Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells

Abstract

We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine.

Figure 1.

A: Box plots of cell areas according to cell type. Each box has its ends at the quartiles, and the median of distribution is marked by a line within the box. The whiskers at either end extend to the 10th and 90th percentiles. A significant difference (P < 0.001) in the whole cell area was found between tumor cells from any of the carcinoma cell lines and PBL. B: Sensitivity test. One MCF-7 (a and a′) or LNCaP (b and b′) cell was micropipetted under the microscope, mixed with 1 ml of blood, and recovered using ISET. a and b: Low magnification (×10) showing the area of filtration and the single cell (arrow). a′ and b′: The same cell as in a and b, but at a higher magnification (×100). Immunostaining was with KL1 antibody. Note the well-preserved morphological details. Arrows: 1, tumorous cell; 2, membrane pore; 3, leukocyte (trapped in a pore lumen).

Figure 2.

Immunocytochemical characterization of cells mixed with peripheral blood and recovered using ISET. a–d: Cells from tumorous cell lines. a: HepG2 cells stained with anti-KL1 (×60). b: Anti-albumin-positive Hep3B cell (×40). c: Double staining of HepG2 cells and leukocytes with anti-AFP and anti-LCA (×40). d: LNCaP cells positive to the anti-MMP3 antibody (×40). f–h: Anti-AFP-positive tumorous cells isolated from patients with hepatocellular carcinoma before tumor resection (f, ×100) and during surgery (g and h, ×100). A tumor embolus is shown in f (arrow 4) to reproduce the histological lobular structure observed in the tumorous tissue from the same patient (e, H&E staining, ×20). Cell nuclei were counterstained with Mayer’s hematoxylin. Arrows: 1, tumorous cells; 2, membrane pores; 3, leukocytes.

Figure 3.

A: Laser microdissection of a Hep3B cell and p53 gene analysis. a: H&E-stained cells on the filter. The membrane around a single cell is precisely cut by the laser microbeam (b) and catapulted by laser pressure (c). d: Inversion of the optical device allows a check that the cell has now been collected into the microfuge lid. B: Gel electrophoresis and ethidium bromide staining of DNA from single HepG2 cells (lanes 1 and 2) and Hep3B cells (lanes 3 and 4) coamplified with p53 primers on exon 8 to 9 and HLA primers. Lane M: 100-bp molecular weight marker. Lane B: PCR-negative control (buffer only). The figure shows that only Hep3B cells carry an homozygous deletion of the p53 gene. C: FISH performed with centromeric chromosome 1-specific probes on HepG2 (a) and MCF-7 (b) cells. a: Biotinylated probe revealed using avidin-fluorescein isothiocyanate. The nucleus is counterstained with propidium iodide. Two spots are clearly visible, indicating two chromosomes one. b: Three red spots (fluorophore-labeled probe) reveal the presence of three chomosomes 1 in MCF-7 cells. Nuclei are counterstained using 4,6-diamidino-2-phenyl-indole (DAPl, 1 μg/ml).