Abstract
BackgroundTo improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.Methodology/Principal FindingsWe purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.Conclusions/SignificanceFor the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery.
Highlights
To cure epithelial-based cancers–such as cancers of the breast, prostate, lung, colon, and pancreas–therapies need to be directed toward those cells that cause metastases
We demonstrated that high dimensional single cell analysis reliably characterizes tumor cells using 96.96 Dynamic Arrays to measure the expression of 87 cancer-associated and reference genes in individual cells isolated from primary and metastatic breast cancer cell lines
This exploratory panel of genes was selected from the published literature and our previous work in breast cancer gene expression for their role in molecular pathways relevant to breast cancer and to represent breast cancer biomarkers, prognostic markers, and phenotypes associated with cancer signaling pathways, epithelial-mesenchymal transition (EMT), cancer stem cells, and metastasis, as well as phenotypes indicative of contaminating leukocytes (Table S1)
Summary
To cure epithelial-based cancers–such as cancers of the breast, prostate, lung, colon, and pancreas–therapies need to be directed toward those cells that cause metastases. Secondary tumors may reseed additional metastatic cells into the bloodstream [1,2], causing subsequent tumor spread that result in multiple metastatic tumors within the same organ and colonization of tumor cells in additional organs, generally leading to patient demise. While considerable progress has been made towards elucidating the basic biology of primary tumors to guide therapy, the molecular characterization of metastatic disease, which generally occurs months or years after primary tumor excision, remains limited. The treatment of patients with metastatic disease continues to be based largely on biomarkers from their primary tumor, despite frequent discordance between primary and metastatic cancer [3,4]. Some patients with disseminated disease may undergo biopsy of a single metastatic focus even though multiple foci are concurrently present. We demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery
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