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.
Highlights
Most cancer-related deaths are associated with metastasis
These changes are accompanied by a process described as epithelial-mesenchymal transition (EMT) [3], which is a complicated process that plays an essential role in metastasis [4]
HepG2 cells spiked into 5 ml of blood from the healthy volunteers and processed as per the patient samples were used as positive controls for the detection of epithelial cell adhesion molecule (EpCAM), CK8/18/19, vimentin and twist (Fig 1)
Summary
Most cancer-related deaths are associated with metastasis. Metastasis is a multi-step process with the presence of circulating tumor cells (CTCs) in the blood stream and disseminated tumor cells (DTCs) that home to the bone marrow [1]. CTCs disseminate from primary tumors by undergoing phenotypic changes that allow the cells to penetrate blood vessels [2, 3]. These changes are accompanied by a process described as epithelial-mesenchymal transition (EMT) [3], which is a complicated process that plays an essential role in metastasis [4]. Recent studies have demonstrated that this technique has failed to detect CTC subpopulations that have undergone EMT [6, 7] These studies suggested that EMT markers could be used for the detection or capture of CTCs
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