Fig 1.
(A) Use of ATPS to isolate EVs in urine. Red particles: proteins; black particles: EVs. After the polymers are dissolved, the solution forms an ATPS, and particles segregate depending on their surface property to one of the phases. Subsequent purification steps yield highly pure EVs. (B) Cancer cells and EVs in prostate release to urine. EVs are isolated by ATPS from 5 ml urine. The diagnostic ability of serum-PSA, sediments, and EVs was compared using samples from 20 patients who have prostate cancer (PCA) and 10 patients who have benign prostate hyperplasia (BPH).
Fig 2.
Characterization and conventional identification of ATPS compared to conventional methods.
(A) Additional purification steps of serial protein-depletion. After the procedure, the recovery efficiency of EVs was almost unchanged, whereas the recovery efficiency of proteins decreased. (B) Recovery efficiency of EVs and protein in U/C-once, U/C-twice, and ATPS. Recovery efficiency of EVs by ATPS was four times larger than by U/C-once, and 14 times larger than by U/C-twice. (C) Purity of EVs in U/C-once, U/C-twice, and ATPS. Purity is the number of isolated EVs divided by amount of isolated protein. Amount of EVs was measured by NTA and ELISA. Protein was measured by Bradford assay. (D) Size distribution of EVs isolated from urine by U/C-twice and ATPS was measured using NTA. Size distributions of total EVs in 5 ml urine: grey line; black line, estimated using ATPS; dotted line, estimated using U/C-twice. Grey line and black line are similar; i.e., ATPS isolated ~100% of EVs. U/C twice isolated only a small fraction of EVs. (E, F) TEM did not show any morphological differences between EVs isolated using U/C-twice and ATPS. The results were analyzed by ANOVA with a post-significance Tukey’s test. pairwise significances: *p < 0.05, **p <0.001.
Fig 3.
ELISA and PCR of EVs isolated by ATPS and U/C-twice.
(A) Existence of EV surface marker was analyzed by CD9, CD81, and CD63 western blot. Total protein isolated from 5 ml urine using ATPS and U/C-twice was used for ATPS and U/C-twice samples. Final volume of isolated EVs samples by the methods was 250 μl, and 40 μl of the samples were used in western blots; 1.5 μg (ATPS) and 0.2 μg (U/C-twice) of protein was used in each well. (B) RNA profile comparison between ATPS and U/C-twice. The total RNA isolated from 5 ml urine using ATPS and U/C was compared. RNA profiles of ATPS and U/C differed significantly. (C) PCR of actin was performed using RNA extracted from EVs isolated by ATPS and U/C-twice from 5 ml urine. Isolated total RNA was used for PCR; ~800 ng (ATPS), and ~ 50 ng (U/C 1, 2, 3, and 4) of RNA was used.
Fig 4.
Prostate cancer-associated gene expression using ATPS and sediments.
(A) Amounts of prostate-specific membrane antigen (PSMA) (normalized by creatinine concentration in urine) obtained using sedimentation and EVs isolated by ATPS in prostate cancer (PCA) and prostate hyperplasia (BPH) patients. (B) Amounts of prostate-specific membrane antigen (PSMA) (normalized by CD9) obtained using EVs isolated by ATPS in PCA and BPH groups. (C) RNA expression level of prostate cancer antigen 3 (PCA3) (normalized by actin) obtained using sedimentation in PCA and BPH groups; y-axis scale is log2. Large value means that PCA3 was highly expressed. (D) RNA expression level of PCA3 (normalized by actin) in EVs obtained using ATPS, in PCA and BPH groups. qPCR was performed to measure gene expression levels.
Fig 5.
Diagnostic ability of conventional methods and ATPS.
(A) Receiver operating characteristic (ROC) curve based on ELISA for diagnosis using ATPS and sediments. PSMA/creatinine or PSMA/CD9 was measured for diagnosis. (B) ROC curve based on qPCR for diagnosis using ATPS and sediments. PCA3/actin was measured for diagnosis. (C) ROC curve for diagnosis by combining ELISA and qPCR using ATPS and sediments. (D) ROC curve for serum-PSA.
Table 1.
AUC, sensitivity, and specificity of methods to distinguish PCA from BPH.