Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation.

Affiliation

Bhandary L(#)(1), Bailey PC(#)(1)(2), Chang KT(1)(3), Underwood KF(4), Lee CJ(1), Whipple RA(1), Jewell CM(5), Ory E(1), Thompson KN(1), Ju JA(1), Mathias TM(1), Pratt SJP(1)(2), Vitolo MI(6)(7)(8), Martin SS(9)(10)(11)(12).
Author information:
(1)Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine
(UMGCCC), 22 S. Greene St., Baltimore, MD, 21201, USA.
(2)Graduate Program in Biochemistry, University of Maryland School of Medicine, 800 W. Baltimore St., Baltimore, MD, 21201, USA.
(3)Graduate Program in Life Sciences, University of Maryland School of Medicine, 800 W. Baltimore St., Baltimore, MD, 21201, USA.
(4)UMGCCC Flow Cytometry Shared Service, 655 West Baltimore Street, BRB 7-022, Baltimore, MD, 21201, USA.
(5)Fischell Department of Bioengineering, 3102 A. James Clark Hall, College Park, MD, 20742, USA.
(6)Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine
(UMGCCC), 22 S. Greene St., Baltimore, MD, 21201, USA. [Email]
(7)Graduate Program in Biochemistry, University of Maryland School of Medicine, 800 W. Baltimore St., Baltimore, MD, 21201, USA. [Email]
(8)Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD, 21201, USA. [Email]
(9)Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine
(UMGCCC), 22 S. Greene St., Baltimore, MD, 21201, USA. [Email]
(10)Graduate Program in Biochemistry, University of Maryland School of Medicine, 800 W. Baltimore St., Baltimore, MD, 21201, USA. [Email]
(11)Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD, 21201, USA. [Email]
(12), Bressler Research Building Room 10-29, 655 West Baltimore Street, Baltimore, MD, 21201, USA. [Email]
(#)Contributed equally

Abstract

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 μm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.