Degree and site of chromosomal instability define its oncogenic potential

Abstract

Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. Using a conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine. In mice predisposed to intestinal cancer (Apc^Min/+), moderate CIN causes a remarkable increase in adenoma burden in the entire intestinal tract and especially in the distal colon, which resembles human disease. Strikingly, a higher level of CIN promotes adenoma formation in the distal colon even more than moderate CIN does, but has no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.

Fig. 1. An allelic series for graded increases in CIN in vivo.

a Theoretical inverse correlation of decreased spindle assembly checkpoint function with increased severity of CIN. b Overview of the CiMKi alleles: the targeting vectors harbor a cDNA cassette of wild-type (WT) exons 17–22, flanked by lox-P sites, and the mutated exon 17* (TA or KD). In the targeted CiMKi alleles, wild-type Mps1 is replaced with mutant Mps1 upon Cre-mediated loxP recombination. c Quantification of time in mitosis (prophase to anaphase) by time lapse imaging of immortalized MEFs of the CiMKi;Rosa26-CreER^T2 genotypes 56 h after 4-OHT addition. DNA was visualized by H2B-mNeon. Two independent untreated lines (CiMKi^WT/WT and CiMKi^KD/KD, both expressing wild-type Mps1) were treated with MPS1 inhibitor CPD5. Error bars indicate ± SEM of three independent MEF lines per genotype; 50 cell divisions per line. See also Supplementary Movies 1, 2. d Quantification of chromosome segregation fidelity of samples described in c. Missegregations of chromosomes were categorized as indicated: severe (≥three), mild (one or two). Scale bars 5 μm. See also Supplementary Movies 1, 2. Analysis as in c. e Chromosome segregation fidelity in situ in small intestine of CiMKi;Rosa26-CreER^T2 mice one week after tamoxifen injection. DAPI (green) and anti-phospho-Histone H3 (Ser10) (pHH3; magenta) were used to identify anaphases (white arrowhead; weak staining) and prophases (magenta arrowhead; strong staining)), scale bars 5 μm. Graph shows quantification by category as in d, for at least 47 anaphases per small intestine. Bars indicate means ± SD (n = 12 (WT/WT), 8 (WT/TA), 11 (WT/KD), 7 (TA/TA), 5 (TA/KD), or 3 (KD/KD) mice per genotype; WT/WT group includes vehicle controls of other CiMKi genotypes). f scKaryo-seq (bin size 5 MB) showing ploidy in individual cells of small intestine 7 days after CiMKi induction. Graphs show individual cells (horizontal lines) of one example per genotype, see also Supplementary Fig. 1I. Average aneuploidy and heterogeneity scores are given for each genotype (n = 3). Colors indicate copy number state for a given chromosome. Statistics for panels c, d, and e: ordinary one-way ANOVA, uncorrected Fisher’s LSD test, exact p-values are indicated when p < 0.05. Source data for panels c, d, and e are provided as a Source Data file.

Fig. 2. Moderate CIN causes early onset spontaneous tumor initiation in the intestine.

a H&E of CiMKi;Rosa26-CreER^T2 small intestines one week after tamoxifen injection, showing aberrant crypt and cell size, hyperproliferation (black arrowheads indicate mitotic cells) and apoptotic bodies in crypt (yellow arrowheads), scale bars 100 μm. b Methylene blue stained, formalin-fixed whole mount small intestine of 12-week old CiMKi;Villin-Cre mice. Zoom boxes indicate normal (CiMKi^WT/WT) and aberrant (CiMKi^TA/TA) mucosa. Scale bars 1 mm. c Quantification of adenomas as determined on whole mount methylene blue staining in small intestine tissue in of 12-week-old CiMKi;Villin-Cre mice. Data represents mean ± SD, (n = 6 (WT/WT), 5 (WT/TA), 5 (WT/KD), 5 (TA/TA), 6 (TA/KD), or 4 (KD/KD) mice per genotype. d Quantification of adenomas as in c, but for small intestines of 8-month-old CiMKi;Villin-Cre mice (n = 8 (WT/WT), 14 (WT/TA), 12 (WT/KD), 5 (TA/TA), 5 (TA/KD), or 3 (KD/KD) mice per genotype. e H&E of small intestine adenomas from 12-week old CiMKi^TA/TA;Villin-Cre mice (moderate CIN), scale bars 100 μm. f ß-catenin immunohistochemistry on small intestine lesions in 12-week old CiMKi^TA/TA;Villin-Cre mice. Arrowheads indicate nuclear ß-catenin, scale bars 100 μm. g H&E staining of low-grade colon adenoma from a 12-week old CiMKi^TA/TA;Villin-Cre mice (moderate CIN). Scale bar 1 mm. Statistics for panels c and d: one-tailed Welch’s t-test, comparing each group to CiMKi^WT/WT;Villin-Cre, exact p-values are indicated when p < 0.05. Source data for panels c and d are provided as a Source Data file.

