Abstract 2230: Wnt signaling in prostate cancer stem like cells
Authors: Davda Reena , Thrasivoulou Christopher , Masters John , Ahmed Aamir 
Published: August 2015

Abstract
Introduction: Cancer stem like cells (CSLCs) are characterized by features of tumorigenicity, serial passages and the expression of specific stem cell markers. CSLCs possess characteristics of adult stem cells, including self-renewal. CSLCs can be isolated from cancer cell lines, including prostate, and form morphologically distinct colonies (e.g. holo, mero and para clones). Colony formation has been used as a surrogate biological assay to identify and characterize CSLCs and holo and meroclones from prostate cell lines have been proposed to contain CSLCs. Wnt signaling through the action of two major intracellular transducers, calcium ([Ca2+]i) and the transcription factor ß-catenin, is a key regulator of stem cell self-renewal. In whole populations of cancer cell lines (e.g. PC3, MCF7), Wnt signaling operates via a convergent rather than distinct canonical (Wnt/ß-catenin) and non-canonical (Wnt/Ca2+) pathways. Little is known about the characteristics of Wnt signaling (e.g. Wnt induced [Ca2+]i release or ß-catenin expression) in prostate CSLCs.
The aim of this study was to characterize the clonal expansion of PC3 cell line and to investigate Wnt signaling in these colonies.
Methods: PC3 cells, in a single cell suspension, were plated at low density (6 cells/cm2) in 6 well plates (Nunc) and imaged over 10 days with Incucyte (Essen Bioscience) and analysed using Image J software. Some colonies were loaded with Fluo4 and FuraRed dyes (Invitrogen) and used in a live [Ca2+]i imaging assay ± Wnt 5A or 9B (200ng/ml) with an Olympus FV1000 confocal system; the same colonies were then used in immunostaining for the ß-catenin protein (Abcam 22656) followed by confocal microscopy. Data is presented as means ± SE; Mann Whitney U test was used for statistical analysis.
Results: IncuCyte imaging captured colony formation from single cells plated and two morphologically distinct types of PC3 colonies were observed at day 10, termed holoclone/meroclone (h/m clone) and paraclone (p clone). Mean colony diameter of those categorized as h/m clone was 838 ± 22μm (n = 102) and 1191 ± 28μm for p clone (n = 96); mean cell density (%) showed a significant difference (p&lt;0.01) in h/m compared to p clones: 27.2 ± 3.2(n = 8) vs 4.7 ± 0.4 (n = 5), respectively. Cells in h/m and p clones showed [Ca2+]i release in response to both Wnt 5A and 9B; Cells in h/m clones demonstrated a significantly (p&lt;0.05) longer dwell time for [Ca2+]i release in response to Wnt 9B than 5A, with mean dwell times (seconds) of 45.3 ± 2.6 (n = 52) and 38.0 ± 4.3 (n = 52), respectively.ß-catenin was expressed in both h/m and p clones from PC3 cell line.
Conclusions: Distinct colony types can be observed in cultures of PC3 prostate cancer cell line. These colonies have quantifiable characteristics in addition of observable morphologies. A majority of cells within different colonies show Wnt induced [Ca2+]i release and ß-catenin expression. A convergent model of Wnt signaling is likely to operate in prostate CSLCs.
Citation Format: Reena Davda, Christopher Thrasivoulou, John Masters, Aamir Ahmed. Wnt signaling in prostate cancer stem like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2015-2230