Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models
October 28th, 2021
The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.
This protocol describes the technique to generate mouse models bearing primary osteosarcoma and the pulmonary metastasis lesions. By intratibial injection of osteosarcoma cells, this model best mimics the clinical development characteristics of tumor osteosarcoma. Accurate numbers of osteosarcoma cells are directly injected into the tibia using a microvolume syringe, allowing for identical tumor formation rates and tumor volumes.
On the day of osteosarcoma cell injection, wash the cells twice with PBS. Trypsinize the cells with 1.5 milliliters of 0.25%trypsin for three minutes. Then add six milliliters of 10%serum-containing MEM media.
Collect the cells in a 15-milliliter centrifuge tube. Aspirate 20 microliters of cell suspension into the chamber of the cell counting plate, and calculate cell concentration using an automatic cell counter. Centrifuge the cells at 800 times g for five minutes at room temperature.
Aspirate the supernatant with a pipette, resuspend the cell pellet in an 8.5-milligram-per-milliliter basement membrane matrix, and place them on ice. Place the mouse in the supine position. Hold the ankle of the mouse using the thumb and index finger, and disinfect the injection site of the tibia with a 70%ethanol swab.
Rotate the ankle joint of each mouse outward to move the tibia and fibula, and bend the knee joint to a suitable position until the proximal tibia plateau is visible through the skin. Attach the needle to a one-milliliter syringe, and point the needle tip toward the injection site, parallel to the axis of the tibia. Percutaneously insert the needle to drill a hole through the tibia platform toward the distal end of the tibia.
Load the osteosarcoma cell suspension previously placed on ice into a microvolume syringe, and replace the one-milliliter syringe in the tibia with the osteosarcoma cell-loaded microvolume syringe. Slowly inject approximately 10 microliters of osteosarcoma cell suspension into each athymic mouse's tibia without applying much pressure. Then remove the microvolume syringe from the injection site, and press the site with the cotton swab for about 30 seconds.
Place the mouse back into a clean cage, and observe its recovery for 10 minutes. This figure depicts the mouse model with orthotopic osteosarcoma developed from intratibial osteosarcoma cell injection and its isolated form. A gradual increase in the tumor volume is observed every week.
The X-ray images obtained from the first through the sixth week after injection of luciferase-labeled osteosarcoma cells showed a gradual increase in volume. Furthermore, the image obtained after the injection into the mouse tibia showed orthotopic osteosarcoma growth in vivo. The pulmonary metastasis caused by intratibial injection of the osteosarcoma cells was successfully tracked in vivo using a bioluminescence live imaging system.
Observation under a stereomicroscope revealed metastatic colonies in the isolated lung tissues. HE staining on paraffin-embedded lung tissues showed metastatic lesions. The needle should be percutaneously inserted into the joint capsule through the patellar ligament to drill a hole through the tibia platform toward the digital tibia by rotating the syringe.
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Overview
This protocol describes the technique to generate mouse models bearing primary osteosarcoma and pulmonary metastasis lesions. By intratibial injection of osteosarcoma cells, this model best mimics the clinical development characteristics of tumor osteosarcoma.
Key Study Components
Area of Science
- Neuroscience
- Oncology
- Animal Models
Background
- Osteosarcoma is a common bone cancer in children and adolescents.
- Understanding its metastasis is crucial for developing effective treatments.
- Animal models are essential for studying tumor behavior and treatment responses.
- Intratibial injection allows for localized tumor growth and metastasis study.
Purpose of Study
- To establish a reliable mouse model for osteosarcoma research.
- To investigate the characteristics of primary tumors and their metastases.
- To facilitate the testing of therapeutic strategies in a controlled environment.
Methods Used
- Intratibial injection of osteosarcoma cells using a microvolume syringe.
- Washing cells with PBS prior to injection.
- Trypsinization of cells to prepare for injection.
- Monitoring tumor formation rates and volumes post-injection.
