Abstract 5202: Human colon cancer cell lines contain subsets of cells with the capacity to initiate highly prolific clonal growth in soft agar culture and to form transplantable tumor xenografts in vivo
Authors: Alley Michael C. , Hollingshead Melinda G. , Borgel Suzanne D. , Plater Kevin , Burkett Mark W. , Anver Miriam R. , Yen Brandon R. , Hite Karen M. , Scudiero Dominic A. , Monks Anne , Mertins Susan D. , Shoemaker Robert H. , Niederhuber John E. 
Published: June 2012

Abstract
Various studies underscore the key role of cancer ‘stem cells’ in the initiation of experimental tumor xenografts from cells derived from patient tumor specimens. Implantation of limited numbers of cancer cells – even ‘single cells’ – which exhibit putative cancer ‘stem cell’ markers were reported to form tumors in mice, although several months were required to achieve palpable tumor masses. In the current study we assessed whether established human colon cancer cell lines still possess the functional capacity to initiate tumor formation from ‘single cell’ preparations. Results indicate that each of seven colon cancer cell lines was capable of soft agar colony formation in RPMI-1640 medium containing either 5-10% FBS or 15% serum substitute. Inoculation of 96-well plates by single cell sorting resulted in highly prolific colony formation by a subset of cells derived from each cell line. While the large majority of colonies achieving a size &gt;60μ in diameter exhibited growth arrest within 3 weeks (&lt;&lt;10 cell mass doublings), a minority of clones (&lt;2%) exhibited sustained growth over the course of 3 – 8 weeks. Colony measurements and cell number / colony calculations indicated that these colonies accumulated cells exceeding 10 mass doublings. According to one ‘stem cell’ model of tumor growth (Mackillop et al, JNCI 70: 9-16, 1983), such prolific colony growth is attributed to the self-renewal of cancer ‘stem cells’, whereas limited colony growth is achieved by partially differentiated cells. To assess whether prolific colonies form tumors in vivo, 10 single colonies derived from each colon cancer cell line were implanted into each of 10 athymic nu/nu mice. Testing of 5 tumor models completed to date shows that 75 – 100% (86% overall) of these colonies form tumor xenografts (median times to 700 mg tumor formation ranged from 40 to 72 days). Subsequent in vivo passage of 3 tumor xenografts per cell line resulted in tumor formation in 85 – 92% of host animals (median times ranging from 29 to 67 days). Thus, despite extensive in vitro propagation, a subset of individual cells in each cell line retained the functional capacity to initiate and sustain cancer cell growth in vitro and in vivo. Single cell cloning in soft agar provides an effective means to isolate individual colonies formed by cancer ‘stem cells’ and to propagate human tumor xenografts with modest numbers of tumor cells, host mice, and tumor formation times. The cell surface marker profile and drug sensitivity of prolific colonies are currently under investigation. (Funded in part by NCI Contract No. HHSN261200800001E)
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5202. doi:10.1158/1538-7445.AM2011-5202