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.

<ol>
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The authors declare no conflicts of interest.

&#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><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 &amp;micro;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 &amp;micro;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 &amp;micro;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 &amp;micro;g/mL, final concentration 0.5 &amp;micro;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>CO&lt;sub&gt;2 &lt;/sub&gt;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&amp;ndash;5 U/mL</td></tr><tr><td>Dissecting scissors</td><td>VWR</td><td>25870-002</td><td></td></tr><tr><td>Dulbecco&#039;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/&amp;micro;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 &amp;micro;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 &amp;micro;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). ...

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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 ...

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JoVE Journal

Cancer Research

3.2K Views.  Columbia University. Oesofageaal plaveiselcelcarcinoom, ESCC, is dodelijk en komt wereldwijd veel voor. Driedimensionale organoïden kunnen worden gebruikt om de vooruitgang in het veld te versnellen om de zware last van ESCC te bestrijden. Single cell-derived 3D-organoïden van muizen behandeld met 4NQO kunnen gemakkelijk en goedkoop worden gemanipuleerd voor functionele annotatie van de moleculaire veranderingen die gepaard gaan met ESCC-initiatie en -ontwikkeling.Dit model legt de genetische heterogenit...

Video: Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Medicine

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 ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

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 ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Bioengineering

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Developmental Biology

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Biology

Preparing Organoids from Tumors: A 3D In Vitro Tumor Model

Source: Wadosky, K. M., et al. <a href="https://www.jove.com/video/59710/" target="_blank">Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer</a>. J. Vis. Exp. (2019).
Organoids derived from tumors are a type of 3D cell culture model that can maintain the pathological features and organ-specific cell lineage of tumors. In the example protocol, we will see scientists generate organoids from a prostate tumor from a genetically engineered mouse model.

Source: Wadosky, K. M., et al. Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer. J. Vis. Exp. (2019).
Organoids ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

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