Login to Access Video or Poster Abstract: MP48-09
Sources of Funding: The Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Ferdinand C. Valentine Fellowship Award from the New York Academy of Medicine, and the National Institute of Health.

Introduction

Integrin signaling plays an important role in cellular proliferation and migration via interactions with extracellular matrix proteins. Prior studies indicate that integrin signaling facilitates tumor invasion and metastasis, and there are several ongoing clinical trials using agents that modulate this pathway. We recently identified clonal enrichment in mutations in the integrin cell surface interactions pathways in advanced urothelial carcinoma. An ideal strategy for investigating integrin signaling is via 3D organoid culture, maintaining intercellular interactions that replicate the epithelial microenvironment. We hypothesize that pharmacologic integrin signaling modulation will impair organoid growth in bladder cancer cells and demonstrate a therapeutic utility for this approach.

Methods

RT4 human bladder cancer cell line was used as well as a second cell line established from a patient-derived bladder cancer sample (PM748). Cells were grown in 3D organoid culture as previously described. For in vitro integrin modulation, defactinib, an orally-bioavailable selective inhibitor of focal adhesion kinase (FAK, a convergent and conserved enzyme activated by integrin ligand binding), was used. SDS-PAGE and immunoblotting were performed to show in vitro FAK inhibition. Single cell suspensions and organoids were plated in the presence of various concentrations of defactinib to determine the impact on organoid formation and regression.

Results

Defactinib caused a dose-dependent decrease in autophosphorylation of FAK for both cell lines, demonstrating effective FAK inhibition. 3D culture of single cells with defactinib produced a dose-dependent decrease in organoid size after 96 hours (mean size for DMSO only, 100nM, 1uM, and 10uM were 128um, 75um, 48um, and 26um, respectively; p<0.0001 versus DMSO for all dilutions). Established organoids showed a dose-dependent regression in size after 72 hours of defactinib exposure (mean size for DMSO, 100nM, 1uM, and 10uM were 225um, 96um, 70um, and 34um, respectively; p<0.0001 versus DMSO). Experiments utilizing Crispr-Cas9-mediated FAK knock-out as well as in vivo studies with FAK inhibitors in xenograft models are currently underway.

Conclusions

Integrin modulation via FAK inhibition with defactinib causes both inhibition of organoid formation as well as regression of formed organoids, and the effects are seen at concentrations well below the cytotoxic range for the drug. This study suggests a utility for these agents in bladder cancer treatment.

Funding

The Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Ferdinand C. Valentine Fellowship Award from the New York Academy of Medicine, and the National Institute of Health.