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Esophageal 3D Culture Systems as Modeling Tools in Esophageal Epithelial Pathobiology and Personalized Medicine

  • Kelly A. Whelan
    Affiliations
    Pathology and Laboratory Medicine, Fels Institute for Cancer Research and Molecular Biology, Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania
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  • Amanda B. Muir
    Correspondence
    Correspondence Address correspondence to: Amanda B. Muir, MD, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Abramson Research Center 902E, Philadelphia, Pennsylvania 19103. fax: (267) 426–7814.
    Affiliations
    Division of Pediatric Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

    Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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  • Hiroshi Nakagawa
    Affiliations
    Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

    Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania
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Open AccessPublished:February 03, 2018DOI:https://doi.org/10.1016/j.jcmgh.2018.01.011
      The stratified squamous epithelium of the esophagus shows a proliferative basal layer of keratinocytes that undergo terminal differentiation in overlying suprabasal layers. Esophageal pathologies, including eosinophilic esophagitis, gastroesophageal reflux disease, Barrett's esophagus, squamous cell carcinoma, and adenocarcinoma, cause perturbations in the esophageal epithelial proliferation-differentiation gradient. Three-dimensional (3D) culture platforms mimicking in vivo esophageal epithelial tissue architecture ex vivo have emerged as powerful experimental tools for the investigation of esophageal biology in the context of homeostasis and pathology. Herein, we describe types of 3D culture that are used to model the esophagus, including organotypic, organoid, and spheroid culture systems. We discuss the development and optimization of various esophageal 3D culture models; highlight the applications, strengths, and limitations of each method; and summarize how these models have been used to evaluate the esophagus under homeostatic conditions as well as under the duress of inflammation and precancerous/cancerous conditions. Finally, we present future perspectives regarding the use of esophageal 3D models in basic science research as well as translational studies with the potential for personalized medicine.

      Keywords

      Abbreviations used in this paper:

      BE (Barrett’s esophagus), COX (cyclooxygenase), CSC (cancer stem cell), EADC (esophageal adenocarcinoma), EGF (epidermal growth factor), EGFR (epidermal growth factor receptor), EMT (epithelial-mesenchymal transition), EoE (eosinophilic esophagitis), ESCC (esophageal squamous cell carcinoma), FEF3 (primary human fetal esophageal fibroblast), GERD (gastroesophageal reflux disease), OTC (organotypic 3-dimensional culture), STAT3 (signal transducer and activator of transcription-3), 3D (3-dimensional)
      Pathology of the esophageal epithelium leads to perturbations of the normal proliferation-differentation gradient. Three-dimensional culture of esophageal epithelial tissue provides a unique platform to study diseases of the esophageal epithelium. Herein, we examine various types of three-dimensional esophageal culture and discuss the future of these applications for translational research and personalized medicine.
      The esophageal mucosa comprises stratified squamous epithelium in which esophageal epithelial cells (keratinocytes) show a proliferation-differentiation gradient and provide a barrier against the chemical and biological milieu of luminal contents. Disruption of this differentiation gradient or barrier function is linked to multiple human pathologies. Eosinophilic esophagitis (EoE), gastroesophageal reflux disease (GERD), and intestinal metaplasia (Barrett’s esophagus [BE]) are benign esophageal conditions featuring aberrant epithelial cell proliferation and differentiation. In addition, adenocarcinoma (EADC) and squamous cell carcinoma (ESCC) represent the 2 primary types of malignancies arising within the esophageal epithelium and progressing via dissemination and invasion toward the underlying subepithelial stromal compartment. Three-dimensional (3D) cell culture model systems have been used as near-physiological experimental platforms to study esophageal biology under homeostatic and pathologic conditions. These 3D platforms include organotypic 3D culture (OTC) and the more recently developed 3D organoid system. In this review, we highlight the historical background of these technologies while also discussing differences among 3D culture model systems as well as applications and current limitations. Finally, we address potential future directions for these 3D model systems as they relate to esophageal epithelial biology, tumor biology, and translation in personalized medicine.

      Esophageal Stratified Squamous Epithelium: Structure and Physiological Function

      As a hollow muscular organ, the esophagus serves the passage of ingested food and liquid from the oral cavity to the stomach. Its luminal surface is lined by the mucosa, comprising stratified squamous epithelium and the underlying lamina propria and muscularis mucosa. The esophageal epithelium consists of proliferative basal keratinocytes and suprabasal keratinocytes, the latter undergoing postmitotic terminal differentiation, passive migration toward the luminal surface, and, ultimately, desquamation into the lumen. Through this dynamic process, a proliferation-differentiation gradient is generated while epithelial renewal occurs over a period of 2 weeks.
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      proposed that the esophageal epithelium is maintained by a single population of basal keratinocytes that give rise stochastically to proliferating and differentiating daughters with equal probability; however, functional cell heterogeneity has been postulated among basal esophageal keratinocytes. A minor subset of basal keratinocytes divide slowly or rarely and may have properties of quiescent stem cells.
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      Species differences exist between rodents and human beings with regard to anatomic esophageal structure. Foremost, the rodent esophagus lacks esophageal glands and papillae, both of which are present in the human esophagus. In addition, the rodent esophagus shows more explicit keratinization in the superficial cell layers, also known as stratum corneum of the squamous epithelium, as compared with its human counterpart. The rodent stomach consists of 2 compartments: the forestomach and distal stomach, featuring squamous epithelium and columnar epithelium, respectively, and some regard the forestomach as the counterpart of the human lower esophagus. This is important to note because Barrett’s esophagus (ie, intestinal metaplasia of the esophagus) is a human mucosal lesion involving the esophagogastric junction and has been modeled at the squamocolumnar junction within the murine stomach.
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      Organ Culture and Multiple 3D Culture Models: What Are the Differences?

      Throughout a long history of cell culture, various forms of 3D culture methodologies have been developed along with unique scaffolds, matrices, and cell culture media. In the esophagus, 3D culture systems have provided unique platforms to study multiple biological processes, including epithelial cell proliferation, differentiation, motility, stress response, and both homotypic and heterotypic cell–cell communications. Cellular interactions involve a variety of cell types (eg, fibroblasts, endothelial cells, and inflammatory cells) in the esophageal tissue microenvironment under homeostatic and pathologic conditions (eg, inflammatory milieu), and are mediated via cell surface molecules (eg, integrins and receptors such as Notch) as well as extracellular matrix proteins (eg, matrix metalloproteinases), as discussed in this review. The ability to experimentally manipulate 3D cultures has greatly enhanced our understanding of the molecular mechanisms and signaling pathways underlying esophageal physiology and pathophysiology.
      Organ (explant) culture was a major tool for in vitro live esophageal tissue analyses before primary esophageal epithelial cell culture
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      became available in late 1970s and early 1980s, respectively. The foremost advantage of organ culture is the maintenance of natural tissue architecture in situ. Organ culture may be used to study cross-talk between epithelial cells and nonepithelial cells in a live tissue-like context. Given the potential importance of a variety of cell types (ie, epithelial cells, fibroblasts, nerve cells, immune cells, and endothelial cells) present in the tissue microenvironment, organ culture indeed may be more physiologically relevant than other 3D culture systems because co-culturing multiple cell types remains difficult.
      Early organ culture studies have shown that esophageal explants from animals and human beings remained viable for 3–14 days ex vivo
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      Unlike organ culture, most 3D culture systems involve dissociation of originating tissue samples or preparation of cells grown in monolayer cultures before 3D reconstitution in vitro. Such 3D culture systems can be largely classified into 2 categories. One category is represented by OTC in which esophageal epithelial cells are grown over a collagen matrix containing fibroblasts that mimics the subepithelial lamina propria. The second category features spherical 3D structures of esophageal epithelial cells typically generated under submerged conditions. This includes multicellular spheroid culture, sphere formation assays, and 3D organoids.
      OTC, also known as raft culture, is a form of tissue engineering with recapitulation of esophageal physiology and pathology (Figure 1). OTC was first established in the field of skin biology in the 1980s when the dermal equivalent comprising contracted type I collagen and fibroblasts
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      Figure thumbnail gr1
      Figure 1Recapitulation of stratified squamous epithelium in OTC and 3D esophageal organoids. In both 3D culture systems, esophageal epithelial cells (keratinocytes) and fibroblasts are dissociated from tissues. In OTC, primary culture or cell lines are prepared in monolayer 2-dimensional culture before 3D reconstruction in the Transwell insert with a bottom filter that permits medium in the bottom chamber feeds the entire system. The trapezoid-shaped collagen matrix containing fibroblasts support keratinocyte growth. Note that keratinocytes are incorporated sequentially, but not simultaneously, into the OTC system because fibroblasts are first grown in the collagen matrix. The air–liquid interface triggers epithelial stratification. In 3D esophageal organoids, keratinocytes are mixed into Matrigel at a low density and allowed to grow as single-cell–derived spherical 3D structures with a proliferation-differentiation gradient. After enzymatic digestion of Matrigel, the 3D structures are recovered and subjected to analyses or can be continued in the subsequent passages. Note the cornification seen in the outmost superficial cell layer in OTC while cornification occurs in the inmost core in 3D organoids.
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      Of note, commonly used HET1A, an oncogenic simian-virus-40 T-antigen-immortalized human esophageal epithelial cell line, did not recapitulate normal squamous esophageal epithelium in OTC.
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      • Thomas G.J.
      Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.
      we have interrogated gene and molecular functions as well as cell–cell and cell–extracellular matrix interactions in broad contexts related to the esophageal physiological and pathologic microenvironment,
      • Ohashi S.
      • Natsuizaka M.
      • Yashiro-Ohtani Y.
      • Kalman R.A.
      • Nakagawa M.
      • Wu L.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Katz J.P.
      • Pear W.S.
      • Seykora J.T.
      • Nakagawa H.
      NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.
      • Andl C.D.
      • Mizushima T.
      • Nakagawa H.
      • Oyama K.
      • Harada H.
      • Chruma K.
      • Herlyn M.
      • Rustgi A.K.
      Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo.
      • Harada H.
      • Nakagawa H.
      • Oyama K.
      • Takaoka M.
      • Andl C.D.
      • Jacobmeier B.
      • von Werder A.
      • Enders G.H.
      • Opitz O.G.
      • Rustgi A.K.
      Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation.
      • Grugan K.D.
      • Miller C.G.
      • Yao Y.
      • Michaylira C.Z.
      • Ohashi S.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Han M.
      • Nakagawa H.
      • Rustgi A.K.
      Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion.
      • Kalabis J.
      • Wong G.S.
      • Vega M.E.
      • Natsuizaka M.
      • Robertson E.S.
      • Herlyn M.
      • Nakagawa H.
      • Rustgi A.K.
      Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
      • Okawa T.
      • Michaylira C.Z.
      • Kalabis J.
      • Stairs D.B.
      • Nakagawa H.
      • Andl C.D.
      • Johnstone C.N.
      • Klein-Szanto A.J.
      • El-Deiry W.S.
      • Cukierman E.
      • Herlyn M.
      • Rustgi A.K.
      The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Takaoka M.
      • Andl C.D.
      • Kim S.H.
      • Klein-Szanto A.
      • Diehl J.A.
      • Herlyn M.
      • El-Deiry W.
      • Rustgi A.K.
      AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.
      • Stairs D.B.
      • Nakagawa H.
      • Klein-Szanto A.
      • Mitchell S.D.
      • Silberg D.G.
      • Tobias J.W.
      • Lynch J.P.
      • Rustgi A.K.
      Cdx1 and c-Myc foster the initiation of transdifferentiation of the normal esophageal squamous epithelium toward Barrett's esophagus.
      • Michaylira C.Z.
      • Wong G.S.
      • Miller C.G.
      • Gutierrez C.M.
      • Nakagawa H.
      • Hammond R.
      • Klein-Szanto A.J.
      • Lee J.S.
      • Kim S.B.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.
      • Rustgi A.K.
      Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor-invasive signature in esophageal cancer.
      • Natsuizaka M.
      • Ohashi S.
      • Wong G.S.
      • Ahmadi A.
      • Kalman R.A.
      • Budo D.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Nakagawa H.
      Insulin-like growth factor-binding protein-3 promotes transforming growth factor-{beta}1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells.
      • Ohashi S.
      • Natsuizaka M.
      • Naganuma S.
      • Kagawa S.
      • Kimura S.
      • Itoh H.
      • Kalman R.A.
      • Nakagawa M.
      • Darling D.S.
      • Basu D.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Nakagawa H.
      A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors.
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      • Vega M.E.
      • Giroux V.
      • Natsuizaka M.
      • Liu M.
      • Klein-Szanto A.J.
      • Stairs D.B.
      • Nakagawa H.
      • Wang K.K.
      • Wang T.C.
      • Lynch J.P.
      • Rustgi A.K.
      Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4.
      as detailed later in this article.
      Table 1Published Esophageal Epithelial 3D Culture Studies
      3D culture systemEsophageal epithelial tissue/cell
      All of esophageal origin.
      Cell designationResearch focusReference
      OTCTissue/normal: mFACS-purified keratinocytesStem/progenitor cells
      • Cheng E.
      • Zhang X.
      • Wilson K.S.
      • Wang D.H.
      • Park J.Y.
      • Huo X.
      • Yu C.
      • Zhang Q.
      • Spechler S.J.
      • Souza R.F.
      JAK-STAT6 pathway inhibitors block eotaxin-3 secretion by epithelial cells and fibroblasts from esophageal eosinophilia patients: promising agents to improve inflammation and prevent fibrosis in EoE.
      Normal: hPrimary keratinocytesEpithelial reconstitution
      • Okawa T.
      • Michaylira C.Z.
      • Kalabis J.
      • Stairs D.B.
      • Nakagawa H.
      • Andl C.D.
      • Johnstone C.N.
      • Klein-Szanto A.J.
      • El-Deiry W.S.
      • Cukierman E.
      • Herlyn M.
      • Rustgi A.K.
      The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Takaoka M.
      • Andl C.D.
      • Kim S.H.
      • Klein-Szanto A.
      • Diehl J.A.
      • Herlyn M.
      • El-Deiry W.
      • Rustgi A.K.
      AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.
      Immortalized cell line: hHET-1AEpithelial reconstitution
      • Sato F.
      • Kubota Y.
      • Natsuizaka M.
      • Maehara O.
      • Hatanaka Y.
      • Marukawa K.
      • Terashita K.
      • Suda G.
      • Ohnishi S.
      • Shimizu Y.
      • Komatsu Y.
      • Ohashi S.
      • Kagawa S.
      • Kinugasa H.
      • Whelan K.A.
      • Nakagawa H.
      • Sakamoto N.
      EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition.
      Immortalized cell line: hEoE2-TEpithelial reconstitution
      • Lehman H.L.
      • Yang X.
      • Welsh P.A.
      • Stairs D.B.
      p120-catenin down-regulation and epidermal growth factor receptor overexpression results in a transformed epithelium that mimics esophageal squamous cell carcinoma.
      Immortalized cell line: h

