Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Summary

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Abstract

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Introduction

Osteosarcoma is a primary bone malignancy derived from interosseous lobe cells and is most common in adolescents as well as children. It often occurs in the epiphysis of the long diaphysis and is characterized by high malignancy, early metastasis, and poor prognosis^1,2. Lung metastasis is the main cause of death in osteosarcoma patients. The 5-year survival rate of patients with non-metastatic osteosarcoma is 65%-70%³. However, over the last 40 years, the 5-year survival rate (only 20%) of patients with metastatic osteosarcoma has not significantly improved, and 25% of osteosarcoma patients have metastases at the time of diagnosis⁴. Currently, the first-line drugs for osteosarcoma treatment have reached a consensus, but there are still significant differences in chemotherapy regimen and treatment time⁵. It is important to perform preclinical experiments based on appropriate animal models to obtain more effective chemotherapy regimens.

Currently, models commonly used for osteosarcoma preclinical experiments include cell line-based in vitro cell culture and in vivo cell-derived xenografts (CDX), as well as patient-derived xenografts (PDX)^6,7.

The cell lines are convenient for culturing and for use in in vitro studies, or for transplantation into immunodeficient mice to establish CDX models⁸. However, cell lines cultured in vitro may not accurately reflect the heterogeneity of malignancies and the individual characteristics of patients due to potential mutations that occur to adapt to the in vitro culture environment during repeated passages. Additionally, they lack the microenvironment and immune system necessary for tumor growth and development in vivo. While CDX models offer some advantages over in vitro cell culture, they still may not fully reflect the individual characteristics of osteosarcoma patients, although tumor tissues obtained from CDX models have limited intratumoral heterogeneity and immune system representation compared to cell lines cultured in vitro⁹. Therefore, establishing a preclinical model with high fidelity is crucial.

PDX models involve the immediate transplantation of freshly resected human cancer tissues into immunodeficient mice. This method allows for the faithful preservation of genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient, even after successive passages in mice. Furthermore, PDX models are known to accurately predict later clinical outcomes¹⁰, making them valuable tools for creating individualized treatments and advancing precision medicine research¹¹.

This work describes the procedure for establishing a PDX model in immunodeficient mice by transplanting human osteosarcoma tissue. Such models serve as platforms for conducting preclinical experiments for osteosarcoma.

Protocol

All studies involving human tissues have been approved by the Institutional Ethics Review Committee of Longhua Hospital, affiliated with Shanghai University of Traditional Chinese Medicine (Shanghai, China) (2013LC52), and written informed consent was obtained from the patients in accordance with the Helsinki Declaration. The IACUC number for this animal study is PZSHUTCM221017013. Four-week-old male CAnN.Cg-Foxn1nu/Crl mice were provided with double lion Irradiated Rodent Diet GB 14924.3 and sterile water, and were housed in IVCs mice cage with five mice per cage, under SPF conditions with a 12-h light/dark cycle. The Table of Materials includes detailed information about all materials, reagents, and instruments used in this protocol.

1. Preparation of human osteosarcoma tissue

NOTE: In this study, the human osteosarcoma tissue was resected¹² from the femur lesion of a 15-year-old osteosarcoma patient before chemotherapy.

1. Immediately store the freshly resected osteosarcoma tissue in a tissue-protective solution to maximize the preservation of osteosarcoma cell activation after rinsing with sterile physiological saline.
   NOTE: Freshly resected osteosarcoma tissues must be transplanted into mice as soon as possible. They can be stored in the tissue protective solution for a maximum of 24 h before transplantation. Osteosarcoma tissues used for modeling must be from patients who have not received chemotherapy. The activity of tumor cells from patients who received chemotherapy will be poor, leading to the failure of model establishment and loss of high fidelity.
2. Transfer the freshly resected osteosarcoma tissues stored in the tissue protective solution to the laboratory as soon as possible.
3. Prepare experimental instruments and materials for establishing the PDX model: scalpels (Figure 1A); ophthalmic tweezers (Figure 1B,C); ophthalmic scissor (Figure 1D); suture needle (Figure 1E); suture line (Figure 1F); straight needle holder (Figure 1G); marking pen (Figure 1H).
   NOTE: Ensure that all surgical instruments are sterilized by autoclaving before use.
4. Rinse the osteosarcoma tissues twice with pre-cooled sterile physiological saline in a sterile hood.
5. Remove areas of the osteosarcoma tissue with bleeding and necrosis.
6. Cut the cleaned osteosarcoma tissue into 3 mm³ pieces with a scalpel in a culture dish containing pre-cooled sterile physiological saline and keep it on ice.

