Abstract 21: A novel bladder cancer syngeneic mouse model using gene-edited organoids derived from bladder urothelium

Abstract

Abstract In recent years, immuno-checkpoint inhibitors have expanded the range of treatment options in advanced bladder cancer, but there are still many issues to be overcome, such as drug resistance, effect of combination therapy and prediction of adverse events. To address these issues, there is an urgent need for useful animal models with normal immune system. About half of the human muscle invasive bladder cancers (MIBCs) show p53 mutations in which over 60% are missense mutations. Additionally, the downregulation of tumor suppressor gene PTEN is known in human MIBC. We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have been working on establishing MIBC mouse model using genetically engineered mouse (GEM) in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system. We have also used CRISPR/Cas9 gene editing technique and 3D organoid culture system. We generated organoids from the GEM bladder urothelium with Krt5CreERT2/+; LSL-Cas9; LSL-Trp53R172H/+. For further gene editing, we infected adeno-associated virus with sgRNA to the organoids. The organoid with Trp53R172H/+ and Pten knock-out showed tumorigenicity in immunodeficient mice. The organoid-derived tumors are histologically similar with the basal-squamous subtype of human bladder cancer. Moreover, these tumors exhibited wild type (WT) Trp53 loss of heterozygosity in genomic level. Similarly, we confirmed tumorigenicity in immunodeficient mice with organoids from Trp53flox/flox; Ptenflox/flox and Trp53R172H/flox; Ptenflox/flox mice, but not from Trp53R172H/flox;Pten+/+ mice or Trp53R172H/+; Ptenflox/flox mice, suggesting that both loss of WT Trp53 and Pten loss are required for tumor formation in this setting. RNA sequencing showed Pten loss is associated with up-regulation of metabolism related gene sets and loss of WT Trp53 is associated with up-regulation of proliferation related gene sets. Furthermore, Trp53R172H/flox; Ptenflox/flox organoids showed higher engraftment rate in immunocompetent mice with less infiltration of CD8+ cells compared with Trp53flox/flox; Ptenflox/flox organoids, suggesting that gain of function of mutant Trp53 may affect immune microenvironment. Our data illustrates the usefulness of clinically relevant syngeneic bladder cancer mouse model from GEM-derived organoids for the research of tumor immunity. Citation Format: Akihiro Hamada, Yuki Kita, Takeshi Sano, Ryosuke Ikeuchi, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. A novel bladder cancer syngeneic mouse model using gene-edited organoids derived from bladder urothelium [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 21.