Prospective derivation of a living organoid biobank of colorectal cancer patients.

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.

Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Identification of multipotent luminal progenitor cells in human prostate organoid cultures.

The prostate gland consists of basal and luminal cells arranged as pseudostratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here, we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays, and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal-cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.

Correction: The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/ß-Catenin Coactivators Essential for Intestinal Homeostasis.

[This corrects the article DOI: 10.1371/journal.pbio.1000539.].

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

Centralized intestinal organoid generation is a feasible and safe approach for personalized medicine as demonstrated in the HIT-CF Europe Organoid Study.

BACKGROUND: Patient-derived intestinal organoids (PDIOs) show great potential as in vitro drug testing platform for personalised medicine in Cystic Fibrosis and oncology. PDIOs can be generated by culturing adult stem cells obtained through rectal forceps biopsy or suction biopsy, but the safety of these procedures and the success rates of generating organoids after shipment to a centralized lab using these procedures has not been studied in this context. We here report the safety and success rates of both biopsy procedures and the subsequent generation of PDIOs in the international multicentre HIT-CF Organoid Study. METHODS: 502 biopsy procedures were conducted, on 489 adult people with Cystic Fibrosis from 33 different hospitals across 12 countries. Depending on the preference of the hospital, either rectal forceps biopsies or suction biopsies were obtained and internationally shipped to a central laboratory for organoid generation. RESULTS: No adverse events were reported for 280 forceps biopsy procedures, while 222 rectal suction biopsy procedures resulted in 2 adverse events, namely continued bleeding and a probably nonrelated gastroenteritis. The success rate of organoid generation from all biopsies was 95%, and the main reason for failure was insufficient sample viability (3.2%). CONCLUSION: Our results indicate that both rectal suction biopsy and forceps biopsy procedures are safe procedures. The high success rates of PDIO generation from the obtained tissue samples demonstrate the feasibility of the organoid technology for personalised in vitro testing in an international setting.

Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis.

BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.

The kinase TNIK is an essential activator of Wnt target genes.

Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/beta-catenin-activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/beta-catenin transcriptional programme and tumourigenesis. The mechanisms by which TCF4/beta-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the germinal centre kinase family as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a beta-catenin-dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential function of the kinase activity in Wnt target gene activation. In vitro binding and kinase assays show that TNIK directly binds both TCF4 and beta-catenin and phosphorylates TCF4. siRNA depletion of TNIK followed by expression array analysis showed that TNIK is an essential, specific activator of Wnt transcriptional programme. This kinase may present an attractive candidate for drug targeting in colorectal cancer.

The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/ß-catenin coactivators essential for intestinal homeostasis.

Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/ß-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/ß-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and ß-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/ß-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a ß-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published ß-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.

Molecular and Functional Characterization of Human Intestinal Organoids and Monolayers for Modeling Epithelial Barrier.

BACKGROUND: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier. METHODS: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib. RESULTS: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib. CONCLUSIONS: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.

Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

A living biobank of matched pairs of patient-derived xenografts and organoids for cancer pharmacology.

Patient-derived tumor xenograft (PDX)/organoid (PDO), driven by cancer stem cells (CSC), are considered the most predictive models for translational oncology. Large PDX collections reflective of patient populations have been created and used extensively to test various investigational therapies, including population-trials as surrogate subjects in vivo. PDOs are recognized as in vitro surrogates for patients amenable for high-throughput screening (HTS). We have built a biobank of carcinoma PDX-derived organoids (PDXOs) by converting an existing PDX library and confirmed high degree of similarities between PDXOs and parental PDXs in genomics, histopathology and pharmacology, suggesting "biological equivalence or interchangeability" between the two. Here we demonstrate the applications of PDXO biobank for HTS "matrix" screening for both lead compounds and indications, immune cell co-cultures for immune-therapies and engineering enables in vitro/in vivo imaging. This large biobank of >550 matched pairs of PDXs/PDXOs across different cancers could become powerful tools for the future cancer drug discovery.

Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

BACKGROUND & AIMS: We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. METHODS: Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. RESULTS: Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. CONCLUSIONS: We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.

Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR × LGR5 bispecific antibody with efficacy in epithelial tumors.

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport, antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

G970R-CFTR Mutation (c.2908G>C) Results Predominantly in a Splicing Defect.