2. Establishment of PDX models by osteosarcoma tissue transplantation at mouse flank region

1. Administer preoperative analgesia of meloxicam (5 mg/kg/24 h) by subcutaneous injection in mice and place the nude mice on sterile surgical drapes. Provide thermal support throughout the procedure.
2. Induce anesthesia in mice by exposing them to 3% isoflurane and 97% oxygen. After the mice are completely anesthetized, transfer them to the nose cone and maintain anesthesia with 1.5% isoflurane and 98.5% oxygen.
3. Pinch the toes to ensure the mice are completely anesthetized, and wait longer if there are still spasms or convulsive reactions.
   NOTE: All procedures must be carried out using sterile equipment in a sterile hood. Avoid dryness of mice during anesthesia by applying ophthalmic ointment to their eyes.
4. Secure the mouse in a lateral decubitus position (Figure 2A). Disinfect one side of the mouse flank region for osteosarcoma tissue transplantation 3 times with alternating rounds of povidone iodine and 70% ethanol disinfectant cotton balls.
   NOTE: Surgical disinfection is performed in a circular pattern, beginning in the center and spiraling outward.
5. Mark the surgical incision site on the skin with a marker pen.
6. Make a 5 mm incision from the skin to the subcutaneous tissues with a scalpel (Figure 2B).
7. Lift the upper side skin of the incision margin with ophthalmic tweezers using the left hand, and perform blunt dissection upward under the dermis of the mice with a straight needle holder using the right hand (Figure 2C).
8. Hold the upper side skin of the incision with the ophthalmic tweezers using the left hand, and place the osteosarcoma tissue under the skin approximately 5 mm above the incision margin with ophthalmic tweezers using the right hand to transplant the osteosarcoma tissue (Figure 2C).
   NOTE: Ensure the osteosarcoma tissue is transplanted just below the dermis of the skin. Blunt dissection is performed with a straight and blunt dissection forceps, such as a straight needle holder, to avoid penetrating the mouse thoracic cavity while finding the dermis layer of the mouse skin.
9. Suture the incision using absorbable suture with 2-3 stitches for a 5 mm incision (Figure 2D).
10. Return the mice to clean cages and monitor them until they completely recover from anesthesia.
    NOTE: PDX established by transplanting the human osteosarcoma tissue is designated as passage 0 (P0), PDX established by transplanting the PDX tissue at P0 is designated as passage 1 (P1), followed by P2 and P3.

3. Collection of PDX tumor tissues

1. Measure the tumor volume once a week. When the tumor size reaches 1500 mm³, euthanize the mice using the cervical dislocation method after CO₂ inhalation. Humane endpoints include tumor ulceration or issues with front limb mobility.
   NOTE: Measure the long diameter (a) and short diameter (b) of the tumor using a caliper. Calculate the tumor volume (V) using the formula: V = 1/2 × a × b².
2. Position the mouse laterally to expose the tumor, and disinfect the skin at the tumor site with an alcohol-soaked cotton ball.
3. Use ophthalmic scissors to separate the entire tumor. Weigh the tumor mass using an electronic scale.

4. Pathological examination of primary clinical and PDX tumor tissues

1. Fix the tumor tissues¹³ in a 50 mL tube containing 30 mL of 10% neutral buffered formalin solution for 24 h. Rinse the tumor tissues thoroughly with flowing water to remove the fixative.
2. Dehydrate and embed the tumor tissues in paraffin¹⁴.
3. Slice the tumor tissues and conduct the routine histological examination¹⁵.

Results

This protocol describes the detailed procedure for establishing a PDX mouse model, preserving the morphological features of human osteosarcoma after immediate transplantation of freshly resected human osteosarcoma tissue and successive passages in mice. Here, a PDX mouse model was successfully established using human osteosarcoma tissue.

