Aptamer–polymer functionalized silicon nanosubstrates for enhanced recovered circulating tumor cell viability and in vitro chemosensitivity testing

Abstract

Selection of the optimal chemotherapy regimen for an individual cancer patient is challenging. The existing chemosensitivity tests are costly, time-consuming, and not amenable to wide utilization within a clinic. This limitation might be addressed by the recently proposed use of circulating tumor cells (CTCs), which provide an opportunity to noninvasively monitor response to therapy. Over the past few decades, various techniques were developed to capture and recover CTCs, but these techniques were often limited by a capture and recovery performance tradeoff between high viability and high efficiency. In this work, we used anti-epithelial cell adhesion molecule coated aptamer–poly (N-isopropylacrylamide) functionalized silicon nanowire substrates to capture and release epithelial cell adhesion molecule-positive CTCs at 32°C and 4°C, respectively. Then, we applied the nuclease to digest the aptamer to release the captured CTCs (near or at the end of the polymer brush), which cannot be released by heating/cooling process. High viability and purity CTCs could be achieved by decreasing the heating/cooling cycles and enzymatic treatment rounds. Furthermore, the time-saving process is helpful to maintain the morphology and enhance vitality of the recovered CTCs and is beneficial to the subsequent cell culture in vitro. We validated the feasibility of chemosensitivity testing based on the recovered HCC827 cells using an adenosine triphosphate–tumor chemosensitivity assay, and the results suggested that our method can determine which agent and what concentration have the best chemosensitivity for the culturing recovered CTCs. So, the novel method capable of a highly effective capture and recovery of high viability CTCs will pave the way for chemosensitivity testing.

Keywords:

• circulating tumor cells
• aptamer–PNIPAM coating
• capture and recovery
• cell culture in vitro
• chemosensitivity testing

Supplementary materials

Figure S1 SEM image of a patterned SiNWS (A). SEM image of biotin-aptamer-PNIPAM growth on SiNWS (B).

Abbreviations: PNIPAM, poly (N-isopropylacrylamide); SEM, scanning electron microscopy; SiNWS, silicon nanowire substrates.

Figure S2 Dynamic ranges of the anti-EpCAM-coated Ap-P-SiNWS chips using a series of artificial NSCLC CTC samples that were prepared by spiking PBS and healthy donor’s blood with DIO-stained HCC827 cells.

Abbreviations: Ap-P-SiNWS, aptamer–PNIPAM-SiNWS; CTC, circulating tumor cell; DIO, 3,3′-dioctadecyloxacarbocyanine; EpCAM, epithelial cell adhesion molecule; PBS, phosphate-buffered saline; PNIPAM, poly (N-isopropylacrylamide); SiNWS, silicon nanowire substrates.

Figure S3 The cell release performance of the Ap-P-SiNWS chips as the foundation of the concentrations (1.0 to 40 μM) of Benzonase.

Notes: The 20 μM of Benzonase concentration is determined for releasing the captured CTCs onto Ap-P-SiNWS.

Abbreviations: Ap-P-SiNWS, aptamer–PNIPAM-SiNWS; CTC, circulating tumor cell; h, hours; PNIPAM, poly (N-isopropylacrylamide); SiNWS, silicon nanowire substrates.

Figure S4 Heating/cooling cycles affected the viability of recovered cells.

Figure S5 The purity study and molecular analysis of recovered HCC827 cells.

Notes: The scatter plot summarizes the HCC827/WBC cell distribution (with a purity of 93.8%) in one of the cell suspensions obtained from the heating/cooling process and enzyme digestion study (A). Mutation analyses of KRAS on the HCC827 cells recovered from the heating/cooling process and enzyme digestion studies using the anti-EpCAM-coated Ap-P-SiNWS chips (B).

Abbreviations: Ap-P-SiNWS, aptamer–PNIPAM-SiNWS; EpCAM, epithelial cell adhesion molecule; FITC, fluorescein isothiocyanate; PNIPAM, poly (N-isopropylacrylamide); SiNWS, silicon nanowire substrates; WBC, white blood cell.

Acknowledgments

The authors thank the National Natural Science Foundation of China (81472748 and 81301084), Natural Science Foundation of Hubei (2014CFB155), and Applied Basic Research Program of Wuhan Science and Technology Bureau (2014062801011263) for supporting this work.

Disclosure

The authors report no conflicts of interest in this work.