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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: Cancer Lett. 2018 Aug 23;436:109–118. doi: 10.1016/j.canlet.2018.08.017

Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids

Xi Liu a,b, Yulan Cheng b, John M Abraham b, Zhixiong Wang c, Zhe Wang b, Xiquan Ke d, Rong Yan e, Eun Ji Shin b, Saowanee Ngamruengphong b, Mouen Khashab b, Guanjun Zhang a, George McNamara f, Andrew J Ewald g, DeChen Lin h, Zhengwen Liu i, Stephen J Meltzer b
PMCID: PMC6152930  NIHMSID: NIHMS1505664  PMID: 30144514

Abstract

Primary organoid cultures generated from patient biopsies comprise a novel improved platform for disease modeling, being genetically stable and closely recapitulating in vivo scenarios. Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma. There has been a dearth of long-term in vitro expansion models of BE neoplastic transformation. We generated a long-term virus-free organoid expansion model of BE neoplasia from patient biopsies. Both wild-type and paired APC-knockout (APCKO) BE organoids genome-edited by CRISPR-Cas9 showed characteristic goblet cell differentiation. Autonomous Wnt activation was confirmed in APCKO organoids by overexpression of Wnt target genes and nuclear-translocated β-catenin expression after withdrawal of Wnt-3A and R-spondin-1. Wnt-activated organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, and prolonged culturability. Wnt-activated organoids also showed sustained protrusive migration ability accompanied by disrupted basement membrane reorganization and integrity. This CRISPR-Cas9 editing human-derived organoid model recapitulates the critical role of aberrant Wnt/β-catenin signaling activation in BE neoplastic transformation. This system can be used to study other ‘driver’ pathway alterations in BE-associated neoplasia, avoiding signaling noise presented in immortalized or cancer-derived cell lines.

Keywords: Organoids, Barrett esophagus, Wnt signaling, CRISPR/Cas9, neoplastic transformation

1. INTRODUCTION

Barrett esophagus (BE) is the major risk factor for esophageal adenocarcinoma (EAC), a deadly cancer that has increased more than 7-fold over the past four decades [1]. Mechanisms underlying progression of BE to EAC remain incompletely understood. Canonical Wnt/β-catenin (CTNNB1) signaling is critical to cell proliferation, survival, migration and stem cell polarity [2, 3]. Constitutive Wnt signaling activation has been definitively shown in BE neoplasia [4–8]. This is not surprising, since BE is a form of intestinal metaplasia and Wnt activation is an integral event in intestinal neoplasia [9–12]. Moreover, APC inactivation via promoter hypermethylation (occurring in 96% of EACs), and other epigenetic events within the Wnt pathway, activate Wnt signaling in EAC [5, 13]. Indeed, the role of Wnt signaling in this cancer has been so definitively established that it was proposed as a molecular treatment target in BE neoplasia [7]. Nevertheless, our ability to model the role of APC inactivation (a seminal event within the Wnt/β-catenin pathway) in BE-associated neoplasia remains limited. Organoids, a major breakthrough of the last decade, are self-organizing 3D structures grown from organ-specific stem cells which mimic in vivo architecture and biology [14, 15]. Because organoids resemble in vivo tissues, they comprise valuable tools for modeling human diseases, including cancers [16]. Long-term expansion of human BE organoids revealscharacteristic goblet cell differentiation [17, 18]. Furthermore, colon cancer models can be engineered in normal colonic organoids, by editing APC, TP53, KRAS, and SMAD4 and/or PIK3CA using CRISPR/Cas9 technology [12, 19].

We therefore generated constitutively Wnt-activated human BE organoid models using CRISPR-Cas9 genome editing torecapitulate and study the role of Wnt signaling in BE-associated neoplastic transformation. This strategy offers potential in other studies of ‘driver’ pathway alterations in all types of human tumorigenesis, avoiding pitfalls from signaling noise caused by preexisting passenger alterations in other models, such as immortalized or cancer-derived cell lines.

2. MATERIALS AND METHODS

2.1. Human samples for organoid cultures

Primary human endoscopic biopsy samples were acquired at the Johns Hopkins Hospital under written informed consent within protocols approved by the JHU Institutional Review Board. Tissue samples were pathologically confirmed as nondysplastic BE.

