Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma

Journal Pre-proof Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma Kenta Kawasaki, Masayuki Fujii, Shinya Sugimoto, Keiko Ishikawa, Mami Matano, Yuki Ohta, Kohta Toshimitsu, Sirirat Takahashi, Naoki Hosoe, Shigeki Sekine, Takanori Kanai, Toshiro Sato PII: DOI: Reference:

S0016-5085(19)41446-7 https://doi.org/10.1053/j.gastro.2019.10.009 YGAST 62949

To appear in: Gastroenterology Accepted Date: 8 October 2019 Please cite this article as: Kawasaki K, Fujii M, Sugimoto S, Ishikawa K, Matano M, Ohta Y, Toshimitsu K, Takahashi S, Hosoe N, Sekine S, Kanai T, Sato T, Chromosome Engineering of Human ColonDerived Organoids to Develop a Model of Traditional Serrated Adenoma, Gastroenterology (2019), doi: https://doi.org/10.1053/j.gastro.2019.10.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 by the AGA Institute

Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma Kenta Kawasaki1, 2, Masayuki Fujii1, 2, Shinya Sugimoto1, 2, Keiko Ishikawa1, 2, Mami Matano1, 2, Yuki Ohta1, 2, Kohta Toshimitsu1, 2, Sirirat Takahashi1, 2, Naoki Hosoe3, Shigeki Sekine4, Takanori Kanai1, Toshiro Sato1, 2. 1

Department of Gastroenterology, 2Department of Organoid Medicine, 3Center for Diagnostic and Therapeutic Endoscopy, Keio University School of Medicine, Tokyo, Japan. 4Division of Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan. Short title: Chromosome-engineered organoids mimic TSA

Grant support: This research was supported by the Japan Agency for Medical Research and Development (AMED)(Grant numbers JP19cm0106206, JP19bm0304001), by the JSPS KAKENHI (Grant numbers JP17H06176). M.F. and K.T. were supported by the Japan Society for the Promotion of Science Research Fellowships for Young Scientists. Abbreviations: ANXA10, AnnexinA10; B/ER/G, BRAFV600E/EIF3E-RSPO2/GREM1 overexpression; B/G, BRAFV600E/ GREM1 overexpression; BMP, bone morphogenetic protein; B/T/ER, BRAFV600E/TP53 KO/ EIF3E-RSPO2; B/T/ER/G, BRAFV600E/TP53 KO/ EIF3E-RSPO2 /GREM1-overexpression; cDNA, complementary DNA; CRC, colorectal cancer; CRISPR, clustered regularly interspaced short palindromic repeat; DPBS, Dulbecco’s phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EGFR-i, EGFR-inhibitor; ER, EIF3E-RSPO2; FGF-2, fibroblast growth factor -2; H&E, hematoxylin and eosin; iCT, iCaspase9-tdTomato; IGF-1, insulin-like growth factors-1; ISH, in situ hybridization; KO, knock out; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; mRNA, messenger RNA; NOG, NOD/Shi-scid, IL-2Rγ null; OE, overexpression; PCA, principal component analysis; PCR, polymerase chain reaction; RIN, RNA integrity number; RT-qPCR, real-time quantitative PCR; sgRNA, single guide RNA; SEM, standard error of the mean; SSA/P, sessile serrated adenoma/polyp; SD, standard deviation; SuSA, superficially serrated adenoma; T/ER, TP53KO/EIF3E-RSPO2; TSA, 1

traditional serrated adenoma; UPL, universal probe library. Correspondence: Toshiro Sato, MD, PhD, Department of Gastroenterology, Department of Organoid Medicine, Keio University School of Medicine, Tokyo, Japan. e-mail address: [email protected] Conflicts of Interests: T.S. is named as an inventor on several patents related to organoids. Author Contributions: Conceptualization: K.K. and T.S., Methodology: K.K., M.F. and T.S., Investigation: K.K., M.F., S. Sugimoto, K.I., M.M., Y.O., K.T., S.Sekine, and S.T., Pathological assessment: S.Sekine, Resources: N.H., S.Sekine, T.K., Writing: K.K., M.F. and T.S., Funding Acquisition: T.S.

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Abstract: Background & Aims: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9– mediated chromosome and genetic modification of human colonic organoids. Methods: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells. Results: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had similar histologic features to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells. Conclusions: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression. KEY WORDS:

intestinal stem cells, ectopic crypt, BMP signaling, colorectal cancer

Introduction

3

Wnt

signaling

is

essential

for

the

self-renewal

of

Leucine-rich

+

repeat-containing G protein-coupled receptor 5 (LGR5) colonic stem cells residing at the crypt bottom, whereas its excessive activation leads to colon tumorigenesis1. Two independent intestinal stem cell niche factors, Wnt ligands and R-spondin, stringently control Wnt signal activation to maintain gut homeostasis2. Engagement of Wnt ligands to their receptors temporarily induces Wnt activation and upregulates their targets RNF43 and ZNRF3, which in turn restrict further signal persistence by ubiquitin-proteasome degradation of Wnt receptors2. Wnt activation also induces LGR5 expression, and binding of R-spondin to LGR5 leads to stabilization of Wnt receptors by counteracting the RNF43/ZNRF3-mediated negative feedback regulation2. Constitutive Wnt activation by APC or CTNNB1 mutations is regarded as a hallmark of colorectal cancer (CRC) 3. As another form of Wnt signal activation, recent deep sequencing efforts have demonstrated that 1–5% of sporadic CRCs acquire chromosomal rearrangements involving R-spondin genes in the absence of APC/CTNNB1 mutations 4. Interestingly, later studies found recurrent R-spondin gene fusions in more than 30% of colorectal traditional serrated adenoma (TSA), a rare subtype of colorectal serrated lesions, suggesting that TSA is a precursor of CRC with R-spondin gene fusions5, 6. TSAs differ markedly from conventional tubular adenoma and the major subtype of serrated lesions, sessile serrated adenoma/polyp (SSA/P) 7, 8. TSAs are characterized by unique structural aberrations, including slit-like serration, villiform configuration and ectopic crypt formation, which are rarely observed in tubular adenoma and SSA/P8. Despite these histological distinctions, TSAs often co-exist with other types of polyps including SSA/P, hyperplastic polyp and adenomatous polyp, suggesting that TSAs develop from these precursor lesions9,10. Genetic analysis identified concordant R-spondin gene fusions in TSAs and their adjacent precursors11. These findings suggest that R-spondin gene fusions are necessary for tumorigenesis but insufficient for histological progression into TSA. A recent report using inducible clustered regularly interspaced short palindromic repeat (CRISPR) transgenic mice introduced R-spondin fusions in the mouse intestine and intestinal organoids, but these models did not phenocopy human TSA12. Thus, faithful disease modeling systems for human TSA has been lacking, and it remains uncertain to what extent R-spondin gene fusion is implicated in TSA tumorigenesis and whether additional genetic aberrations are required for histological progression. In this study, we developed a chromosome-engineering system for human colon organoids to implement genetic reconstruction and functional modeling of human TSA. In combination with our 4

