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• W2783122422 abstract "•39 patient-derived pancreas adenocarcinomas (PDACs) form a tumor organoid library•PDAC segregates into 3 subtypes with distinct dependency on Wnt niche signals•Cancer-associated fibroblasts provide a Wnt niche for PDAC•CRISPR-Cas9 gene engineering recapitulates multistep carcinogenesis of human pancreas Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression. Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease that has an extremely poor prognosis, with a median survival of <1 year and a 5-year overall survival rate of <9% (National Cancer InstituteNational Cancer Institute. Cancer Stat Facts: Pancreas Cancer. https://seer.cancer.gov/statfacts/html/pancreas.html.Google Scholar). Whereas PDACs are generally chemo-resistant, a fraction of patients benefit from current therapeutics, underscoring the importance of understanding inter-patient tumor heterogeneity in depth and stratifying PDACs to predict clinical behaviors. Recent gene-expression-based classification identified 2 major prognosis-predicting molecular subtypes in PDAC, namely the classical and quasi-mesenchymal (QM) subtypes (Bailey et al., 2016Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. Miller D.K. Christ A.N. Bruxner T.J. Quinn M.C. et al.Australian Pancreatic Cancer Genome InitiativeGenomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (2000) Google Scholar, Collisson et al., 2011Collisson E.A. Sadanandam A. Olson P. Gibb W.J. Truitt M. Gu S. Cooc J. Weinkle J. Kim G.E. Jakkula L. et al.Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy.Nat. Med. 2011; 17: 500-503Crossref PubMed Scopus (1069) Google Scholar, Moffitt et al., 2015Moffitt R.A. Marayati R. Flate E.L. Volmar K.E. Loeza S.G. Hoadley K.A. Rashid N.U. Williams L.A. Eaton S.C. Chung A.H. et al.Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma.Nat. Genet. 2015; 47: 1168-1178Crossref PubMed Scopus (1016) Google Scholar, Noll et al., 2016Noll E.M. Eisen C. Stenzinger A. Espinet E. Muckenhuber A. Klein C. Vogel V. Klaus B. Nadler W. Rösli C. et al.CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.Nat. Med. 2016; 22: 278-287Crossref PubMed Scopus (147) Google Scholar). The classical subtype was characterized by differentiated duct cell marker expression and favorable prognosis, whereas the QM subtype was characterized by aggressive clinical behavior and by gene silencing of definitive endoderm specification genes, including GATA6, FOXA2, and HNF4A. Similar QM-like subtypes regulated by GATA6 expression were also reported as squamous (Bailey et al., 2016Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. Miller D.K. Christ A.N. Bruxner T.J. Quinn M.C. et al.Australian Pancreatic Cancer Genome InitiativeGenomic analyses identify molecular subtypes of pancreatic cancer.Nature. 2016; 531: 47-52Crossref PubMed Scopus (2000) Google Scholar) and basal-like (Moffitt et al., 2015Moffitt R.A. Marayati R. Flate E.L. Volmar K.E. Loeza S.G. Hoadley K.A. Rashid N.U. Williams L.A. Eaton S.C. Chung A.H. et al.Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma.Nat. Genet. 2015; 47: 1168-1178Crossref PubMed Scopus (1016) Google Scholar) subtypes. Despite the robust identification of gene expression subtypes, whether these subtypes reflect genetically distinct cell-of-origin or tumor-progression statuses has remained elusive, owing to a paucity of functional assay systems for human PDAC. Functional analyses of PDAC have mainly relied on genetically engineered mice, cell lines, and patient-derived tumor xenograft models. The genetic tractability of mouse models and cell lines has contributed to the understanding of pancreas tumorigenesis, yet its relevance to the clinical traits of human PDAC, including histological and gene expression subtypes, remains unknown (Hwang et al., 2016Hwang C.I. Boj S.F. Clevers H. Tuveson D.A. Preclinical models of pancreatic ductal adenocarcinoma.J. Pathol. 2016; 238: 197-204Crossref PubMed Scopus (76) Google Scholar, Hruban et al., 2006Hruban R.H. Adsay N.V. Albores-Saavedra J. Anver M.R. Biankin A.V. Boivin G.P. Furth E.E. Furukawa T. Klein A. Klimstra D.S. et al.Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations.Cancer Res. 2006; 66: 95-106Crossref PubMed Scopus (317) Google Scholar). Xenograft models can be efficiently generated from clinical samples with preserved histological and molecular subtypes, whereas the labor-intensive and genetically intractable natures of such models have limited large-scale analyses and prospective genetic approaches for PDAC. The organoid culture system has recently emerged as a technology that can propagate epithelial tissues as 3D structures using artificial stem cell niche environments (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (4154) Google Scholar). A defined niche factor combination of R-spondin, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF10), and Noggin (a BMP4 inhibitor) promoted the expansion of normal mouse pancreatic duct cells (Huch et al., 2013Huch M. Bonfanti P. Boj S.F. Sato T. Loomans C.J. van de Wetering M. Sojoodi M. Li V.S. Schuijers J. Gracanin A. et al.Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.EMBO J. 2013; 32: 2708-2721Crossref PubMed Scopus (464) Google Scholar) and was later applied to patient-derived PDAC organoids (Boj et al., 2015Boj S.F. Hwang C.I. Baker L.A. Chio I.I. Engle D.D. Corbo V. Jager M. Ponz-Sarvise M. Tiriac H. Spector M.S. et al.Organoid models of human and mouse ductal pancreatic cancer.Cell. 2015; 160: 324-338Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar). To date, a dozen lines of PDAC organoids have been generated in which the preservation of histological traits and genetic-mutation profiles of the parental tumors were confirmed. Despite these advances, large-scale transcriptome analysis of human PDAC organoids has not been conducted, posing a bottleneck in connecting biological behavior with gene expression subtypes (Boj et al., 2015Boj S.F. Hwang C.I. Baker L.A. Chio I.I. Engle D.D. Corbo V. Jager M. Ponz-Sarvise M. Tiriac H. Spector M.S. et al.Organoid models of human and mouse ductal pancreatic cancer.Cell. 2015; 160: 324-338Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar, Huang et al., 2015Huang L. Holtzinger A. Jagan I. BeGora M. Lohse I. Ngai N. Nostro C. Wang R. Muthuswamy L.B. Crawford H.C. et al.Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.Nat. Med. 2015; 21: 1364-1371Crossref PubMed Scopus (456) Google Scholar). In this study, by refining organoid culture conditions, we established 39 lines of PDAC organoids and performed comprehensive molecular characterization, which illuminated different modes of Wnt/R-spondin niche dependency in association with gene expression subtypes. Furthermore, CRISPR-Cas9-mediated engineering of pancreas organoids demonstrated the stepwise tumorigenesis of PDAC with progressive acquisition of niche independency. Using surgical, fine-needle aspiration (FNA) and ascites specimens, we established organoids from pancreatic tumors as well as normal organoids when adjacent normal pancreatic tissues were available (Figure 1A). Using previously published culture conditions (Huch et al., 2013Huch M. Bonfanti P. Boj S.F. Sato T. Loomans C.J. van de Wetering M. Sojoodi M. Li V.S. Schuijers J. Gracanin A. et al.Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.EMBO J. 2013; 32: 2708-2721Crossref PubMed Scopus (464) Google Scholar), normal pancreas duct organoids ceased proliferation within 2 or 3 months (Boj et al., 2015Boj S.F. Hwang C.I. Baker L.A. Chio I.I. Engle D.D. Corbo V. Jager M. Ponz-Sarvise M. Tiriac H. Spector M.S. et al.Organoid models of human and mouse ductal pancreatic cancer.Cell. 2015; 160: 324-338Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar). We found that the replacement of serum-stabilized Wnt3A-conditioned medium with serum-free Afamin-stabilized Wnt3A (Mihara et al., 2016Mihara E. Hirai H. Yamamoto H. Tamura-Kawakami K. Matano M. Kikuchi A. Sato T. Takagi J. Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.eLife. 2016; 5: e11621Crossref PubMed Scopus (109) Google Scholar) enabled stable culture for over 8 months. Notably, the addition of serum triggered senescence, suggesting that serum in standard Wnt3A-conditioned medium is detrimental for the long-term maintenance of human pancreas organoids (Figure S1A). Using this culture protocol, we established a pancreatic tumor organoid library (PTOL) consisting of 49 organoid lines generated from patient-derived pancreatic tumors (Table S1). In our initial experiments, we often observed the outgrowth of normal pancreas organoids from PDAC specimens. The selective growth of “contaminating” normal organoids has been previously reported during the establishment of