We thank the Shared Resources (Flow Cytometry, Molecular Pathology, and Confocal &#38; Specialized Microscopy) at the Herbert Irving Comprehensive Cancer Center at Columbia University for technical support. We thank Drs. Alan Diehl, Adam J. Bass, and Kwok-Kin Wong (NCI P01 Mechanisms of Esophageal Carcinogenesis) and members of the Rustgi and Nakagawa laboratories for helpful discussions. This study was supported by the following NIH Grants: P01CA098101 (H.N. and A.K.R.), R01DK114436 (H.N.), R01AA026297 (H.N.), L30CA264714 (S.F.), DE031112-01 (F.M.H.), KL2TR001874 (F.M.H.),3R01CA255298-01S1 (J.G.), 2L30DK126621-02
(J.G.) R01CA266978 (C.L.), R01DK132251 (C.L.), R01DE031873 (C.L.), P30DK132710 (C.M. and H.N.), and P30CA013696 (A.K.R.). H.N. and C.L. are recipients of the Columbia University Herbert Irving Comprehensive Cancer Center Multi-PI Pilot Award. H.N. is a recipient of the Fanconi Anemia Research Fund Award. F.M.H. is the recipient of The Mark Foundation for Cancer Research Award (20-60-51-MOME) and an American Association for Cancer Research Award. J.G. is the recipient of the American Gastroenterological Association (AGA) award.

The murine experiments were planned and performed in accordance with regulations and under animal protocol #AABB1502, reviewed and approved by Columbia University&#39;s Institutional Animal Care and Use Committee. The mice were housed at a proper animal care facility that ensures the humane treatment of mice and provides appropriate veterinary care for the mice and laboratory safety training for the laboratory personnel.
1. Treatment of mice with 4NQO to induce esophageal IEN and ESCC le...