recently established orthotopic xenotransplantation method, chromosome-engineered human colon organoids illustrated essential steps for the initiation and progression of TSA at the level of histopathology. Materials & Methods Organoid Culture All organoids were established from patients with written informed consent under the approval of the ethical committee of Keio University School of Medicine. TP53 knock out (KO) human colonic organoids used for chromosome engineering were previously described13. The organoids were embedded in Matrigel and cultured with the previously described basal culture medium, specifically Advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with penicillin/streptomycin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific) and 2 mM GlutaMAX (Thermo Fisher Scientific). The basal medium was further supplemented with 1× B27 Supplement (Thermo Fisher Scientific), 10 nM gastrin I (Sigma-Aldrich), 1 mM N-acetylcysteine (Sigma-Aldrich), 50 ng/ml recombinant mouse epidermal growth factor (EGF) (Thermo Fisher Scientific), 100 ng/ml recombinant mouse Noggin (PeproTech), 1 µg/ml recombinant human R-spondin1 (R&D), 500 nM A83-01 (Tocris), 10 µM SB202190 (Sigma-Aldrich) and 20% Afamin-Wnt-3a serum-free conditioned medium14. For niche-based selection, R-spondin, EGF or Noggin was omitted from the culture medium to enrich organoids with R-spondin fusions, BRAFV600E knock in or GREM1 overexpression (OE), respectively. The EGF-removed medium was further supplemented with 1 µM EGFR/ErbB-2/ErbB-4 inhibitor (Merck Millipore) for complete epidermal growth factor receptor (EGFR) signal blockade. For specific experiments, 1 µg/ml recombinant human Gremlin1 (Peprotech), 100 ng/ml recombinant human insulin-like growth factor-1 (IGF-1) (BioLegend) or 50 ng/ml recombinant human fibroblast growth factor-2 (FGF-2) (PeproTech), 100 ng/ml human recombinant bone morphogenetic protein (BMP4) (PeproTech) was used. For dimerization of iCaspase9, organoids were treated with 0.5 µM dimerizer (AP20187, Clontech) in vitro. Construction of CRISPR-Cas9 and Plasmid Vectors To generate CRISPR-Cas9 plasmids, we cloned 20-bp single guide RNA (sgRNA) target sequences into the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene #42230) and obtained vectors bicistronically expressing an sgRNA and human codon-optimized Cas9 nuclease. At least two sgRNAs were designed for each 5

target breakpoints for efficient genome cleavage. For construction of a BRAFV600E donor vector, a custom double strand 1000-bp gene fragment harboring BRAFV600E mutation flanked by homology arms and non-synonymous mutations at CRISPR target sites was synthesized (GeneArt, Thermo Fisher Scientific). The gene fragment was cloned into the pCR2.1-TOPO vector (Thermo Fisher Scientific). A GREM1 OE vector was generated by cloning polymerase chain reaction (PCR) -amplified GREM1 complementary DNA (cDNA) into the multiple cloning site of the PB-CMV-MCS-EF1α-GFP-Puro vector (System Biosciences). The template cDNA was reverse transcribed using the Omniscript RT Kit (QIAGEN), according to the manufacturer's instructions. CRISPR-Cas9 plasmids targeting LGR5 and the knock-in vector were previously described15. Custom oligonucleotides and double strand DNA sequences are listed in Supplementary Table 1. Gene Engineering of Organoids For generation of organoids with R-spondin fusions and BRAFV600E mutation, human normal colon organoids were electroporated with CRISPR-Cas9 plasmids using a NEPA21 electroporator (Nepagene) using a previously reported method with slight modifications16. The PB-CMV-MCS-EF1α-GFP-Puro vector, Super PiggyBAC Transposase Expression Vector (System Biosciences) and CRISPR-Cas9 plasmids were co-delivered, and the organoids were cultured at 30 °C for two days after electroporation to facilitate genome editing17, 18. Eight days after the electroporation, the organoids were selected with 2 µg/ml puromycin for two days. The organoids were further subjected to niche-based selection as mentioned above. Single organoids were manually picked up and expanded to obtain organoid clones. Genomic DNA was isolated using the QIAamp DNA blood mini kit (QIAGEN). Fusion genes were confirmed using PCR primer sets listed in Supplementary Table 1. GREM1-overexpressing organoids were generated following the similar procedures using the GREM1 OE PiggyBAC vector mentioned previously. GREM1 OE organoids were functionally selected by Noggin withdrawal. Knock-in of the LGR5 reporter was performed by electroporation following the above procedures using LGR5 sgRNAs and the template vector. Five days after electroporation, organoids were treated with 2 µg/ml puromycin for two days for selection. Puromycin-resistant clones were manually picked up, expanded and subjected to PCR-based genotyping. The puromycin-RFP cassette flanked by loxP sequences was deleted by a transient infection of Cre-expressing adenovirus (Takara Bio) at a multiplicity of infection of 10. We again expanded each organoid clone and checked for proper excision of the selection cassette using 6

PCR-based diagnostics. Real-Time Quantitative PCR(RT-qPCR) and Microarray Analysis For RT-qPCR, total RNA was extracted with RNeasy Mini Kit (QIAGEN) and reverse-transcribed using the Omniscript RT Kit, according to the manufacturer's instructions. Real-time qPCR was performed using Universal Probe Library (UPL) probes and FastStart Essential DNA Probes Master (Roche) on a LightCycler 96 device (Roche). Relative gene expression levels were calculated using the delta–delta Ct method. Primer sets and probes used for real-time quantitative PCR are listed in Supplementary Table 1. For gene expression microarray analysis, total RNA was extracted with the RNeasy Plus Mini Kit (QIAGEN) and the RNA quality was determined based on the RNA Integrity Number (RIN) value using the RNA6000 assay (Agilent). Only specimens with RIN values > 7.0 were used in this study. Gene expression levels of organoids were determined by microarray (Prime View Human Gene Expression Array, Thermo Fisher Scientific) according to the manufacturer’s instructions. Signal intensities were normalized with the RMA function implemented in the R bioconductor package affy (version 1.60.0). For each gene, the probe with the highest standard deviation (SD) was selected. Variably expressed genes (SD > 2) were used for principal component analysis (PCA). Raw gene expression data of 4 normal colon, 2 SSA/P and 4 adenoma organoids were retrieved from our previous dataset (GSE74843) 19. The gene expression data of TSA organoids and engineered organoids in this paper is available on the accession number of GSE137336. Xenotransplantation of Organoids and Endoscopic Procedures All animal procedures were approved by the Animal Care Committee of Keio University School of Medicine. NOD/Shi-scid, IL-2Rγ null (NOG) mice (7– 12-week-old male) were obtained from the Central Institute for Experimental Animals (CIEA, Japan). Orthotopic xenografts of organoids were generated as previously described20. Briefly, mice were anesthetized with inhalation of 2–3% isoflurane, and their luminal contents were removed with manual abdominal compression. For removal of the mouse colonic epithelium, hot ethylenediaminetetraacetic acid (EDTA) (50 ℃, 250mM) was flushed into the mouse colon lumen. To prevent EDTA passage to the proximal colon and retain EDTA in the rectum, we used a homemade thin catheter (1 mm in diameter) with a small balloon. After a 2 min of EDTA exposure, the lumen was washed with Dulbecco’s phosphate-buffered saline (DPBS), and epithelial abrasion was 7