PDAC and prostate cancer organoids (Boj et al., 2015Boj S.F. Hwang C.I. Baker L.A. Chio I.I. Engle D.D. Corbo V. Jager M. Ponz-Sarvise M. Tiriac H. Spector M.S. et al.Organoid models of human and mouse ductal pancreatic cancer.Cell. 2015; 160: 324-338Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar, Gao et al., 2014Gao D. Vela I. Sboner A. Iaquinta P.J. Karthaus W.R. Gopalan A. Dowling C. Wanjala J.N. Undvall E.A. Arora V.K. et al.Organoid cultures derived from patients with advanced prostate cancer.Cell. 2014; 159: 176-187Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar). Considering the high prevalence of KRAS mutations that occur among PDACs, the organoids were first placed in an EGF-depleted condition to enrich the KRAS mutant organoids (Figure 1B). In contrast to the standard Wnt-conditioned medium that could activate EGFR and other cognate receptors by serum-derived growth factors (Wang et al., 2013Wang M. Maier P. Wenz F. Giordano F.A. Herskind C. Mitogenic signalling in the absence of epidermal growth factor receptor activation in a human glioblastoma cell line.J. Neurooncol. 2013; 115: 323-331Crossref PubMed Scopus (8) Google Scholar), EGF removal from the serum-free medium efficiently selected KRAS mutant organoids. The efficient enrichment of PDAC organoids can be readily visualized when spherical PDAC organoids grew along with normal cystic organoids; after EGF-based selection, KRAS mutant spherical PDAC organoids dominated whereas cystic organoids with wild-type KRAS disappeared (Figures 1C, S1B, and S1C). KRAS mutant spherical organoids, but not normal cystic organoids, displayed phospho-ERK expression in the absence of EGF, confirming the ligand-independent activation of Ras signaling in PDAC organoids (Figure S1D). Organoids that were susceptible to EGF removal were alternatively treated with Nutlin3 (an MDM2 inhibitor) or with Noggin removal/BMP4 to select potentially existing TP53 or SMAD4 mutant organoids, respectively (Figure 1B). Together with the aforementioned EGF-based selection, the niche-based selection was used to diagnose 39 out of 49 organoids in the PTOL as PDAC organoids. The remaining 10 organoids exhibited strict niche dependencies indistinguishable from those of normal pancreas organoids and were referred to as normal-like (NL) organoids. To determine the accuracy of niche-factor-based diagnosis, we analyzed the genetic status of the established organoids using whole-exome sequencing and comparative genomic-hybridization microarray analyses. Consistent with previous large-scale deep-sequencing analyses (Waddell et al., 2015Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. Johns A.L. Miller D. Nones K. Quek K. et al.Australian Pancreatic Cancer Genome InitiativeWhole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1701) Google Scholar), PDAC organoids harbored common driver-gene alterations at the expected frequencies: KRAS (36/38); CDKN2A (31/38); TP53 (29/38); and SMAD4 (14/38; Table S2). GNAS hotspot mutations (GNASR201H) were detected in two organoid lines (2/49), one of which was derived from intraductal papillary mucinous neoplasm (IPMN). Of note, no authenticated IPMN cell line with a GNAS mutation has been derived to date (Furukawa et al., 2011Furukawa T. Kuboki Y. Tanji E. Yoshida S. Hatori T. Yamamoto M. Shibata N. Shimizu K. Kamatani N. Shiratori K. Whole-exome sequencing uncovers frequent GNAS mutations in intraductal papillary mucinous neoplasms of the pancreas.Sci. Rep. 2011; 1: 161Crossref PubMed Scopus (345) Google Scholar). We did not detect recurrent driver-gene mutations in normal-like organoids, corroborating the accurate selection of PDAC organoids (Figure 1D). Whereas detailed copy number analyses of PDACs have been hampered by the low tumor content of clinical PDAC samples (Shain et al., 2012Shain A.H. Giacomini C.P. Matsukuma K. Karikari C.A. Bashyam M.D. Hidalgo M. Maitra A. Pollack J.R. Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer.Proc. Natl. Acad. Sci. USA. 2012; 109: E252-E259Crossref PubMed Scopus (177) Google Scholar, Witkiewicz et al., 2015Witkiewicz A.K. McMillan E.A. Balaji U. Baek G. Lin W.C. Mansour J. Mollaee M. Wagner K.U. Koduru P. Yopp A. et al.Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets.Nat. Commun. 