2.2. BE organoid cultures

Barrett epithelium was isolated and cultured as described, with key new modifications [17]. Growth medium for human wild-type (WT) BE organoids was as follows: Advanced DMEM/F12 (Gibco, Grand Island, NY) was supplemented with 50% Wnt-3A conditioned medium, 20% R-spondin-1 conditioned medium, 10 nM prostaglandin E2 (Cayman Chemical, Arbor, Michigan), 100 ng/ml human recombinant FGF-10 (Peprotech, Rocky Hill, NJ), 1× B27 (Gibco, Grand Island, NY), 1 mM N-acetylcysteine (Sigma-Aldrich, Louis, MO), 50 ng/ml mouse recombinant EGF (R&D Systems, Minneapolis, MN), 100 ng/ml mouse recombinant noggin (Peprotech), 10 mM Nicotinamide (Sigma-Aldrich), 10 nM Gastrin I (Sigma-Aldrich), 500 nM A83–01 (Tocris, Bristol, UK), 10 μM SB202190 (Sigma-Aldrich), 10 μM Y-27632 (Sigma-Aldrich), 1× Primocin (InvivoGen, San Diego, California) [12, 20]. This medium for WT BE organoids is referred to as WENR-supplemented medium (W, Wnt-3A; E, EGF; N, noggin; R, R-spondin-1). BE organoids were embedded in Matrigel (BD Biosciences, San Jose, CA); they were grown in rat-tail collagen I (BD Biosciences) only for epithelial migration assays.

2.3. CRISPR/Cas9-mediated organoid transfection

The strategy of using CRISPR/Cas9 to induce APC inactivating mutations was applied to BE organoids [12, 20]. The human codon-optimized Cas9 expression plasmid (Addgene 41815) and a guide RNA (sgRNA)-GFP plasmid targeting APC were obtained from Drs. Jarno Drost and Hans Clevers (Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, 3584CT Utrecht, Netherlands, and Cancer Genomics Netherlands, UMC Utrecht, 3584CX Utrecht, Netherlands). The APC sgRNA sequence was 5’-GTTTGAGCTGTTTGAGGAGGGTTTTA-3’. The sgRNA-GFP plasmid used as a sgRNA-transfected control was obtained from Addgene (41819). BE organoids were dissociated into single cells using TrypLE Express (Gibco) plus 10 μM Y-27632 incubated at 37°C for 8–10 min, and then re suspended in BE organoid growth medium and plated in 96-well plates. Cells were transfected with 10 μl DNA-lipid complex per well according to the manufacturer’s guidelines, containing 0.5 μl Lipofectamine® 2000 (Invitrogen, Carlsbad, CA) in 5 μl Opti-MEM medium (Gibco) and 100 ng of a plasmid expressing Cas9 and 80 ng of a sgRNA targeting APC or sgRNA control in 5 μl Opti-MEM medium. The plate was centrifuged at 3000rpm for 60 min at 32 °C, then incubated for 5 h at 37 °C. Single cells were re-plated in Matrigel and cultured with BE organoid growth medium in 24-well plates. Three days after transfection, selective medium made without Wnt-3A and R-spondin-1-conditioned media, referred to as EN-supplemented medium (E, EGF; N, noggin), was used to select for APC knockout (APCKO) BE organoids. Two to three weeks later, single surviving organoids were picked manually with a pipette for clonal expansion.

2.4. APCKO BE organoid genotyping

Genotyping was performed for validation of targeted APC mutation. Genomic DNA was isolated from clonal expanded organoids after transfection using DNeasy® Blood & Tissue Kit (Qiagen, Valencia, CA). The targeted APC locus was PCR-amplified using the primers 5’-TGTAATCAGACGACACAGGAAGCAGA-3’ (sense) and 5’-TGGACCCTCTGAACTGCAGCAT-3’ (antisense). PCR products were cloned into the pCR 4-TOPO TA vector (Life Technologies, Carlsbad, CA) and transformed into Escherichia coli (Thermo-Fisher). Plasmid DNA was extracted and sequenced using a T7 sequencing primer.

2.5. RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Organoids were harvested in Cell recovery solution (Corning Incorporated, Corning, NY). Total RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy Mini Kits (Qiagen). Reverse transcriptions were performed using High-Capacity cDNA RT Kits (Life Technologies). The resulting cDNA was then amplified by qRT–PCR using IQ™ SYBR green mix (Bio-Rad, Hercules, CA). Threshold cycle (Ct) was normalized to GAPDH expression. Fold change between APCKO and WT BE organoids were calculated using the 2-ΔΔCt method. Primer sequences were: AXIN2, 5’-AGCTTACATGAGTAATGGGG-3’ (sense) and 5’-AATTCCATCTACACTGCTGTC-3’ (antisense); C-MYC, 5’-AATGAAAAGGCCCCCAAGGTAGTTATCC-3’ (sense) and 5’-GTCGTTTCCGCAACAAGTCCTCTTC-3’ (antisense); CYCLIN D1, 5’-CCGTCCATGCGGAAGATC-3’ (sense) and 5’-GAAGACCTCCTCCTCGCACT-3’ (antisense); GAPDH, 5’-GGTATCGTGGAAGGACTCATGAC-3’ (sense) and 5’-ATGCCAGTGAGCTTCCCGTTCAG-3’ (antisense).