performed with electric toothbrush. The toothbrush head was inserted into the colon, and the half side of the luminal surface was scratched to scrape off the colonic epithelium. For organoid transplantation, the organoids were grown for 3–4 days and suspended in 10% Matrigel in DPBS. 70 µL of the cell suspension (equivalent to at least 1 × 106 cells) was instilled into the colonic lumen of each recipient mouse using a 200-µL pipette. After infusion, the anal verge was attached with an adhesive (AronAlpha, Toagosei) and left for 3–6 h to promote retention of transplanted cells at the rectum. The endoscopic procedure was carried out under general anesthesia with inhalation of 2–3% isoflurane. A high-definition 3-charge coupled device camera system (Image 1 Hub HD H3-Z; Karl Storz) and 300 Watt Xenon light source (D-Light C; Karl Storz) were utilized. Air infusion using an air pump was replaced with water infusion using a 5-ml syringe filled with DPBS. A rigid HOPKINS telescope covered with an endoscopic sheath (Karl Storz) was inserted into the rectum. Fluorescent endoscopic examination was performed as previously described20. For LGR5 ablation in vivo, xenografts of engineered organoids grown for 4-6 weeks after transplantation were used. After randomization based on endoscopically determined tumor size, tumor-bearing mice were treated with intraperitoneal injection of either Dimerizer (40 µg/body) or Vehicle for 7 consecutive days. Proliferating cells were labelled by intraperitoneal administration of EdU (10 mg/kg, Thermo Fisher Scientific) one hour before euthanasia. Immunohistochemistry and In Situ Hybridization (ISH) Archived human TSA samples obtained from patients with written informed consent were used for histological analysis of clinical tumors. For organoid xenografts, rectum tissues were immediately fixed with 4% paraformaldehyde and 5-µm paraffin embedded tissue sections were processed for immunohistochemistry, and 7-µm paraffin embedded tissue sections were processed for ISH. For LGR5 ablation, we prepared 8-µm frozen section. Hematoxylin and eosin (H&E) staining was performed using a standard histological protocol. For immunohistochemistry, rabbit anti-Ki67 (Clone SP6, Thermo Fisher Scientific, 1:300) and anti-c-Myc (ab32072, abcam, 1:1000) antibodies were used. For ISH, we used an RNAscope 2.5HD kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. Probes for RSPO2, RSPO3, AXIN2, LGR5 and GREM1 were designed by Advanced Cell Diagnostics. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). For EdU staining, the Click-IT Plus EdU Imaging kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Images were captured with a Leica SP8 confocal microscope or BZX-700 8

fluorescence microscope (Keyence). Statistics Gene expressions in quantitative PCR data were analyzed using unequal variances t-test. In all three cases, P values were less than 0.01 (*P < 0.01). Data were presented as mean ± standard error of the mean(SEM). Results Generation of fusion genes in human colonic organoids using CRISPR-Cas9-mediated chromosome engineering For accurate genetic modeling of TSA with R-spondin gene fusions, we adopted CRISPR-Cas9 and niche factor-based selection to generate fusion genes in human normal colonic organoids (Figure. 1A). EIF3E-RSPO2 (ER) gene fusions are produced by intra-chromosomal deletion of a relatively small genetic locus (113 kb), and we first set out to model this fusion using CRISPR-Cas9 (Figure. 1B). Electroporation of human colonic organoids with sgRNAs targeting the fusion breakpoints generated intra-chromosomal deletions corresponding to the ER fusion that existed for at least 48 hours after electroporation (Figure. S1A). Nonetheless, we failed to propagate organoids with detectable intra-chromosomal deletions, and organoids with insertions or deletions at both ends of the target grew out. This observation suggested that the organoids subjected to chromosomal deletion underwent rapid apoptosis or cell cycle arrest by chromosomal rearrangement-induced genomic stress, consistent with recent reports demonstrating p53-mediated growth arrest following sgRNA delivery in cell lines21, 22. To mitigate this potential unfavorable effect, we employed previously generated TP53 KO colonic organoids13. Interestingly, the TP53 KO background allowed efficient introduction of intra-chromosomal deletions, as judged by PCR-based diagnostics (Figure. S1B). A previous study reported that mouse intestinal organoids with the Eif3e-Rspo2 fusion gene did not survive in the absence of R-spondin, suggesting that the Eif3e-Rspo2 fusion was biologically inactive in mice12. To determine whether the human ER fusion can render exogenous R-spondin dispensable, we placed sgRNA-treated bulk organoids in R-spondin-removed culture condition. In contrast to Eif3e-Rspo2 fusion mouse organoids, numerous organoids recovered from sgRNA-treated organoids in the absence of R-spondin (Figure. 1C). Strikingly, PCR-diagnostics revealed legitimate ER genetic fusions in six out of the seven examined clones that were enriched by the R-spondin selection (Figure. S1B). Thus, the niche-based selection efficiently enriched ER fusion organoids and the ER fusion 9