      Nontransformed and transformed
      EPC1-hTERT EPC2-hTERT and derivativesNotch in squamous-cell differentiation and Notch
      • Ohashi S.
      • Natsuizaka M.
      • Yashiro-Ohtani Y.
      • Kalman R.A.
      • Nakagawa M.
      • Wu L.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Katz J.P.
      • Pear W.S.
      • Seykora J.T.
      • Nakagawa H.
      NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.
      AKT in squamous-cell differentiation
      • Michaylira C.Z.
      • Wong G.S.
      • Miller C.G.
      • Gutierrez C.M.
      • Nakagawa H.
      • Hammond R.
      • Klein-Szanto A.J.
      • Lee J.S.
      • Kim S.B.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.
      • Rustgi A.K.
      Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor-invasive signature in esophageal cancer.
      EGFR in basal cell hyperplasia
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Takaoka M.
      • Andl C.D.
      • Kim S.H.
      • Klein-Szanto A.
      • Diehl J.A.
      • Herlyn M.
      • El-Deiry W.
      • Rustgi A.K.
      AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.
      EGFR, cyclin D1, p53, and Notch in malignant transformation
      • Stairs D.B.
      • Nakagawa H.
      • Klein-Szanto A.
      • Mitchell S.D.
      • Silberg D.G.
      • Tobias J.W.
      • Lynch J.P.
      • Rustgi A.K.
      Cdx1 and c-Myc foster the initiation of transdifferentiation of the normal esophageal squamous epithelium toward Barrett's esophagus.
      • Croagh D.
      • Cheng S.
      • Tikoo A.
      • Nandurkar S.
      • Thomas R.J.
      • Kaur P.
      • Phillips W.A.
      Reconstitution of stratified murine and human oesophageal epithelia in an in vivo transplant culture system.
      EGFR and p53 in ESCC cell invasion
      • Stairs D.B.
      • Nakagawa H.
      • Klein-Szanto A.
      • Mitchell S.D.
      • Silberg D.G.
      • Tobias J.W.
      • Lynch J.P.
      • Rustgi A.K.
      Cdx1 and c-Myc foster the initiation of transdifferentiation of the normal esophageal squamous epithelium toward Barrett's esophagus.
      • Kidacki M.
      • Lehman H.L.
      • Green M.V.
      • Warrick J.I.
      • Stairs D.B.
      p120-Catenin downregulation and PIK3CA mutations cooperate to induce invasion through MMP1 in HNSCC.
      EGFR and p120 in ESCC invasion
      • Wong G.S.
      • Lee J.S.
      • Park Y.Y.
      • Klein-Szanto A.J.
      • Waldron T.J.
      • Cukierman E.
      • Herlyn M.
      • Gimotty P.
      • Nakagawa H.
      • Rustgi A.K.
      Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment.
      PI3K and p120 in ESCC invasion
      • Long A.
      • Giroux V.
      • Whelan K.A.
      • Hamilton K.E.
      • Tetreault M.P.
      • Tanaka K.
      • Lee J.S.
      • Klein-Szanto A.J.
      • Nakagawa H.
      • Rustgi A.K.
      WNT10A promotes an invasive and self-renewing phenotype in esophageal squamous cell carcinoma.
      Periostin and ESCC invasion
      • Ohashi S.
      • Natsuizaka M.
      • Naganuma S.
      • Kagawa S.
      • Kimura S.
      • Itoh H.
      • Kalman R.A.
      • Nakagawa M.
      • Darling D.S.
      • Basu D.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Nakagawa H.
      A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors.
      STAT1 and ESCC invasion
      • Whelan K.A.
      • Chandramouleeswaran P.M.
      • Tanaka K.
      • Natsuizaka M.
      • Guha M.
      • Srinivasan S.
      • Darling D.S.
      • Kita Y.
      • Natsugoe S.
      • Winkler J.D.
      • Klein-Szanto A.J.
      • Amaravadi R.K.
      • Avadhani N.G.
      • Rustgi A.K.
      • Nakagawa H.
      Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.
      Wnt in ESCC invasion
      • Croagh D.
      • Cheng S.
      • Tikoo A.
      • Nandurkar S.
      • Thomas R.J.
      • Kaur P.
      • Phillips W.A.
      Reconstitution of stratified murine and human oesophageal epithelia in an in vivo transplant culture system.
      • Grugan K.D.
      • Vega M.E.
      • Wong G.S.
      • Diehl J.A.
      • Bass A.J.
      • Wong K.K.
      • Nakagawa H.
      • Rustgi A.K.
      A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion.
      Notch in ESCC invasion
      • Vega M.E.
      • Giroux V.
      • Natsuizaka M.
      • Liu M.
      • Klein-Szanto A.J.
      • Stairs D.B.
      • Nakagawa H.
      • Wang K.K.
      • Wang T.C.
      • Lynch J.P.
      • Rustgi A.K.
      Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4.
      • Croagh D.
      • Cheng S.
      • Tikoo A.
      • Nandurkar S.
      • Thomas R.J.
      • Kaur P.
      • Phillips W.A.
      Reconstitution of stratified murine and human oesophageal epithelia in an in vivo transplant culture system.
      IGFBP3 in ESCC invasion
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      Activin A in ESCC invasion
      • Kong J.
      • Crissey M.A.
      • Stairs D.B.
      • Sepulveda A.R.
      • Lynch J.P.
      Cox2 and beta-catenin/T-cell factor signaling intestinalize human esophageal keratinocytes when cultured under organotypic conditions.
      CD44 and ESCC invasion
      • Kalabis J.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Michaylira C.Z.
      • Stairs D.B.
      • Figueiredo J.L.
      • Mahmood U.
      • Diehl J.A.
      • Herlyn M.
      • Rustgi A.K.
      A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.
      Autophagy and EMT in ESCC
      • Laczko D.
      • Wang F.
      • Johnson F.B.
      • Jhala N.
      • Rosztoczy A.
      • Ginsberg G.G.
      • Falk G.W.
      • Rustgi A.K.
      • Lynch J.P.
      Modeling esophagitis using human three-dimensional organotypic culture system.
      c-Met and p53 in ESCC invasion
      • Muir A.B.
      • Dods K.
      • Noah Y.
      • Toltzis S.
      • Chandramouleeswaran P.M.
      • Lee A.
      • Benitez A.
      • Bedenbaugh A.
      • Falk G.W.
      • Wells R.G.
      • Nakagawa H.
      • Wang M.L.
      Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition.
      Wnt and COX2 in columnar-cell differentiation
      • Muir A.B.
      • Lim D.M.
      • Benitez A.J.
      • Modayur Chandramouleeswaran P.
      • Lee A.J.
      • Ruchelli E.D.
      • Spergel J.M.
      • Wang M.L.
      Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro.
      Cdx1, c-Myc, and Notch in columnar-cell differentiation
      • Natsuizaka M.
      • Ohashi S.
      • Wong G.S.
      • Ahmadi A.
      • Kalman R.A.
      • Budo D.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Nakagawa H.
      Insulin-like growth factor-binding protein-3 promotes transforming growth factor-{beta}1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells.
      • Wang D.H.
      • Tiwari A.
      • Kim M.E.
      • Clemons N.J.
      • Regmi N.L.
      • Hodges W.A.
      • Berman D.M.
      • Montgomery E.A.
      • Watkins D.N.
      • Zhang X.
      • Zhang Q.
      • Jie C.
      • Spechler S.J.
      • Souza R.F.
      Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.
      Modeling esophagitis
      • Davis B.P.
      • Stucke E.M.
      • Khorki M.E.
      • Litosh V.A.
      • Rymer J.K.
      • Rochman M.
      • Travers J.
      • Kottyan L.C.
      • Rothenberg M.E.
      Eosinophilic esophagitis-linked calpain 14 is an IL-13-induced protease that mediates esophageal epithelial barrier impairment.
      EMT in EoE
      • D'Mello R.J.
      • Caldwell J.M.
      • Azouz N.P.
      • Wen T.
      • Sherrill J.D.
      • Hogan S.P.
      • Rothenberg M.E.
      LRRC31 is induced by IL-13 and regulates kallikrein expression and barrier function in the esophageal epithelium.
      • Lioni M.
      • Noma K.
      • Snyder A.
      • Klein-Szanto A.
      • Diehl J.A.
      • Rustgi A.K.
      • Herlyn M.
      • Smalley K.S.
      Bortezomib induces apoptosis in esophageal squamous cell carcinoma cells through activation of the p38 mitogen-activated protein kinase pathway.
      Epithelial barrier functions in EoE
      • Sherrill J.D.
      • Kc K.
      • Wu D.
      • Djukic Z.
      • Caldwell J.M.
      • Stucke E.M.
      • Kemme K.A.
      • Costello M.S.
      • Mingler M.K.
      • Blanchard C.
      • Collins M.H.
      • Abonia J.P.
      • Putnam P.E.
      • Dellon E.S.
      • Orlando R.C.
      • Hogan S.P.
      • Rothenberg M.E.
      Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis.
      • Kosoff R.E.
      • Gardiner K.L.
      • Merlo L.M.
      • Pavlov K.
      • Rustgi A.K.
      • Maley C.C.
      Development and characterization of an organotypic model of Barrett's esophagus.
      • Whelan K.A.
      • Merves J.F.
      • Giroux V.
      • Tanaka K.
      • Guo A.
      • Chandramouleeswaran P.M.
      • Benitez A.J.
      • Dods K.
      • Que J.
      • Masterson J.C.
      • Fernando S.D.
      • Godwin B.C.
      • Klein-Szanto A.J.
      • Chikwava K.
      • Ruchelli E.D.
      • Hamilton K.E.
      • Muir A.B.
      • Wang M.L.
      • Furuta G.T.
      • Falk G.W.
      • Spergel J.M.
      • Nakagawa H.
      Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis.
      ESCC cell line: hTE9, TE10, KYSE70ESCC invasion
      • Kalabis J.
      • Wong G.S.
      • Vega M.E.
      • Natsuizaka M.
      • Robertson E.S.
      • Herlyn M.
      • Nakagawa H.
      • Rustgi A.K.
      Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
      OE21ESCC invasion
      • Sato F.
      • Kubota Y.
      • Natsuizaka M.
      • Maehara O.
      • Hatanaka Y.
      • Marukawa K.
      • Terashita K.
      • Suda G.
      • Ohnishi S.
      • Shimizu Y.
      • Komatsu Y.
      • Ohashi S.
      • Kagawa S.
      • Kinugasa H.
      • Whelan K.A.
      • Nakagawa H.
      • Sakamoto N.
      EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition.
      TE12p38 MAPK in ESCC invasion
      • Okano J.
      • Gaslightwala I.
      • Birnbaum M.J.
      • Rustgi A.K.
      • Nakagawa H.
      Akt/protein kinase B isoforms are differentially regulated by epidermal growth factor stimulation.
      TE7, TE12Fibroblast HGF in ESCC invasion
      • Grugan K.D.
      • Miller C.G.
      • Yao Y.
      • Michaylira C.Z.
      • Ohashi S.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Han M.
      • Nakagawa H.
      • Rustgi A.K.
      Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion.
      Barrett’s esophagus cell line: hCP-A, CP-B, CP-C, CP-DModeling Barrett’s esophagus, all-trans retinoic acid in columnar differentiation
      • Yan Y.X.
      • Nakagawa H.
      • Lee M.H.
      • Rustgi A.K.
      Transforming growth factor-alpha enhances cyclin D1 transcription through the binding of early growth response protein to a cis-regulatory element in the cyclin D1 promoter.
      EADC cell line: hMFD-1, OE19, OE33, FLO-1Modeling invasive EADC,

