Unveiling urinary extracellular vesicle mRNA signature for early diagnosis and prognosis of bladder cancer

Figures

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  Figure 1. The overall workflow of the study. (A) The discovery phase: systematic analysis involved transcriptomic profiling of EVs obtained from the cell lines: normal cells (SVHUC1, HK-2), low-grade BC cells (5637, RT4), and high-grade BC cells (T24, EJ). Then, candidate genes were screened by RT-qPCR, from which SRGN, FLI1, and MACROH2A2 in EVs were identified for the subsequent investigations. (B) The validation phase: uEVs isolated from both healthy (n = 28) and BC (n = 69) urine samples to affirm the diagnostic efficacy of SRGN, FLI1, and MACROH2A2. (C) The supplementary phase: uEVs obtained from healthy cohort and BC patients with different stages, as well as those from pre- and post-surgery BC patients, were employed to assess the ability of SRGN, FLI1, and MACROH2A2 for early diagnosis and prognosis of BC.

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  Figure 2. Characterization and transcriptomic analysis of BC-related EVs. Characterization of EVs derived from T24 cells by (A) cryo-TEM, (B) NTA, and (C) WB. Transcriptomic profiling of EVs derived from normal cells (SVHUC1, HK-2), low-grade BC cells (5637, RT4), and high-grade BC cells (T24, EJ). Volcano plot of differentially expressed genes (DEGs) in EVs derived from (D) low-grade BC cells relative to normal cells, (E) high-grade BC cells relative to normal cells, and (F) high-grade BC cells relative to low-grade cells. The differential expression degrees of hub genes in the EVs derived from (G) low-grade BC cells relative to normal cells, (H) high-grade BC cells relative to normal cells, and (I) high-grade BC cells relative to low-grade cells.

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  Figure 3. Discovery of candidate BC biomarkers. (A) Venn diagram of significantly upregulated genes in EVs derived from low-grade BC cells and high-grade BC cells relative to those derived from normal cells. (B) Expression heatmap of the 10 gene panel with EVs derived from SVHUC1, HK-2, 5637, RT4, T24, and EJ cells from sequencing results. Scattering plots (C) and heatmap (D) of the relative expression levels of EV-RNAs derived from SVHUC1, RT4 and T24 cells. Relative mRNA level was analyzed by RT-qPCR.

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  Figure 4. BC diagnosis by SRGN, FLI1 and MACROH2A2 in uEVs. (A) Heatmap of the three mRNAs in uEVs collected from HC (n = 28) and patients with BC (n = 69). (B) Scattering plots of the mRNA expression levels of SRGN, FLI1 and MACROH2A2 in uEVs collected from HC and patients with BC. (C) ROC curves of the individual markers for BC diagnosis. (D) Scattering plots of the mRNA expression levels of the weighted sum of the three markers in uEVs collected from HC and patients with BC. (E) ROC curves of the weighted sum calculated by logistic regression algorithm of the three markers for BC diagnosis. (F) PCA plot demonstrates the obvious separation in mRNA expression between HC and BC. (G) Confusion matrix of the weighted sum of three biomarkers. (H) BC subjects were divided into two subgroups of high and low mRNA expression levels by the weighted sum of the three markers. A cut-off value of 0.612 was determined by Youden index (YI). All statistical analyses were performed at 95% CIs.

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  Figure 5. Early diagnosis and prognosis of BC by SRGN, FLI1 and MACROH2A2. (A) Scattering plots of the mRNA expression levels of SRGN, FLI1 and MACROH2A2 in uEVs collected from HC (n = 28), early-stage (ES) BC patients (n = 56), and advanced-stage (AS) BC patients (n = 13). (B) ROC curves of the individual markers for early-stage BC diagnosis. (C) Scattering plots of the mRNA expression levels of the weighted sum of the three markers in uEVs collected from HC, early-stage BC patients, and advanced-stage BC patients. (D) ROC curves of the weighted sum calculated by logistic regression algorithm of the three markers for early-stage BC diagnosis. (E) PCA plot demonstrates the obvious separation in mRNA expression between HC and early-stage BC. Sample 20 was not shown due to high expression level of SRGN. (F) Confusion matrix of the weighted sum of three biomarkers. (G-I) Longitudinal urine sample analysis of 5 patients undergoing surgery. All statistical analyses were performed at 95% CIs.