Main Results
- Consistent tumor formation rates were achieved across models.
- Identical tumor volumes were observed, indicating reproducibility.
- Successful establishment of pulmonary metastasis in the models.
- Model reflects clinical characteristics of osteosarcoma progression.
Conclusions
- The intratibial injection method is effective for osteosarcoma modeling.
- This model can be used for future therapeutic studies.
- Findings contribute to understanding osteosarcoma biology and treatment.
Frequently Asked Questions
Executive Industry Relevance
Orthotopic osteosarcoma models generated via intratibial injection enable reproducible tumor formation and pulmonary metastasis, providing a clinically relevant system for target validation and therapeutic efficacy testing. This approach supports mechanistic de-risking by recapitulating human disease progression in immunocompromised mice, allowing for longitudinal monitoring of primary and metastatic burden. The model’s sensitivity to bioluminescence and X-ray imaging facilitates quantitative readouts essential for preclinical go/no-go decisions in oncology pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses in a disease-relevant system that mimics human osteosarcoma carcinogenesis and lung metastasis.
- Operational Value: Provides consistent tumor take rates and measurable growth kinetics for reliable target engagement and pathway modulation studies.
- Predictive Value: Supports preclinical model selection by reflecting clinical metastatic patterns, improving confidence in target prioritization.
Screening & Assay Development
- Scientific Value: Generates standardized orthotopic tumors suitable for preparing validated biological systems used in downstream drug screening.
- Operational Value: Enables reproducible quantification of tumor volume and metastatic burden via imaging, supporting assay standardization across studies.
- Scalability: Short incubation period and rapid growth allow for timely compound evaluation and platform reuse in lead identification campaigns.
Translational & Preclinical Research
- Translational Continuity: Mirrors clinical osteosarcoma progression with pulmonary metastasis as the dominant lethal phenotype, aligning with patient outcomes.
- Mechanistic De-risking: Allows evaluation of tumor microenvironment interactions and metastatic drivers in a physiologically contextualized model.
- Therapeutic Assessment: Facilitates in vivo efficacy testing of candidates, with primary tumor and metastasis lesions isolatable for endpoint analysis.
Pipeline & Workflow Integration
The model fits within the discovery continuum from target validation through lead identification to preclinical efficacy, particularly for bone-tropic and metastasis-modulating agents.
- Discovery Biology: Supports hypothesis testing on osteosarcoma drivers and metastasis suppressors using an orthotopic, immunocompromised host system.
- Screening: Delivers quantitative, imaging-based outputs (bioluminescence, X-ray) that enable comparison of treatment effects on tumor growth and dissemination.
- Analytics: Provides longitudinal, quantifiable metrics on tumor burden and metastatic incidence, aiding cross-functional data interpretation.
- Translational Research: Connects early mechanistic findings to preclinical validation through clinically relevant metastatic patterns.
- Enterprise Reuse: Establishes a reusable platform for osteosarcoma oncology programs, reducing model redevelopment across projects.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence by modeling the full spectrum of osteosarcoma progression, including metastatic dissemination to lungs.
- Operational Value: Ensures reproducibility through standardized cell preparation, injection technique, and imaging-based monitoring.
- Strategic Value: Improves capital efficiency by enabling early detection of ineffective candidates via metastasis-sensitive endpoints.
- Portfolio Impact: Informs risk-adjusted advancement decisions by providing mechanistic insight into tumor-stroma interactions and metastatic potential.
Implementation Considerations
- Requires expertise in orthotopic injection techniques and handling of immunocompromised mouse models.
- Dependent on access to microvolume syringes, basement membrane matrix, and bioluminescence/X-ray imaging systems.
- Necessitates cross-team standardization of cell preparation, injection volume, and endpoint criteria for reproducible results.
- Adaptation to alternative osteosarcoma cell lines may require optimization of matrix concentration and injection volume.
- Practical limitations include model variability due to injection precision and the need for IACUC-compliant tumor size endpoints.