<ol>
	<li>Rustgi, A. K., El-Serag, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esophageal+carcinoma.">Esophageal carcinoma.</a> The New England Journal of Medicine. 371 (26), 2499-2509 (2014).</li><li>Dotto, G. P., Rustgi, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Squamous+cell+cancers:+A+unified+perspective+on+biology+and+genetics.">Squamous cell cancers: A unified perspective on biology and genetics.</a> Cancer Cell. 29 (5), 622-637 (2016).</li><li>Giroux, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+keratin+15++esophageal+progenitor+cells+contribute+to+homeostasis+and+regeneration.">Long-lived keratin 15+ esophageal progenitor cells contribute to homeostasis and regeneration.</a> The Journal of Clinical Investigation. 127 (6), 2378-2391 (2017).</li><li>DeWard, A. D., Cramer, J., Lagasse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+heterogeneity+in+the+mouse+esophagus+implicates+the+presence+of+a+nonquiescent+epithelial+stem+cell+population.">Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.</a> Cell Reports. 9 (2), 701-711 (2014).</li><li>Kinugasa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+SOD2+regulates+epithelial-mesenchymal+transition+and+cell+populations+defined+by+differential+CD44+expression.">Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression.</a> Oncogene. 34 (41), 5229-5239 (2015).</li><li>Whelan, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+supports+generation+of+cells+with+high+CD44+expression+via+modulation+of+oxidative+stress+and+Parkin-mediated+mitochondrial+clearance.">Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.</a> Oncogene. 36 (34), 4843-4858 (2017).</li><li>Natsuizaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Notch1+and+Notch3+promotes+EMT+and+tumor+initiation+in+squamous+cell+carcinoma.">Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma.</a> Nature Communications. 8 (1), 1758 (2017).</li><li>Whelan, K. A., Muir, A. B., Nakagawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esophageal+3D+culture+systems+as+modeling+tools+in+esophageal+epithelial+pathobiology+and+personalized+medicine.">Esophageal 3D culture systems as modeling tools in esophageal epithelial pathobiology and personalized medicine.</a> Cellular and Molecular Gastroenterology and Hepatology. 5 (4), 461-478 (2018).</li><li>Sachdeva, U. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+cellular+origin+and+progression+of+esophageal+cancer+using+esophageal+organoids.">Understanding the cellular origin and progression of esophageal cancer using esophageal organoids.</a> Cancer Letters. 509, 39-52 (2021).</li><li>Nakagawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+targeting+of+the+cyclin+D1+oncogene+by+an+Epstein-Barr+virus+promoter+in+transgenic+mice+causes+dysplasia+in+the+tongue,+esophagus+and+forestomach.">The targeting of the cyclin D1 oncogene by an Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue, esophagus and forestomach.</a> Oncogene. 14 (10), 1185-1190 (1997).</li><li>Andl, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+mediates+increased+cell+proliferation,+migration,+and+aggregation+in+esophageal+keratinocytes+in+vitro+and+in+vivo.">Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo.</a> The Journal of Biological Chemistry. 278 (3), 1824-1830 (2003).</li><li>Opitz, O. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+human+oral-esophageal+cancer.">A mouse model of human oral-esophageal cancer.</a> The Journal of Clinical Investigation. 110 (6), 761-769 (2002).</li><li>Stairs, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+p120-catenin+results+in+a+tumor+microenvironment+with+inflammation+and+cancer+that+establishes+it+as+a+tumor+suppressor+gene.">Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene.</a> Cancer Cell. 19 (4), 470-483 (2011).</li><li>Tang, X. -. H., Knudsen, B., Bemis, D., Tickoo, S., Gudas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+cavity+and+esophageal+carcinogenesis+modeled+in+carcinogen-treated+mice.">Oral cavity and esophageal carcinogenesis modeled in carcinogen-treated mice.</a> Clinical Cancer Research. 10, 301-313 (2004).</li><li>Fong, L. Y. Y., Mancini, R., Nakagawa, H., Rustgi, A. K., Huebner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+cyclin+D1+overexpression+and+zinc+deficiency+disrupts+cell+cycle+and+accelerates+mouse+forestomach+carcinogenesis.">Combined cyclin D1 overexpression and zinc deficiency disrupts cell cycle and accelerates mouse forestomach carcinogenesis.</a> Cancer Research. 63 (14), 4244-4252 (2003).</li><li>Zheng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+murine+esophageal+organoid+culture+method+and+organoid-based+model+of+esophageal+squamous+cell+neoplasia.">A new murine esophageal organoid culture method and organoid-based model of esophageal squamous cell neoplasia.</a> iScience. 24 (12), 103440 (2021).</li><li>Hisha, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+lingual+organoid+culture+system:+generation+of+organoids+having+mature+keratinized+epithelium+from+adult+epithelial+stem+cells.">Establishment of a novel lingual organoid culture system: generation of organoids having mature keratinized epithelium from adult epithelial stem cells.</a> Scientific Reports. 3, 3224 (2013).</li><li>Kalabis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mouse+and+human+esophageal+epithelial+cells+in+3D+organotypic+culture.">Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.</a> Nature Protocols. 7 (2), 235-246 (2012).</li><li>Nguyen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;1+alters+esophageal+epithelial+barrier+function+by+attenuation+of+claudin-7+in+eosinophilic+esophagitis.">TGF-&#946;1 alters esophageal epithelial barrier function by attenuation of claudin-7 in eosinophilic esophagitis.</a> Mucosal Immunology. 11 (2), 415-426 (2018).</li><li>Sherrill, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+expansion+of+the+eosinophilic+esophagitis+transcriptome+by+RNA+sequencing.">Analysis and expansion of the eosinophilic esophagitis transcriptome by RNA sequencing.</a> Genes and Immunity. 15 (6), 361-369 (2014).</li><li>Ruffner, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+2+stimulation+augments+esophageal+barrier+integrity.">Toll-like receptor 2 stimulation augments esophageal barrier integrity.</a> Allergy. 74 (12), 2449-2460 (2019).</li><li>Kabir, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+transcriptomic+analysis+reveals+cellular+diversity+of+murine+esophageal+epithelium.">Single cell transcriptomic analysis reveals cellular diversity of murine esophageal epithelium.</a> Nature Communications. 13 (1), 1-15 (2022).</li><li>Shimonosono, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+metabolism+enriches+squamous+cell+carcinoma+cancer+stem+cells+that+survive+oxidative+stress+via+autophagy.">Alcohol metabolism enriches squamous cell carcinoma cancer stem cells that survive oxidative stress via autophagy.</a> Biomolecules. 11 (10), 1479 (2021).</li><li>Flashner, S., Yan, K. S., Nakagawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+organoids:+An+untapped+platform+for+studying+host-microbiome+interactions+in+esophageal+cancers.">3D organoids: An untapped platform for studying host-microbiome interactions in esophageal cancers.</a> Microorganisms. 9 (11), 2182 (2021).</li><li>Liu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2+cooperates+with+inflammation-mediated+stat3+activation+in+the+malignant+transformation+of+foregut+basal+progenitor+cells.">Sox2 cooperates with inflammation-mediated stat3 activation in the malignant transformation of foregut basal progenitor cells.</a> Cell Stem Cell. 12 (3), 304-315 (2013).</li></ol>

