Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Abstract

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness.

A motor-based approach to induce chromosome-specific mis-segregations in human cells

“…employed dCas9-CENP-T fusion proteins to build an ectopic kinetochore on targeted chromosomes, a strategy that also increased the frequency of chromosome-specific CIN and aneuploidies. When for instance combined with microscopy-based single-cell isolation methods and subsequent sequencing, such as described for Look-Seq 57 , Live-Seq 58 , or Photostick 59 , our methods open up the possibility to investigate the immediate cellular responses to specific (arm level) aneuploidies in different cell types; an important step towards understanding how recurrent aneuploidy patterns arise in different cancer types. Moreover, by taking advantage of the poleward pulling forces exerted by Kin14VIb on the targeted chromosome, our method can also be applied to study biophysical properties of mitotic human chromosomes in living cells.…”

A DNA damage response-like phenotype defines a novel subset of colon cancer

“…We reasoned that the DDR + tumor areas might be the site of punctuated karyotipic evolution 37 in COAD with ongoing copy number variation. To prove that, we used a FISH probe for the most represented chromosome alteration in COAD, chr20qAmp 27 and we targeted the analysis to the DDR + phenotypecontaining areas (and controls).…”

Recent advances in targeted drug delivery systems for resistant colorectal cancer

“…Aneuploidy is also common feature of cancers and copy number alteration (CNA) studies on organoid culture revealed de novo emergence of whole-chromosome and sub-chromosomal changes during tumor growth with chromatin errors acting as underlying reasons; as chromatin bridge led to subchromosomal CNAs while, lagging chromatin result in whole chromosomal CNAs. Multipolar spindle defect and acentric chromosome fragment replication are other karyotype alteration reasons [ 51 ]. Indeed chromosomal instability result in cancer cell population diversity and thereafter immune escape, inflammation and is a negative indicator for survival rate of cancers [ 27 ].…”