Human Pancreatic Tumor Organoids

Resource
Human Pancreatic Tumor Organoids Reveal Loss of

Stem Cell Niche Factor Dependence during Disease
Progression
Graphical Abstract Authors
Takashi Seino, Shintaro Kawasaki,
39 Patient-derived Pancreas Tumors Mariko Shimokawa, ..., Yuko Kitagawa,
Takanori Kanai, Toshiro Sato
Metastasis
Correspondence
t.sato@keio.jp
In Brief
CAF
Sato and colleagues established a library
of patient-derived pancreas cancer
Malignant Transformation
organoids and identified heterogeneous
Mesenchymal Wnt Epithelial Wnt patterns of dependency on Wnt ligands
among pancreas cancers. Biological and
genetic analyses highlighted GATA6 as a
mediator of the Wnt niche requirement,
which links pancreatic tumor progression
Normal Wnt non-secreting Wnt secreting Wnt/Rspo
independent
to independence from the stem cell niche.
GATA6 Expression
Wnt Niche Independency

Pancreas Tumor Organoid Library
Highlights
d 39 patient-derived pancreas adenocarcinomas (PDACs) form
a tumor organoid library
d PDAC segregates into 3 subtypes with distinct dependency

on Wnt niche signals
d Cancer-associated fibroblasts provide a Wnt niche for PDAC
d CRISPR-Cas9 gene engineering recapitulates multistep

carcinogenesis of human pancreas
Seino et al., 2018, Cell Stem Cell 22, 1–14

March 1, 2018 ª 2017 Elsevier Inc.
https://doi.org/10.1016/j.stem.2017.12.009
Please cite this article in press as: Seino et al., Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Dis-
ease Progression, Cell Stem Cell (2017), https://doi.org/10.1016/j.stem.2017.12.009
Cell Stem Cell
Resource
Human Pancreatic Tumor Organoids Reveal Loss

of Stem Cell Niche Factor Dependence
during Disease Progression
Takashi Seino,1 Shintaro Kawasaki,1 Mariko Shimokawa,1 Hiroki Tamagawa,1 Kohta Toshimitsu,1 Masayuki Fujii,1
Yuki Ohta,1 Mami Matano,1 Kosaku Nanki,1 Kenta Kawasaki,1 Sirirat Takahashi,1 Shinya Sugimoto,1 Eisuke Iwasaki,1
Junichi Takagi,3 Takao Itoi,4 Minoru Kitago,2 Yuko Kitagawa,2 Takanori Kanai,1 and Toshiro Sato1,5,*
1Department of Gastroenterology, Keio University School of Medicine, Tokyo 160-8582, Japan
2Department of Surgery, Keio University School of Medicine, Tokyo 160-8582, Japan
3Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita 565-0871, Japan
4Department of Gastroenterology, Tokyo Medical University, Tokyo 160-0023, Japan
5Lead Contact
*Correspondence: t.sato@keio.jp
https://doi.org/10.1016/j.stem.2017.12.009
SUMMARY apeutics, underscoring the importance of understanding inter-

patient tumor heterogeneity in depth and stratifying PDACs to
Despite recent efforts to dissect the inter-tumor predict clinical behaviors. Recent gene-expression-based clas-
heterogeneity of pancreatic ductal adenocarcinoma sification identified 2 major prognosis-predicting molecular
(PDAC) by determining prognosis-predictive gene subtypes in PDAC, namely the classical and quasi-mesen-
expression signatures for specific subtypes, their chymal (QM) subtypes (Bailey et al., 2016; Collisson et al.,
functional differences remain elusive. Here, we 2011; Moffitt et al., 2015; Noll et al., 2016). The classical sub-
type was characterized by differentiated duct cell marker
established a pancreatic tumor organoid library
expression and favorable prognosis, whereas the QM subtype
encompassing 39 patient-derived PDACs and iden-
was characterized by aggressive clinical behavior and by
tified 3 functional subtypes based on their stem cell gene silencing of definitive endoderm specification genes,
niche factor dependencies on Wnt and R-spondin. including GATA6, FOXA2, and HNF4A. Similar QM-like sub-
A Wnt-non-producing subtype required Wnt from types regulated by GATA6 expression were also reported as
cancer-associated fibroblasts, whereas a Wnt- squamous (Bailey et al., 2016) and basal-like (Moffitt et al.,
producing subtype autonomously secreted Wnt 2015) subtypes. Despite the robust identification of gene
ligands and an R-spondin-independent subtype expression subtypes, whether these subtypes reflect geneti-
grew in the absence of Wnt and R-spondin. Tran- cally distinct cell-of-origin or tumor-progression statuses has
scriptome analysis of PDAC organoids revealed remained elusive, owing to a paucity of functional assay sys-
gene-expression signatures that associated Wnt tems for human PDAC.
Functional analyses of PDAC have mainly relied on geneti-
niche subtypes with GATA6-dependent gene
cally engineered mice, cell lines, and patient-derived tumor
expression subtypes, which were functionally
xenograft models. The genetic tractability of mouse models
supported by genetic perturbation of GATA6. and cell lines has contributed to the understanding of
Furthermore, CRISPR-Cas9-based genome editing pancreas tumorigenesis, yet its relevance to the clinical traits
of PDAC driver genes (KRAS, CDKN2A, SMAD4, of human PDAC, including histological and gene expression
and TP53) demonstrated non-genetic acquisition subtypes, remains unknown (Hwang et al., 2016; Hruban
of Wnt niche independence during pancreas tumor- et al., 2006). Xenograft models can be efficiently generated
igenesis. Collectively, our results reveal functional from clinical samples with preserved histological and molecu-
heterogeneity of Wnt niche independency in PDAC lar subtypes, whereas the labor-intensive and genetically
that is non-genetically formed through tumor pro- intractable natures of such models have limited large-scale
gression. analyses and prospective genetic approaches for PDAC. The
organoid culture system has recently emerged as a technol-
ogy that can propagate epithelial tissues as 3D structures
using artificial stem cell niche environments (Sato et al.,
INTRODUCTION 2009). A defined niche factor combination of R-spondin,
epidermal growth factor (EGF), fibroblast growth factor 10
Pancreatic ductal adenocarcinoma (PDAC) is a devastating (FGF10), and Noggin (a BMP4 inhibitor) promoted the expan-
disease that has an extremely poor prognosis, with a median sion of normal mouse pancreatic duct cells (Huch et al.,
survival of <1 year and a 5-year overall survival rate of <9% 2013) and was later applied to patient-derived PDAC organo-
(National Cancer Institute). Whereas PDACs are generally ids (Boj et al., 2015). To date, a dozen lines of PDAC organoids
chemo-resistant, a fraction of patients benefit from current ther- have been generated in which the preservation of histological
Cell Stem Cell 22, 1–14, March 1, 2018 ª 2017 Elsevier Inc. 1
Figure 1. Establishment of the PTOL Using Niche-Based Selection

(A) Overview of the procedure used to develop the PTOL. Pancreatic tumor organoids and CAFs were established from surgically resected-, FNA-, or punctured
ascites specimens.
(B) Strategy to enrich PDAC organoids. Tumor organoids were selected using the indicated culture conditions. Tumor organoids were judged as originating from
PDAC based on the retention of growth capacity under at least one of the indicated selection conditions.
(C) Representative images before and after eliminating contaminating normal organoids by EGF removal. Pancreatic tumors (top) occasionally include both
normal (black arrowheads) and PDAC organoids (white arrowheads), and PDAC organoids were selectively expanded in culture in the absence of EGF (bottom).
Of note, this selection medium did not include Nutlin3 or BMP4.
(D) Niche factor requirement (top), genetic mutations (middle), and chromosomal alterations (bottom) of PDAC and normal-like organoids. The mutations are
indicated as follows: protein-damaging mutations or oncogenic mutations (black); deletions (light blue); and wild-type (light gray). Squares and triangles indicate
homozygosity and heterozygosity, respectively. Gene alterations with heterozygous deletion-only patterns are omitted (specified later in Figure S2). Hotspot
missense mutations in KRAS (codons 12, 13, and 61) and GNAS (codon 201) are demonstrated. *KRAS mutation was not detected in the whole-exome
sequencing due to a low coverage but confirmed by Sanger sequencing. The bar plots on the right depict the alteration frequency of each gene in the PTOL (black)
and the TCGA database (white; data derived from cBioPortal). Copy number alteration status is shown: gain (red) and loss (blue). The overall chromosomal status
of each sample is shown in the right panel.
See also Figure S1 and Tables S1, S2, and S5.
traits and genetic-mutation profiles of the parental tumors RESULTS

were confirmed. Despite these advances, large-scale tran-
scriptome analysis of human PDAC organoids has not been Establishment of a Human Pancreatic Tumor Organoid
conducted, posing a bottleneck in connecting biological Library
behavior with gene expression subtypes (Boj et al., 2015; Using surgical, fine-needle aspiration (FNA) and ascites speci-
Huang et al., 2015). In this study, by refining organoid culture mens, we established organoids from pancreatic tumors as
conditions, we established 39 lines of PDAC organoids and well as normal organoids when adjacent normal pancreatic
performed comprehensive molecular characterization, which tissues were available (Figure 1A). Using previously published
illuminated different modes of Wnt/R-spondin niche depen- culture conditions (Huch et al., 2013), normal pancreas duct
dency in association with gene expression subtypes. Further- organoids ceased proliferation within 2 or 3 months (Boj et al.,
more, CRISPR-Cas9-mediated engineering of pancreas orga- 2015). We found that the replacement of serum-stabilized
noids demonstrated the stepwise tumorigenesis of PDAC with Wnt3A-conditioned medium with serum-free Afamin-stabilized
progressive acquisition of niche independency. Wnt3A (Mihara et al., 2016) enabled stable culture for over
2 Cell Stem Cell 22, 1–14, March 1, 2018

