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

This protocol helps establish ATC and HNSCC PDX models which better reflect tumor heterogeneity, molecular diversity, and predict clinic outcomes. The technique is simple to operate, has a high success rate, and can also be suitable for other PDX models. This technique can be extended toward anti-cancer drug sensitivity screening to guide the personalized treatment of cancer patient.

An individual who has never performed this technique may encounter some difficulties and paying attention to every detail of the scheme is recommended. After obtaining ATC and HNSCC tumor samples in a 50-millimeter centrifuge tube containing sterile HTK solution, disinfect them with 75%alcohol. Use sterilized ophthalmic forceps to transfer the tumor tissue to a six-centimeter Petri dish filled with saline, and cut them into small pieces using a blade.

Then, transfer the pieces into a new six-centimeter Petri dish containing the saline and wrap it with sealing film. Place the wrapped dish in an ice box, carry it to the pathogen-free animal room with scissors, forceps, and inoculation needles. After anesthetizing the mouse, remove the hair on the right lateral thorax and disinfect the area with 75%alcohol.

Use scissors to make a two-millimeter incision in the middle of the right lateral thorax. With forceps, take the tissue from the Petri dish and place it into the trocar needle. Hold the mouse and tighten the skin at the puncture site.

Insert the trocar with the tumor piece into the incision to the subcutaneous space, then move to the back of the shoulder and push the trocar core. To reserve the remaining tissue using a sterile gauze, remove saline from the tumor surface, ensuring it's not excessively wet. Put four to six tissue pieces in a two-milliliter cell cryopreservation tube and add one milliliter of cryopreservation solution into the tube.

Then, place the tube in a gradient cooling box and freeze it at minus 80 degrees Celsius overnight. Finally, transfer it to liquid nitrogen. For resuscitating the PDX model tumors, use Vernier calipers to measure the length and width of the the subcutaneous tumor once a week.

Calculate the tumor volume and draw the tumor growth curve. Then, using scissors, cut the euthanized mouse skin surrounding the tumor. Remove the tumor with forceps and place it in a Petri dish.

Perform the tumor transplantation till the fifth generation of mice, as previously demonstrated. Select the tumor tissue of a P5 generation ATC PDX model mouse. Cut it into two to four cubic millimeter pieces.

Inoculate these pieces subcutaneously as previously demonstrated, to the right back of a nude, four to six week female BALB/c mouse. When the tumor grows 50 to 150 cubic millimeters, divide the mice into three groups. After administering lenvatinib, cisplatin, and control twice a week, measure the mouse's body weight.

Also, measure the tumor volume. The correlation analysis show that the success rate of ATC PDX model construction was not dependent on age, gender, tumor diameter, tumor grade, and differentiation. The tumor take rate in the HNSCC PDX model construction demonstrated that the degree of differentiation was associated with model success rate, while the other parameters did not affect the tumor take rate.

The tumor growth curves for each PDX model illustrated that the ATC samples were stably passed to the P3 generation, while two cases of HNSCC samples failed to form tumors after passing to the P1 generation. The tumor growth rate of the P0 generation for most PDX models was relatively slower than for the later passages, likely due to the adaptation of the mouse microenvironment. Histopathological examination demonstrated that the PDX tumors retain the morphological characteristics of the primary tumors.

Treatment with lenvatinib significantly inhibited tumor growth and showed lower tumor weight in the ATC PDX model compared to the control group. Additionally, lenvatinib-treated mice did not exhibit discernible changes in their overall weight. Excessive cisplatin dosage resulted in significant toxicity, as evidenced by weight loss and even death.

Fresh tumors must be washed several times with normal saline before being cut into pieces. Mixing by biopsied tumor samples with and inoculating them will likely cause tumor formation. This model can be used for tumor biology research, drug screening, personalized therapy.