Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.
The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.
Osteosarcoma (OS) is the most common primary bone tumor in children, adolescents, and young adults. The most recent advance in the treatment of OS occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone1. OS develops during rapid bone growth, typically occurring in long tubular bones such as femur, tibia, and humerus. They are characterized by an osteolytic, osteoblastic, or mixed appearance with notable periosteal reaction2. Chemotherapy and surgical resection can improve the outcome for patients with a 5-year survival for 65% of patients2,3. Unfortunately, high grade OS patients with metastatic disease have 20% survival. OS invades regionally and metastasizes primarily to the lungs or other bones and is more prevalent in males. The most compelling need for these young patients is a novel therapy that prevents and eliminates viability of distant metastases.
OS pre-clinical models have been reviewed4,5,6,7 and few available immunocompetent models using amputation of orthotopic OS have been developed. In 2000, an important model was developed using BALB/c mice with orthotopic syngeneic OS and amputation8. Compared to this mouse model, the rat model is based on genetically outbred and 10 times larger animals leading to some advantages. The rat UMR106 model was developed from a 32P induced OS in a Sprague Dawley (SD) rat, which was derived into a cell line9. In 2001, orthotopic implantation of UMR106-01 was first described in implanted tibias of athymic mice with rapid, consistent primary tumor development and radiological, histologic features in common with OS in humans. Pulmonary metastases developed and were dependent on orthotopic placement of UMR106 into the bone microenvironment10. In 2009, Yu et al.11 established a reproducible orthotopic femur OS rat model using UMR106 cells in larger male SD rats. The successful tumor implantations and lung metastasis rate in rats without amputation were similar to the data presented here. In this study, an added amputation to the model using young rats was performed, which suggested that the timing of primary tumor removal is crucial in modeling OS, especially related to metastatic progression. With this refinement, amputation and in vivo imaging improve this model for pre-clinical studies for novel drug assessment for OS.
All the procedures and experiments involving rats were performed according to protocols approved by Johns Hopkins Animal Care and Use Committee.
1. The SD rat OS cell line UMR-106 cell culture protocol
2. Intratibial injection of OS cells protocol
NOTE: Time-mated pregnant SD rats give birth in the animal facility and at 3 weeks of age, litters are used (since UMR 106 cell line is syngeneic to SD rats, no irradiation is needed).
3. Measurement and monitoring
4. Doxorubicin intravenous administration
5. Hind limb amputation protocol
6. Imaging with X-ray
7. Necropsy procedure
8. Immunohistochemistry
9. Western blotting
Immunocompetent SD outbred rats are used for these OS studies, which offers an animal model with an intact immune system. We have used the UMR106 cell line from ATCC, developed from cells that were initially isolated from an OS from a SD rat. We implanted the cells into SD rats, thus providing a syngeneic model for OS. UMR106 cells are implanted into the tibia of 3-week-old male and female SD rats, simulating a pediatric OS model. Moreover, the orthotopic implantation of UMR106 cells directly into the tibia metaphysis/diaphysis gives a relevant tumor microenvironment.
When implanting tumor cells, a needle must be inserted correctly through the tibial plateau (Figure 1) at the correct angle (parallel to the bone shaft) extending the needle tip approximately 10 mm into the central cavity of the bone. With this procedure, 95% (52/55) of rats developed tumors in tibias distal to the knee. With tibial injection experience, 100% of rats developed tumors. In a group of rats that were not amputated, the average tumor volumes in males was 504 mm3 at 3 weeks and 1195 mm3 at 5 weeks post-implantation. In females, tumor volumes average at 285 mm3 at 3 weeks and 495 mm3 at 5 weeks post-implantation.
Two cohorts of rats were compared, including those with amputation (23 rats) (Figure 2) and those without amputation (29 rats). Both cohorts were euthanized at 7 weeks post implantation to examine tumor metastasis to the lungs. In the amputation group (3/23) rats developed pulmonary metastases. These three rats died or were euthanized within 24 h of surgery due to post surgery complications. Two rats died from prolonged anesthesia as the surgeon was learning the method. One rat developed a dehiscence and was euthanized the following day. Lungs of these three rats were evaluated and three small metastases (>1 mm) were found histologically. The surviving 20 rats did not have lung metastases 7 weeks post-implantation. This indicated that 3 weeks post-implantation amputations are adequate to decrease the number of rats with pulmonary metastasis. In a second group of 29 rats that did not have the amputation procedure, 26/29 rats had lung metastases consistent with the previously published data11. We saw no pattern in the size or number of metastases in these rats. Most rats have more than 10 grossly visible 2-7 mm diameter metastases that were easily sampled during necropsy. Occasionally, rats had even larger metastases of up to 10 mm in diameter. It is important to implant UMR106 cells with a low passage number, as the studies demonstrated that the cells with 10 or higher passage number become more aggressive and metastasize as early as 2-3 weeks post-implantation. The reason for the nature is not known but the speculation is that the cells in culture could develop mutations that favor metastasis.
