Ewing's Sarcoma Spheroids as a 3-D Model of In Vivo Biology Useful for High-Throughput Preclinical Screening of Antineoplastic Drug Candidates

Emily Burdett1, Antonios G. Mikos1, and Joseph Ludwig2. (1) Bioengineering, Rice University, MS-142, P.O. Box 1892, Houston, TX 77251, (2) Sarcoma Medical Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030

Pre-clinical screening for candidate drugs with potential antineoplastic activity typically relies upon high-throughput screens across cancer cell lines grown in monolayer culture, but this method often fails to accurately predict whether compounds eventually prove to be successful in later human clinical trials. In part this may be due to dedifferentiation and change in cellular phenotype that occurs upon removal from a native in vivo 3-D cellular microenvironment. Subsequent animal models do involve cell placement within a tissue microenvironment, but they are not a high-throughput platform, and do not allow for repeated biopsies for analysis during testing. A high-throughput screening method that can recreate this microenivoronment would offer greater predictive power, streamline the preclinical antineoplastic drug discovery process, and by extension, decrease the time required to move a drug from bench to bedside. In this study toward the development of such a screening model, we compare Ewing's Sarcoma cell lines grown in monolayers to 3-D cellular aggregates called spheroids in order to determine the effect that a 3-D architecture has upon cellular phenotype. The response of several different Ewing's Sarcoma cell lines to cancer therapeutic drugs was monitored through the use of cellular proliferation assays in order to evaluate the degree of therapeutic resistance conferred to cells by three dimensional growth. Drugs tested include several different chemotherapeutics that are currently used in standard treatment for Ewing's Sarcoma, including doxorubicin, vincristine, and etoposide. The inhibitory effects of insulin-like growth factor I receptor (IGF-1R) agonists were also tested to investigate the hypothesis that these multicellular Ewing's spheroids could serve as an intermediate model that is more representative of the in vivo signaling cascade. These tests revealed that growth within spheroids does confer resistance to many of these therapies; the drug concentration required to achieve 50 % growth inhibition within spheroids is about tenfold higher than that for monolayer growth. These results suggest cancer cells grown within a 3-D environment have a more clinically accurate cellular response to therapeutics, and show potential for development of a more predictive high-throughput preclinical screen for cancer therapies through the use of 3-D culturing techniques.