Abstract
Here, we report a model system using in vitro 7,12-dimethylbenz[a]anthracene (DMBA; 0.6 μM)-treated mammary tissue-derived organoids generated from heterozygous BALB/c-Trp53 knockout mice to induce tumors after injection into the nude mouse subcutis. In parallel, a single oral dose of DMBA (50 mg/kg bodyweight) to the same murine strain induced mammary adenocarcinomas, characterized by biphasic structures differentiated into luminal and myoepithelial lineages and frequent Hras mutations at codon 61. In the present study, the genetic and histological characteristics of DMBA-induced tumors in the organoid-based model were evaluated to validate its similarities to the in vivo study. The organoid-derived tumors were low-grade adenocarcinomas composed of luminal and basal/myoepithelial cells. When the organoid-derived carcinomas were passaged to other nude mice, they partly progressed to squamous cell carcinomas (SCCs). Whole exome sequencing revealed no mutations at Hras codon 61 in the organoid-derived tumors. However, various mutations were detected in other genes such as Tusc3 and Tgfbr2, which have been reported as cancer-associated or homeostatic squamous cell genes. The most common mutational pattern observed in these genes were the G:C to T:A transversions and G:C to A:T transitions, which are not typical of the mutations caused by DMBA treatment. In conclusion, DMBA exhibited carcinogenicity in the both the ex vivo and in vivo mammary carcinogenesis models, albeit with distinct histological and genetical alterations. Further studies are needed to clarify whether organoid-based carcinogenesis models generated following chemical treatment in vitro could be applied to the clarification of the novel mode of action of chemical carcinogenesis.
Highlights
A method for the long-term expansion of three dimensional (3D) organoids derived from healthy intestinal tissue was first introduced by Sato and Clevers et al (Sato et al, 2009)
To evaluate the genetic alterations associated with DMBAinduced carcinogenesis in mammary tissue-derived organoids after injection into the subcutis of nude mice, whole exome sequencing analysis (WES) analysis was conducted on the 0.6 μM DMBA-treated organoids and DMBAtreated organoid-derived subcutaneous adenocarcinoma tissue in comparison with the DMBA-untreated organoid negative control
single nucleotide variants (SNVs) shared between the DMBA-induced organoid-derived adenocarcinomas and 0.6 μM DMBA-treated organoids were limited to 10 genes, including Tnrc6b and Vps13d, which were selected by the Integrative Genomics Viewer (IGV) (Figure 1B)
Summary
A method for the long-term expansion of three dimensional (3D) organoids derived from healthy intestinal tissue was first introduced by Sato and Clevers et al (Sato et al, 2009). Liver organoids with bile transport function were developed from pluripotent stem cell lines, in which the hepatocyte transcriptomic state relative to primary hepatocytes was confirmed (Shinozawa et al, 2021). These in vitro organoid systems should be more readily applied to the development of mechanism-based toxicological studies. To this end, we recently reported an organoid-based chemical carcinogenesis model employing organoids derived from healthy murine tissue (Naruse et al, 2020). The four chemicals exhibited tumorigenicity or carcinogenic histopathological characteristics accompanied by the activation of oncogenic kinases, echoing previous reports from corresponding animal studies
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