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.
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.
Ein natürlicher Multivalenzeffekt wurde bei der Modifizierung einer Zellen einfangenden Oberfläche mit Dendrimeren genutzt. Die verstärkte Bindung aufgrund dieses Multivalenzeffekts verbessert den Nachweis von Tumorzellen deutlich, was für klinische Tests auf zirkulierende Tumorzellen im Blut von Krebspatienten interessant sein könnte.
Multivalent interactions, simultaneous binding of multiple ligands toward their multiple receptors, commonly occur in physiological and pathological events, which have been utilized to enhance targeting efficacy. The multivalent binding can be leveraged to develop an extremely sensitive device for detection of rare cells such as circulating tumor cell (CTC). Accurate enumeration of CTCs is of clinical importance in diagnosis and prognosis of cancer metastasis; however the rareness of CTCs, comprising as few as one tumor cell in the background of one million-one billion of normal blood cells, has made the effective detection of the cells extremely difficult. Here we report a novel surface chemistry that utilizes the multivalent effect for a highly stable cancer cell capture. Poly(amidoamine) (PAMAM) dendrimers have been reported as an excellent multivalent binding mediator due to their deformability and multiple functional groups. PAMAM dendrimers were conjugated with anti-epithelial cell adhesion molecules (aEpCAM), a commonly used antibody for CTC detection. A direct, quantitative analysis using surface plasmon resonance (SPR) revealed that the aEpCAM-dendrimer conjugates exhibited a dramatically enhanced binding avidity by a million-fold, compared to free aEpCAM. Dendrimers and aEpCAM were successfully immobilized on the surfaces, as confirmed by x-ray photoelectron spectroscopy and fluorescence microscopy. The surfaces were tested using in vitro cell lines (MDA-MB-361, MCF-7, and MDA-MB-231 cells as a CTC model). Surface immobilization of the dendrimer-aEpCAM conjugates resulted in a significantly enhanced tumor cell detection than the surfaces with the linear PEG-aEpCAM conjugates by ∼1.9 fold with higher binding stability over agitation (up to 69.1-fold). The enhancement through dendrimer-mediated multivalent binding effect was further increased up to 7-fold with addition of E-selectin that recruits the cancer cells onto the surface and induces their rolling under flow. This study demonstrates a novel surface engineering approach to exploiting the strong multivalent binding for sensitive detection of tumor cells, which has great potential for clinically significant detection of CTCs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2388. doi:1538-7445.AM2012-2388
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