Selection of Metastatic Breast Cancer Cells Based on Adaptability of Their Metabolic State

A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary tumor growth was impaired in mice injected with parental cell line, but not in mice injected with Gln-ind cells

Celecoxib resistance in Gln-ind cells.

<p>One million cells of parental SUM149-Luc cell line or Gln-ind cells were treated with 50 µM celecoxib and rare surviving cells were allowed to grow into colonies. Then cells were dispersed by trypsinization and plated in dishes in medium with glutamine (complete medium) or without glutamine.</p

Selection of Gln-ind variants from breast cancer cell lines.

<p>(A–C) Half a million cells were plated in a 10-cm dish. The next day, the medium was changed to a Gln-free medium containing dialyzed fetal bovine serum. Colonies of cells growing under these conditions for 2–4 weeks were photographed. (D, E) Dishes of colonies stained with crystal violet. (F) SUM149-Luc-Gln-ind cells growing in Gln-free medium.</p

Model summarizing western blot analysis and additional data.

<p>Adaptable Gln-ind cells have high levels of COX-2 and Myc proteins, but they are able to dissociate Myc expression and GLS expression. The majority of cells lacking such adaptability may have perished in the initial selection in Gln-free medium. Broken arrows indicate adaptability to dissociate COX-2 function from Myc function, and Myc function from GLS level in Gln-ind cells, as identified in this study.</p

A reduction in COX-2 level upon treatment with N-acetyl cysteine.

<p>We exposed parental SUM149-Luc and Gln-ind cell lines, both growing in a medium with glutamine, to N-acetyl cysteine for 24 hours before subjecting them to western blotting using equal volumes of cell lysates. Since β-actin was not a reliable indicator of protein loading, we ran a gel in parallel and stained it with Coomassie blue. Lane numbers 1–6 at the bottom in left panel correspond to lane numbers 1–6 in the right panel.</p

Increased resistance to doxorubicin and paclitaxel in Gln-ind cells.

<p>Parental SUM149-Luc cell line or Gln-ind cells were treated with 200 nM doxorubicin (top) or 5 nM paclitaxel (bottom) for 7 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036510#s4" target="_blank">Materials and Methods</a>, and allowed to recover in a drug-free medium for 2 weeks (doxorubicin-treated) or 1 week (paclitaxel treated) before staining the colonies.</p

Glutamine-independent phenotype is adaptable but stable.

<p><b>Top panel:</b> Induction of GLS upon growth in glutamine-containing medium. SUM149-Gln-ind cells growing in Gln-free medium were shifted to Gln-containing medium, and GLS was analyzed by western blotting at different time points after the medium change. <b>Bottom panel:</b> Morphologies of cells growing under different conditions are shown. (A) Gln-ind cells growing without Gln. (B) Gln-ind cells growing in the presence of Gln (they exhibited faster growth and altered morphology upon growth with Gln). (C) Gln-ind cells were maintained in Gln-containing medium for 8 passages and then switched to Gln-free medium for 7 days.</p

COX-2 function is linked with Myc in regulating glycolysis and glutaminolysis in the COX-2-overexpressing, celecoxib-resistant SUM149-CER cell line.

<p>We transfected SUM149-CER cells with a COX-2-specific siRNA or with a control siRNA and analyzed the levels of Myc and some proteins involved in Myc-mediated glycolysis (hexokinase II and lactate dehydrogenase A) and glutaminolysis (glutaminase) by western blotting.</p

Impairment of primary tumor growth in mice injected with parental SUM149-Luc cells, but not with Gln-ind cells, upon reducing the number of injected cells.

<p>Cells (200 to 2 million) were injected into thoracic fat pads of 44-day-old nude mice (decreasing cell number from left to right) in duplicate. The luciferase images collected at day 62 show that primary tumor growth in the mice injected with the parental cell line was significantly reduced or absent upon reducing the number of injected cells below 2,000. In contrast, all mice injected with Gln-ind cells developed tumors, including both mice injected with only 200 cells. Empty slots on the left correspond to the mice that needed to be sacrificed because of high tumor burden.</p