StemCell Therapy

Abstract Introduction

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis.

Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low-dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power.

A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. A total of 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis.

Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC.

Breast cancer is a highly heterogeneous disease broadly classified on the basis of clinical parameters such as size, grade and node status, as well as histopathological criteria, primarily expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [1]. While defined targeted therapeutic strategies have been developed for patients with ER+/PR+ and HER2+ diseases, chemotherapy is currently the mainstay of systemic treatment for triple-negative (ER/PR/HER2) breast cancer (TNBC) patients, which represents approximately 20% of all breast cancers [2]. Clinically, TNBC encompasses a heterogeneous group of aggressive tumours with poor prognosis [1],[3]-[7], partly due to high recurrence within the first years and limited targeted therapy options. Although chemotherapy is often initially beneficial in these tumours, especially in the neoadjuvant setting, many TNBCs have a high risk of relapse [8]. Since there is currently no means of predicting which TNBC will relapse, identification of subpopulations of TNBC that are most at risk is vital for the clinical management of these breast cancer patients.

Strong evidence is emerging supporting the hypothesis that cancer stem cells with similar features to normal tissue stem cells are resistant to standard chemotherapy and drive tumour regrowth after therapy finishes [9]. We hypothesised that biological properties of normal stem cells are reactivated in tumour cells to facilitate metastasis. Genes expressed in stem cells of the normal mammary gland might therefore carry prognostic information for relapse and metastasis in breast cancer. However, the development of such gene sets depends on the ability to isolate highly pure stem cells for analysis.

The mammary epithelium consists of two main layers, the luminal and basal layers. The luminal layer consists of ER- cells (mainly proliferative progenitors) and ER+ cells (mainly non-proliferative differentiated cells). The basal layer consists of myoepithelial cells (MYOs) and mammary stem cells (MaSCs), the latter characterised by their robust outgrowth activity in the cleared fat pad transplant assay. The relationship between these populations is summarised in Additional file 1A. Previous studies have analysed total basal breast epithelial cells, without further purification of the minority stem cell fraction [10] or used a dye label-retention strategy to identify asymmetrically dividing cells (putative stem cells) in non-adherent mammosphere cultures [11]. Only one previous study has attempted to freshly purify basal stem cells and compare their gene expression profile to MYOs [12]; however, that study identified only four genes expressed >2-fold more highly in stem cells compared to MYOs, and none of these achieved statistical significance. Here, we have defined the first gene signature specific for highly purified, freshly isolated MaSCs and further enriched the stem cell specificity by excluding basal-associated genes common to both the stem and myoepithelial populations. Pathway analysis revealed that this signature was enriched in genes associated with cell migration, adhesion and tissue morphogenesis. Single cell resolution gene expression analysis showed that the stem cell population included cells that expressed both epithelial- and mesenchymal-associated genes. Strikingly, when the expression of the stem cell gene signature was interrogated in two large independent TNBC cohorts, tumours with an activated stem cell signature showed a higher propensity to relapse in the first years after diagnosis in comparison to TNBC with lower activation scores for the stem cell gene signature. In contrast, in three large independent ER+ breast cancer data sets, an activated stem cell signature identified tumours least likely to metastasise. The prognostic power of the stem cell gene signature when applied to expression profiling of total tumour material implies that in poor prognosis TNBC the cancer stem cell-like genetic programme is not restricted to a minority cell population but rather is driving the behaviour of the bulk of tumour cells.

Our findings show that the biology of normal MaSCs, as reflected in their gene expression profiles, is highly relevant for understanding the drivers of aggressive disease in TNBC.

All animal work was carried out under UK Home Office project and personal licences following local ethical approval by the Institute of Cancer Research Animal Ethics Committee and in accordance with local and national guidelines. Single cells were prepared from fourth mammary fat pads of 8- to 10-week-old virgin female FVB mice as described [13] and stained with anti-CD24-FITC, anti-Sca-1-APC, anti-CD45-PE-Cy7, anti-CD49f-PE-Cy5 and anti-c-Kit-PE. Mammary epithelial cell subpopulations were defined as shown in Figure1 and Additional file 1.

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Mouse mammary stem cells express prognostic markers for ...

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