LAUSR

Colorectal cancer is a deadly disease with a 14% five-year survival rate once it metastasizes. The development of colorectal cancer involves a stepwise accumulation of genetic alterations in APC, KRAS, DCC, and p53 that is often described as a “Vogelgram,” named after the Vogelstein laboratory which laid the groundwork for our understanding of the molecular events leading to colorectal cancer. Organoids are a highly physiologically relevant laboratory tool that can provide valuable information regarding the tumor microenvironment. Here, we describe ex vivo establishment of APC fl/fl, CRE+, KRAS G12D +/-, P53 fl/fl colorectal organoids, which behave normally before tamoxifen treatment but acquire malignant behaviors after tamoxifen-induced loss of APC and p53. Colonic crypts from mice with colon-specific expression of APC fl/fl, CRE+, KRAS G12D +/-, P53 fl/fl were isolated and grown in Matrigel and WENRAS medium following the Organoid Core Facility protocol of the Legorreta Cancer Center. Western blot was used to measure levels of KRAS, KRAS G12D, APC, and p53 after 12 days of tamoxifen treatment and images were taken after 6 days of tamoxifen treatment. The organoid growth medium was collected after 3-4 days of incubation and run on the Luminex 200 platform to measure the levels of 50+ cytokines. Organoids were embedded in OCT and cryosectioned for IF staining with p53, p21, Ki67 and Lgr5 antibodies. IF stained organoid slides were imaged by confocal microscope at 40X magnification. Tamoxifen treatment (12 days) induces overexpression of KRAD G12D protein, reduces expression of APC, and abolishes p53 expression. At day 6, distinct morphological changes indicate malignant transformation. This transformation was accompanied by a trend toward decreased BAFF and CCL20. H&E staining revealed clearly visible intra-organoid structures and florescent labeling of Ki67, Lgr5, and p21 visualized by confocal microscope revealed distinct localization of these proteins within the organoid structures. Here, we describe Vogelgram-mimicking organoids that may be used as a valuable laboratory tool to study the processes involved in the stepwise transformation into colorectal cancer. We plan to use this validated system to evaluate drug sensitivity, cytokine secretion, and immune cell interactions along colorectal cancer progression. We also plan to characterize cell populations with single-cell RNA-seq, establish triangulated cultures, and conduct in vivo studies using this transgenic mouse model. Citation Format: Leiqing Zhang, Lindsey Carlsen, Lanlan Zhou, Liz Hernandez Borrero, Wafik S. El-Deiry. Establishment and characterization of Vogelgram-mimic colorectal cancer organoids as a laboratory tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 165.