Figure 3A shows a representative mouse of PDX at P0, two months after the transplantation of femoral osteosarcoma tissue from a ...

Discussion

The PDX models can simulate the characteristics of human cancers and retain more similarity with the primary tumor, including genetic and genomic alterations, histology, heterogeneity, and gene expression profile^16,17,18,19. Therefore, they preserve the molecular phenotypes and genotypes of cancer patients, providing innovative approaches for studying biology and evaluating potential therapeuti...

Materials

Name	Company	Catalog Number	Comments
10% formalin neutral solution	Wuhan Saiweier Biotechnology Co., Ltd	G1101-500ml	Fix the tissues
Autoclave	Japan Hiryama Company	HVE-50	Sterilization surgical instruments
CAnN.Cg-Foxn1nu/Crl	Shanghai SLAC Laboratory Animal Co, Ltd.	/	Animal
Caliper	Yantai Green Forest Tools Co., Ltd.	034180A	Measure the tumor volume
Dish (60mm)	Shanghai NianYue Biotechnology Co., Ltd	430166, Corning	Sample placment during transplantation
Disinfectant cotton balls	Shanghai Honglong Industrial Co., Ltd.	20230627	Disinfect the skin of mice
Disposable sterile gloves	Guilin Hengbao Health Protection Co., Ltd.	YT21131	Sterile operation
Double lion Irradiated Rodent Diet	Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.	GB 14924.3	Animal feed
Electronic scale	Shanghai NianYue Biotechnology Co., Ltd	1-2000	Weigh the weight of the tumor
Eosin	Shanghai Gengyun Biotechnology Co., Ltd	E4009-25G	Hematoxylin eosin stain
Hematoxylin	Shanghai Gengyun Biotechnology Co., Ltd	H3136-25G	Hematoxylin eosin stain
Isoflurane	Shenzhen RWD Life Technology Co., Ltd	VETEASY	Mouse anesthesia
IVCs mice cage	Suzhou Monkey King Animal Experimental Equipment Technology Co., Ltd.	HH-MMB-2	Animal barrier
Mark pen	Zebra Trading (Shenzhen) Co., Ltd.	YYST5	Mark the surgical incision
Olympus Optical microscope	Japanese Olympus Company	BH20	Scan tissue slices
Ophthalmic ointment	Shanghai Gengyun Biotechnology Co., Ltd	SOICOEYEGRL	Avoid dry eyes of mice during anesthesia
Ophthalmic scissors	Shanghai NianYue Biotechnology Co., Ltd	Y00030 JZ	Cut the skin
Ophthalmic tweezers	Shanghai NianYue Biotechnology Co., Ltd	BS-ZER-S-100 Biosharp	Hold osteosarcoma tissues during transplantation
Paraffin	Jiangsu Shitai Experimental Equipment Co., Ltd.	80200-0015	Buried osteosarcoma tissue
Paraffin slicing machine	Lyca Microsystem (Shanghai) Trading Co., Ltd.	RM2235	Osteosarcoma tissue section
physiological saline	Guangzhou Jinsheng Biotechnology Co., Ltd.	605-004057	Rinse and temporary storage of osteosarcoma tissue
Scalpels	Surgical Instrument Factory of Shanghai Medical Devices (Group) Co., Ltd.	J11010-10# JZ	Separation of osteosarcoma tissue and making surgical incisions
Sterile hood	Thermo Fisher Technology (China) Co., Ltd.	ECO0.9	Surgical operation table
sterile surgical drapes	Henan Huayu Medical Equipment Co., Ltd.	20160090	Provide sterile surgery area
Straight needle holder	Shanghai Gengyun Biotechnology Co., Ltd	J31050 JZ	Suture the wound
Suture line	Shanghai Pudong Jinhuan Medical Products Co., Ltd	F3124	Suture the wound
Suture needle	Shanghai Pudong Jinhuan Medical Products Co., Ltd	F3124	Suture the wound
Tissue protective solution	Nanjing Shenghang Biotechnology Co., LTD	BC-CFM-03	Maintain the activity of tissue cells
Tube (50 mL)	Shanghai Baisai, Biotechnology Co., Ltd.	BLD-BL2002500	Install formalin fixation solution