In previous work, participants with a G970R mutation in cystic fibrosis transmembrane conductance regulator (CFTR) (c.2908G>C) had numerically lower sweat chloride responses during ivacaftor treatment than participants with other CFTR gating mutations. The objective of this substudy was to characterize the molecular defect of the G970R mutation in vitro and assess the benefit of ivacaftor in participants with this mutation. This substudy assessed sweat chloride, spirometry findings, and nasal potential difference on and off ivacaftor treatment in three participants with a G970R/F508del genotype. Intestinal organoids derived from rectal biopsy specimens were used to assess ivacaftor response ex vivo and conduct messenger RNA splice and protein analyses. No consistent or meaningful trends were observed between on-treatment and off-treatment clinical assessments. Organoids did not respond to ivacaftor in forskolin-induced swelling assays; no mature CFTR protein was detected in Western blots. Organoid RNA analysis demonstrated that 3 novel splice variants were created by G970R-CFTR: exon 17 truncation, exons 13-15 and 17 skipping, and intron 17 retention. Functional and molecular analyses indicated that the c.2908G>C mutation caused a cryptic splicing defect. Organoids lacked an ex vivo response with ivacaftor and supported identification of the mechanism underlying the CFTR defect caused by c.2908G>C. Analysis of CFTR mutations indicated that cryptic splicing was a rare cause of mutation misclassification in engineered cell lines. This substudy used organoids as an alternative in vitro model for mutations, such as cryptic splice mutations that cannot be fully assessed using cDNA expressed in recombinant cell systems.

Lumacaftor/ivacaftor in people with cystic fibrosis with an A455E-CFTR mutation.

BACKGROUND: Previous in vitro organoid data showed A455E-CFTR, a rare CFTR mutation with 4.1% prevalence in the Netherlands, responds to lumacaftor/ivacaftor (LUM/IVA). We explored LUM/IVA's clinical efficacy in people with CF and ≥1 A455E-CFTR mutation. METHODS: Participants aged ≥12 years were randomized to 1 of 2 treatment sequences (LUM/IVA→placebo or placebo→LUM/IVA) with an 8-week washout period between. Primary endpoint was absolute change in ppFEV1 from study baseline through 8 weeks. Additional endpoints were change in sweat chloride concentration (SwCl) and CFQ-R respiratory domain score. Correlations between organoid-based measurements and clinical endpoints were investigated. RESULTS: Twenty participants were randomized at 2 sites in the Netherlands. Mean absolute change in ppFEV1 from study baseline through Week 8 showed a treatment difference of 0.1 percentage points (95% CI, -2.5 to 2.7; P = 0.928) between LUM/IVA (within-group mean change, 2.7) and placebo (within-group mean change, 2.6). The mean absolute change in SwCl concentration from study baseline through Week 8 showed a treatment difference of -7.8 mmol/L between LUM/IVA and placebo (P = 0.004), while the absolute change in CFQ-R respiratory domain score showed a treatment difference of 3.5 between LUM/IVA and placebo (P = 0.469). The in vitro organoid-based assay demonstrated a concentration-dependent swelling increase with LUM/IVA. Exploratory correlation analyses between organoid swelling and ppFEV1 and SwCl outcomes showed correlation coefficients of 0.49 and -0.11, respectively. CONCLUSIONS: In this exploratory study, LUM/IVA elicited an in vitro response in organoid swelling and in vivo response in SwCl in participants with CF and ≥1 A455E-CFTR mutation. The primary endpoint (ppFEV1) did not show a statistically significant difference between LUM/IVA and placebo; correlations between in vitro and in vivo responses were not established (NCT03061331).

An FBXW7-ZEB2 axis links EMT and tumour microenvironment to promote colorectal cancer stem cells and chemoresistance.

Colorectal cancer (CRC) patients develop recurrence after chemotherapy owing to the survival of stem cell-like cells referred to as cancer stem-like cells (CSCs). The origin of CSCs is linked to the epithelial-mesenchymal transition (EMT) process. Currently, it remains poorly understood how EMT programmes enable CSCs residing in the tumour microenvironment to escape the effects of chemotherapy. This study identifies a key molecular pathway that is responsible for the formation of drug-resistant CSC populations. Using a modified yeast-2-hybrid system and 2D gel-based proteomics methods, we show that the E3-ubiquitin ligase FBXW7 directly binds and degrades the EMT-inducing transcription factor ZEB2 in a phosphorylation-dependent manner. Loss of FBXW7 induces an EMT that can be effectively reversed by knockdown of ZEB2. The FBXW7-ZEB2 axis regulates such important cancer cell features, as stemness/dedifferentiation, chemoresistance and cell migration in vitro, ex vivo and in animal models of metastasis. High expression of ZEB2 in cancer tissues defines the reduced ZEB2 expression in the cancer-associated stroma in patients and in murine intestinal organoids, demonstrating a tumour-stromal crosstalk that modulates a niche and EMT activation. Our study thus uncovers a new molecular mechanism, by which the CRC cells display differences in resistance to chemotherapy and metastatic potential.

Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids.

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.

Rectal Organoids Enable Personalized Treatment of Cystic Fibrosis.

In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.