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. 2023 Jan;12(1):e12300.
doi: 10.1002/jev2.12300.

Identification of faecal extracellular vesicles as novel biomarkers for the non-invasive diagnosis and prognosis of colorectal cancer

Zhaowei Zhang  1 , Xuehui Liu  1   2 , Xiaoqing Yang  3 , Ying Jiang  1 , Ang Li  1 , Jiying Cong  4 , Yuwei Li  4 , Qinjian Xie  5 , Chen Xu  4 , Dingbin Liu  1
Affiliations

Identification of faecal extracellular vesicles as novel biomarkers for the non-invasive diagnosis and prognosis of colorectal cancer

Zhaowei Zhang et al. J Extracell Vesicles. 2023 Jan.

Abstract

Colorectal cancer (CRC) is one of the most common malignancies that is usually detected late in the clinic. The currently available diagnostic tools for CRC are either invasive or insensitive to early lesions due to the dearth of reliable biomarkers. In this study, we discovered that the extracellular vesicles (EVs) in the faeces of CRC patients can act as a potent biomarker for the non-invasive diagnosis and prognosis of CRC. This finding is based on the identification of two transmembrane proteins-CD147 and A33-on faeces-derived EVs (fEVs) that are intrinsically associated with CRC. The detection results show that the levels of CD147 and A33 on fEVs were upregulated in the CRC patients (n = 48), dramatically distinguishing them from the healthy donors (n = 16). The CD147/A33-enriched EVs offer a clinical sensitivity of 89%, much higher than that (40%) of carcinoembryonic antigen (CEA), a clinically-established serum biomarker for CRC diagnosis. In addition, the analysis of longitudinal faeces samples (n = 29) demonstrated that the CD147/A33-enriched fEVs can be utilized to track the prognosis of CRC. Due to the high compliance of faeces-based detection, the CD147/A33-enriched fEVs could serve as new-generation CRC biomarkers for large-scale, non-invasive CRC screening as well as real-time monitoring of patient outcomes during clinical interventions.

Keywords: biomarkers; clinical diagnosis; colorectal cancer; extracellular vesicles; faeces.

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Conflict of interest statement

All the authors declare no competing interests.

Figures

FIGURE 1
FIGURE 1
Isolation and characterization of fEVs. (a) Scheme of biogenesis and isolation of fEVs. (b) Particle size distribution of fEVs by NTA and representative morphology of fEVs by cryo‐TEM. (c) Western blotting analysis for EV general markers of fEVs collected from three CRC patients. (d) Western blotting analysis of CRC‐related proteins in the fEVs and tumour tissues collected from two patients.
FIGURE 2
FIGURE 2
Screening of biomarker candidates and confirmation of CD147 and A33 on fEVs and tissues. (a) Procedures for screening biomarker candidates. (b) Western blotting analysis of fEVs from CRC patients and healthy donors (left) and the relative band intensity (right). (c) Schematic of immunogold labelling to confirm CD147 and A33 on fEVs. (d) Representative IG‐TEM pictures of CD63, CD147, and A33 labelled by 10 nm‐gold on fEVs. fEVs without primary antibody treatment were used as control. Scale bar: 100 nm. (e) Immunofluorescence (red) of CD147 and A33 in colorectal carcinoma (CRC) and para‐carcinoma (N) tissues. Scale bar: 50 μm
FIGURE 3
FIGURE 3
Analysis of CD147 and A33 in EVs from clinical faeces specimens. (a) Schematic of ELISA system to detect proteins on fEVs. Heat map (b) and scatter diagrams (c) of the CD147 and A33 levels on fEVs collected from healthy donors (n = 16) and CRC patients (n = 48). (d) The levels of CD147 and A33 were combined into a single index scatter plot by binary logistic regression analysis (CD147 & A33). (e) ROC curves of CD147, A33, and CD147 & A33 to differentiate CRC patients from healthy donors. (f) Levels of CD147 and A33 on fEVs across CRC stages. (g) Levels of CD147 and A33 in plasma‐derived EVs collected from healthy donors and CRC patients.
FIGURE 4
FIGURE 4
Analysis of CD147 and A33 in a longitudinal study. (a) Levels of CD147 and A33 on fEVs from CRC patients before and after surgery. (b) Longitudinal faeces collection preoperatively and postoperatively (month 2). (c) Analysis of longitudinal faeces specimens from seven CRC patients who underwent surgery. (d) Representative MRI of a CRC patient before and after surgery, with tumour and post‐resection site encircled in red and green, respectively.
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