Fig 1: Isolation and identification of human pancreatic stellate cells (PSCs). (A) Schematic diagram of human PSCs isolation. The image of the pancreas was drawn by Figdraw (www.figdraw.com). The lipid droplets were observed under the microscope with or without oil-red O staining. Bars, 50 µm. (B, C) Immunofluorescence staining of PSCs for fibroblast (a-SMA, FAP) and epithelial markers (EpCAM, E-cadherin). Bars, 50 µm
Fig 2: Urinary EpCAM-CD9-positive EV is a potential biomarker for PCa diagnosis. (A) The uEVEpCAM-CD9 in different urine volumes was detected by WB. (B) The uEVEpCAM-CD9 from different urine volumes was detected by our chemiluminescent immunoassay. (C) The uEVEpCAM-CD9 of the pooled samples from PCa and healthy controls were detected by EV assay. (D) A standard curve was for urinary EV quantification using our EV assay. (E) The uEVEpCAM-CD9 from nine randomly selected donors including five PCa and four HDs was assayed by the chemiluminescent immunoassay. (F) The levels of uEVEpCAM-CD9 was observed from men with PCa (n = 112) and without PCa (n = 81). (G) The ROC curve of uEVEpCAM-CD9 and PSA. (H) The uEVEpCAM-CD9 was detected before and after prostatectomy in 20 PCa patients. (I) The correlation between the uEVEpCAM-CD9 and PSA. DensityEpCAM-CD9, EpCAM-CD9 protein density; RCU, relative chemiluminescent unit; EVEpCAM-CD9, EpCAM-CD9-positive extracellular vesicles; Ctrl, control; PCa, prostate cancer; ROC, receiver operating characteristic; uEVEpCAM-CD9, urinary EpCAM-CD9-positive extracellular vesicles; PSA, prostate-specific antigen; AUC, area under the curve; HD, healthy donor. *P < 0.05,**P < 0.01,****P < 0.0001.
Fig 3: EVEpCAM-CD9 is ultrasensitively detected by chemiluminescent immunoassay and oversecreted under simulated tumor microenvironment. (A) Groups of PC3 EVs, non-EVs, non-streptavidin-labeled magnetic beads, non-biotin-labeled anti-EpCAM antibodies, and non-ACE-labeled anti-CD9 antibodies were detected by our assay. (B) EVs were penetrated by Triton X-100. (C) A standard curve was for EVs from cell line supernatant quantification using our EV assay. (D) EVs derived from FBS, BPH cell line RWPE-1, and human prostate cancer cell lines PC3 and LNCaP were detected by our EV assay and WB. (E) The changes of EVEpCAM-CD9 secretion index during the growth of PC3 cells. (F) The changes of EVEpCAM-CD9 secretion index when the PC3 cells were cultured under hypoxia. (G) The changes of EVEpCAM-CD9 secretion index when the PC3 cells were cultured under serum starvation. (H) The changes of EVEpCAM-CD9 secreted by PC3 cells with the treatment of 10 and 20 µM GW4869. RCU, relative chemiluminescent unit; EVs, extracellular vesicles; FBS, fetal bovine serum; EVEpCAM-CD9, EpCAM-CD9-positive extracellular vesicles. *P < 0.05,**P < 0.01,****P < 0.0001.
Fig 4: The scheme of workflow for urinary EpCAM-CD9-positive extracellular vesicle (uEVEpCAM-CD9) detection. (A) EpCAM-CD9-positive EVs diffused in urine are bound with acridinium ester (ACE)-labeled anti-CD9 antibodies and captured by magnetic microbeads labeled anti-EpCAM antibodies. After incubation for 60 min, uEVEpCAM-CD9 binding with magnetic microbeads can be easily isolated under an external magnetic field and quantitatively analyzed by a chemiluminescent immunoassay analyzer to diagnose prostate cancer. (B) TEM images of EVs isolated by ultracentrifugation (white arrow). (C) EVs are characterized by NTA. (D) The expression of CD63, CD9, EpCAM, calnexin, and APO in PC3 cell lysates and the EV fraction from PC3 by WB analysis. (E–G) Flow cytometry assay identified that approximately 80% of EVs released by PC3 carried EpCAM and CD9. EpCAM, epithelial cell adhesion molecule; uEVEpCAM-CD9, urinary EpCAM-CD9-positive extracellular vesicles; EVs, extracellular vesicles; ACE, acridinium ester; TEM, transmission electron microscope; NTA, nanoparticle tracking analysis; WB, western blot.
Fig 5: A multivariate diagnostic model based on uEVEpCAM-CD9 for PCa. (A) The nomogram was constructed according to the results of multivariate logistic regression. (B) The ROC curve analysis of the multivariable diagnostic model in the training set and validation set. (C) The multivariable diagnostic model was calibrated in the internal validations. (D) The decision curve analysis of the multivariable diagnostic model and uEVEpCAM-CD9. (E) The diagnostic performance of the model, uEVEpCAM-CD9, and PSA in patients with PSA gray zone (4–10 ng/ml) including 23 PCa and 31 BPH. BMI, body mass index; uEVEpCAM-CD9, Log urinary EpCAM-CD9-positive extracellular vesicles concentration (n.u); PV, prostate volume; PSA, prostate-specific antigen; ROC, receiver operating characteristic; AUC, area under the curve.
Supplier Page from Abcam for Anti-EpCAM antibody [EGP40/1372]