Name: a syngeneic orthotopic osteosarcoma sprague dawley rat model with amputation to control metastasis rate
Uploaded: 2021-05-03T13:00:00.000+00:00
Duration: 7 min 31 s
Description: Watch this Scientific Journal Video about A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate at JoVE.com

NIH funding through National Cancer Institute, grant # CA228582. Shun Ishiyama is currently receiving a grant from Toray Medical Co., Ltd.

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
<ol>
	<li>Grow cells in DMEM, supplemented with 10% (v/v) FBS, penicillin (10 U/mL)-streptomycin (10 U/mL) at 37 &#176;C in humidified 5% CO2 atmosphere. Perform experiments using cells with passages of 2-812.</li>
</ol>
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).
<ol>
	<li>Induction
	<ol>
		<li>Place the rat in a medium-sized induction chamber and induce anesthesia with 2%-3% isoflurane. Monitor the animal continuously for the depth of anesthesia by reflex to toe pinch, respiratory rate, and character.</li>
		<li>Insert the nose into the nosecone. Secure with tape, if necessary.</li>
		<li>Remove the hair on the right leg up to the ventral and the dorsal lower abdomen with clippers or use depilatory agent. Place the rat in a supine position.</li>
		<li>Scrub the surgical area aseptically using 70% ethanol and dilute chlorhexidine acetate or dilute betadine. Begin around the area of the knee and scrub in a circular motion both proximally and distally. Repeat this step three times. No drape is used for tumor implantation.</li>
		<li>Apply eye lubricant in both the eyes of the rat to prevent corneal desiccation caused by anesthesia. Place the rat on a low heat setting heating pad. Ensure that the rat has normal body temperature (37 &#176;C) and normal respiratory rate.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Turn on isoflurane at ~1.5%-2% (for maintenance). Ensure that the animal is at an adequate plane of anesthesia via lack of a toe-pinch reflex. If not, increase the isoflurane percentage to 2.5%.</li>
		<li>Mark a sterile needle (approximately 22 G) at 10 mm from the tip for guidance of depth to insert.</li>
		<li>Insert the needle 10 mm down into the diaphysis of the tibia by entering the bent knee in the middle of the tibial plateau, extending the needle through the metaphysis into the diaphysis using a light drill-like motion to make an opening. Remove the needle.</li>
		<li>Load the cell suspension into the Hamilton syringe directly before injection into the tibia. To do this, gently mix the cells before drawing into syringe as gravity makes the cells settle in the bottom of the tube. 
		NOTE: Cells can be stored in a 1.5 to 2 mL tube (at room temperature) before drawing into the Hamilton syringe (100 &#181;L) after careful mixing. Cells can be kept at room temperature for 2-3 h during the implantation procedure. Always check the cell viability of cells in the tube before and then after the implantation session. Trypan blue exclusion is the easiest method for cell viability assessment.</li>
		<li>Once the bone is traversed with the first needle, insert a second (smaller diameter needle also marked at 10 mm) needle attached to the 100 &#181;L Hamilton syringe loaded with cells. Ensure to insert the needle up to the 10 mm mark into the same hole extending into the diaphysis.</li>
		<li>Gently discharge 20 &#181;L of 75,000 OS cells suspension in PBS into the diaphysis and marrow cavity. 
		NOTE: The needle should not wobble in the bone and should feel secure. If the needle easily moves, the cortex may have been traversed at the diaphysis. Repeat the insertion again to get a firmer placement before the injection of cells.</li>
		<li>Remove the Hamilton needle from the bone. 
		NOTE: If a small drop of blood forms, apply light pressure. If clear fluid drop forms at the puncture site, the needle may not have been extended far enough in the bone and the tumor cell suspension may have leaked back through the hole. Record this in the notes but generally, tumor implantation will be successful. With experience, the tumor implantation procedure should take 5 min per rat. With experience, the tumor implantation of cells in the bone will become easier. Accidental injection of cells in the muscle around the bone, may not lead to the tumor microenvironment needed for pulmonary metastasis.</li>
	</ol>
	</li>
	<li>Recovery
	<ol>
		<li>Ensure that the rat is normothermic. Place the rat in a cage with a heating pad placed under the cage for recovery.</li>
		<li>After fully awake, mobile, and breathing well, inject the rats with Buprenorphine (1.0-1.2 mg/kg SC). 
		NOTE: To get access to Buprenorphine, check with the institution to see options for approval through the veterinary staff to place the order.</li>
		<li>Move the rats back to clean cages and monitor once a day, each week.</li>
	</ol>
	</li>
</ol>
3. Measurement and monitoring
<ol>
	<li>Measure the tumor size 9-10 days post-implantation and then every 2 days until 3 weeks post-implantation to establish a growth rate. Measure the maximum diameter of the tibia using an electronic or manual caliper. Store the data in a spreadsheet with a formula to calculate tumor volume. Measure the contralateral (not implanted tibia) as the baseline. 
