Aptamer-Mediated Transparent-Biocompatible Nanostructured Surfaces for Hepotocellular Circulating Tumor Cells Enrichment

Abstract

Circulating tumor cells (CTCs) have been considered as the origin of cancer metastasis. Thus, detection of CTCs in peripheral blood is of great value in different types of solid tumors. However, owing to extremely low abundance of CTCs, detection of them has been technically challenging. To establish a simple and efficient method for CTCs detection in patients with hepatocellular carcinoma (HCC), we applied biocompatible and transparent HA/CTS (Hydroxyapatite/chitosan) nanofilm to achieve enhanced topographic interactions with nanoscale cellular surface components, and we used sLex-AP (aptamer for carbohydrate sialyl Lewis X) to coat onto HA/CTS nanofilm for efficient capture of HCC CTCs, these two functional components combined to form our CTC-(BioT)Chip platform. Using this platform, we realized HCC CTCs' capture and identification, the average recovery rate was 61.6% or more at each spiking level. Importantly, our platform identified CTCs (2±2 per 2 mL) in 25 of 42 (59.5%) HCC patients. Moreover, both the positivity rate and the number of detected CTCs were significantly correlated with tumor size, portal vein tumor thrombus, and the TNM (tumor-node-metastasis) stage. In summary, our CTC-(BioT)Chip platform provides a new method allowing for simple but efficient detection of CTCs in HCC patients, and it holds potential of clinically usefulness in monitoring HCC prognosis and guiding individualized treatment in the future.

Keywords: Aptamer; Cell capture; Circulating tumor cells; Hepatocellular carcinoma; Hydroxyapatite/chitosan.; Nanomaterial.

Figure 1

(A-B) Schematic workflow of the CTC-^BioTChip. The biotinylated-sLeX-AP coated on HA/CTS substrate for HCC CTCs capture. (C) SEM image of HA/CTS substrate. (D) Conceptual illustration of HCC CTC interacting with sLeX-AP coated CTC-^BioTChip. (E) SEM image of HepG2 cell captured on sLeX-AP coated CTC-^BioTChip. (F) Conceptual illustration of the molecular mechanism of capturing HCC CTC by sLeX-AP coated CTC-^BioTChip. Abbreviations: CTCs, circulating tumor cells; SEM, scanning electron microscope; HA/CTS, hydroxyapatite/chitosan.

Figure 2

(A) Schematic workflow of CTC-^BioTChip fabrication. HepG2 capture yields of sLeX-AP coated CTC-^BioTChip (B) at different sLeX-AP concentration (with concentration of 10, 20, 30, 40, 50, 60 μM) and (C) at different incubation time (15, 30, 45, 60, 90min). The error bar represents standard deviation from three repeats.

Figure 3

(A-B) Five control studies and their corresponding fluorescent micrographs (DAPI nuclear staining only): 1) sLex-AP coated CTC-^BioTChip, 2) random DNA coated CTC-^BioTChip, 3) SA coated CTC-^BioTChip, 4) bare CTC-^BioTChip, 5)sLeX-AP coated glass were examined. (C) Cell capture efficiences on sLeX-AP and anti-EpCAM coated CTC-^BioTChip were validated using HepG2 (EpCAM-), MCF7 (EpCAM+), HCT116 (EpCAM+), MGC803 (EpCAM+), HeLa (EpCAM-). (D)The captured cell number of CTCs was validated from two kinds of artificial blood samples. (E)The fluorescent micrographs of cancer cells captured from the artificial blood samples. Three-color immunocytochemistry method based on FITC-labeled anti-CD45, PE-labeled anti-CK, and DAPI nuclear staining was applied to identify and enumerate CTCs from non-specially trapped WBCs. Scale bars are 20μm. All capture efficiency experiments were under optimal condition. The error bar represents standard deviation from three repeats. Abbreviations: CTCs, circulating tumor cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; DAPI, 4 ,6-diamidino-2-phenylindole dihydrochloride; WBCs, white blood cells.

Figure 4

(A) CTCs isolated from a HCC patient. Three-color immunocytochemistry method based on FITC-labeled anti-CD45, PE-labeled anti-CK, and DAPI nuclear staining was applied to identify and enumerate CTCs from non-specifically trapped WBCs. (B) CTC enumeration results obtained from 42 cancer patients. Scatter plot for CTCs number of HCC patients(C) with and without portal vein tumor thrombus, (D) different tumor size and (E) different TNM stage, each red dots stand for one HCC patient. The error bar represents standard error of the mean. Abbreviations: CTCs, circulating tumor cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; DAPI, 4 ,6-diamidino-2-phenylindole dihydrochloride; WBCs, white blood cells.