Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Summary

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Abstract

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

Introduction

Osteosarcoma is the most common primary bone cancer in children and adolescents^1,2, which mainly infiltrates the surrounding tissue, and even metastasizes to the lungs when the patients are diagnosed. Pulmonary metastasis is the main challenge for osteosarcoma therapy, and the five-year survival rate of osteosarcoma patients with pulmonary metastasis remains as low as 20%-30%^3,4,5. However, the five-year survival rate of primary osteosarcoma has been increased to about 70% since the 1970s due to the introduction of chemotherapy⁶. Therefore, it's urgently needed to understand the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapies. The application of mouse models that best mimic the osteosarcoma progression in humans is of great significance⁷.

The osteosarcoma animal models are generated by spontaneous, induced genetic engineering, transplantation, and other techniques. The spontaneous osteosarcoma model is rarely used due to the long tumor formation time, inconsistent tumor occurrence rate, low morbidity, and poor stability^8,9. Although the induced osteosarcoma model is more accessible to obtain than the spontaneous osteosarcoma, the application of the induced osteosarcoma model is limited because the inducing factor will affect the microenvironment, the pathogenesis, and pathological characteristics of osteosarcoma¹⁰. Transgenic models are helping to understand the pathogenesis of cancers since they can better simulate the human physiological and pathological environments; however, the transgenic animal models also have their limitations due to the difficulty, long-term, and high cost of transgenic modification. Moreover, even in the most widely accepted transgenic animal models generated by p53 and Rb gene modification, only 13.6% of sarcoma occurred in the four limb bones^11,12.

Transplantation is one of the most commonly used primary and distant metastatic cancer model-producing methods in recent years due to its simple maneuver, stable tumor formation rate, and better homogeneity¹³. Transplantation includes heterotopic transplantation and orthotopic transplantation according to the transplantation sites. In osteosarcoma heterotopic transplantation, the osteosarcoma cells are injected outside the primary osteosarcoma sites (bone) of the animals, commonly under the skin, subcutaneously¹⁴. Although the heterotopic transplantation is straightforward without the necessity to perform surgery in animals, the sites where the osteosarcoma cells are injected do not represent the actual human osteosarcoma microenvironment. Osteosarcoma orthotopic transplantation is when the osteosarcoma cells are injected into animals' bones, such as tibia^15,16. Compared to the heterotopic grafts, orthotopic osteosarcoma grafts are characterized by a short incubation period, rapid growth, and strong erosive nature; therefore, they are ideal animal models for osteosarcoma-related studies¹⁷.

The most commonly used animals are mice, dogs, and zebrafish^18,19. The spontaneous model of osteosarcoma is usually used in canines because osteosarcoma is one of the most common tumors in canines. However, the application of this model is limited because of the long tumor formation time, the low tumorigenesis rate, poor homogeneity, and stability. Zebrafishes are often used to construct transgenic or knockout tumor models because of their rapid reproduction²⁰. But zebrafish genes are different from human genes, so their applications are limited.

This work describes the detailed procedures, precautions, and representative images for producing the primary osteosarcoma in the tibia with pulmonary metastasis via intratibia injection of osteosarcoma cells in athymic mice. This method was applied to create the primary osteosarcoma in mouse tibia for therapeutic efficacy evaluation, which showed a high reproducibility^21,22.

Protocol

All animal experiments were approved by the animal welfare committee of Shanghai University of Traditional Chinese Medicine. Four-week-old male BALB/c athymic mice were acclimated for a week before the surgery for orthotopic injection of osteosarcoma cells. Mice were housed in individually ventilated mice cages with five mice per cage in a 12-hour light/dark cycle with ad libitum access to SPF feed and sterile water.

