Abstract 322: Establishment and genomic characterization of enteroid cultures from human colonic adenomas and adenocarcinomas
Authors: Dame Michael K. , McClintock Shannon D. , Attili Durga , Simon Becky , Copley Kelly , Finkbeiner Stacy , Altheim Christopher , Spence Jason , Appelman Henry , Turgeon D Kim , Samuelson Linda C. , Brenner Dean E. , Varani James 
Published: August 2015

Abstract
Introduction Characterized enteroid cultures of human colon cancer can more precisely model the diversity of colonic neoplasia for the study of cancer initiation, progression and potentially prevention. Using tissue from colon resections and endoscopic biopsies, we have successfully isolated and cultured 14 colorectal adenomas and 2 adenocarcinomas to date. We have maintained these enteroid cultures for up to 2 years and established a working cryorepository. Specific epithelial cell lineage markers and the stem cell marker Lgr5 can be detected throughout the culture period.
Methods Enteroid cultures have been initiated and maintained in a serum-free medium containing EGF and pituitary extract. However, approximately half of all neoplasms do not establish in this reduced medium. In contrast, most neoplasms develop and expand in an enriched culture medium containing serum, Wnt, R-spondin, Noggin, and EGF. We have also created a mouse xenograft from an adenoma expanded in the reduced media; this graft was then successfully reintroduced into culture. Using whole exome sequencing, we are investigating how the genetic background of individual patients contributes to 1) variability in the establishment and expansion of enteroid cultures 2) tumor heterogeneity in neoplasms and xenografts, and 3) the stability of genomic signatures in enteroids over time in culture.
Results Twenty-two damaging somatic variants identified in a single colon tumor were preserved in enteroid culture after 2 months in reduced medium. Variants included a frameshift mutation in APC and missense mutations in KRAS and TP53. The allele frequency of most variants increased in enteroid culture, suggesting that cells lacking these mutations failed to propagate (including stromal/immune cells), or cells carrying these mutations expanded at a faster rate. Ten mutations not present in the original tumor were acquired over time in enteroid culture. These mutations included a missense variant of TRPS1, a putative prognosticator of colon cancer. The mutations acquired in enteroid culture may reflect genetic instability in the source neoplastic tissue, or the emergence of subpopulations that were below level of detection in the source tissue. Three mutations were present in the tumor and lost in enteroid culture; this presumably reflects the loss of a subset of cells expressing these variants.
Conclusion Changes in allele frequencies suggest that neoplasms are heterogeneous, with shifting cell populations that are differentially affected by culture conditions. This heterogeneity can be further interrogated by comparing allele frequencies in the original tumor with those in enteroids established in reduced or enriched media. This platform can provide further understanding of genetic determinants that underlie the risk for colorectal cancer, as well as strategic insights into the enteroid model as a sophisticated system for the study of tumor biology.
Citation Format: Michael K. Dame, Shannon D. McClintock, Durga Attili, Becky Simon, Kelly Copley, Stacy Finkbeiner, Christopher Altheim, Jason Spence, Henry Appelman, D Kim Turgeon, Linda C. Samuelson, Dean E. Brenner, James Varani. Establishment and genomic characterization of enteroid cultures from human colonic adenomas and adenocarcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2015-322