Abstract 3357: Isolation of tumor cell subpopulations using semi-automated tissue dissociation and magnetic cell separation
Authors: Hardt Olaf , Parker Linda L. , Dudley Mark E. , Bosio Andreas 
Published: February 2011

Abstract
The analysis of tumor resident cell and stem cell populations requires proper methods for tissue dissociation and cell purification. Over the past few years, there has been a particular focus on the study of tumor infiltrating lymphocytes (TIL) and cancer stem cells (CSC). TIL can be isolated and characterized using conventional lymphocyte markers whereas CSC-specific cell surface markers are currently established (1). Two important markers, namely CD24 and CD44, have been shown to define CSC in various neoplasms such as breast cancer (2) or colon cancer (3).
We have established methods for the enzymatic and mechanical dissociation of solid tumors and optimized them according to the specific needs of a given tissue or cell type. Solid tumors are built up of a mixture of cell types, which are interconnected to each other and surrounded by an extracellular matrix composed of a variety of proteins and polysaccharides. The major goal was to disrupt the extracellular matrix and cell adhesion components without harming the integrity of the cell membrane. This was achieved by a combination of varying enzyme mixes, mechanical forces, incubation periods, and temperatures. The automation of all mechanical steps (gentleMACS™ dissociator) led to reproducible and moreover user independent results with reduced overall processing times. Viable tumor infiltrating lymphocytes (TIL) as well as tumor cells were obtained from melanoma metastases in high numbers. The isolated TIL expanded significantly faster than those from manually dissociated tumors and showed functionality in vitro and in vivo. As for cancer stem cells, we present novel tools for the immunomagnetic isolation of CD44+ and CD44+/CD24- cells. Using magnetic cell sorting (MACS®), a subpopulation of human teratocarcinoma cells could be isolated to a purity of 92% based on their expression of CD44. Furthermore, a combined depletion and positive selection approach allowed for the enrichment of a CD44+/CD24- human CML subpopulation from a starting frequency of 34% to final purity of 95%.
The separation of pure tumor cell populations will be necessary for their further characterization and also targeted drug screening approaches. The methods presented here are broadly applicable as they can be easily adapted to other cancer tissues and cell types and should help to optimize and standardize the future basic and clinical cancer research.
1. Trumpp, A. and Wiestler, O. D. (2008); Nature Clin. Prac. Oncology.; Vol. 5 No. 6
2. Al-Hajj, M. et al. (2003); Proc. Natl. Acad. Sci. USA; Vol. 100 No. 7
3. Du, L. et al. (2008); Clin. Cancer Res.; 6751 14 (21)
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
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 3357.