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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., Analysis of live-cell imaging and single-cell genome sequencing data of colorectal cancer organoids identifies temporal dynamics of sub-chromosomal copy-number amplifications.

Aneuploidy, defined as the loss and gain of whole and part chromosomes, is a near-ubiquitous feature of cancer genomes, is prognostic, and likely an important determinant of cancer cell biology. In colorectal cancer (CRC), aneuploidy is found in virtually all tumours, including precursor adenomas. However, the temporal evolutionary dynamics that select for aneuploidy remain broadly uncharacterised. Here we perform genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that cross-sectionally represent the distinct stages of tumour evolution, and longitudinally track individual tumours through metastasis and treatment. Precancer lesions (adenomas) exhibited low levels of aneuploidy but high intra-tumour heterogeneity, whereas cancers had high aneuploidy but low heterogeneity, indicating that progression is through a genetic bottleneck that suppresses diversity. Individual CRC glands from the same tumour have similar karyotypes, despite prior evidence of ongoing instability at the cell level. Pseudo-stable aneuploid genomes were observed in metastatic lesions sampled from liver and other organs, after chemo- or targeted therapies, and late recurrences detected many years after the diagnosis of a primary tumour. Modelling indicates that these data are consistent with the action of stabilising selection that ‘traps’ cancer cell genomes on a fitness peak defined by the specific pattern of aneuploidy. These data show that the initial progression of CRC requires the traversal of a rugged fitness landscape and subsequent genomic evolution, including metastatic dissemination and therapeutic resistance, is constrained by stabilising selection.

The aim of our study is to follow the temporal and spatial interactions of proteins within different signalling networks in live cells. To map protein-protein interactions, we apply Forster resonant energy transfer (FRET) readout using fluorescence lifetime imaging (FLIM) and we present an optically sectioned FLIM microscope capable of time-lapse readouts of multiplexed FRET pairs. We note that FLIM FRET provides robust measurements, permits the use of dark acceptors and helps minimise the impact of spectral cross-talk.Wide-field FLIM is implemented using a gated optical intensifier synchronized with the excitation pulses from a fibre-laser pumped supercontinuum source or a frequency doubled Ti:Sapphire laser and enables FLIM FRET in live cells with acquisition times below ∼10 seconds. Optical sectioning is implemented using a Nipkow disk unit and improves quantitation compared to wide-field imaging, e.g. permitting separation of signals from the plasma membrane and the cytosol.To demonstrate the capabilities of this instrument, we have mapped the intracellular changes of calcium levels using interleaved time lapse FLIM acquisitions of live cells labelled with two calcium probes following stimulation with ionomycin and calcium solutions. HEK293 cells were labelled with the genetically expressed biosensor, Troponin TN-XL, for which changes in calcium levels are read out by FRET between the CFP and YFP fluorophores, and with a calcium sensing dye, GFP-Certified™ FluoForte™. The emission profile of the latter is spectrally separate from CFP and YFP, making it useful for multiplexing with the many available CFP/YFP FRET biosensors.We aim to apply this instrument to follow the spatial and temporal changes of simultaneous activation of signalling pathways during fibroblast migration in a concentration gradient of a chemo-attractant (PDGF), particularly using specifically designed FRET biosensors for small GTPases (Rac), phosphoinositide pathway (IP3) and calcium.