Takahiro Yoshida1, Max Kates2, Nikolai A Sopko2, Xiaopu Liu2, Alok K Singh3, William R Bishai3, Gregory Joice2, David J McConkey4, Trinity J Bivalacqua4. 1. The James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD. Electronic address: yakinikugohan@gmail.com. 2. The James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD. 3. Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, MD. 4. The James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins School of Medicine, Baltimore, MD; The Johns Hopkins Greenberg Bladder Cancer Institute, Baltimore, MD.
Abstract
OBJECTIVE: We ex vivo cultured primary tumor cells from N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and established an immortalized cell line from them. MATERIALS AND METHODS: Bladder tumors in rats were induced by instillation of MNU into the murine bladder. Primary tumor cells were prepared by the cancer-tissue originated spheroid method. An immortalized cell line was established by co-culture with fibroblasts. The cultured tumor cells were molecularly and functionally characterized by quantitative real-time polymerase chain reaction, Western blot, growth assay, and transwell migration assay. RESULTS: Primary tumor cells were successfully prepared as multicellular spheroids from MNU-induced bladder tumors. The differentiation marker expression patterns observed in the original tumors were largely retained in the spheroids. We succeeded in establishing a cell line from the spheroids and named it T-MNU-1. Although basal markers (CK14 and CK5) were enriched in T-MNU-1 compared to the spheroids, T-MNU-1 expressed both luminal and basal markers. T-MNU-1 was able to migrate through a transwell. CONCLUSIONS: Tumor cells in MNU-induced bladder tumors were successfully cultured ex vivo as organoids, and an immortalized cell line was also established from them. The ex vivo models offer a platform that enables analysis of intrinsic characteristics of tumor cells excluding influence of microenvironment in MNU-induced bladder tumors.
OBJECTIVE: We ex vivo cultured primary tumor cells from N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and established an immortalized cell line from them. MATERIALS AND METHODS:Bladder tumors in rats were induced by instillation of MNU into the murine bladder. Primary tumor cells were prepared by the cancer-tissue originated spheroid method. An immortalized cell line was established by co-culture with fibroblasts. The cultured tumor cells were molecularly and functionally characterized by quantitative real-time polymerase chain reaction, Western blot, growth assay, and transwell migration assay. RESULTS:Primary tumor cells were successfully prepared as multicellular spheroids from MNU-induced bladder tumors. The differentiation marker expression patterns observed in the original tumors were largely retained in the spheroids. We succeeded in establishing a cell line from the spheroids and named it T-MNU-1. Although basal markers (CK14 and CK5) were enriched in T-MNU-1 compared to the spheroids, T-MNU-1 expressed both luminal and basal markers. T-MNU-1 was able to migrate through a transwell. CONCLUSIONS:Tumor cells in MNU-induced bladder tumors were successfully cultured ex vivo as organoids, and an immortalized cell line was also established from them. The ex vivo models offer a platform that enables analysis of intrinsic characteristics of tumor cells excluding influence of microenvironment in MNU-induced bladder tumors.