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Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

ABSTRACT: We have developed a bladder cancer-on-a-chip model which supports the 3D growth of cells and can be used to assess and quantify bladder cancer cell invasiveness in a physiologically appropriate environment. Three bladder cancer cell lines (T24, J82, and RT4) were resuspended in 50% Matrigel^® and grown within a multi-channel organ-on-a-chip system. The ability of live cells to invade across into an adjacent 50% Matrigel^®-only channel was assessed over a 2-day period. Cell lines isolated from patients with high-grade bladder cancer (T24 and J82) invaded across into the Matrigel^®-only channel at a much higher frequency compared to cells isolated from a patient with low-grade cancer (RT4) (p < 0.001). The T24 and J82 cells also invaded further distances into the Matrigel^®-only channel compared to the RT4 cells (p < 0.001). The cell phenotype within the model was maintained as assessed by cell morphology and immunohistochemical analysis of E-cadherin. Treatment with ATN-161, an α5β1 integrin inhibitor and well-known migrastatic drug, caused a dose-dependent decrease in the invasiveness of the J82 cells (p < 0.01). The combined data demonstrate that our bladder cancer-on-a-chip model supports the retention of the bladder cancer cell phenotype and can be used to reproducibly assess and quantify the invasiveness of live bladder cancer cells.

SUBMITTER: Ewell DJ

PROVIDER: S-EPMC11311651 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

Ewell Desiree J DJ Vue Nita N Moinuddin Sakib M SM Sarkar Tanoy T Ahsan Fakhrul F Vinall Ruth L RL

Cancers 20240726 15

We have developed a bladder cancer-on-a-chip model which supports the 3D growth of cells and can be used to assess and quantify bladder cancer cell invasiveness in a physiologically appropriate environment. Three bladder cancer cell lines (T24, J82, and RT4) were resuspended in 50% Matrigel<sup>®</sup> and grown within a multi-channel organ-on-a-chip system. The ability of live cells to invade across into an adjacent 50% Matrigel<sup>®</sup>-only channel was assessed over a 2-day period. Cell li ...[more]

PMID: 39123388

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Project description:BackgroundBladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways.MethodsRT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly.ResultsHerein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a 'high E2F1/APLF/DCLRE1C' signature, which was found to be particularly favorable for BC patient survival.ConclusionSuppression of the 'out-of-context' activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.

Dataset Information

Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

Publications

Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

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