Abstract
The aim of the present study was to isolate and investigate the genetic heterogeneities in single circulating tumour cells (CTCs) from patients with colorectal carcinoma (CRC). Twenty-eight single CTCs were collected from eight patients with CRC using a negative immunomagnetic enrichment method. After validation with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in 3 colon cancer cell lines, a panel of 19 genes were used to analyse the single CTCs (n = 28), primary colorectal carcinoma tissues (n = 8) and colon carcinoma cells (n = 6) using real-time qPCR. Genetic heterogeneities were assessed by comparing gene expression profiles of single CTCs from the different patients and in the same patient, respectively. Genetic profiling of the single CTCs showed extensive heterogeneities of the selected genes among the CTCs. Hierarchical clustering analyses exhibited two clusters of CTCs with differentially expressed genes, which highlighted different modifications from the primary carcinomas. Further, the genetic heterogeneities were observed between different patients or in the same patient. Finally, AKT1 expression was significantly (p = 0.0129) higher in single CTCs from CRC of advanced pathological stages (III or IV) CRC than in CTCs from CRC of early stages (I or II). Our findings suggest that single-cell genetic analysis can monitor the genetic heterogeneities and guide the personalised therapeutic targets in clinical sectors.
Highlights
Circulating tumour cells (CTCs) have been considered as a minimally or non-invasive diagnostic and prognostic biomarker in patients with colorectal carcinoma (CRC) [1,2]
Recent studies showed that heterogeneous CTCs were often detected from the blood and were phenotypically and genetically different from the primary tumour [18,19,20]
We examined the gene expression of the CTCs at a single-cell resolution with comparison to the matched primary tumour tissues
Summary
Circulating tumour cells (CTCs) have been considered as a minimally or non-invasive diagnostic and prognostic biomarker in patients with colorectal carcinoma (CRC) [1,2]. Many groups have confirmed the presence of heterogeneous CTC phenotypes (such as epithelial, epithelium–mesenchymal transition (EMT) and mesenchymal/stem) in patients with different cancers, including CRC [6,7]. Immunoaffinity-dependant negative selection techniques demonstrated higher efficacy in isolating heterogeneous CTCs from the blood of patients with cancer [8,9]. Molecular characterisation of the heterogeneous CTCs showed that they were more concordant with metastatic tumours than primary tumours in patients with colorectal cancers, suggesting the acquired characteristics of the CTCs in the blood microenvironment [10]
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