Specific Capture And Release Of Circulating Tumor Cells Using Aptamer Modified Nanosubstrates

Lung cancer is the leading cause of cancer deaths in the worldwide. The most common cause of deaths in patients with lung cancer is metastases, while the molecular mechanisms of cancer metastases are still largely unknown, there is a considerable body of evidence indicating that tumor cells are shed from a primary tumor mass at the earliest stages of malignant progression. These ’break-away’ cancer cells enter the blood stream and travel to different tissues of the body as the cellular origin of metastases. The cells that escape from the primary tumor and are implicated in metastases are known as circulating tumor cells (CTCs). The gold standard of examining disease status of tumors is through the genetic and proteomic analysis of the biopsy samples. In early stage metastasis, it is difficult to identify the metastatic sites for biopsy. CTCs can be regarded as the ’liquid’ biopsy of the tumor, thus providing convenient access to primary tumor cells, and more important, lethal metastases. Over the past decade, a diversity of technologies capable of counting and isolating CTCs have been developed based on different operation mechanisms. However, detection and characterization of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds of cells per mL) of CTCs among a high number (109cells/mL) of haematologic cells in the blood.Objective The aim of this study is to investigate the specificity and affinity of Ap-1and Ap-2that generated by an in vitro cell-SELEX (systematic evolution of ligands by exponential enrichment) process to A549NSCLC cells,to test the feasibility of specific capture and release of CTCs using aptamer modified nanosubstrates,and to examine the feasibility of performing subsequent molecular and functional analyses on the CTCs recovered from the two rounds of specific capture and release processes.Methods In this study, we screen two single-stranded oligonucleotides Ap-1and Ap-2with high specificity and affinity to A549NSCLC cells by an in vitro cell-SELEX process. We perform specific capture CTCs in patients blood samples using aptamer modified nanosubstrates. We carry out specific release immobilized cells by treatment nuclease.We performe viability studies by AO/EB staining and culturing the recovered A549cells.We identify KRASGI2S mutation in A549cells recovered from the two rounds of capture/release processes, by performing PCR amplifications, followed by Sanger sequencing.Results 1. The results demonstrated that the screened two DNA-aptamers had high specifity and affinity with A549cells and could replace the antibody-based capture agent anti-EpCAM.2. The results implied that flow rates at1.0mL h-1had the best capture efficiency. More than70%of the cells were captured in the first8cm of the aptamer-coated NanoVelcro Chips, the results suggest that a22-cm-long microchannel is sufficient to achieve the desired cell-capture performance. By integrating a NanoVelcro substrate with a polydimethylsiloxane (PDMS)-based chaotic mixer further improved CTC capture efficiency. The results suggest that the aptamer-coated NanoVelcro Chips were capable of specifically capturing NSCLC cells, while there were less then10%the other cells (i.e., Hela, PC3, U87, and Jurkat cells, and WBCs) non-specifically trapped in the devices.3. The results showed that the substrate-immobilized A549cells were then released via enzyme treatment, more than85%of A549cell release and less than15%WBC release performance from the experimental data, and after the1st round of capture/release process the final purities of A549cells are in the rage of15±5%(n=5). But after two rounds of capture/release processes the final purities of A549cells are greater than95%.4. The results suggest that the recovered A549cells exhibited78-83%viability and could be continuously passaged. The KRASGI2S mutation in A549cells recovered from the two rounds of capture/release processes can be detected by performing PCR amplifications and Sanger sequencing.Conclusions We have demonstrated the NanoVelcro Chip that is capable of not only capturing NSCLC CTCs from blood with high efficiency, but also recovering the nanosubstrate-immobilized NSCLC CTCs upon treatment of a nuclease solution. Two single-stranded DNA-aptamers (Ap-1and Ap-2) were generated via Cell-SELEX process to replace conventional anti-body based capture agents, allowing specific capture and release of NSCLC cells from whole-blood samples using aptamer-grafted NanoVelcro Chips. The capturing and releasing features enable isolation of circulating tumor cells (CTCs) with minimum contamination of the surrounding white blood cells (WBCs) and negligible disruption to CTCs’ viability and functions, thus paving the way toward molecular and functional analyses of CTCs. It is conceivable that, by this new diagnostic platform, the CTC-derived molecular signatures and functional readouts will provide valuable insight into tumor biology during the critical window where therapeutic intervention could make a significant difference.