      cancer-associated fibroblasts and periostin in EADC cell invasion
      • Underwood T.J.
      • Derouet M.F.
      • White M.J.
      • Noble F.
      • Moutasim K.A.
      • Smith E.
      • Drew P.A.
      • Thomas G.J.
      • Primrose J.N.
      • Blaydes J.P.
      A comparison of primary oesophageal squamous epithelial cells with HET-1A in organotypic culture.
      • Underwood T.J.
      • Hayden A.L.
      • Derouet M.
      • Garcia E.
      • Noble F.
      • White M.J.
      • Thirdborough S.
      • Mead A.
      • Clemons N.
      • Mellone M.
      • Uzoho C.
      • Primrose J.N.
      • Blaydes J.P.
      • Thomas G.J.
      Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.
      • Sato F.
      • Kubota Y.
      • Natsuizaka M.
      • Maehara O.
      • Hatanaka Y.
      • Marukawa K.
      • Terashita K.
      • Suda G.
      • Ohnishi S.
      • Shimizu Y.
      • Komatsu Y.
      • Ohashi S.
      • Kagawa S.
      • Kinugasa H.
      • Whelan K.A.
      • Nakagawa H.
      • Sakamoto N.
      EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition.
      OrganoidNormal, disease tissues: mFACS-purified keratinocytesStem/progenitor cells
      • DeWard A.D.
      • Cramer J.
      • Lagasse E.
      Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.
      • Jeong Y.
      • Rhee H.
      • Martin S.
      • Klass D.
      • Lin Y.
      • Nguyen le X.T.
      • Feng W.
      • Diehn M.
      Identification and genetic manipulation of human and mouse oesophageal stem cells.
      Bulk or primary keratinocytesBasal cell hyperplasia and autophagy
      • Tanaka K.
      • Whelan K.A.
      • Chandramouleeswaran P.M.
      • Kagawa S.
      • Rustgi S.L.
      • Noguchi C.
      • Guha M.
      • Srinivasan S.
      • Amanuma Y.
      • Ohashi S.
      • Muto M.
      • Klein-Szanto A.J.
      • Noguchi E.
      • Avadhani N.G.
      • Nakagawa H.
      ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds.
      Alcohol and autophagy
      • Nakagawa H.
      • Wang T.C.
      • Zukerberg L.
      • Odze R.
      • Togawa K.
      • May G.H.
      • Wilson J.
      • Rustgi A.K.
      The targeting of the cyclin D1 oncogene by an Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue, esophagus and forestomach.
      EMT and Notch in ESCC
      • Kalabis J.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Michaylira C.Z.
      • Stairs D.B.
      • Figueiredo J.L.
      • Mahmood U.
      • Diehl J.A.
      • Herlyn M.
      • Rustgi A.K.
      A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.
      Bulk keratinocytesCommon culture conditions for murine and human organoids
      • Kasagi Y.
      • Chandramouleeswaran P.M.
      • Whelan K.A.
      • Tanaka K.
      • Giroux V.
      • Sharma M.
      • Wang J.
      • Benitez A.J.
      • DeMarshall M.
      • Tobias J.W.
      • Hamilton K.E.
      • Falk G.W.
      • Spergel J.M.
      • Klein-Klein-Szanto A.J.
      • Rustgi A.K.
      • Muir A.B.
      • Nakagawa H.
      The esophageal organoid system reveals functional interplay between notch and cytokines in reactive epithelial changes.
      Normal, GERD, EoE biopsy specimens: h
      Immortalized cell line: hEPC2-hTERT and derivativesBasal cell hyperplasia and Notch in squamous-cell differentiation
      • Kasagi Y.
      • Chandramouleeswaran P.M.
      • Whelan K.A.
      • Tanaka K.
      • Giroux V.
      • Sharma M.
      • Wang J.
      • Benitez A.J.
      • DeMarshall M.
      • Tobias J.W.
      • Hamilton K.E.
      • Falk G.W.
      • Spergel J.M.
      • Klein-Klein-Szanto A.J.
      • Rustgi A.K.
      • Muir A.B.
      • Nakagawa H.
      The esophageal organoid system reveals functional interplay between notch and cytokines in reactive epithelial changes.
      Barret’s esophagus biopsy specimens: hBarrett’s esophagusHuman Barrett’s esophagus 3D organoids
      • Matano M.
      • Date S.
      • Shimokawa M.
      • Takano A.
      • Fujii M.
      • Ohta Y.
      • Watanabe T.
      • Kanai T.
      • Sato T.
      Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.
      SphereESCC cell line: hKYSE70, KYSE140, KYSE150, KYSE520, TE1ESCC CSC
      • Almanaa T.N.
      • Geusz M.E.
      • Jamasbi R.J.
      A new method for identifying stem-like cells in esophageal cancer cell lines.
      • Zhang G.
      • Ma L.
      • Xie Y.K.
      • Miao X.B.
      • Jin C.
      Esophageal cancer tumorspheres involve cancer stem-like populations with elevated aldehyde dehydrogenase enzymatic activity.
      • Honjo S.
      • Ajani J.A.
      • Scott A.W.
      • Chen Q.
      • Skinner H.D.
      • Stroehlein J.
      • Johnson R.L.
      • Song S.
      Metformin sensitizes chemotherapy by targeting cancer stem cells and the mTOR pathway in esophageal cancer.
      CE81T, CE146TICAM1 in ESCC CSC
      • Wang J.L.
      • Yu J.P.
      • Sun Z.Q.
      • Sun S.P.
      Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines.
      TE4, TE8JARID1B in ESCC CSC
      • Wang Z.
      • Da Silva T.G.
      • Jin K.
      • Han X.
      • Ranganathan P.
      • Zhu X.
      • Sanchez-Mejias A.
      • Bai F.
      • Li B.
      • Fei D.L.
      • Weaver K.
      • Carpio R.V.
      • Moscowitz A.E.
      • Koshenkov V.P.
      • Sanchez L.
      • Sparling L.
      • Pei X.H.
      • Franceschi D.
      • Ribeiro A.
      • Robbins D.J.
      • Livingstone A.S.
      • Capobianco A.J.
      Notch signaling drives stemness and tumorigenicity of esophageal adenocarcinoma.
      YES-2, Eca109ALDH and ESCC CSC
      • Song S.
      • Ajani J.A.
      • Honjo S.
      • Maru D.M.
      • Chen Q.
      • Scott A.W.
      • Heallen T.R.
      • Xiao L.
      • Hofstetter W.L.
      • Weston B.
      • Lee J.H.
      • Wadhwa R.
      • Sudo K.
      • Stroehlein J.R.
      • Martin J.F.
      • Hung M.C.
      • Johnson R.L.
      Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties.
      • Yue D.
      • Zhang Z.
      • Li J.
      • Chen X.
      • Ping Y.
      • Liu S.
      • Shi X.
      • Li L.
      • Wang L.
      • Huang L.
      • Zhang B.
      • Sun Y.
      • Zhang Y.
      Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer.
      • Li B.
      • Xu W.W.
      • Han L.
      • Chan K.T.
      • Tsao S.W.
      • Lee N.P.Y.
      • Law S.
      • Xu L.Y.
      • Li E.M.
      • Chan K.W.
      • Qin Y.R.
      • Guan X.Y.
      • He Q.Y.
      • Cheung A.L.M.
      MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.
      KYSE270, T.TnmiR-377 in ESCC CSC
      • Sato 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.
      Eca109, Eca9706miR-181b and STAT3 in ESCC CSC
      • Zhao J.S.
      • Li W.J.
      • Ge D.
      • Zhang P.J.
      • Li J.J.
      • Lu C.L.
      • Ji X.D.
      • Guan D.X.
      • Gao H.
      • Xu L.Y.
      • Li E.M.
      • Soukiasian H.
      • Koeffler H.P.
      • Wang X.F.
      • Xie D.
      Tumor initiating cells in esophageal squamous cell carcinomas express high levels of CD44.
      KATO-TNYAP1 in ESCC
      • Kano Y.
      • Konno M.
      • Ohta K.
      • Haraguchi N.
      • Nishikawa S.
      • Kagawa Y.
      • Hamabe A.
      • Hasegawa S.
      • Ogawa H.
      • Fukusumi T.
      • Noguchi Y.
      • Ozaki M.
      • Kudo T.
      • Sakai D.
      • Satoh T.
      • Ishii M.
      • Mizohata E.
      • Inoue T.
      • Mori M.
      • Doki Y.
      • Ishii H.
      Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth.
      EADC

