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Impact of spheroid culture on molecular and functional characteristics of bladder cancer cell lines.

ABSTRACT: The three-dimensional cell culture system is an increasingly important technique for discovering new biological aspects of cancer cells. In the present study it was demonstrated that bladder cancer cell lines, RT4 and 5637, spontaneously formed round multicellular spheroids (MCSs) in suspension by the aggregation method. MCSs consisted of cells differentially expressing luminal/basal markers. Western blotting showed that PPAR? and forkhead box A1 (FOXA1)of luminal markers were expressed to a lesser extent in MCSs than in parental cells grown in two-dimensional (2D) adherent culture. Cells in MCSs in suspension proliferated less efficiently, and were more resistant to cisplatin (CDDP) and gemcitabine than parental cells grown in 2D culture. Culturing cell lines as MCSs in suspension is a notable platform to decipher alternative biological aspects of bladder cancer cells, which could not be unraveled by the conventional 2D adherent culture.

SUBMITTER: Yoshida T

PROVIDER: S-EPMC6781760 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Impact of spheroid culture on molecular and functional characteristics of bladder cancer cell lines.

Yoshida Takahiro T Sopko Nikolai A NA Kates Max M Liu Xiaopu X Joice Gregory G Mcconkey David J DJ Bivalacqua Trinity J TJ

Oncology letters 20190829 5

The three-dimensional cell culture system is an increasingly important technique for discovering new biological aspects of cancer cells. In the present study it was demonstrated that bladder cancer cell lines, RT4 and 5637, spontaneously formed round multicellular spheroids (MCSs) in suspension by the aggregation method. MCSs consisted of cells differentially expressing luminal/basal markers. Western blotting showed that PPARγ and forkhead box A1 (FOXA1)of luminal markers were expressed to a les ...[more]

PMID: 31612003

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Project description:We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

Dataset Information

Impact of spheroid culture on molecular and functional characteristics of bladder cancer cell lines.

Publications

Impact of spheroid culture on molecular and functional characteristics of bladder cancer cell lines.

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