Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells

Abstract

Whole chromosome gains or losses (aneuploidy) are a hallmark of ~70% of human tumors. Modeling the consequences of aneuploidy has relied on perturbing spindle assembly checkpoint (SAC) components, but interpretations of these experiments are clouded by the multiple functions of these proteins. Here, we used a Cre recombinase-mediated chromosome loss strategy to individually delete mouse chromosomes 9, 10, 12, or 14 in tetraploid immortalized murine embryonic fibroblasts. This methodology also involves the generation of a dicentric chromosome intermediate, which subsequently undergoes a series of breakage-fusion-bridge (BFB) cycles. While the aneuploid cells generally display a growth disadvantage in vitro, they grow significantly better in low adherence sphere-forming conditions and three of the four lines are transformed in vivo, forming large and invasive tumors in immunocompromised mice. The aneuploid cells display increased chromosomal instability and DNA damage, a mutator phenotype associated with tumorigenesis in vivo Thus, these studies demonstrate a causative role for whole chromosome loss and the associated BFB-mediated instability in tumorigenesis and may shed light on the early consequences of aneuploidy in mammalian cells.

Keywords: aneuploidy; chromosomal instability; chromosome losses; tetraploidy; tumorigenesis.

Figure 1. Generation of ICL in MEFs by Cre recombination

1. Schematic of the inverted lox P (iLoxP) site, flanked by sortable GFP and hCD2 markers.

2. Model of reciprocal recombination yielding parental configurations 1/2′ and 1′/2 and dicentric/acentric configurations as a result of inverted recombination yielding configurations 1/1′ and 2/2′.

3. Representative FACS plot showing loss of hCD2 marker expression of Ch14 ICL MEFs after treatment with 4‐hydroxy tamoxifen (4OHT) for 14 days to induce Cre, compared to ethanol‐treated control cells (See also Appendix Fig S1A).

4. Shallow whole‐genome sequencing (copy number profiles) of large T antigen immortalized MEFs after exposure to Cre recombinase and sorted for control (hCD2 Plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10, without any in vitro culturing, post‐FACS sort.

Figure 2. Validation and tumorigenic potential of early passage aneuploid cells

1. Shallow whole‐genome sequencing (copy number profiles) of large T antigen immortalized, early passage MEFs after exposure to Cre recombinase and sorted for control (hCD2 Plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10.

2. Tumor growth curve after early passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SEM, *P < 0.05 for Ch9 and Ch14). Two‐tailed unpaired t‐test was used to determine statistical significance.

Figure 3. Karyotypic characterization of the late passage ICL lines

1. A–D

   Representative karyotypes of late passage, large T antigen immortalized MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 Plus) and ICL (hCD2 Minus) cells for chromosome lines (A) Ch12, (B) Ch14, (C) Ch9, and (D) Ch10. Boxed panels denote targeted chromosome loss; arrows and asterisks denote shared and unshared, non‐targeted chromosomal copy number variations, respectively.

Figure 4. Comprehensive karyotypic characterization of the late passage ICL lines

1. A–D

   Karyotypic analysis of later passage, control, and ICL of a replicate (#1) of Ch12 (A), Ch10 (B), Ch14 (C), and Ch9 (D) lines. All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.

Figure EV1. Comprehensive karyotypic characterization of the late passage ICL lines

1. A–D

   Karyotypic analysis of later passage, control and ICL of a replicate (#2) of Ch12 (A), Ch10 (B), Ch14 (C) and Ch9 (D) lines. All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red.

Figure 5. Tumorigenic potential of late passage aneuploid cells

1. Tumor growth curve after later passage ICL cells were injected into flanks of athymic nude mice (n = 5 per group and error bars denote SD, ***P < 0.0005 for Ch9, *P < 0.05 for Ch10 and **P < 0.005 for Ch14). Two‐tailed unpaired t‐test was used to determine statistical significance.

2. Tumor sections were stained with H&E to assess tumor histology (scale bars denote 5 mm for the inset and 200 μm for the zoomed image). Arrows in the ICL tumors sections indicate regions where tumors have invaded into the adjacent tissue.

3. Tumor growth curve after later passage ICL cells were injected into flanks of NOD/SCID mice (n = 5 per group and error bars denote SD, ***P < 0.0005 for Ch10 and **P < 0.005 for Ch14). Two‐tailed unpaired t‐test was used to determine statistical significance.

Figure EV2. Comprehensive karyotypic characterization of the late passage ICL tumor explants

1. A–C

   Karyotypic analysis of later passage, control, and ICL tumor explants derived from the injecting the late passage control and ICL cells of Ch10 (A), Ch14 (B), and Ch9 (C). All chromosomes are shown for 20 metaphases. Target chromosomes are boxed in red. No karyotypes are shown for the Ch9 controls, as they did not form tumors in nude mice.

Figure EV3. Global copy number profiles of the late passage ICL MEFs

1. A–C

   Shallow whole‐genome sequencing (copy number profiles) of large T antigen immortalized, late passage MEFs after exposure to Cre recombinase and serially sorted for control (hCD2 Plus) and ICL (hCD2 Minus) cells for chromosomes 12, 14 and 9 and 10.

