Enhanced Tumor Cell Isolation by a Biomimetic Combination of E-selectin and anti-EpCAM: Implications for the Effective Separation of Circulating Tumor Cells (CTCs)

Abstract

Selective detection of circulating tumor cells (CTCs) is of significant clinical importance for the clinical diagnosis and prognosis of cancer metastasis. However, largely due to the extremely low number of CTCs (as low as one in 10 9 hematologic cells) in the blood of patients, effective detection and separation of the rare cells remain a tremendous challenge. Cell rolling is known to play a key role in physiological processes such as recruitment of leukocytes to sites of inflammation and selectin-mediated CTC metastasis. Furthermore, as CTCs typically express epithelial-cell adhesion molecule (EpCAM) on the surface whereas normal hematologic cells do not, substrates with immobilized antibody against EpCAM may specifically interact with CTCs. In this paper, we created biomimetic surfaces functionalized with P-and E-selectin and anti-EpCAM that induce different responses of HL-60 (used as a model of leukocytes in this study) and MCF-7 (a model of CTCs) cells. HL-60 and MCF-7 cells showed different degrees of interaction with P-/E-selectin and antiEpCAM at a shear stress of 0.32 dyn/cm 2 . HL-60 cells exhibited rolling on P-selectin-immobilized substrates at a velocity of 2.26 ± 0.28 μm/sec whereas MCF-7 cells had no interaction with the surface. Both cell lines, however, showed interactions with E-selectin, and the rolling velocity of MCF-7 cells (4.24 ± 0.31 μm/sec) was faster than that of HL-60 cells (2.12 ± 0.15 μm/sec). On the other hand, only MCF-7 cells interacted with anti-EpCAM-coated surfaces, forming stationary binding under flow. More importantly, the combination of the rolling (E-selectin) and stationary binding (antiEpCAM) resulted in substantially enhanced separation capacity and capture efficiency (more than 3-fold enhancement), as compared to a surface functionalized solely with anti-EpCAM which has been commonly used for CTC capture. Our results indicate that cell-specific detection and separation may be achieved through mimicking the biological processes of combined dynamic cell rolling and stationary binding, which will likely lead to a CTC detection device with significantly enhanced specificity and sensitivity without any complex fabrication process.

Identification of novel molecular markers for detection of gastric cancer cells in the peripheral blood circulation using genome-wide microarray analysis

“…Magnetic beads or structures coated with EPCAM monoclonal antibodies have been recently used for circulating cancer cell separation (29)(30)(31). Although we did not compare the accuracy of the detection of gastric cancer cells by these methods to that of nested RT-PCR since we did not conduct the former assays, 30% of patients with gastric cancer exhibited EPCAM-positivity in PBMCs by nested RT-PCR.…”

Microchips and their Significance in Isolation of Circulating Tumor Cells and Monitoring of Cancers

“…The mechanism behind was the fact that combined use of the two antigens enhances the marginalization of CTCs on blood vessels, a process which facilitates the CTC isolation. This micro-fluidic system is referred to as "E-selectin biomimetic chip" (Myung et al, 2010). A study in 2011, in order to detect the KRAS gene mutation in colorectal cancer, used a special sort of chips named "Integrated CTC selection chips" with a remarkable capacity for tumor cell isolation (Dharmasiri et al, 2011).…”

Isolation of viable cancer cells in antibody-functionalized microfluidic devices

“…27 By combining E-selectin and anti-EpCAM molecules, efficient capture of target cells was reported in microfluidic chambers. 29 Highly efficient capture of CTCs was reported by using nanostructured silicon substrates with integrated chaotic micromixers. 30 Label-free cancer cell separation techniques, such as the size-based separation using deterministic lateral displacement structure 31 or using dielectrophoretic techniques, 32 were also reported.…”