      cell line: h
      OE33, JH-EsoAd1EADC CSC
      • Tang K.H.
      • Dai Y.D.
      • Tong M.
      • Chan Y.P.
      • Kwan P.S.
      • Fu L.
      • Qin Y.R.
      • Tsao S.W.
      • Lung H.L.
      • Lung M.L.
      • Tong D.K.
      • Law S.
      • Chan K.W.
      • Ma S.
      • Guan X.Y.
      A CD90(+) tumor-initiating cell population with an aggressive signature and metastatic capacity in esophageal cancer.
      JH-EsoAd1YAP1 in EADC CSC
      • Kano Y.
      • Konno M.
      • Ohta K.
      • Haraguchi N.
      • Nishikawa S.
      • Kagawa Y.
      • Hamabe A.
      • Hasegawa S.
      • Ogawa H.
      • Fukusumi T.
      • Noguchi Y.
      • Ozaki M.
      • Kudo T.
      • Sakai D.
      • Satoh T.
      • Ishii M.
      • Mizohata E.
      • Inoue T.
      • Mori M.
      • Doki Y.
      • Ishii H.
      Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth.
      JH-EsoAd1ALDH in EADC CSC
      • Almanaa T.N.
      • Geusz M.E.
      • Jamasbi R.J.
      Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines.
      OE33, OE19, FLO1, JH-EsoAd1Notch in EADC CSC
      • Xu D.D.
      • Zhou P.J.
      • Wang Y.
      • Zhang L.
      • Fu W.Y.
      • Ruan B.B.
      • Xu H.P.
      • Hu C.Z.
      • Tian L.
      • Qin J.H.
      • Wang S.
      • Wang X.
      • Li Y.C.
      • Liu Q.Y.
      • Ren Z.
      • Zhang R.
      • Wang Y.F.
      Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.
      Organotypic sphere cultureTissue/normal:

      m, h
      Primary

      keratinocytes
      Stem/progenitor cells
      • Garcia E.
      • Hayden A.
      • Birts C.
      • Britton E.
      • Cowie A.
      • Pickard K.
      • Mellone M.
      • Choh C.
      • Derouet M.
      • Duriez P.
      • Noble F.
      • White M.J.
      • Primrose J.N.
      • Strefford J.C.
      • Rose-Zerilli M.
      • Thomas G.J.
      • Ang Y.
      • Sharrocks A.D.
      • Fitzgerald R.C.
      • Underwood T.J.
      consortium O
      Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.
      Multicellular spheroid/

      aggregate culture
      Normal

      (bovine)
      Primary

      keratinocytes
      Morphologic comparison with spheroids formed with other cell types and detection of cell death in the inner cell mass within the resulting spheroid structures
      • Li M.L.
      • Aggeler J.
      • Farson D.A.
      • Hatier C.
      • Hassell J.
      • Bissell M.J.
      Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.
      ESCC

      cell line (h)
      OSC-1, OSC-2Confirmation of spheroid formation
      • Andl C.D.
      • Fargnoli B.B.
      • Okawa T.
      • Bowser M.
      • Takaoka M.
      • Nakagawa H.
      • Klein-Szanto A.
      • Hua X.
      • Herlyn M.
      • Rustgi A.K.
      Coordinated functions of E-cadherin and transforming growth factor beta receptor II in vitro and in vivo.
      Immortalized