&#160; &#160; &#160;There are several critical steps and considerations for the generation and analysis of MEOs in the protocols described here. To ensure reproducibility and rigor in MEO experiments, biological and technical replicates are both important. For biological replicates, two to three independent mice bearing ESCC are generally sufficient per experimental condition. However, the appropriate number of biological replicates may vary depending on the parameters to be tested in individual studies. For example, it ...

Esophageal squamous cell carcinoma (ESCC) is the deadliest of human squamous cell carcinomas, owing to its late diagnosis, therapy resistance, and metastasis1,2. ESCC arises from the stratified squamous epithelium, which lines the luminal surface of the esophagus. The squamous epithelium is comprised of proliferative basal cells and differentiated cells within the suprabasal cell layer. Under physiologic conditions, basal cells express markers such as p63, Sox2, and cytokeratin K5 and K14, while differentiated cells express K4, K13, and IVL. Basal cells themselves are heterogeneous and include putative stem cells defined by markers such as K153 and CD734. In homeostasis, basal cells undergo post-mitotic terminal differentiation within the suprabasal cell layer, whereas differentiated cells migrate and desquamate into the lumen to complete epithelial renewal. Reminiscent of their cells of origin, ESCC displays squamous cell differentiation to varying degrees. ESCC is often accompanied by multifocal histologic precursor lesions, known as intraepithelial neoplasia (IEN) or dysplasia, comprising atypical basaloid cells. In addition to epithelial changes, ESCC displays tissue remodeling within the subepithelial compartment, where the activation of cancer-associated fibroblasts (CAFs) and the recruitment of immune/inflammatory cells take place to foster the tumor-promoting microenvironment.
The pathogenesis of ESCC involves genetic changes and exposure to environmental risk factors. Key genetic lesions include the inactivation of the tumor suppressor genes TP53 and CDKN2A (p16INK4A) and the activation of the CCND1 (cyclin D1) and EGFR oncogenes, which culminate in impaired cell cycle checkpoint function, aberrant proliferation, and survival under genotoxic stress related to exposure to environmental carcinogens. Indeed, genetic changes interact closely with behavioral and environmental risk factors, most commonly tobacco and alcohol use. Tobacco smoke contains human carcinogens such as acetaldehyde, which is also the major metabolite of alcohol. Acetaldehyde induces DNA adducts and interstrand DNA crosslinks, leading to DNA damage and the accumulation of DNA mutations and chromosomal instability. Given excessive mitogenic stimuli and aberrant proliferation from oncogene activation, the malignant transformation of esophageal epithelial cells is facilitated by mechanisms to cope with genotoxic stress, including the activation of antioxidants, autophagy, and epithelial-mesenchymal transition (EMT). Interestingly, these cytoprotective functions are often activated in ESCC cancer stem cells (CSCs) that are characterized by high CD44 (CD44H) expression and have the capabilities of tumor initiation, invasion, metastasis, and therapy resistance5,6,7.
ESCC has been modeled in cell culture and in rodent models8,9. In the last three decades, robust genetically engineered mouse models of ESCC have been developed. These include CCND1 and EGFR transgenic mice10,11 and p53 and p120Ctn knockout mice12,13. However, single genetic changes do not typically result in rapid-onset ESCC. This challenge has been overcome with the use of esophageal carcinogens that recapitulate well the human genetic lesions in ESCC14. For example, 4-nitroquinoline-1-oxide (4NQO) accelerates ESCC development in CCND1 transgenic mice15. In recent years, putative esophageal epithelial stem cells, progenitor cells, and their respective fates have been investigated in cell lineage-traceable mouse models3,4. Furthermore, these cell lineage-traceable mice have been utilized to explore the cells of origin of ESCC and how such cells give rise to CD44H CSCs via conventional histology and omics-based molecular characterization7.
One emerging area related to these mouse models is the novel application of cell culture techniques to analyze live ESCC and precursor cells in a three-dimensional (3D) organoid system in which the architecture of the original tissues is recapitulated ex vivo7,8,9. These 3D organoids are rapidly grown from a single-cell suspension isolated from murine tissues, including primary and metastatic tumors (e.g., lymph node, lung, and liver lesions). The cells are embedded in basement membrane extract (BME) and fed with a well-defined serum-free cell culture medium. The 3D organoids grow within 7-10 days, and the resulting spherical structures are amenable for subculture, cryopreservation, and assays for analyzing a variety of cellular properties and functions, including CSC markers, EMT, autophagy, proliferation, differentiation, and apoptotic cell death.
These methods can be broadly applied to 3D organoid cultures established from any stratified squamous epithelial tissue, such as the head and neck mucosa (oral cavity, tongue, pharynx, and larynx) and even the forestomach. The head and neck mucosa are contiguous with the esophagus, and the two tissues share similar tissue organization, function, and susceptibility to disease. Both head and neck squamous cell carcinoma (HNSCC) and ESCC share genetic lesions and lifestyle-related environmental risk factors such as tobacco and alcohol exposure. Underscoring this similarity, mice treated with the tobacco smoke mimetic 4NQO readily develop both HNSCC and ESCC. Given the ease with which the protocols described below can be applied to modeling HNSCC, we include specific instructions for establishing 3D organoid cultures from these lesions.