8 months. Notably, the addition of serum triggered senescence, of copy number alterations. NL organoids invariably showed
suggesting that serum in standard Wnt3A-conditioned medium euploidy, reinforcing their non-cancer origins, whereas most
is detrimental for the long-term maintenance of human pancreas PDAC organoids acquired well-defined chromosomal alter-
organoids (Figure S1A). Using this culture protocol, we estab- ations, namely losses of 6p, 9p, 17p, and 18q (Su et al., 1998).
lished a pancreatic tumor organoid library (PTOL) consisting of Of note, four PDAC lines exhibited euploidy or near euploidy,
49 organoid lines generated from patient-derived pancreatic of which three harbored KRAS mutations, confirming their can-
tumors (Table S1). cer origin (Figure 1D). The remaining near-euploidy line (PC8)
In our initial experiments, we often observed the outgrowth of was devoid of driver-gene mutations, including those in KRAS,
normal pancreas organoids from PDAC specimens. The selec- and its tumorigenic mechanism was unclear.
tive growth of ‘‘contaminating’’ normal organoids has been pre- Upon xenografting into orthotopic pancreata of NOD.Cg-
viously reported during the establishment of PDAC and prostate PrkdcscidIl2rgtm1Sug/Jic (NOG) mice, PDAC organoids (including
cancer organoids (Boj et al., 2015; Gao et al., 2014). Considering the wild-type KRAS organoid) formed tumors resembling their
the high prevalence of KRAS mutations that occur among parental PDACs, whereas NL organoids did not successfully
PDACs, the organoids were first placed in an EGF-depleted con- engraft (Figure S1F). Taken together, these data indicate that
dition to enrich the KRAS mutant organoids (Figure 1B). In niche-based selection efficiently propagated PDAC organoids
contrast to the standard Wnt-conditioned medium that could with the accurate exclusion of potentially contaminating normal
activate EGFR and other cognate receptors by serum-derived organoids.
growth factors (Wang et al., 2013), EGF removal from the
serum-free medium efficiently selected KRAS mutant organoids. Identification of Three Functional PDAC Organoid
The efficient enrichment of PDAC organoids can be readily visu- Subtypes with Distinct Wnt Niche Requirements
alized when spherical PDAC organoids grew along with normal Once PDAC organoids were established, each organoid was
cystic organoids; after EGF-based selection, KRAS mutant subjected to successive niche-based treatments to determine
spherical PDAC organoids dominated whereas cystic organoids minimally essential niche factors, which demonstrated that
with wild-type KRAS disappeared (Figures 1C, S1B, and S1C). driver-gene alterations largely dictated the requirements for the
KRAS mutant spherical organoids, but not normal cystic organo- corresponding niche factors. Specifically, sensitivity to EGF
ids, displayed phospho-ERK expression in the absence of EGF, removal, Noggin removal/BMP4 treatment, A83-01 removal/
confirming the ligand-independent activation of Ras signaling in transforming growth factor b1 (TGF-b1) treatment, and Nutlin3
PDAC organoids (Figure S1D). treatment were associated with KRAS, SMAD4, TGFBR2, and
TP53 mutations/in-del alterations, respectively (Figures S2A–
Determination of the Cancer Origin in Established PDAC S2H). In contrast to these mutation-driven adaptations, we noted
Organoids that Wnt/R-spondin dependency was mostly unrelated to Wnt-
Organoids that were susceptible to EGF removal were alterna- signaling mutations in PDAC organoids (Figures 2A and 2B).
tively treated with Nutlin3 (an MDM2 inhibitor) or with Noggin Akin to the niche dependency of normal pancreas organoids,
removal/BMP4 to select potentially existing TP53 or SMAD4 14 PDAC organoids required both Wnt3A and R-spondin for their
mutant organoids, respectively (Figure 1B). Together with the growth. Interestingly, the remaining R-spondin-dependent
aforementioned EGF-based selection, the niche-based selec- PDAC organoids grew in the absence of Wnt3A. Because
tion was used to diagnose 39 out of 49 organoids in the PTOL R-spondin is known to potentiate Wnt signaling through stabili-
as PDAC organoids. The remaining 10 organoids exhibited strict zation of Wnt receptors (Koo et al., 2012), this phenotype sug-
niche dependencies indistinguishable from those of normal gested that R-spondin-dependent PDAC organoids harness
pancreas organoids and were referred to as normal-like (NL) or- either exogenously supplied or endogenously produced Wnt
ganoids. To determine the accuracy of niche-factor-based ligands. Therefore, we divided R-spondin-dependent PDAC
diagnosis, we analyzed the genetic status of the established organoids into two subtypes, namely Wnt-non-secreting (W)
organoids using whole-exome sequencing and comparative and Wnt-secreting (W+) PDAC organoids, based on their require-
genomic-hybridization microarray analyses. Consistent with ments for exogenous Wnt3A.
previous large-scale deep-sequencing analyses (Waddell et al., To validate the Wnt-producing capacity of W+ PDAC organo-
2015), PDAC organoids harbored common driver-gene alter- ids, we tested the effect of a porcupine inhibitor (Porcn-i; C59)
ations at the expected frequencies: KRAS (36/38); CDKN2A (Proffitt et al., 2013), which abrogates the production of biologi-
(31/38); TP53 (29/38); and SMAD4 (14/38; Table S2). GNAS cally active Wnt ligands. Notably, Porcn-i treatment suppressed
hotspot mutations (GNASR201H) were detected in two organoid the growth of W+ PDAC organoids in parallel with the reduction of
lines (2/49), one of which was derived from intraductal papillary their Wnt target gene-expression levels, and these effects were
mucinous neoplasm (IPMN). Of note, no authenticated IPMN reversed by the supplementation with exogenous Wnt3A (Fig-
cell line with a GNAS mutation has been derived to date (Furu- ures 2C and 2D). Furthermore, W+ PDAC organoids exhibited
kawa et al., 2011). We did not detect recurrent driver-gene higher expression of Wnt target genes than W PDAC organoids
mutations in normal-like organoids, corroborating the accurate in the absence of Wnt3A (Figure S3A). The potent niche function
selection of PDAC organoids (Figure 1D). of Wnt ligands secreted from W+ PDAC organoids was further
Whereas detailed copy number analyses of PDACs have been validated by their growth-promoting effects on co-cultured
hampered by the low tumor content of clinical PDAC samples W PDAC organoids (Figure S3B). These results collectively indi-
(Shain et al., 2012; Witkiewicz et al., 2015), the purely epithelial cated that W+ PDAC organoids autonomously create their own
composition of PDAC organoids enabled accurate assessment Wnt niche.
Cell Stem Cell 22, 1–14, March 1, 2018 3

Figure 2. PDAC Organoids Exhibit Distinct Wnt Niche Requirements

(A) Representative images depicting Wnt and R-spondin dependency of PDAC organoids.
(B) R-spondin/Wnt dependency profiles (green) and Wnt pathway mutations (black) in PDAC organoids and NL organoids. Triangles indicate heterozygosity. Bars
on the top represent organoid subtypes as follows: gray, normal like (NL); pink, W; red, W+; and purple, WRi.
(C) The potent effect of a Porcn-i on W+ PDAC organoid growth. Supplementation of exogenous Wnt3A blocks this negative effect.
(D) qPCR analyses of Wnt target genes (LGR5 and AXIN2) in 3 Wnt-independent PDAC organoid lines cultured under the indicated conditions (n = 3 per each line
per for each condition). mRNA expression levels relative to b-actin (log10 value) are shown. Upon Porcn-i treatment, LGR5 and AXIN2 mRNA expression levels
were significantly reduced (p < 0.05; Student’s t test).
(E) Schematic representation of the proposed Wnt niche subtypes. NL and Wnt-non-producing (W) organoids require for exogenous Wnt and R-spondin ligand;
Wnt-producing organoids are independent of exogenous Wnt ligand but rely on R-spondin; and Wnt and R-spondin-independent organoids (WRi) have no
requirement for Wnt signal activation.
(A and C) The values shown indicate organoid areas relative to the optimal growth conditions (the mean ± SEM; n = 4 per each line for each condition). See also
Figure S3 and Tables S2, S5, and S6.
We next characterized six Wnt and R-spondin-independent ment for Wnt-signal activation. In contrast, the growth of WRi
(WRi) PDAC organoids. To determine whether WRi PDAC orga- PDAC organoids was maintained, suggesting that Wnt-signal
noids require Wnt-signal activation for their growth, we tested activation itself was not essential for maintaining these organo-
the effect of ICG001, a small-molecule inhibitor that blocks ids (Figure S3C). Though WRi PDAC organoids also tolerated
downstream Wnt-b-catenin signaling (Emami et al., 2004). Porcn-i treatment corroborating their dispensability of Wnt-
Upon ICG001 treatment, both W and W+ PDAC organoids irre- signal activation, some WRi PDAC organoids were responsive
versibly terminated their proliferation in line with their require- to the Porcn-i treatment, suggesting their partial dependency

