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 prognosis1,2. 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.
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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 BALB/c thymus-free mice were provided with specific pathogen-free (SPF) feed and sterile water, and were housed in a single ventilated mouse 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.
- 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. - Transfer the freshly resected osteosarcoma tissues stored in the tissue protective solution to the laboratory as soon as possible.
- 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 disinfected by autoclaving before use. - Rinse the osteosarcoma tissues twice with pre-cooled sterile physiological saline in a sterile hood.
- Remove areas of the osteosarcoma tissue with bleeding and necrosis.
- 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
- Place the nude mice on sterile surgical drapes.
- Anesthetize the mice with 2% isoflurane by supplying oxygen at a flow rate of 2 L/min in a well-ventilated area.
- 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. - Secure the mouse in a lateral decubitus position (Figure 2A). Disinfect one side of the mouse flank region for osteosarcoma tissue transplantation with 70% ethanol disinfectant cotton balls.
NOTE: Surgical disinfection is performed in a circular pattern, beginning in the center and spiraling outward. - Mark the surgical incision site on the skin with a marker pen.
- Make a 5 mm incision from the skin to the subcutaneous tissues with a scalpel (Figure 2B).
- 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).
- 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. - Suture the incision using surgical lines with 2-3 stitches for a 5 mm incision (Figure 2D).
- 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
- Sacrifice mice using the cervical dislocation method after CO2 inhalation when the tumor size reaches 1500 mm3.
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. - Position the mouse laterally to expose the tumor, and disinfect the skin at the tumor site with an alcohol-soaked cotton ball.
- 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
- 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.
- Dehydrate and embed the tumor tissues in paraffin14.
- Slice the tumor tissues and conduct the routine histological examination15.
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Representative 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 15-year-old patient. Figure 3B depicts a representative mouse of PDX at P1, one month after transplantation of PDX tissue at P0. Figure 3C displays a representative mouse of PDX at P2, one month after transplantation of PDX tissue at P1. Figure 3D illustrates a representative mouse of PDX at P3, one month after transplantation of PDX tissue at P2.
Mice were sacrificed using the cervical dislocation method after CO2 inhalation when the tumor size reached 1,500 mm3 to collect the PDX tumor tissues. The tumor tissues from clinical patients and mice were then embedded in paraffin and subjected to H&E staining. A representative H&E staining image of clinical patient tumor tissue is shown in Figure 4A. Representative H&E staining images of PDX mouse tumor tissues at P0, P1, and P2 are shown in Figure 4B,C,D. The osteosarcoma features, characterized by small cells with round, hyperchromatic nuclei in the parental clinical patient tumor tissues, were maintained in established PDXs at P0, P1, and P2.
Figure 1: Surgical instruments. (A) Scalpel. (B,C) Ophthalmic tweezers. (D) Ophthalmic scissor. (E) Suture needle. (F) Suture line. (G) Straight needle holder. (H) Marking pen. Please click here to view a larger version of this figure.
Figure 2: Establishment of PDX model. (A) The osteosarcoma tissue transplantation site at the mouse flank region is labeled with a marking pen. (B) A 5 mm length incision was cut from the skin to the subcutaneous tissue. (C) Blunt dissection and osteosarcoma tissue transplantation. (D) Sutured incision. Please click here to view a larger version of this figure.
Figure 3: Growth of PDX transplanted originally with femoral osteosarcoma tissue from a 15-year-old patient. (A) PDX established by transplanting the human osteosarcoma tissue (P0). (B) PDX established by transplanting the PDX tissue at P0 (P1). (C) PDX established by transplanting the PDX tissue at P1 (P2). (D) PDX established by transplanting the PDX tissue at P2 (P3). Please click here to view a larger version of this figure.
Figure 4: Hematoxylin-Eosin (H&E) staining of tissue sections from primary clinical and PDX tissues. H&E staining detected histological features of (A) Femoral osteosarcoma from the 15-year-old patient, (B) PDX at P0, (C) PDX at P1, (D) PDX at P2. Scale bars: (A-D) 100 µm; (E-H) 20 µm. Please click here to view a larger version of this figure.