2.6. Alcian blue staining

Sections were stained in Alcian blue (TMS-010-C, MilliporeSigma, Darmstadt, Germany) for 30 min and counterstained with Nuclear Fast Red (N8002, Sigma).

2.7. Organoid formation and proliferation assays

Single cells were obtained from WT and APCKO human BE organoids by TrypLE Express plus 10 μM Y-27632 dissociation. 50 μl of cells and Matrigel mixture were seeded onto 24-well plates at 1.2×105 cells/ml at day 0. Organoid formation and proliferation were assessed at days 0, 2, 5 and 7. Organoid-forming efficiency equaled the number of formed organoids at day 2, 5 and 7 divided by the number of single cells at day 0 per 100X field. Organoid replicative capacity was assayed by passaging. Each experiment was performed in quintuplicate from 2 independently derived organoids per group.

2.8. Epithelial protrusive migration assay

2.8.1. Preparation of organoids in acid-solubilized collagen I

Organoid protrusive invasion was assayed in a collagen I-rich microenvironment [21, 22]. Rat-tail collagen I gels containing 3 mg collagen/mL at pH 7–7.5 were prepared by neutralizing collagen I with 1.0 N NaOH (Sigma) and 10× DMEM (Sigma) [21]. Neutralized collagen I solution was then kept on ice for 45–60 min until the gels turned slightly fibrous. Then, single cells from organoids were resuspended in collagen I solution and seeded onto 24-well plates. Plates were incubated at 37 °C for 45 min to allow collagen I polymerization, then organoid culture medium was added for time-lapse imaging.

2.8.2. Time-lapse live-cell imaging and quantification of protrusive migration

Live imaging of organoids embedded in collagen I was conducted using the Keyence BZX700 microscope with Tokai Hit Incubator at 37°C and CO 2 at 5%. Images were collected at 60-min intervals continuously for 6 days. Organoids showing protrusive migration were characterized by sharp-edged protrusive strands invading into collagen I, unlike the smooth edges usually observed during growth in Matrigel. From 50 randomly selected organoids per group, organoids exhibiting protrusive migrations were counted manually, frame-by-frame, over the entire course of each time-lapse movie. The percentage of organoids showing protrusive migration was calculated at days 1 and 6.

2.9. Western blotting

Cell lysates (80 μg) were electrophoresed on 4–15% polyacrylamide gels (Bio-Rad, Hercules, CA), then transferred to 0.45 μm supported nitrocellulose transfer membranes (Bio-Rad). Membranes were blocked with TBS containing 3% BSA and 0.1% Tween-20, then probed with rabbit anti-APC (2504; Cell Signaling Technology, Danvers, MA) and anti-β-actin-peroxidase (AC-15, Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL) and ECL Western blotting detection kits (Amersham-Pharmacia Biotech, Buckinghamshire, UK) were used.

2.10. Immunohistochemistry and immunofluorescence staining

Immunohistochemistry and immunofluorescence staining of organoids were performed as described previously [21]. The following primary antibodies were used: rabbit anti-MUC2 (1:200; sc15334, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-TFF3 (5 μg/ml; ab101099, Abcam, Cambridge, MA), rabbit anti-Ki67 (1:150; RM-9106, Thermo Fisher, Carlsbad, CA), rabbit anti-β-catenin (1:200; #9582, Cell Signaling Technology), mouse anti-C-myc (1:200; sc40, Santa Cruz Biotechnology), and rabbit anti-cyclin D1 (1:150; MA5–16356, Thermo Fisher Scientific).

For immunofluorescence staining, primary antibodies were rabbit anti-cleaved caspase-3 (1:400; #9661, Cell Signaling Technology), rabbit anti-laminin 332 (1:500; kind gift from Peter Marinkovich, Stanford University, Stanford, CA), and goat anti-collagen IV (1:100; AB769, Millipore). Secondary antibodies were AlexaFluor 568 goat anti-rabbit IgG (1:500; A-11036, Invitrogen), AlexaFluor 488 donkey anti-goat IgG (1:500; A-11055, Invitrogen), and AlexaFluor 488 goat anti-rabbit IgG (1:500; A-11034, Invitrogen). Nuclei were counterstained with Hoechst 33342 (Thermo-Fisher Scientific).

2.11. Statistical analysis

Mean and SD were displayed as representative values for data in figures. Student’s unpaired t-test was used to evaluate statistical significance, unless otherwise noted.