on self-producing Wnt ligands (Figure S3D). In sum, our results niche environments and that Wnt-targeting therapeutics could
revealed 3 functional subtypes of PDAC organoids that dis- be potent against wild-type W PDACs, regardless of the
played unique requirements for Wnt and R-spondin niche RNF43 mutation status.
environments (Figure 2E).
Epithelial Wnt Ligand Expression Is Associated with the
Cancer-Associated Fibroblasts Provide Wnt Ligands Clinical Progression of PDACs
for PDACs In contrast to W PDAC organoids, W+ PDAC organoids
W PDAC cells critically depend on exogenous Wnt ligands for harnessed their self-produced Wnt ligands. To determine which
their survival and growth, yet the source of the Wnt ligands Wnt ligands were expressed in PDAC organoids, their expres-
remains obscure. Because PDAC is characterized by abundant sion levels were assessed by microarray analyses. An unbiased
stromal cell infiltration, we inferred that these stromal cells could criterion was set to select Wnt ligands that were expressed at
support the growth of PDACs through Wnt production. To deter- significant levels in at least one W+ PDAC organoid line (>5 SD
mine the functional role of stromal Wnt ligands, we established expression from the mean expression levels of NL organoids;
patient-derived cancer-associated fibroblasts (CAFs) (Figure Figure 4A), which nominated 6 Wnt ligands from 19 Wnt-ligand
1A). Immunostaining of fibroblast activation protein alpha (FAP) family members. To determine whether any of these Wnt ligands
and a smooth muscle actin (aSMA), characteristic markers for could serve as an epithelial Wnt niche, we overexpressed each
CAFs in PDAC (Öhlund et al., 2017), confirmed that the estab- Wnt ligand in NL organoids and examined their potential for
lished CAFs were of stromal origin (Figure S4A). In contrast to driving niche function. In this functional assay, 4 Wnt ligands
the quiescence induction in CAFs embedded in Matrigel (Öhlund (WNT3, WNT7A, WNT7B, and WNT10A) were found to substitute
et al., 2017), CAFs in a collagen type I Matrigel mixture showed for Wnt3A and, thus, were designated as ‘‘epithelial’’ Wnt ligands
proliferation potency. To investigate whether these CAFs can (Figures 4A and S5A). Importantly, the expression levels of these
functionally support the growth of W PDAC organoids, we epithelial Wnt genes were significantly higher in WRi and W+
generated single stroma-attached organoids by aggregating PDAC organoids than in W PDAC organoids, indicating the as-
dissociated PDAC cells and CAFs (Figures 3A and 3B). Interest- sociation between epithelial Wnt-ligand expression and Wnt
ingly, this physical stroma attachment enabled W PDAC orga- niche independency (Figure 4B).
noids to grow without exogenous Wnt3A, and as expected, To determine whether epithelial Wnt ligands are expressed in
Porcn-i treatment abrogated this growth-promoting effect (Fig- clinical specimens, we analyzed their expression levels in whole
ures 3C and 3D). Consistent with the short-range Wnt gradient PDAC tissues using a publicly available transcriptome dataset.
in intestinal organoids (Farin et al., 2016), this growth-promoting Hierarchical clustering of Wnt-ligand gene-expression levels
effect on W PDAC organoids was not observed in conditioned aggregated the epithelial Wnt-ligand genes in a single cluster
medium from CAFs or when CAFs and W PDAC organoids were (Figure S5B). WNT2, WNT2B, WNT4, and WNT5A were ex-
co-cultured without physical attachment (Figure S4B). These re- pressed in PDAC tissues but were rarely detected in organoids,
sults demonstrated that the juxtacrine interaction with PDAC suggesting their stromal origins. Real-time qPCR analyses of
cells was critical for CAFs to support the growth of W PDACs. Wnt ligand gene-expression levels in organoids and CAFs
To further validate the pro-tumorigenic effects of CAFs on confirmed these tissue-specific expression patterns (Figure 4C).
PDAC organoids in vivo, PDAC organoids were subcutaneously Of note, the functional assay revealed that only WNT2 and
transplanted, either alone or with CAFs. In the absence of CAFs, WNT2B served as potent niche factors among stromal Wnt
W+ and WRi PDAC organoids efficiently engrafted, whereas two ligands (Figure 4A).
out of the three examined W PDAC organoid lines were poorly To determine the differential expression of epithelial Wnts in
tumorigenic, suggesting the requirement for Wnt niche during patients, we next performed in situ hybridization of the clinical
tumor formation. Interestingly, when interfaced with CAFs prior specimens for the epithelial Wnt genes. WNT7B and WNT10A
to transplantation, these organoids successfully formed subcu- were markedly expressed in the epithelial component of W+
taneous tumors (Figures 3E and 3F). We observed eventual PDACs, whereas no or subtle expression of these genes was
replacement of transplanted CAFs with host-derived fibroblasts observed in W PDACs and adjacent normal pancreas tissues
(Figure S4C), suggesting that the effect of co-transplanted CAFs (Figure 4D). Stromal expression of WNT2B was detected in close
was limited to the initial phase of xenotransplantation. Indeed, proximity to PDAC tissue (Figure 4D), consistent with the short-
co-transplantation with CAFs increased the engraftment rate of range activity of Wnt ligands. These results suggested that the
W PDAC organoids but did not enhance the tumorigenic growth expression of epithelial Wnts could serve as a surrogate marker
of xenograft-competent organoids (Figure 3F). to define Wnt-producing PDACs. In addition, the high expression
To investigate whether the pro-tumorigenic effect of CAFs was of epithelial Wnts in clinical PDACs was associated with signifi-
mediated by their stromal Wnt production, we next treated xeno- cantly poor survival and metastatic progression (Figures 4E and
grafts with a Porcn-i (Figure S4D). Whereas the therapeutic effect S5C). These results demonstrated that W+ PDACs cell autono-
of Porcn-i has only been observed in RNF43 mutant PDAC cell mously activated their own Wnt signaling by expressing epithelial
lines in previous studies (Jiang et al., 2013), Porcn-i treatment Wnts, which also predicts aggressive clinical behaviors.
significantly reduced the growth of xenografts from two indepen-
dent RNF43-wild-type W PDAC organoids (Figure S4E). Wnt Niche Dependency Is Associated with Gene
Conversely, Porcn-i treatment did not affect the growth of WRi Expression Subtypes
PDAC organoids in vivo. These results demonstrated that the To explore the mechanisms underlying each functional PDAC
CAF-dependent growth of PDACs was driven by stromal Wnt subtype, we performed transcriptome analyses of the PTOL.

Figure 3. CAFs Support W– PDAC Growth via Wnt Ligand Secretion

(A) Overview of the PDAC organoid-CAF co-culture system.
(B) Time course images of a hybrid organoid during formation.
(C and D) EdU incorporation (red) in PDAC organoids (PC8) cultured in the above protocol. EdU was pulsed 1 hr before the analyses. The aggregated organoids
without CAFs ceased their proliferation in the absence of Wnt3A supplementation (C). When co-cultured with CAFs, PDAC organoids started to incorporate EdU.
EdU incorporation was decreased by Porcn-i treatment and was maintained by exogenous Wnt3A supplementation in W PDAC organoids (PC8) (D). Similar
result was obtained with another line of W PDAC organoids (PC11). The scale bar represents 100 mm.
(E) Schematic representation of the transplantation of a co-cultured organoid (left). Representative images of co-cultured organoids (PC11) xenotransplanted into
the subcutaneous space of nude mice (right) are shown. (Top) bright field is shown; (bottom) GFP fluorescence is shown.
(F) Tumor volumes of the organoids transplanted with or without CAFs. W, W+, and WRi PDAC organoids were subcutaneously xenotransplanted, either alone or
after CAF attachment. Tumor sizes were calculated from GFP-positive regions. n = 6–12 per each group. Each dot indicates the size of an individual tumor. The
take rates of xenografts are indicated below. Bars represent mean volumes of the grafts. *p < 0.05; **p < 0.01; N.S., not significant; determined by Wilcoxon’s rank
sum test.
See also Figure S4.
An unbiased projection of global gene expression with t-distrib- was directed from normal organoids toward W, W+, and WRi
uted stochastic neighbor embedding (tSNE) analysis illustrated subtypes, suggesting serial transition of gene expression signa-
linearly connected gene expression clusters corresponding to ture in line with acquisition of Wnt niche independency. To gain
the Wnt niche subtypes (Figure 5A). Notably, the linear trajectory insights into the transcriptional programs regulating this

Figure 4. Expression of Epithelial Wnts Defines the Wnt-Producing PDAC Subtype

(A) Identification of the potent epithelial Wnt ligands. Dot plots depicting expression levels of Wnt ligands in PDAC and NL organoids. Dots represent individual
organoids (gray, NL; pink, W PDAC; red, W+ PDAC; purple, WRi PDAC). The dashed line indicates the threshold of 5 SD used to determine differentially ex-
pressed Wnt ligands. The niche potency of each Wnt ligand was evaluated by measuring the growth of NL organoids overexpressing each ligand (right). Ratios
relative to the organoid area in the presence of Wnt3A are demonstrated as bars. NE, not examined.
(B) Boxplot showing the epithelial Wnt score in NL samples and the indicated PDAC subtypes. *p < 0.05; Student’s t test.
(C) Heatmap showing mRNA expression (Z score) of the indicated Wnt ligands, as determined by qPCR analysis. WRi (PC1), W+ (PC3) PDAC organoids, W (PC7
and PC8) PDAC organoids, and CAFs were analyzed. ND, not detected by qPCR. The raw values are shown in Table S6.
(D) In situ hybridization for WNT10A, WNT7B, and WNT2B in parental PDACs or normal pancreas tissues. Nuclear counter staining, hematoxylin. The scale bar
represents 100 mm. Insets, magnified views.
(E) Kaplan-Meier plots showing overall survival based on data deposited in the ICGC Data Portal (left) or reported by Moffitt et al. (2015; right). Tumor samples
were stratified based on the epithelial Wnt score (sum of the Z score, calculated using the mean and SD of all samples with survival information). p value; log
rank test.
See also Figure S5 and Tables S5 and S6.
process, we generated gene sets consisting of differentially ex- along with the loss of Wnt niche dependency, i.e., in the
pressed genes between Wnt-dependent (NL and W) and sequence of NL, W, W+, and WRi PDAC organoids (Figures
Wnt-independent (W+ and WRi) organoids (Table S3). Interest- 5B and S6B). These results implicated common transcriptional
ingly, the total expression of each gene set, Wnt-independent programs in operating both Wnt niche subtypes and gene
or dependent score, either monotonously increased or expression subtypes in PDACs.
decreased along with the Wnt niche independency statuses, The gene expression subtypes correlated with GATA6 expres-
suggesting the presence of transcriptional programs regulating sion, and a recent report (Bailey et al., 2016) showed the epige-
Wnt niche dependency (Figures 5B and S6A). Previous reports netic silencing of GATA6 in the QM-like subtype, suggesting the
have demonstrated distinct molecular PDAC subtypes based pivotal role of GATA6 expression in determining the gene
on gene-expression patterns, and thus, we next sought to inves- expression subtype. Consistently, GATA6 expression was asso-
tigate whether the Wnt niche subtypes reflected these gene ciated with the extent of Wnt niche independency (Figure 5C).
expression subtypes. When the ratio of basal/QM gene expres- Furthermore, GATA6 expression was regulated by DNA methyl-
sion levels to those of classical genes were calculated, PDAC ation, as a methylation microarray revealed progressive DNA
organoids showed incremental up-regulation of these indices methylation of GATA6 in parallel with Wnt niche subtypes

