Towards High Performance Detection of Circulating Tumor Cells in ...

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ScienceDirect Procedia Engineering 120 (2015) 380 – 383

EUROSENSORS 2015

Towards high performance detection of circulating tumor cells in whole blood Marc Zinggeler, Thomas Brandstetter, Jürgen Rühe* Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, GeorgesKöhler-Allee 103, 79110 Freiburg, Germany

Abstract A novel dead-end affinity filtration system is presented for the enrichment of circulating tumor cells (CTCs) from whole blood with high efficiency, selectivity and throughput. At the heart of the system lies an affinity membrane, which was prepared by chemical surface modification of a commercial micro-filter to exclusively allow for the specific adhesion of CTCs. First filtration experiments showed that the prepared membranes are highly permeable for blood cells and allow the efficient capture of cancer cells at high filtration pressure. This promising combination is expected to enable high quality enrichments, which are required for subsequent CTC identification and characterization. © 2015 2015Published The Authors. Published by isElsevier © by Elsevier Ltd. This an open Ltd. access article under the CC BY-NC-ND license Peer-review under responsibility of the organizing committee of EUROSENSORS 2015. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of EUROSENSORS 2015

Keywords: Circulating tumor cells; enrichment; capture; detection; affinity filtration

1. Introduction Circulating tumor cells (CTCs) can be found in patient´s blood at typical concentrations of 0.3-100 cells per mL [1] and are becoming increasingly important as biomarkers for various cancers [2]. Accordingly, it is of high interest to develop an automatable, high performance cell enrichment technology, which allows to analyze large blood volumes of up to 100 mL. Such a method would allow for the reliable detection and characterization of those rare cells by immunocytochemistry (ICC) or molecular methods like polymerase chain reaction (PCR). Existing filtration devices,

* Corresponding author. Tel.: +49-761-203-7160; fax: +49-761-203-7162. E-mail address: [email protected]

1877-7058 © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of EUROSENSORS 2015

doi:10.1016/j.proeng.2015.08.645

Marc Zinggeler et al. / Procedia Engineering 120 (2015) 380 – 383

which separate CTCs from blood cells based on physical differences (i.e. cell size and deformability) are simple and achieve high throughputs. However, they suffer from low selectivity since CTCs and the abundant white blood cells (WBCs) often show overlapping size distribution profiles [3]. The bio-chemical recognition of specific cell surface markers in contrast can be highly selective and is exploited by various affinity (adhesion) based separation methods [4]. In this group, the commercially available fluorescence- or immunomagnetic-activated cell sorting technologies suffer from extensive sample pre-treatment and the complexity of their procedures, while the recently developed, much simpler chip-technologies, are mainly lacking in throughput [1]. To address this limitations, we study a novel approach based on the combination of classical dead-end filtration with affinity separation. First, a surface engineering process was developed to generate highly selective capture surfaces on commercial micro-filters. The obtained affinity membranes should solely allow for the specific adhesion of target cells and can be used in a simple dead-end filtration setup (e.g. applied as syringe filters). To achieve a high capture efficiency, the pore size of the used membrane is chosen to be smaller than the smallest CTCs under investigation. Applying sufficient filtration pressure should even force the largest WBCs to pass through the pores, while CTCs interacting strongly with the pore walls should remain tightly captured on the filter (see figure 1).

Figure 1. Schematic depiction of the developed dead-end affinity filtration system for the selective capture of CTCs from whole blood.

2. Surface engineering process Water-swellable, surface-attached, neutral polymer networks (hydrogels) were found to protect surfaces effectively from non-specific adhesion of proteins and cells [5]. However, when functionalized with specific groups, the coatings allowed to trigger the specific adhesion of cells [6]. Based on this concept a surface engineering process was developed to generate a CTC-specific capture surface on plastic substrates (see figure 2A). A

B

Figure 2. A) Schematic of the developed surface engineering process to generate highly selective capture surfaces on plastic substrates. B) Obtained surface concentration and distribution of immobilized streptavidin (fluorescence labelled) using the developed modification process.

1. The substrate is coated with polydimethyl acrylamide co methacryloyl benzophenon (PDMAA-MABP), which contains the photoactive cross-linker benzophenone (BP), by dip coating and subsequently illuminated with UV-light