3. RESULTS

3.1. Establishment and characterization of BE organoids

BE is replacement of the normal esophageal squamous epithelium by metaplastic columnar epithelium, with histologic intestinal metaplasia, as shown by intestinal goblet cells in BE patient tissue samples in our study (Fig. 1A) [23]. Under WT organoid culture conditions, 7 organoids from 10 human BE samples were established (Fig. 1B and C), yielding an organoid establishment efficiency of 70%. These 7 organoids were continuously propagated for up to 4–6 months ex vivo, whereas normal esophageal squamous cells could not be passaged as organoids (data not shown). Organoids were confirmed as BE organoids by the presence of goblet cells positive for acidic mucin (Alcian blue +) (Fig. 1D) and expressing the goblet cell markers TFF3 (Fig. 1E) and MUC2 (Fig. 1F) [18], with cytoplasmic staining in both goblet cells and columnar epithelial cells.

Figure 1.

Figure 1.

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Characterization of Expanded BE Organoids. A, Haematoxylin and eosin (H&E) staining of paired one of BE patient biopsy tissue sample from which the organoids were derived. Intestinal metaplasia with goblet cells are presented. (Magnification, 200X). B, Visible light image of BE organoids. (Magnification, 400X). C, H&E staining of WT BE organoids shows single-layered columnar epithelium with uniform architecture and retained nuclear polarity (Magnification, 400X). D, In BE organoids, goblet cells contain large cytoplasmic vacuoles of mucin staining positively with Alcian blue (Magnification, 400X). E, TFF3 is expressed in columnar epithelium containing goblet cells in the BE organoids (Magnification, 200X). F, MUC2 is expressed in columnar epithelium containing goblet cells (Magnification, 200X).

3.2. Constitutive Wnt signaling activation in APCKO BE organoids induced by CRISPR-Cas9 genome editing

We applied CRISPR/Cas9 genome editing to introduce APC-inactivating mutations into BE organoids, based on known negative regulation by APC of the Wnt pathway (Fig. 2A) [2, 3]. Subsequently, under selection conditions created by withdrawing Wnt-3A and R-spondin-1, only 2 of 7 original BE organoids survived; these 2 organoids were clonally expanded for more than 6 months. These were co-transfected with a plasmid expressing Cas9 plus an sgRNA targeting APC. In contrast, control sgRNA-transfected WT BE organoids began dying at 7 days, and most WT BE organoids died by 14 days after transfection. Control sgRNA-transfected WT BE organoids survived for, at most, only 1 month in selective medium. (Fig. 2B). Notably, the efficiency of selecting surviving organoids post-CRISPR/Cas9 editing was less than 1 organoid/well.

Figure 2.

Figure 2.

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APC-knockout (APCKO) BE organoids created by CRISPR-Cas9 genome editing. A, Schematic of genome engineering using the CRISPR-Cas9 system: sgRNA is designed to target the human APC gene locus. B, Cas9 and the control sgRNA are successfully co-transfected into WT BE organoids expressing green fluorescence protein (GFP) (upper panel, left), but most WT BE organoids died within 14 days after transfection in selective medium lacking Wnt-3A and R-spondin-1 (upper panel, right). In contrast, WT BE organoids co-transfected Cas9 and an sgRNA targeting APC (lower panel, left) survive and sustain growth in medium that functionally selects for APC loss by removing Wnt and R-spondin (lower panel, right). Efficiency of transfection and survival of selected organoids was less than 1 organoid per well. C, Verification of APC mutation in BE organoids. Left panel, DNA sequencing of APCKO BE organoids showing a 2-bp deletion at nt 530 (between 2 T’s, located in center of sequence, upper panel) or 4-bp deletion at nt 524 (also between 2 T’s, lower panel). Right panel, corresponding frameshift deletions in APCKO BE organoids (top) vs. WT (bottom).

Sequencing of the edited targeted APC exon confirmed two types of frameshift mutation, either a 2-bp or a 4-bp deletion at the target site (Fig. 2C). Westerns demonstrated absence of full-length APC protein in both APCKO BE organoids (Fig. 3A). Messenger RNA expression levels of the known Wnt target genes AXIN2, C-MYC, CYCLIN D1 in the two independent APCKO BE organoids were significantly upregulated, even after withdrawing Wnt-3A- and R-spondin-1 (Fig. 3B-D). These qRT-PCR results confirmed constitutive activation of Wnt signaling in both APCKO BE organoids.

Figure 3.

Figure 3.

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Analysis of Wnt target genes in APCKO vs. WT BE organoids. A, Western blotting reveals absence of APC protein in APCKO BE organoids. B-D, Expression levels of AXIN2, C-MYC, and CYCLIN D1 messenger RNAs are significantly upregulated in two independently derived APCKO BE organoids grown in selective medium lacking Wnt-3A and R-spondin-1 (W, Wnt-3A; E, EGF; N, noggin; R, R-spondin-1). Each experiment was repeated independently 3 times.

