Fig. 2

VHL-KO cells cooperate with VHL-WT cells to cause metastasis. a Mice were implanted with a total of 1 × 10^6 tumor cells, either RC-VHL-WT, RC-VHL-KO, or a 1:1 mixture of the two cell types, into their left kidney (n = 6 per group). Images of three representative animals assessed by BLI on week 4 post-implantation are shown. b Primary tumor indicated by BLI in different groups are shown at the end point. c Lung metastases are indicated by elevated BLI signals in the thoracic cavity in the mixed tumor group. One-way ANOVA was used to test the statistical significance in (b) and (c) with n = 6, presented with mean ± SD. d H&E-stained sections (low magnification) of lung and heart of each animal in the RC-VHL-WT tumor group and mixed (1:1) tumor group. Arrowhead indicates the heart. Scale bar: 3 mm. e RC tumor growth was assessed in the CAM tumor system through longitudinal observation. Day 0 is the day of tumor cell implantation, which occurred on day 7 postfertilization. Scale bar: 1 cm. One-way ANOVA was used in the comparison with n = 9, presented with mean ± SD. f Flow cytometric analyses of CTC from chick embryos, bearing either VHL-WT cells (marked with mStrawberry) or 1:1 VHL-WT and VHL-KO (marked with EGFP) mixed CAM tumor, harvested on day 21 post-fertilization. g In the CAM tumor system, the metastasis in the duck liver can only be seen in mixed implantation of primary tumor cells from #22 and its VHL-KO counterpart as analyzed by TaqMan probes for human alu sequence and avian β-actin. n = 5 with “no signal” sample dots not shown and Ct value differences are presented with mean ± SEM. h Heatmap of the top 200 genes based on conserved gene expression patterns across comparisons of #22 VHL⁺ vs. VHL⁻ cells in single-cell sequencing (as noted in Fig. 1f), VHL KO vs. VHL WT ACHN models, and VHL KO vs. VHL WT RENCA models. Heatmap colors represent log2 fold changes z-score scaled by column. Detailed information is listed in Supplementary Table 2. i Top 30 gene sets based on conserved enrichment scores across the same three comparisons are listed. The color of points represents the differential expression comparison (ACHN VHL-KO vs VHL-WT as red, RENCA VHL-KO vs VHL-WT as green, and the single cell comparison of #22-VHL⁺ vs VHL⁻ as blue) and the size of the points corresponds to the adjusted p-value from GSEA, in which the bigger circles indicate up/down-regulation with lower p-values. (*p < 0.05, **p < 0.01) (not consistent with adjusted p vals in Fig. 2i)