      cell line (h)
      EPC2-hTERT and derivativesE-cadherin and cell adhesion
      • Pastrana E.
      • Silva-Vargas V.
      • Doetsch F.
      Eyes wide open: a critical review of sphere-formation as an assay for stem cells.
      NOTE. In OTC, primary human FEF3 and other fibroblasts (eg, cancer-associated fibroblasts) were used to form subepithelial collagen matrix. Extensive lists of fibroblast cell lines and epithelial cell lines validated in 3D OTC are available in Kalabis et al.
      • Kalabis J.
      • Wong G.S.
      • Vega M.E.
      • Natsuizaka M.
      • Robertson E.S.
      • Herlyn M.
      • Nakagawa H.
      • Rustgi A.K.
      Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
      FACS, fluorescence-activated cell sorter; h, human; HGF, hepatocyte growth factor; ICAM1, intercellular adhesion molecule 1; m, murine; MAPK, mitogen-activated protein kinase.
      a All of esophageal origin.
      In vivo transplant culture has served as another platform to reconstitute esophageal epithelium in 3D.
      • Croagh D.
      • Cheng S.
      • Tikoo A.
      • Nandurkar S.
      • Thomas R.J.
      • Kaur P.
      • Phillips W.A.
      Reconstitution of stratified murine and human oesophageal epithelia in an in vivo transplant culture system.
      According to this method, which may not be categorized as cell culture in a strict sense, epithelial cells and fibroblasts are injected into the devitalized and denuded trachea tube of rats, which then is further transplanted into immunodeficient mice. Esophageal keratinocytes are allowed to grow for 4 weeks inside the xenografted trachea and the resulting epithelial structure may be analyzed morphologically. Wang et al
      • Wang D.H.
      • Tiwari A.
      • Kim M.E.
      • Clemons N.J.
      • Regmi N.L.
      • Hodges W.A.
      • Berman D.M.
      • Montgomery E.A.
      • Watkins D.N.
      • Zhang X.
      • Zhang Q.
      • Jie C.
      • Spechler S.J.
      • Souza R.F.
      Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.
      • Wang D.H.
      • Clemons N.J.
      • Miyashita T.
      • Dupuy A.J.
      • Zhang W.
      • Szczepny A.
      • Corcoran-Schwartz I.M.
      • Wilburn D.L.
      • Montgomery E.A.
      • Wang J.S.
      • Jenkins N.A.
      • Copeland N.A.
      • Harmon J.W.
      • Phillips W.A.
      • Watkins D.N.
      Aberrant epithelial-mesenchymal Hedgehog signaling characterizes Barrett's metaplasia.
      used this system successfully to characterize the contribution of Hedgehog signaling to esophageal development and metaplasia with esophageal keratinocytes isolated from genetically engineered mice.
      Multicellular spheroid culture emerged in the early 1970s, stemming from dissociation-aggregation experiments in which dissociated tissue-derived cells undergo aggregation and self-organization under free-floating conditions with gentle stirring via spinner flasks or roller bottles. This technique was applied to a variety of cell types with primary culture and established cell lines recapitulating histologic tissue architecture of the originating organ.
      • Moscona A.
      Rotation-mediated histogenetic aggregation of dissociated cells. A quantifiable approach to cell interactions in vitro.
      • Moscona A.
      • Moscona H.
      The dissociation and aggregation of cells from organ rudiments of the early chick embryo.
      • Weiss P.
      • Taylor A.C.
      Reconstitution of complete organs from single-cell suspensions of chick embryos in advanced stages of differentiation.
      This platform has been used to study cross-talk between tumor cells and other cell types (eg, immune cells and endothelial cells) in co-culture experiments.
      • Dangles V.
      • Validire P.
      • Wertheimer M.
      • Richon S.
      • Bovin C.
      • Zeliszewski D.
      • Vallancien G.
      • Bellet D.
      Impact of human bladder cancer cell architecture on autologous T-lymphocyte activation.
      • Dangles-Marie V.
      • Richon S.
      • El-Behi M.
      • Echchakir H.
      • Dorothee G.
      • Thiery J.
      • Validire P.
      • Vergnon I.
      • Menez J.
      • Ladjimi M.
      • Chouaib S.
      • Bellet D.
      • Mami-Chouaib F.
      A three-dimensional tumor cell defect in activating autologous CTLs is associated with inefficient antigen presentation correlated with heat shock protein-70 down-regulation.
      • Timmins N.E.
      • Dietmair S.
      • Nielsen L.K.
      Hanging-drop multicellular spheroids as a model of tumour angiogenesis.
      Although multicellular spheroid culture was performed with nontransformed bovine esophageal epithelial cells as well as ESCC cell lines (Table 1), these studies were limited to morphologic comparison or analysis of cell viability in resulting spheroids.
      • Korff T.
      • Augustin H.G.
      Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation.
      • Sarbia M.
      • Bosing N.
      • Hildebrandt B.
      • Koldovsky P.
      • Gerharz C.D.
      • Gabbert H.E.
      Characterization of two newly established cell lines derived from squamous cell carcinomas of the oesophagus.
      In the history of 3D culture, the importance of extracellular matrix components for self-organization of functional epithelial cells has been recognized by studies using mammary epithelial cells grown under floating conditions in type I collagen or Matrigel (Corning, Tewksbury, MA) rather than on a plastic surface. Under such conditions, mammary epithelial cells showed not only polarity but also luminal formation with milk protein secretion, which occurred more efficiently in the presence of Matrigel compared with type I collagen.
      • Li M.L.
      • Aggeler J.
      • Farson D.A.
      • Hatier C.
      • Hassell J.
      • Bissell M.J.
      Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells.
      Andl et al
      • Andl C.D.
      • Fargnoli B.B.
      • Okawa T.
      • Bowser M.
      • Takaoka M.
      • Nakagawa H.
      • Klein-Szanto A.
      • Hua X.
      • Herlyn M.
      • Rustgi A.K.
      Coordinated functions of E-cadherin and transforming growth factor beta receptor II in vitro and in vivo.
      embedded esophageal epithelial cell aggregates into either type I collagen or Matrigel (Table 1) to observe that cell migration and invasion from the resulting spheroids were coupled with loss of E-cadherin–mediated cell adhesion. They further identified activin in the tumor microenvironment as a regulator of spheroid growth and invasion.
      • Le Bras G.F.
      • Loomans H.A.
      • Taylor C.J.
      • Revetta F.L.
      • Andl C.D.
      Activin A balance regulates epithelial invasiveness and tumorigenesis.
      Sphere formation assays gained popularity in the 2000s as an excellent tool to characterize stem cells from multiple tissue types,
      • Pastrana E.
      • Silva-Vargas V.
      • Doetsch F.
      Eyes wide open: a critical review of sphere-formation as an assay for stem cells.
      including tumors (see Weiswald et al
      • Weiswald L.B.
      • Bellet D.
      • Dangles-Marie V.
      Spherical cancer models in tumor biology.
      for an excellent review regarding tumor spheres and other spherical cancer models). Sphere formation assays were first used to show proliferation, self-renewal, and multipotent capabilities of neural stem cells in serum-free medium supplemented with epidermal growth factor (EGF) and differentiation-inducing other growth factors.
      • Reynolds B.A.
      • Weiss S.
      Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.
      To form 3D spherical structures, a single cell or a small number (<101–102) of cells are inoculated per well and allowed to proliferate under free-floating conditions. This permits cell fate determination by tracking cell lineage within resulting spherical 3D structures. Unlike multicellular spheroid culture, cell aggregation is deliberately avoided at the onset of sphere formation assays to ensure a proliferative expansion of single-cell derivatives. In cancer research, sphere formation was used to explore cancer stem cells (CSCs) or tumor-initiating cells in conjunction with a variety of putative CSC markers and evaluation of therapeutic sensitivity for such cell populations.
      As summarized in Table 1, sphere formation assays have been used to characterize EADC and ESCC CSCs expressing a variety of markers, including CD54 (intercellular adhesion molecule 1), CD49f, CD44, CD271, CD90, and aldehyde dehydrogenase, either alone or in combination.
      • Tsai S.T.
      • Wang P.J.
      • Liou N.J.
      • Lin P.S.
      • Chen C.H.
      • Chang W.C.
      ICAM1 is a potential cancer stem cell marker of esophageal squamous cell carcinoma.
      • Zhao R.
      • Quaroni L.
      • Casson A.G.
      Identification and characterization of stemlike cells in human esophageal adenocarcinoma and normal epithelial cell lines.
      • Wang J.L.
      • Yu J.P.
      • Sun Z.Q.
      • Sun S.P.
      Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines.
      • Tang K.H.
      • Dai Y.D.
      • Tong M.
      • Chan Y.P.
      • Kwan P.S.
      • Fu L.
      • Qin Y.R.
      • Tsao S.W.
      • Lung H.L.
      • Lung M.L.
      • Tong D.K.
      • Law S.
      • Chan K.W.
      • Ma S.
      • Guan X.Y.
      A CD90(+) tumor-initiating cell population with an aggressive signature and metastatic capacity in esophageal cancer.
      • Almanaa T.N.
      • Geusz M.E.
      • Jamasbi R.J.
      A new method for identifying stem-like cells in esophageal cancer cell lines.
      • Zhang G.
      • Ma L.
      • Xie Y.K.
      • Miao X.B.
      • Jin C.
      Esophageal cancer tumorspheres involve cancer stem-like populations with elevated aldehyde dehydrogenase enzymatic activity.
      • Song S.
      • Ajani J.A.
      • Honjo S.
      • Maru D.M.
      • Chen Q.
      • Scott A.W.
      • Heallen T.R.
      • Xiao L.
      • Hofstetter W.L.
      • Weston B.
      • Lee J.H.
      • Wadhwa R.
      • Sudo K.
      • Stroehlein J.R.
      • Martin J.F.
      • Hung M.C.
      • Johnson R.L.
      Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties.
      Increased sphere formation corroborated CSC attributes such as chemoresistance, invasiveness, and tumorigenicity, and expression of stemness markers (SOX2, ALDH1A1, and KLF4) induced by transforming growth factor-β1.
      • Yue D.
      • Zhang Z.
      • Li J.
      • Chen X.
      • Ping Y.
      • Liu S.
      • Shi X.
      • Li L.
      • Wang L.
      • Huang L.
      • Zhang B.
      • Sun Y.
      • Zhang Y.
      Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer.
      Sphere formation assays were used to document the requirement of H3K4 demethylase Jumonji/Arid1b (Jarid1b) in the maintenance of ESCC CSCs
      • Kano Y.
      • Konno M.
      • Ohta K.
      • Haraguchi N.
      • Nishikawa S.
      • Kagawa Y.
      • Hamabe A.
      • Hasegawa S.
      • Ogawa H.
      • Fukusumi T.
      • Noguchi Y.
      • Ozaki M.
      • Kudo T.
      • Sakai D.
      • Satoh T.
      • Ishii M.
      • Mizohata E.
      • Inoue T.
      • Mori M.
      • Doki Y.
      • Ishii H.
      Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth.
      and the role of Hippo coactivator yes-associated protein 1-mediated transcriptional regulation of SOX9 in EADC CSCs.
      • Song S.
      • Ajani J.A.
      • Honjo S.
      • Maru D.M.
      • Chen Q.
      • Scott A.W.
      • Heallen T.R.
      • Xiao L.
      • Hofstetter W.L.
      • Weston B.
      • Lee J.H.
      • Wadhwa R.
      • Sudo K.
      • Stroehlein J.R.
      • Martin J.F.
      • Hung M.C.
      • Johnson R.L.
      Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties.
      Sphere formation also has been used to test pharmacologic therapeutic effects upon putative esophageal CSCs. Metformin appeared to inhibit sphere formation by Aldehyde dehydrogenase (ALDH)1+ EADC CSCs by targeting phosphatidylinositol 3-kinase/AKT and mammalian target of rapamycin.
      • Honjo S.
      • Ajani J.A.
      • Scott A.W.
      • Chen Q.
      • Skinner H.D.
      • Stroehlein J.
      • Johnson R.L.
      • Song S.
      Metformin sensitizes chemotherapy by targeting cancer stem cells and the mTOR pathway in esophageal cancer.
      Pharmacologic inhibition of Notch signaling by γ-secretase inhibitors impaired tumor initiation as well as sphere formation by EADC CSCs, and Notch appeared to regulate genes such as SOX2, which is essential in stemness.
      • Wang Z.
      • Da Silva T.G.
      • Jin K.
      • Han X.
      • Ranganathan P.
      • Zhu X.
      • Sanchez-Mejias A.
      • Bai F.
      • Li B.
      • Fei D.L.
      • Weaver K.
      • Carpio R.V.
      • Moscowitz A.E.
      • Koshenkov V.P.
      • Sanchez L.
      • Sparling L.
      • Pei X.H.
      • Franceschi D.
      • Ribeiro A.
      • Robbins D.J.
      • Livingstone A.S.
      • Capobianco A.J.
      Notch signaling drives stemness and tumorigenicity of esophageal adenocarcinoma.
      The plant-derived agent curcumin also was found to decrease the sphere formation capability of ESCC cell lines.
      • Almanaa T.N.
      • Geusz M.E.
      • Jamasbi R.J.
      Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines.
      In sphere formation assays, microRNA miR-181b was implicated in signal transducer and activator of transcription-3 (STAT3)-mediated transcriptional regulation of CSCs,
      • Xu D.D.
      • Zhou P.J.
      • Wang Y.
      • Zhang L.
      • Fu W.Y.
      • Ruan B.B.
      • Xu H.P.
      • Hu C.Z.
      • Tian L.
      • Qin J.H.
      • Wang S.
      • Wang X.
      • Li Y.C.
      • Liu Q.Y.
      • Ren Z.
      • Zhang R.
      • Wang Y.F.
      Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.
      whereas miR-377–mediated regulation of CD133+ ESCC CSCs has been shown.
      • Li B.
      • Xu W.W.
      • Han L.
      • Chan K.T.
      • Tsao S.W.
      • Lee N.P.Y.
      • Law S.
      • Xu L.Y.
      • Li E.M.
      • Chan K.W.
      • Qin Y.R.
      • Guan X.Y.
      • He Q.Y.
      • Cheung A.L.M.
      MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.
      It must be noted that sphere formation assays in the studies described earlier were performed with cell lines, but not cancer cells isolated from primary tumors. In 1 study, sphere formation assays failed to detect ESCC CSCs expressing CD44 despite the high tumor-initiating capability of these cells being validated in mice.
      • Zhao J.S.
      • Li W.J.
      • Ge D.
      • Zhang P.J.
      • Li J.J.
      • Lu C.L.
      • Ji X.D.
      • Guan D.X.
      • Gao H.
      • Xu L.Y.
      • Li E.M.
      • Soukiasian H.
      • Koeffler H.P.
      • Wang X.F.
      • Xie D.
      Tumor initiating cells in esophageal squamous cell carcinomas express high levels of CD44.
      In addition, high densities (103–104 cells/well) of cells were seeded to form spheres in many of the described studies,
      • Tsai S.T.
      • Wang P.J.
      • Liou N.J.
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      A CD90(+) tumor-initiating cell population with an aggressive signature and metastatic capacity in esophageal cancer.
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      precluding determination of whether resulting spherical 3D structures represented cell aggregates or single-cell–derivative products. Because this represents a deviation from the original neurosphere assay principle,
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      data obtained in these studies must be interpreted carefully.
      The 3D organoid system has emerged in the past several years as a robust tool in basic research with the potential for personalized medicine. 3D organoids have been defined as a miniature organ-like 3D structure derived from single cells or a small number of cell clusters containing stem/progenitor cells grown in basement membrane (ie, Matrigel) under submerged conditions (Figure 1). Overcoming the difficulty to culture intestinal cell types, Sato et al