product functionally activates Wnt signaling in human cells without exogenous R-spondin. Although specific mechanisms underlying the species difference in R-spondin sensitivity remains unknown, our data highlighted the advantage of the human organoid-based approach using CRISPR-mediated chromosomal engineering for disease modeling. PTPRK-RSPO3 fusions were more prevalent in TSAs than ER fusions5. Encouraged by the successful introduction of the ER fusion in human colonic organoids, we next sought to reconstruct the PTPRK-RSPO3 fusion in TP53 KO colonic organoids using the similar strategy. In contrast to ER fusions, one of the PTPRK-RSPO3 fusions involves an inversion between the first introns of PTPRK and RSPO3 genes spanning a 1 Mega base pair genomic locus4 (Figure. 1D). Despite this seemingly difficult chromosomal rearrangement pattern, we detected both intra-chromosomal deletions and PTPRK-RSPO3 fusion genes after sgRNA delivery in one organoid line (Figure. S1C). Interestingly, although the R-spondin-removed condition enriched PTPRK-RSPO3 fusion organoids, intra-chromosomal deletions were concurrently detected in some of the selected clones (Figure. 1E and Figure. S1C). These results indicated that CRISPR-targeting stochastically generates clones with intra-chromosomal deletions or inversions, and organoids with a competitive growth advantage selectively expand. To further extend the applicability of the CRISPR-based chromosomal engineering, we next aimed to create colonic organoids with chromosomal translocation (Figure. 1F). Recent deep sequence analysis revealed rare DLG1-BRAF inter-chromosomal fusions in sporadic CRCs23, but their functional significance remains unexplored. Notably, our strategy successfully established DLG1-BRAF fusion organoids which selectively propagated under the EGF-removed condition (Figure. 1G and Figure. S1D). This result not only corroborated efficient chromosome-engineering in human organoids but also provided biological evidence that the DLG1-BRAF fusion leads to EGF-independent growth. R-spondin gene fusion colonic organoids histologically resemble TSA precursors For further implementation of TSA genetic modeling, we knocked-in BRAFV600E mutation to normal or TP53 KO/ER (T/ER) colonic organoids based on the prevalence of BRAF mutation in TSA24 (Figure. S2A, B). The resulting organoids grew in the absence of EGF, but did not exhibit discernible morphological alterations in vitro (Figure. S2C). To investigate histological phenotypes of BRAFV600E and BRAFV600E/T/ER (B/T/ER) organoids, we turned to the orthotopic xenotransplantation system in which normal colonic organoids can repopulate the epithelium-depleted 10

mouse colon and form crypt structures20. Prior to transplantation, organoids were labelled with GFP for endoscopic detection of engraftment. For some unknown reason, BRAFV600E organoids were inefficiently engrafted, even to a lesser extent than normal colonic organoids, suggesting a detrimental effect of constitutive BRAF activation during colon epithelial regeneration. Nevertheless, we observed aberrant crypt formation with surface serration in BRAFV600E xenografts in support of the role of the BRAFV600E mutation in the serrated histopathology (Figure. S2D, E). In contrast to the poor engraftment capacity of BRAFV600E organoids, B/T/ER organoids were efficiently engrafted and formed flatly elevated lesions (Figure. S2D). Histology of B/T/ER organoid xenografts demonstrated hyperplastic crypt structures with V-shaped serration and basal dilation, common features of SSA/P rather than TSA (Figure. 2A, B). ISH confirmed uniform RSPO2 and AXIN2 messenger RNA (mRNA) expression in the B/T/ER organoid xenografts, indicating that EIF3E promoter activity persistently induced ectopic RSPO2 expression, thereby activating Wnt signal throughout the transplanted epithelium (Figure. 2B). These results suggested that ER-mediated Wnt signaling amplification contributed to the enhanced engraftment capacity and pathological progression into SSA/P-like lesions, but not TSA. To assess the clinical relevance of B/T/ER organoids, we analyzed five TSA specimens with RSPO gene fusions and their adjacent SSA/P-like lesions, referred to as superficially serrated adenoma (SuSA), which is observed in 12% of TSAs 11 (Figure. 2C-E). SuSAs exhibited V-shaped serration as well as broad RSPO2 and AXIN2 expression throughout the epithelium, which underscored the histological and molecular resemblance between B/T/ER organoids and SuSA (Figure. 2B, D). Nonetheless, this result also implied that fusion-driven R-spondin OE alone was insufficient for histological progression to TSA. To gain insights into molecular mechanisms mediating TSA progression, we analyzed the expression of LGR5, another Wnt target gene and an intestinal stem cell marker. In contrast to SuSAs and B/T/ER xenografts that expressed LGR5 predominantly at the crypt bottom, TSAs exhibited diffuse LGR5 expression (Figure. 2E). These results suggested that LGR5+ cells are ectopically positioned during transition from SuSA to TSA, despite the similar distribution of AXIN2+ cells in these two lesions. GREM1 is activated in TSAs and increases LGR5 expression in colonic organoids The discrepancy between LGR5 and AXIN2 expression patterns in SuSAs and B/T/ER xenografts and the emergence of ectopic LGR5+ cells in TSAs prompted us to investigate alternative molecular mechanisms regulating LGR5 expression in TSA. 11

AXIN2 expression is mainly regulated by canonical Wnt signaling, whereas LGR5 expression requires both Wnt activation and BMP inhibition25. Supporting this notion, Davis et al. have shown ectopic expression of GREM1 in TSAs26. To investigate its functional relevance in human TSA tissue, we established two organoid lines from independent TSA specimens using a previously defined niche factor condition for normal colonic organoids19. Sequencing analysis detected BRAFV600E and RNF43 truncating mutations in these organoids and confirmed their tumor origin (Figure. S3A). The TSA organoids lacked detectable RSPO fusions but were capable of growing without R-spondin (Figure. S3B), suggesting that these cases have acquired R-spondin independency through RNF43 mutations and not RSPO fusion genes. Interestingly, gene expression microarray revealed substantive GREM1 mRNA expression in one of the two TSA organoid lines (Figure. 3A). ISH confirmed epithelial GREM1 expression in its parental clinical tumor (Figure. 3B). The preserved expression of GREM1 in organoids indicated that GREM1 expression was regulated by an epithelial cell-autonomous mechanism. The absence of detectable genetic alterations and chromosomal rearrangements in the GREM1 gene and other BMP signaling molecules in exome and RNA sequencing data (data not shown) implied that ectopic GREM1 expression is mediated by mutation- or fusion-independent mechanisms. Importantly, GREM1+ TSA organoids grew independent of Noggin, demonstrating functional effect of ectopic GREM1 expression (Figure. 3C). These observations suggested the role of GREM1 expression during TSA tumorigenesis. All five TSA samples with SuSA precursors also exhibited ectopic GREM1 expression (Figure. 3D, S3C). These TSA specimens harbored R-spondin gene fusions in both TSA and SuSA compartments, whereas GREM1 was predominantly expressed in the TSA compartment with weak or no expression in counterpart SuSAs. Together, ectopic GREM1 expression occurs subsequent to R-spondin fusions and potentially contribute to the polypoid growth of the tumor. Xenografts of GREM1-overexpressing R-spondin gene fusion colonic organoids mimic TSA To evaluate the phenotypic impact of GREM1 upregulation during TSA progression, we overexpressed GREM1 in B/T/ER organoids, termed as B/T/ER/G organoids. B/T/ER/G organoids were readily selected and expanded in a Noggin-deficient condition, which phenocopied the Noggin independency of GREM1+ TSA organoids. (Figure. 3E, S3D). Consistent with the negative regulation of LGR5 expression by BMP signaling, B/T/ER/G organoids showed higher LGR5 expression as 12