	NOTE: The implanted hindlimb diameters are used as a surrogate for tumor size. The hind limbs are measured perpendicular to the tibia long axis at the largest diameter for two measurements, ventral/dorsal and media/ lateral on the limb. The estimated tumor volume is calculated by the formula11: Tumor volume (mm3) = largest diameter (mm) x smallest diameter (mm)2/2.</li>
	<li>Consider the rats for survival amputation or chemotherapy treatment when tumors approach 15 mm in the largest dimension or 3 weeks post tumor implantation. The contralateral limb measures about 7-9 mm in most rats at this age.</li>
</ol>
4. Doxorubicin intravenous administration
<ol>
	<li>Anesthetize the rats with 2% isoflurane. Prepare the skin over jugular vein with three surgical washes using betadine and alcohol as described13.</li>
	<li>Under careful dissection, visualize the right or left jugular vein. Insert the needle into the overlying muscle and then direct toward the rat&#39;s head into the jugular vein lumen as it is visualized in the jugular vein anterior to the pectoralis muscle.
	<ol>
		<li>When the needle is inserted into the jugular vein, gently draw blood into the syringe to ensure proper insertion. It is possible to believe that the needle is through the vein, but the needle is under the vein and not in the lumen. If the jugular vein becomes too small for injections when blunt dissecting to expose the jugular vein, use the other jugular vein for the injection.</li>
	</ol>
	</li>
	<li>Inject doxorubicin (2 mg/kg) slowly over 1 min in a volume of 100-150 &#181;L. The solution can be visualized intravenously in the jugular vein during delivery.</li>
	<li>Inject similar volumes of normal saline in control rats.</li>
	<li>Remove the needle and apply gentle pressure on the vein with a sterile gauze.</li>
	<li>Close the skin incision using 3-4 wound clips. Remove the clips at 7-10 days post injection. 
	NOTE: Rats do not usually try to remove metal clips but will bite and remove sutures. The jugular injection method is preferable to tail vein injections for doxorubicin since any drug that leaks outside the vessel causes necrosis of tail that may require tail amputation.</li>
</ol>
5. Hind limb amputation protocol
<ol>
	<li>Induction
	<ol>
		<li>Place the rat in a medium-sized induction chamber and induce anesthesia with 2%-4% isoflurane. Monitor the rat continuously for the depth of the anesthesia. 
		NOTE: The induction chamber is scavenged to a charcoal canister and any other gases are removed by a down-draft table used for surgery.</li>
		<li>Insert the nose into a nosecone. Secure with tape, if necessary. 
		NOTE: This procedure is done on a down-draft table to scavenge excess volatile gases (i.e., isoflurane).</li>
		<li>Remove the hair on the right leg up to the ventral and the dorsal lower abdomen with clippers or use depilatory agent. Place the rat in a supine position.</li>
		<li>Scrub the surgical area aseptically using 70% ethanol and dilute chlorhexidine acetate or dilute betadine. Prepare the skin for surgery from middle of the calf to skin area just above the hip joint of the right lower abdomen. Scrub the leg proximal and the distal area circumferentially. Repeat this step three times.</li>
		<li>Apply the eye lubricant in both the eyes of the rat. Ensure that the rat has normal body temperature (37 &#176;C) and normal vital signs. Monitor and regulate body temperature using a heating pad that is connected to a rectal temperature probe monitor.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Turn on isoflurane at 1.5%-3% (maintenance). Monitor for anesthetic depth, including reaction to toe pinch, respiratory rate, and character. Adjust the rate of isoflurane as needed to maintain an appropriate plane of anesthesia. 
		NOTE: Respiratory rate while under anesthesia should be between 50-100 breaths per minute. Deep, infrequent breaths are an indication that the rat is too deeply anesthetized.</li>
		<li>Open sterile instrument packs and drapes and don sterile gloves. Take care to maintain sterility through the duration of the procedure. Basic sterile surgical instruments needed include, scalpel blade holder, forceps, hemostats, needle holder, scissors, and wound clip applier.</li>
		<li>Pull the leg of the rat through the vent of the sterile surgical drape. Ensure that the animal is at an adequate plane of anesthesia via toe-pinch reflex.</li>
		<li>Using a scalpel blade or surgical scissors, make a circumferential, cutaneous incision just proximal to the stifle (knee joint).</li>
		<li>Deglove the hind limb using gauze or blunt dissection to expose the femoral artery and vein on the ventral-medial surface of the hind limb.</li>
		<li>Ligate the vessels using 4-0 absorbable sutures at the level of the mid-femur and transect distally.</li>
		<li>Clamp the vein distally to reduce leakage during muscle dissection. 
