Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis
Venkata Lokesh Battula, Yuexi Shi, Kurt W. Evans, Rui-Yu Wang, Erika L. Spaeth, Rodrigo O. Jacamo, Rudy Guerra, Aysegul A. Sahin, Frank C. Marini, Gabriel Hortobagyi, Sendurai A. Mani, Michael Andreeff
Venkata Lokesh Battula, Yuexi Shi, Kurt W. Evans, Rui-Yu Wang, Erika L. Spaeth, Rodrigo O. Jacamo, Rudy Guerra, Aysegul A. Sahin, Frank C. Marini, Gabriel Hortobagyi, Sendurai A. Mani, Michael Andreeff
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Research Article Oncology

Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis

Abstract

Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have increased resistance to conventional therapies and are capable of establishing metastasis. However, only a few biomarkers of CSCs have been identified. Here, we report that ganglioside GD2 (a glycosphingolipid) identifies a small fraction of cells in human breast cancer cell lines and patient samples that are capable of forming mammospheres and initiating tumors with as few as 10 GD2+ cells. In addition, the majority of GD2+ cells are also CD44hiCD24lo, the previously established CSC-associated cell surface phenotype. Gene expression analysis revealed that GD3 synthase (GD3S) is highly expressed in GD2+ as well as in CD44hiCD24lo cells and that interference with GD3S expression, either by shRNA or using a pharmacological inhibitor, reduced the CSC population and CSC-associated properties. GD3S knockdown completely abrogated tumor formation in vivo. Also, induction of epithelial-mesenchymal transition (EMT) in transformed human mammary epithelial cells (HMLER cells) dramatically increased GD2 as well as GD3S expression in these cells, suggesting a role of EMT in the origin of GD2+ breast CSCs. In summary, we identified GD2 as a new CSC-specific cell surface marker and GD3S as a potential therapeutic target for CSCs, with the possibility of improving survival and cure rates in patients with breast cancer.

Authors

Venkata Lokesh Battula, Yuexi Shi, Kurt W. Evans, Rui-Yu Wang, Erika L. Spaeth, Rodrigo O. Jacamo, Rudy Guerra, Aysegul A. Sahin, Frank C. Marini, Gabriel Hortobagyi, Sendurai A. Mani, Michael Andreeff

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Figure 2

GD2 identifies CD44hiCD24lo stem cell phenotype in breast cancer cells.

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GD2 identifies CD44hiCD24lo stem cell phenotype in breast cancer cells. ...
(A) HMLER cells were stained with anti-GD2 antibody and with CD44-APC and CD24-FITC using the 4-step staining protocol described in Methods. Cells were electrically gated on GD2+/– cells and displayed in a pseudocolor dot plot with CD44 on the y axis and CD24 on the x axis using FlowJo data analysis software. (B) In an identical experiment, CD44hi/loCD24lo/hi cells were displayed on a pseudocolor dot plot with GD2 on the y axis and FSC on the x axis. (C) Primary breast tumor samples were processed as described in Methods, and the single cells in suspension were stained with anti-GD2, CD44-APC, CD24-FITC, CD45-FITC, and DAPI using the 4-step staining protocol. Cells were initially gated on DAPI-negative cells to exclude dead cells, and the cells were then gated on CD45– cells to exclude hematopoietic cells. GD2+CD45– cells were displayed on a dot plot, with CD44 on the y axis and CD24 on the x axis. Analysis was perfumed using an LSR II flow cytometer. Data were analyzed using FlowJo software.