Abstract 3757: Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells.
Authors: Agorku David , Parker Greg , Chelius Nadine , Bosio Andreas , Hardt Olaf 
Published: August 2014

Abstract
Cancer stem cells (CSCs), also called tumor initiating cells, have gained substantial interest in the research field over the past few years. CSCs have been isolated from multiple tumor entities and were shown to play a crucial role during tumor growth and metastasis. However, there is still a major debate about specific cell surface markers capable of identifying CSCs in most tumor entities. The leucine-rich repeat-containing G-protein-coupled receptor (LGR) Lgr5 and its close homologues Lgr4 and Lgr6 associate with Wnt-receptors and act as R-spondin receptors thereby playing a central role in the modulation of Wnt/β-catenin signaling in normal and neoplastic stem cells (de Lau et al., 2011, Carmon et al., 2011). Initially described as a highly specific marker for stem cells in the small intestine, colon, hair follicle, stomach, and during kidney development (Barker et al., 2007, Jaks et al., 2008, Barker et al., 2010, Barker et al., 2012), Lgr5 positive cells were also shown to be crucial during the development and progression of cancer. It was shown that Lgr5 positive crypt stem cells are the cells-of-origin in intestinal cancer and that CSCs in human colorectal cancer can be identified and isolated based on Lgr5 expression (Barker et al., 2009, Kemper et al., 2012). However, the analysis of Lgr5 expressing cells is hampered by the lack of highly specific monoclonal antibodies. We have developed rabbit monoclonal antibodies against peptide sequences of Lgr5. After generating cell lines stably expressing Lgr5, we screened for high affinity antibodies by flow cytometry. Using stable transfectants for Lgr4 and Lgr6, we could prove that there is no cross reactivity with its close homologues. This is of particular importance for Lgr4, as this protein is also expressed on more differentiated progenitor cells. To allow for identification, analysis, and isolation of Lgr5 positive cells from solid tissues, automated and therefore user-independent methods for tissue dissociation were developed. This resulted in enzyme mixtures which allow dissociating different tissues without harming the target epitopes. To detect cells expressing Lgr5 at low levels in flow cytometry based assays we combined our novel high affinity antibodies with fluorescence amplification systems. In addition, we generated a new magnetic cell separation (MACS) protocol that can be used as an easy and fast method to isolate Lgr5 positive cells from primary human tissue. Taken together, we have developed highly specific monoclonal antibodies allowing for the analysis and isolation of Lgr5 positive cells from cell lines and primary tissues.
Citation Format: David Agorku, Greg Parker, Nadine Chelius, Andreas Bosio, Olaf Hardt. Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3757. doi:10.1158/1538-7445.AM2013-3757