compared to B/T/ER organoids either in the presence or absence of Noggin and BMP4 (Figure. 3F). These data collectively indicated that GREM1 activation foster TSA progression by maintaining LGR5 expression that are otherwise attenuated by BMP signaling. We next orthotopically engrafted B/T/ER/G organoids to study their histological phenotypes. To our surprise, one month post-transplantation, endoscopic surveillance revealed a pine-cone like polyp formation, reminiscent of the macroscopic appearance of clinical TSAs (Figure. 4A). Further histopathological assessment confirmed TSA-like morphological aberrations in B/T/ER/G xenografts, including villiform configuration, eosinophilic cytoplasm, slit-like serration and penicillate nuclei (Figure. 4A). ISH revealed expansion of LGR5+ cells at the crypt base as well as diffuse expression of RSPO2, AXIN2, GREM1 in the xenografts (Figure. 4B). Although ectopic crypt formation was not evident in B/T/ER/G xenografts, we could identify ectopic LGR5 expression at intermediate crypt positions (Figure. 4C). These results suggested that GREM1 OE dampened the BMP gradient and induced LGR5 expression at ectopic positions, underscoring the role of GREM1 upregulation during TSA development. IGF-1 and FGF-2 enable generation of R-spondin gene fusion in human normal colonic organoids The above experiments demonstrated genetic reconstitution of human TSA. However, this model was initiated from TP53 KO organoids for efficient introduction of chromosomal fusions, raising a possibility that the TP53 mutation was necessary for the phenotypes we observed. We recently found that a p38 inhibitor had detrimental effects on organoid colony formation, and its replacement with IGF-1 and FGF-2 improved gene editing efficiency of human intestinal organoids27. This refined culture protocol also mitigated cellular demise following the electroporation procedure27. Of note, this culture protocol enabled introduction of the ER fusion in another donor-derived normal colonic organoid line (Figure. 5A). Using these ER fusion organoids, we subsequently generated BRAF/GREM1 OE (B/G) and B/ER/G organoids without a TP53 mutation (Figure. S4A, B). B/G xenografts invariably formed flat lesions, while B/ER/G organoids formed polyps resembling TSA, demonstrating that the TP53 mutation was dispensable for the histopathological traits of TSA (Figure. 5B, S4C, D). We next analyzed the gene expression profile of normal colon, SSA/P, TSA, adenoma and engineered (B/G, B/ER, B/ER/G) organoids. TSA and SSA/P organoids showed a similar gene expression pattern presumably reflecting the CpG island methylator phenotype, whereas the engineered organoids did not, suggesting that 13

genetic engineering did not induce epigenetic alterations. Nevertheless, the global gene expression signature of engineered organoids showed a trend toward the SSA/P and TSA pattern in PCA. Hierarchical clustering using 40 genes with highest and lowest loadings in the primary principal component demonstrated upregulation of AnnexinA10 (ANXA10), a marker for SSA/P and TSA, in engineered organoids and highlighted the similarity of the gene expression patterns between TSA organoids and engineered organoids (Figure 5C). Ablation of LGR5+ cells in engineered organoid xenografts LGR5 is expressed in normal colonic stem cells and CRC stem cells1. LGR5+ cells are also frequently observed in the ectopic crypts of TSA, suggesting their tumorigenic capacity. However, the absence of LGR5+ cells in some ectopic crypts raised a possibility that LGR5- cells have tumorigenic potential (Figure S5). To address this possibility, we set out to perform LGR5+ cell-specific ablation in xenografts of engineered organoids. We previously constructed a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker15, and inserted this iCaspase9-tdTomato (iCT) cassette downstream of the LGR5 gene in normal colonic organoids (LGR5-iCT). B/ER/G genetic alterations were subsequently introduced into the LGR5-iCT knock-in organoids (Figure. 6A, S6). Dimerizer treatment effectively eradicated LGR5+ cells and diminished organoid growth in vitro (Figure. 6B). Xenografts of B/ER/G LGR5-iCT organoids formed polypoid tumors in the mouse colon, as visualized by endoscopy. Following stable engraftment, we randomized tumor-bearing mice based on endoscopically evaluated tumor size and treated them with either vehicle or dimerizer for 7 days (Figure. 6C). After the treatment, blinded researchers measured the tumor area and calculated the frequency of EdU-marked proliferating cells in the xenografts (Figure. 6D-G). Interestingly, LGR5 ablation did not affect the tumor architecture, tumor size or the proportion of proliferating cells (Figure. 6D-G), suggesting that LGR5+ cells are dispensable for the maintenance of B/ER/G tumors. Despite the loss of LGR5, an R-spondin receptor, the B/ER/G xenografts sustained AXIN2 expression (Figure. 6H) which suggested the activation of the Wnt pathway via other R-spondin receptors or R-spondin-independent mechanisms. Taken together, our results demonstrated that self-production of niche factors by B/ER/G organoids facilitated polypoid tumor formation and conferred tumorigenic potential on broad cellular populations, including LGR5- cells. Discussion 14

Human colorectal tumors are classified into a variety of histological subtypes, yet establishing genetic bases differentiating these subtypes has been challenging. Large efforts have been dedicated for the generation of genetically engineered mouse models to study genetic mutations identified in human colorectal tumors. However, most of these genetic models beget tumors with clinically-irrelevant histology. This drawback is partly attributable to the difficulty in introducing multiple genetic mutations to mouse colons, as well as the differing genetic makeups between humans and mice. For instance, Ptprk-Rspo3 fusion mice developed tumors histologically compatible with Apc-deficient tumors predominantly in the small intestine, and the Eif3e-Rspo2 fusion gene was functionally inert in mouse intestinal organoids12. To circumvent these caveats, we and others have previously developed a prospective genetic approach using human organoids and CRISPR-Cas9. This strategy allowed us to introduce multiple genetic mutations to human normal colon organoids in a sequential fashion13, 28. Nonetheless, chromosomal engineering in human organoids has been difficult largely owing to the low genome editing efficiency. In the current study, we harnessed a niche-based selection to selectively enrich organoids that underwent successful chromosomal engineering. The usage of the refined culture condition further enabled efficient chromosome engineering in normal colonic organoids. Encouraged by a recent report on serrated tumor formation by orthotopic transplantation of Braf engineered mouse organoids29, we successfully engrafted human BRAF engineered organoids in the mouse colon using EDTA-based epithelial depletion, although their engraftment efficiency was modest. Additional introduction of R-spondin rearrangement improved the engraftment efficiency without a dramatic change in histological appearance. Further induction of GREM1 promoted polypoid tumor formation and recapitulated histological traits of TSA, indicating the significance of ectopic GREM1 expression in pathologic transformation. GREM1 upregulation in clinical TSAs in comparison to their adjacent SuSAs further highlighted the pathological impact of GREM1 during SuSA-TSA transition. Despite the tumorigenic effect, constitutive GREM1 expression in our engineered organoid failed to show typical ectopic crypt formation, which is in contrast to the salient formation of ectopic crypts in a villin promoter-driven Gremlin1 transgenic mouse model26. This difference is reminiscent of the phenotypic discrepancy between a total epithelial Bmpr1a KO model and a villin promoter-driven Noggin transgenic model25, 30. These findings collectively suggested that ectopic crypt formation might require BMP inhibition in the differentiated compartment rather than the entire epithelium. Although the molecular mechanism mediating ectopic GREM1 activation in 15