3.3. Elevated proliferative and replicative activity in Wnt-activated organoids

To investigate proliferative and replicative potential of APCKO BE organoids upon WNT and R-spondin withdrawal, organoid formation and proliferation assays were performed. Significantly more APCKO BE organoids (grown without WNT/R-spondin) formed from single cells than did WT organoids (grown with WNT/R-spondin) at day 7; moreover, APCKO BE organoids exhibited larger, more complex multicellular structures (Fig. 4A and B). Average organoid-forming efficiency was 87% and 48% for APCKO vs. WT BE organoids, respectively (P < 1×10−4; Fig. 4C). In particular, APCKO BE organoids demonstrated significantly higher proliferation at days 5 and 7 vs. WT BE organoids (P = 1.5×10−4 and P = 2.9×10−6, respectively; Fig. 4D). Furthermore, replicative capacity was significantly greater in APCKO vs. WT BE organoids, with prolongation of total culture period from 5 months to 10 months, when cultured with a weekly 1:3 split (P < 1×10−4) (Fig. 4E).

Figure 4.

Figure 4.

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Proliferative and replicative potential of APCKO vs. WT BE organoids. A, WT BE organoids grown in WENR medium (left panel) and APCKO BE organoids in EN medium (right panel) at day 1 (Magnification, 100X). B, At day 7, APCKO BE organoids derived from single dissociated organoid cells (right panel) exhibit more complex structures than do WT organoids derived similarly (Magnification, 100X; Box inset magnification, 200X). C, At day 7, average organoid-forming efficiency was 87% for APCKO and 48% for WT BE organoids derived from single dissociated organoid cells (P < 1×10−4). D, APCKO BE organoids exhibit significantly higher proliferation than WT BE organoids at days 5 and 7 (P=1.5×10−4 and P = 2.9×10−6, respectively). E, The lifespan (total number of passages) of APCKO BE organoids is significantly higher than that of WT BE organoids (P < 1×10−4).

3.4. Association of constitutively activated Wnt Signaling with neoplastic features in APCKO BE organoids

Next, histological and immunohistochemical analysis was used to evaluate the phenotypic effect of constitutively activated canonical Wnt signaling on neoplasia-related characteristics in BE organoids. Histologically, WT BE organoids showed a single-layered epithelium with regular architecture and non/mild cytologic atypia with retained nuclear polarity (Fig. 1C). In contrast, APCKO BE organoids demonstrated a multilayered epithelium characterized by architectural irregularity and cytologic atypia, with loss of nuclear polarity (Fig. 5A). Glands in APCKO BE organoids were irregularly sized, shaped, and crowded, with back-to-back glands consistent with severely dysplastic or cancerous BE; moreover, nuclei were enlarged, pleomorphic, and hyperchromatic, with increased mitoses (Fig. 5A), additional features of dysplastic or cancerous BE [23]. Furthermore, these APCKO organoids retained BE differentiation characteristics, such as Alcian blue positivity and expression of the goblet cell markers TFF3 and MUC2 (Fig. 5A).

Figure 5.

Figure 5.

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Neoplastic and dysplastic features of APCKO BE organoids. A, In APCKO BE organoids, a multilayered epithelium is seen, showing architectural and cytologic atypia. Specifically, glands are crowded, located back-to-back, and of irregular sizes and shapes. Moreover, nuclei are enlarged, showing hyperchromasia and loss of nuclear polarity with increased mitosis (e.g., arrow) (H&E staining, 400X). Alcian blue (AB) staining remains positive in APCKO BE organoids (Magnification, 400X). TFF3 is expressed in APCKO BE organoids (Magnification, 200X). MUC2 is expressed in APCKO BE organoids (Magnification, 200X). B, Proliferation in APCKO vs. WT BE organoids. Nuclear expression of Ki67 is increased in APCKO BE organoids (middle panel) relative to WT BE organoids (left panel) (immunohistochemical staining, 400X). The percentage of Ki67-positive cells was 79.7% vs. 24.7%, respectively (P < 1×10−4; right panel). C, B-catenin expression in APCKO vs. WT BE organoids. Mostly membranous staining, but very little nuclear staining of β-catenin are seen in WT BE organoids (left panel), while much more abundant nuclear staining and much less membranous staining are seen in APCKO BE organoids (middle panel; immunohistochemical staining, 400X). The percentage of nuclear-positive cells increased from 1.3% to 22.9%, respectively (P < 1×10−4; right panel). D, C-myc expression in APCKO vs. WT BE organoids. Weak, uncommon expression of C-myc occurs in WT BE organoids (left panel), while stronger and more abundant higher expression occurs in APCKO BE organoids (middle panel; immunohistochemical staining, 400X). The percentage of nuclear-positive cells increased from 10.6% to 14.4%% (P =0.0124; right panel). E, Cyclin D1 expression in APCKO vs. WT BE organoids. Cyclin D1 expression was higher in APCKO BE organoids (middle panel) than in WT BE organoids (left panel; immunohistochemical staining, 400X). Nuclear expression of Cyclin D1 was 16.9% in WT BE organoids vs. 29.2% in APCKO BE organoids (P < 1×10−4; right panel). The number of positive cells and total cells were counted using Image J, and the percentages of positive cells were calculated. Data is presented as mean, and error bars indicate standard deviation (SD), for 6 slides made from 2 independently derived organoids for each group.

