Live-cell imaging integrated with genome sequencing enabled de novo karyotype alteration analysis.
Major Finding: Live-cell imaging integrated with genome sequencing enabled de novo karyotype alteration analysis.
Concept: Evolutionary paths of karyotype alterations in colorectal tumor organoids were reconstructed using this method.
Impact: This examination of the dynamics of karyotype alteration provides support for punctuated evolution in cancer.
Aneuploidy and copy number alterations (CNA) are common features of cancer, but technical limitations have hindered the study of aneuploid tumor genome evolution over time at single-cell resolution. To understand how de novo karyotype alterations emerge, Bollen, Stelloo, and colleagues assessed chromosomal changes during clonal outgrowth in tumor organoids derived from patients with colorectal cancer. Single-cell whole-genome sequencing analysis of the entire cell population in each tumor organoid revealed the emergence of de novo CNAs during clonal outgrowth, including gains and losses at whole-chromosome and subchromosomal levels, as well as extreme cases of large deviations from core karyotypes, likely to substantially alter fitness. To reconstruct the evolutionary history of karyotype alterations across cell generations, a novel method called 3D Live-Seq was developed, integrating confocal live-cell imaging and single-cell whole-genome sequencing. When applied in patient-derived colorectal cancer tumor organoids, 3D Live-Seq analysis found that most de novo CNAs resulted from either chromatin bridges, associated with subchromosomal CNAs, or lagging chromatin, associated with whole-chromosome missegregation. Mitotic trees were reconstructed across multiple consecutive cell divisions within tumor organoids, mapping the propagation of various de novo CNAs through lineages and suggesting that acentric chromosomal fragments often went through cycles of replication before undergoing collective missegregation into one daughter cell. Mitotic slippage was shown to enable whole-genome doubling, often resulting in multipolar spindle defects that led to the generation of daughter cells with aberrant, genome-wide karyotype alterations. Through modeling of CNA evolution and selection pressures, novel karyotypes seemed to frequently remain proliferative, experiencing neutral or slightly negative changes in fitness, whereas large deviations from the core karyotype were least fit. In agreement with data from organoids, freshly frozen human colorectal cancer samples displayed substantial karyotype diversity, supporting the emergence and propagation of de novo CNAs in vivo. In summary, this work elucidates patterns of gradual and punctuated karyotype alterations and sheds light on mechanisms that contribute to aneuploid genome evolution in colorectal cancer.
Bollen Y, Stelloo E, van Leenen P, van den Bos M, Ponsioen B, Lu B, et al. Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns. Nat Genet 2021 Jul 1 [Epub ahead of print].
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