ABSTRACT
The intestine is maintained by stem cells, marked by LGR5 expression, located at the base of crypts. Genetically engineered mouse models have provided information about marker genes and stem cell pathways. 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 for LGR5 expression by immunohistochemistry. LGR5-positive cells were isolated from 4 adenoma organoid lines and analyzed by RNA-sequencing. LGR5 expression in epithelium and stroma was associated with tumor stage. Integrating functional experiments with RNA-seq data from LGR5-positive adenoma organoid cells and normal colon, we associated expression of CRC-specific genes, including DKK4, with LGR5 expression. This system can be used to study LGR5-expressing cells in human tissue homeostasis and carcinogenesis.
Footnotes
Abbreviations DKK4, dickkopf WNT signaling pathway inhibitor 4; FGF20, fibroblast growth factor 20; FFPE, formalin fixed paraffin embedded; ISC, intestinal stem cell; ISH, in situ hybridization; LGR5, leucine-rich repeat-containing g-protein coupled receptor 5; MACS, magnetic-activated cell sorting; OLFM4, olfactomedin 4; TCGA, The Cancer Genome Atlas
Funding This study was supported by University of Michigan Comprehensive Cancer Center (M.S.W.) and UMCCC Fund for Discovery Pilot Project Award (J.A.C.) P30CA046592; MCubed (J.A.C.), a seed funding program at the University of Michigan; NIDDK 5P30DK034933 [The University of Michigan Center for Gastrointestinal Research (UMCGR)] and NIH NIAID U19AI116482 (J.R.S.); NCI CA148828 (Y.M.S.); NIH 5R01GM068848 and NIH P50CA130810 (GI SPORE, D.E.B., PI), the Kutsche Family Endowed Chair in the Department of Internal Medicine and the Geriatrics Research Education and Clinical Center at the Ann Arbor VA Medical Center (D.E.B.); NCI 3P30CA046592-26-S3 (M.S.W.); NIH R21 CA181855- 01/02/03A1 and NCI R21CA201782-01(J.V.); American College of Surgeons, T32HD007505 (P.H.D.); X.X. was supported by the Research Scholar Award from American Gastroenterological Association; and A.M. was supported by the NIH Cellular and Molecular Biology Training Grant T32GM007315; the University of Washington Laboratory of Developmental Biology was supported by NIH 5R24HD000836 (Ian Glass, PI) from the Eunice Kennedy Shriver National Institute of Child Health & Human Development.
Disclosures Olaf Hardt, David Agorku, and Andreas Bosio are full-time employees of Miltenyi Biotec GmbH. Julie Laliberte and Jay Stoerker are full-time employees of Progenity, Inc. The remaining co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.