This study investigated three phenomena: i) two proteins with distinct biological functions can be co-immobilized; ii) rolling and stationary binding of tumor cells can be controlled by immobilized proteins; and iii) the protein combination enhances overall capture efficiency of tumor cells.
Tables and demonstrate co-immobilization of the two proteins. The red fluorescence from APC and the green fluorescence from fluorescein from the surfaces immobilized with either anti-EpCAM or E-selectin have little to no spectral overlap (supplementary figure 1
). Furthermore, the even distribution of detected fluorescence (supplementary figure 1
) indicates that uniform immobilization of both anti-EpCAM and E-selectin was achieved. The specific correlation between fluorescence and protein presentation on the slides was confirmed by two experiments. First, the control surfaces treated with BSA exhibited neither red nor green fluorescence, indicating that non-specific protein adsorption was minimal, which is consistent with a previous report.33
Second, although E-selectin/APC-anti-EpCAM combinations showed a slightly decreased red fluorescence intensities (by ~30%) compared to the surfaces functionalized solely with anti-EpCAM at the same concentration, the decrease was marginal. By way of contrast, the green fluorescence intensities from fluorescein-anti-E-selectin substantially increased, in a non-linear fashion, with an increase of the immobilized amount of E-selectin in the protein mixtures.
Rolling and stationary binding were individually assessed to test the second phenomenon. As shown in Figure , , and , we have found that the MCF-7 response on different surfaces can be controlled from no interaction (P-selectin) and the rolling response (E-selectin) to stationary binding (anti-EpCAM). The rolling velocities of the HL-60 cells that have a high level of PSGL-1 or sialyl Lewis X (sLex
) expression were less shear stress-dependent than those of the MCF-7 cells (carcinoma cells).34
It is most likely caused by the regulation mechanisms by which leukocytic cells such as HL-60 maintain constant rolling velocities under varying flow conditions () whereas carcinoma cells do not.35, 36
Although the transient binding for rolling is a state between firm adhesion and the lack of the adhesion (i.e. no interaction), the rolling of leukocytic cells through selectins is highly stable due to a high density of selectin ligands presented on the leukocytic cells and their resistance against hydrodynamic force applied on the cells.37
It may be also related to rigidity of cells. One can easily imagine that rigid cells are typically more sensitive to shear stress than deformable cells. As a result, leukocytic cells are known to have a nearly constant rolling speed in vivo
over a wide range of shear stresses.38
It is also suspected that leukocytic cells maintain a constant rolling speed by shear dependent compensation mechanisms such as increasing the number of tethers and the number of selectin bonds so that they can be uniformly exposed to activating stimuli.35
MCF-7 cells (Carcinoma cells), on the other hand, seem to lack these mechanisms, given that they are more susceptible to changes in shear stress (). Moreover, the formation of metastatic cancers often exhibits the organ selectivity because of the different interactions between the ligands of cancer cells and the organ-specific selectins of endothelial cells for the extravasation of CTCs, which does not require CTCs to adapt the controlling mechanism of the leukocytic cells.39
MCF-7 cells exhibit the rolling behavior only on E-selectin, and as reported by Aigner et al.,34
MCF-7 cells do not interact with P-selectin. Although MCF-7 cells express CD24, a P-selectin ligand, a lack of decoration with sLex
results in weak interactions that are not strong enough to stably support rolling on P-selectin.34
E-selectin-mediated rolling of MCF-7 cells under flow was reported by Toezeren et al.40
Under the presence of laminar flow, they reported that the adhesion capacity and rolling behavior of MCF-7 cells on human umbilical endothelial cells (HUVECs) were blocked by treatment with antibody against E-selectin on the surface of HUVECs, without providing clear evidence. We have shown that clear interaction of MCF-7 cells with immobilized E-selectin in , and the behavior of MCF-7 cells was compared with HL-60. However, it is still unclear which interaction induces the observed rolling response. As a ligand of MCF-7 cells against E-selectin needs to be identified because MCF-7 cells lack most of the known ligands against E-selectin such as PSGL-1,34
There have been no definitive reports that clearly identify ligands of MCF-7 cells against E-selectin in the literature.
Adherent proteins that are involved in the metastasis process are randomly co-distributed on the endothelium.42
Thus, our hypothesis was that cooperation of adherent proteins to trap tumor cells would be more efficient than the activity of one of them alone. The surfaces with the protein mixtures (anti-EpCAM and E-selectin) indeed more efficiently recognize DsRED-MCF-7 cells out of the cell mixture with HL-60 cells than the surfaces functionalized solely with anti-EpCAMs (Figures and ). The protein combinations used in this study clearly demonstrate great potential to improve sensitivity and specificity of CTC separation and capturing from the whole blood. The capture efficiency achieved in this study is as high as approximately 35%. Enhancing hydrodynamic efficiency of the device will likely further increase the capture efficiency. That is, introduction of turbulent flow in lieu of the laminar flow we used in this study will increase the chance of cells to interact with the surface, thereby maximizing the capture efficiency. It was previously reported that microposts in a microfluidic channel18
or a chaotic mixer43
substantially increase interactions between flowing particles (cells) with microfluidic channel surfaces.
One can argue that an increase of E-selectin composition in the protein mixture may lead pre-occupation of the surface by abundant cells such as leukocytes (HL-60 in this study), resulting in binding interruption of CTCs (MCF-7 in our study). However, it would not be the case because HL-60 cells exhibit the continuous dynamic rolling response whereas MCF-7 cells remain statically adhered on the surface. That is, a thorough washing step will remove all the rolling cells, leaving only captured cells behind on the surface. Further, it is shown that the enhanced capture efficiency of MCF-7 cells by addition of E-selectin to anti-EpCAM is not interrupted by competitive binding of HL-60 cells. Instead, it is our expectation that E-selectin would be effective in pulling CTCs (MCF-7 cells in this research) along with leukocytes out of the blood flow, inducing rolling, thereby reducing the velocities of the flowing cells, which would facilitate stationary binding of CTCs by adjacent anti-EpCAM on the surface. Furthermore, given that cells exhibit significantly different rolling velocities and different levels of interactions with various proteins, the surface responses of different types of cells are expected to be easily controlled by various combinations of proteins. Another potential problem of our CTC detection method as a prognostic tool is that tumor cells are known to alter their adhesiveness and expression of various proteins on their surfaces upon therapeutic intervention.44
If the surface property alterations result in a substantial decrease in the capture efficiency of our device, a mix-and-match approach using various ratios between E-selectin and anti-EpCAM would be necessary. That is, a thorough study on the relationship between surface properties of CTCs during treatments and sensitivity/specificity of various protein combinations should be well understood prior to implementation of this method into clinics.
Taken together, it is obvious that the addition of E-selectin can induce the rolling of various cell types to be readily accessible by anti-EpCAM that recognizes/captures tumor cells, resulting in substantially enhanced capture efficiency of tumor cells by the surface – more than 3-fold enhancement as compared to the surface with anti-EpCAM alone. The E-selectin-induced tumor cell rolling most likely maximizes the chance of the tumor cells to interact with anti-EpCAM on the surface, resulting in effective stationary binding.