Extended Data Fig. 4. WGD and polyploidization are critical events in metastatic RCC.
a) Summary of segment-level amplification or deletion frequency across murine primary tumors or metastatic lesions as determined by GISTIC2. b) Representative scatter plots of exon-level log2(Read-Depth Ratios) as calculated by CNVkit from primary tumor derived cell lines from tumor within Cluster #1 (left panel) and Cluster #2 (right panel), where different patterns of chromosomal alterations can be appreciated. c) Representative sections of TdT stained tumor tissues. Images representative of N = 2 experiments. d) Cellularity estimation of primary and metastatic samples as assessed through TdT positive cell quantification, data are presented as mean values +/- SD (N = 4 fields per tumor). e) Most probable ploidy by log posterior probability at given sample’s cellularity as predicted by Sequenza from WGS data (representative mouse #7 and #8. f) Chromosome counts in RCC SM-GEMM–derived short-term cultures. Malignant cells are characterized by prominent polyploidy, data are represented as median values, minimum, maximum (M#7: 72, 42, 85; M#1: 60, 48, 84; M#8: 58, 55, 94; M#3: 52, 50, 78; M#5: 54, 40, 80; M#4: 58, 44, 64) with boundaries at the 25th and 75th percentile (N = 5/line tested). g) Costaining of chromosomes (DAPI) and centromeres in representative nuclei of metaphase short-term cultures, established from Nf2KO-Setd2KO-Trp53KO-4q9p21 tumor-bearing mice. Images representative of N = 2 experiments. h) Comparison of primary tumor sample and matched normal B-allele frequencies (BAF) of heterozygous SNPs derived from WGS in the matched normal tissue sample (0.2 ≤ normal sample SNP BAF ≤ 0.8). The analysis was performed on chromosomes undergoing gains (5q, 11q) or losses (12q, 16q). A copy-neutral chromosome was used as control (6q). Correlation of SNP BAFs between tumor and matched normal samples. The BAFs of heterozygous SNPs suggest that WGD precedes somatic CNVs. Error bars represent the standard deviation of technical replicates. Scale bar: 100 μm.