Figure 5. Transcriptome Analyses of the PTOL Revealed an Association of Wnt Niche Subtypes with GATA6 Expression and the Gene
Expression Subtypes
(A) A tSNE plot of the PTOL showing trajectory formation along with Wnt niche independency, in the sequence of NL (gray), W (pink), W+ (red), and WRi (purple)
PDAC organoids.
(B) Boxplots of gene expression score (sum of Z score) depicted gradual increase of Wnt-niche-independent genes or basal to classical subtype gene signatures
along with Wnt niche independency. Basal and classical gene sets were retrieved from data generated by Moffitt et al. (2015).
(C) Boxplots of GATA6 expression (Z score) depicted gradual decreases of GATA6 expression along with Wnt niche independency.
(D) Hierarchical clustering of differentially methylated genes among the W, W+, and WRi subtypes. Methylation values are shown as M values. The gene lists and
corresponding M values are shown in Table S4.
(E) Plots showing significant correlations between gene expression (log2 values) and gene methylations (M values) in indicated probes for GATA6 and FOXA2.
Pearson’s correlation coefficients and adjusted p values are indicated.
(B and C) *p < 0.05; **p < 0.005; determined by Student’s t test. See also Figure S6 and Table S5.
(Figures 5D and 5E; Table S4). We also observed similar trends in form) and GATA6S (short form) (Brewer et al., 1999). Although
other endoderm lineage-specific genes, FOXA2 and HNF4A GATA6L was reported to have a higher transactivation potential
(Figures 5E and S6C). Conversely, W PDAC organoids ex- than GATA6S, the functional difference between these isoforms
hibited higher methylation of WNT10A than the other subtypes has remained unexplored in pancreas tissues (Brewer et al.,
(Figures 5D and S6C). These results suggested the existence 1999). As our sgRNAs targeted only GATA6L, we observed a
of GATA6-dependent epigenetic regulation of Wnt niche complete loss of GATA6L, but GATA6S expression remained
independency. detectable in the KO experiments. Nevertheless, we confirmed
the reduction of total GATA6 expression at the protein level in
GATA6 Expression Regulates Epithelial Wnt Expression both GATA6-KD and GATA6L-KO lines (Figure 6A). Furthermore,
in W+ PDAC Organoids both lines consistently showed the reduction of FOXA2, a GATA6
To investigate whether GATA6 expression levels are functionally target gene, indicating successful GATA6 loss of function
relevant in differentiating molecular subtypes and Wnt produc- (Figure 6B). Importantly, upon the downregulation of GATA6,
tion capacities, we further performed GATA6 short hairpin RNA W PDAC organoids acquired Wnt self-activation capacity along
(shRNA)-based knockdown (KD) and CRISPR-Cas9-based with WNT7B upregulation (Figures 6C and 6D). The gene set
knockout (KO) experiments with W PDAC organoids. Of note, enrichment analysis (GSEA) revealed a significant enrichment
two GATA6 translational isotypes exist, namely GATA6L (long of the signature genes associated with the Wnt-independent

Figure 6. GATA6 Expression Regulated Wnt Dependency of PDAC Organoids

(A) Western blotting analysis showing GATA6 protein levels in W PDAC (PC24) with GATA6L knockout (-KO) and GATA6 knockdown (-KD) organoids. The upper
and the lower band depict the long and the short isoform of GATA6, respectively. The expression levels of NL (PC4) and W+ PDAC (PC3) organoids were included
as references.
(B and C) qPCR analysis of FOXA2 (B) and WNT7B (C) in parental W PDAC (PC24), GATA6-KD, and GATA6L-KO organoids. mRNA expression relative to b-actin
is shown. n = 3 per genotype. The expression levels of W+ (PC3) and WRi (PC1) PDAC organoids were included as references.
(D) GATA6-KD and GATA6L-KO enabled organoids to expand in Wnt3A-depleted culture medium. +Wnt, culture medium including Wnt3A; Wnt, culture
medium without Wnt3A. The values shown indicate organoid area relative to the +Wnt condition (the mean ± SEM; n = 4 per each line for each condition).
(E) Immunohistochemistry of GATA6 (top, brown) and in situ hybridization of WNT10A and WNT7B (bottom, red) in the clinical specimens of W+ PDACs (PC3 and
PC14). Nuclear counterstaining, hematoxylin (blue). The scale bar represents 100 mm.
(F) GATA6-high PDACs expressed higher levels of epithelial Wnt ligands (epithelial Wnt score) than GATA6-low PDACs in the ICGC (left) and GSE71729 (reported
by Moffitt et al., 2015; right) datasets. The expression value was indicated as the Z score. The GATA6-high and GATA6-low statuses were determined by Z score
of 0.
*p < 0.005; Student’s t test. See also Figure S6 and Tables S3, S6, and S7.
subtype in GATA6-engineered organoids (Figure S6E). In and epithelial Wnt ligands using public datasets. Notably,
contrast, the overexpression of GATA6 rendered W+ PDAC or- GATA6 expression inversely correlated with the expression of
ganoids dependent on exogenous Wnt ligands in conjunction epithelial Wnt ligands in two independent datasets (Figure 6F).
with reduced WNT7B expression (Figures S6F–S6H). These re- These results collectively indicate the presence of GATA6-regu-
sults suggested the presence of a GATA6-dependent transcrip- lated Wnt niche dependency in patient PDACs.
tional program that regulates Wnt dependency in PDAC.
We also investigated the clinical relevance of GATA6-regu- Transformation of Human Pancreatic Organoids into
lated expression of Wnt ligands. In the parental specimens of W– PDAC by Driver Gene Engineering
two independent W+ PDAC lines, GATA6 immunostaining The observations above demonstrated that patient-derived
presented a heterogeneous pattern with predominant GATA6- PDAC organoids acquired niche independency through driver
negative compartments and GATA6-expressing (GATA6+) sub- gene mutations and GATA6-mediated transcriptional reprog-
populations. Interestingly, these GATA6+ lesions were devoid ramming. To verify this functionality in a prospective manner,
of WNT10A or WNT7B mRNA expression, corroborating the we generated genetically engineered pancreas organoids using
negative regulation of Wnt ligands by GATA6 in clinical speci- CRISPR-Cas9. Our previously reported electroporation and se-
mens (Figure 6E). To confirm this relationship on a larger scale, lective culture protocol (Matano et al., 2015) enabled efficient
we analyzed gene expression correlations between GATA6 introduction of TP53 or SMAD4 in-del mutations to organoids

Figure 7. CRISPR-Cas9-Engineered Human

Pancreas Organoids Demonstrated Muta-
tion-Independent Adaptation to Wnt-free
Environments
(A) Strategy for generating CRISPR-Cas9 genome-
edited organoids carrying driver gene mutations in
KRAS (K), TP53 (T), CDKN2A (C), and/or SMAD4 (S).
(B) KT or KCTS organoids were subcutaneously
transplanted to nude mice. Representative histo-
logical images of xenografted tumors are shown.
Arrows indicate tumor budding formation. The
scale bar represents 100 mm.
(C) Various engineered organoids were cultured
with or without Wnt3A after a passage; +Wnt P1
(left) and Wnt P1 (middle), respectively. When the
organoids can be passaged in the absence of
Wnt3A, the image of PDAC organoids in passage 4
is shown (Wnt P4, right). The schematic repre-
sentation is shown in Figure S7G. After removing
Wnt3A from culture medium, parental wild-type,
KC, and KT organoids ceased their proliferation
and became extinct within 1–3 weeks. KCT and
KCTS organoids stopped their proliferation but
survived in the Wnt3A removed culture medium
(middle). After multiple passages, KCT and KCTS
organoids grew without exogenous Wnt3A (right).
(D and E) qPCR analysis of WNT7B and GATA6
expression in KCT (D) and KCTS (E) organoids
grown in the presence or absence of exogenous
Wnt3A for four passages.
(F) GSEA analysis revealed that adapted KCTS
organoids (versus control KCTS organoids without
adaptation) were positively and negatively en-
riched for Wnt-independent and dependent gene
signatures, respectively.
(G) Schematic representation of PDAC progres-
sion. GATA6 expression regulated both Wnt niche
subtype and gene expression (GE) subtype. EGF,
Noggin, and A83-01 (ENA) niche requirements
were regulated by driver gene mutations, whereas
Wnt and R-spondin (WR) niche dependency was
associated with GATA6 expression.
*p < 0.05; **p < 0.01; Student’s t test. See also
Figure S7 and Tables S3, S6, and S7.
driver gene mutations and the alteration of

their target gene expression in both PDAC
organoids and the engineered organoids,
corroborating the validity of the intro-
duced mutations (Figure S7E). To deter-
(not shown). Nevertheless, KRASG12V mutation knockin clones mine the tumorigenic potential of the engineered organoids,
could not be obtained, presumably due to the relatively low elec- KC, KT, and KCTS organoids were xenotransplanted into the
troporation efficiency. To improve the genome-editing effi- subcutaneous space of immunodeficient mice. KC organoids
ciency, we used a ‘‘cold shock’’ method (Doyon et al., 2011), failed to engraft. KT organoids formed subcutaneous tumors
where organoids were cultured at 30 C for 3 days after only when co-transplanted with CAFs and showed benign histo-
electroporation. This method substantially improved the pathology reminiscent of pancreatic intraepithelial neoplasia
electroporation efficiency, enabling KRASG12V (K) mutation (PanIN) (Figure 7B). In contrast, KCTS organoids engrafted
knockin. KRASG12V mutant organoids exhibited slow prolifera- without CAFs and exhibited severe nuclear atypia, structural de-
tion, whereas simultaneous engineering of CDKN2A (C) allowed formations, and tumor budding phenotypes corresponding to
efficient establishment of KC organoids. KCT and KCTS organo- the PDAC histology, which indicated that the quadruple muta-
ids were generated by subsequently introducing TP53 (T) and/or tions induced histological transformation (Figure 7B). Of note,
SMAD4 (S) mutations, respectively (Figures 7A and S7A–S7D). KCTS tumors exhibited heterogeneous histological appearance
Transcriptome analyses confirmed robust associations between comprising PanIN-like lesions and invasive PDACs, implicating

