Abstract 4894: Sorafenib enriches for sarcoma cancer stem cells in vitro.

Abstract

Abstract Background: Cancer stem cells (CSCs) are a putative source of relapse in many cancers following anti-proliferative therapies. We hypothesized that tyrosine kinase inhibition (TKI) and radiotherapy (RT) would enrich for CSCs in sarcoma cell lines. Methods: A673 Ewing's sarcoma and SW982 synovial sarcoma cells were exposed to sorafenib and RT in a dose- and time-dependent fashion. Cultured cells were harvested and stained with fluorochrome-conjugated antibodies against human CSC markers including CD133, CD24, CD44, and aldehyde dehydrogenase (ALDH). Cell viability was analyzed using 7-Aminoactinomycin (7-AAD). Data were acquired using a BD Fortessa cell sorter (BD Biosciences) and analyzed with FlowJo software version 7.2. Parametric and non-parametric statistical tests were performed as appropriate. Results: Short-term exposure (≤1 day) to sorafenib demonstrated a linear dose-dependent cytotoxicity for A673 cells at doses ≥ 4 μM, while SW982 cells required doses ≥ 16 μM. At 24 hours, A673 cell viability decreased from 100% at 1 μM sorafenib to 0% at 32 μM (P<0.05). By day 5, A673 cells exposed to sorafenib doses < 32 μM recovered log-phase growth, while cells exposed to doses > 32 μM remained non-viable. Baseline CSC phenotyping of A673 cells demonstrated 55±5% CD133+, 12±7% ALDH+, 14% CD44+, and 0% CD24+ sub-populations. Baseline CSC phenotyping of SW982 cells demonstrated 0.2±0.9% CD133+, 42±8% ALDH+, 95±5% CD44+, and 40±7% CD24+. A673 cells exposed to 24h sorafenib increased the ALDH+ fraction to 40±3% (> 3-fold increase) at sorafenib 16 μM (P<0.05). SW982 cells showed a non-significant increase in the ALDH+ fraction at doses ≤ 16 μM. Although single fractions of RT were anti-proliferative to A673 cells starting at doses ≥5 Gy, there was not an additive effect of 2.5 Gy RT with sorafenib on cell viability or CSC marker expression. Conclusion: Sorafenib exerts an anti-proliferative effect on sarcoma cells but enriches for sarcoma CSC. The magnitude of these effects appears to be inversely correlated to baseline ALDH+ levels. Sustained anti-sarcoma therapeutic effects may require targeting of the CSC population following anti-proliferative therapy. Citation Format: Robert J. Canter, Erik Ames, Joseph Tellez, Rachel C. Smith, William J. Murphy. Sorafenib enriches for sarcoma cancer stem cells in vitro. [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 4894. doi:10.1158/1538-7445.AM2013-4894 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.