Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation

초록

Background: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. Methods: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vec-tors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. Results: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) stain-ing, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. Conclusions: This study developed a radi-ation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

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