2015; 6: 6744Crossref PubMed Scopus (726) Google Scholar), the purely epithelial composition of PDAC organoids enabled accurate assessment of copy number alterations. NL organoids invariably showed euploidy, reinforcing their non-cancer origins, whereas most PDAC organoids acquired well-defined chromosomal alterations, namely losses of 6p, 9p, 17p, and 18q (Su et al., 1998Su G.H. Hilgers W. Shekher M.C. Tang D.J. Yeo C.J. Hruban R.H. Kern S.E. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene.Cancer Res. 1998; 58: 2339-2342PubMed Google Scholar). Of note, four PDAC lines exhibited euploidy or near euploidy, of which three harbored KRAS mutations, confirming their cancer origin (Figure 1D). The remaining near-euploidy line (PC8) was devoid of driver-gene mutations, including those in KRAS, and its tumorigenic mechanism was unclear. Upon xenografting into orthotopic pancreata of NOD.Cg-PrkdcscidIl2rgtm1Sug/Jic (NOG) mice, PDAC organoids (including the wild-type KRAS organoid) formed tumors resembling their parental PDACs, whereas NL organoids did not successfully engraft (Figure S1F). Taken together, these data indicate that niche-based selection efficiently propagated PDAC organoids with the accurate exclusion of potentially contaminating normal organoids. Once PDAC organoids were established, each organoid was subjected to successive niche-based treatments to determine minimally essential niche factors, which demonstrated that driver-gene alterations largely dictated the requirements for the corresponding niche factors. Specifically, sensitivity to EGF removal, Noggin removal/BMP4 treatment, A83-01 removal/transforming growth factor β1 (TGF-β1) treatment, and Nutlin3 treatment were associated with KRAS, SMAD4, TGFBR2, and TP53 mutations/in-del alterations, respectively (Figures S2A–S2H). In contrast to these mutation-driven adaptations, we noted that Wnt/R-spondin dependency was mostly unrelated to Wnt-signaling mutations in PDAC organoids (Figures 2A and 2B ). Akin to the niche dependency of normal pancreas organoids, 14 PDAC organoids required both Wnt3A and R-spondin for their growth. Interestingly, the remaining R-spondin-dependent PDAC organoids grew in the absence of Wnt3A. Because R-spondin is known to potentiate Wnt signaling through stabilization of Wnt receptors (Koo et al., 2012Koo B.K. Spit M. Jordens I. Low T.Y. Stange D.E. van de Wetering M. van Es J.H. Mohammed S. Heck A.J. Maurice M.M. Clevers H. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors.Nature. 2012; 488: 665-669Crossref PubMed Scopus (638) Google Scholar), this phenotype suggested that R-spondin-dependent PDAC organoids harness either exogenously supplied or endogenously produced Wnt ligands. Therefore, we divided R-spondin-dependent PDAC organoids into two subtypes, namely Wnt-non-secreting (W−) and Wnt-secreting (W+) PDAC organoids, based on their requirements for exogenous Wnt3A. To validate the Wnt-producing capacity of W+ PDAC organoids, we tested the effect of a porcupine inhibitor (Porcn-i; C59) (Proffitt et al., 2013Proffitt K.D. Madan B. Ke Z. Pendharkar V. Ding L. Lee M.A. Hannoush R.N. Virshup D.M. Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.Cancer Res. 2013; 73: 502-507Crossref PubMed Scopus (265) Google Scholar), which abrogates the production of biologically active Wnt ligands. Notably, Porcn-i treatment suppressed the growth of W+ PDAC organoids in parallel with the reduction of their Wnt target gene-expression levels, and these effects were reversed by the supplementation with exogenous Wnt3A (Figures 2C and 2D). Furthermore, W+ PDAC organoids exhibited higher expression of Wnt target genes than W− PDAC organoids in the absence of Wnt3A (Figure S3A). The potent niche function of Wnt ligands secreted from W+ PDAC organoids was further validated by their growth-promoting effects on co-cultured W− PDAC organoids (Figure S3B). These results collectively indicated that W+ PDAC organoids autonomously create their own Wnt niche. We next characterized six Wnt and R-spondin-independent (WRi) PDAC organoids. To determine whether WRi PDAC organoids require Wnt-signal activation for their growth, we tested the effect of ICG001, a small-molecule inhibitor that blocks downstream Wnt-β-catenin signaling (Emami et al., 2004Emami K.H. Nguyen C. Ma H. Kim D.H. Jeong K.W. Eguchi M. Moon R.T. Teo J.L. Kim H.Y. Moon S.H. et al.A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].Proc. Natl. Acad. Sci. USA. 2004; 101: 12682-12687Crossref PubMed Scopus (710) Google