Abstract
Large external antigen (LEA) is considered as a colorectal cancer (CRC)-associated antigen, which was found via mAb ND-1 generated using hybridoma technology, but its molecular features remain unknown. To facilitate the clinical application of LEA, we identified LEA as a podocalyxin-like protein 1 (PODXL) with molecular weight of approximately 230 kDa, a hyperglycosylated protein, using immunoprecipitation and mass spectrometry in combination, and verified that ND-1-recognized epitope is on the terminal sialic acid of LEA. Correlation analysis between LEA and PODXL in molecular weight, immunological cross-reactivity, and gene expression dependence supported the PODXL identity of the LEA. Moreover, we assessed the clinical significance of the LEA in 89 pairs of primary CRC tissues and adjacent nontumor colorectal tissues using ND-1 by quantum dot-based immunohistochemistry (QD-IHC). High LEA expression was correlated significantly with T stage (P = 0.010). Patients with high LEA expression showed significantly poorer prognosis than those with LEA low expression (P = 0.007). Multivariate analysis indicated LEA expression as an independent predictor. Furthermore, the comparative analysis showed that mAb ND-1-based IHC analysis toward sugar residue of PODXL has higher sensitivity and specificity to evaluate the LEA/PODXL expression than mAb 3D3-based method toward core protein of PODXL in CRC cell lines and clinical samples. In addition, we first found that LEA/PODXL can be secreted in exosomes from cancer cells and CRC patient peripheral blood. Our results demonstrate that LEA is an independent predictor for CRC progression and has the potential to be applied for clinical setting with high sensitivity, high specificity, and noninvasive access.