In addition to the amputation surgery, another refinement of the methods included the X-ray imaging for tumor surveillance or at necropsy. This method allows the researcher to confirm bone tumor invasion in rats under anesthesia. The planar radiography method can also be used on recently amputated limbs or formalin fixed limbs. The method is fast (5 min per rat) and inexpensive ($2-5/rat) compared to Computed Tomography (CT). For in vivo monitoring, it requires the rats to be anesthetized during imaging. Figure 3 demonstrates the detailed morphology seen by X-ray imaging of two previously amputated limbs. This method illuminates the osteolytic and osteoblastic nature of these tumors. Note the disruption of normal bone cortical architecture of the tibia and fibula in both examples (white arrows). Figure 4 illustrates the radiographic morphology of lungs with and without metastases. Imaging by X-ray can quickly reveal to the laboratory, the need for euthanasia to prevent unnecessary spontaneous deaths.
Primary and metastatic tumors to the lung are histologically similar to human OS exhibiting both osteolytic and osteoblastic tumor morphology. In the rat OS, both osteolytic and osteoblastic tumor morphology is confirmed by histopathology of amputated limb in Figure 5 and Figure 6. Note that the cortical bone is absent in this example and the adjacent bone is also replaced or fortified by new woven bone (exostoses) that is oriented perpendicular to the existing shaft of the cortex. Islands of immature osteoid (amorphous extracellular material) are shown within the tumor example. Additionally, the microscopic morphology of the lung metastases, some with mineralized bone, and tumor vascular emboli are shown in Figure 7.
Limb amputation with OS increases survival in rats. Rats can die spontaneously due to pulmonary metastasis housed for longer than 7 weeks post implantation. The use of amputation can allow the further investigation of standard or targeted cancer therapy in this model. Lengthening the time between tumor implantation and amputation will increase the incidence of metastasis.
Doxorubicin is a chemotherapeutic agent used to treat OS in humans. In rats, doxorubicin can be given via jugular injections13 or a catheter15 as described here. The jugular injection does require 5-10 min per rat but assures the delivery of the dose in the exposed vein. Overall, jugular injections are much more reproducible compared to the rat tail vein injections. If doxorubicin leaks into the dermis during tail vein injections, necrosis of the tail can occur and prevent further treatments. In this study, five rats were treated with 2 mg/kg dose of doxorubicin and euthanized 48 h post injection to investigate cell death in the tumors as shown in Figure 5A,B.
Five control rats treated with saline were also evaluated to select antibodies that can be used to immunostain immune cells in rat OSs. Here, two antibodies were tested for immune reactivity. For immunohistochemistry studies, tumors were fixed in formalin for 48-72 h and then moved to 70% ethanol to reduce protein cross-linking that occurs in formalin. Immunohistochemistry was performed for immune cell infiltrates in primary OS tumors and immunostained for macrophages (CD68) and T cells (CD3). Figure 8 shows two examples of immunostains of immune cell infiltrates within the tumor microenvironment.
The potential targets for therapeutic intervention were also explored. After amputation of limbs with tumors, rat OS samples were frozen for future protein isolation. In this study, we discovered that UMR106 cells express the ErbB family pathway proteins. Western blots performed on UMR106 cell protein lysates demonstrate the expression of ErbB2, EGFR, ErbB4, and other proteins that interact with these pathways (Figure 9).
Figure 1: Tibia with tumor implantation needle inserted. Please click here to view a larger version of this figure.
Figure 2: Tibia during amputation procedure with skin removed (A), exposed femoral artery and vein (B), with muscle elevated from the femur (C), and in a rat 3 weeks post amputation surgery (D). Please click here to view a larger version of this figure.
Figure 3: X-ray radiograph of right legs after amputation (ex vivo) from two rats with OS. Note the osteolytic and osteoblastic nature of the tumor. Please click here to view a larger version of this figure.
Figure 4: X-ray images of the rat lungs. (A) with no lung metastases. (B) with OS pulmonary metastasis. (C) correlation to gross pathology of metastases in an inflated lung. Please click here to view a larger version of this figure.
Figure 5: (A) Histopathology of OS with 90% of cells showing cell death in the tibia tumor 48 h after a dose of 2 mg/kg doxorubicin. (B) Tumor cell death (arrow) in tibial primary OS at 48 h after a dose of 2mg/kg doxorubicin. Note the top right and left corner has viable cells. (C) OS invasion in cortical bone. Please click here to view a larger version of this figure.
