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Background Patients with peritoneal metastases from colorectal cancer have a poor prognosis. If the intraperitoneal tumour load is limited, patients may be eligible for cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment has improved overall survival, but recurrence rates are high. The aim of this study was to create a preclinical platform for the development of more effective intraperitoneal chemotherapy strategies. Methods Using organoid technology, five tumour cultures were generated from malignant ascites and resected peritoneal metastases. These were used in an in vitro HIPEC model to assess sensitivity to mitomycin C (MMC) and oxaliplatin, the drugs used most commonly in HIPEC. The model was also used to test a rational combination treatment involving MMC and inhibitors of the checkpoint kinase ATR. Results MMC was more effective in eliminating peritoneal metastasis‐derived organoids than oxaliplatin at clinically relevant concentrations. However, the drug concentrations required to eliminate 50 per cent of the tumour cells (IC50) were higher than the median clinical dose in two of five organoid lines for MMC, and all five lines for oxaliplatin, indicating a general resistance to monotherapy. ATR inhibition increased the sensitivity of all peritoneal metastasis‐derived organoids to MMC, as the IC50 decreased 2·6–12·4‐fold to well below concentrations commonly attained in clinical practice. Live‐cell imaging and flow cytometric analysis showed that ATR inhibition did not release cells from MMC‐induced cell cycle arrest, but caused increased replication stress and accelerated cell death. Conclusion Peritoneal metastasis‐derived organoids can be used to evaluate existing HIPEC regimens on an individual‐patient level and for development of more effective treatment strategies. Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has improved prognosis of patients with peritoneal metastases from colorectal cancer, but disease recurrence is common. More effective and personalized HIPEC is urgently needed. Organoid technology is frequently used for drug screens, as patient‐derived organoids can accurately predict clinical therapeutic response in vitro.A panel of organoids was established from peritoneal metastases from colorectal cancer and used to develop a model for testing HIPEC regimens in vitro. Patient‐derived organoids differed in sensitivity to commonly used chemotherapeutics, in line with variable clinical outcomes following cytoreductive surgery–HIPEC. Combining MMC with an ATR inhibitor improved the efficacy of MMC.Peritoneal metastasis‐derived organoids can be used as a platform to test novel (combination) strategies that increase HIPEC efficacy. In the future, organoids could be used to select patent‐tailored HIPEC regimens.

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 Biosensor for the Mitotic Kinase MPS1 Reveals Spatiotemporal Activity Dynamics and Regulation Highlights d Development of a FRET-based biosensor of MPS1 kinase activity d Active MPS1 detected at centromeres and chromatin is derived from kinetochores d MPS1 activity is initiated 12 min before NEB in a PP2A-B56dependent manner d Colon cancer cell lines and organoids have lower MPS1 activity than healthy lines

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Chromosomal instability (CIN) drives the formation of karyotype aberrations in cancer cells and is a major contributor to intra-tumour heterogeneity, metastasis, and therapy resistance. Understanding how CIN contributes to tumour karyotype evolution requires quantification of CIN rates in primary tumours. Single-cell sequencing-based technologies enable the detection of karyotype heterogeneity, however deducing the actual CIN rates that underlie intra-tumour heterogeneity is still complicated. We have developed an in-silico model, called CINsim, to simulate the karyotype dynamics and validated our model in a murine mouse model for T-cell lymphoma (T-ALL) in which CIN is introduced by mutation of the Mps1 spindle assembly checkpoint protein. CINsim can simulate karyotype evolution within physiologically relevant timescales, across a range of CIN rates, and across a range of karyotype-imposed survival and proliferation effects. We find that CINsim can accurately predict the CIN rates in chromosomal instable mouse T-ALLs as well as in human colon cancer organoids as observed by live-cell time-lapse imaging. We conclude that CINsim is a powerful tool to estimate CIN rates from static single-cell DNA sequencing data by finding the most likely path from euploid founder cell to a heterogeneous tumour cell population.