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Research
GCC2 is a New Biomarker for Diagnosis of Early Non-Small Cell Lung Cancer and A Potential Target to Reverse Epithelial to Mesenchymal Transition
Sunghoi Hong
Korea University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4926-1044
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Nano-sized exosomes (30–150 nm) are cell membrane-encapsulated vesicles that contain nucleic acids and proteins. Specific markers detecting non-small cell lung cancer (NSCLC) cell-derived exosomes in the blood circulation remain unidentified. Here, we report a new biomarker distinguishing cancer from non-cancer exosomes that also involved in epithelial to mesenchymal transition for cancer treatment.
Methods: Exosomes were isolated from plasma of patients with various pathological stages of NSCLC and NSCLC cell lines, human pulmonary alveolar epithelial cells by size exclusion chromatography and characterized by Nanoparticle Tracking Analysis and western-blotting. The exosomes were lysed and applied to proteomic analysis. The expression levels of the GCC2 proteins from NSCLC patients were analyzed by ELISA assays, and the effects by GCC2 shRNA were analyzed by real-time RT-PCR, cell migration and colony formation assays.
Results: A protein GRIP and coiled-coil domain-containing 2 (GCC2), which is involved in endosome-to-Golgi transport, was identified by the proteomics analysis of exosomes isolated from NSCLC cell lines. The GCC2 protein expression levels were increased in the exosomes derived from patients with early-stage NSCLC compared with healthy controls. The receiver operating characteristic curve of exosomal GCC2 revealed 94.74% sensitivity and 75.00% specificity, and AUC of 0.875. GCC2 knockdown experiments by GCC2 shRNA showed reduced exosome secretion in cancer cell lines, which altered the molecular and cellular properties, such as the expression levels of mesenchymal-to-epithelial genes, and cellular growth and motility.
Conclusion: GCC2 represents a promising biomarker for early diagnosis of NSCLC and a therapeutic target for future cancer treatment.
Keywords
Exosomes, non-small cell lung cancer, GCC2, biomarker, therapeutic target
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This is a list of supplementary files associated with this preprint. Click to download.
- supplefigure.docx
Supplementary Figure 1S. GCC2 expression and exosome secretion in lung cancer cell lines and patients with lung cancer. (a) Expression levels of cellular GCC2 mRNA. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3, technical replicates). (b) The exosome number secreted from HPAEpiC and cancer cell lines. Cells (1.0×105, 35-mm dish) were seeded in a cell culture dish. After 2 days, exosomes were isolated and measured by NTA. (c) Concentration of the exosomes in healthy controls and patients with different pathological stages of lung cancer by NTA. An independent Student’s t-test and the Jonckheere–Terpstra test were used for statistical validation. Supplementary Figure S2. Relative expression levels of the exosomal GCC2, CD63, and CD9 proteins in healthy controls and patients with different pathological stages of lung cancer by western blot. An independent Student’s t-test and the Jonckheere–Terpstra test were used for statistical validation. Supplementary Figure S3. Concentration of the exosomal GCC2 protein and ROC curve analysis by ELISA assay in healthy controls and patients with different pathological stages of lung cancer. (a) Concentration of the exosomal GCC2 protein in healthy controls (n = 16) and patients with stage T2aN0-T2bN0 lung cancer (n = 24). (b) ROC curve analysis for the concentration of the exosomal GCC2 protein in patients with stage T2aN0-T2bN0 lung cancer. (c) Concentration of the exosomal GCC2 protein in healthy controls (n = 16) and patients with stage T2aN1-T2bN1-T2aN2-T2bN2 lung cancer (n = 10). (d) ROC curve analysis for the concentration of the exosomal GCC2 protein in patients with stage T2aN1-T2bN1-T2aN2-T2bN2 lung cancer. Supplementary Figure S4. Construction of the stable cell lines with GFP reporter under the control of the CD63 gene promoter. (a) A scheme for construction of the stable cell lines after transfection of CD63-GFP plasmid by Lipofectamine 2000. (b) FACS sorting and analysis of the transfected cell lines. (c) The established stable cell lines expressing GFP. Scale bar = 50 µm.
- Supplementarytable1.PrimersequenceGCC2.docx
Supplementary Table S1. Primer sequence for qRT-PCR.
- Supplementarytable2.Proetomicanalysis.xlsx
Supplementary Table S2. List of the total proteins identified from proteomic analysis
- supplementarytable3.sizeofexosomes.docx
Supplementary Table S3. Size of exosomes derived from the cell lines and plasmas of patients with lung cancer.
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This is a preprint. It has not completed peer review.
Research
GCC2 is a New Biomarker for Diagnosis of Early Non-Small Cell Lung Cancer and A Potential Target to Reverse Epithelial to Mesenchymal Transition
Sunghoi Hong
Korea University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-4926-1044
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 29 Dec, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Nano-sized exosomes (30–150 nm) are cell membrane-encapsulated vesicles that contain nucleic acids and proteins. Specific markers detecting non-small cell lung cancer (NSCLC) cell-derived exosomes in the blood circulation remain unidentified. Here, we report a new biomarker distinguishing cancer from non-cancer exosomes that also involved in epithelial to mesenchymal transition for cancer treatment.
Methods: Exosomes were isolated from plasma of patients with various pathological stages of NSCLC and NSCLC cell lines, human pulmonary alveolar epithelial cells by size exclusion chromatography and characterized by Nanoparticle Tracking Analysis and western-blotting. The exosomes were lysed and applied to proteomic analysis. The expression levels of the GCC2 proteins from NSCLC patients were analyzed by ELISA assays, and the effects by GCC2 shRNA were analyzed by real-time RT-PCR, cell migration and colony formation assays.
Results: A protein GRIP and coiled-coil domain-containing 2 (GCC2), which is involved in endosome-to-Golgi transport, was identified by the proteomics analysis of exosomes isolated from NSCLC cell lines. The GCC2 protein expression levels were increased in the exosomes derived from patients with early-stage NSCLC compared with healthy controls. The receiver operating characteristic curve of exosomal GCC2 revealed 94.74% sensitivity and 75.00% specificity, and AUC of 0.875. GCC2 knockdown experiments by GCC2 shRNA showed reduced exosome secretion in cancer cell lines, which altered the molecular and cellular properties, such as the expression levels of mesenchymal-to-epithelial genes, and cellular growth and motility.
Conclusion: GCC2 represents a promising biomarker for early diagnosis of NSCLC and a therapeutic target for future cancer treatment.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5