Liver Colonization by Colorectal Cancer Metastases Requires YAP-Controlled Plasticity at the Micrometastatic Stage

Micrometastases of colorectal cancer can remain dormant for years prior to the formation of actively growing, clinically detectable lesions (i.e., colonization). A better understanding of this step in the metastatic cascade could help improve metastasis prevention and treatment. Here we analyzed liver specimens of patients with colorectal cancer and monitored real-time metastasis formation in mouse livers using intravital microscopy to reveal that micrometastatic lesions are devoid of cancer stem cells (CSC). However, lesions that grow into overt metastases demonstrated appearance of de novo CSCs through cellular plasticity at a multicellular stage. Clonal outgrowth of patient-derived colorectal cancer organoids phenocopied the cellular and transcriptomic changes observed during in vivo metastasis formation. First, formation of mature CSCs occurred at a multicellular stage and promoted growth. Conversely, failure of immature CSCs to generate more differentiated cells arrested growth, implying that cellular heterogeneity is required for continuous growth. Second, early-stage YAP activity was required for the survival of organoid-forming cells. However, subsequent attenuation of early-stage YAP activity was essential to allow for the formation of cell type heterogeneity, while persistent YAP signaling locked micro-organoids in a cellularly homogenous and growth-stalled state. Analysis of metastasis formation in mouse livers using single-cell RNA sequencing confirmed the transient presence of early-stage YAP activity, followed by emergence of CSC and non-CSC phenotypes, irrespective of the initial phenotype of the metastatic cell of origin. Thus, establishment of cellular heterogeneity after an initial YAP-controlled outgrowth phase marks the transition to continuously growing macrometastases. Significance: Characterization of the cell type dynamics, composition, and transcriptome of early colorectal cancer liver metastases reveals that failure to establish cellular heterogeneity through YAP-controlled epithelial self-organization prohibits the outgrowth of micrometastases

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

Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells

<div><p>The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.</p></div

Specific Labeling of Stem Cell Activity in Human Colorectal Organoids Using an ASCL2-Responsive Minigene

Summary: Organoid technology provides the possibility of culturing patient-derived colon tissue and colorectal cancers (CRCs) while maintaining all functional and phenotypic characteristics. Labeling stem cells, especially in normal and benign tumor organoids of human colon, is challenging and therefore limits maximal exploitation of organoid libraries for human stem cell research. Here, we developed STAR (stem cell Ascl2 reporter), a minimal enhancer/promoter element that reports transcriptional activity of ASCL2, a master regulator of LGR5+ intestinal stem cells. Using lentiviral infection, STAR drives specific expression in stem cells of normal organoids and in multiple engineered and patient-derived CRC organoids of different genetic makeup. STAR reveals that differentiation hierarchies and the potential for cell fate plasticity are present at all stages of human CRC development. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research into human adult stem cell biology

Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

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

Specific Labeling of Stem Cell Activity in Human Colorectal Organoids Using an ASCL2-Responsive Minigene

Organoid technology provides the possibility of culturing patient-derived colon tissue and colorectal cancers (CRCs) while maintaining all functional and phenotypic characteristics. Labeling stem cells, especially in normal and benign tumor organoids of human colon, is challenging and therefore limits maximal exploitation of organoid libraries for human stem cell research. Here, we developed STAR (stem cell Ascl2 reporter), a minimal enhancer/promoter element that reports transcriptional activity of ASCL2, a master regulator of LGR5+ intestinal stem cells. Using lentiviral infection, STAR drives specific expression in stem cells of normal organoids and in multiple engineered and patient-derived CRC organoids of different genetic makeup. STAR reveals that differentiation hierarchies and the potential for cell fate plasticity are present at all stages of human CRC development. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research into human adult stem cell biology. Oost et al. present an ASCL2-responsive minigene (STAR) that enables stem cell labeling in patient-derived colorectal cancer organoids, as well as in normal and benign colorectal tumor samples. The user-friendly nature of STAR applications in combination with organoid technology will facilitate basic research into human adult stem cell biology

CRISPR/Cas9 baculovirus mediated Cas9 expression in U-2 OS cells.

