Image: The Becton Dickinson fluorescent activated cell sorter FACScan (Photo courtesy of the Albert Einstein College of Medicine).
A clinically proven, noninvasive fluorescence virus-guided capture system of human colorectal circulating tumor cells (CTCs) from blood samples for genetic testing has been introduced.
This noninvasive companion diagnostics is important for personalized targeted cancer therapy because current CTC detection strategies mainly depend on epithelial cell-surface markers, and the presence of heterogeneous populations of CTCs with epithelial and/or mesenchymal characteristics may pose obstacles to the detection of CTCs.
Scientists at Okayama University (Japan) developed a new approach to capture live CTCs among millions of peripheral blood leukocytes using a green fluorescent protein (GFP)-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication. The team used different cell lines and a recombinant adenovirus.
Immunochemical staining was performed on cells seeded on tissue culture chamber slides. The cells were labelled with primary mouse antibodies for various receptors and were analyzed using single cell flow cytometry (FACS; Becton Dickinson, Mountain View, CA, USA). DNA was extracted from CTC models and clinical samples and gene mutation analysis was carried out by direct sequencing and the sequence of each gene was analyzed with an ABI PRISM 3100 Genetic Analyzer (Life Technologies; Carlsbad, CA, USA).
The blood samples obtained from eight patients with gene-mutated colorectal cancers were analyzed by the replication competent adenovirus OBP-401-based CTC capture system and by allele-specific blocker polymerase chain reaction (ASB-PCR) technology. In preliminary experiments, the number of GFP-positive cells at the P3 gate was less than 10 cells in some clinical blood samples and, therefore, they performed ASB-PCR analysis using GFP-positive cells at the P2 gate. Among the eight blood samples from patients with various stages of colorectal cancer, the same gene mutations as in the primary tumors were detected in the CTCs of two patients with advanced colorectal cancer.
The authors concluded that they have established a telomerase-dependent biological CTC capture system for genotyping of epithelial, mesenchymal, and epithelial-mesenchymal transition (EMT)-induced types of CTCs using telomerase-specific replication-competent adenovirus variant OBP-401 and fluorescent activated cell sorting (FACS) analysis. This technology facilitates the surveillance of genetic alterations in viable CTCs in patients with cancer. The study was published on July 8, 2014, in the journal Gut