TSA epithelium remains to be investigated, establishment of patient-derived TSA organoids which has not been reported previously may provide a unique opportunity for studying the origin and regulation of GREM1 expression in an epithelium-centered context. LGR5 expression is positively or negatively controlled by stem cell niche signaling including Wnt and BMP signals and reflects the abundance of niche factors in the surrounding tissue. In agreement with this, self-production of niche factors driven by those signal alterations induced ectopic LGR5 expression. Although the overrepresentation of LGR5+ cells suggested their potential in TSA tumorigenesis, LGR5-ablation experiments demonstrated that LGR5+ cells were dispensable at least temporarily in B/ER/G tumors. In addition, the remaining tumor cells had intact AXIN2 expression despite the absence of LGR5, suggesting that LGR5- cells were capable of activating Wnt signaling in an LGR5-independent manner. These results represented the advantage of prospective genetic analysis over marker-based inference in understanding the function of genetic alterations and the role of specific tumor cell populations in human diseased tissues. Collectively, our organoid engineering technology not only enabled precise disease modeling but also provided pathological insights into human colon tumorigenesis that can bridge the gap between tumor genotypes and histological phenotypes.

16

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of homology directed repair gene editing in induced pluripotent stem cells. Sci Rep 2018;8:2080. Seino T, Kawasaki S, Shimokawa M, et al. Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression. Cell Stem Cell 2018;22:454-467.e6.

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juvenile polyposis on BMP inhibition in mouse intestine. Science 2004; 303: 1684-1686. Author names in bold designate shared co-first authorship.

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Figure legends Figure. 1: CRISPR-Cas9-mediated chromosome engineering of human colonic organoids. (A) A strategy for implementing chromosome engineering in human colonic organoids. Human colonic organoids are electroporated with sgRNAs targeting fusion breakpoints, followed by enrichment with niche factor-based selection. (B) Generation of the ER fusion in TP53 KO colonic organoids via chromosomal deletion. Successful fusion was confirmed by Sanger sequencing (bottom). (C) Organoids with the ER fusion selectively grow in the absence of R-spondin. (D, E) Generation of PTPRK-RSPO3 fusion in TP53 KO colonic organoids via chromosomal inversion and its confirmation by sequencing (D). Organoids with the PTPRK-RSPO3 fusion selectively grow in the R-spondin-removed condition (E). (F, G) Generation of the DLG1-BRAF fusion in TP53 KO colonic organoids by chromosomal translocation between chromosomes 3 and 7 (F). Organoids with the DLG1-BRAF fusion selectively grow in the EGF-removed condition (G). Scale bar: 500 µm. Figure. 2: B/T/ER organoids phenocopy TSA precursors. (A, B) Representative histology, c-MYC immunohistochemistry, and RSPO2, LGR5, and AXIN2 ISH images of normal (A) and B/T/ER (B) organoid xenografts. (C-E) C-MYC immunohistochemistry, and RSPO2, LGR5, and AXIN2 ISH images of clinical normal colon (C), SuSA (D), and TSA (E) samples. Representative data of one of the five analyzed patient’s samples are shown. Scale bar: 100 µm. Figure. 3: GREM1 elevates LGR5 expression. (A) GREM1 expression in normal, SSA/P and TSA organoids analyzed by gene expression microarray. Expression values are normalized to the mean GREM1 expression in normal organoids. (B) GREM1 ISH of a clinical TSA sample from which the GREM1+ TSA organoid line was established. Scale bar: 100 µm. (C) GREM1+ TSA organoids survive in the absence of Noggin. Normal colonic organoids are shown for comparison. Scale bar: 500 µm. (D) GREM1 ISH images of clinical normal colon, SuSA, and TSA. The images are the serial sections from Figure 3C-E samples. Scale bar: 100 µm. (E) GREM1-overexpressing (OE) organoids grow in the absence of Noggin. Scale bar: 500 µm. (F) LGR5 expression in B/T/ER and B/T/ER/G organoids in the presence or absence of Noggin or BMP4. Expression values are shown as relative values to ACTB expression. Data are presented as mean ± SEM.

20

Figure. 4: Genetic reconstruction of human TSA. (A) Endoscopic images (left) and histology (right) of clinical adenoma (top), clinical TSA (middle), and B/T/ER/G organoid xenografts (bottom). Insets show high magnification images with slit-like serrations (red arrowheads) and penicillate nuclei. The area surrounded by blue dots indicates the recipient mouse epithelium. Scale bar: 100 µm. (B) Immunohistochemistry (c-MYC) and ISH (LGR5, GREM1, RSPO2 and AXIN2) images of a B/T/ER/G organoid xenograft shown in Figure 4A. The area framed by a red dotted line in Figure 4A is used. Scale bar indicates 100 µm. (C) Immunohistochemistry (Ki67, c-MYC) and ISH (LGR5, AXIN2 and GREM1) of a B/T/ER/G organoid xenograft with insets showing high magnification images of ectopic LGR5 expression. Scale bar indicates 100 µm. Figure. 5: Generation of R-spondin gene fusion in human normal colonic organoids using the IGF-1 and FGF-2 condition. (A) Introduction of the ER fusion in normal or TP53 KO colonic organoids. The refined condition using IGF-1 and FGF-2, but not the conventional method using a p38 inhibitor, enables successful fusion introduction in normal colonic organoids. Scale bar: 500 µm. (B) White light (top left) and fluorescent (top right) endoscopic images and histology (bottom) of a B/ER/G organoid xenograft with wild-type TP53. Red arrowheads in the inset indicate slit-like serrations. Scale bar: 100 µm. (C) Gene expression analysis of normal colon, SSA/P, TSA, and engineered organoids. PCA analysis using variably expressed genes. Hierarchical clustering using 40 genes with top and bottom loadings in the first principal component (PC1). Figure. 6: Ablation of LGR5+ cells in engineered organoid xenografts. (A) Strategy for the construction of B/ER/G LGR5-iCT organoids. LGR5-iCT organoids was constructed based on normal colon organoids by inducing iCT vector (triangle shows LoxP) targeting the exon 18 of LGR5.