		NOTE: Circumferential musculature will be transected distal to the level of femoral artery vessel ligation and muscles elevated from the femur to the level of the coxofemoral joint.</li>
		<li>Using blunt dissection, abduct the hip joint with lateral outward rotation.</li>
		<li>Find the head of the femur and disarticulate it from the acetabulum. Cut any remaining tissue keeping the leg attached to the body.</li>
		<li>Give a splash block to the acetabulum and sciatic nerve with approximately 6 mg/kg Ropivacaine.</li>
		<li>Close the musculature over the acetabulum using a simple interrupted suture (4-0, absorbable suture). 
		NOTE: Additional 0.5% bupivacaine or lidocaine (0.15-0.2 mg total) may be injected in several sites along the closed muscle layer (local infiltration/splash block).</li>
		<li>Oppose and close the edges of the skin using wound clips placed apart every 5-10 mm.</li>
	</ol>
	</li>
	<li>Recovery
	<ol>
		<li>Place the rat in a clean recovery cage with heat support using a heating pad placed under the cage. 
		NOTE: To prevent hyperthermia, the heating pad should not be in direct contact with the animal and should not exceed 40 &#176;C.</li>
		<li>Monitor the animal until it fully recovers and is normothermic (37.5-39 &#176;C). 
		NOTE: The animal should not be left unattended until fully conscious and sternally recumbent and moving easily around the cage.</li>
		<li>After fully awake, mobile, and breathing well, inject the rats with Buprenorphine (1.0-1.2 mg/kg SC). 
		NOTE: Giving Buprenorphine in an anesthetized rat may impair recovery.</li>
		<li>Administer 10 mL of warm lactated Ringer&#39;s solution subcutaneously between the shoulder blades.</li>
		<li>Move the rats back to the clean cage and reunite with conspecifics.</li>
		<li>Monitor all the animals twice daily for the next month for signs of pain and distress, including piloerection, hunched posture, or inappetence or signs of incision site infection, including erythema, purulent discharge, or wound dehiscence. 
		NOTE: To date we have not observed clinical signs (such as infections) following post-operative recovery in any of the animals. Only one rat had a dehiscence with sutures, after which wound clips were used to close wounds with no further problems.</li>
		<li>Administer buprenorphine or meloxicam to animals presenting clinical signs of pain at published doses in consultation with a laboratory animal veterinarian.</li>
		<li>Administer antibiotics (i.e., cephalosporin) to animals presenting clinical signs of pain at published doses in consultation with a laboratory animal veterinarian. 
		NOTE: Any animals exhibiting protracted signs of pain or distress that do not improve with analgesics or animals exhibiting signs of infection that do not respond to antibiotics need to be humanely euthanized.</li>
	</ol>
	</li>
</ol>
6. Imaging with X-ray
<ol>
	<li>After tumor implantation, image the tibias and lungs non-invasively to detect tumor growth using X-ray with a machine designed for rodents.</li>
	<li>Anesthetize the rats as previously done.</li>
	<li>Take images at 3x magnification for 6 s at 25 kV.</li>
	<li>Process the film using the X-ray processor. Radiographs can be digitally scanned as well.</li>
</ol>
7. Necropsy procedure
<ol>
	<li>Euthanize the rats with CO2. Confirm death via lack of heartbeat and immediately draw 3 mL blood from the heart for serum or plasma samples.</li>
	<li>Open the thorax and abdomen for examination.</li>
	<li>Isolate the trachea and cannulate with a (18 G) catheter. To secure the catheter in the trachea, tie a silk suture around both the trachea with the catheter.</li>
	<li>Connect the infusion catheter to a 3 or 5 mL syringe. Infuse formalin or saline to gently inflate the lung lobes for better histology specimens. On infusion, the lungs will puff up and enable a better visualization of pulmonary metastases.</li>
	<li>Examine, dissect, and weigh all the selected thoracic and abdominal organs (such as liver, kidneys).</li>
	<li>Fix the organs in formalin for histopathology or freeze on dry ice, 2-methylbutane, or liquid nitrogen.</li>
	<li>For evaluation of protein expression using western blot, make lysates of frozen tissues. Antibodies that react with rat tissues are detailed13.</li>
</ol>
8. Immunohistochemistry
<ol>
	<li>Process the primary OS tissue, embed in paraffin, and section it for immunohistochemical staining.</li>
	<li>Retrieve the antigen after deparaffinization using citrate buffer (pH 6.0). Incubate in 0.3% H2O2 in methanol for 30 min to quench endogenous peroxidase.</li>
	<li>Block the 5 &#181;m thick paraffin sections using normal serum.</li>
	<li>Incubate with primary anti-CD68 and CD3 antibodies (see table) overnight at 4 &#176;C.</li>
	<li>Rinse the sections in PBS and incubate them in HRP polymer using a detection kit. 
