A naturally occurring multivalent binding effect is manipulated by engineering cell capture surfaces using dendrimers. The enhanced binding through the multivalent effect significantly improves detection of tumor cells. This improvement can be potentially translated into clinically significant detection of circulating tumor cells from the blood of cancer patients.

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.

Effective quantification and in situ identification of circulating tumor cells (CTCs) in blood are still elusive because of the extreme rarity and heterogeneity of the cells. In our previous studies, we developed a novel platform that captures tumor cells at significantly improved efficiency in vitro using a unique biomimetic combination of two physiological processes: E-selectin-induced cell rolling and poly(amidoamine) (PAMAM) dendrimer-mediated strong multivalent binding. Herein, we have engineered a novel multifunctional surface, on the basis of the biomimetic cell capture, through optimized incorporation of multiple antibodies directed to cancer cell-specific surface markers, such as epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor-2 (HER-2), and prostate specific antigen (PSA). The surfaces were tested using a series of tumor cells, MDA-PCa-2b, MCF-7, and MDA-MB-361, both in mixture in vitro and after being spiked into human blood. Our multifunctional surface demonstrated highly efficient capture of tumor cells in human blood, achieving up to 82% capture efficiency (∼10-fold enhancement than a surface with the antibodies alone) and up to 90% purity. Furthermore, the multipatterned antibodies allowed differential capturing of the tumor cells. These results support that our multifunctional surface has great potential as an effective platform that accommodates virtually any antibodies, which will likely lead to clinically significant, differential detection of CTCs that are rare and highly heterogeneous.

Tumor cell rolling on the endothelium plays a key role in the initial steps of cancer metastasis, i.e. extravasation of circulating tumor cells (CTCs). Identification of the ligands that induce the rolling of cells is thus critical to understand how cancers metastasize. We have previously demonstrated that MCF-7 cells, human breast cancer cells, exhibit the rolling response selectively on E-selectinimmobilized surfaces. However, the ligand that induces rolling of MCF-7 cells on E-selectin has not yet been identified, as these cells lack commonly known E-selectin ligands. Here we report, for the first time to our knowledge, a set of quantitative and direct evidence demonstrating that CD24 expressed on MCF-7 cell membranes is responsible for rolling of the cells on E-selectin. The binding kinetics between CD24 and E-selectin was directly measured using surface plasmon resonance (SPR), which revealed that CD24 has a binding affinity against E-selectin (K D = 3.4 ± 0.7 nM). The involvement of CD24 in MCF-7 cell rolling was confirmed by the rolling behavior that was completely blocked when cells were treated with anti-CD24. A simulated study by flowing microspheres coated with CD24 onto E-selectin-immobilized surfaces further revealed that the binding is Ca 2+ dependent. Additionally, we have found that actin filaments are involved in the CD24-mediated cell rolling, as observed by the decreased rolling velocities of the MCF-7 cells upon treatment with cytochalasin D (an inhibitor of actin-filament dynamics) and the stationary binding of CD24-coated microspheres (the lack of actins) on the E-selectin-immobilized slides. Given that CD24 is known to be directly related to enhanced invasiveness of cancer cells, our results imply that CD24-based cell rolling on E-selectin mediates, at least partially, cancer cell extravasation, resulting in metastasis.

Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.