(B) In vitro ablation of LGR5+ cells by

dimerizer. Scale bar: 500 µm. (C) Treatment schedule for LGR5 ablation experiment in vivo. (D) Endoscopic images of xenograft tumors pre, and post vehicle or dimerizer administration. (E) Tumor area (mm2) of vehicle- or dimerizer-treated B/ER/G LGR5-iCT organoid xenografts. (F) Histology of B/ER/G LGR5-iCT organoid xenografts after vehicle (top) or dimerizer (bottom) administration. The left image shows a GFP+ organoid xenograft (green) and the LGR5-TdTomato reporter (red). The right image shows EdU+ proliferating cells (green). Scale bar: 100 µm. (G) The proportion of EdU+ cells in B/ER/G LGR5-iCT xenografts as a relative value compared 21

to GFP+ cells post vehicle or dimerizer administration. (H) AXIN2 ISH of LGR5-iCT organoid xenografts post vehicle (top) or dimerizer (bottom) administration. Scale bar: 100 µm.

22

23

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Figure 1

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Figure 2

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CTTGCCCTGTCCGTGGTCCT CTTGCCCTGTCCGTGGTCCT

Figure S1

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C

D GREM1

GFP

B/G

B/G

Bright field

Figure S4

GREM1 ISH

LGR5 ISH

AXIN2 ISH

C-MYC

Patient sample 1

TSA

SuSA

Patient sample 2

TSA

SuSA

Figure S5

T/EIF3E-RSPO2 (T/ER)

Normal organoid #1

TP53 KO (T)

BRAFV600E/T/ER (B/T/ER)

B/T/ER/GREM1 OE (B/T/ER/G)

(N=3)

(N=6)

T/PTPRK-RSPO3

T/BRAF-DLG1

Normal organoid #2

EIF3E-RSPO2 (ER)

BRAFV600E/ER/GREM1 OE (B/ER/G) (N=1)

BRAFV600E (B) (N=3) BRAFV600E/GREM1 OE (B/G) (N=4) Normal LGR5-iCT

BRAFV600E/ER/GREM1 OE LGR5-iCT (B/ER/G LGR5-iCT) Dimerizer (N=6) Vehicle (N=4)

Figure S6

WHAT YOU NEED TO KNOW BACKGROUND AND CONTEXT: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. It is not clear how fusions in Rspondin genes and other genetic alterations found in TSAs affect their development. NEW FINDINGS: The authors developed a chromosome engineering system to introduce TSAassociated genetic changes into human colon organoids. Colon organoids with R-spondin gene fusions that express a mutant form of BRAF and overexpress GREM1 formed xenograft tumors in mice with histopathologic features of TSAs. LIMITATIONS: Some features of TSAs were not observed in the tumors grown from the engineered organoids, and the mechanisms that regulate epithelial expression of GREM1 are not known. IMPACT: Chromosomes in human colonic organoids can be engineered to introduce TSAassociated genetic changes. When these are transplanted into mice, they form tumors with histologic features of TSAs. Lay Summary: We introduced genetic changes into colon tissue organoids that cause them to grow into tumors that resemble TSAs in mice. These mice can be used to study TSA development.

Supplemental Information: Supplemental information includes six figures and one table. Supplementary Figure Legends Supplementary Figure 1. Sequencing analysis of fusion breakpoints in chromosome-engineered organoids. (A) CRISPR-Cas9-mediated ER fusion modeling in human normal colonic organoids. Three sgRNAs were designed for each breakpoint (top). Genotyping PCR primers are indicated in red and blue arrows. The existence of the ER fusion gene was detected at 48 hr post electroporation (bottom). (B) Introduction of the ER fusion in TP53 KO colonic organoids. PCR products corresponding to the expected chromosomal deletion were confirmed in six out of seven clones analyzed (top). Legitimate ER fusions were further confirmed by sequencing (bottom). Red and blue arrows indicate primer positions. (C) Generation of the PTPRK-RSPO3 fusion in TP53 KO organoids. Chromosomal inversion was detected in the bulk electroporated population and in all of the six clones analyzed (top). Chromosomal inversion was also observed in the bulk organoids as well as in two of the six clones (bottom). Red and blue arrows indicate primer positions. (D) Generation of the DLG1-BRAF fusion in TP53 KO organoids. Expected PCR products were confirmed in four out of five clones tested, which were further validated by sequencing. Red and blue arrows indicate primer positions. Supplementary Figure 2. Generation of B/T/ER organoids and their orthotopic xenotransplantation. (A) A strategy for introducing BRAFV600E mutation in T/ER (or normal) organoids. BRAFV600E mutation was knocked-in using CRISPR-Cas9 and a template vector, and the organoids were selected with an EGFR-inhibitor (EGFR-i). (B) Confirmation of BRAFV600E mutation knock-in by sequencing. The template vector for homologous recombination harbors BRAFV600E mutation and non-synonymous mutations at CRISPR targets highlighted in red. The genomic sequence of the organoids was analyzed by bacterial cloning (bottom). (C) BRAFV600E organoids grow in the absence of EGF and tolerate EGFR-i treatment. Scale bar: 500 µm. (D) Bright field (left) and fluorescent (right) endoscopic images of BRAFV600E (top) and B/T/ER (bottom) organoid xenografts. (E) Histology of orthotopically engrafted BRAFV600E organoids. Scale bar: 100 µm. Supplementary Figure 3. Characterization of TSA precursors and TSA by GREM1 expression. (A) Independency of patient-derived TSA organoids on R-spondin. Normal 1

colonic organoids are dependent on exogenous R-spondin (top right), whereas TSA organoids grow in the absence of R-spondin (bottom right). Scale bar: 500 µm. (B) Identification of BRAFV600E mutation and RNF43 truncating mutation in TSA organoid lines. (C) GREM1 ISH images of clinical normal colon, SuSA, and TSA in additional four samples. Scale bar: 100 µm. (D) Recombinant Gremlin1 substitutes Noggin. The growth of B/T/ER organoids in the presence of Noggin (left), after Noggin removal (middle) and in the presence of recombinant Gremlin1. Recombinant Gremlin1 rescues growth arrest by Noggin withdrawal. Scale bar: 500 µm. Supplementary Figure 4. Generation of B/G organoids and their orthotopic xenotransplantation. (A) A strategy for introducing BRAFV600E mutation and GREM1 overexpression in normal organoids. (B) The organoids were selected with a removal of EGF and Noggin from the media. (C) Bright field (left) and fluorescent (right) endoscopic images of B/G organoid xenografts. (D) Histology and GREM1 ISH of orthotopically engrafted B/G organoids. Scale bar: 100 µm. Supplementary Figure 5. Immunohistochemistry (c-MYC) and ISH (LGR5, GREM1 and AXIN2) of SuSA and TSA in two patient samples indicating LGR5+ and LGR5- ectopic crypts. Surrounded area with blue and black dots are shown in insets. LGR5+ cells and LGR5- cells are shown indicated in white arrowhead and red arrowhead. Scale bar: 1000 µm. Supplementary Figure 6. Genetically engineered human colon organoids generated in the study. The normal organoid line #1 was used for TP53-initiated genetic engineering. The normal organoid line #2 was used for genetic engineering on a TP53 mutation-null background. The LGR5-iCT organoid line was also derived from this normal line. N shows the number of xenografts made from each genetically engineered organoid line. All the engineered organoids except for B/T/ER/G organoids were cloned. OE stands for overexpression. Supplementary Table1. Sequences of oligonucleotides and double-stranded DNA used in the study.

