Development of CRISPR/Cas9 in vivo bladder cancer models

Author(s): Stefanson, Ofir | Advisor(s): Wang, Zhu | Abstract: Bladder cancer prevalence is high, and patients diagnosed with this malignancy incur a high economic burden and a poor-quality lifestyle. The majority of bladder cancers are urothelial carcinomas, with two subtypes: papillary non-invasive and muscle-invasive bladder cancer. The muscle-invasive subtype is associated with a poor prognosis and a high mutation frequency. Nevertheless, there are no efficient in vivo bladder cancer models in the field to study disease initiation and progression. Thus far, most in vivo bladder cancer models rely on the Cre-LoxP system. However, this technique is expensive and time-consuming. Furthermore, it depends on available mice with floxed alleles for target genes. Recently, CRISPR/Cas9 has been used to study cancer and overcomes the hurdles of Cre-LoxP models. The objective of this work is to determine if CRISPR/Cas9 can be coopted for bladder cancer research in vivo. As-proof-of concept CRISPR/Cas9 was used to recapitulate a Cre-LoxP muscle-invasive bladder mouse model produced by knockout of tumor suppressors PTEN and TP53. Single guide RNAs targeting TP53 and PTEN were delivered into the bladder urothelium of Cas9 expressing mice by a novel electroporation approach. Histological and pathological characterization of bladders indicated tumor development in 2 mice, with a tumor penetrance of 33%. Tumors phenotypically resembled hyperplasia and papillary carcinoma, with an increase of CK5 positive basal layer. Papillary tumors exhibited an increase in cell proliferation compared to controls. Overall, optimizations are required in order to use the CRISPR/Cas9 technique for in vivo bladder cancer models.