Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

Abstract

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

Functional Optical Imaging of Primary Human Tumor Organoids: Development of a Personalized Drug Screen

“…Organoids retain the organ structure, morphology, stromal composition, genetic mutations, and heterogeneity of the original tumor (5,(7)(8)(9)(10). When organoids are transplanted into mice, they form carcinomas that resemble the histology of the tumor of origin (5,7,10).…”

“…Organoids are amenable to genetic, biochemical, and imaging assays and have been used to study many aspects of cancer, including carcinogenesis, cancer invasion, drug response, and drug resistance mechanisms. Genetic manipulation of organoids allows study of genetic mutations that drive or suppress cancer progression (7,8,11). Furthermore, studies of organoids have revealed characteristics and mechanisms of tumor-extracellular matrix interactions and cell invasion (12)(13)(14)(15).…”

“…Optical imaging technologies used to study organoids include bright-field microscopy, light microscopy, phase microscopy, fluorescence microscopy, confocal microscopy, time-lapse microscopy, multiphoton fluorescence imaging, fluorescence lifetime imaging, and optical coherence tomography (OCT) (8,12,(18)(19)(20)29,30). In wide-field or whole-field microscopy, an entire sample or field of view is illuminated by a light source, and images are captured by a camera.…”

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“…Approximately 30-50 mg of minced tissue was mixed with 400 µL of ice-cold Matrigel by pipetting using a 2.0 ml pipette and transfer to the cell culture insert. The tissue was allowed to solidify for 10 minutes at 37 o C. The outer dish was subsequently filled to cover the acellular layer with WENRAS media [14,30] [15]. Tumoroid cultures were prepared by washing the tissue fragments 6-8 times in 10 mL of ice-cold PBS allowing the tissue to settle after each wash and removing the supernatant.…”

The myeloma stem cell concept, revisited: from phenomenology to operational terms

“…[84][85][86][87] Bmi-1, Notch, Hedgehog, and Wnt, which were initially identified based on their roles in tumor formation, have been shown to be involved in the regulation of self-renewal in normal stem cells in many tissues. [88][89][90] Since myeloma cells are influenced by the host, the microenvironment may play a key role in the initiation of myeloma and associated phenotypic changes-a phenomenon called "plasticity", defined as altered cellular phenotype and function during deregulated differentiation.…”