Nanostructured Polystyrene Well Plates Allow Unbiased High-Throughput Characterization of Circulating Tumor Cells

Abstract

Rapid, reliable and unbiased circulating tumor cell (CTC) isolation and molecular characterization methods are urgently required for implementation in routine clinical diagnostic and prognostic procedures. We report on the development of a novel unbiased CTC detection approach that combines high-throughput automated microscopy with a simple yet efficient approach for achieving a high level of tumor cell binding in standard tissue culture polystyrene (PS) well plates. A single 5 min high-power oxygen plasma treatment was used to create homogeneous nanoscale roughness on standard PS tissue culture plates and, in turn, drastically enhance the binding of a range of tumor cells. After physical adsorption of an adlayer of poly-l-lysine, binding yields above 97% were obtained at 2 h for all tumor cell lines used in the study. Morphological analysis of the cells confirmed strong adherence to the nanorough PS substrates. Clinically relevant concentrations of a highly metastatic breast cancer cell line, used as model for CTCs, could be reliably detected among blood cells on the nanorough polystyrene plates using an automated microscopy system. The approach was then successfully used to detect CTCs in the blood of a stage IIIc colorectal cancer patient. By combining the high binding abilities of nanorough PS well plates with the high-throughput nature of high-content analysis systems, this methodology has great potential toward enabling unbiased routine clinical analysis of CTCs. It could be applied, once clinically validated, in any clinical center equipped with an automated microscopy facility at a fraction of the cost of current CTC isolation technologies.

Current detection technologies for circulating tumor cells

“…37 A single 5 min high-power oxygen plasma treatment was used to create homogeneous nanoscale roughness on standard PS tissue culture plates and, in turn, drastically enhanced the binding of a range of tumor cells. After physical adsorption of a layer of poly-L-lysine, binding yields above 97% were obtained at 2 h for all tumor cell lines used in the study.…”

“…However, the isolation purity from the physical methods is typically lower than that of biological methods with ligand capture. 37 …”

Biotin-Triggered Decomposable Immunomagnetic Beads for Capture and Release of Circulating Tumor Cells

“…13,18,45 Consequently, new approaches that can target Furthermore, the reaction between aldehyde group and amino group is also helpful to obtain high labeling efficiency of Strep-tag II to secondary antibody. 47 Meanwhile, Strep-tag II was controllably linked to the Fc region of IgG, allowing active Fab zone fully exposed for connecting primary antibody.…”

Detection and Clinical Significance of Circulating Tumor Cells in Colorectal Cancer—20 Years of Progress

“…Recently our group has described the use of nanoroughened polystyrene tissue-culture plates for the capture of CTCs from blood following lysis of red blood cells. The captured cells were stained using fluorophore-tagged antibodies to cytokeratins, CD45 (to preclude detection of white blood cells), vimentin, and DAPI nuclear stain (to exclude cell fragments) and detected using the Operetta automated high-content imaging system (PerkinElmer) (64). This study showed that nanoscale roughness significantly increased the binding of a range of tumor cells (up to 97%), and morphological analysis demonstrated strong adherence of the cells to the nano-rough polystyrene substrates.…”

“…This study showed that nanoscale roughness significantly increased the binding of a range of tumor cells (up to 97%), and morphological analysis demonstrated strong adherence of the cells to the nano-rough polystyrene substrates. By using these plates and the Operetta automated high-content analysis system, CTCs were detected in the blood of a stage III CRC patient, demonstrating the feasibility of this method in a clinical setting (64). However, recent technological advances in the field of imaging flow cytometry (IFC) circumvent altogether the need for CTC capture on solid substrates.…”