Figure 6: (A) Histopathology of OS that has replaced the bone marrow cells and infiltrated into the cortices of the tibia. Notice the accompanied reactive new bone growth as it is layered outside and perpendicular to the pre-existing cortex. (B) Higher power examination of OS tumor cells adjacent to an island of bone. (C) Higher power examination of OS cells embedded in pink to blue extracellular matrix (osteoid). Please click here to view a larger version of this figure.
Figure 7: (A) Multiple pulmonary metastases in rat with tibia tumor implantation. (B) Tumor OS cells in an embolus in small pulmonary artery branch vessel adjacent to a bronchiole below the vessel. (C) Some pulmonary metastases contain islands of bone while other metastases are more cellular. (D) Higher power of metastases with OS cells admixed with islands of mineralized bone. Please click here to view a larger version of this figure.
Figure 8: Immunohistochemistry of (A) CD68 immunostaining macrophages and (B) immunostaining T cells showing CD3 positive cells in OS in the tibia. Please click here to view a larger version of this figure.
Figure 9: ErbB pathway proteins expressed in UMR106 OS cells from tibia tumors. Lysates from primary tumors were examined for protein expression from ErbB family signal transduction pathway, including ErbB2, EGFR, ErbB4, AKT, ERK1/2, and β2-adrenergic receptors with actin as a loading control. Please click here to view a larger version of this figure.
Rats with OS tibial implants develop measurable tumors by 3 weeks post-implantation. If limbs with tumors are amputated 3 weeks post-implantation, the incidence of lung metastasis is reduced significantly. OSs are both osteolytic and osteoblastic. Rats without amputation develop lung metastases that are multiple and variably sized, observed by radiography or at necropsy by 7 weeks post-implantation.EGFR, ErbB2, and ErbB4 are expressed in rat UMR106 OS, similar to human OS16,17,18. CD3 T cells and macrophages are easily detected in OS by immunohistochemistry methods. Jugular vein injections are preferred to tail vein for delivery of chemotherapy doxorubicin, a drug given to OS patients. The method described here is a complete coxofemoral amputation. This procedure is a refinement and could be considered to replace the tumor removing surgical method (femoral osteotomy) where the bone is cut leaving a stump for the patient8. The study suggests a complete limb removal to reduce the likelihood of post-surgical pain and complications.
There are a number of critical steps in this protocol. First, it is important to note the passage of tumor cells and to use lower passage of cells for the studies to keep the model consistent from experiment to experiment. The older passage cells become more aggressive with time in culture. Second, using the needle of appropriate size and the Hamilton syringe will help in correctly injecting the cells in tibia at a very small volume of 20 µL, a volume determined as optimal and did not cause leakage. Third, the surgeon needs to initially practice disarticulation when doing necropsies on similar aged rats to learn the mechanics of the procedure. Fourth, for the success of amputation, maintain thermoregulation and limit the surgery time. An experienced surgeon can complete the amputation in 15 min.
It was observed that implantation of cells in the tibia greatly improved when a larger bore needle was used to make the initial opening followed by the insertion of a smaller bore Hamilton syringe needle. This protects the Hamilton syringe from breakage and dulling over time. Hamilton syringes can have volumes as small as 10 µL. The 1 mL tuberculin syringes would not be accurate enough for the implantation of 20 µL. The same Hamilton syringe was used for all the rats implanted on a day but were washed between the surgical procedures of each rat. Avoid autoclaving the Hamilton syringe syringes as they are prone to breakage. At the end of the procedure, wash it with saline (10 times) and then with 100% ethanol (10 times) and let it dry with plunger removed to store.
Skin and subcutaneous sutures were initially used to close the incision, yet one rat was found with dehiscence the day after the surgery. The use of wound clips and surgical glue to close the incision improved the method. With this refinement, no other rats had any such post-surgery complication. The inclusion of the radiography of the lungs by X-ray refines this model to demonstrate lung metastasis in rats allowing for euthanasia that is timely and prevents unexpected deaths. X-ray images allow us to determine the osteolytic and osteoblastic nature of these rat OSs, similar to human OSs.
A moderate level of surgical expertise is necessary to perform the amputation procedure. The most difficult step is the dissection into the musculature to locate the coxofemoral joint. Magnification and good lighting are important during this step. Surgical expertise can be achieved with practice on animals that have been euthanized. After about 10 rats, the surgeon should be confident to amputate a limb with OS from a live rat under anesthesia.
Existing methods to remove limbs with sarcomas in mice and rats are based on removing the tibia by cutting the femur bone and musculature mid-shaft and leaving the stump8. Although, this may be useful for some investigations, in this study, the complete leg removal was attempted. The procedure was found to be satisfactory and offered no post-surgical complications. In rats with a hind limb stump, there could be more post-surgical skin, muscle, or nerve pain. Leaving a stump, rats could reach around and access the surgery site. Rats do very well post-amputation and ambulate well in the cage with one hindlimb.
