The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.
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.
Osteosarcoma is the most common primary bone cancer in children and adolescents1,2, 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 stability8,9. 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 bones11,12.
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 tibia15,16. 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 zebrafish18,19. 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 reproducibility21,22.
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
2. Surgery for orthotopic injection of the osteosarcoma cells
NOTE: The surgery tools are shown in Figure 1.
3. Pathologic examination (collecting primary and pulmonary metastatic osteosarcoma specimen for analysis)
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 once a week with a caliper and calculated as described in step 2.11 (Figure 3C). The orthotopic (primary) osteosarcoma growth in vivo was tracked by both the X-ray and the bioluminescence (when the injected cells were labeled with luciferase) live imaging system. The X-ray images were obtained from the first week to the sixth week after 143B osteosarcoma cell injection (Figure 3D). Furthermore, the image of orthotopic (primary) osteosarcoma growth in vivo was obtained after luciferase labeled 143B cells were injected into the mouse tibia (Figure 3E).
The pulmonary metastasis caused by the intratibial injection of luciferase labeled osteosarcoma cells was successfully tracked in vivo by a bioluminescence live imaging system (Figure 4A). The metastatic colonies in the isolated lung tissues were also visualized under the stereomicroscope (Figure 4B). The metastatic lesions were further confirmed by H&E staining on paraffin-embedded lung tissues (Figure 4C).
Figure 1: Surgery Tools. (A) 1 mL scale syringe. (B) Micro-volume syringe. Please click here to view a larger version of this figure.
Figure 2: Representation of the intratibial injection surgery. (A) The intratibial injection site of an athymic mouse. (B) A sterile 1 mL syringe with an accompanied needle was percutaneously inserted into the tibia toward the distal end via the proximal tibia plateau (the top of the tibia). (C) A lateral view of the drilling process. The syringe needle was parallel to the long tibia axis (solid line). (D) Intratibial injection with osteosarcoma cell loaded micro-volume syringe. Please click here to view a larger version of this figure.
Figure 3: Visualization of the osteosarcoma growth in mice. (A) Successful mouse orthotopic osteosarcoma model. (B) Isolated orthotopic osteosarcoma. (C) Tumor volume was measured with a caliper and calculated using the following formula: tumor volume = 0.5 x longer diameter x short diameter x short diameter. Error bars stand for standard deviation (n = 8). (D) X-ray images were obtained from the same mouse at a different time (from 1-6 weeks). (E) Image obtained on the 28th day after luciferase labeled 143B cells were injected into the mouse tibia. The red arrows indicated the luminescence intensity of the orthotopic (primary) osteosarcoma. Please click here to view a larger version of this figure.
Figure 4: Pulmonary metastasis of osteosarcoma. (A) Image obtained on the 28th day after luciferase labeled 143B cells were injected into the mouse tibia. The red arrows indicated the luminescence intensity of the pulmonary metastasis. (B) The isolated lungs with osteosarcoma metastases. The red arrows indicated the metastatic colonies (x20). (C) H&E staining showed metastatic lesions in lung tissues (scale bar = 200 µm). Please click here to view a larger version of this figure.
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 numbers being injected into the tibia of each mouse, the cell suspension needs to be appropriately mixed up and down with a pipette to avoid the formation of cell clumps.
Another critical aspect that must be taken into consideration is the resuspended solution for osteosarcoma cells. The injected cells are resuspended in a basement membrane matrix instead of in PBS or culture medium. Moreover, a high concentration of basement membrane matrix is challenging to be pipetted and affects the accurate volume; thus, an appropriate concentration of basement membrane matrix is required26. To drill a hole through the tibia platform for osteosarcoma cell injection, the needling moves forward with the syringe rotation rather than being directly pushed forward until about half of the needle is in the tibia. More particularly, immunodeficient mice are applied to establish an orthotopic osteosarcoma model using human osteosarcoma cells27. Meanwhile, the injection procedure is performed in biological safety cabinet using sterile surgical tools. Since mice may experience uneasiness after anesthesia and surgery, the mice must be closely monitored on the first week postsurgery.
Intratibia injection of osteosarcoma cells labeled with fluorescent protein or luciferase enables the tracking of primary and metastatic lesions using optical imaging28. Osteosarcoma is never allowed beyond the size limit as in the IACUC-approved protocol; meanwhile, ulcerations may occur in enormous size tumor mass, which may lead to failed immunohistochemical analyses. Although the primary bone tumors and bone metastasis have been recently reported to be achieved by implantation of solid tumor graft into bone, and the animals developed reproducible growth, as well as lung metastasis eventually29; however, the authors directly implanted fresh or cryopreserved tumor fragments into the proximal tibia, which showed the disadvantage of open surgery caused potential infection and failure of developing tumor engraftment. Moreover, the volume of implanted tumor fragments without strict control will result in a significant difference in produced tumor volume, which is difficult in following application, such as evaluating the therapeutic efficacy in vivo. Here, a simple and reproducible technique is reported to establish the intratibia primary osteosarcoma with later pulmonary metastasis mouse models via intratibia injection of osteosarcoma cells. This showed the advantages of best mimicking the clinical development characteristics of osteosarcoma in humans; accurate numbers of osteosarcoma cells being directly injected into tibia using micro-volume syringe allowing identical tumor formation rate (100%) and tumor volume. The method ensures avoiding the possibilities of infection or even death using open surgery techniques and allowing lively monitor and quantifying osteosarcoma growth and metastasis using the bioluminescence live imaging system after the injected osteosarcoma cells are labeled with bioluminescence. This prevents the injected osteosarcoma cells from directly reaching the bloodstream and colonizing in the lungs to form pulmonary embolism and/or false-positive pulmonary metastasis by resuspending the injected osteosarcoma cells in appropriate concentration of basement membrane matrix since the basement membrane matrix has the property of coagulation above room temperature. The immediate coagulation support and restrict osteosarcoma cells within the basement membrane matrix after being injected into the mouse tibia.
Another literature has reported the bone metastasis model establishment by intracardiac inoculation or intratibial inoculation of breast cancer cells30; however, cells used in this literature are breast cancer cells, which have different biological and clinical characteristics with osteosarcoma cells; moreover, both the intracardiac and the intratibial inoculation established cancer models in bone are formed by cancer cell colonizing directly or reaching through bloodstream rather than metastasis lesions formed by cancer cell dissemination from the primary cancer lesions.
There are several limitations of the current protocol. Mice used in this protocol are genetic immune system defect nude mice without thymus that prevents them from immunologically rejecting human cells and are widely used in preclinical trials, which are not applicable for immune functional research. Furthermore, we found that not all osteosarcoma cell lines are identically relevant in these models, and the tumorigenesis abilities of 143B, MNNG, MG-63, and U-2 OS cells are higher than the Saos-2 cells.
In conclusion, the present primary and pulmonary metastatic osteosarcoma models generated by orthotopic osteosarcoma cell injection are handy tools to study the tumor microenvironment, efficacy of therapeutics on osteosarcoma growth and/or metastasis. In addition, by intratibia injection of the genetically modified osteosarcoma cells specifically targeting a gene, the models are helpful to explore the key oncogenes and tumor suppressors in osteosarcoma growth and pulmonary metastasis.
The authors have nothing to disclose.
This study was supported by grants from (1) National Key R&D Program of China (2018YFC1704300 and 2020YFE0201600), (2) National Nature Science Foundation (81973877 and 82174408).
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 |