Scholar). Upon ICG001 treatment, both W− and W+ PDAC organoids irreversibly terminated their proliferation in line with their requirement for Wnt-signal activation. In contrast, the growth of WRi PDAC organoids was maintained, suggesting that Wnt-signal activation itself was not essential for maintaining these organoids (Figure S3C). Though WRi PDAC organoids also tolerated Porcn-i treatment corroborating their dispensability of Wnt-signal activation, some WRi PDAC organoids were responsive to the Porcn-i treatment, suggesting their partial dependency on self-producing Wnt ligands (Figure S3D). In sum, our results revealed 3 functional subtypes of PDAC organoids that displayed unique requirements for Wnt and R-spondin niche environments (Figure 2E). W− PDAC cells critically depend on exogenous Wnt ligands for their survival and growth, yet the source of the Wnt ligands remains obscure. Because PDAC is characterized by abundant stromal cell infiltration, we inferred that these stromal cells could support the growth of PDACs through Wnt production. To determine the functional role of stromal Wnt ligands, we established patient-derived cancer-associated fibroblasts (CAFs) (Figure 1A). Immunostaining of fibroblast activation protein alpha (FAP) and α smooth muscle actin (αSMA), characteristic markers for CAFs in PDAC (Öhlund et al., 2017Öhlund D. Handly-Santana A. Biffi G. Elyada E. Almeida A.S. Ponz-Sarvise M. Corbo V. Oni T.E. Hearn S.A. Lee E.J. et al.Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.J. Exp. Med. 2017; 214: 579-596Crossref PubMed Scopus (1094) Google Scholar), confirmed that the established CAFs were of stromal origin (Figure S4A). In contrast to the quiescence induction in CAFs embedded in Matrigel (Öhlund et al., 2017Öhlund D. Handly-Santana A. Biffi G. Elyada E. Almeida A.S. Ponz-Sarvise M. Corbo V. Oni T.E. Hearn S.A. Lee E.J. et al.Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer.J. Exp. Med. 2017; 214: 579-596Crossref PubMed Scopus (1094) Google Scholar), CAFs in a collagen type I Matrigel mixture showed proliferation potency. To investigate whether these CAFs can functionally support the growth of W− PDAC organoids, we generated single stroma-attached organoids by aggregating dissociated PDAC cells and CAFs (Figures 3A and 3B ). Interestingly, this physical stroma attachment enabled W− PDAC organoids to grow without exogenous Wnt3A, and as expected, Porcn-i treatment abrogated this growth-promoting effect (Figures 3C and 3D). Consistent with the short-range Wnt gradient in intestinal organoids (Farin et al., 2016Farin H.F. Jordens I. Mosa M.H. Basak O. Korving J. Tauriello D.V. de Punder K. Angers S. Peters P.J. Maurice M.M. Clevers H. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche.Nature. 2016; 530: 340-343Crossref PubMed Scopus (327) Google Scholar), this growth-promoting effect on W− PDAC organoids was not observed in conditioned medium from CAFs or when CAFs and W− PDAC organoids were co-cultured without physical attachment (Figure S4B). These results demonstrated that the juxtacrine interaction with PDAC cells was critical for CAFs to support the growth of W− PDACs. To further validate the pro-tumorigenic effects of CAFs on PDAC organoids in vivo, PDAC organoids were subcutaneously transplanted, either alone or with CAFs. In the absence of CAFs, W+ and WRi PDAC organoids efficiently engrafted, whereas two out of the three examined W− PDAC organoid lines were poorly tumorigenic, suggesting the requirement for Wnt niche during tumor formation. Interestingly, when interfaced with CAFs prior to transplantation, these organoids successfully formed subcutaneous tumors (Figures 3E and 3F). We observed eventual replacement of transplanted CAFs with host-derived fibroblasts (Figure S4C), suggesting that the effect of co-transplanted CAFs was limited to the initial phase of xenotransplantation. Indeed, co-transplantation with CAFs increased the engraftment rate of W− PDAC organoids but did not enhance the tumorigenic growth of xenograft-competent organoids (Figure 3F). To investigate whether the pro-tumorigenic effect of CAFs was mediated by their stromal Wnt production, we next treated xenografts with a Porcn-i (Figure S4D). Whereas the therapeutic effect of Porcn-i has only been observed in RNF43 mutant PDAC cell lines in previous studies (Jiang et al., 2013Jiang X. Hao H.X. Growney J.D. Woolfenden S. Bottiglio C. Ng N. Lu B. Hsieh M.H. Bagdasarian L. Meyer R. et al.Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma.Proc. Natl. Acad. Sci. USA. 2013; 110: 12649-12654Crossref PubMed Scopus (287) Google Scholar), Porcn-i treatment significantly reduced the growth of xenografts from two independent RNF43-wild-type W− PDAC organoids (Figure S4E). Conversely, Porcn-i treatment did not affect the growth of WRi PDAC organoids in vivo. These results demonstrated that the CAF-dependent growth of PDACs was driven by stromal Wnt niche environments and that Wnt-targeting therapeutics could be potent against wild-type W− PDACs, regardless of the RNF43 mutation status. In contrast to W− PDAC organoids, W+ PDAC organoids harnessed their self-produced Wnt ligands. To determine which Wnt ligands were expressed in PDAC organoids, their expression levels were assessed by microarray analyses. An unbiased criterion was set to select Wnt ligands that were expressed at significant levels in at least one W+ PDAC organoid line (>5 SD expression from the mean expression levels of NL organoids; Figure 4A), which nominated 6 Wnt ligands from 19 Wnt-ligand family members. To determine whether any of these Wnt ligands could serve as an epithelial Wnt niche, we overexpressed each Wnt ligand in NL organoids and examined their potential for driving niche function. In this functional assay, 4 Wnt ligands (WNT3, WNT7A, WNT7B, and WNT10A) were found to substitute for Wnt3A and, thus, were designated as “epithelial” Wnt ligands (Figures 4A and S5A). Importantly, the expression levels of these epithelial Wnt genes were significantly higher in WRi and W+ PDAC organoids than in W− PDAC organoids, indicating the association between epithelial Wnt-ligand expression and Wnt niche independency (Figure 4B). To determine whether epithelial Wnt ligands are expressed in clinical specimens, we analyzed their expression levels in whole PDAC tissues using a publicly available transcriptome dataset. Hierarchical clustering of Wnt-ligand gene-expression levels aggregated the epithelial Wnt-ligand genes in a single cluster (Figure S5B). WNT2, WNT2B, WNT4, and WNT5A were expressed in PDAC tissues but were rarely detected in organoids, suggesting their stromal origins. Real-time qPCR analyses of Wnt ligand gene-expression levels in organoids and CAFs confirmed these tissue-specific expression patterns (Figure 4C). Of note, the functional assay revealed that only WNT2 and WNT2B served as potent niche factors among stromal Wnt ligands (Figure 4A). To determine the differential expression of epithelial Wnts in patients, we next performed in situ hybridization of the clinical specimens for the epithelial Wnt genes. WNT7B and WNT10A were markedly expressed in the epithelial component of W+ PDACs, whereas no or subtle expression of these genes was observed in W− PDACs and adjacent normal pancreas tissues (Figure 4D). Stromal expression of WNT2B was detected in close proximity to PDAC tissue (Figure 4D), consistent with the short-range activity of Wnt ligands. These results suggested that the expression of epithelial Wnts could serve as a surrogate marker to define Wnt-producing PDACs. In addition, the high expression of epithelial Wnts in clinical PDACs was associated with significantly poor survival and metastatic progression (Figures 4E and S5C). These results demonstrated that W+ PDACs cell autonomously activated their own Wnt signaling by expressing epithelial Wnts, which also predicts aggressive clinical behaviors. To explore the mechanisms underlying each functional PDAC subtype, we performed transcriptome analyses of the PTOL. An unbiased projection of global gene expression with t-distributed stochastic neighbor embedding (tSNE) analysis illustrated linearly connected gene expression clusters corresponding to the Wnt niche subtypes (Figure 5A). Notably, the linear trajectory was directed from normal organoids toward W−, W+, and WRi subtypes, suggesting serial transition of gene expression signature in line with acquisition of Wnt niche independency. To gain insights into the transcriptional programs regulating this process," @default.
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• W2783122422 modified "2023-10-16" @default.
• W2783122422 title "Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression" @default.
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