Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells
There has been increasing awareness of the importance of three-dimensional culture of cancer cells. Tumor cells growing as multicellular spheroids (organoids) are believed to more closely mimic solid tumors in situ. Meanwhile, Wnt/β-catenin pathway was reported to be upregulated in human bladder cancer specimens. However, no clear evidence has been reported that the pathway is directly involved in proliferation of bladder cancer cells. In this study, we assessed the involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells using organoid culture.
Organoids from bladder cancer cell lines RT4 (luminal phenotype) and 5637 (basal phenotype) were generated by an aggregation method. A partial-digestion method was applied to prepare organoids directly from patient bladder cancer specimens. Wnt/β-catenin pathway was activated by using a small molecule CHIR99021 (GSK3 inhibitor, Wnt activator) and inhibited by siRNA against β-catenin. Activation of Wnt/β-catenin pathway was confirmed by upregulated AXIN2 mRNA expression and translocation of β-catenin into nucleus. Proliferation of cancer cells were evaluated by growth assay under microscope and ATP viability assay. Differentiation status of organoids over growth was characterized by qRT-PCR and western blot.
CHIR99021 inhibited proliferation of cell lines in conventional monolayer adherent culture, but promoted proliferation in three-dimensional organoid culture with activation of Wnt/β-catenin pathway. Enhanced proliferation of cancer cells with activation of Wnt/?-catenin pathway by CHIR99021 was also observed in patient-derived organoids. When β-catenin was knockdowned in cell lines, the growth of organoids was significantly suppressed. Cytokeratin 20, a terminal differentiation marker, was less expressed over CHIR99021-enhanced cell proliferation.
We showed for the first time that Wnt/β-catenin pathway was directly involved in proliferation of bladder cancer cells, suggesting that Wnt/β-catenin pathway may be a potential target for the treatment of a subset of bladder cancer. The above finding could not be observed when the same cells were grown in conventional two-dimensional adherent cultures, providing a concrete example of why organoid culture is important for cancer research.
Funding: The Greenberg Bladder Cancer Institute Research Grant and the Urology Care Foundation