Abstract B45: A novel reporter system with potential for in situ assessment of tumor microenvironmental effects on cancer stem cells

Many tumors consist of a hierarchy of cells with different proliferative and developmental potential. A small number of cancer stem cells (CSCs) give rise to a larger population of highly proliferative, committed progenitor cells, which may then undergo limited differentiation. Importantly, CSCs are uniquely capable of initiating and sustaining tumorigenesis, and they have been implicated in driving disease recurrence after cancer therapy. Thus understanding CSC biology will be critical to the development of more effective therapies. CSCs are most commonly identified by FACS analysis but the optimal marker combinations are very dependent on the tissue and specific cell-of-origin of the tumor, and they cannot be used to monitor the CSCs in situ, with all the microenvironmental cues intact. Such markers cannot readily be used for real-time assessment of stem cell behavior at a single cell rather than a population level. To address this problem, we have developed and validated a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of the cells with CSC properties. The construct consists of a tandemly repeated composite Sox2-Oct4 response element (SORE6) driving expression of a destabilized green fluorescent protein reporter. The reporter responds to the presence of the core stem cell transcription factors Oct4 and Sox2, with further stem cell selectivity and kinetic resolution coming from the use of a proteosome-targeting degron on the fluorescent protein. Using the human MCF10CA1h breast cancer cell line, we have shown that SORE6-GFP+ cells within the cell population are undifferentiated and enriched for stem cell markers. These cells can self-renew and regenerate GFP- cells, show enhanced asymmetric division, and are enriched for tumorsphere formation in vitro. Most importantly the SORE6-GFP+ cells are enriched for tumor-initiating and metastasis-initiating ability in vivo and they are relatively resistant to chemotherapeutics both in vitro and in vivo. Thus by a number of criteria, the reporter is marking a cell population that is substantially enriched for CSCs. The reporter works in primary human breast cancer cultures and patient-derived xenografts in addition to established cell line models. Our novel imaging approach opens up the possibility of assessing the spatial distribution of CSCs and temporal changes in CSC properties in experimental settings that retain the complex microenvironmental and structural cues of the tumor bed. Citation Format: Binwu Tang, Asaf Raviv, Dominic Esposito, Catherine Daniel, Bao Tram Nghiem, Susan Garfield, Langston Lim, Poonam Mannan, Ana Robles, William Smith, Joshua Zimmerberg, Rea Ravin, Lalage Wakefield. A novel reporter system with potential for in situ assessment of tumor microenvironmental effects on cancer stem cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B45. doi:10.1158/1538-7445.CHTME14-B45