Immunohistochemically, expression of Ki67, a marker of cell proliferation, was significantly higher in APCKO vs. WT BE organoids to (i.e., with 79.9% vs. 24.7% positive cells; P < 1×10−4; Fig. 5B).

Because activation of Wnt signaling is associated with accumulated expression of nuclear β-catenin at the expense of membranous staining [24], we compared the percentage of nuclear β-catenin-positive cells in WT vs. APCKO organoids. Nuclear β-catenin expression was 1.3% in WT BE organoids vs. 22.9% in APCKO BE organoids (P < 1×10−4, Fig. 5C). We also observed decreased membranous expression of β-catenin in APCKO vs. WT BE organoids (Fig. 5C).

Subsequently, the expression of two Wnt target genes – c-myc and cyclin D1 - were analyzed to assess Wnt signaling in BE-associated neoplastic transformation. C-myc was weakly and infrequently expressed in WT BE organoids, while stronger and more frequent C-myc expression was seen in APCKO BE organoids (i.e., c-myc expression increased from 10.6% to 14.4%%, P =0.0124; Fig. 5D). The expression of cyclin D1 also showed an increased pattern of staining similar to nuclear Ki67 and β-catenin. Nuclear expression of cyclin D1 was 16.9% in WT vs. 29.2% in APCKO BE organoids (P < 1×10−4; Fig. 5E).

These results confirmed that constitutively activated Wnt signaling caused by CRISPR/Cas9 editing of APC is associated with dysregulated cell proliferation and neoplastic phenotypic changes in APCKO BE organoids.

3.5. Inhibition of apoptosis in Wnt-activated organoids

We next assessed effects of Wnt activation on apoptosis by analyzing the expression of cleaved caspase-3 in the two groups of organoids [25]. APCKO BE organoids exhibited significantly lower cleaved caspase-3 levels than did WT BE organoids (5.9% vs. 10%, P = 9.3×10−3; Fig. 6A and B).

Figure 6.

Figure 6.

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A, Suppression of apoptosis in Wnt-activated APCKO BE organoids (immunofluorescent staining, 400X). Apoptosis rates (red staining) were higher WT than in APCKO BE organoids. B, The number of caspase-positive cells and total cells were counted; error bars indicate SD for 6 slides from 2 different organoids of each group. Apoptotic cells comprised 10% of WT BE organoids vs. 5.9% of APCKO BE organoids (P = 9.3×10−3). C, Loss-of-function mutation in APC contributes to enhanced migration ability of BE organoids, visualized by time-lapse live-cell imaging. In WT organoids re-embedded in collagen I (upper panel), protrusive migration evidenced by sharp-edged cellular strands (red arrows) is observed at 24h and 54h but then retracts, evinced by smooth-edged strands (yellow arrows) at 114h and 145h. In contrast, in APCKO BE organoids (lower panel), sharp-edged cellular strands (red arrows) demonstrate sustained protrusive migration in collagen I, even at 114h and 145h (magnification, 200X). D, The percentage of APCKO BE organoids showing protrusive migration is significantly higher than that of WT BE organoids at collagen-re-embedded day 1 (64.6% vs. 39.3%, respectively, P = 4×10−3), and at re-embedded day 6 (22.7% vs. 0.67%, respectively, P = 3×10−4).

3.6. Sustained protrusive migration into collagen I in Wnt-activated organoids

Collective cell migration is a hallmark of normal epithelial development, tissue repair and cancer invasion [21, 26]. In collective migration, a cohesive cell group moves as a unit in sheets, strands or clusters, as opposed to invasion, which is considered a single-cell process [26]. Since collective cell migration is not only crucial for epithelial morphogenesis but also relevant to cancer biology [27], we hypothesized that loss-of-function mutations in APC might contribute to enhanced collective migration in BE organoids. WT and APCKO BE organoids were re-embedded in collagen I (instead of Matrigel) for 2 months. At re-embedded day 1, we observed protrusive migration, characterized by sharp-edged cell strands, into the collagen I-rich microenvironment in both WT and APCKO BE organoids. Protrusive migration was seen in 39.3% or 64.6% of the WT or APCKO organoids, respectively (P = 4×10−3) (Fig. 6C and D). However, at day 6, almost all sharp-edged cell strands were retracted, becoming smooth edges, in WT BE organoids (Fig. 6C). Only 0.67% of WT organoids, but 22.7% of APCKO organoids, still exhibited protrusive migration at day 6 (Fig. 6C and D). Thus, Wnt-activated organoids demonstrated enhanced collective migration.

