Abstract 1392: ALDH1A1 is the best marker by a composite approach for cancer stem cells in ovarian cancer whose integrity is controlled by the hedgehog pathway
Authors: Rohit P. Nagare, S Sneha, S Krishnapriya, Trivadi S. Ganesan
Journal: Cancer Research (2015)

Abstract Cancer Stem Cells (CSCs) express distinct surface markers and have unique functional properties that distinguish them from the rest of the cells in a tumour. There have been variability in the identity of CSC markers in serous adenocarcinoma of the ovary. We have taken a composite approach for evaluating surface markers together with functional assays in primary tumours (n = 20). Expression of CSC markers were evaluated by flow cytometry, immunofluorescence (IF) and immunohistochemistry (IHC) in cell lines (n = 8), primary malignant cells (PMC, n = 20) and normal ovary (n = 2). The ALDEFLUOR assay was used to identify CSCs individually (n = 20) and together with surface markers (n = 6). PMC (n = 5) were grown as spheroids to evaluate expression of markers and ALDEFLUOR assay (n = 3). The role of signaling by Notch, Wnt and Hedgehog pathways were evaluated to ascertain maintenance of CSCs in the spheroid model (n = 3) using specific small molecule inhibitors. PMC (n = 20) were grown in the presence or absence of inhibitors for 2 weeks and CSC markers evaluated to ascertain specificity of effect along with motility assays. We identified 3 novel surface markers in silico (CD9, CD24 and EPHA1) in addition to those previously detected (CD133, CD117, CD44). The surface markers CD117 and CD9 were expressed in ovarian surface epithelium. CD117, CD9 and ALDH1A1 also showed expression in the fallopian tube by IHC. PMC expressed individually CD44 (57.7±3.2), CD117 (12.5±6.1), CD133 (09.7±3.9), CD9 (11.1±7.2), CD24 (34.6±4.9), Epha1 (16.4±3.1) by flow cytometry. Co expression of markers in the same cells (n = 20) were (11.33±5.32). PMC expressed ALDH1A1 (2.5±0.2) and a fraction of these cells co-expressed CSC surface markers (2.09 ± 0.08). ALDH1A1 expressing malignant cells (n = 5) were predominantly in G0/G1 phase (67.2±2.6) of the cell cycle. Spheroids expressed all the above CSC markers by IF. In addition, ALDH1A1 was expressed in spheroids suggesting that these are enriched for CSCs. Spheroid formation in OVCAR3 and UC1101 cells was inhibited completely by the Hedgehog pathway inhibitor (SMO) GDC0449 (3μM). Flow cytometry analysis of PMC showed significant reduction in expression of ALDH1A1 (2.5±0.9), CD44 (18.6±5.5), CD9 (1.6±0.8), CD133 (0.5±0.3), CD117 (18.8±13.4), CD24 (0.1±0) after incubation with GDC0449. Similar results were obtained with two other inhibitors, LDE225 (SMO, 2.5nM) and GANT61 (Gli, 5μM). Significant reduction in migration of PMC was observed in the scratch assay in the presence of inhibitors. These results demonstrate that ALDH1A1 expression is consistently low in PMC and is the most reliable marker for CSCs. There was wide variability of expression of the surface markers in PMC and none uniquely co-expressed with ALDH1A1. The hedgehog signaling pathway is important in maintaining the integrity of CSCs. Targeting components of this pathway may be an alternate approach for treating ovarian cancer. Citation Format: Rohit P. Nagare, S Sneha, S Krishnapriya, Trivadi S. Ganesan. ALDH1A1 is the best marker by a composite approach for cancer stem cells in ovarian cancer whose integrity is controlled by the hedgehog pathway. [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 1392. doi:10.1158/1538-7445.AM2015-1392