Epithelial-mesenchymal transitioned circulating tumor cells capture for detecting tumor progression

Abstract

Purpose: This study aimed to detect cell-surface vimentin (CSV) on the surface of epithelial-mesenchymal transitioned (EMT) circulating tumor cells (CTC) from blood of patients with epithelial cancers.

Experimental design: In this study, 101 patients undergoing postsurgery adjuvant chemotherapy for metastatic colon cancer were recruited. EMT CTCs were detected from blood of patients using the 84-1 monoclonal antibody against CSV as a marker. EMT CTCs isolated were characterized further using EMT-specific markers, fluorescent in situ hybridization, and single-cell mutation analysis.

Results: Using the 84-1 antibody, we detected CSV exclusively on EMT CTCs from a variety of tumor types but not in the surrounding normal cells in the blood. The antibody exhibited very high specificity and sensitivity toward different epithelial cancer cells. With this antibody, we detected and enumerated EMT CTCs from patients. From our observations, we defined a cutoff of <5 or ≥5 EMT CTCs as the optimal threshold with respect to therapeutic response using ROC curves. Using this defined threshold, the presence of ≥5 EMT CTCs was associated with progressive disease, whereas patients with <5 EMT CTCs showed therapeutic response.

Conclusion: Taken together, the number of EMT CTCs detected correlated with the therapeutic outcome of the disease. These results establish CSV as a universal marker for EMT CTCs from a wide variety of tumor types and thus provide the foundation for emerging CTC detection technologies and for studying the molecular regulation of these EMT CTCs.

©2014 American Association for Cancer Research.

Figures

Figure 1. Cell surface vimentin expression is limited to cancer cells

(A) Immunological assessment of CSV expression in cancer (top) and normal (bottom) cell lines using flow cytometry. CSV was detected by the 84-1 antibody in only the cancer cell lines. Isotype controls were used as negative controls. (B) Cell surface staining analysis for CSV in liver cancer cell line HLM-3 and normal colon cell line NCM-356 using confocal microscopy. Cells were stained for CSV (with the 84-1 antibody; green), nuclei (with DRAQ5; blue), and the cell-surface marker WGA (red). Arrows, cell surface vimentin co-localized with WGA. Scale bar, 10 μm. (C) Immunological assessment of CSV in human primary colon cancer (HPC) cells, human colon cells metastasized to the liver (HLM), and human colon cells metastasized to the lung (CPM-1). Metastatic cancers are outlined in black; primary tumors are outlined in orange. (D) CSV detection in HPC-1-derived spheres on thin Geltrex-coated wells using confocal microscopy. CSV (green) was detectable in a few cells at the periphery of the sphere (arrows). DRAQ5 was used to demarcate the cells. Scale bar, 20 μm. (E) Vimentin and β-catenin expression in HPC-1-derived spheres on thin Geltrex-coated wells using confocal microscopy. Total vimentin (green) was detectable in a few cells at the periphery of the sphere, which correlated with the increased nuclear accumulation of β-catenin (red). DRAQ5 was used to demarcate the cells. Scale bar, 20 μm. Inset A in “Merge” image is enlarged at right.

Figure 2. Spiking assay and detection of CTCs from human colorectal cancer patients

(A) Detection of HLM-3 cells labeled with tracker dye Calcein-AM (CAM) (green) that were spiked into 7.5 mL of blood using fluorescence microscopy. Cells were also stained for nuclei (with DRAQ5; blue) and vimentin (with the 84-1 antibody; red). (B) Regression analysis of capture efficiency for up to 25 HLM-3 cells spiked in human blood. (C) Analysis of 84-1+ CD45− CTCs from human colon cancer samples that were isolated and stained for nuclei (with DRAQ; blue), total vimentin (with 84-1; green), β-catenin (red), and c-myc (red). The results indicated complete nuclear localization of β-catenin and c-myc. Scale bar, 10 μm. (D) Mutational analysis of KIT in colon cancer-derived CTC. (E) Dual-probe FISH with a centromere probe for chromosome 8 (red) and region-specific probe for KRAS (green). Three chromosome 8 signals and three KRAS signals were detected in the cell at top, while two chromosome 8 signals and four KRAS signals were detected in the cell at bottom. Nuclei were counterstained with DAPI (blue). (F) Analysis of colon cancer-derived 84-1+ CD45− CTCs from patient samples for specific molecular EMT markers. CTCs were stained for vimentin (with 84-1 antibody) and for FOXC2, SNAIL, TWIST-1, SLUG, EpCAM, and E-Cadherin with the respective antibodies. Scale bar, 5 μm.

Figure 3

(A) EMT CTCs isolated from breast, bladder, and liver cancers. CTCs were stained for vimentin (green) and nuclei (DRAQ5). (B) Enumeration of CSV+ CTCs from blood specimens from healthy volunteers and from patients with metastatic colorectal cancer. Patients were categorized based on whether the disease was stable or progressive. Horizontal bar in the graph represents the mean value of the CTC.