Three-dimensional imaging of upper tract urothelial carcinoma improves diagnostic yield and accuracy

Abstract

Upper tract urothelial carcinoma (UTUC) is a rare form of urothelial cancer with a high incidence of recurrence and a low survival rate. Almost two-thirds of UTUCs are invasive at the time of diagnosis; therefore, improving diagnostic methods is key to increasing survival rates. Histopathological analysis of UTUC is essential for diagnosis and typically requires endoscopy biopsy, tissue sectioning, and labeling. However, endoscopy biopsies are minute, and it is challenging to cut into thin sections for conventional histopathology; this complicates diagnosis. Here, we used volumetric 3-dimensional (3D) imaging to explore the inner landscape of clinical UTUC biopsies, without sectioning, revealing that 3D analysis of phosphorylated ribosomal protein S6 (pS6) could predict tumor grade and prognosis with improved accuracy. By visualizing the tumor vasculature, we discovered that pS6+ cells were localized near blood vessels at significantly higher levels in high-grade tumors than in low-grade tumors. Furthermore, the clustering of pS6+ cells was associated with shorter relapse-free survival. Our results demonstrate that 3D volume imaging of the structural niches of pS6 cells deep inside the UTUC samples improved diagnostic yield, grading, and prognosis prediction.

Keywords: Oncology; Signal transduction; Urology.

Figure 1. Pipeline for 3D imaging and gelatin embedding of UTUC samples.

(A and B) Schematic representation of the endoscopic examination of patients with UTUC (A) and the DIPCO 3D light-sheet microscopy pipeline modified for gel embedding of small samples (B). (C) UTUC sample #16HG before and after gel embedding and clearing. Grid lines: 1 mm. The red dashed line indicates the cleared sample.

Figure 2. Whole-mount multiplex immunostaining of UTUC samples.

(A) Schematic indicating where on the ureter UTUC sample #21LG was taken and which 2D layer showing the immunostaining for histone (blue), AE1/AE3 (cyan), pS6 (red), CD34 (green), and merged. (B) Cross section images of UTUC sample #5HG immunostained and segmented for AE1/AE3 and tumor region, respectively. Nuclei stained with histone nanobody. Segmentation lines in cyan. (C) Volume rendering of UTUC sample #5HG immunostained for pS6. Bounding box, 1,034 × 1,124 × 540 μm. (D) Cell-by-cell analysis of the percentage of pS6⁺ cells of the whole sample versus the tumor region (n = 21, ****P < 0.001 by Wilcoxon signed rank test). (E) Violin plot of the percentage of pS6⁺ cells in low-grade (n = 8) and high-grade (n = 11) UTUC samples (P = 0.002). (F) Volume rendering and cell-by-cell analysis of pS6⁺ cells in #13LG (left) and #3HG (right) UTUC samples. Bounding boxes, 786 × 540 × 470 μm (#13LG) and 1443 × 1304 × 644 μm (#3HG). (G) Violin plots of pS6⁺ cells in low-grade (n = 8) and high-grade (n = 11) UTUC samples analyzed for 5 randomly selected cross sections (z1-z5): z1, P = 0.026; z2, P = 0.083; z3, P = 0.302; z4, P = 0.021; and z5, P = 0.021.(H) ROC analysis of the pS6⁺ cell ratio in five 2D data sets (red, blue, green, magenta, and cyan) and the 3D data set (black). Scale bars: 100 μm (yellow), 250 μm (white), and x, y, z indicators 500 μm (white). The pseudocolors indicate low (blue) and high (red) expression levels of pS6. For the violin and box plots, the violin illustrates the distribution, the box center line indicates the median, the upper and lower boundaries of the indicate the upper and lower quartiles, and the whiskers indicate the minimum and maximum values. *P < 0.05, ***P < 0.005, ****P < 0.001 by Mann-Whitney U tests unless stated otherwise.

Figure 3. Cluster analysis of pS6⁺ cells in UTUC samples.