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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 profile16,17,18,19. Therefore, they preserve the molecular phenotypes and genotypes of cancer patients, providing innovative approaches for studying biology and evaluating potential therapeutics. These approaches include preclinical screening of novel anticancer drugs with highly similar responses to real clinical responses, detecting drug resistance biomarkers, screening sensitive drugs in personalized clinical treatment regimen selection, and predicting patient outcomes20. Furthermore, the establishment process of PDX models is relatively simple and does not require special equipment.
This study successfully established a PDX mouse model using human osteosarcoma tissues. Several key aspects of successfully establishing a PDX model with human osteosarcoma tissues were revealed. Firstly, the transplantability of human osteosarcoma tissues in mice is feasible, enabling the study of human osteosarcoma biology in a murine system. The high tumor formation rate may be attributed to the use of immunodeficient mice lacking functional immune systems, allowing transplanted osteosarcoma cells to proliferate without rejection. Moreover, this study demonstrated that the PDX model maintained the histology of the original human osteosarcoma tissues. The tumor morphology and histopathological features were similar between the primary patient tumor and the PDX tumors. This indicates that the PDX model accurately recapitulates osteosarcoma, making it a valuable tool for studying osteosarcoma progression, metastasis, and response to treatments.
However, several key points need to be addressed during PDX modeling. Firstly, human osteosarcoma tissues used for transplantation must be freshly resected and should be stored in a tissue protective solution as soon as possible. It's preferable to complete the transplantation within 12 h after resection, although the activity of tumor cells in the human osteosarcoma tissues stored in the tissue protective solution can be maintained for a maximum of 24 h16. Secondly, it's preferable to use tumor tissues from patients who have not received chemotherapy to maximally preserve the molecular phenotypes and genotypes of patients, although human osteosarcoma tissues from patients having received chemotherapy once or twice can be successfully transplanted into immunodeficient mice. Moreover, tumor cell activity will be poor if a patient receives multiple chemotherapies, leading to transplantation modeling failure; however, osteosarcoma tissues from relapsed patients may have a higher successful tumor formation rate after being transplanted into immunodeficient mice than osteosarcoma tissues from patients diagnosed initially. Thirdly, transplanting osteosarcoma tissues just below the dermis of the skin will improve the tumor formation rate in immunodeficient mice21.
It is important to acknowledge the limitations of this study. Firstly, the establishment cycle of the PDX model is long and takes 4-12 weeks from transplantation to tumor formation22. Secondly, information related to tumor initiation and etiology may be absent because fully developed cancer cell osteosarcoma tissues are transplanted, and rare subclones or genomic alterations may be lost during the transplantation process. Whole-genome sequencing of PDX tumors and comparison with patient samples would be valuable to assess the genetic fidelity of the model23. Thirdly, mouse host cells can infiltrate tumors, potentially affecting tumor cell activity9. Fourthly, the PDX model has a failure rate of about 50%24,25. Finally, in some cases, cancer metastasis cannot be studied due to the absence of tumor cell metastasis after being transplanted into immunodeficient mice26.
In conclusion, this study validates the successful establishment of a PDX mouse model using human osteosarcoma tissues. This model faithfully recapitulates the histopathological features of the original tumor, providing a valuable tool for exploring osteosarcoma biology and testing potential therapeutics. Further studies using this PDX model will enhance our understanding of osteosarcoma and result in the development of more effective strategies for these patients.
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Disclosures
The authors declare no competing financial interests.
Acknowledgments
This work is supported by grants from (1) the National Nature Science Foundation (81973877 and 82174408); (2) Shanghai Top Priority Research Center construction project (2022ZZ01009); (3) National Key R&D Program of China (2020YFE0201600); (4) Shanghai Collaborative Innovation Center of Industrial Transformation of Hospital TCM Preparation and (5) Research Projects within Budget of Shanghai University of Traditional Chinese Medicine (2021LK047).
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 |
| BALB/c athymic mice | 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 |
| 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 |
| 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 |
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