Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

• Published in: PloS one Vol. 12; no. 1; p. e0169427
• Main Authors: Laget, Sophie, Broncy, Lucile, Hormigos, Katia, Dhingra, Dalia M, BenMohamed, Fatima, Capiod, Thierry, Osteras, Magne, Farinelli, Laurent, Jackson, Stephen, Paterlini-Bréchot, Patrizia
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.01.2017; Public Library of Science (PLoS)
• Subjects: Animals; Antigens, Neoplasm - immunology; Antigens, Neoplasm - metabolism; Biology and Life Sciences; Biomarkers, Tumor; Blood; Breast cancer; Cancer; Cancer patients; Care and treatment; Cell culture; Cell Line, Tumor; Cell Separation - methods; Cell Survival; Cytoskeleton; Cytoskeleton - metabolism; Early Detection of Cancer - methods; Early Detection of Cancer - standards; Enrichment; Extreme values; Feasibility studies; Fluorescence; Genetic Testing - methods; Genetic Testing - standards; Genomes; High-Throughput Nucleotide Sequencing; Humans; Identification; Immunohistochemistry; Immunomagnetic Separation - methods; In Situ Hybridization, Fluorescence; Lung cancer; Medicine and Health Sciences; Metastasis; Mice; Neoplasm Invasiveness; Neoplasms - diagnosis; Neoplasms - metabolism; Neoplastic Cells, Circulating - metabolism; Neoplastic Cells, Circulating - pathology; Prostate cancer; Reproducibility of Results; Research and Analysis Methods; Sensitivity; Tumor antigens; Tumor cells
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0169427

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.