Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

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.