Advantages of full limb amputation include removing the primary tumor before it becomes too large and painful for the rat. Importantly, removal of the primary tumor will help control primary tumor metastasis to the lung. Rats with amputation can be further studied in order to test efficacy of novel therapeutics on circulating tumor cells in the blood or in the micrometastases in capillaries of the lungs or other bones.
There is a substantial need for development of new cancer therapeutics for OS and other sarcomas, especially therapeutics that have drug activity against metastatic progression. Compared to the novel therapeutics developed for other cancers, therapeutics for OS have unfortunately not progressed in many decades. Responding to this problem, a meeting of key leaders and experts in OS and metastasis convened to develop guidelines for improved OS drug development19. As per the suggestions of the panel, studies were set out to improve the rat pre-clinical model, a lesser-known model of OS. In summary, amputation and imaging refines rat pre-clinical model for further use by the sarcoma research community. The amputation procedure will allow for improved patient survival for multiple months enabling evaluation of efficacy of novel treatments on micrometastases or dormant tumors or to test for toxicity of treatments with a model with better longevity.
In summary, we provide the advantage of this OS model. Immunocompetent SD outbred rats are used to provide a syngeneic model with implanted UMR106 OS cell line isolated from a SD rat OS. The primary and metastatic tumor is histologically similar to OS in humans. Juvenile male and female rats are used for UMR106 tumor implantation studies modeling pediatric sarcoma. Orthotopic placement of implanted cells is done directly into the tibia for a relevant tumor microenvironment. The primary tumor metastasizes to the lung and the metastases can be monitored by in vivo imaging with X-ray method. The rat OS expresses proteins in common with human OS, such as ErbB2. Compared to the dog OS, rat model allows for larger numbers of animal to be used simultaneously. Rats are 10 times larger than mice for ease of tibial injections, surgery, imaging, blood draws, and biopsy. The longevity of rats is more assured with amputation and this model can combine neoadjuvant therapy, amputation and adjuvant therapy allowing for improved patient survival enabling evaluation of efficacy of treatments on micrometastases or dormant tumors. Off target toxicity evaluation can also be assessed in this model where rats can be treated with cancer therapeutic such as doxorubicin and monitored long-term for doxorubicin induced cardiac toxicity or recurrence of OS. This would allow for the testing of cardio-protection agents in a model with OS.
The authors have nothing to disclose.
NIH funding through National Cancer Institute, grant # CA228582. Shun Ishiyama is currently receiving a grant from Toray Medical Co., Ltd.
AKT | Cell Signaling TECHNOLOGY | 4685S | |
absorbable suture | Ethicon | J214H | |
β-actin | SANTA CRUZ BIOTECHNOLOGY | sc-47778 | |
β2-AR antibody | SANTA CRUZ BIOTECHNOLOGY | sc-569 | replaced by β2-AR (E-3): sc-271322 |
Bis–Tris gels | Thermo Fisher | NP0321PK2 | |
Buprenorphine SR Lab | ZooPharm | IZ-70000-201908 | |
CD3 antibody | Dako | #A0452 | |
CD68 antibody | eBioscience | #14-0688-82 | |
Chemiluminescent substrate | cytiva | RPN2232 | |
CL-Xposure film | Thermo Fisher | 34089 | |
Complete Anesthesia System | EVETEQUIP | 922120 | |
diaminobenzidine | VECTOR LABORATORIES | SK-4100 | |
Doxorubicin | Actavis | NDC 45963-733-60 | |
EGFR antibody | SANTA CRUZ BIOTECHNOLOGY | sc-03 | replaced by EGFR (A-10): sc-373746 |
ERBB2 antibody | SANTA CRUZ BIOTECHNOLOGY | sc-284 | replaced by Neu (3B5): sc-33684 |
ERBB4 antibody | SANTA CRUZ BIOTECHNOLOGY | sc-283 | replaced by ErbB4 (C-7): sc-8050 |
ERK antibody | SANTA CRUZ BIOTECHNOLOGY | sc-514302 | |
eye lubricant | PHARMADERM | NDC 0462-0211-38 | |
Hamilton syringe (100 µL) | Hamilton | Model 1710 SN SYR | |
horseradish peroxidase-linked secondary antibody | cytiva | NA934 | |
HRP polymer detection kit | VECTOR LABORATORIES | MP-7401 | |
HRP polymer detection kit | VECTOR LABORATORIES | MP-7402 | |
isoflurane | BUTLER SCHEIN | NDC 11695-6776-2 | |
isoflurane vaporizer | EVETEQUIP | 911103 | |
UMR-106 cell | ATCC | CRL-1661 | |
X-ray | Faxitron | UltraFocus | |
X-ray processor | Hope X-Ray Peoducts Inc | MicroMax X-ray Processor | Hope Processors are not available in USA anymore |
wound clips | BECTON DICKINSON | 427631 |