<p>A) Schematic representation of the recombination of a pAceBac-Cas9 plasmid with a bacmid in EmBacY cells. The resulting bacmids were used for CRISPR/Cas9 baculovirus production in Sf9 cells. B) Representative FACS-profile showing GFP expression in U-2 OS cells treated with CRISPR/Cas9 baculovirus (MOI: 25). C) Western blot showing expression of Cas9 in U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25). α-tubulin was used as a loading control.</p

Introduction of the H250Y mutation in Aurora B.

<p>A) Schematic representation of the introduction of an HDR template carrying point mutations in the pAceBac-Cas9 plasmid. B) Colony formation of HCT116 cells in ZM447439 with cells carrying either wildtype Aurora B alleles, or alleles with homozygous H250Y mutations, or with a heterozygous mutation for H250Y combined with an indel in the other allele. The numbers indicate colony outgrowth in the ZM447439 treated conditions relative to the untreated conditions. Clones were also tested for their capacity to form colonies in the presence of puromycin. C) Sequence chromatograms of the Aurora B locus in the vicinity of H250. Depicted is the Aurora B sequence trace of wildtype HCT116 cells or clones carrying either the homozygous Aurora B H250Y mutation or the heterozygous Aurora B H250Y mutation combined with an indel. The PAM site is indicated and the arrows point out the base substitutions that were introduced. The corresponding amino acid sequence is shown in the top graph. D) Representative immunofluorescence images of prometaphase cells that have either retained or lost histone H3 Serine 10 (H3S10) phosphorylation after treatment with ZM447439 (0.5 μM). E) Quantification of immunofluorescence images of prometaphase cells depicted in (D) Cells were scored for H3S10ph loss following treatment with ZM447439 (0.5 μM). F) Immunofluorescence images of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75) were arrested in mitosis and scored for loss of centromeric Aurora B. G) Representative FACS-profiles showing the DNA content of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations that were treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75). Puromycin treatment was used to select for transduced cells and samples were harvested 4 days after transduction.</p

Effect of CRISPR/Cas9 baculoviral transduction in a panel of cell lines.

<p>A) Representative FACS-profiles showing GFP expression in cells treated with Cas9-GFP baculovirus (MOI: 75). The markers are set such that 2% of the cells treated with Cas9-puro baculovirus are included in this region. The percentage of cells treated with Cas9-GFP baculovirus within the marker region is indicated. B) Immunofluorescence images of mitotic cells treated with the indicated Cas9-GFP baculoviruses (MOI: 75) were scored by eye for the presence or absence of centromeric Aurora B. The bars represent the mean ± SD of 2 experiments. At least 24 cells were analyzed per experiment per condition.</p

Endogenous tagging of Haspin using CRISPR/Cas9 baculovirus.

<p>A) Schematic representation of the introduction of an HDR template for endogenous tagging of <i>GSG2</i> in the pAceBac-Cas9 plasmid. B) Integration of the YFP-tag at the C-terminus of the gene encoding Haspin was confirmed by PCR using the indicated primer pairs, schematically depicted in Fig 5A. The PCR product obtained using primers 1 and 2 is of the untagged allele, based on its size. C) Representative live cell images of U-2 OS-LacO Haspin-YFP cells and RPE-1 Haspin-YFP cells in prometaphase and interphase. D) Schematic overview depicting the binding of LacI-tagRFP-Pds5B to the LacO repeats. Upon interaction between Pds5B and Haspin an YFP signal can be detected at this ectopic locus. E) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B and stained for DAPI, YFP and RFP. F) Quantification of Haspin-YFP at the LacO locus. Depicted is the mean of Haspin-YFP normalized over RFP ± SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. G) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B stained for DAPI, H3T3ph and RFP. H) Quantifications of H3T3ph at the LacO locus. Depicted is the mean of H3T3ph levels normalized over RFP ± the SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. A minimum of 23 cells was analyzed per experiment.</p