Herein, we provide detailed protocols for generating murine esophageal 3D organoids (MEOs) representing normal, preneoplastic, and ESCC lesions that develop in mice treated with 4NQO. Various mouse strains can be utilized, including common laboratory strains such as C57BL/6 and cell lineage-traceable and other genetically engineered derivatives. We emphasize the key steps, including the isolation of normal or diseased murine esophageal epithelium, the preparation of single-cell suspensions, the cultivation and monitoring of the growing 3D organoids, subculture, cryopreservation, and the processing for subsequent analyses, including morphology and other applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>25-200-114</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tuberculin syringe without needle</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L wide bore micropipette tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>212361A</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needles</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-556-006</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Nitroquinoline-1-oxide (4NQO)</td>
			<td>Tokyo Chemical Industry</td>
			<td>NO250</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-565-270</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12556004</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>99.9% ethylene propylene glycol</td>
			<td>SK picglobal</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>15290018</td>
			<td>Stock concentration 250 &#181;g/mL, final concentration 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>Stock concentration 100x, final concentration 1x</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td>Stock concentration 50x, final concentration 1x</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator, e.g.Heracell 150i</td>
			<td>Thermo Fisher Scientific</td>
			<td>51026406</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMQAX1000</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-337-7D</td>
			<td></td>
		</tr>
		<tr>
			<td>DietGel 76A</td>
			<td>Clear H2O</td>
			<td>72-07-5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>MilliporeSigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Corning</td>
			<td>354235</td>
			<td>Stock concentration 50 U/mL, final concentration 2.5&#8211;5 U/mL</td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>VWR</td>
			<td>25870-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190250</td>
			<td>Stock concentration 1x</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>VWR</td>
			<td>82027-386</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing container</td>
			<td>Corning</td>
			<td>432002</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Thermo Fisher Scientific</td>
			<td>G7-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Stock concentration 100x, final concentration 1x</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>Stock concentration 1 M, final concentration 10 mM</td>
		</tr>
		<tr>
			<td>Hot plate/stirrer</td>
			<td>Corning</td>
			<td>PC-420D</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Lab Armor bead bath (or water bath)</td>
			<td>VWR</td>
			<td>89409-222</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Laboratory balance</td>
			<td>Ohaus</td>
			<td>71142841</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Matrigel basement membrane extract (BME)</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Minispin</td>
			<td>Eppendorf</td>
			<td>22620100</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube rack</td>
			<td>Southern Labware</td>
			<td>0061</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td>Stock concentration 100x, final concentration 1x</td>
		</tr>
		<tr>
			<td>N-acetylcysteine (NAC)</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>Stock concentration 0.5 M, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Parafilm M wrap</td>
			<td>Thermo Fisher Scientific</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>MilliporeSigma</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272416</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Recombinant mouse epidermal growth factor (mEGF)</td>
			<td>Peprotech</td>
			<td>315-09-1mg</td>
			<td>Stock concentration 500 ng/&#181;L, final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>RN cell-conditioned medium expressing R-Spondin1 and Noggin (RN CM)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Available through the Organoid and Cell Culture Core upon request, final concentration 2%</td>
		</tr>
		<tr>
			<td>Sorval ST 16R centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004380</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor (STI)</td>
			<td>MilliporeSigma</td>
			<td>T9128</td>
			<td>Stock concentration 250 &#181;g/mL</td>
		</tr>
		<tr>
			<td>ThermoMixer C</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-285-562 PM</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck Chemicals</td>
			<td>S1049</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
	</tbody>
</table>