the histological progression after the acquisition of KCTS muta- mouse model (Heiser et al., 2008), Wnt signal mutations might
tions (Figure 7B). serve as tumor suppressors in the pancreas. Thus, pancreas
The four major driver gene mutations endowed engineered or- duct cells may favor Wnt-signaling activation by ligand stimula-
ganoids with mutation-specific niche independency (Figure S7F). tion during pancreas tumorigenesis.
In contrast, the engineered organoids lost proliferative potential The expression of Wnt ligands was observed in pancreas stro-
after Wnt removal, indicating that the driver gene mutations did mal cells, but whether and how the stromal Wnt ligands
not confer Wnt-niche-independent growth (Figure 7C). Interest- contribute to the PDAC ecosystem have not been determined
ingly, 1–3 weeks after the Wnt removal, KC and KT organoids (Moffitt et al., 2015). In this study, we established a PDAC orga-
became extinct, whereas KCT and KCTS organoids survived noid-CAF co-culture assay and revealed that CAFs transmit a
and slowly expanded in the Wnt-removed culture condition (Fig- pro-tumorigenic niche signal to PDACs through the juxtacrine
ures 7C and S7G). This result suggested that the presence of production of stromal Wnt ligands. Furthermore, we also found
CDKN2A and TP53 mutations allowed organoids to circumvent that CAF-derived Wnt niche signaling could be targeted by
the apoptosis/senescence responses induced by the Wnt Porcn-i treatment, the therapeutic effects of which have previ-
removal. Consistently, though the proportion of PDAC organoids ously been observed exclusively in RNF43 mutant PDAC cell
with four-driver gene mutations was comparable among Wnt lines (Jiang et al., 2013). These results suggested the broad
niche subtypes, TP53 mutation frequency was significantly application of Wnt-targeting therapy for PDACs based on an
higher in W+/WRi PDAC organoids than in W PDAC organoids, organoid-centered, non-genetic screening system. Besides
suggesting the role of TP53 mutations in Wnt niche adaptive Wnt production, CAFs have pleiotropic effects on pancreas
response in patient PDACs (Figures S7H and S7I). This pheno- tumorigenesis (Apte et al., 2012; Sousa et al., 2016). Recently,
type was dependent on endogenous Wnt production, as indi- a similar co-culture assay was established and used to identify
cated by the sensitivity to Porcn-i treatment (Figure S7J). Further- inflammatory CAFs (iCAFs) that promoted tumorigenesis
more, gene expression analyses showed GATA6 downregulation through inflammatory cytokine production (Öhlund et al., 2017).
and acquisition of the Wnt-independent PDAC gene signature Notably, the Wnt-producing CAFs resided in mutual proximity
during the adaptation to a Wnt-free culture condition (Figures to the PDAC cells, whereas the iCAFs were scattered in areas
7D–7F). Taken together, our results suggested that niche inde- distant from PDAC cells, underscoring the distinct pro-tumori-
pendency was mainly acquired through driver gene mutations, genic functions between these CAF subpopulations. In contrast,
whereas the Wnt niche independency was predominantly regu- other recent studies revealed an anti-tumorigenic role of CAFs in
lated by epigenetic mechanisms, highlighting a unique niche- pancreas tumorigenesis (Özdemir et al., 2014; Rhim et al., 2014).
adaptation process during pancreas tumorigenesis (Figure 7G). The conflicting results have been partly reconciled by the hetero-
typic subtypes identified in CAFs and PDACs (Moffitt et al., 2015;
DISCUSSION Öhlund et al., 2017). Indeed, in our study, the pro-tumorigenic ef-
fect of CAFs varied depending on the Wnt niche subtypes of host
In this study, by optimizing the culture method, we established PDAC organoids, supporting the context-dependent role of
an organoid library comprising 39 PDAC organoid lines. The CAFs in pancreas tumorigenesis.
use of serum-free Afamin-stabilized Wnt3A enabled stable prop- The tumor heterogeneity of human PDAC was molecularly
agation of human normal pancreas organoids and exclusion of dissected by the gene expression subtypes. Interestingly, these
contaminating normal organoids. Extensive characterization of gene expression subtypes pertained to the Wnt niche subtypes.
the genotype-phenotype correlation in PDAC organoids identi- The epigenetic regulation of GATA6 identified in both taxonomies
fied 3 novel Wnt niche subtypes that could not be defined by suggested the existence of a common GATA6-dependent tran-
genetic mutations. Of note, two of these subtypes (the W+ and scriptional program driving the subtype heterogeneity. In gene
W subtypes) exhibited stringent dependency on Wnt3A and/or expression classification studies, the subtypes were formulated
R-spondin, indicating that these subtypes were not previously based on snapshots of the gene expression signatures derived
derived as cell lines. Whereas an antecedent study showed from crude tumor samples, and thus, it has not been defined
consistent Wnt and R-spondin dependency in PDAC organoids whether the identified subtypes inherently represent distinct
(Boj et al., 2015), our larger scale PDAC organoid library revealed tumorigenic pathways or transitive statuses during tumor pro-
various Wnt-niche dependencies among human PDACs. gression. In contrast, the organoid platform is amenable to func-
The development of organoid culture systems is based on the tional assays and prospective genetic engineering, by which we
identification of defined niche factors that fuel the self-renewal of substantiated the GATA6-mediated subtype switching in PDAC
epithelial stem cells (Sato and Clevers, 2013). Although the exis- organoids. Furthermore, KCTS organoids showed adaptive re-
tence of pancreatic stem cells in the adult normal pancreas sponses to Wnt-free environments, coinciding with the GATA6-
remains controversial, Wnt signal activation is essential during dependent subtype conversion. These results suggested that
development and in organoid culture, underscoring the niche Wnt niche subtypes reflected dynamic reprogramming during tu-
functions of Wnt ligands and R-spondin (Kopp et al., 2016). We mor progression rather than an intrinsic property of a given
previously proposed that niche factor signaling pathways coin- tumorigenic pathway. The GATA6-mediated subtype switching
cided with recurrently dysregulated pathways in colorectal can- was not unprecedented, as GATA6 downregulation also contrib-
cers (Sato and Clevers, 2013). Contrary to this notion, Wnt uted to squamous subtype commitment and malignant progres-
pathway genes were rarely mutated in PDAC despite its essen- sion in a lung cancer model (Cheung et al., 2013). Of note, KCTS
tiality in niche signaling. Given the fact that b-catenin mutations organoids did not progress to the QM subtype and remained
retarded KRAS-mediated pancreas tumorigenesis in a genetic dependent on R-spondin, suggesting that four-driver mutations

were insufficient to confer on the engineered organoids the full d EXPERIMENTAL MODEL AND SUBJECT DETAILS
spectrum of PDAC phenotypes, including WRi phenotype. B Establishment of human pancreas organoids
Recently, transcriptional reprogramming by another endoderm- B Mice
related transcription factor, FOXA1, was reported to induce met- d METHOD DETAILS
astatic progression in PDAC (Roe et al., 2017). Though how these B Co-culture of PDAC Organoids with CAFs
endodermal-specific genes coordinate the niche requirements B Xenotransplantation of Organoids
and progression of PDACs remains unexplored, the prevalent B Gene Engineering of Organoids
methylation of these genes in QM and WRi PDAC subtypes indi- B Immunohistochemistry and in situ Hybridization
cates that epigenetic regulations drive these processes. Further B Whole-Exome Sequence Analysis
studies are warranted to understand the mechanistic role of these B Copy-Number Analysis
transcription factors in the malignant progression of PDAC. B Methylation Analysis
Decades of pathologic and genetic research on human B Real-Time Quantitative PCR
pancreas tumorigenesis have indicated that PDAC originates B Western blot Analysis
from PanIN lesions (Hruban et al., 2000), but its prospective B Gene-Expression Microarray Analysis
demonstration has not been achieved in human pancreas. In B Data Analysis for Publicly Available PDAC Specimens
this study, using CRISPR-Cas9-based KO and mutation knockin, d QUANTIFICATION AND STATISTICAL ANALYSIS
we generated isogenic KCTS organoids from normal pancreas d DATA AND SOFTWARE AVAILABILITY
organoids, which faithfully replicated the pancreas carcinogenic
process. Recently, Lee et al. (2017) also generated similar engi- SUPPLEMENTAL INFORMATION
neered pancreas organoids using CRISPR-Cas9 system. In
contrast to our KCTS organoid-derived tumors that were histolog- Supplemental Information includes seven figures and seven tables and can be
found with this article online at https://doi.org/10.1016/j.stem.2017.12.009.
ically compatible with PDAC, the pancreas organoids engineered
by Lee et al. (2017) only formed tumors corresponding to PanINs.
This discrepancy might be associated with the different methods ACKNOWLEDGMENTS
used to introduce KRAS mutations (KRAS overexpression by Lee

