Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell

Abstract

Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule) or muc1 (mucin1) expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell) occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon). The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots) and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1) or BHQ2 (black hole quencher2). In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.

Keywords: ALB; EpCAM; aptamer; metastasis; molecular beacon.

Figure 1

Schematic illustration of: QD₅₂₅-muc1 ALB (A); cy5.5-EpCAM ALB (B); and QD₅₆₅-EpCAM ALB (C). The EpCAM expression as an evidence of CTC was determined by the QD signal of ALB. The predicted secondary structures of full-length EpCAM ALB aptamer modeled using Mfold.

Figure 2

Specificity of the QD₅₆₅-EpCAM ALB to sense EpCAM expression of CTCs in vitro: (A) LAS 4000 images of Panc02 cells treated with the QD₅₆₅-EpCAM ALB in PCR tube were shown. A fixed concentration of the QDs was incubated with various cell numbers of the EpCAM-targeting protein to detect the optimal contents of EpCAM needed to perform the best specific effect; (B) Fluorescence intensity of the QD₅₆₅-EpCAM ALB after incubated with the different cell numbers of Panc02 cells. ROI analysis from the fluorescence tube image showed that the fluorescence signal increased in a cells-dependent manner. Data are displayed as mean ± standard deviations of triplicate samples (** p < 0.005). The Confocal images QD525 and QD565 were recorded under excitation of 525 and 565 nm, respectively. The scale bar in the CLSM images is 20 μm. Fluorescence intensity of QD565-EpCAM/QD525-muc1 ALB on Panc02 cells after caerulein treatment (C).

Figure 3

Selectivity study of various probe (RS; random sequence, EpCAM ALB, antibody, and mutant) against different cell lines including EpCAM-positive cell lines: (A) breast cancer cell line MDA-MB-231; (B) human gastric carcinoma cell line Kato III; and (C) negative cell line human kidney epithelial cell line HEK-293T.

Figure 4

Confocal images of CTC existence from the Panc02 cells by the dose-dependent caerulein treatment after incubated with: EpCAM ALB (QD₅₆₅, 1 pmol, specifically recognizes EpCAM), muc1-ALB (QD₅₂₅-labeled, 2 pmol, specifically recognizes muc1) (A); and EpCAM/muc1 mutant probe (QD₅₂₅/QD₅₆₅-labeled, 2.5 pmol, specifically recognizes EpCAM/muc1, respectively) for 2 h (B). The Confocal images QD₅₂₅ and QD₅₆₅ were recorded under excitation of 525 and 565 nm, respectively. The scale bar in the CLSM images is 15 μm. Fluorescence intensity of QD-EpCAM/muc1 ALB on CTC cells after a dose-dependent caerulein. Data are displayed as mean ± standard deviations of triplicate samples (** p < 0.005). P/C: phase contrast.

Figure 5

Confocal microscopy imaging of CTC cells mixed with bloods and NIF-EpCAM/muc1 ALB probes. Either 5 pmol NIF-EpCAM ALB or 10 pmol NIF-muc1 ALB was mixed with isolated-bloods two weeks after caerulein injection in Panc02-implanted mice (A). Fluorescence signals two weeks after caerulein treatment were significantly enhanced compared to those of the untreated caerulein in Panc02-implanted group. Positive cell numbers of QD-EpCAM/muc1 ALB on CTC cells in bloods after caerulein treatment (B). Data are represented as mean ± standard deviations of triplicate samples (** p < 0.005). Quantitative positive cells showed a higher fluorescent positive cells in blood with the caerulein-treated Panc02 cells than in mice without caerulein treatment (control) and normal mice, ** p < 0.005.

Figure 6

In vivo monitoring of the CTC existence pattern in mice with the NIF-EpCAM ALB incorporated into Panc02 cells. (A) The NIF-EpCAM ALB injection into mice was performed in the tail vein. The implanted trial of the Panc02 injection in pancreatic tissues, in the mice treated with caerulein for CTC existence, and placed by surgical implantation. An enhanced fluorescence signal in the CTC existence group (right) was detected compared to the group injected with phosphate-buffered saline (PBS) (left). Fluorescence images indicated that CTC cells of the implanted Panc02 cells in liver had similar observed; (B) Quantitative ROI analysis showed a higher fluorescence intensity in mice with the caerulein-treated Panc02 cells than in mice without caerulein treatment (middle), ** p < 0.005. Dic: differential interference contrast; RFU: relative fluorescence unit.