1. Preparation of cells

1. On the day of osteosarcoma cell (143B-Luciferase) injection, wash 80%-90% confluent cells cultured in a 10 cm cell culture dish twice with PBS (pH 7.4) and trypsinize with 1.5 mL of 0.25% trypsin for 3 min. Then, add 6 mL of 10% serum-containing MEM media to quench the trypsin, and collect the cells in a 15 mL centrifuge tube.
   NOTE: 143B-Luciferase cell line is obtained from 143B cell line transfect with pLV-luciferase vector²³.
2. Aspirate 20 µL of cell suspension into the chamber of cell counting plate and calculate cell concentration using an automatic cell counter (see Table of Materials).
3. Centrifuge the cells at 800 x g for 5 min at room temperature.
4. Aspirate the supernatant with a pipette and resuspend the cell pellet in an 8.5 mg/mL basement membrane matrix (see Table of Materials) to a final concentration of 2 x 10⁷ cells/mL.
5. Keeping the cells on ice, bring them to the surgery room. The cells are to be used within 2 h.
   NOTE: To avoid inaccurate injection doses (for example, due to the dead space in syringes), an extra cell suspension is prepared (usually two times the required volume of cell suspension). The basement membrane matrix is kept on ice all the time since it has coagulation property above room temperature²⁴.

2. Surgery for orthotopic injection of the osteosarcoma cells

NOTE: The surgery tools are shown in Figure 1.

1. Mice were raised in specific pathogen-free conditions. All procedures were done in an aseptic cabinet with sterile tools.
2. Anesthetize the mice by exposing them to 2% isoflurane and 98% oxygen (oxygen flow rate, 2 L/min).
3. Apply a small amount of ophthalmic ointment on the eyes to prevent dryness while under anesthesia.
   NOTE: Perform the entire procedure in a well-ventilated area. Before osteosarcoma cell injection, ensure that each mouse is under deep anesthesia by a toe pinch; if the mouse still has responses, such as twitch or jerk, wait for a long time until the above responses disappear.
4. Keep each mouse in a 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.
   NOTE: To tightly hold the mouse ankle, both the thumb and index fingertips are of great importance for the subsequent procedures.
5. 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 (the top of the tibia) is clearly visible through the skin (Figure 2A).
6. Attach the needle to a 1 mL syringe and point the needle tip toward the injection site. Ensure that the syringe needle is parallel to the long axis of the tibia.
   1. Percutaneously insert the needle through or adjacent to the patellar ligament as it goes through the skin/joint capsule; th0en, rotate the syringe (1/2 to 3/4-circle) to drill a hole through the tibia platform toward the distal end of the tibia (medullary cavity) for osteosarcoma cell injection with a micro-volume syringe (Figure 2B,C).
      NOTE: Simultaneous rotation of the tibia can be felt while drilling if the needle tip is accurate. Ensure the needling moves forward with the syringe rotation rather than being directly pushed forward until about half of the needle is in the tibia.
7. Check whether the syringe needle made a prominent movement into the medullary canal to ensure successful drilling.
   NOTE: Perform an X-ray examination (see Table of Materials) to confirm the proper position of the needle and collect the images.
8. Load 143B osteosarcoma cell suspension (from step 1.5) into a micro-volume syringe and replace the 1 mL syringe in the tibia with the 143B cell-loaded micro-volume syringe (Figure 2D). Slowly inject ~10 µL (ignore pre-existing solution in the needle) of 143B cell suspension into each athymic mouse's tibia (about 2 x 10⁵ cells) without applying high pressure.
9. Press the injection site with a cotton swab for 20-30 s when the micro-volume syringe is removed.
10. Put each mouse back into a clean cage and closely monitor until the mouse is completely recovered from anesthesia (about 10 min).
11. Monitor the tumor growth in vivo using an X-ray imaging system. Measure the longer diameter (a) and the short diameter (b) of the cancer mass every week with a caliper for tumor volume (V) calculation: V = 1/2 x a x b².
    ​NOTE: Anesthetize the mice by exposing them to 2% isoflurane and 98% oxygen. The mice were anesthetized for x-ray imaging. Intratibia injection of luciferase or fluorescent protein labeled osteosarcoma cells enables tracking of primary and metastatic osteosarcoma lesions.
    NOTE: Humane endpoints of the mice with osteosarcoma due to tumor growth of the knee and lung metastasis were based on the following criteria: (1) Body Condition Score, (2) weight loss threshold of 20%, (3) average maximum diameter of tumors of 2 cm, or (4) severely limited animal behavior.