2

Supplementary Table 1: CRISPR target sites, PCR primers and homology arms used in this study. CRISPR

sgRNA Location

Sequence (5' to 3') + PAM

EIF3E-1

chr8: 109,253,596-109,253,615

CCAGCATCATCAGTTCACCT

GGG

EIF3E-2

chr8: 109,253,542-109,253,561

GATTAAGTAGGTCTGGTGTG

TGG

EIF3E-3

chr8: 109,253,514-109,253,533

TAAGTGGTCCTCTTAAGTTT

GGG

RSPO2-1

chr8: 109,128,816-109,128,835

AGAATGTCTAACGGCAAATG

TGG

RSPO2-2

chr8: 109,128,648-109,128,667

AATGGAAGGGTTTTCGGATC

TGG

RSPO2-3

chr8: 109,128,545-109,128,564

CTAGTGTAAATAGTCTAGGG

AGG

PTPRK-1

chr6: 128,840,818-128,840,837

AAGCAGTGCCCGAGCGCACA

GGG

PTPRK-2

chr6: 128,840,952-128,840,971

TGACCTCGGCGCGCTTTGCC

AGG

RSPO3-1

chr6: 127,442,416-127,442,435

GGGGAAAGCTTCCTGCGCCG

TGG

RSPO3-2

chr6: 127,442,472-127,442,491

CTCCTCTCGGCCCAACCTAG

AGG

DLG1-1

chr3: 196,912,536-196,912,555

AAATGCTACGGTAATTGGTC

TGG

DLG1-2

chr3: 196,912,372-196,912,391

CGAATTAGATAGGATCACTC

TGG

BRAFInt8-1

chr7: 140,487,899-140,487,918

GATGTGCAGGGTTGTTACGT

AGG

BRAFInt8-2

chr7: 140,487,830-140,487,849

GGATGCTGTGCTTCATACCG

AGG

BRAFKnock-in CRISPR1

chr7: 140,753,326-140,753,345

TAGCTACAGAGAAATCTCGA

TGG

BRAFKnock-in CRISPR2

chr7: 140,753,321-140,753,340

ACAGAGAAATCTCGATGGAG

TGG

LGR5Knock-in CRISPR1

chr12: 71,978,584-71,978,603

GTAATTAATAAGAAGAGCTG

AGG

LGR5Knock-in CRISPR2

chr12: 71,978,506-71,978,525

TGTCTCTAATTAATATGTGA

AGG

Fusion gene

Genotyping primers

Sequence (5' to 3')

EIF3E F

CTATAGGTTGGTTGCTTGCCATATTGCT

RSPO2 R

GCCAATTATATGCCACTGTATTGGTCT

PTPRK F

CCAAACTCGGCATGGATACGACT

RSPO3 R

AGATTCTGTGGCTATCATTATGGCAA

PTPRK del F

GCTGTCCTCTCACCGTCCT

RSPO3 del R

GCACGCTCTCTCCTTTACCC

DLG1 F

ACACTAAGAGCCTACCATTGACC

BRAF R

CTACTTACCTCCATCACCACGAA

Sequencing primers

Sequence (5' to 3')

BRAF F

TCATAATGCTTGCTCTGATAGGA

BRAF R

GGCCAAAAATTTAATCAGTGGA

RNF43 F

TTAATGCAGTCCCACCCGCTGT CACCACCGAGTCCAAGGAACGA

EIF3E-RSPO2

PTPRK-RSPO3

DLG1-BRAF

Gene BRAF RNF43

RNF43 R

Probes

qPCR Primers LGR5 forward LGR5 reverse ACTB forward ACTB reverse

Sequence (5' to 3') GTGGACTGCTCCGACCTG GCTGACTGATGTTGTTCATACTGAG CCAACCGCGAGAAGATGA CCAGAGGCGTACAGGGATAG

Homology arms

Genomic sequence

BRAF

CAGTGTGCTGGAATTGTAGGAGTTTTTGATTGCTTGCTTACATTTTATCAGCACTATAAA ACTGATAGTTTTGTAGCTATCTATTAGTCCCTTTCAGACCTCTGACCTTGCTCAGTGGT AGTTGAGATATAACTGAAGACTCTAAATTATATAACAATGAGGTGAGAAAAACATAATATTT CTCTTCCCTAAGTGCAGACTAAGATACTATCTGCAGCATCTTCATTCCAATGAAGAGCCT TTACTGCTCGCCCAGGAGTGCCAAGAGAATATCTGGGCCTACATTGCTAAAATCTAATG GGAAAGTTTTAGGTTCTCCTATAAACTTAGGAAAGCATCTCACCTCATCCTAACACATTT CAAGCCCCAAAAATCTTAAAAGCAGGTTATATAGGCTAAATAGAACTAATCATTGTTTTAG ACATACTTATTGACTCTAAGAGGAAAGATGAAGTACTATGTTTTAAAGAATATTATATTACA GAATTATAGAAATTAGATCTCTTACCTAAACTCTTCATAATGCTTGCTCTGATAGGAAAAT GAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCA CAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCACGTTGGAGTGGGTCCCAT CAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCC ACTGATTAAATTTTTGGCCCTGAGATGCTGCTGAGTTACTAGAAAGTCATTGAAGGTCTC AACTATAGTATTTTCATAGTTCCCAGTATTCACAAAAATCAGTGTTCTTATTTTTTATGTA AATAGATTTTTTAACTTTTTTCTTTACCCTTAAAACGAATATTTTGAAACCAGTTTCAGTG TATTTCAAACAAAAATATATGTCTTATAAACAGTGTTTCATATTTTATTCTTAAATAAATAT GAACCCTTAAAACGAATATAATTCTGCAGATATC

Homology arms

Genomic Location

LGR5 5'

chr12: 71,583,717-71,584,731

LGR5 3'

chr12: 71,584,874-71,585,979

Universal Probe Library #8 Universal Probe Library #64