Removal of Circulating Tumor Cells from Blood Samples of Cancer Patients Using Highly Magnetic Nanoparticles: A Translational Research Project

Abstract

The count of circulating tumor cells (CTCs) has been associated with a worse prognosis in different types of cancer. Perioperatively, CTCs detach due to mechanical forces. Diagnostic tools exist to detect and isolate CTCs, but no therapeutic technique is currently available to remove CTCs in vivo from unprocessed blood. The aim of this study was to design and test new magnetic nanoparticles to purify whole blood from CTCs. Novel magnetic carbon-coated cobalt (C/Co) nanoparticles conjugated with anti-epithelial cell adhesion molecule (EpCAM) antibodies were synthesized, and their antifouling and separation properties were determined. The newly developed C/Co nanoparticles showed excellent separation and antifouling properties. They efficiently removed tumor cells that were added to healthy subjects' blood samples, through an anti-EpCAM antibody interaction. The nanoparticles did not interact with other blood components, such as lymphocytes or the coagulation system. In blood samples of carcinoma patients suffering from metastatic disease, on average, ≥68% of CTCs were removed. These nanoparticles could prompt the development of a blood purification technology, such as a dialysis-like device, to perioperatively remove CTCs from the blood of cancer patients in vivo and potentially improve their prognosis.

Keywords: blood purification; circulating tumor cells; nanoparticles.

Figure 1

Overview of working principle in blood from cancer patients. (1) Blood from cancer patients with circulating tumour cells (CTC) (light blue) is drawn, and CTC are deterimined. (2) Nanoparticles, coated with anti-EpCAM antibodies, are added. (3) Through nanoparticles, captured tumor cells are eliminated by a strong magnet, followed by a CTC enumeration.

Figure 2

Nanoparticle synthesis and characterization. (a) Overview of the synthesis of antibody-functionalised Co/C nanoparticles. (b) Transmission electron micrographs, particle size distribution (SI). Black dots represent nanoparticles. Scale bar: 200 nm. (c) Graphic representation of particle size distribution. (d) Scanning electron micrograph of a HT-29 cancer cell after incubation with anti-EpCAM-functionalised Co/C nanoparticles. The blue area delimits a HT-29 cell, and the red area nanoparticles bound on the cell surface. Scale bar: 300 nm.

Figure 3

Nanoparticle optimization. (a) Separability: The graph depicts a representative of several experiments performed with different batches. (b) Antifouling: the fraction of bovine serum albumin (BSA) absorbed was determined in particles with a different number of polymer units. (c) Efficiency: CTC-absorption was determined using nanoparticles with a different polymer unit.

Figure 4

Removal of CTCs. (a) Flow cytometry data of filtrates obtained after removal of CTCs from healthy donors’ blood spiked with tumor cells using IgG isotype and anti-EpCAM-coated nanoparticles. The gate indicates the region where the spiked HT-29 cells appear. (b) Reproducibility of the experiment, which was repeated three times with three different optimized anti-EpCAM nanoparticles batches in comparison to IgG istotype nanoparticles. (c) Flow cytometry results after treatment of blood with anti-EpCAM-functionalized nanoparticles focusing on granulocytes, monocytes and lymphocytes. (d) Percentage of remaining granulocytes, monocytes and lymphocytes after IgG isotope or anti-EpCAM particle treatment.