Abstract 1928: Reporter construct for functional and real-time evaluation of cytokeratin 14+ bladder cancer stem cells
Authors: Ho Philip L. , Kurtova Antonina , Xiao Jing , Krasnow Ross , Lay Erica , Pazhanisamy Senthil , Lerner Seth P. , Chan Keith S. 
Published: May 2015

Abstract
Bladder cancer is the fifth most common malignancy in the US. We recently reported that bladder cancer stem cells isolated by cell surface markers (i.e. CD44/CD90/CD49f) express a higher level of the intermediate filament cytokeratin 14 (CK14). Importantly, patients with a higher fraction of CK14+ cancer cells correlated with worse survival outcomes. Therefore, this CK14+ cancer cell subpopulation warrants further functional evaluation and characterization.
In the current study, we report the generation and characterization of a lentiviral reporter construct that carries the gene promoter region of human KRT14 gene upstream to a red fluorescent protein (tdTomato; Tm). With this reporter stably integrated into the genome of bladder cancer cells, the viable CK14+ cancer cell subpopulation could be isolated by FACS and visualized by fluorescent microscopy. We first validated the reporter by demonstrating that Tm+ cancer cells indeed express a higher level of KRT14 mRNA and relatively lower levels of differentiated cell markers (i.e. KRT18 and UPK1B) by qPCR. Next, we verified both in vitro and in vivo that CK14+/Tm+ cancer cells have functional properties of cancer stem cells by demonstrating their enriched sphere-forming ability and proficient engraftment as xenograft tumors in immunocompromised mice. Since neoadjuvant chemotherapy is a well-established treatment approach in patients with high-risk bladder cancer, we investigated CK14+ and CK14- cancer cells' response to the cytotoxic chemotherapy, gemcitabine/cisplatin (GC). FACS-purified CK14+/Tm+ cancer cells were more resistant to GC chemotherapy in vitro than CK14-/Tm- cancer cells. These findings were confirmed with in vivo studies using both patient-derived xenografts and immortalized bladder cancer xenografts. GC treated xenografts demonstrated a greater expansion of CK14+ cancer cells than vehicle treated controls. Additionally, we obtained a panel of human bladder cancer specimens from patients before and after neoadjuvant GC chemotherapy (n=15) and evaluated the CK14 status. An expansion or persistence of an infiltrating pattern of CK14 in post-neoadjuvant GC chemotherapy tumor specimens was associated with worse overall survival.
Collectively, these findings verified the unique intrinsic biological properties of CK14+/Tm+ bladder cancer cells and their response to GC chemotherapy. Ongoing experiments using fluorescence live imaging will evaluate Tm+/- bladder cancer cells' response to chemotherapy. The capacity to observe this CK14+/Tm+ subpopulation and their trace response to cytotoxic chemotherapy in these studies will open up new avenues to study the mechanisms of chemoresistance.
Citation Format: Philip L. Ho, Antonina Kurtova, Jing Xiao, Ross Krasnow, Erica Lay, Senthil Pazhanisamy, Seth P. Lerner, Keith S. Chan. Reporter construct for functional and real-time evaluation of cytokeratin 14+ bladder cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1928. doi:10.1158/1538-7445.AM2014-1928