GCC2 as a New Early Diagnostic Biomarker for Non-Small Cell Lung Cancer

Abstract

No specific markers have been identified to detect non-small cell lung cancer (NSCLC) cell-derived exosomes circulating in the blood. Here, we report a new biomarker that distinguishes between cancer and non-cancer cell-derived exosomes. Exosomes isolated from patient plasmas at various pathological stages of NSCLC, NSCLC cell lines, and human pulmonary alveolar epithelial cells isolated using size exclusion chromatography were characterized. The GRIP and coiled-coil domain-containing 2 (GCC2) protein, involved in endosome-to-Golgi transport, was identified by proteomics analysis of NSCLC cell line-derived exosomes. GCC2 protein levels in the exosomes derived from early-stage NSCLC patients were higher than those from healthy controls. Receiver operating characteristic curve analysis revealed the diagnostic sensitivity and specificity of exosomal GCC2 to be 90% and 75%, respectively. A high area under the curve, 0.844, confirmed that GCC2 levels could effectively distinguish between the exosomes. These results demonstrate GCC2 as a promising early diagnostic biomarker for NSCLC.

Keywords: GCC2; biomarkers; cancer; early detection; exosomes; liquid biopsy; non-small cell lung cancer.

Figure 1

Characterization of exosomes derived from normal and lung cancer cell lines. (a) Average size of SEC fractions 6–8 determined by NTA. Average sizes of SEC fractions 6, 7, and 8 were 134.131 ± 1.99, 138.73 ± 6.98, and 131 ± 3.2 nm (mean ± SD), respectively. (b) Western blot analysis of the CD63, CD9, and CD81 exosomal proteins in the exosome lysates (6 µg of total protein/well of acrylamide gel). (c) TEM analysis shows the typical cup shape of the exosomes. Scale bar = 100 nm. (d) Average size of SEC fractions 6–8 from healthy and patient plasmas determined by NTA were 135.36 ± 5.31 and 138.71 ± 12.40 (mean ± SD), respectively. (e) Western blot analysis of the CD63 and CD9 exosomal proteins in the exosomal lysates. (f) TEM analysis shows the typical cup shape of the exosomes derived from plasma. Scale bar = 100 nm. (g) TEM analysis shows immunogold labeling using an anti-CD63 antibody for the exosomes isolated from HPAEpiC and human plasmas. Scale bar = 100 nm.

Figure 2

Identification of a potential factor for NSCLC diagnosis and treatment by proteomic analysis. (a) A Venn diagram for the proteomic analysis and Gene Ontology classification of the exosomes derived from six cell lines. (b) GCC2 expression in the exosomal lysates determined by western blotting. (c) GCC2 protein present on both the surface and inside the exosomes. The GCC2 protein disappeared following the exposure of the exosomal surface proteins to proteinase K.

Figure 3

GCC2 levels in NSCLC patient plasma-derived exosomes. (a) Comparison of GCC2, CD63, and CD9 protein levels in the plasma-derived exosomes isolated from the healthy control group (n = 16) and patients suffering from different pathological stages of NSCLC (n = 70) by western blot analysis. Each of the three lanes represents 3 healthy controls and 3 patients at different pathological stages of NSCLC. (b) Comparison of the relative intensities of GCC2, CD63, and CD9 protein levels between the control and T1aN0-T1bN0 patient groups (n = 30). Protein intensities were measured using ImageJ. An independent Student’s t-test and the Jonckheere–Terpstra test were used for statistical validation. (c) Comparison of GCC2 and CD63 protein levels in the plasma-derived exosomes isolated from the control group and patients suffering from different pathological stages of NSCLC by western blotting. The same exosome number determined by NTA was used for each sample during western blotting. Each of three lanes represents 3 healthy controls and 3 patients at different pathological stages of NSCLC. (d) Comparison of the relative intensities of GCC2 and CD63 protein levels. The GCC2 levels, but not CD63 levels, gradually increased as the pathological stage progressed.

Figure 4

Evaluation of the exosomal GCC2 protein as a diagnostic biomarker for patients with early-stage lung cancer. (a) Exosomal GCC2 protein concentrations in the healthy control group (n = 16) and patients with T1aN0-T1bN0 lung cancer (n = 30) determined by ELISA. (b) ROC curve based on the exosomal GCC2 protein levels to distinguish patients with early NSCLC from normal healthy controls. An independent Student’s t-test and the Jonckheere–Terpstra test were used for statistical validation.