1276 A NOVEL PRECISION-ENGINEERED MEMBRANE MICROFILTRATION DEVICE FOR CAPTURE AND CHARACTERIZATION OF BLADDER CANCER CELLS IN URINE

Bladder tumors represent a special therapeutic challenge as they have a high recurrence rate requiring repeated interventions and may progress to invasive or metastatic disease. Exosomes carry proteins implicated in bladder cancer progression and have been implicated in bladder cancer cell survival. Here, we characterized exosome uptake and internalization by human bladder cancer cells using Amnis ImageStreamX, an image cytometer. Exosomes were isolated by ultracentrifugation from bladder cancer culture conditioned supernatant, labeled with PKH-26, and analyzed on the ImageStreamX with an internal standard added to determine concentration. Exosomes were cocultured with bladder cancer cells and analyzed for internalization. Using the IDEAS software, we determined exosome uptake based on the number of PKH-26+ spots and overall PKH-26 fluorescence intensity. Using unlabeled beads of a known concentration and size, we were able to determine concentrations of exosomes isolated from bladder cancer cells. We measured exosome uptake by recipient bladder cancer cells, and we demonstrated that uptake is dose and time dependent. Finally, we found that uptake is active and specific, which can be partially blocked by heparin treatment. The characterization of cellular uptake and internalization by bladder cancer cells may shed light on the role of exosomes on bladder cancer recurrence and progression.

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Novel In Vitro Assays for the Characterization of EMT in Tumourigenesis

Analytical Cellular Pathology ◽

10.1155/2010/985981 ◽

2010 ◽

Vol 32 (1-2) ◽

pp. 67-76

Author(s):

Vincent Koo ◽

Amgad El Mekabaty ◽

Peter Hamilton ◽

Perry Maxwell ◽

Osama Sharaf ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Cells ◽

In Vitro Assays ◽

The Novel ◽

Mesenchymal Transition ◽

Rat Bladder ◽

Bladder Cancer Cells ◽

Bonferroni Test ◽

E Cadherin

Background: Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-β1 treatment of AY-27 rat bladder cancer cells acted as a model of EMT in tumourigenesis.Methods: AY-27 rat bladder cancer cells incubated with 3 ng/ml TGF-β1 or control media for 24 or 48 h were assessed using novel and traditional assays. The Spindle Index, a novel measure of spindle phenotype, was derived from the ratio of maximum length to maximum width of cells. The area covered by cells which migrated from a fixed coverslip towards supplemented agarose was measured in a novel chemoattractant assay. Motility, migration and immunoreactivity for E-cadherin, Vimentin and cytokeratin were assessed.Results: TGF-β1 treated cells had increased “spindle” phenotype together with decreased E-cadherin, decreased Cytokeratin-18 and increased Vimentin immunoreactivity. After 48 h, the mean Spindle Index of TGF-β1 treated cells was significantly higher than Mock (p=0.02, Bonferroni test) and there were significant differences in migration across treatment groups measured using the novel chemoattractant assay (p=0.02, Chi-square). TGF-β1 significantly increased matrigel invasion.Conclusions: The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.

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ARID1A-mutant and deficient bladder cancer is sensitive to EZH2 pharmacologic inhibition

10.1101/2021.01.12.426383 ◽

2021 ◽

Author(s):

James E. Ferguson ◽

Hasibur Rehman ◽

Darshan S. Chandrashekar ◽

Balabhadrapatruni V. S. K. Chakravarthi ◽

Saroj Nepal ◽

...

Keyword(s):

Bladder Cancer ◽

Cancer Cells ◽

Wild Type ◽

Driver Genes ◽

Specific Subset ◽

Bladder Cancer Cells ◽

Metastatic Urothelial Carcinoma ◽

Carcinoma Of The Bladder

AbstractMetastatic urothelial carcinoma of the bladder is generally incurable by current systemic therapy. Molecular characterization of bladder cancer (BLCa) has revealed multiple candidate driver genes for BLCa tumorigenesis. Epigenetic/chromatin modifiers have been shown to be frequently mutated in BLCa, with ARID1A mutations highly prevalent in nearly 20% of early and late stage tumors. EZH2 is a histone methyltransferase that acts as an oncogene. The data herein show that ARID1A deficient tumors, but not ARID1A wild-type tumors are sensitive to EZH2 inhibition. Specifically, EZH2 inhibitor-treated ARID1A deficient bladder cancer cells show significantly reduced cell viability, colony formation, and in vivo tumor growth relative to ARID1A-wild type bladder cancer cells. Thus, our study suggests that a specific subset of bladder cancer patients with ARID1A mutations can be therapeutically treated with pharmacologic inhibitors targeting EZH2.

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