modeling oral-esophageal squamous cell carcinoma in 3d organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the process of generating murine esophageal organoids (MEOs) from normal esophageal tissue or ESCC tumor tissue from 4NQO-treated mice according to a specific treatment regimen consisting of 16 weeks of 4NQO administered in drinking water, followed by a 10 week to 12 week observation period (Figure 1). The mice are then euthanized for the dissection of the tongue or esophageal tissue (Figure 2 and Figure 3). ...

Watch this Scientific Journal Video about Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids at JoVE.com

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Esophageal squamous cell carcinoma, ESCC, is deadly and prevalent worldwide. Three-dimensional organoids can be utilized to accelerate progress in the field to combat the severe burden of ESCC. Single cell-derived 3D organoids from mice treated with 4NQO can be readily and inexpensively manipulated for functional annotation of the molecular changes accompanying ESCC initiation and development.This model captures the genetic heterogeneity of mutagen-induced tumors. Therefore, these organoids provide a physiologically relevant platform to test novel therapeutic strategies or identify the salient genetic changes driving tumorigenesis. Helping to demonstrate the animal dissection and organ collection will be Yasuto Tomita, a staff associate, and demonstrating the preparation of organoid for paraffin-embedding procedure will be Norihiro Matsuura, a postdoctoral research fellow from my laboratory.To begin, open the skin of the euthanized mice by pinching the midabdominal fur and using surgical scissors, make a craniocaudal, ventral midline incision from the lower abdomen to the chin. Starting at the midline incision, make radial cuts extending to the limbs on both sides of the mouse, inflate the skin flaps open. To expose the cervical trachea, using dissecting scissors, divide the salivary glands at the midline.To expose the thoracic trachea, remove the sternum. Gently pinch and lift the peritoneum with forceps and use scissors to divide the peritoneum craniocaudally. and laterally along the rib cage.Gently retract the liver from the caudal surface of the diaphragm and use scissors to make a small incision in the diaphragm at the sternal notch, specifically at the dorsal surface of the xiphoid process. Once the rib cage is separated from the thoracic contents, insert scissors into the incision in the diaphragm and dissect cranially to the cervical girdle adhering closely to the dorsal surface of the sternum to avoid damage to the organs below. Cut the ribs on either side of the sternum using scissors and remove the sternum.To expose the abdominal esophagus, gently lift the stomach anteriorly by holding the antrum with forceps. Using scissors, dissect the spleen, pancreas, and mesentery from the stomach and esophagus. To expose the thoracic esophagus, gently lift the trachea immediately caudal to the thyroid cartilage, and dissect the esophagus of the dorsal side of the trachea using iris scissors.Divide the trachea at the thyroid cartilage with iris scissors. Peel the trachea off the remainder of the esophagus by carefully dissecting it in the caudal direction. Remove the lung, heart, and thymus altogether with the trachea.Divide the stomach at the pylorus with scissors. Separate the esophagus from the vertebra by holding the antrum with forceps and dissecting cranially. Divide the esophagus at the level of the thyroid cartilage and harvest the esophagus and stomach altogether.Separate the stomach and esophagus by dividing the esophagus at the cardia and dissect any fascia on the outer surface of the esophagus. To reserve a sample for histology, split longitudinally with scissors. Place the remaining intact esophagus and cold PBS on ice.Open the stomach along the greater curvature and wash with PBS sufficiently. Separate the forestomach and wash with cold PBS. To harvest the tongue, remove the pinned needle from the nose and pull out the tongue with tweezers.Cut the tongue as long as possible and place it in cold PBS on ice. After transferring the dissociated tissue to a culture dish, carefully remove the muscle layer from the epithelium using forceps. Transfer the epithelium to a microcentrifuge tube containing 500 microliters of 0.25%trypsin and incubate in a thermomixer for 10 minutes at 37 degrees Celsius and 800 rpm.After brief centrifugation, transfer the cell suspension through a 100-micrometer cell strainer kept on a 50-milliliter conical tube with a wide-bore tip using circular motions, then add 3 milliliters of soybean trypsin inhibitor, or STI, through the strainer using