(A) Cross sections of UTUC sample #14HG with clustered pS6⁺ cells at depth z = 620 μm (upper) and sparse pS6⁺ cells at depth z = 594 μm (lower). Nuclei stained with histone. (B) Volume rendering and cell-by-cell analysis of pS6⁺ cells in UTUC samples #3HG (upper) and #6LG (lower). Bounding boxes, 1,548 × 1,500 × 708 μm (#3HG) and 1,600 × 1,260 × 1,100 μm (#6LG). (C–I) Violin plots of pS6⁺ cells in low-grade (LG, n = 8) and high-grade (HG, n = 11) UTUC samples analyzed for nearest distance (C, P = 0.008), number of cells within a 100 μm radius (D, P = 0.003), nearest neighbor index (E, P = 0.004), cluster density (F, P = 0.002), clustered pS6⁺ cell density (G, P < 0.001), cells per cluster (H, P = 0.026), and clustered pS6⁺ cell ratio (I, P = 0.048). For the violin and box plots, the violin indicates the distribution, the box center line indicates the median, the upper and lower boundaries of the box indicate the upper and lower quartiles, and the whiskers indicate the minimum and maximum values. au, arbitrary unit. Scale bars: 50 μm (yellow), and x, y, z indicators 250 μm (white). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by Mann-Whitney U tests.

Figure 4. Assessment of the tumor vasculature and the density of pS6⁺ cells around vessels.

(A) Volume rendering of the tumor vasculature and pS6⁺ cells in low-grade UTUC sample #10LG. The tumor vasculature was stained for CD34. The pseudocolors indicate thin (blue) and thick (red) blood vessels. Bounding box, 1,879 × 1,447 × 920 μm. (B–H) Violin plots of pS6⁺ cells in low-grade (LG, n = 8) and high-grade (HG, n = 11) UTUC samples analyzed for CD34 density in 5 randomly selected 2D data sets (z1–z5) (B, z1, P = 0.36; z2, P = 0.32; z3, P = 0.46; z4, P = 0.41; and z5, P = 0.069), 3D CD34 density (C, P = 0.22), mean vessel length (D, P = 0.22), total vessel length per volume (E, P = 0.14), vessel radius (F, P = 0.008), vessel tortuosity (G, P = 0.46), and pS6⁺ cell density around CD34⁺ vessel (H, P = 0.01). AU, arbitrary unit. For the violin and box plots, the violin indicates the distribution, the box center line indicates the median, the upper and lower boundaries of the box indicate the upper and lower quartiles, and the whiskers indicate the minimum and maximum values. **P < 0.01 by Mann-Whitney U test.

Figure 5. Analysis of intratumoral heterogeneity in UTUC samples.

(A) A 3D rendering of UTUC sample #19HG and the 2D layers at the 10th (z = 188 μm, blue), 25th (z = 924 μm, cyan), 50th (z = 724 μm, green), 75th (z = 584 μm, yellow), and 90th (z = 444 μm, red) percentiles. Bounding box, 1,600 × 1,396 × 1,100 μm. Nuclei stained with histone. (B and C) Heatmap of all UTUC samples and their number of layers with the indicated percentage of pS6⁺ cells (B) and the corresponding hierarchical cluster analysis (C). High and low numbers of layers are indicated by yellow and dark blue, respectively. Red and blue boxes indicate high-grade (HG) and low-grade (LG) samples, respectively, diagnosed by the pathologist. Arrows indicate mismatched samples.

Figure 6. Relapse-free survival analysis in patients with UTUC who underwent focal therapy.

Kaplan-Meier plots of relapse-free survival in patients with UTUC (n = 9) who underwent focal therapy. (A–D) Stratification of UTUC, based on 2D-analysis, into high versus low pS6⁺ cell ratio (A, P = 0.806) and CD34⁺ cell density (B, P = 0.670), and, based on 3D-analysis, into high versus low clustered pS6⁺ cell ratio (C, P = 0.022) and pS6⁺ cell density around CD34⁺ vessel (D, P = 0.003). Statistical analysis was performed using log–rank tests.