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 ...

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

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

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 number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

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

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.