This work was supported by the Project for Cancer Research and Therapeutic
et al., 2017 versus KRAS knockin in our study). The discordant Evolution (P-CREATE) from the Japan Agency for Medical Research and
outcomes between these two genetic approaches has been Development (AMED), a Grant-in-Aid for Scientific Research on Innovative
previously observed in mouse and human pancreas cells, under- Areas Stem Cell Ageing and Disease, and Grants-in-Aid for Scientific
scoring the ability of the KRAS knockin strategy in faithful PDAC Research funded by the Ministry of Education, Culture, Sports, Science and
disease modeling (Arena et al., 2007; Brembeck et al., 2003; Hin- Technology of Japan. Y.O., M.F., K.K., and S.S. were supported by the Japan
Society for the Promotion of Science Research Fellowships for Young Scien-
gorani et al., 2003; Konishi et al., 2007). It should also be noted
tists. We also thank the Collaborative Research Resources, School of Medi-
that, unlike the xenografts generated in orthotopic pancreas by cine, Keio University for the technical assistance provided. The R-spondin-
Lee et al. (2017), we xenografted organoids into subcutaneous producing cell line was a kind gift from C. Kuo (Stanford University).
sites, in which KCTS organoids displayed tumor budding forma-
tion. Therefore, our KCTS organoids provided the first isogenic AUTHOR CONTRIBUTIONS
PDAC model that recapitulated the PanIN-PDAC sequence from
Conceptualization, T. Seino and T. Sato; Methodology, T. Seino, S.K., and
human normal pancreas organoids. Of note, in mouse models,
T. Sato; Investigation, T. Seino, S.K., M.S., Y.O., M.M., K.N., M.F., H.T.,
pancreas acinar cells have been proposed to engender PDACs
K.K., S.T., S.S., and T. Sato; Data Curation, M.S. and K.T.; Writing – Original
through acinar-to-ductal metaplasia (Kopp et al., 2012). As the Draft, T. Seino and T. Sato; Writing – Review & Editing, T. Seino, M.S., M.F.,
current organoid culture cannot propagate or reproduce acinar- and T. Sato; Funding Acquisition, T. Sato; Resources, E.I., J.T., T.I., M.K.,
to-ductal metaplasia (Huch et al., 2013), it is premature to either Y.K., and T.K.
exclude or assert the possibility of tumorigenesis originating
directly from human pancreas acinar cells. DECLARATION OF INTERESTS
In conclusion, we established a PTOL encompassing a
The authors declare no competing interests.
panoply of patient-derived PDACs and engineered organoids,
followed by comprehensive molecular and functional analyses. Received: May 18, 2017
This resource can bridge the gap between cancer genotypes Revised: October 30, 2017
and biological phenotypes that have previously been elusive, Accepted: December 14, 2017
due to a lack of genetically tractable patient-derived PDAC Published: January 11, 2018
models. Our results provide novel insights into PDAC tumorigen-
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit monoclonal anti-Ki67 Thermo Fisher Scientific RM-9106-S, RRID:AB_2335745
Mouse monoclonal anti-a smooth muscle actin Thermo Fisher Scientific MS-113-P, RRID:AB_64000
Sheep polyclonal anti-human FAP R&D AF3715, RRID:AB_2102369
Rabbit monoclonal anti-GATA6 (D61E4) Cell Signaling Technology 5851, RRID:AB_10705521
Rabbit polyclonal anti-p44/42 MAPK (Erk1/2) Cell Signaling Technology 9102, RRID:AB_330744
Rabbit monoclonal anti-phospho-p44/42 Cell Signaling Technology 4370, RRID:AB_2315112
MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)
Mouse monoclonal anti-cytokeratin 19 Leica Biosystems NCL-L-CK19, RRID:AB_563799
Rat monoclonal anti-human/mouse CD49f Biolegend 313602, RRID:AB_345296
(integrin a6)
Mouse monoclonal anti-b-actin Sigma-Aldrich A1978, RRID:AB_476692
Phalloidin, Alexa Fluor 647 (F-actin) Thermo Fisher Scientific A22287, RRID:AB_2620155
Goat polyclonal anti-rat IgG (H+L), Alexa Thermo Fisher Scientific A-11077, RRID:AB_2534121
Fluor 568
Donkey polyclonal anti-mouse IgG (H+L), Thermo Fisher Scientific A10037, RRID:AB_2534013
Alexa Fluor 568
Donkey polyclonal anti-sheep IgG (H+L), Abcam Ab175712
Alexa Fluor 568
Chemicals, Peptides, and Recombinant Proteins
Advanced DMEM/F12 Thermo Fisher Scientific 12634010
Human fibroblasts defined medium (HFDM-1) Cell Science & Technology Institute 2102P05
HEPES Thermo Fisher Scientific 15630080
Penicillin-Streptomycin Thermo Fisher Scientific 15140122
GlutaMAX Supplement Thermo Fisher Scientific 35050061
Matrigel BD Biosciences 356231
Collagen Gel Culturing Kit Nitta Gelatin N/A
B-27 Supplement Thermo Fisher Scientific 17504044
N-Acetyl-L-cysteine Sigma-Aldrich A9165
[Leu15]-Gastrin I human Sigma-Aldrich G9145
Afamin-Wnt-3A serum-free conditioned medium Mihara et al., 2016 N/A
Recombinant Mouse EGF Thermo Fisher Scientific PMG8043
R-spondin-1 conditioned medium Ootani et al., 2009 N/A
Recombinant mouse Noggin Peprotech 250-38
A83-01 Tocris 2939
SB202190 Sigma-Aldrich S7067
Y-27632 Wako 253-00513
CHIR99021 Cayman Chemical 13122
(±)-Nutlin-3 Cayman Chemical 548472-68-0
human recombinant BMP4 Peprotech 120-05ET
Wnt-C59 ShangHai Biochempartner 1243243-89-1
ICG001 Cayman Chemical 16257
Fetal bovine serum Biowest S1820
Liberase TH Research Grade Roche 05401151001
Red Blood Cell Lysis Buffer Roche 11814389001
Cell Lysis Buffer Cell Signaling Technology 9803
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Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018 e1

Continued
Cell Recovery Solution Corning 354253
Hoechst 33342 Thermo Fisher Scientific H3570
DAPI Thermo Fisher Scientific D1306
TrypLE Express Thermo Fisher Scientific 12605010
5-ethynyl-20 -deoxyuridine (EdU) Thermo Fisher Scientific A10044
Puromycin Thermo Fisher Scientific A1113802
Blasticidin Thermo Fisher Scientific A11139-03
BTXpress Solution BTX 45-0805
Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific 31985062
Critical Commercial Assays
Click-iT plus EdU Imaging Kit Alexa Fluor 647 Thermo Fisher Scientific C10640
RNAscope 2.5 HD Reagent Kit Advanced Cell Diagnostics 322350
QIAamp DNA Blood Mini Kit QIAGEN 51106
RNeasy Mini Kit QIAGEN 74104
Wes 12-230 kDa Master Kit ProteinSimple N/A
High-Capacity cDNA Reverse Transcription Kit Thermo Fisher Scientific 4368814
Plasmid Plus Maxi Kit QIAGEN 12965
Deposited Data
Gene expression microarray This study GEO: GSE107610
Gene expression microarray Collisson et al., 2011 GEO: GSE17891
Gene expression microarray and clinical data Moffitt et al., 2015 GEO: GSE71729
Gene expression microarray and clinical data ICGC PACA-AU
Experimental Models: Cell Lines
Human: pancreas organoids: see Table S1 This study N/A
Human: cancer-associated fibloblasts This study N/A
Experimental Models: Organisms/Strains
Mouse: BALB/cAJcl-nu/nu CLEA Japan N/A
Mouse: NOD.Cg-PrkdcscidIl2rgtm1Sug/Jic Central Institute for Experimental N/A
Animals; Ito et al., 2002
Oligonucleotides
Primers: see Table S6 This study and Shimokawa et al., 2017 N/A
RNAscope Probe Hs-LGR5 Advanced Cell Diagnostics 311021
RNAscope Probe Hs-AXIN2 Advanced Cell Diagnostics 400241
RNAscope Probe Hs-WNT10A Advanced Cell Diagnostics 421571
RNAscope Probe Hs-WNT7B Advanced Cell Diagnostics 421561
RNAscope Probe Hs-WNT2B Advanced Cell Diagnostics 453361
RNAscope Positive Control Probe Hs-PPIB Advanced Cell Diagnostics 313901
RNAscope Negative Control Probe DapB Advanced Cell Diagnostics 310043
Recombinant DNA
Donor vectors: see Table S7 This study and Matano et al., 2015 N/A
PiggyBac-CMV-MCS-EF1a-GFP-T2A-Puro SBI PB513B-1
shRNA: GATA6 OriGene TL312832
Software and Algorithms
Rtsne v.0.13 Jesse H. Krijthe (2015). Rtsne: N/A
T-Distributed Stochastic Neighbor
Embedding using a Barnes-Hut
Implementation
GSEA Broad Institute http://software.broadinstitute.org/gsea/
index.jsp
(Continued on next page)
e2 Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018

Continued
Burrows-Wheeler Aligner (BWA) Slashdot Media https://sourceforge.net/projects/bio-bwa/
Genome Analysis Toolkit (GATK) v. 1.6.13 Broad Institute https://software.broadinstitute.org/gatk/
Picard v.1.75 Broad Institute http://broadinstitute.github.io/picard/
Samtools Genome Research http://www.htslib.org/
Compass for SW ProteinSimple http://www.proteinsimple.com/
compass/downloads/
R v.3.3.3 Comprehensive R Archive Network https://cran.r-project.org/
LuminaVision Mitani https://www.mitani-visual.jp/
download/catalogs/
BZ-X analyzer Keyence N/A
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Toshiro
Sato (t.sato@keio.jp).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Establishment of human pancreas organoids

