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The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2–9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids. Currently, genetically encoded calcium indicators are not suitable for direct quantification. Here the authors engineer a fluorescence lifetime imaging calcium biosensor, Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS), and measure intracellular calcium concentrations in human-derived organoids.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.A biobank of ovarian cancer organoids recapitulates the histopathological and molecular hallmarks of patient tumors and provides a resource for preclinical research.

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting. Recent technical advances mean that miniature replicas of many tissues can be grown in the laboratory. These so-called organoids provide scientists with model systems that are not as limited as simple, two-dimensional sheets of cells growing in a petri dish, and less labor and resource intensive than studies using laboratory animals. In particular, organoids grown from tumor cells from cancer patients have been suggested as having numerous advantages over both laboratory-grown cancer cells and mice when it comes to testing potential new anticancer drugs. Mutations in a gene called KRAS are common in many types of cancer including colon cancer. Tumors with these mutations are difficult to treat and so far virtually all attempts to generate compounds that selectively interfere with the KRAS protein encoded by the mutant gene have failed. Instead, drugs that indirectly inhibit this protein’s effects by targeting other proteins in the same signaling pathway are currently being tested on patients. However, there is still a need for better ways to pre-test whether these drugs will be effective in humans without having to expose the patient to side effects or an ineffective drug. Now, Verissimo, Overmeer, Ponsioen et al. have tested clinically-used KRAS pathway inhibitors and drug combinations against normal colon organoids and colon cancer organoids derived from patients with colon cancer. Gene editing techniques were used to introduce KRAS mutations into some of the normal organoids grown from healthy tissue, and into cancer organoids grown from tumors that had a normal copy of the KRAS gene. In all cases, only those organoids with mutant forms of the KRAS gene were resistant to the treatments. Furthermore, when organoids with the KRAS mutation were treated with some combination therapies that are currently being tested in clinical trials, the tumors stopped growing but the tumor cells failed to die. Similar drug treatments on mice carrying human colon cancer organoids confirmed these results, which is in line with previous studies where tumor tissue from human patients was transplanted into mice. These findings show that collections of tumor organoids from multiple patients could help researchers to quickly identify and optimize targeted anticancer therapies before they are incorporated into clinical trials. In the future, clinical studies are needed to verify how accurately the testing of cancer drugs on organoids predicts whether the drug will or will not work in patients.

Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC. Ponsioen et al. use a FRET‐based ERK biosensor EKAREN5 in patient‐derived organoids to show that EGFR activity amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways.

Notch and vascular endothelial growth factor receptor (VEGFR) coordinates endothelial cells behaviour during angiogenesis sprouting although exactly how is uncertain. Endothelial cells dynamically compete for the leading position in a sprout through relative levels of Vegfr1 and Vegfr2 in a Notch dependent manner. Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2 , demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo , indicating that the VEGFR–Dll4–Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

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. The authors use three-dimensional live-cell imaging of colorectal carcinoma organoids to show that chromosomal instability is widespread. Single-cell sequencing identifies heterogeneity of copy number alterations and shows clonal evolution.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs.

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.

Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP–Epac–YFP fusion construct. When expressed in mammalian cells, CFP–Epac–YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP‐raising agents, whereas it fully recovers after addition of cAMP‐lowering agonists. Thus, by undergoing a cAMP‐induced conformational change, CFP–Epac–YFP serves as a highly sensitive cAMP indicator in vivo . When compared with a protein kinase A (PKA)‐based sensor, Epac‐based cAMP probes show an extended dynamic range and a better signal‐to‐noise ratio; furthermore, as a single polypeptide, CFP–Epac–YFP does not suffer from the technical problems encountered with multisubunit PKA‐based sensors. These properties make Epac‐based FRET probes the preferred indicators for monitoring cAMP levels in vivo .

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.