Abstract B24: Differentiation screen identifies small molecules that target histologically divergent subtypes of patient-derived lung cancer stem cells
Authors: Shlyakhter Dina , Boucher Diane , Gu Yong , Hall Amy B. , Krueger Elaine , Lindquist Anna , Murphy Cheryl , Wang Yuxin , Wood Mark , Eustace Brenda 
Published: May 2016

Abstract
Non-small cell lung cancer (NSCLC) is a prevalent and deadly disease because of high incidence and relapse rates. One hypothesis for the high relapse rate is the existence of cancer stem cells (CSCs), a rare subpopulation of cells within these tumors, that are resistant to therapy and thought to be responsible for local and distal recurrence. CSCs are also able to self-renew and differentiate into multiple cell types forming the mass of new tumors. These differentiated progeny that constitute the bulk of the tumor are more sensitive to radiation and chemotherapeutic agents, express tissue of origin markers, and have an intrinsically limited lifespan. While most solid tumors have these properties, the degree of heterogeneity from patient to patient in the multi-lineage potential of the CSC population is not known. In particular, NSCLC CSC populations from primary patient tumors have not been well studied. The goal of this study was to characterize newly derived NSCLC CSC lines to determine the degree of heterogeneity between patient samples, and to provide a starting point for the discovery of novel differentiation therapeutic agents to target CSCs.
We isolated six CSC lines from primary patient NSCLC tumors using an unbiased culture-based enrichment method, rather than a biased marker-based approach. Each of these six CSC lines was tumorigenic in immunodeficient mice at low cell numbers and the resulting tumors recapitulated the original tumor histology. All-trans retinoic acid (ATRA), a well-known differentiation agent for the treatment of acute promyelocytic leukemia (APL), maintains the normal growth and differentiation of human bronchial epithelial cells in culture. Each of the six CSC lines was treated with ATRA for two weeks to induce differentiation and then assessed for transcript and protein levels of candidate CSC and differentiation markers. Pre-treatment, the CSC lines all expressed CD44. Post-ATRA treatment, the majority of the CSC lines expressed Mucin-2, a marker of Goblet cell lineage. In addition, each cell line started to show evidence of the very early stages of differentiation into other lineages including Clara Cells, Neuroendocrine Cells, and Alveolar Type I and Type II cells. In one CSC line, ATRA treatment both in vitro and in vivo delayed tumor cell growth, induced the expression of Mucin-2 and decreased the expression of Nestin, a cancer stem cell marker.
To further characterize CSC differentiation potential, we screened a pilot set of small molecules in two independent CSC lines to identify compounds that induce terminal differentiation, using Mucin-2 expression as readout. Surprisingly, despite the fact that these two CSC lines were from different histological subtypes (adenocarcinoma and squamous cell carcinoma) and displayed different transcriptional profiles post-differentiation, there was a good correlation between hit sets in the screens.
Taken together, these data suggest that while the CSC of origin in tumors differs between patient samples in profile and function, there is promise to identify differentiation agents that are broadly active in different NSCLC subtypes. Further screening is currently being performed to identify these novel differentiation agents for therapeutic use.
Citation Format: Dina Shlyakhter, Diane Boucher, Yong Gu, Amy B. Hall, Elaine Krueger, Anna Lindquist, Cheryl Murphy, Yuxin Wang, Mark Wood, Brenda Eustace. Differentiation screen identifies small molecules that target histologically divergent subtypes of patient-derived lung cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B24.