Clinical samples used for organoid establishment and biological analyses were obtained from patients at Keio University Hospital or
Tokyo Medical University Hospital with informed consent after approval by the ethical committees. Healthy and neoplastic pancreatic
specimens were collected by surgical resection, endoscopic ultrasound guided-fine needle aspiration (EUS-FNA), or ascites punc-
ture. Organoids derived from surgically resected and EUS-FNA tissues were established as reported previously, with modifications
(Fujii et al., 2015). Surgical specimens were washed vigorously and minced into 10 mm3-size fragments with surgical scissors. The
fragments were digested with Liberase TH (Roche) at 37 C for 30 min. Undigested pellets were further digested with TrypLE Express
(Thermo Fisher Scientific) at 37 C for 10 min. The digested cells were washed with phosphate-buffered saline (PBS) containing 10%
fetal bovine serum (FBS) to inactivate the digestive enzyme, and then they were cultured. FNA samples were incubated with Red
Blood Cell Lysis Buffer (Roche) to eliminate red blood cells and washed 3 times with ice-cold PBS. Ascites samples were centrifuged
and washed 3 times with ice-cold PBS, and the sedimented cells were used for organoid culture. The obtained pancreatic cells were
embedded in Matrigel and overlaid with a previously described basal culture medium (Fujii et al., 2015), namely Advanced Dulbecco’s
Modified Eagle’s Medium/F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1 3 B27 (Thermo Fisher
Scientific), 10 nM gastrin I (Sigma), and 1 mM N-acetylcysteine (Wako, Japan). The basal culture medium was supplemented with the
following niche factors: 50 ng/ml mouse recombinant EGF, 100 ng/ml mouse recombinant noggin (Peprotech), 10% R-spondin-1
conditioned medium (Ootani et al., 2009), 25% Afamin-Wnt-3A serum-free conditioned medium (Mihara et al., 2016), 500 nM
A83-01 (Tocris), and 10 mM SB202190 (Sigma). To suppress endogenous Wnt production, the cells were treated with 100 nM C59
(Shanghai Biochempartner Co., Ltd). Each plate was incubated at 5% CO2 in 20% O2, and the media were changed every 3 or
4 days. To enrich for PDAC-derived organoids, we cultured the organoids in EGF-removed medium. Once tumor-derived organoids
were established, niche factors were subsequently removed to determine the minimal requirements of the niche factors. When the
organoids survived without a given niche factor for at least 1 month, the organoids were judged as being independent of the niche
factor. When the organoids were survived, but their growth declined after the removal of a niche factor, we judge their niche factor
dependency by the viability after 3 months in culture. The established organoids were subjected to molecular analyses (Table S5) and
cryopreserved, as previously described (Fujii et al., 2015).
All established pancreatic tumor-derived organoids were expanded for at least 3 months and stored as cryo-stocks for on-demand
usage. Detailed clinical information is available in Table S1.
Mice
All animal procedures were approved by the Keio University School of Medicine Animal Care Committee. Male NOD/Shi-scid,
IL-2Rgnull (NOG) mice (7–12 weeks old) were obtained from the Central Institute for Experimental Animals (CIEA, Japan) and female
BALB/c-nude mice (7–12 weeks old) were obtained from CLEA Japan, and housed under specific pathogen-free conditions.

METHOD DETAILS
Co-culture of PDAC Organoids with CAFs

To establish CAFs, the liberase-digested PDAC samples described above were resuspended in HFDM-1 (Cell Science & Technology
Institute.) supplemented with penicillin/streptomycin and 5% FBS. The suspended tissues/cells were placed on a gelatin-treated cul-
ture plate and incubated in 5% CO2 and 20% O2 at 37 C. Under this culture condition, CAF selectively expanded, whereas remnant
PDAC cells were depleted after a few passages.
For co-culture, PDAC organoids and CAFs were independently dissociated into single cells using TrypLE Express and mixed at a 1:
2 ratio (1 3 105 cells of organoid and 2 3 105 cells of CAF). The cell mixture was suspended in PDAC culture medium supplemented
with 10% matrigel and 5% FBS, and was dispensed into the round-bottom wells of ultra-low attachment plates. The plates were
centrifuged at 400 3 g for 3 min to facilitate aggregation and incubated for 1 day. The aggregates were transferred into 20 mL of a
25% Matrigel/75% type-I collagen gel (Nitta Gelatin) mixture and solidified as a droplet on a 48-well culture plate (Corning). Upon
solidification, basal media with culture medium containing 5% FBS and minimal niche factors but Wnt was overlaid. For control ex-
periments, the same number of PDAC cells were aggregated and transferred to the Matrigel/collagen gel.
Xenotransplantation of Organoids
Prior to the assay, the organoids were labeled by electroporation with a GFP-puro PiggyBac vector (System Biosciences), as previ-
ously described (Matano et al., 2015). PDAC organoids were isolated from Matrigel using Cell Recovery Solution (BD Biosciences)
and mechanically dissociated into cell clusters. For orthotopical transplantation, cell clusters (equivalent to 1 3 105 cells) were
resuspended in cold Matrigel and injected into the pancreas of NOG mice. For subcutaneous transplantation, a small incision
was created in the flank of BALB/c-nude mice and an organoid co-cultured in collagen gel was placed into each pocket. The incision
was surgically sutured. To control the number of PDAC organoids, we aggregated the same number of PDAC cells (1 3 105 cells) with
or without CAFs (2 3 105 cells) for 1 day, and cultured the aggregates in Matrigel/collagen mixture for 1 day in medium containing
Wnt3A. The numbers of PDAC cells in the aggregates were nearly equivalent, regardless of CAF association.
At 2 months post-transplantation, the pancreas or subcutaneous engrafts were isolated, and tumor sizes were measured as the
area of green fluorescent protein (GFP) fluorescence (Nikon Multi-zoom microscopy, LuminaVision software). For Porcn-i-treatment
experiments, tumor-bearing mice were randomized before treatment. The volumes of subcutaneous tumors were calculated as 1/2
(length 3 width2). A blinded investigator measured the tumor sizes. In cases where no tumor was detected, the volume was set to
0.01 to avoid arithmetic error during log transformation. The grafts were fixed for subsequent histological analysis. Each line of PDAC
organoids was transplanted into 4 independent NOG mice or 10–12 independent BALB/c-nude mice.
Gene Engineering of Organoids

Gene specific single-guide RNA (sgRNA) were cloned into px330 expression vector (Addgene #42230) or lentiGuide-Puro vector
(Addgene #52963). The sgRNA sequences are shown in Table S7. GATA6-shRNA lentiviral vector was obtained from OriGene
(TL312832). For GATA6-OE, we cloned a coding region of GATA6 into pLVSIN-CMV-Pur Vector (TaKaRa #6183). We engineered
organoids using electroporation (Nepa Gene) or lentiviral transduction as previously reported with slight modifications (Matano
et al., 2015). GFP-Puro PiggyBac and single-guide RNA (sgRNA) vectors (constructed from px330, Addgene #42230) were co-elec-
troporated for visualization and selecting engineered organoids. After electroporation, the organoids were cultured at 30 C for 3 days
to induce cold shock. The organoids were thereafter cultured at 37 C. Engineered organoids were selected in puromycin (2 mg/ml) for
2 days, followed by selection in EGF-removed or Nutlin3-including culture medium to isolate KRAS mutant or TP53 mutant organo-
ids, respectively. For SMAD4 and CDKN2A engineering, organoids were first transduced with a lentiviral Cas9-expression vector
(lentiCas9-Blast, Addgene #52962) and selected with Blasticidin (10 mg /ml) for 7 days. The selected Cas9-expressing organoids
were further infected with sgRNA-lentivirus targeting SMAD4 or CDKN2A. The sgRNA-expressing organoids were selected with
puromycin (2 mg/ml). SMAD4 mutant organoids were further selected by Noggin withdrawal. We do not have a selection method
for CDKN2A mutations; thus, we cloned single-cell-derived, puromycin-resistant organoids and sequenced them for biallelic frame-
shift mutations of CDKN2A. To validate each targeted mutation, PCR-amplified genomic DNA was cloned into a TOPO-TA cloning
vector. Plasmid DNAs isolated from 8 bacterial clones were analyzed by Sanger sequencing to confirm the introduction of mutations.
Immunohistochemistry and in situ Hybridization

Xenografts were isolated from mice and immediately fixed in 4% paraformaldehyde. Five micrometer paraffin-embedded tissue
sections were used for further histological analyses. Hematoxylin and eosin (H&E) staining was performed following a standard his-
tological protocol. For immunohistochemistry, rat anti-integrin-a6 (313602, Biolegend, 1:100), mouse anti-aSMA (#MS-113-P,
Thermo Fisher Scientific, 1:800), sheep anti-human FAP (#AF3715, R&D systems, 1:50), rabbit anti-GATA6 (#5851, CST, 1:300),
anti-cytokeratin19 (#NCL-L-CK19, Novocastra, 1:100) were used. The secondary antibodies were horseradish peroxidase-conju-
gated anti-rabbit antibodies (Nichirei Bioscience) or Alexa Fluor 568-conjugated anti-rat, mouse, sheep antibodies (Thermo Scientific
or Abcam). Alexa Fluor 647-phalloidin (A22287, Thermo Scientific) was used to visualize F-actin. For immunocytochemistry, the or-
ganoids and CAFs were fixed in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton-X. For EdU staining, we used
the Click-IT Plus EdU Imaging kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Nuclei were counterstained
with Hoechst 33342 or DAPI. For in situ hybridization, we used an RNAscope 2.5 HD Kit (Advanced Cell Diagnostics), according to the

manufacturer’s instructions. Probes for WNT7B, WNT10A, and WNT2B were designed by Advanced Cell Diagnostics. The PPIB and
DapB genes were used as positive and negative controls, respectively. Images were taken with a BZ-X710 digital microscope
(Keyence) or an SP8 confocal microscope (Leica).
Whole-Exome Sequence Analysis

