Abstract 3320: Head & neck cancer stem cells express drug resistance transporters but do not display a resistant phenotype
Authors: Steadman Kenneth , Robey Robert , Creswell Karen , Slack Rebecca , Deeken John 
Published: February 2011

Abstract
Background: The cancer stem cell (CSC) hypothesis holds that CSCs generate tumor daughter cells via asymmetric cell division, evade immune surveillance, and are resistant to anticancer treatment, and thus remain viable to regenerate a population of malignant clones after therapy. While CSCs’ inherent resistance to chemotherapy is a central tenet of the cancer stem cell hypothesis, little research has focused on this critical question.
Methods: Identification and separation of putative head and neck cancer squamous cell carcinoma (H&N SCC) CSCs was performed via flow cytometry of the SCC9 cell line using both Hoechst 33342 dye (‘side population’) and antibodies against CD44, CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b (‘CD44+/lineage-‘). Expression of genes characterizing a stem cell phenotype as well as drug transporters known to mediate chemotherapy drug resistance was measured via RT-PCR and compared between subpopulations. The SCC9 cell line was incubated with various chemotherapy agents in attempt to enrich the CSC subpopulation, including cisplatin, paclitaxel, ixabepilone, depsipeptide, methotrexate, SN38, and topotecan, followed by flow cytometric sorting. The resultant CSC subpopulation was compared between drug treated and untreated controls to determine whether the previous drug exposure enriched the CSC subpopulation.
Results: Use of Hoechst dye did not identify a ‘side population,’ likely due to low expression of the ABCG2 transporter in H&N CSCs. Using CD44+/lineage- markers, a CSC subpopulation representing 8.5% on average of total cells was identified. Variant expression of stem cell genes in this subpopulation included ACTC1, BMP3, CCND2, COL9A1, DLL3, FGF3, GDF2, GDF3, PDX1, KRT15, SOX1, TERT, and WNT1. Significant expression of drug transporters in the CSC subpopulation included ABCA3, SLC16A2, SLC16A3, SLC22A1, SLC28A2, SLC2A1, SLC25A13, and SLCO1B3. ABCB1 expression was increased in the CSC subpopulation but still showed relatively low RNA expression. There was no difference in expression of ABCC1 and ABCG2. Incubation with cytotoxic agents at various concentrations (IC25 to IC75) for 24 to 96 hours showed no clear pattern of enriching or depletion of the CSC subpopulation.
Conclusion: CD44+/lineage- can identify a putative CSC subpopulation in H&NSCC which preferentially expresses stem cell pathway genes as well as drug transporters. This increased transporter expression does not, however, induce a drug resistance phenotype in this in vitro cell line model. Other in vitro and/or in vivo methodologies should be explored to better analyze and characterize treatment resistance of CSCs in order to further test the validity of the cancer stem cell hypothesis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3320.