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(pre-exposure) to initiate both cross-linking and surface-attachment by C-H insertion reactions [5]. 2. The surface, which still contains active BP units, is saturated with streptavidin, followed by another UV-treatment and a washing step to remove non-bound protein. 3. The surface is completed with target cell specific biotinylated capture molecules (e.g. antibodies) by simple incubation. The developed process yielded a homogeneous distribution of stably immobilized streptavidin and allowed to control the degree of functionalization by varying the energy dose of the preexposure (see figure 2B). 3. Selectivity of the capture surface The selectivity of the developed capture surface was studied both on the level of proteins and cells. The interaction with proteins was measured by surface plasmon resonance spectroscopy (SPR). To this a gold chip was coated with the streptavidin functionalized hydrogel (with the highest degree of functionalization) using the process described in the previous section. The surface was then incubated with different protein solutions, while the reflectivity, which is proportional to an increase in layer thickness, was tracked in real-time (see figure 3A). It was found that the functional hydrogel coating was resistant against non-specific protein adsorption, which was measured by incubation with concentrated fibrinogen (Fg) solution. The functionality of the surface was confirmed by immobilizing a biotinylated antibody against the epithelial cell adhesion molecule (EpCAM), which is the most frequently used marker for CTC enrichment [7]. Finally, the completed capture surface was used successfully to detect the EpCAM protein in solution. The interaction of cells with the anti-EpCAM based capture surface was studied by incubation under shear flow. Figure 3B shows fluorescence micro-graphs of the capture surface after incubation with stained WBCs or MCF-7 breast cancer cells (CTC-model) under identical shear conditions (0.9 dyn/cm2). It was observed that the cancer cells were captured selectively on the surface and no permanently deposited WBCs were observed even at shear stresses < 0.05 dyn/cm2 (not shown). A

B WBCs

MCF-7

Figure 3. Selectivity of the developed capture surface on A) the protein- and B) cell-level.

4. Preparation and characterization of affinity membranes Track-etched polycarbonate micro-filters (PCTE) have shown superior performance in size-selective CTC enrichment compared to other filter types [8] and were therefore selected as support for the developed capture surface. Affinity membranes were prepared by modifying the surface of commercial PCTE (with 10 μm pores) using the described process (see section 2). After modification, the surfaces were found to be completely covered by the functional hydrogel coating (see figure 4A).


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Figure 4. A) Fluorescence micro-graph of a modified PCTE membrane (10 μm pores). The surface on the right (bright) side was functionalized with fluorescent streptavidin. It is visible that both the cover surface and the pores (bright rings) are covered by the functional network. B) Measured (optical) size distribution profiles for MCF-7 cells and WBCs.

First filtration experiments with whole blood showed that the prepared membranes are highly permeable for blood cells and just minor pressures of 10-20 mbar were required to achieve remarkable throughputs of 10-20 ml/min (filter area = 1.5 cm2). Additionally, capture experiments with MCF-7 cells suspended in viscous PVP solution were performed. These cells were selected as a model for breast cancer CTCs and show an overlapping size distribution profile with WBCs (see figure 4B). Using filtration pressures between 70-100 mbar > 90 % of cancer cells were retained on the affinity membrane, while the capture efficiency of a hydrogel coated PCTE lacking the anti-EpCAM (pure size selection) dropped below 50 %. This results support our hypothesis that 1. target cells interacting with the capture surface are retained on the affinity membrane even at high filtration pressures and 2. increased filtration pressure clears the filter surface from non-interacting cells (i.e. background cells). 5. Conclusion and outlook Cell selective affinity membranes were prepared by chemical surface modification of commercial PCTE microfilters. The generated membranes can be used in a simple dead-end filtration setup and could enable the enrichment of CTCs from whole blood with high selectivity, efficiency and throughput. Currently, the performance of the system is being optimized for the capture of cancer cells spiked into whole blood and a combination of ICC followed by whole cell PCR is studied for target cell detection and characterization directly on the membrane surface. Acknowledgements We gratefully acknowledge Ms. Birgit Holzwarth for her assistance with cell culture and the Zentrales Innovationsprogramm Mittelstand - ZIM (KF2162028AJ3) for financial support. References [1] U. Dharmasiri, M.A. Witek, A.A. Adams, S.A. Soper, Microsystems for the capture of low-abundance cells, Annu. Rev. Anal. Chem. 3 (2010) 409–431. [2] S.A. Joosse, T.M. Gorges, K. Pantel, Biology, detection, and clinical implications of circulating tumor cells, EMBO Mol. Med. 7 (2015) 1-11. [3] M.S. Kim, T.S. Sim, Y.J. Kim, S.S. Kim, H. Jeong, J. Park, H. Moon, S.I. Kim, O. Gurel, S.S. Lee, J.C. Park, SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter, Lab Chip 12 (2012) 2874-2880. [4] T.F. Didar, M. Tabrizian, Adhesion based detection, sorting and enrichment of cells in microfluidic Lab-on-Chip devices., Lab Chip 10 (2010) 3043–3053. [5] A. Wörz, B. Berchtold, K. Moosmann, O. Prucker, J. Rühe, Protein-resistant polymer surfaces, J. Mater. Chem. 22 (2012) 19547–19561. [6] S. Loschonsky, K. Shroff, A. Wörz, O. Prucker, J. Rühe, M. Biesalski, Surface-attached PDMAA-GRGDSP hybrid polymer monolayers that promote the adhesion of living cells, Biomacromolecules 9 (2008) 543–552. [7] C. Alix-Panabières, K. Pantel, Challenges in circulating tumour cell research, Nat. Rev. Cancer. 14 (2014) 623-631. [8] F.A.W. Coumans, G. van Dalum, M. Beck, L.W.M.M. Terstappen, Filter characteristics influencing circulating tumor cell enrichment from whole blood., PLoS One 8 (2013) e61770.