3. Pathologic examination (collecting primary and pulmonary metastatic osteosarcoma specimen for analysis)

1. Six weeks after osteosarcoma cell injection, sacrifice the mice by cervical dislocation after exposing them for CO₂ inhalation.
2. Keep the mouse in a supine position and stretch both the hind limbs.
3. Separate the whole legs bearing osteosarcoma from the inguinal area.
   NOTE: Ensure that all legs are separated from the same anatomical site.
4. Prepare the histological specimen of legs bearing osteosarcoma by removing the skin, muscles, and feet, and then fix the specimen of each mouse in a 50 mL tube with 20 mL formalin solution (10%) for 24 h, followed by decalcification in 10% EDTA solution for 14 days with occasional buffer change.
5. Embed the specimen in paraffin and prepare sections for histological examination following previously published work²⁵.
6. Gently separate the lungs and put them into a 50 mL tube filled with 20 mL formalin solution (10%). After 24 h, transfer the lungs of each mouse into a 15 mL tube with 70% ethanol. Embed the lungs in paraffin for Hematoxylin and Eosin (H&E) staining and immunohistochemistry assay²⁵.

Results

Successful orthotopic (primary) osteosarcoma and metastatic pulmonary models depend on the accurate orthotopic injection of osteosarcoma cells. Here, an orthotopic (primary) osteosarcoma model via intratibial osteosarcoma cell injection was successfully developed. Figure 3A shows a representative mouse bearing orthotopic (primary) osteosarcoma, and Figure 3B shows a representative isolated orthotopic (primary) osteosarcoma. The tumor volume was measured...

Discussion

Orthotopic injection of osteosarcoma cells is an ideal model to study the function and mechanism of specific factors in osteosarcoma carcinogenesis and development to evaluate the therapeutic efficacy. To avoid differences in tumor growth, most active osteosarcoma cells at 80%-90% confluent with the same number are carefully injected into the tibia of each mouse, and the cell trypsinization time is strictly controlled without affecting the cell viability. As cell clumps affect cell counting leading to inaccurate cell num...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automatic cell counter	Shanghai Simo Biological Technology Co., Ltd	IC1000	Counting cells
Anesthesia machine	Shenzhen RWD Life Technology Co., Ltd	R500IP	The Equipment of Anesthesia mice
BALB/c athymic mice	Shanghai SLAC Laboratory Animal Co, Ltd.	/	animal
Basement Membrane Matrix	Shanghai Uning Bioscience Technology Co., Ltd	356234, BD, Matrigel	re-suspende cells
Bioluminescence imaging system	Shanghai Baitai Technology Co., Ltd	Vieworks	tracking the tumor growth and pulmonary metastasis, if the injection cell is labeled by luciferase
Centrifuge tube (15 mL)	Shanghai YueNian Biotechnology Co., Ltd	430790, Corning	Centrifuge the cells
isoflurane	Shenzhen RWD Life Technology Co., Ltd	VETEASY	Anesthesia mice
MEM media	Shanghai YueNian Biotechnology Co., Ltd	LM-E1141	Cell culture medium
Micro-volume syringe	Shanghai high pigeon industry and trade Co., Ltd	0-50 μL	Inject precise cells into the tibia
Phosphate-buffered saline	Beyotime Biotechnology	ST447	wash the human osteosarcoma cells
1ml syringes	Shandong Weigao Group Medical Polymer Co., Ltd	20200411	drilling
143B cell line	ATCC	CRL-8303	osteosarcoma cell line
Trypsin (0.25%)	Shanghai YueNian Biotechnology Co., Ltd	25200056, Gibco	trypsin treatment of cells
Trypan blue	Beyotime Biotechnology	ST798	Staining cells to assess activity
vector (pLV-luciferase)	Shanghai YueNian Biotechnology Co., Ltd	VL3613	Plasmid
Lipofectamine 2000	Shanghai YueNian Biotechnology Co., Ltd	11668027,Thermo fisher	Plasmid transfection reagent
X-ray imaging system	Brook (Beijing) Technology Co., Ltd	FX PRO	X-ray images were obtained to detect tumor growth