Expression profiling of circulating tumor cells in metastatic breast cancer

Abstract

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients’ blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.

Signatures of breast cancer metastasis at a glance

“…When coupled to high-throughput microarray or RNAsequencing technologies and unbiased bioinformatics analyses, the aforementioned obstacles are significantly surmounted (Curtis, 2015;Magbanua and Park, 2014). Overall, these new-generation studies have allowed single-cell and bulk-cell analyses of CTCs, which efficiently capture the transcriptional heterogeneity and identify the molecular portraits of metastasizing cells (Ascolani et al, 2015;Cassatella et al, 2012;De Mattos-Arruda et al, 2013;Fina et al, 2015;Lang et al, 2015;Polzer et al, 2014;Powell et al, 2012). For example, one study has demonstrated that CTCs express various biomarkers and mediators of EMT (Yu et al, 2013), a phenotypic change crucial for the completion of the metastatic cascade (Bill and Christofori, 2015;Hanahan and Weinberg, 2011;Kalluri, 2009;Kalluri and Weinberg, 2009;Radaelli et al, 2009).…”

“…with regard to their epidermal growth factor receptor 2 (EGFR2) status. Strikingly, CTC incidence might be observed in the peripheral blood of breast cancer patients, even after removal of the primary tumor, posing additional layers of complexity with regard to the specific origin of these cells and their relationship to dissemination from the primary tumor (Baccelli et al, 2013;Bidard et al, 2014;Curtis, 2015;Lang et al, 2015;Magbanua and Park, 2014;Pukazhendhi and Glück, 2014). As such, molecular signatures derived from CTCs should always be interpreted with caution.…”

Tumour Heterogeneity: The Key Advantages of Single-Cell Analysis

“…Importantly, they found high transcript levels of genes associated with metastases: NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFβ1, ZEB2, FOXC1, CXCR4 [103]. More recently, Lang et al published a study of RNA expression profiling of breast cancer CTCs using cDNA microarrays [104]. …”

“…Robust technologies have been developed to provide genome-wide maps of most epigenetic marks. ChIP-seq, suitable for studying histone marks at the single-cell level, combines chromatin precipitation with next generation sequencing [104]. To assess methylation levels, the most comprehensive picture can be obtained from whole-genome bisulphite sequencing (WGBS) [89], but precipitation techniques (methylated-DNA immunoprecipitation sequencing and methylated–DNA-binding domain sequencing) or reduced-representation bisulphite sequencing (RRBS) [90] are also used.…”

Disseminated tumour cells in bone marrow are the source of cancer relapse after therapy

“…Most DTCs in the bone marrow display properties of epithelial‐mesenchymal transition, during which they gain expression of the mesenchymal marker N‐cadherin and lose expression of the epithelial marker E‐cadherin (Figure ) . The synergistic effect of Snail and β‐catenin confers cancer cells the ability to survive during dissemination and invasion .…”