Ablation of breast cancer stem cells with radiation

Abstract

Tumor radioresistance leads to recurrence after radiation therapy. The radioresistant phenotype has been hypothesized to reside in the cancer stem cell (CSC) component of breast and other tumors and is considered to be an inherent property of CSC. In this study, we assessed the radiation resistance of breast CSCs using early passaged, patient-derived xenografts from two separate patients. We found a patient-derived tumor in which the CSC population was rapidly depleted 2 weeks after treatment with radiation, based on CD44(+) CD24(-) lin(-) phenotype and aldehyde dehydrogenase 1 immunofluorescence, suggesting sensitivity to radiotherapy. The reduction in CSCs according to phenotypic markers was accompanied by a decrease in functional CSC activity measured by tumor sphere frequency and the ability to form tumors in mice. In contrast, another patient tumor sample displayed enrichment of CSC after irradiation, signifying radioresistance, in agreement with others. CSC response to radiation did not correlate with the level of reactive oxygen species in CSC versus non-CSC. These findings demonstrate that not all breast tumor CSCs are radioresistant and suggest a mechanism for the observed variability in breast cancer local recurrence.

Figure 1

Effect of radiation on CSCs. MC1 and UM2 xenografts were treated with radiation and the proportion of CSCs analyzed after 2 weeks using flow cytometry. (A) Flow cytometric detection of CD44⁺ CD24^- cells (upper left quadrant) in untreated and treated UM2 xenografts. (B) Flow cytometric detection of CD44⁺ CD24^- cells in untreated and treated MC1 xenografts. *P < .05 compared with untreated, n = 2–15.

Figure 2

Immunofluorescent detection of ALDH1. Control and irradiated UM2 and MC1 tumor sections were stained for ALDH1 2 weeks after treatment. ALDH1 staining is in green on the upper panels, and DAPI staining of nuclei is in blue on the lower panels. Irradiated tumors displayed fewer ALDH1-stained cells than untreated tumors.

Figure 3

Tumor spheres in control and irradiated tumors. The frequency of tumor sphere-forming cells in control or irradiated UM2 and MC1 xenografts was determined. *P < .01, n = 3–4.

Figure 4

Functional assay for CSC activity. (A) MC1 cells from control and two different irradiated tumors (2 weeks after irradiation) were injected into the mammary fat pad of mice at the indicated cell quantities. The number of days required for tumor formation was recorded and plotted. (B) Measurement of MC1 tumor growth. There was no statistical difference between the growth rate of each group (P < .05).

Figure 5

Analysis of CSC in recurrent tumors. Recurrent tumor xenografts derived from control or two irradiated primary xenografts (IR#1 and IR#2) were subjected to flow cytometric detection of CD44⁺ CD24^- CSC. *P < .05 compared with control.

Figure 6

Reactive oxygen species in CSC. MC1 and UM2 cells were stained with H₂DFFDA to detect ROS levels by flow cytometry. CSC had statistically lower ROS levels than non-CSC (P < .05, N = 2). (+)ctrl signifies positive controls treated with H₂O₂.