circular motions to wash, then scrub the strainer with the base of a 1-milliliter tuberculin syringe plunger to push the cells through. Wash the strainer with 3 milliliters of PBS 3 to 5 times, scrubbing the strainer with the base of the syringe between washes. After pelleting down the cells by centrifugation, remove the supernatant leaving 1 milliliter of solution in the tube for re-suspension.Once the pellet is resuspended, transfer the cell suspension through a 70-micrometer cell strainer into a new 50-milliliter conical tube. After centrifuging the tube again, resuspend the pellet in 100 microliters of mouse organoid medium, or MOM, and perform an automated cell count. Plate 5, 000 viable cells in 75%basement membrane extract, or BME, and MOM with 50 microliters total volume per well.Using a 200-microliter wide-bore tip, slowly add a 50 microliter droplet to the center of each well avoiding contact between the tip and the bottom or sides of the well. Allow the BME to solidify for 30 minutes in an incubator at 37 degrees Celsius with 5%carbon dioxide and 95%relative humidity. After incubation, carefully add 500 microliters of MOM per well.Change the MOM on day 3 or 4, and then every 2 to 3 days after that until ready for passage. Keep the thawed BME on ice, prewarm MOM, 0.05%trypsin, and STI to 37 degrees Celsius before use. Using a wide-bore micropipette tip, collect the organoids in the BME dome along with the supernatant and disrupt the BME by pipetting up and down.After obtaining the organoid pellet by brief centrifugation, once it is dislodged, resuspend it in 500 microliters of 0.05%trypsin. Incubate the tube in a thermomixer at 37 degrees Celsius and 800 rpm for 10 minutes. Inactivate the trypsin with 600 microliters of STI.After centrifuging and discarding the supernatant, resuspend the cell pellet in 100 microliters of MOM. Perform an automated cell count by trypan blue exclusion. To fix the organoids, gently dislodge the pellet and resuspended in 300 microliters of 4%paraformaldehyde overnight at 4 degrees Celsius.Next, prepare an embedding surface by inverting a microcentrifuge tube rack and covering the surface with a sheet of ceiling film, then label with the corresponding organoid id. Carefully overlay the paraformaldehyde removed in PBS-washed organoid pellet by adding 50 microliters of agar down the side of the tube. Repeat this step.Without disturbing the pellet, transfer the pellet in the agar gel droplet to the ceiling film on the embedding surface for solidification at 4 degrees Celsius for 45 minutes. Using forceps, carefully transfer the droplet containing the solidified organoid pellet to a labeled pathology cassette. The normal murine esophageal tongue and forestomach organoids were analyzed morphologically by brightfield imaging and histological staining.The esophageal squamous cell carcinoma from a 4NQO-treated mouse displayed nuclear atypia and abrupt keratinization. The self-renewal capacity of organoids assessed by determining the organoid formation rate upon subculture showed that the formation rate decreases as a function of time reflecting decreased proliferative basaloid cells in the matured organoids. The growth kinetics of the organoids are revealed by their morphology at various time points.Keratinization of the inner core of organoids becomes prominent by day 10. The proliferative basaloid cells remain in the outermost cell layer, even at the day 14 and day 21 time points. A population doubling curve of normal mouse tongue organoids generated from 4NQO-untreated mice demonstrates their steady growth over multiple passages and long-term culture.Organoids are platforms for high-throughput screening to identify novel therapeutic targets, CRISPR-based editing to interrogate specific gene function or co-culture to define which interactions in the tumor microenvironment influence transformation. This technique has enabled us to investigate phenomena such as partial EMT and HPV infection, along with their interaction with the immune system in the biology of aerodigestive tract squamous cell cancers.

modeling-oral-esophageal-squamous-cell-carcinoma-in-3d-organoids

Research

JoVE Journal

Cancer Research

3.0K Views.  Columbia University. Esophageal squamous cell carcinoma, ESCC, is deadly and prevalent worldwide. Three-dimensional organoids can be utilized to accelerate progress in the field to combat the severe burden of ESCC. Single cell-derived 3D organoids from mice treated with 4NQO can be readily and inexpensively manipulated for functional annotation of the molecular changes accompanying ESCC initiation and development.This model captures the genetic heterogeneity of mutagen-induced tumors. Therefore, these orga...