Fig. 3. Degree and site define oncogenic potential of CIN in tumor-prone intestines.

a Methylene blue stained whole mount small intestines of CiMKi;Apc^Min/+;Villin-Cre mice, showing mucosal architecture and abnormalities. Scale bars 1 mm. b H&E sections of small intestines of mice from panel a. c Quantification of small intestine adenomas from CiMKi;Apc^Min/+;Villin-Cre mice. Open dots represent mice euthanatized at 6–8 weeks of age, closed dots represent mice at 12 weeks of age (n = 10 (WT/WT), 25 (WT/TA), 16 (WT/KD), 4 (TA/TA), or 7 (TA/KD) mice per genotype), data represents mean ± SD. d Formalin-fixed whole mount colons of CiMKi;Apc^Min/+;Villin-Cre mice, with adenomas predominantly located in the distal colon. Zooms indicate adenoma(s) in both genotypes. Scale bars 1 mm. e H&E sections of colons of mice from panel d. f Quantification of colon adenomas from CiMKi;Apc^Min/+;Villin-Cre mice. Representation as in c. g Quantification of chromosome segregation fidelity by time lapse imaging of colon adenoma organoid lines from different mice per genotype (N = 3 (WT/WT, WT/TA, TA/TA), or N = 5 (TA/KD). Percentages of missegregations per organoid were quantified (n = 29 (WT/WT), n = 27 (WT/TA), n = 24 (TA/TA), or n = 51 (TA/KD), from at least 5 divisions per organoid. Box-plot: each dot is one organoid, center line is median, box extends from 25th to 75th percentile, whiskers show min-to-max. h scKaryo-seq (bin size 5 MB) showing ploidy in individual cells of colon adenomas per genotype: aneuploidy and heterogeneity scores are given for each sample (n = 2 (TA/TA, TA/KD), or n = 3 (WT/WT, WT/TA), see also Supplementary Fig. 3H). Graphs show individual cells (horizontal lines) of one example per genotype. Colors indicate copy number state for a given chromosome. Statistics for panels c, f, and g: one-tailed Welch’s t-test, comparing each group to CiMKi^WT/WT;Apc^Min/+;Villin-Cre, exact p-values are indicated when p < 0.05, and p < 0.0001 is indicated by asterisks (****). Source data for panels c, f, and g are provided as a Source Data file.

Fig. 4. Enhanced proliferation in colon but not in small intestine.

a, b Quantification of chromosome segregation fidelity by time lapse imaging of colon organoids (n = 11 (WT/WT), n = 10 (WT/TA), n = 3 (TA/TA), n = 16 (TA/KD)) (a) and small intestine organoids (n = 21 (WT/WT), n = 16 (WT/TA), n = 15 (TA/TA), n = 10 (TA/KD)) (b), with at least 5 divisions per organoid, from various CiMKi;Apc^Min/+;Villin-CreER^T2 genotypes. Data represents mean ± SD, each dot is one organoid. c, d Proliferative compartment in colon (c) and small intestine (d) of 4-week old CiMKi;Apc^Min/+;Villin-Cre mice as determined on ki67 stained tissue sections by scoring the number of cells between the first positive cell at the bottom of the crypt and the last positive cell in the transit amplifying zone (for example images see Supplementary Fig. 2G, H). Dot plots show the average size of the compartment for each mouse (10 crypts (with normal appearance, selected from similar regions (~2/3 from proximal site) per mouse), and mean ± SD of 3 mice (WT/WT, TA/TA, TA/KD), or 4 mice (WT/TA) per genotype. Percentages indicate proliferative index (percentage of ki67 positive cells within compartment) for each genotype. e, f PCNA staining of tissue sections from colon (e) and small intestine (f) of 4-week old CiMKi;Apc^Min/+;Villin-Cre mice, as a proxy for proliferative activity, showing increased size and PCNA positivity of colon crypts from CiMKi^TA/TA;Apc^Min/+;Villin-Cre and CiMKi^TA/KD;Apc^Min/+;Villin-Cre mice. Images are from one of three mice per genotype (see Supplementary Fig. 4A, B). Alternating gray and white scale bars 50 μm. Crypts were selected from similar regions (~2/3 from proximal site). Statistics for panels a–d: one-tailed Welch’s t-test, comparing each group to CiMKi^WT/WT;Apc^Min/+;Villin-Cre(ER^T2), exact p-values are indicated when p < 0.05, and p < 0.0001 is indicated by asterisks (****). Source data for panels a–d are provided as a Source Data file.