Login to Access Video or Poster Abstract: MP48-19
Sources of Funding: Johns Hopkins Greenberg Bladder Cancer Institute

Introduction

Introduction and Objectives: Human urothelial cancers can be classified into luminal or basal subtypes based on RNA and protein expression that have distinct clinical behaviors and responsiveness to chemotherapy. Therefore, it is essential to classify experimental animal models of bladder cancer based on molecular subtypes. In this study, we characterized N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and ex vivo-cultured spheroids derived from them to determine their similarities to the luminal and basal subtypes observed in human cancers.

Methods

Female Fisher 344 rats at age of 7 weeks received 4 intravesical doses of 1.5mg/kg MNU. By week 8, the MNU bladders displayed evidence of dysplasia and by week 16 small animal ultrasounds revealed papillary tumors with associated CIS, high grade non-invasive disease, or invasion into the lamina propria. In order to assess the subtype memberships of mature invasive tumors, rats were sacrificed at 30 weeks. Bladder tumors were evaluated by H&E staining and immunohistochemistry using antibodies against basal marker CK14 and luminal CK20. Ex-vivo culture of tumor cells was carried out using a spheroidal culture method. Partially digested tumor fragments were cultured in StemPro® medium. Viability of ex-vivo cultured cells was evaluated by Ki67 immunohistochemical staining and growth assay under microscope. Cultured cells were also tested by immnohistochestry using antibodies of CK14 and CK20.

Results

By about 30 weeks after exposure to MNU, rats developed 5-10-mm, protruding tumors in their bladders. Immunohistochemistry revealed enrichment of CK14-stained cells in CIS lesions and invasive tumors compared to normal urothelium, while the number of CK20-stained cells declined. Papillary tumors had a mixed staining pattern with both CK14 and CK20 positive staining. Partially digested fragments of tumors formed clear spheroids 24 hours after digestion. Immunohistochemistry revealed CK14-positive cells comprised 88% of spheroids and CK20-positive cells less than 5%.

Conclusions

The results of the present study demonstrate a pre-clinical model of urothelial cancer associated with progression of papillary non-muscle invasive bladder tumors with associated CIS to invasive cancers. CIS, invasive tumors, and ex-vivo cultured spheroids had the appearance of a basal subtype membership, while papillary tumors from the same model had staining patterns consistent with the urobasal/luminal tumor subtype.

Funding

Johns Hopkins Greenberg Bladder Cancer Institute