Fig. 3

VHL⁻ cells induce the proliferation and the metastasis of VHL⁺ cells. a The growth rate of RC-VHL-WT (red line), RC-VHL-KO cells (green line), and a 1:1 mixture of the two cells (orange line) in a transwell setting. b The growth rate of ACHN VHL-WT cells (red line), its VHL-KO counterpart (green line) or VHL-WT cells with the addition of VHL-KO condition media (orange line). c The growth rate of the Caki-1 -VHL-WT (red line), Caki-1-VHL-KO (green line), and the Caki-1-VHL-WT in the conditioned media from Caki-1-VHL-KO (orange line). d The growth rate of the VHL + primary cell line from case #22 (red line), its VHL-KO derivative (green line) and VHL + co-culture with its VHL-KO derivative in a transwell setting. One-way ANOVA was used in the comparisons in (a), (b), (c) and (d) with triplicate repeats, presented with mean ± SD. e A section of primary tumor derived from implantation of 1:1 RC-VHL-KO:RC-VHL-WT cells was stained with IF to detect VHL (red), Ki67 (green), and nuclei (DAPI, blue). The dash lines demarcate VHL-negative areas with intact nuclei. Scale bar: 200 μm. f VHL IHC and Ki-67 IF in serial sections of human ccRCC (case #22). High-magnification images show cytoplasmic VHL expression specifically in area (#) and not in area (*). The bar graph shows the average percentage of Ki-67 positivity in the VHL-positive and VHL-negative regions. Scale bar for low magnification field: 3 mm; for high magnification field: 50 μm. Student t-test was used in the comparisons (e) and (f) with triplicate repeats and presented with mean ± SD. g The spatial relationship between VHL and Ki-67 expression in case #22 was assessed with IF. Fluorescent images were analyzed with HALO software. In the first and fifth panels, VHL-positive cells and Ki-67-positive cells are represented by blue dots, respectively. The second panel shows a heatmap of VHL-positive cell density. The third panel shows a boundary map of the VHL-positive tumor regions as topographic contour lines indicating the distance from the tumor boundary. For distance measurements of Ki67-positive cells, contour lines were placed up to 2000 μm from the tumor edge toward the inside of the tumor and up to 4000 μm away from the tumor edge of VHL-positive tumor regions. Regions between the contour lines are shown as different colors from the innermost red to farthest blue. Ki-67-positive cells in each region were counted, normalized to the area, and plotted in a histogram that is shown in the fourth panel. Scale bar: 3 mm. h IF staining of the CD31 (yellow), VHL (red), Ki-67(white) as well as DAPI (blue) in mixed implanted RENCA-VHL-WT and VHL-KO cells mouse tissues. Left: the primary tumor. Right: lung metastasis. Scale bar for the low magnification field: 100 μm; for high magnification field: 30 μm. i VHL IHC staining of a large, lung metastasis from the mixed implanted group with RC-VHL-WT and RC-VHL-KO cells. Scale bar for low magnification field: 1 mm; for high magnification field: 200 μm. j IF of a small, lung metastatic nodule from a mouse implanted with the mixture with HA-positive RC-VHL-WT cells shown in red and the few flag-positive VHL-KO cells shown in green. Scale bar: 100 μm. k Flow cytometry analysis of the lung metastasis showing the relative proportion of RC-VHL-WT (TRITC+) and RC-VHL-KO(FITC+) cells. l IF of CD31(yellow), VHL (red) and DAPI (blue) on the primary tumor tissue from the case #22. Scale bar for low magnification field: 100 μm; for high magnification field: 30 μm. m H&E staining and (n) VHL IHC staining of the lung metastasis from the case #22. Scale bar for the low magnification field: 5 mm; for high magnification field: 100 μm. (*p < 0.05, **p < 0.01)