Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Esophageal squamous cell carcinoma (ESCC) is the deadliest of human squamous cell carcinomas, owing to its late diagnosis, therapy resistance, and metastasis^1,2. 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 ce....

Protokół

The murine experiments were planned and performed in accordance with regulations and under animal protocol #AABB1502, reviewed and approved by Columbia University'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 lesions (time consideration: up to 28 weeks)

NOTE: To generate MEOs representing neoplastic esophageal lesions, the....

Wyniki

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). .......

Dyskusje

     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 .......

Materiały

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