Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe

Abstract

The large external antigen (LEA) is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC) as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC) combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605) conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage ( P <0.05), indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC) combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of the cell lines, which was confirmed by flow cytometry. The results of this study indicate that QD-ICC has the potential to noninvasively detect rare circulating tumor cells in clinical samples in real clinical applications.

Identification of LEA, a podocalyxin‐like glycoprotein, as a predictor for the progression of colorectal cancer

“…Previous studies have shown that LEA expression level is related to T stage of CRC patients . To further verify the clinical significance of PODXL recognized by ND‐1 in CRC, we detected LEA expression level in TMA, which contains 89 CRC tissues and adjacent nontumor colorectal tissues, by QD‐based IHC with mAb ND‐1.…”

“…ND‐1, the mAb of LEA, was obtained and purified as described previously . Mouse anti‐PODXL mAb (3D3, cat#sc‐23904) and normal mouse IgG (cat#sc‐2025) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).…”

“…Previous researches showed that LEA was a glycoprotein mainly expressed on CRC cell membrane . IHC primarily revealed that LEA expression was associated with T stage of CRC . Notably, LEA was detectable in the serum and ascites of CRC patients, leading to a convenient clinical access .…”

Recent insights into nanotechnology development for detection and treatment of colorectal cancer

“…[31][32][33] In a recent study, researchers qualitatively analyzed the expression level of large external antigen in tissue samples from CRC patients using QD-based immunohistochemistry (QD-IHC) and conventional IHC. 34 Compared with conventional IHC, QD-IHC offered several noticeable advantages for protein marker quantification. These included higher sensitivity, simpler operation, less human interference, and increased capability for simultaneous multifactor analysis, which would lead to more accurate clinical evaluations.…”

“…This result suggested that this method is for noninvasive molecular analysis of rare CTCs and provided a rationale for expanding the application of QD probes to clinical practice. 34 In another study, a simple and highly sensitive technique was developed to detect aldo-ketoreductase family 1 member B10 (AKR1B10), a prognostic predictor and therapeutic target for CRC in the serum, using QDs with a high-fluorescence quantum yield against photo bleaching and size-controlled luminescence. 35 This immunofluorescence assay to detect serum AKR1B10 using anti-AKR1B10-conjugated CdTe/CdS QDs is a promising approach for the early prediction of CRC.…”