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.

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.

Metastasis is an important hallmark of malignant tumors. In this study, we developed a microfluidic system to screen highly metastatic sublines via differential resolution of cell invasiveness. The system was composed of a PDMS-glass device connected with a syringe pump and a Petri dish. To facilitate the selection process, a long-term cell invasion driving force based on a chemotactic factor gradient was created using the Petri dish-based liquid supply pattern, and the invasive cells were collected for round-by-round selection via an open region in the chip. Using the system, we established an SGC-7901/B2 subline from the human gastric cancer SGC-7901 cell line by only two rounds of selection. In vitro assays showed that the SGC-7901/B2 cells were superior to the parental cells in proliferation and invasiveness. Furthermore, an in vivo tumorigenicity assay demonstrated that compared with the parental cells, the subline had stronger spontaneous metastatic and proliferative capability, which led to a shorter survival duration. Additionally, the protein expression differences including E-cadherin and Smad3 between the subline and parental cells were revealed. In conclusion, this microfluidic system is a highly effective tool for selecting highly metastatic sublines, and SGC-7901/B2 cells could serve as a potential model for tumor metastasis research.

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Natural products are becoming increasingly important in chemoprevention and for cancer therapy. Silene viscidula (S. viscidula), a traditional Chinese herb, has long been used as an anti-inflammatory and neuroleptic agent. However, the anticancer activity of S. viscidula has remained unclear. In this study, 16 compounds were extracted from S. viscidula. Among those compounds, sinocrassulosides VI/VII, an inseparable isomer mixture, possess the strongest inhibitory activity on HeLa cells with the IC 50 value of 2.37 M. Mechanism studies found that sinocrassulosides VI/VII downregulated the expression of cyclin D1 and decreased retinoblastoma (Rb) phosphorylation, which arrested HeLa cells in the G1 phase. Also, sinocrassulosides VI/VII could induce senescence via the upregulation of p16 and a significant increase of -galactosidase ( -gal) staining. Our results suggest that sinocrassulosides VI/VII may be a new therapeutic potential agent for cervical cancer. In addition, we explored the structure-activity relationships of three groups of the configurational isomer with similar chemical structure from S. viscidula. We first demonstrated that the length of the ester chains linked to the carboxyl group of the glucuronic acid residue could affect the potent cytotoxicity. This finding will open new avenues for developing effective anticancer compounds by modifying the components derived from plants in nature.