Survival and self-renewing capacity of breast cancer initiating cells during fractionated radiation treatment

Abstract Introduction Recent data indicate a hierarchical organization of many solid cancers, including breast cancer, with a small number of cancer initiating cells (CICs) that have the ability to self-renew and exhibit multi-lineage potency. We, and others, have demonstrated that CICs in breast cancer and glioma are relatively resistant to ionizing radiation if compared to their non-tumorigenic counterparts. However, the extent of the remaining self-renewing capacity of CICs after fractions of radiation is currently unknown. We hypothesized that CICs, in contrast to their non-tumorigenic counterparts, not only survive fractions of ionizing radiation but also retain the CIC phenotype as defined by operational means. Methods We used two marker systems to identify breast CICs (CD24-/low/CD44high, or lack of proteasome activity) and performed sphere-forming assays after multiple clinical fractions of radiation. Lineage tracking was performed by membrane staining. Cell cycle distribution and RNA content were assessed by flow cytometry and senescence was assessed via β-galactosidase staining. Results We demonstrated that irradiated CICs survived and retained their self-renewal capacity for at least four generations. We show that fractionated radiation not only spared CICs but also mobilized them from a quiescent/G0 phase of the cell cycle into actively cycling cells, while the surviving non-tumorigenic cells were driven into senescence. Conclusions The breast CIC population retains increased self-renewal capacity over several generations and therefore, we conclude that increases in the number of CICs after sublethal doses of radiation have potential clinical importance. Prevention of this process may lead to improved clinical outcome

Oxygen Levels Do Not Determine Radiation Survival of Breast Cancer Stem Cells

For more than a century oxygen has been known to be one of the most powerful radiosensitizers. However, despite decades of preclinical and clinical research aimed at overcoming tumor hypoxia, little clinical progress has been made so far. Ionizing radiation damages DNA through generation of free radicals. In the presence of oxygen these lesions are chemically modified, and thus harder to repair while hypoxia protects cells from radiation (Oxygen enhancement ratio (OER)). Breast cancer stem cells (BSCSs) are protected from radiation by high levels of free radical scavengers even in the presence of oxygen. This led us to hypothesize that BCSCs exhibit an OER of 1. Using four established breast cancer cell lines (MCF-7, T47D, MDA-MB-231, SUM159PT) and primary breast cancer samples, we determined the number of BCSCs using cancer stem cell markers (ALDH1, low proteasome activity), compared radiation clonogenic survival and mammosphere formation under normoxic and hypoxic conditions, and correlated these results to the expression levels of key members of the free radical scavenging systems. The number of BCSCs increased with increased aggressiveness of the cancer. This correlated with increased radioresistance (SF8Gy), and decreasing OERs. When cultured as mammospheres, breast cancer cell lines and primary samples were highly radioresistant and not further protected by hypoxia (OER∼1)

Radiation response of BCSCs.

<p>Mammosphere assay using the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034545#pone-0034545-g002" target="_blank">Figure 2</a>. Data was fitted using a linear quadratic model.</p

Radiation response of differentiated cells.

<p>Clonogenic survival assays of established breast cancer cells lines and primary patient-derived breast cancer samples under normoxic and hypoxic conditions. Data was fitted using a linear quadratic model.</p

Correlation of free radical scavengers and OER.

<p>Correlation of free radical scavenger mRNA expression and oxygen enhancement ratios found in breast cancer cell lines and patient samples.</p

Expression of free radical scavenger mRNAs in breast cancer cells.

<p>qRT-PCR using primers for 13 mRNAs involved in free radical scavenging. Expression levels are expressed relative to expression levels found in MCF-7 cells.</p

Plating efficiency and percentage of stem cells.

<p>(<b>A</b>) Plate efficiency of breast cancer cell lines and patient-derived tumor samples in clonogenic survival assays under normoxic and hypoxic conditions. (<b>B</b>) Percentage of ZsGreen-cODC⁺ BCSCs in established breast cancer cell lines. (<b>C</b>) Percentage of ALDH⁺ cells in established breast cancer cell lines and patient-derived tumor samples.</p