To further characterize whether these histologic changes (i.e., from sharp protrusive to smooth edges) were related to basement membrane (BM) reorganization, we compared expression of the BM proteins, collagen IV and laminin 332, by immunofluorescence staining at both timepoints in the two groups of organoids. At day 1, expression of both collagen IV and laminin 332 were incomplete in WT and APCKO BE organoids (Fig. 7A and C), involving only small portions of each type of organoid. These findings agreed with the protrusive cell strands observed in both types of organoids. However, at day 6, expression of collagen IV and laminin 332 became complete, covering the entire extent of each WT organoid, but remaining incomplete in APCKO BE organoids (Fig. 7B and D). These results were consistent with retraction of almost all protrusive cell strands and their transition to smooth edges in WT BE organoids after 6 days of culture, suggesting BM reorganization. The sustained incomplete expression of collagen IV and laminin 332 in APCKO BE organoids (Fig. 7B and D) supports enhanced protrusive migration and disruption of BM reorganization and integrity.

Figure 7.

Figure 7.

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A, Completeness and extent of collagen IV expression in APCKO vs. WT BE organoids re-embedded in collagen I. At day 1 after re-embedding, collagen IV expression remains incomplete and limited in extent in both WT (upper panel) and APCKO (lower panel) BE organoids. B, However, by day 6, collagen IV expression is now complete and extends all the way around WT organoids (upper panel), while remaining incomplete and limited in extent in APCKO BE organoids (lower panel). (Immunofluorescence; magnification, 400X). C, Completeness and extent of laminin 332 expression in APCKO vs. WT BE organoids re-embedded in collagen I. At day 1, the expression of laminin 332 remains incomplete and limited in extent in both WT (upper panel) and APCKO (lower panel) BE organoids. D, However, by day 6, the expression of laminin 332 involves the entire extent of WT organoids (upper panel) while remaining incomplete and limited in extent in APCKO BE organoids (lower panel). (Immunofluorescence; magnification, 400X).

4. DISCUSSION

In this study, we used CRISPR-Cas9 genome editing combined with a niche-based selection system to introduce APC gene mutations into human BE organoids. We thereby generated a three-dimensional model recapitulating activated Wnt signaling seen during human in vivo BE neoplastic transformation. In addition, we showed that Wnt-activated BE organoids exhibit higher proliferative and replicative capacity and less apoptosis than WT BE organoids, features associated with neoplastic transformation [28–30]. Moreover, Wnt-activated BE organoids exhibited sustained and enhanced protrusive migration ability, associated with disruption of BM reorganization and integrity.

Organoids have greatly enhanced human disease modeling with improved precision [15]. Neoplastic transformation is a multifactorial process, involving complex genetic and environmental factors [28]. The genomic landscape of BE and EAC, now defined by next-generation genomic technologies, has implicated somatic alterations, including mutations in APC, CTNNB1, and other genes and pathways [31, 32]. Robust in vitro models generated from primary adult tissues with accurate representation of the human in vivo situation are now needed to functionally test these genomic alterations. Organoids generated from adult organ-specific stem cells are well-suited to such functional validations [15, 29], offering long-term growth, similarity to primary tissue architecture, and genetic stability, while avoiding signaling “noise” introduced by complex passenger alterations, unlike immortalized normal or cancerous cell lines [17, 33]. As with other organ systems [17, 18], our results reveal that human BE organoids expand long-term in niche factor–based culture conditions, with intestinal-type goblet cell differentiation (Alcian Blue positivity, MUC2 cytoplasmic staining) in vitro, with high efficiency (an establishment success rate of 70%).

The CRISPR/Cas9 system has developed rapidly as an efficient genome-editing method [34]. Among studies modeling oncogene or tumor suppressor gene function in neoplastic transformation of human organoids [12, 19, 35–38], however, few studies have engineered models directly using CRISPR-Cas9 genome modification [12, 19, 35]. Recently, Drost et al. [12] and Matano et al. [19] modeled multistep carcinogenesis in human intestinal organoids by mutating APC, KRAS, TP53, SMAD4, and PIK3CA using CRISPR-Cas9 editing.. Our results demonstrate that a human APCKO BE organoid model can be successfully generated by lipid-mediated Cas9-sgRNA complex delivery, confirmed by frameshift mutations (2- or 4-bp deletions) at the target APC locus.

