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. 2015 Apr 24;10(4):e0123976.
doi: 10.1371/journal.pone.0123976. eCollection 2015.

Classification of circulating tumor cells by epithelial-mesenchymal transition markers

Affiliations

Classification of circulating tumor cells by epithelial-mesenchymal transition markers

Shiyang Wu et al. PLoS One. .

Abstract

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107 (65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.

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Conflict of interest statement

Competing Interests: SW, SL, ZL, JH, XP and JX are employees of SurExam Bio-Tech, whose company funded this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. EpCAM, CK8/18/19, vimentin and twist expression in HepG2 tumor cells and leukocytes.
A: negative control, leukocytes stained for CD45 expression (bright blue fluorescence); B: HepG2 cells stained for EpCAM expression (red fluorescence); C: HepG2 cells stained for CK8 expression(red fluorescence); D: HepG2 cells stained for CK18 expression(red fluorescence); E: HepG2 cells stained for CK19 expression(red fluorescence); F: HepG2 cells stained for vimentin expression (green fluorescence); G: HepG2 cells stained for twist expression(green fluorescence); H: HepG2 cells stained for EpCAM, CK8/18/19, vimentin and twist expression (red/green fluorescence). The cells were analyzed using a 100x oil objective
Fig 2
Fig 2. Calibration curve obtained using the optimized CanPatrol CTC enrichment technique in the spiking experiment (n = 8) using HepG2 cells at different dilutions.
Fig 3
Fig 3. The average ratio of mesenchymal CTCs in each positive sample in cancers at different stages.
Compared with the earlier stages of cancer, the average ratio of mesenchymal CTCs in each positive sample increased in the metastatic stages of cancer. The error bars indicate standard deviations.
Fig 4
Fig 4. CTCs detected in a blood sample from a liver cancer patient.
A total of 10 CTCs were detected in this sample; 3 single migratory biophenotypic epithelial/mesenchymal CTCs, 3 single migratory mesenchymal CTCs and a tumor microembolus containing 4 mesenchymal CTCs were observed (epithelial biomarkers are indicated by red fluorescence; mesenchymal biomarkers are indicated by green fluorescence).

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References

    1. Fidler IJ. The pathogenesis of cancer metastasis: the seed and soil hypothesis revisited. Nat Rev Cancer. 2003; 3: 453–458. - PubMed
    1. Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer. 2004; 4: 448–456. - PubMed
    1. Ksiazkiewicz M, Markiewicz A, Zaczek AJ. Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells. Pathobiology. 2012; 79: 195–208. 10.1159/000337106 - DOI - PubMed
    1. Guarino M. Epithelial-mesenchymal transition and tumour invasion. Int J Biochem Cell Biol. 2007; 39: 2153–2160. - PubMed
    1. Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 2009; 119: 1429–1437. 10.1172/JCI36183 - DOI - PMC - PubMed

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