Genetic Heterogeneity of Single Circulating Tumour Cells in Colorectal Carcinoma

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.

Gene Expression Analysis of Immune Regulatory Genes in Circulating Tumour Cells and Peripheral Blood Mononuclear Cells in Patients with Colorectal Carcinoma

“…Hence, gene expression profiling of single CTCs would be more beneficial in providing more accurate information. Nowadays, the molecular profiling of single CTCs in different cancers, including colorectal carcinomas, is receiving more attention [34,35,41,42]. As this study is an ongoing project, we have obtained some preliminary results with single CTCs isolated from individual patients, which further confirm our findings.…”

“…Steinert et al noted that downregulation of c-Myc may indicate the state of dormancy of CTCs (if Ki-67 expression is also low) [34]. Though no significant variations were found between high vs. low CTC-positive groups, possibly due to the extensive heterogenous nature of CTCs [35], we found comparatively higher expression of these genes in patients detected with low CTC counts. Taken together, our findings suggest that changes in the mRNA expression level of tumour suppressor genes and oncogenes, especially KRAS in CTCs, may become more aggressive at the early stages; thus, CTCs could play a significant role in predicting targeted therapy at the onset of the disease.…”

“…However, our data showed decreasing expression level of CD47 in patients with CRC compared with healthy donors, though positive expression were seen in a number of individual patients. This may have happened because of the heterogeneous characteristics of CTCs [35]. The above findings may suggest that CTLA-4 is also expressed in CTCs along with other immune checkpoint molecules, so blocking these inhibitory molecules could improve their therapeutic efficacy.…”

Detection and clinical significance of circulating tumor cells in colorectal cancer

“…A comprehensive analysis of the information shared by the CTC population can be used to judge the origin and diagnosis of tumors. CTCs also carry acquired mutations different from those found at the sites of exfoliated, which can be used to monitor tumor drug resistance and the acquisition of metastatic potential [ 76 ]. Combining these two types of information can provide unique insights on tumor heterogeneity at the level of the tumor genome, transcriptome, proteome and function [ 77 , 78 ].…”

Editorial for the Special Issue “Molecular Biomarkers in Colorectal Adenocarcinoma”

“…Based on the great potential of CTCs as a non-invasive prognostic or diagnostic approach, another interesting study, conducted by Hamid et al, investigated the genetic heterogeneities in CRC patients’ CTCs [ 6 ]. The genetic profiling of single CTCs revealed the heterogenous expression of particular genes among CTCs from different patients but in the same individual, as well.…”