Name: modeling oral-esophageal squamous cell carcinoma in 3d organoids
Uploaded: 2022-12-23T14:30:00
Description: Watch this Scientific Journal Video about Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids at JoVE.com

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...

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&#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 ...

Research

JoVE Journal

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Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

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Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

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