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      Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.
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      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      were successfully able to grow stem and progenitor cells from either isolated intestinal crypts or single-cell suspensions prepared from the small or large intestine into lobulated 3D structures containing crypt and villus compartments, the latter containing terminally differentiated secretory and absorptive cell lineages. By passaging dissociated primary structures to generate secondary 3D organoids, this system has been used to validate the self-renewal activities of putative stem cells. Because this can be performed using live tissue pieces from biopsy specimens or even frozen tissues, this novel cell culture method has been transformative with great potential to advance personalized medicine, for example, by testing the chemotherapeutic sensitivity of colorectal cancer–derived 3D organoids from individual patients.
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      Intestinal organoids have been coupled successfully with genetically engineered mouse models of colorectal cancer,
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      Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.
      facilitating functional studies into the biology of this organ in the context of health and disease. Application of the 3D organoid system has been extended to a variety of cell types from digestive (intestines, stomach, liver, pancreas) and nonintestinal organs (eg, brain, lung, breast, kidney, prostate, and ovary) from both human and murine tissue sources as well as embryonic or induced pluripotent stem cells.
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      SnapShot: growing organoids from stem cells.
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      Besides colorectal cancer,
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      cancer cells in primary and metastatic lesions and blood samples, with a goal of translation into personalized therapy.
      3D organoids have been cultured in Dulbecco's modified Eagle medium:nutrient mixture F12-based serum-free medium supplemented with transferrin, selenium, ethanolamine, insulin, antioxidants, and vitamins.
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      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      This medium is supplemented with pharmacologic agents, such as transforming growth factor-β kinase/activin receptor-like kinase inhibitor A83-01, the p38 mitogen-activated protein kinase inhibitor SB202190 and the rho-associated kinase inhibitor Y-27632 to facilitate the establishment of organoids. Besides Matrigel, addition of growth factors and hormones, including noggin, EGF, Wnt3A, R-spondin, and gastrin, to 3D organoid media provides essential niche factors present in the tissue microenvironment in situ. Unique niche factors may influence organoid formation differentially from different cell types as pioneered by Sato et al
      • Sato T.
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      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      in intestinal cell types. For example, Wnt3A is essential for stem cell maintenance in colonic, but not small intestinal, 3D organoids, in mice in which withdrawal of Wnt3A facilitates differentiation in murine colonic organoids.
      • Sato T.
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      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      Optimization of 3D organoid culture medium has broadened the cell types of human and rodent origin that can be grown successfully and passaged as 3D organoids.
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      Prospective derivation of a living organoid biobank of colorectal cancer patients.
      3D organoids featuring the stratified squamous epithelium of the esophagus were first established from murine esophageal mucosa by DeWard et al
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      Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.
      to investigate esophageal stem and progenitor cells. This group used medium similar to that used for human intestinal 3D organoids.
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      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      We have used simplified medium components to generate single-cell–derived murine esophageal 3D organoids for multiple passages,
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      Long-lived keratin 15+ esophageal progenitor cells contribute to homeostasis and regeneration.
      indicating that certain agents and factors are dispensable for murine esophageal 3D organoid culture. The conditions that we have optimized were permissive for generation of murine 3D organoids from normal esophageal epithelium as well as chemical carcinogen-induced precancerous and ESCC lesions.
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      showed successful generation of clonally formed spherical 3D structures from both murine and human esophageal mucosa in a method referred to as “3D organotypic sphere culture.” Although esophageal keratinocytes were suspended in Matrigel, this method used 2 different types of keratinocyte serum-free media to maintain undifferentiated progenitor cells and induce terminal differentiation. In addition, the esophageal keratinocyte-containing Matrigel compartment was placed atop a cell culture insert and exposed to the air–liquid interface, although the contribution of this exposure to sphere formation and/or differentiation was not discussed.
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      Among the earlier-described esophageal 3D cell culture systems, OTC and 3D esophageal organoids have been used most extensively to model epithelial physiological as well as pathologic conditions (Table 1). As a form of tissue engineering, OTC has been most broadly used in studies focusing on epithelial cell behaviors, including epithelial-stromal interaction after pharmacologic and genetic manipulations of either epithelial or stromal cells
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      • Robertson E.S.
      • Herlyn M.
      • Nakagawa H.
      • Rustgi A.K.
      Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
      • Okawa T.
      • Michaylira C.Z.
      • Kalabis J.
      • Stairs D.B.
      • Nakagawa H.
      • Andl C.D.
      • Johnstone C.N.
      • Klein-Szanto A.J.
      • El-Deiry W.S.
      • Cukierman E.
      • Herlyn M.
      • Rustgi A.K.
      The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Takaoka M.
      • Andl C.D.
      • Kim S.H.
      • Klein-Szanto A.
      • Diehl J.A.
      • Herlyn M.
      • El-Deiry W.
      • Rustgi A.K.
      AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.
      • Stairs D.B.
      • Nakagawa H.
      • Klein-Szanto A.
      • Mitchell S.D.
      • Silberg D.G.
      • Tobias J.W.
      • Lynch J.P.
      • Rustgi A.K.
      Cdx1 and c-Myc foster the initiation of transdifferentiation of the normal esophageal squamous epithelium toward Barrett's esophagus.
      • Michaylira C.Z.
      • Wong G.S.
      • Miller C.G.
      • Gutierrez C.M.
      • Nakagawa H.
      • Hammond R.
      • Klein-Szanto A.J.
      • Lee J.S.
      • Kim S.B.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.
      • Rustgi A.K.
      Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor-invasive signature in esophageal cancer.
      • Natsuizaka M.
      • Ohashi S.
      • Wong G.S.
      • Ahmadi A.
      • Kalman R.A.
      • Budo D.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Nakagawa H.
      Insulin-like growth factor-binding protein-3 promotes transforming growth factor-{beta}1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells.
      • Ohashi S.
      • Natsuizaka M.
      • Naganuma S.
      • Kagawa S.
      • Kimura S.
      • Itoh H.
      • Kalman R.A.
      • Nakagawa M.
      • Darling D.S.
      • Basu D.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Nakagawa H.
      A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors.
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      • Vega M.E.
      • Giroux V.
      • Natsuizaka M.
      • Liu M.
      • Klein-Szanto A.J.
      • Stairs D.B.
      • Nakagawa H.
      • Wang K.K.
      • Wang T.C.
      • Lynch J.P.
      • Rustgi A.K.
      Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4.
      • Andl C.D.
      • Fargnoli B.B.
      • Okawa T.
      • Bowser M.
      • Takaoka M.
      • Nakagawa H.
      • Klein-Szanto A.
      • Hua X.
      • Herlyn M.
      • Rustgi A.K.
      Coordinated functions of E-cadherin and transforming growth factor beta receptor II in vitro and in vivo.
      • Natsuizaka M.
      • Whelan K.A.
      • Kagawa S.
      • Tanaka K.
      • Giroux V.
      • Chandramouleeswaran P.M.
      • Long A.
      • Sahu V.
      • Darling D.S.
      • Que J.
      • Yang Y.
      • Katz J.P.
      • Wileyto E.P.
      • Basu D.
      • Kita Y.
      • Natsugoe S.
      • Naganuma S.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Bass A.J.
      • Wong K.K.
      • Rustgi A.K.
      • Nakagawa H.
      Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma.
      • Kalabis J.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Michaylira C.Z.
      • Stairs D.B.
      • Figueiredo J.L.
      • Mahmood U.
      • Diehl J.A.
      • Herlyn M.
      • Rustgi A.K.
      A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.
      • Garcia E.
      • Hayden A.
      • Birts C.
      • Britton E.
      • Cowie A.
      • Pickard K.
      • Mellone M.
      • Choh C.
      • Derouet M.
      • Duriez P.
      • Noble F.
      • White M.J.
      • Primrose J.N.
      • Strefford J.C.
      • Rose-Zerilli M.
      • Thomas G.J.
      • Ang Y.
      • Sharrocks A.D.
      • Fitzgerald R.C.
      • Underwood T.J.
      consortium O
      Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.
      • Cheng E.
      • Zhang X.
      • Wilson K.S.
      • Wang D.H.
      • Park J.Y.
      • Huo X.
      • Yu C.
      • Zhang Q.
      • Spechler S.J.
      • Souza R.F.
      JAK-STAT6 pathway inhibitors block eotaxin-3 secretion by epithelial cells and fibroblasts from esophageal eosinophilia patients: promising agents to improve inflammation and prevent fibrosis in EoE.
      • Sato F.
      • Kubota Y.
      • Natsuizaka M.
      • Maehara O.
      • Hatanaka Y.
      • Marukawa K.
      • Terashita K.
      • Suda G.
      • Ohnishi S.
      • Shimizu Y.
      • Komatsu Y.
      • Ohashi S.
      • Kagawa S.
      • Kinugasa H.
      • Whelan K.A.
      • Nakagawa H.
      • Sakamoto N.
      EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition.
      • Lehman H.L.
      • Yang X.
      • Welsh P.A.
      • Stairs D.B.
      p120-catenin down-regulation and epidermal growth factor receptor overexpression results in a transformed epithelium that mimics esophageal squamous cell carcinoma.
      • Kidacki M.
      • Lehman H.L.
      • Green M.V.
      • Warrick J.I.
      • Stairs D.B.
      p120-Catenin downregulation and PIK3CA mutations cooperate to induce invasion through MMP1 in HNSCC.
      • Wong G.S.
      • Lee J.S.
      • Park Y.Y.
      • Klein-Szanto A.J.
      • Waldron T.J.
      • Cukierman E.
      • Herlyn M.
      • Gimotty P.
      • Nakagawa H.
      • Rustgi A.K.
      Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment.
      • Long A.
      • Giroux V.
      • Whelan K.A.
      • Hamilton K.E.
      • Tetreault M.P.
      • Tanaka K.
      • Lee J.S.
      • Klein-Szanto A.J.
      • Nakagawa H.
      • Rustgi A.K.
      WNT10A promotes an invasive and self-renewing phenotype in esophageal squamous cell carcinoma.
      • Whelan K.A.
      • Chandramouleeswaran P.M.
      • Tanaka K.
      • Natsuizaka M.
      • Guha M.
      • Srinivasan S.
      • Darling D.S.
      • Kita Y.
      • Natsugoe S.
      • Winkler J.D.
      • Klein-Szanto A.J.
      • Amaravadi R.K.
      • Avadhani N.G.
      • Rustgi A.K.
      • Nakagawa H.
      Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.
      • Grugan K.D.
      • Vega M.E.
      • Wong G.S.
      • Diehl J.A.
      • Bass A.J.
      • Wong K.K.
      • Nakagawa H.
      • Rustgi A.K.
      A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion.
      • Kong J.
      • Crissey M.A.
      • Stairs D.B.
      • Sepulveda A.R.
      • Lynch J.P.
      Cox2 and beta-catenin/T-cell factor signaling intestinalize human esophageal keratinocytes when cultured under organotypic conditions.
      • Laczko D.
      • Wang F.
      • Johnson F.B.
      • Jhala N.
      • Rosztoczy A.
      • Ginsberg G.G.
      • Falk G.W.
      • Rustgi A.K.
      • Lynch J.P.
      Modeling esophagitis using human three-dimensional organotypic culture system.
      • Muir A.B.
      • Dods K.
      • Noah Y.
      • Toltzis S.
      • Chandramouleeswaran P.M.
      • Lee A.
      • Benitez A.
      • Bedenbaugh A.
      • Falk G.W.
      • Wells R.G.
      • Nakagawa H.
      • Wang M.L.
      Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition.
      • Muir A.B.
      • Lim D.M.
      • Benitez A.J.
      • Modayur Chandramouleeswaran P.
      • Lee A.J.
      • Ruchelli E.D.
      • Spergel J.M.
      • Wang M.L.
      Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro.
      • Davis B.P.
      • Stucke E.M.
      • Khorki M.E.
      • Litosh V.A.
      • Rymer J.K.
      • Rochman M.
      • Travers J.
      • Kottyan L.C.
      • Rothenberg M.E.
      Eosinophilic esophagitis-linked calpain 14 is an IL-13-induced protease that mediates esophageal epithelial barrier impairment.
      • D'Mello R.J.
      • Caldwell J.M.
      • Azouz N.P.
      • Wen T.
      • Sherrill J.D.
      • Hogan S.P.
      • Rothenberg M.E.
      LRRC31 is induced by IL-13 and regulates kallikrein expression and barrier function in the esophageal epithelium.
      • Lioni M.
      • Noma K.
      • Snyder A.
      • Klein-Szanto A.
      • Diehl J.A.
      • Rustgi A.K.
      • Herlyn M.
      • Smalley K.S.
      Bortezomib induces apoptosis in esophageal squamous cell carcinoma cells through activation of the p38 mitogen-activated protein kinase pathway.
      • Kosoff R.E.
      • Gardiner K.L.
      • Merlo L.M.
      • Pavlov K.
      • Rustgi A.K.
      • Maley C.C.
      Development and characterization of an organotypic model of Barrett's esophagus.
      (see Kalabis et al
      • Kalabis J.
      • Wong G.S.
      • Vega M.E.
      • Natsuizaka M.
      • Robertson E.S.
      • Herlyn M.
      • Nakagawa H.
      • Rustgi A.K.
      Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
      for detailed protocols and resources available). OTC has been better characterized using human esophageal cells as compared with cells of rodent origin, although this method is possible with the latter.
      • Kalabis J.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Michaylira C.Z.
      • Stairs D.B.
      • Figueiredo J.L.
      • Mahmood U.
      • Diehl J.A.
      • Herlyn M.
      • Rustgi A.K.
      A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.
      Although OTC requires preparation of monolayer cell culture before epithelial and subepithelial tissue reconstitution in vitro, the 3D organoids system is initiated directly after dissociation of live tissues and has been applied to both murine and human esophageal cells (Figure 1). In the following section, we review several normal and disease conditions in which OTC or 3D organoids have served as modeling tools.

