The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation.
BACKGROUND
If cancer arises and is maintained by a small population of cancer-initiating cells within every tumor, understanding how these cells react to cancer treatment will facilitate improvement of cancer treatment in the future. Cancer-initiating cells can now be prospectively isolated from breast cancer cell lines and tumor samples and propagated as mammospheres in vitro under serum-free conditions.


METHODS
CD24(-/low)/CD44+ cancer-initiating cells were isolated from MCF-7 and MDA-MB-231 breast cancer monolayer cultures and propagated as mammospheres. Their response to radiation was investigated by assaying clonogenic survival and by measuring reactive oxygen species (ROS) levels, phosphorylation of the replacement histone H2AX, CD44 levels, CD24 levels, and Notch-1 activation using flow cytometry. All statistical tests were two-sided.


RESULTS
Cancer-initiating cells were more resistant to radiation than cells grown as monolayer cultures (MCF-7: monolayer cultures, mean surviving fraction at 2 Gy [SF(2Gy)] = 0.2, versus mammospheres, mean SF(2Gy) = 0.46, difference = 0.26, 95% confidence interval [CI] = 0.05 to 0.47; P = .026; MDA-MB-231: monolayer cultures, mean SF(2Gy) = 0.5, versus mammospheres, mean SF(2Gy) = 0.69, difference = 0.19, 95% CI = -0.07 to 0.45; P = .09). Levels of ROS increased in both mammospheres and monolayer cultures after irradiation with a single dose of 10 Gy but were lower in mammospheres than in monolayer cultures (MCF-7 monolayer cultures: 0 Gy, mean = 1.0, versus 10 Gy, mean = 3.32, difference = 2.32, 95% CI = 0.67 to 3.98; P = .026; mammospheres: 0 Gy, mean = 0.58, versus 10 Gy, mean = 1.46, difference = 0.88, 95% CI = 0.20 to 1.56; P = .031); phosphorylation of H2AX increased in irradiated monolayer cultures, but no change was observed in mammospheres. Fractionated doses of irradiation increased activation of Notch-1 (untreated, mean = 10.7, versus treated, mean = 15.1, difference = 4.4, 95% CI = 2.7 to 6.1, P = .002) and the percentage of the cancer stem/initiating cells in the nonadherent cell population of MCF-7 monolayer cultures (untreated, mean = 3.52%, versus treated, mean = 7.5%, difference = 3.98%, 95% CI = 1.67% to 6.25%, P = .009).


CONCLUSIONS
Breast cancer-initiating cells are a relatively radioresistant subpopulation of breast cancer cells and increase in numbers after short courses of fractionated irradiation. These findings offer a possible mechanism for the accelerated repopulation of tumor cells observed during gaps in radiotherapy.
Phillips
Tiffany M
TM
Department of Radiation Oncology, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave., Los Angeles, CA 90095-1714, USA.
McBride
William H
WH
Pajonk
Frank
F
D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology / Q000473:pathology* / Q000532:radiotherapy*; D051927:CD24 Antigen / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002470:Cell Survival / Q000528:radiation effects; D019583:Dose Fractionation, Radiation; D005260:Female; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000276:immunology* / Q000528:radiation effects*; D010766:Phosphorylation; D011836:Radiation Tolerance; D017382:Reactive Oxygen Species / Q000378:metabolism; D051881:Receptor, Notch1 / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000528:radiation effects; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D051927:CD24 Antigen; D002135:Calcium-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D018960:Hyaluronan Receptors; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D017382:Reactive Oxygen Species; D051881:Receptor, Notch1; D000072099:Serrate-Jagged Proteins