Study of the Effect of Micro-Environment Stresses on Breast Tumor Initiating Cells by In Vitro Cell Tracking.

Increasing clinical as well as experimental evidence supports the existence of a small population of tumor initiating cells (TIC) that possesses enhanced self-renewal capacity, and the ability to form tumors upon transplantation. These TIC, which highly resemble normal stem cells, are believed to be responsible for tumor initiation and propagation. Evidence also suggests that TIC reside in unique micro-environment which may account for their resistance to traditional chemo- and radiotherapy. Understanding the effect of micro-environment on TIC will have dramatic implications for breast cancer prevention, treatment, and drug development.It is known that tumor micro-environment is characterized by various degrees of chemical imbalance, including oxygen depletion (hypoxia), lactic acidosis, glucose deprivation and high oxidative stresses caused by elevated reactive oxygen species (ROS) [1]. These physiological stresses have significant clinical impact and are often related to poor patient prognosis [2]. Interestingly, recent studies revealed that there are molecular connections between physiological stress regulated transcription factors and pathways known to control stem cell function [3], suggesting that the micro-environment stresses may either promote TIC cell transformation or proliferation [4]. However, despite these molecular level findings, direct evidence on the effect of physiological stresses on the proliferation and differentiation of TIC is still missing.We use in vitro live cell tracking to study the influence of micro-environment on TIC. Primary cultured cells are prepared from breast cancer surgery specimen. They are cultured in suspension in serum free medium as mammospheres which have been demonstrated to be highly enriched with TIC [5]. Various physiological stresses are mimicked by growing cells in tissue culture incubators with 2% O 2 (hypoxia), acidic medium (pH 6.7) with 25mM lactic acid, glucose deprivation condition by using glucose free DMEM medium, high ROS condition with 100uM H 2 O 2 . Cells are also infected with lentivirus to express H2B-GFP to label the cell nuclei so that they can be tracked by fluorescence imaging. Experiments are carried out in a full environmental controlled (CO 2 , O 2 , temperature, humidity) chamber and monitored by confocal imaging over prolonged time up to 96 hours. Since mammosphere cells grow in 3D instead of monolayer, they were scanned by confocal to reconstruct the 3D structure. We also developed computer programs for 3D segmentation to separate each cell, so that each cell within the mammosphere can be labeled and monitored individually. Such system allows us to draw a lineage tree for all the cells in the final mammosphere and detect the active dividing cells (which represent the differentiated cells) and the cells which reenter the quiescent status (which represent the progenitor cells), and hence allow us to study the effect of various micro-environment stresses on them.1. Vaupel, P., Semin Radiat Oncol, 2004. 14(3): p. 198-206.2. Schwickert, G., et al., Cancer Res, 1995. 55(21): p. 4757-9.3. Keith, B. and M.C. Simon, Cell, 2007. 129(3): p. 465-72.4. Li, Z., et al., Cancer Cell, 2009. 15(6): p. 501-13.5. Ponti, D., et al., Cancer Res, 2005. 65(13): p. 5506-11. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1163.