      Esophageal Normal Stem/Progenitor Cell Proliferation and Differentiation in 3D

      Because the esophageal 3D organoid system features single-cell–derived clonal expansion, establishment of a squamous cell differentiation gradient, and the maintenance of 3D structure upon serial passaging, this experimental platform has been used to test the self-renewal capability of putative esophageal stem cells. DeWard et al
      • DeWard A.D.
      • Cramer J.
      • Lagasse E.
      Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.
      have shown that esophageal basal keratinocytes defined by Sox2 expression comprise stem cells and transit-amplifying cells defined by distinct levels of cell surface CD73, α6 integrin (CD49f), and β4 integrin expression with differential 3D organoid formation capabilities. Jeong et al
      • Giroux V.
      • Lento A.A.
      • Islam M.
      • Pitarresi J.R.
      • Kharbanda A.
      • Hamilton K.E.
      • Whelan K.A.
      • Long A.
      • Rhoades B.
      • Tang Q.
      • Nakagawa H.
      • Lengner C.J.
      • Bass A.J.
      • Wileyto E.P.
      • Klein-Szanto A.J.
      • Wang T.C.
      • Rustgi A.K.
      Long-lived keratin 15+ esophageal progenitor cells contribute to homeostasis and regeneration.
      showed that undifferentiated esophageal keratinocytes defined by positive/high CD49f and low CD24 expression show high 3D sphere formation capability where the transcription factor p63 appeared to regulate self-renewal and gene expression of basal cell markers. Giroux et al
      • Pauli C.
      • Hopkins B.D.
      • Prandi D.
      • Shaw R.
      • Fedrizzi T.
      • Sboner A.
      • Sailer V.
      • Augello M.
      • Puca L.
      • Rosati R.
      • McNary T.J.
      • Churakova Y.
      • Cheung C.
      • Triscott J.
      • Pisapia D.
      • Rao R.
      • Mosquera J.M.
      • Robinson B.
      • Faltas B.M.
      • Emerling B.E.
      • Gadi V.K.
      • Bernard B.
      • Elemento O.
      • Beltran H.
      • Demichelis F.
      • Kemp C.J.
      • Grandori C.
      • Cantley L.C.
      • Rubin M.A.
      Personalized in vitro and in vivo cancer models to guide precision medicine.
      identified a long-lived stem/progenitor cell population characterized by expression of keratin 15 in murine esophageal epithelium, which showed self-renewal, proliferation, and differentiation capabilities in 3D organoid assays. As has been postulated as a weakness in conventional neurosphere assays,
      • Pastrana E.
      • Silva-Vargas V.
      • Doetsch F.
      Eyes wide open: a critical review of sphere-formation as an assay for stem cells.
      organoid formation involves cell proliferation, and, thus, may not necessarily determine quiescent stem cells. In addition, the frequency of stem cells detected by these assays may not necessarily be representative of that found in originating tissues, potentially owing to loss of subsets of cells during tissue dissociation and cell isolation. The proliferation-differentiation gradient has been analyzed in esophageal stem cell–derived 3D organoids using molecular markers of basal keratinocytes and differentiated suprabasal keratinocytes in the earlier-described studies.
      • DeWard A.D.
      • Cramer J.
      • Lagasse E.
      Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.
      • Giroux V.
      • Lento A.A.
      • Islam M.
      • Pitarresi J.R.
      • Kharbanda A.
      • Hamilton K.E.
      • Whelan K.A.
      • Long A.
      • Rhoades B.
      • Tang Q.
      • Nakagawa H.
      • Lengner C.J.
      • Bass A.J.
      • Wileyto E.P.
      • Klein-Szanto A.J.
      • Wang T.C.
      • Rustgi A.K.
      Long-lived keratin 15+ esophageal progenitor cells contribute to homeostasis and regeneration.
      • Jeong Y.
      • Rhee H.
      • Martin S.
      • Klass D.
      • Lin Y.
      • Nguyen le X.T.
      • Feng W.
      • Diehn M.
      Identification and genetic manipulation of human and mouse oesophageal stem cells.
      • Whelan K.A.
      • Merves J.F.
      • Giroux V.
      • Tanaka K.
      • Guo A.
      • Chandramouleeswaran P.M.
      • Benitez A.J.
      • Dods K.
      • Que J.
      • Masterson J.C.
      • Fernando S.D.
      • Godwin B.C.
      • Klein-Szanto A.J.
      • Chikwava K.
      • Ruchelli E.D.
      • Hamilton K.E.
      • Muir A.B.
      • Wang M.L.
      • Furuta G.T.
      • Falk G.W.
      • Spergel J.M.
      • Nakagawa H.
      Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis.
      Because the most proliferative and undifferentiated basal-like (or basaloid) keratinocytes are present in the outmost cell layer of 3D organoid structures and less proliferative differentiated cells represent the inner cell mass, one caveat of this model system is that the former does not migrate toward the center of the 3D structure to become the latter, unlike generation of the differentiation gradient in vivo or in OTC. Namely, the proliferative stem/progenitor cells form first the inner cell mass, which undergoes terminal differentiation because organoids grow in an outward fashion, and, thus, the outmost cell layers represent the cells that were generated last.
      OTC also was used to show that putative esophageal stem cells are capable of reconstituting stratified squamous epithelia in vitro
      • Jeong Y.
      • Rhee H.
      • Martin S.
      • Klass D.
      • Lin Y.
      • Nguyen le X.T.
      • Feng W.
      • Diehn M.
      Identification and genetic manipulation of human and mouse oesophageal stem cells.
      ; however, a limitation of the use of OTC for the assessment of esophageal stem cells is that this technique fails to generate single-cell–derived 3D structures. Esophageal epithelial cell proliferation and differentiation have been characterized extensively along with their regulatory signaling pathways in OTC using either primary human esophageal keratinocytes or telomerase-immortalized cell lines.
      • Ohashi S.
      • Natsuizaka M.
      • Yashiro-Ohtani Y.
      • Kalman R.A.
      • Nakagawa M.
      • Wu L.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Katz J.P.
      • Pear W.S.
      • Seykora J.T.
      • Nakagawa H.
      NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.
      • Andl C.D.
      • Mizushima T.
      • Nakagawa H.
      • Oyama K.
      • Harada H.
      • Chruma K.
      • Herlyn M.
      • Rustgi A.K.
      Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo.
      • Harada H.
      • Nakagawa H.
      • Oyama K.
      • Takaoka M.
      • Andl C.D.
      • Jacobmeier B.
      • von Werder A.
      • Enders G.H.
      • Opitz O.G.
      • Rustgi A.K.
      Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation.
      Esophageal keratinocytes engineered to overexpress epidermal growth factor receptor (EGFR) showed hyperproliferation in an EGFR tyrosine kinase activity–dependent manner in OTC.
      • Andl C.D.
      • Mizushima T.
      • Nakagawa H.
      • Oyama K.
      • Harada H.
      • Chruma K.
      • Herlyn M.
      • Rustgi A.K.
      Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo.
      This study showed that EGFR overexpression induces translocation of p120 catenin to the cell membrane, which mediates epithelial cell–cell adhesion.
      • Andl C.D.
      • Mizushima T.
      • Nakagawa H.
      • Oyama K.
      • Harada H.
      • Chruma K.
      • Herlyn M.
      • Rustgi A.K.
      Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo.
      Among downstream effectors of EGFR is the phosphatidylinositol 3-kinase/AKT signaling pathway.
      • Okano J.
      • Gaslightwala I.
      • Birnbaum M.J.
      • Rustgi A.K.
      • Nakagawa H.
      Akt/protein kinase B isoforms are differentially regulated by epidermal growth factor stimulation.
      In OTC, EGFR overexpression led to AKT activation via phosphatidylinositol 3-kinase. Although inducible activation of AKT increased cell size concurrent with a decreased level of keratohyalin granules, a marker of terminal differentiation, AKT activation did not increase esophageal cell proliferation in OTC.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Takaoka M.
      • Andl C.D.
      • Kim S.H.
      • Klein-Szanto A.
      • Diehl J.A.
      • Herlyn M.
      • El-Deiry W.
      • Rustgi A.K.
      AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.
      EGFR signaling regulates the cell cycle by inducing cyclin D1, a key G1 cyclin.
      • Yan Y.X.
      • Nakagawa H.
      • Lee M.H.
      • Rustgi A.K.
      Transforming growth factor-alpha enhances cyclin D1 transcription through the binding of early growth response protein to a cis-regulatory element in the cyclin D1 promoter.
      Cyclin D1 was found to be responsible for basal keratinocyte proliferation because tetracycline-inducible cyclin D1 overexpression resulted in basal cell hyperplasia in OTC.
      • Ohashi S.
      • Natsuizaka M.
      • Naganuma S.
      • Kagawa S.
      • Kimura S.
      • Itoh H.
      • Kalman R.A.
      • Nakagawa M.
      • Darling D.S.
      • Basu D.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Nakagawa H.
      A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors.
      The transcription factor KLF4 facilitates esophageal differentiation via activation of noncanonical Wnt5A signaling.
      • Wong G.S.
      • Lee J.S.
      • Park Y.Y.
      • Klein-Szanto A.J.
      • Waldron T.J.
      • Cukierman E.
      • Herlyn M.
      • Gimotty P.
      • Nakagawa H.
      • Rustgi A.K.
      Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment.
      Notch signaling plays a critical role in esophageal epithelial cell fate decisions. In studies using OTC coupled with RNA interference, genetic or pharmacologic pan-Notch inhibition showed that activated Notch signaling drives terminal differentiation by transcriptionally activating early differentiation markers such as involucrin and cytokeratin K13 in a Notch3-dependent manner.
      • Ohashi S.
      • Natsuizaka M.
      • Yashiro-Ohtani Y.
      • Kalman R.A.
      • Nakagawa M.
      • Wu L.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Katz J.P.
      • Pear W.S.
      • Seykora J.T.
      • Nakagawa H.
      NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      These observations in OTC were recapitulated in genetically engineered mice with esophageal epithelium-targeted pan-Notch inhibition showing Notch3 down-regulation and impaired terminal differentiation.
      • Ohashi S.
      • Natsuizaka M.
      • Yashiro-Ohtani Y.
      • Kalman R.A.
      • Nakagawa M.
      • Wu L.
      • Klein-Szanto A.J.
      • Herlyn M.
      • Diehl J.A.
      • Katz J.P.
      • Pear W.S.
      • Seykora J.T.
      • Nakagawa H.
      NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.
      In our recent attempt to dissect Notch signaling in normal human esophageal 3D organoids, a similar approach confirmed the role of Notch signaling in basal keratinocyte exit toward terminal differentiation.
      • Kasagi Y.
      • Chandramouleeswaran P.M.
      • Whelan K.A.
      • Tanaka K.
      • Giroux V.
      • Sharma M.
      • Wang J.
      • Benitez A.J.
      • DeMarshall M.
      • Tobias J.W.
      • Hamilton K.E.
      • Falk G.W.
      • Spergel J.M.
      • Klein-Klein-Szanto A.J.
      • Rustgi A.K.
      • Muir A.B.
      • Nakagawa H.
      The esophageal organoid system reveals functional interplay between notch and cytokines in reactive epithelial changes.
      Thus, the OTC and 3D organoid models may complement studies in genetically engineered mouse models.

      Impaired Epithelial Homeostasis and Barrier Defect Modeled in 3D

      A variety of stressors in the tissue microenvironment influence esophageal homeostasis. Little has been explored as to how esophageal epithelial cells respond to these stressors in 3D culture. In esophageal 3D organoids, we recently investigated epithelial response to oxidative stress induced by alcohol and its toxic metabolite acetaldehyde or inflammatory cytokines. These stressors were found to induce autophagy, a homeostatic cytoprotective mechanism to decrease oxidative stress, recapitulating epithelial changes observed in mice subjected to excessive alcohol drinking in the presence of dysfunctional Aldh2, a mitochondrial acetaldehyde metabolizing enzyme
      • Tanaka K.
      • Whelan K.A.
      • Chandramouleeswaran P.M.
      • Kagawa S.
      • Rustgi S.L.
      • Noguchi C.
      • Guha M.
      • Srinivasan S.
      • Amanuma Y.
      • Ohashi S.
      • Muto M.
      • Klein-Szanto A.J.
      • Noguchi E.
      • Avadhani N.G.
      • Nakagawa H.
      ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds.
      or EoE-like esophageal inflammation.
      • Whelan K.A.
      • Merves J.F.
      • Giroux V.
      • Tanaka K.
      • Guo A.
      • Chandramouleeswaran P.M.
      • Benitez A.J.
      • Dods K.
      • Que J.
      • Masterson J.C.
      • Fernando S.D.
      • Godwin B.C.
      • Klein-Szanto A.J.
      • Chikwava K.
      • Ruchelli E.D.
      • Hamilton K.E.
      • Muir A.B.
      • Wang M.L.
      • Furuta G.T.
      • Falk G.W.
      • Spergel J.M.
      • Nakagawa H.
      Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis.
      An epithelial barrier defect has been implicated in the pathogenesis of esophageal diseases including GERD and EoE. Squamous epithelial stratification is less mature and suboptimal in OTC lacking the subepithelial matrix compartment containing fibroblasts,
      • Okawa T.
      • Michaylira C.Z.
      • Kalabis J.
      • Stairs D.B.
      • Nakagawa H.
      • Andl C.D.
      • Johnstone C.N.
      • Klein-Szanto A.J.
      • El-Deiry W.S.
      • Cukierman E.
      • Herlyn M.
      • Rustgi A.K.
      The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.
      underscoring the cross-talk between stromal fibroblasts and epithelial cells in epithelial homeostasis.
      • Muir A.B.
      • Lim D.M.
      • Benitez A.J.
      • Modayur Chandramouleeswaran P.
      • Lee A.J.
      • Ruchelli E.D.
      • Spergel J.M.
      • Wang M.L.
      Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro.
      Interestingly, OTC-like air–liquid interface was used in the absence of fibroblasts to study the role of desmoglein-1, calpain 14, and LRRC31 in epithelial barrier functions.
      • Sherrill J.D.
      • Kc K.
      • Wu D.
      • Djukic Z.
      • Caldwell J.M.
      • Stucke E.M.
      • Kemme K.A.
      • Costello M.S.
      • Mingler M.K.
      • Blanchard C.
      • Collins M.H.
      • Abonia J.P.
      • Putnam P.E.
      • Dellon E.S.
      • Orlando R.C.
      • Hogan S.P.
      • Rothenberg M.E.
      Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis.
      • Davis B.P.
      • Stucke E.M.
      • Khorki M.E.
      • Litosh V.A.
      • Rymer J.K.
      • Rochman M.
      • Travers J.
      • Kottyan L.C.
      • Rothenberg M.E.
      Eosinophilic esophagitis-linked calpain 14 is an IL-13-induced protease that mediates esophageal epithelial barrier impairment.
      • D'Mello R.J.
      • Caldwell J.M.
      • Azouz N.P.
      • Wen T.
      • Sherrill J.D.
      • Hogan S.P.
      • Rothenberg M.E.
      LRRC31 is induced by IL-13 and regulates kallikrein expression and barrier function in the esophageal epithelium.
      In these studies, genetic modulations of these molecules resulted in impaired epithelial stratification as corroborated by altered transepithelial resistance or dextran flux,
      • Sherrill J.D.
      • Kc K.
      • Wu D.
      • Djukic Z.
      • Caldwell J.M.
      • Stucke E.M.
      • Kemme K.A.
      • Costello M.S.
      • Mingler M.K.
      • Blanchard C.
      • Collins M.H.
      • Abonia J.P.
      • Putnam P.E.
      • Dellon E.S.
      • Orlando R.C.
      • Hogan S.P.
      • Rothenberg M.E.
      Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis.
      • Davis B.P.
      • Stucke E.M.
      • Khorki M.E.
      • Litosh V.A.
      • Rymer J.K.
      • Rochman M.
      • Travers J.
      • Kottyan L.C.
      • Rothenberg M.E.
      Eosinophilic esophagitis-linked calpain 14 is an IL-13-induced protease that mediates esophageal epithelial barrier impairment.
      • D'Mello R.J.
      • Caldwell J.M.
      • Azouz N.P.
      • Wen T.
      • Sherrill J.D.
      • Hogan S.P.
      • Rothenberg M.E.
      LRRC31 is induced by IL-13 and regulates kallikrein expression and barrier function in the esophageal epithelium.
      however, the absence of fibroblasts in the OTC-like system may have potentially exaggerated the observed barrier defects because the reconstituted epithelia contained much fewer regular basal cells than the typical OTC with fibroblasts.