Figure 6. In vitro characterization of later passage ICL MEFs

1. Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n = 3 for each data set and error bars denote SD, **P < 0.005 for Ch10 and ***P < 0.0001 for Ch14). See also Appendix Fig S3A. Two‐tailed unpaired t‐test was used to determine statistical significance.

2. Colony formation in ICL and control MEFs. 5,000 cells were seeded in 10‐cm plates and stained with methylene blue after 3 days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n = 3 for each data set and error bars denote SD, **P < 0.005 for Ch10, Ch12, and Ch14 and ns for Ch9). Two‐tailed unpaired t‐test was used to determine statistical significance.

3. Quantification of growth in ultra‐low adherent, sphere‐forming conditions for the four ICL lines (n = 3 for Ch10 and Ch14, n = 6 for Ch9 and Ch12 and error bars denote SD, ***P < 0.0001 for Ch9, **P < 0.001 for Ch10 and *P < 0.05 for Ch12 and Ch14). Two‐tailed unpaired t‐test was used to determine statistical significance.

4. Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL, and control line was grown in culture for 3 days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples (n = 3 for each data set and error bars denote SD; Glucose consumed: ***P < 0.0005 for Ch9 and Ch14, ***P = 0.0005 for Ch10 and *P < 0.05 for Ch12; Lactate produced: ***P < 0.0005 for Ch9 and Ch12, ***P = 0.0008 for Ch10, and ***P = 0.0005 for Ch14; Glutamine consumed: ***P < 0.0005 for Ch9 and Ch14). Two‐tailed unpaired t‐test was used to determine statistical significance.

Figure 7. Effects of ploidy on the tumorigenic potential of ICL cells

1. Flow cytometry analysis of tail blood from Ch10/+ (control) at 30 days post‐tamoxifen administration, Ch10/+; Id1 Cre/+ at 30 days post‐tamoxifen administration, and Ch10/+; Id1Cre/+ at 80 days post‐tamoxifen administration.

2. Analysis from the Mitelman database for the fraction of human solid tumors that have lost (top) or gained (bottom) individual chromosomes that have modal chromosome number > 2N or ˜2N (n = 3,459 cases for modal number > 2N and n = 12,464 cases for modal number ˜2N, top: ***P < 0.0001 for chromosomes 1–22, X and *P < 0.05 for chromosome Y; bottom: ***P < 0.0001 for all chromosomes). Two‐tailed two‐sample t‐test to compare sample means was used to determine statistical significance.

3. Analysis from the Mitelman database for the fraction of human solid tumors that have lost or gained any chromosome that have modal chromosome number > 2N or ˜2N (n = 3,459 cases for modal number > 2N and n = 12,464 cases for modal number ˜2N, ***P < 0.0001 for both modal number > 2N and ˜2N). Two‐tailed two‐sample t‐test to compare sample means was used to determine statistical significance.

Figure 8. Increased chromosomal instability and DNA damage in ICL in vitro lines and tumors

1. Chromosomal instability readouts, of later passage lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n = 20 for each data set and error bars denote SD, average chromosome loss: **P < 0.005 for Ch9, ***P < 0.0001 for Ch10 and Ch14, and **P < 0.05 for Ch12; average structural rearrangements: ***P < 0.0001 for Ch9 and Ch10, **P < 0.05 for Ch12, and *P < 0.05 for Ch14; percentage of metaphases with marker chromosomes: **P < 0.0005 for Ch10). Two‐tailed unpaired t‐test was used to determine statistical significance for average chromosome loss and structural breaks. Two‐tailed Z‐test was used to determine statistical significance for the percentage of cells with marker chromosomes.

2. Determination of number of γH2AX foci in ICL and control nuclei and quantification (scale bars denote 10 μm, ***P < 0.0001 for Ch9, Ch10, and Ch12, *P < 0.05 for Ch14; n = 25 for each data set, and error bars denote SD). See also Appendix Fig S5A and B. Two‐tailed unpaired t‐test was used to determine statistical significance.

3. Number of γH2AX‐positive cells in formalin fixed ICL and control tumor sections by immunofluorescence staining and quantification (Scale bars denote 20 μm, n = 8 fields for Ch10 and 5 fields for Ch14 with at least 200 cells in each field, ***P < 0.0001 for Ch10 and *P < 0.05 for Ch14). Two‐tailed Z‐test was used to determine statistical significance.

Figure EV4. Increased chromosomal instability in ICL tumors

Chromosomal instability readouts, of tumor lines, assessed by average chromosomes loss per metaphase, average number of structural rearrangements per metaphase and percentage of metaphases with marker chromosomes (n = 20 for each data set and error bars denote SD, average chromosome loss: ****P < 0.0001 for Ch10; average structural rearrangements: *P < 0.05 for Ch10 and Ch14; percentage of metaphases with marker chromosomes: *P < 0.05 for Ch10). Two‐tailed unpaired t‐test was used to determine statistical significance for average chromosome loss and structural breaks. Two‐tailed Z‐test was used to determine statistical significance for the percentage of cells with marker chromosomes.