recent advances, and their implications for new treatment approaches.
Epigenomic changes are commonly observed in cancer. We developed a next-generation sequencing (NGS) assay which can identify subtle changes in global methylation as well as copy number alterations (CNAs). The assay measures the methylation level of some repeat elements, covering over 25% of all CpG in the genome. Genomic DNA extracted from 18 pairs of matched colorectal tumor to normal tissue was tested (four adenocarcinomas, including one associated with inflammatory bowel disease, and fourteen adenomas, including one adenoma from a Lynch syndrome patient, one familial adenomatous polyposis (FAP) and two sessile serrated adenomas (SSA)). We also assayed organoids established from these tissues at early (2 month) and late (more than 6 months) timepoints in culture. Briefly, bisulfite conversion and enrichment of the repeats was performed. The Illumina compatible products were sequenced on a HiSEQ 2500. Analysis was performed using Bismark (Krueger F., Babraham institute) and Nexus copy number (BioDiscovery) to determine the global methylation and the CNAs, respectively. Finally, somatic and germline variants were determined by targeted sequencing of 71 genes using the QIAseq colorectal cancer panel (QIAGEN). Our data showed that all adenocarcinoma presented hypomethylation and CNAs as well as multiple somatic variants in APC, KRAS, TP53, SMAD4 and PIK3CA. Six out of the fourteen adenomas presented CNAs and nine showed hypomethylation. The most frequently observed copy number gain affected the chromosomes 8q and 13. Eight adenomas presenting somatic mutations in APC also exhibited distinct global hypomethylation. Previous publications have reported that mutations in APC precede global hypomethylation. Interestingly, one adenoma showed significant hypomethylation but no detectable CNAs or APC mutations. The two SSA samples showed the characteristic BRAF V600E mutations and the FAP sample presented germline mutation in APC. The FAP and the SSA samples did not show hypomethylation or CNAs. Most organoids presented CNAs and somatic mutations similar to their matched tissue even after 2 years in culture. However, few organoids developed new CNAs and somatic variants. Monitoring the changes in organoid may provide important information on tumor progression. This assay measured CNAs and methylation in colorectal cancer samples, findings which may assist in determining the status of the disease and provide guidance to the appropriate action. Our NGS assay interrogates a significant portion of the genome leading to a more accurate and sensitive evaluation of the state of these cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Julie C. Laliberte, Michael K. Dame, Durga Attili, Bodrul Islam, Kevin Kim, Jessica Zhang, Erica L. Katz, Gina M. Newsome, Priya H. Dedhia, Adele Kruger, Tobias Mann, Tom Goodman, Jeffrey Buis, Dean E. Brenner, James Varani, Jason R. Spence, Justin A. Colacino, Jay Stoerker. Simultaneous measurement of global methylation and copy number alterations in human colorectal cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-093. doi:10.1158/1538-7445.AM2017-LB-093
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