      Modeling Inflammatory Disease Conditions in 3D

      Inflammation is pertinent to multiple esophageal pathologies including GERD, EoE, radiation-induced esophagitis, as well as preneoplastic and neoplastic conditions; however, modeling inflammation in 3D culture systems remains largely unexplored. Laczko et al
      • Laczko D.
      • Wang F.
      • Johnson F.B.
      • Jhala N.
      • Rosztoczy A.
      • Ginsberg G.G.
      • Falk G.W.
      • Rustgi A.K.
      • Lynch J.P.
      Modeling esophagitis using human three-dimensional organotypic culture system.
      created an OTC esophagitis model by incorporating human peripheral blood mononuclear cells and exogenous cytokines into OTC, recapitulating the T-helper cell type 1 acute inflammatory response found in human GERD. Indeed, esophageal epithelial cells showed aberrant proliferation, differentiation, oxidative stress, DNA damage, and apoptosis in response to peripheral blood mononuclear cells and stimulation by cytokines in OTC.
      • Laczko D.
      • Wang F.
      • Johnson F.B.
      • Jhala N.
      • Rosztoczy A.
      • Ginsberg G.G.
      • Falk G.W.
      • Rustgi A.K.
      • Lynch J.P.
      Modeling esophagitis using human three-dimensional organotypic culture system.
      EoE is characterized by long-term inflammation mediated by eosinophils and other immune cell types that lead to substantial tissue remodeling affecting both epithelial and subepithelial stromal compartments. In EoE, esophageal epithelial cells show basal cell hyperplasia, which features not only expansion of undifferentiated basal keratinocytes but morphologic and functional changes such as epithelial-mesenchymal transition (EMT),
      • Muir A.B.
      • Dods K.
      • Noah Y.
      • Toltzis S.
      • Chandramouleeswaran P.M.
      • Lee A.
      • Benitez A.
      • Bedenbaugh A.
      • Falk G.W.
      • Wells R.G.
      • Nakagawa H.
      • Wang M.L.
      Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition.
      • Muir A.B.
      • Lim D.M.
      • Benitez A.J.
      • Modayur Chandramouleeswaran P.
      • Lee A.J.
      • Ruchelli E.D.
      • Spergel J.M.
      • Wang M.L.
      Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro.
      • Kagalwalla A.F.
      • Akhtar N.
      • Woodruff S.A.
      • Rea B.A.
      • Masterson J.C.
      • Mukkada V.
      • Parashette K.R.
      • Du J.
      • Fillon S.
      • Protheroe C.A.
      • Lee J.J.
      • Amsden K.
      • Melin-Aldana H.
      • Capocelli K.E.
      • Furuta G.T.
      • Ackerman S.J.
      Eosinophilic esophagitis: epithelial mesenchymal transition contributes to esophageal remodeling and reverses with treatment.
      contributing to the inflammatory milieu as well as decreased epithelial barrier functions. In the stromal compartment of EoE, activation of myofibroblasts leads to fibrostenotic disease, the most serious functional consequence in EoE inflammation. These alterations in keratinocytes and fibroblasts have been hypothesized to create a unique epithelial-stromal cross-talk mediated by EoE-relevant cytokines. We have used OTC to investigate the role of transforming growth factor-β1, tumor necrosis factor-α, and interleukin-1β as essential mediators in EMT and myofibroblast activation in the context of EoE to corroborate their molecular profiling of endoscopic biopsy specimens from EoE patients.
      • Muir A.B.
      • Dods K.
      • Noah Y.
      • Toltzis S.
      • Chandramouleeswaran P.M.
      • Lee A.
      • Benitez A.
      • Bedenbaugh A.
      • Falk G.W.
      • Wells R.G.
      • Nakagawa H.
      • Wang M.L.
      Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition.
      • Muir A.B.
      • Lim D.M.
      • Benitez A.J.
      • Modayur Chandramouleeswaran P.
      • Lee A.J.
      • Ruchelli E.D.
      • Spergel J.M.
      • Wang M.L.
      Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro.
      Cheng et al
      • Garcia E.
      • Hayden A.
      • Birts C.
      • Britton E.
      • Cowie A.
      • Pickard K.
      • Mellone M.
      • Choh C.
      • Derouet M.
      • Duriez P.
      • Noble F.
      • White M.J.
      • Primrose J.N.
      • Strefford J.C.
      • Rose-Zerilli M.
      • Thomas G.J.
      • Ang Y.
      • Sharrocks A.D.
      • Fitzgerald R.C.
      • Underwood T.J.
      consortium O
      Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.
      tested the effect of esophageal fibroblasts from EoE patient biopsy specimens upon epithelial stratification in OTC.
      The role of cytokines in EoE has been evaluated in a 3D organoid system in which transforming growth factor-β1, tumor necrosis factor-α, and interleukin-13 stimulated the expansion of basal cells in single-cell–derived murine esophageal 3D organoids, recapitulating reactive epithelial changes and basal cell hyperplasia as found in human EoE biopsy specimens.
      • Whelan K.A.
      • Merves J.F.
      • Giroux V.
      • Tanaka K.
      • Guo A.
      • Chandramouleeswaran P.M.
      • Benitez A.J.
      • Dods K.
      • Que J.
      • Masterson J.C.
      • Fernando S.D.
      • Godwin B.C.
      • Klein-Szanto A.J.
      • Chikwava K.
      • Ruchelli E.D.
      • Hamilton K.E.
      • Muir A.B.
      • Wang M.L.
      • Furuta G.T.
      • Falk G.W.
      • Spergel J.M.
      • Nakagawa H.
      Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis.
      We envision the field moving toward optimized 3D organoid culture to allow expansion and passaging of human and murine organoids from single cells. Such protocols will allow for generation of organoids from patients with normal mucosa, GERD, and EoE, as well as correlation with human pathology found on biopsy specimens. Our group recently optimized 3D organoid culture conditions to allow expansion and multiple passages of both murine and human esophageal epithelial single-cell–derived organoids in a common and simplified culture medium (Kasagi et al, CMGH in press). In this study, human esophageal organoids were generated from biopsy specimens of patients with normal mucosa, GERD, and EoE. These 3D organoids showed hyperplasia in the presence of tumor necrosis factor-α or interleukin-13, indicating that reactive epithelial changes in situ may indeed occur in response to the inflammatory milieu.

      Modeling Neoplastic and Preneoplastic Conditions in 3D

      ESCC and EADC, 2 major esophageal cancers, and their precursor lesions have been modeled in 3D OTC while 3D organoid models of esophageal cancers are emerging (Table 1). Both OTC and 3D organoids permit morphologic assessment of a broad range of cancer cell characteristics, including proliferation (Ki67),
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      DNA damage (γH2AX),
      • Tanaka K.
      • Whelan K.A.
      • Chandramouleeswaran P.M.
      • Kagawa S.
      • Rustgi S.L.
      • Noguchi C.
      • Guha M.
      • Srinivasan S.
      • Amanuma Y.
      • Ohashi S.
      • Muto M.
      • Klein-Szanto A.J.
      • Noguchi E.
      • Avadhani N.G.
      • Nakagawa H.
      ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds.
      epithelial-mesenchymal transition (E-cadherin and vimentin),
      • Ohashi S.
      • Natsuizaka M.
      • Naganuma S.
      • Kagawa S.
      • Kimura S.
      • Itoh H.
      • Kalman R.A.
      • Nakagawa M.
      • Darling D.S.
      • Basu D.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Nakagawa H.
      A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT-competent cells that express the ZEB transcription factors.
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      and CSC markers (eg, CD44),
      • Whelan K.A.
      • Chandramouleeswaran P.M.
      • Tanaka K.
      • Natsuizaka M.
      • Guha M.
      • Srinivasan S.
      • Darling D.S.
      • Kita Y.
      • Natsugoe S.
      • Winkler J.D.
      • Klein-Szanto A.J.
      • Amaravadi R.K.
      • Avadhani N.G.
      • Rustgi A.K.
      • Nakagawa H.
      Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.
      in conjunction with genetic or pharmacologic modifications of cancer cells and fibroblasts. In OTC, pharmacologic treatment is not selective for either cancer cells or fibroblasts, although genetic modifications (eg, RNA interference or tetracycline- or tamoxifen-inducible systems) can be performed in a cell type–specific manner.
      • Grugan K.D.
      • Miller C.G.
      • Yao Y.
      • Michaylira C.Z.
      • Ohashi S.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Han M.
      • Nakagawa H.
      • Rustgi A.K.
      Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion.
      • Okawa T.
      • Michaylira C.Z.
      • Kalabis J.
      • Stairs D.B.
      • Nakagawa H.
      • Andl C.D.
      • Johnstone C.N.
      • Klein-Szanto A.J.
      • El-Deiry W.S.
      • Cukierman E.
      • Herlyn M.
      • Rustgi A.K.
      The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.
      • Oyama K.
      • Okawa T.
      • Nakagawa H.
      • Takaoka M.
      • Andl C.D.
      • Kim S.H.
      • Klein-Szanto A.
      • Diehl J.A.
      • Herlyn M.
      • El-Deiry W.
      • Rustgi A.K.
      AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.
      • Naganuma S.
      • Whelan K.A.
      • Natsuizaka M.
      • Kagawa S.
      • Kinugasa H.
      • Chang S.
      • Subramanian H.
      • Rhoades B.
      • Ohashi S.
      • Itoh H.
      • Herlyn M.
      • Diehl J.A.
      • Gimotty P.A.
      • Klein-Szanto A.J.
      • Nakagawa H.
      Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma.
      Because cancer cells invade collectively or individually, cell invasion is evaluated quantitatively by measuring the area of invasive tumor islands within the stromal compartment.
      • Grugan K.D.
      • Miller C.G.
      • Yao Y.
      • Michaylira C.Z.
      • Ohashi S.
      • Klein-Szanto A.J.
      • Diehl J.A.
      • Herlyn M.
      • Han M.
      • Nakagawa H.
      • Rustgi A.K.
      Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion.
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