Karyotype diversification in colorectal cancer

From the moment of conception, every human being is in the process of developing cancer. Whether or not you will ultimately be diagnosed with this disease is simply a matter of whether something else kills you first. Cancer is not one disease. Rather, the radical growth of malignant cells represents a phenotypical extreme of cells that escape homeostasis. There are astronomically many potential combinations of genetic, epigenetic and environmental influences that can push cells into malignancy. Therefore, not only is every type of cancer different, but every instance of cancer is its own unique disease. The standardized treatments given to patients with similar types of malignancy are simplified abstractions, born from our lack of understanding of optimal treatment. The development of personalized cancer treatment options is one of the defining goals of modern medicine. Fundamental research plays an important role in progressing toward this goal, since without a true understanding of cancer biology we cannot hope to develop truly personalized treatment protocols. This thesis describes technological advances aimed at investigating fundamental cancer biology using patient derived organoids as a model system. Patient derived organoid culture protocols allow in vitro culture of three-dimensional (3D) wild-type and malignant tissue structures. By allowing differentiated outgrowth in 3D space, organoid culture protocols more accurately recapitulate tissue composition, density and cell division dynamics of human tumors in vivo. When combined with fluorescent live cell imaging, patient derived tumor organoids provide spectacular tools to support fundamental cancer research.One of the protocols we developed is called 3D Live-Seq, which combines imaging of tumor organoid outgrowth and single-cell sequencing of each imaged cell to reconstruct evolving tumor karyotypes (the number of chromosomes or sub chromosomal fragments per cell) across consecutive cell generations. By using 3D Live-Seq, we showed that advanced colorectal cancer cells continuously generate both singular and very complex chromosome errors during outgrowth of a tumor. Collectively, these cells represent a body of genetic diversity from which treatment resistant or more aggressive subclones may emerge during disease progression. <br/

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

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

How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing

Model systems with defined genetic modifications are powerful tools for basic research and translational disease modelling. Fortunately, generating state-of-the-art genetic model systems is becoming more accessible to non-geneticists due to advances in genome editing technologies. As a consequence, solely relying on (transient) overexpression of (mutant) effector proteins is no longer recommended since scientific standards increasingly demand genetic modification of endogenous loci. In this review, we provide up-to-date guidelines with respect to homology-directed repair (HDR)-mediated editing of mammalian model systems, aimed at assisting researchers in designing an efficient genome editing strategy

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids

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

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