Abstract LB-266: Identification and enrichment of tumor-initiating cells from human prostate cancers using lentiviral vectors that can detect activated Wnt, Notch, or Hedgehog signaling
Authors: Pudi Renuka , Pinto Peter A. , Vogel Jonathan C. 
Published: February 2011

Abstract
In the cancer stem cell hypothesis, tumors are initiated and maintained by a small subset of tumor initiating cells (TIC) that possess stem cell properties of self-renewal and long-term repopulation. However, TIC have not been well characterized in human prostate cancer with previous reports describing enrichment of TIC in prostate cancer based on expression of CD133 and a2b1 integrin cell surface markers. A significant problem has been the relative lack of cell surface markers that could be used to define subsets of prostate cancer cells. Rather than rely on cell surface markers, we wanted to identify subsets based on the presence or absence of the activated Wnt, Notch, and Sonic Hedgehog (Hh) signaling pathways that have been associated with stem cell maintenance and self-renewal, and also implicated in several cancers. We hypothesized that these signaling pathways may be more highly activated in TIC and may allow for the identification and enrichment of human prostate cancer TIC. To detect these signaling pathways, we generated lentiviral vectors with reporters downstream of specific enhancer-promoter sequences that can detect activated Wnt, Notch, or Hh signaling. Since established in vivo models that can recapitulate human prostate cancer from freshly isolated cancer cells do not exist, we developed in vitro clonogenic sphere formation assays, both in a 3-dimensional matrix and on non-adherent plates, to detect prostate cancer TIC. We first established the prostate cancer cells origin of the cells by demonstrating their invasive capacity in a matrigel cell invasion assay; their lack of CD10 and CD13 expression; and their ability to form non-adherent spheres when compared to normal prostate cells. The transduction efficiency of the prostate cancer cells by the lentiviral vectors was approximately 70%, with activated Wnt, Notch, and Hh signaling detected in 39%, 61%, and 34% of the transduced cells, respectively. We found that human prostate cancer cells with activated Wnt signaling produced approximately 10-fold more clonogenic spheres than Wnt-negative cells indicating enrichment of prostate cancer TIC within the Wnt-positive fraction. In contrast, prostate cancer cells lacking activated Notch signaling formed 2-fold more clonogenic spheres than cells with activated Notch signaling. The presence or absence of activated Hh signaling did not affect clonogenic sphere formation. When prostate cancer cells were transduced with both Wnt and Notch lentiviral vectors, the Wnt-postive/Notch-negative populations gave rise to 6-fold more clonogenic spheres than the Wnt-positive/Notch-positive cells. Finally, CD133+ and CD117+ prostate cancer cells were only detected within the Wnt-positive and Notch-negative populations. These results indicate that prostate cancer TIC are enriched within prostate cancer cells that have activated Wnt signaling but lack activated Notch signaling and suggest that prostate cancer subsets of interest can be identified based on the presence of activated signaling pathways.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-266.