Abstract 1928: Reporter construct for functional and real-time evaluation of cytokeratin 14+ bladder cancer stem cells

Abstract

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