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.
Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Single-Cell Analysis/methods , Cell Proliferation/genetics , Chromatin/genetics , Chromosomes, Human , Gene Dosage , Humans , Karyotype , Karyotyping , Microscopy, Confocal , Mitosis , Organoids/growth & development , Organoids/pathology , Spindle Apparatus/geneticsABSTRACT
Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation occurs and how MPS1 signaling is modulated during mitosis, we developed MPS1sen, a sensitive and specific FRET-based biosensor for MPS1 activity. By placing MPS1sen at different subcellular locations, we show that MPS1 activity initiates in the nucleus â¼9-12 min prior to nuclear envelope breakdown (NEB) in a kinetochore-dependent manner and reaches the cytoplasm at the start of NEB. Soon after initiation, MPS1 activity increases with switch-like kinetics, peaking at completion of NEB. We further show that timing and extent of pre-NEB MPS1 activity is regulated by Aurora B and PP2A-B56. MPS1sen phosphorylation declines in prometaphase as a result of formation of kinetochore-microtubule attachments, reaching low but still detectable levels at metaphase. Finally, leveraging the sensitivity and dynamic range of MPS1sen, we show deregulated MPS1 signaling dynamics in colorectal cancer cell lines and tumor organoids with diverse genomic instability phenotypes.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosome Segregation/physiology , M Phase Cell Cycle Checkpoints/physiology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Anaphase , Aurora Kinase B/metabolism , Cell Cycle/physiology , Cell Cycle Proteins/physiology , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Nucleus/physiology , Chromosome Segregation/genetics , Fluorescence Resonance Energy Transfer/methods , HeLa Cells , Humans , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints/genetics , Metaphase , Microtubules/metabolism , Mitosis/genetics , Mitosis/physiology , Organoids/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/physiology , Protein-Tyrosine Kinases/physiology , Signal Transduction , Spatio-Temporal Analysis , Spindle Apparatus/genetics , Spindle Apparatus/metabolismABSTRACT
Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.
Subject(s)
Chromosomal Instability , Colorectal Neoplasms/genetics , Aneuploidy , Cell Line, Tumor , Chromosome Segregation , Colorectal Neoplasms/pathology , DNA Copy Number Variations , Humans , Imaging, Three-Dimensional , Karyotype , Karyotyping , Microsatellite Instability , Mitosis/genetics , Mutation , Organoids/pathology , Single-Cell AnalysisABSTRACT
Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids.
