Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Derivation of functional thymic epithelial organoid lines from adult murine thymus.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Tubuloid differentiation to model the human distal nephron and collecting duct in health and disease.

Organoid technology is rapidly gaining ground for studies on organ (patho)physiology. Tubuloids are long-term expanding organoids grown from adult kidney tissue or urine. The progenitor state of expanding tubuloids comes at the expense of differentiation. Here, we differentiate tubuloids to model the distal nephron and collecting ducts, essential functional parts of the kidney. Differentiation suppresses progenitor traits and upregulates genes required for function. A single-cell atlas reveals that differentiation predominantly generates thick ascending limb and principal cells. Differentiated human tubuloids express luminal NKCC2 and ENaC capable of diuretic-inhibitable electrolyte uptake and enable disease modeling as demonstrated by a lithium-induced tubulopathy model. Lithium causes hallmark AQP2 loss, induces proliferation, and upregulates inflammatory mediators, as seen in vivo. Lithium also suppresses electrolyte transport in multiple segments. In conclusion, this tubuloid model enables modeling of the human distal nephron and collecting duct in health and disease and provides opportunities to develop improved therapies.

Druggable growth dependencies and tumor evolution analysis in patient-derived organoids of neuroendocrine neoplasms from multiple body sites.

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Treatment options for patients with NENs are limited, in part due to lack of accurate models. We establish patient-derived tumor organoids (PDTOs) from pulmonary NETs and derive PDTOs from an understudied subtype of NEC, large cell neuroendocrine carcinoma (LCNEC), arising from multiple body sites. PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through hypothesis-driven drug sensitivity analyses, we identify ASCL1 as a potential biomarker for response of LCNEC to treatment with BCL-2 inhibitors. Additionally, we discover a dependency on EGF in pulmonary NET PDTOs. Consistent with these findings, we find that, in an independent cohort, approximately 50% of pulmonary NETs express EGFR. This study identifies an actionable vulnerability for a subset of pulmonary NETs, emphasizing the utility of these PDTO models.

Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification.

BACKGROUND: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas. METHODS: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. FINDINGS: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. CONCLUSIONS: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation. FUNDING: This work was funded by Oncode PoC 2018-P0003.

Drug-repurposing screen on patient-derived organoids identifies therapy-induced vulnerability in KRAS-mutant colon cancer.

Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids.

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation.

Opposing roles have been proposed for IL-22 in intestinal pathophysiology. We have optimized human small intestinal organoid (hSIO) culturing, constitutively generating all differentiated cell types while maintaining an active stem cell compartment. IL-22 does not promote the expansion of stem cells but rather slows the growth of hSIOs. In hSIOs, IL-22 is required for formation of Paneth cells, the prime producers of intestinal antimicrobial peptides (AMPs). Introduction of inflammatory bowel disease (IBD)-associated loss-of-function mutations in the IL-22 co-receptor gene IL10RB resulted in abolishment of Paneth cells in hSIOs. Moreover, IL-22 induced expression of host defense genes (such as REG1A, REG1B, and DMBT1) in enterocytes, goblet cells, Paneth cells, Tuft cells, and even stem cells. Thus, IL-22 does not directly control the regenerative capacity of crypt stem cells but rather boosts Paneth cell numbers, as well as the expression of AMPs in all cell types.

BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states.

Intestinal epithelial cells derive from stem cells at the crypt base and travel along the crypt-villus axis to die at the villus tip. The two dominant villus epithelial cell types, absorptive enterocytes and mucous-secreting goblet cells, are mature when they exit crypts. Murine enterocytes switch functional cell states during migration along the villus. Here, we ask whether this zonation is driven by the bone morphogenetic protein (BMP) gradient, which increases toward the villus. Using human intestinal organoids, we show that BMP signaling controls the expression of zonated genes in enterocytes. We find that goblet cells display similar zonation involving antimicrobial genes. Using an inducible Bmpr1a knockout mouse model, we confirm that BMP controls these zonated genes in vivo. Our findings imply that local manipulation of BMP signal strength may be used to reset the enterocyte "rheostat" of carbohydrate versus lipid uptake and to control the antimicrobial response through goblet cells.

Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism.

The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves' disease patient sera, demonstrating that organoids can model human autoimmune disease.

Modelling of primary ciliary dyskinesia using patient-derived airway organoids.

Patient-derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long-term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient-specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient-specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.

Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer.

Cervical cancer is a common gynecological malignancy often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study the cervical epithelium and the cancers derived thereof. Here we describe a long-term culturing protocol for ecto- and endocervical epithelia that generates 3D organoids that stably recapitulate the two tissues of origin. As evidenced for HSV-1, organoid-based cervical models may serve to study sexually transmitted infections. Starting from Pap brush material, a small biobank of tumoroids derived from affected individuals was established that retained the causative human papillomavirus (HPV) genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutic agents and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Exploring the human lacrimal gland using organoids and single-cell sequencing.

The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland.

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.