Aberrant activation of Wnt signaling was confirmed in our APCKO human BE organoid model by finding significantly upregulated expression of AXIN2, C-MYC, CYCLIN D1 and nucleartranslocated β-catenin expression. These Wnt-activated organoids displayed higher proliferative activity, lower levels of apoptosis, and a prolonged culture period relative to WT organoids (10 vs. 5 months, respectively). These changes were associated with known features of BE neoplasia, including histologic and architectural abnormalities as well as overexpression of the markers Ki67, C-myc, and cyclin D1, which are associated with neoplastic progression of BE to EAC [24, 39, 40]. Notably, Wnt/β-catenin signaling restricts expression of two chemokine receptors, cxcr4b and cxcr7b to control collective cell migration, a hallmark of embryonic morphogenesis and cancer metastasis [27]. Collagen I, which is enriched in the tumor microenvironment, can be used to model the stromal extracellular matrix and induce collective migration in organoids [21, 22]. We used this model to measure collective migration of Wnt-activated vs. WT BE organoids. We found that Wnt-activated BE organoids exhibited greater and more sustained protrusive migration into collagen I, associated with disruption of basement membrane integrity. In addition, we observed that the number and length of protrusive cellular strands was lower than in previous studies [21, 22]; this discrepancy may have occurred because our organoids were passaged and embedded in Matrigel for 2 months before being cultured in collagen I. Indeed, other investigators reported that protrusive migration was reduced upon switching from Matrigel to collagen I and repeated passaging [21].

Canonical Wnt/β-catenin signaling is essential during early gut morphogenesis, with a lower level in the anterior (esophagus and stomach) than the posterior gut (small intestine and colon) [3, 41]. Canonical Wnt signaling activation leads to characteristic accumulated nuclear translocation of dephosphorylated β-catenin, which binds to TCF/LEF-1 transcription factors that initiate transcription of target oncogenes such as AXIN2, c-myc, and cyclin D1 [3, 42]. In tissues from patients with EAC and dysplasia developing from BE, Bian et al. [43] demonstrated that Wnt signaling was activated and nuclear accumulation of β-catenin occurred during neoplastic progression. Moyes et al. [24] revealed that abnormal Wnt signaling activation is critical in the development of BE dysplasia, based both on patient’s tissues and in a transgenic mouse model. However, these models may suffer from potential signaling noise arising within the context of multiple preexisting “passenger” alterations in EAC tissues, or from interspecies differences in transgenic mouse models. Furthermore, although the mechanism of Wnt pathway activation in BE-associated neoplastic transformation remains incompletely understood [5], inactivation of APC as a negative regulator of β-catenin stability occurs by LOH, aberrant methylation, or mutation in the majority of EACs [31, 32, 44–48]. In the current study, constitutively activated Wnt signaling models were directly generated from human WT BE organoids by CRISPR/Cas9 genome-editing of APC. Thus, we combined organoid and CRISPR/Cas9 genome-editing system technologies to model activated Wnt signaling in BE-associated neoplasia. Our model enables precise study of individual pathway alterations in driving BE neoplastic transformation, while avoiding potential signaling noise inherent in other model systems.

In conclusion, we have generated a novel human-derived organoid model by using CRISPR-Cas9 genome editing to recapitulate aberrantly activated Wnt signaling in BE neoplastic transformation in both Matrigel and collagen I. This novel virus-free tool may promote future studies of ‘driver’ pathway alterations in human tumorigenesis and progression ex vivo.

Highlights:

  • A long-term expansion Wnt-activated human BE organoid model has been established using CRISPR-Cas9 combined with a niche-based selection system.

  • Compared to wild-type BE organoids, Wnt-activated BE organoids demonstrated histologic atypia, higher proliferative and replicative activity, reduced apoptosis, overexpression of Ki67, c-myc and cyclin D1, and enhanced protrusive migration ability, which disrupted reorganization and integrity of the basement membrane.

  • This CRISPR-Cas9 editing human-derived organoid model recapitulates the role of activated Wnt signaling during BE neoplastic transformation.

Acknowledgements:

The authors thank Drs. Jarno Drost and Hans Clevers (Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, 3584CT Utrecht, Netherlands, and Cancer Genomics Netherlands, UMC Utrecht, 3584CX Utrecht, Netherlands) for plasmids, guide RNAs, and technical support. The authors also thank Dr. Peter Marinkovich (Stanford University, Stanford, CA) for primary antibody to rabbit anti-laminin 332.

Financial support: this work was supported by the National Institutes of Health (CA190040 and CA211457 to Dr. Meltzer); the Johns Hopkins University Discovery Fund (Dr. Meltzer); the Key Science and Technology Program of Shaanxi Province, China (2015SF128 to Xi Liu); and the International Scientific and Technological Cooperation and Exchange Program of Shaanxi Province, China (2015KW-030 to Guanjun Zhang). Dr. Meltzer is the Harry and Betty Myerberg Professor of Gastroenterology and an American Cancer Society Clinical Research Professor.

Footnotes

CONFLICT OF INTEREST: The authors declare no conflict of interest.

Patient consent Obtained.

Ethics approval Specimens for organoid culture were obtained under approved Johns Hopkins University human subjects protocol NA_00004336.

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