DNA was extracted from organoids or blood with the QIAamp Blood Mini Kit (QIAGEN) and treated with RNase Cocktail (Ambion). The
quality of DNA specimens was confirmed by agarose gel electrophoresis within the expected range of DNA fragmentation. Three
micrograms of genomic DNA was fragmented for whole-exome sequencing analyses, as previously described (Fujii et al., 2016).
Paired-end libraries were prepared using the SureSelect Human All Exon V5 or V6 kit (Agilent) according to the manufacturer’s
instructions and were sequenced using an Illumina HiSeq2500 (outsourced to Hokkaido System Science Co., Ltd.) or HiSeq4000
(outsourced to BGI JAPAN K.K.) instrument. The generated fastq files were mapped onto human reference genome version
GRCh37 (hg19) using the Burrowsrrowsent. The generated n Illumina HiSeq2500 HiSeq2500NA specimens wstological protocol.
For immunohistochemistry, introductition of reads were performed with the Genome Analysis Toolkit (GATK, version 1.6.13). Somatic
mutations, single-nucleotide variations (SNVs), insertions, and deletions were detected using GATK UnifiedGenotyper. Variants were
called by removing those registered in SNV databases (dbSNP build 131 or lower) or a frequency in the 1000 Genomes Project
of > 0.01. When normal organoids or blood samples were available, variants were further filtered by altering the allele frequency
(< 0.08) of the counterpart normal organoids or blood DNA. Otherwise, variants were filtered based on Japanese SNVs registered
in the Human Genetic Variation Database (Higasa et al., 2016).
Copy-Number Analysis
For copy-number analysis, 500 ng of genomic DNA was applied to CGH/SNP microarray (CytoScan HD, Affymetrix), according to the
manufacturer’s instructions. Probe-level copy numbers was determined using Affymetrix Power Tools (version 1.19.0). Segmentation
analysis was performed with the R package, using the ‘‘rCGH’’ workflow (Commo et al., 2016).
Methylation Analysis
For methylation analysis, 500 ng of genomic DNA was subjected to bisulfite conversion and applied to an Infinium MethylationEPIC
microarray (Illumina), according to the manufacturer’s instructions. The obtained data were normalized using ‘‘methylumi.bgcorr’’
and ‘‘normalizeMethylumiSet’’ in the R package ‘‘methylumi.’’ Microarray data for probes recognizing single-nucleotide polymor-
phisms and genes located on sex chromosomes were filtered out before analysis. Probe-level M-values were mapping to the
same gene that showed the largest variance in probes mapped to the same gene. Genes with small variance (less than 5) were
removed from analysis. Differentially methylated genes among three subtypes (W-, W+ and WRi) were determined using the
R ‘‘limma’’ package and filtered with a false-discovery rate > 0.15. A list of differentially methylated genes was shown in Table S4.
Hierarchical clustering was performed using hclust (R package stats) with Spearman distance and ward.D linkages.
Real-Time Quantitative PCR

Total RNA was extracted from PDAC organoids or CAF using the RNeasy Mini Kit (QIAGEN), and cDNA was synthesized using the
High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Real-time qPCR was performed using the Universal Probe
Library (UPL) and FastStart Essential DNA Probes Master (Roche), and LightCycler 96 System (Roche). Relative gene-expression
levels were calculated using the delta–delta CT method. The primer sequences and UPL probe numbers are shown in Table S6.
Western blot Analysis

For GATA6 expression analysis, organoids were grown under optimal growth culture conditions. For phospho-ERK-production anal-
ysis, organoids were grown under optimal growth culture conditions, followed by cultured without EGF for 3 days. Proteins were
extracted from organoids using Cell Lysis Buffer (#9803, CST) according to the manufacturer’s instructions. Protein separation
and detection were performed using an automated capillary electrophoresis system (Simple Western system and Compass software;
proteinsimple). Antibodies against the following proteins were used; anti-ERK-1/2 (#9102, CST, 1:50) and anti-phospho ERK1/2
(T202/Y204) (#4370, CST, 1:50), GATA6 (#5851, CST, 1:50) and anti-b actin (#A1978, Sigma, 1:50). Signals were detected by
HRP-conjugated secondary anti-rabbit antibody and were visualized using proteinsimple software.
Gene-Expression Microarray Analysis

Organoids were cultured from single cells for 5 days in the indicated culture condition (Table S1). RNA was extracted from organoids
using the RNeasy Plus Mini Kit (QIAGEN). RNA quality was evaluated based on the RNA Integrity Number (RIN) value determined in
RNA6000 assays (Agilent). Only specimens with an RIN > 7.0 were used in this study. Gene-expression profiling of 32 PDACs, 6
normal-like, and 1 normal organoids was performed using the Prime View Human Gene Expression Array (Affymetrix), according
to the manufacturer’s instructions. Raw data were normalized using robust multi-array analysis implemented in the R software pack-
age affy. We detected non-biological batch effects related to the date of experiments, and employed the ComBat algorithm (R pack-
age sva) to compensate for batch effects (Johnson et al., 2007). The probes were summarized into a gene by selecting probes with
the highest Median Absolute Deviation (MAD) value per gene. Normalized microarray data are publically available from GEO data-
sets. Z-score for PDAC and normal-like organoids was calculated using mean and s.d. of normal-like organoids. Epithelial Wnt

(epiWNT) score was calculated as the Z-score sum of epithelial WNT genes (WNT3, WNT7A, WNT7B, WNT10A). t-distributed
Stochastic Neighbor Embedding was performed for all genes using Rtsne (R package ‘‘Rtsne’’), with perplexity = 10. Basal-classical
gene scores (Moffitt et al.) and QM-Clasical gene scores (Collisson et al.) were defined as the Z-score sum by subtracting scores for
classical genes from those of basal/QM genes. The Wnt niche (in)dependency related gene sets were determined by differentially
expressed genes between Wnt dependent organoids (normal-like and W- PDAC organoids) and Wnt independent organoids
(W+ and WRi PDAC organoids) using sam (R software package siggenes) (Fold change S 5 or < 0.2, p % 0.01, false-discovery
rate % 0.01). The genes list are shown in Table S3. Gene set enrichment analysis (GSEA) was performed using the GSEA module
in GenePattern (v17, Broad Institute) using Wnt niche (in)dependency related genes as determined above.
Data Analysis for Publicly Available PDAC Specimens

Gene expression microarray data from 125 primary PDAC samples deposited by Moffitt et al. were obtained from Gene Expression
Omnibus (GEO; GSE71729), and its associated clinical data were extracted from the original report. International Cancer Genome
Consortium (ICGC) gene-expression data and its associated clinical data from 267 primary PDACs were obtained from the ICGC
Data Portal (project PACA-AU). Z-score was calculated using mean and s.d. of all PDAC samples for each dataset. When normal
pancreas data were included, Z-score was calculated using mean and s.d. of the expression values of the gene expression in normal
samples. Epithelial Wnt score for each sample was calculated as the sum of Z-score expression values of epithelial Wnt genes
(WNT3, WNT7A, WNT7B, and WNT10A). epiWNThigh and epiWNTlow patients were defined as epithelial Wnt score R 0, or < 0,
respectively. Overall survival was analyzed by the Kaplan–Meier method with survfit (R package survival), and the significance
was measured by the log-rank test. Hierarchical clustering was performed using hclust (R package stats) with spearman distance
and ward.D linkage.
QUANTIFICATION AND STATISTICAL ANALYSIS
Pairwise analyses for tumor volumes and gene expression values were performed using unpaired two-tailed Student’s t test or
Wilcoxon’s rank sum test. The data are presented as mean ± s.e.m. For survival analyses, distribution of two groups were compared
using Log-rank test. For further statistical details, refer to each figure legend.
DATA AND SOFTWARE AVAILABILITY
The accession number for the data for gene expression microarray reported in this study is GEO: GSE107610.

Human Pancreatic Tumor Organoids

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Human Pancreatic Tumor Organoids Reveal Loss of

Wnt Niche Independency

d PDAC segregates into 3 subtypes with distinct dependency

d Cancer-associated fibroblasts provide a Wnt niche for PDAC

d CRISPR-Cas9 gene engineering recapitulates multistep

Seino et al., 2018, Cell Stem Cell 22, 1–14

Cell Stem Cell

Human Pancreatic Tumor Organoids Reveal Loss

SUMMARY apeutics, underscoring the importance of understanding inter-

Figure 1. Establishment of the PTOL Using Niche-Based Selection

traits and genetic-mutation profiles of the parental tumors RESULTS

2 Cell Stem Cell 22, 1–14, March 1, 2018

Cell Stem Cell 22, 1–14, March 1, 2018 3

Figure 2. PDAC Organoids Exhibit Distinct Wnt Niche Requirements

4 Cell Stem Cell 22, 1–14, March 1, 2018

Cell Stem Cell 22, 1–14, March 1, 2018 5

Figure 3. CAFs Support W– PDAC Growth via Wnt Ligand Secretion

6 Cell Stem Cell 22, 1–14, March 1, 2018

Figure 4. Expression of Epithelial Wnts Defines the Wnt-Producing PDAC Subtype

Cell Stem Cell 22, 1–14, March 1, 2018 7

8 Cell Stem Cell 22, 1–14, March 1, 2018

Figure 6. GATA6 Expression Regulated Wnt Dependency of PDAC Organoids

Cell Stem Cell 22, 1–14, March 1, 2018 9

Figure 7. CRISPR-Cas9-Engineered Human

driver gene mutations and the alteration of

10 Cell Stem Cell 22, 1–14, March 1, 2018

Cell Stem Cell 22, 1–14, March 1, 2018 11

used to introduce KRAS mutations (KRAS overexpression by Lee

12 Cell Stem Cell 22, 1–14, March 1, 2018

Cell Stem Cell 22, 1–14, March 1, 2018 13

14 Cell Stem Cell 22, 1–14, March 1, 2018

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018 e1

e2 Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018

CONTACT FOR REAGENT AND RESOURCE SHARING

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Establishment of human pancreas organoids

Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018 e3

Co-culture of PDAC Organoids with CAFs

Gene Engineering of Organoids

Immunohistochemistry and in situ Hybridization

e4 Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018

Whole-Exome Sequence Analysis

Real-Time Quantitative PCR

Western blot Analysis

Gene-Expression Microarray Analysis

Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018 e5

Data Analysis for Publicly Available PDAC Specimens

QUANTIFICATION AND STATISTICAL ANALYSIS

DATA AND SOFTWARE AVAILABILITY

e6 Cell Stem Cell 22, 1–14.e1–e6, March 1, 2018

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