	NOTE: Immunostaining was developed with diaminobenzidine as a chromogen.</li>
</ol>
9. Western blotting
<ol>
	<li>Lyse the UMR-106 cells in 200-300 &#181;L of lysis buffer14 to perform standard gel electrophoresis and western blotting.</li>
	<li>Use 4% to 12% Bis-Tris gels.</li>
	<li>Incubate in anti-ErbB2, anti-ErbB4, anti-EGFR, anti-ERK, &#946;-actin or anti-mouse &#946;2-AR primary antibody (see table) and horseradish peroxidase-linked secondary antibody.</li>
	<li>Add the chemiluminescent substrate. Expose the membranes to X-ray film. 
	NOTE: &#946;-actin levels are used as loading controls.</li>
</ol>

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No disclosure to declare.

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 &#181;L, 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 &#181;L. The 1 mL tuberculin syringes would not be accurate enough for the implantation of 20 &#181;L. 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.

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.

<table><tbody><tr><td>AKT</td><td>Cell Signaling TECHNOLOGY</td><td>4685S</td><td></td></tr><tr><td>absorbable suture</td><td>Ethicon</td><td>J214H</td><td></td></tr><tr><td>&amp;beta;-actin</td><td>SANTA CRUZ BIOTECHNOLOGY</td><td>sc-47778</td><td></td></tr><tr><td>&amp;beta;2-AR antibody</td><td>SANTA CRUZ BIOTECHNOLOGY</td><td>sc-569</td><td>replaced by &amp;beta;2-AR (E-3): sc-271322</td></tr><tr><td>Bis&amp;ndash;Tris gels</td><td>Thermo Fisher</td><td>NP0321PK2</td><td></td></tr><tr><td>Buprenorphine SR Lab</td><td>ZooPharm</td><td>IZ-70000-201908</td><td></td></tr><tr><td>CD3 antibody</td><td>Dako</td><td>#A0452</td><td></td></tr><tr><td>CD68 antibody</td><td>eBioscience</td><td>#14-0688-82</td><td></td></tr><tr><td>Chemiluminescent substrate</td><td>cytiva</td><td>RPN2232</td><td></td></tr><tr><td>CL-Xposure film</td><td>Thermo Fisher</td><td>34089</td><td></td></tr><tr><td>Complete Anesthesia System</td><td>EVETEQUIP</td><td>922120</td><td></td></tr><tr><td>diaminobenzidine</td><td>VECTOR LABORATORIES</td><td>SK-4100</td><td></td></tr><tr><td>Doxorubicin</td><td>Actavis</td><td>NDC 45963-733-60</td><td></td></tr><tr><td>EGFR antibody</td><td>SANTA CRUZ BIOTECHNOLOGY</td><td>sc-03</td><td>replaced by EGFR (A-10): sc-373746</td></tr><tr><td>ERBB2 antibody</td><td>SANTA CRUZ BIOTECHNOLOGY</td><td>sc-284</td><td>replaced by Neu (3B5): sc-33684</td></tr><tr><td>ERBB4 antibody</td><td>SANTA CRUZ BIOTECHNOLOGY</td><td>sc-283</td><td>replaced by ErbB4 (C-7): sc-8050</td></tr><tr><td>ERK antibody</td><td>SANTA CRUZ BIOTECHNOLOGY</td><td>sc-514302</td><td></td></tr><tr><td>eye lubricant</td><td>PHARMADERM</td><td>NDC 0462-0211-38</td><td></td></tr><tr><td>Hamilton syringe (100 &amp;micro;L)</td><td>Hamilton</td><td>Model 1710 SN SYR</td><td></td></tr><tr><td>horseradish peroxidase-linked secondary antibody</td><td>cytiva</td><td>NA934</td><td></td></tr><tr><td>HRP polymer detection kit</td><td>VECTOR LABORATORIES</td><td>MP-7401</td><td></td></tr><tr><td>HRP polymer detection kit</td><td>VECTOR LABORATORIES</td><td>MP-7402</td><td></td></tr><tr><td>isoflurane</td><td>BUTLER SCHEIN</td><td>NDC 11695-6776-2</td><td></td></tr><tr><td>isoflurane vaporizer</td><td>EVETEQUIP</td><td>911103</td><td></td></tr><tr><td>UMR-106 cell</td><td>ATCC</td><td>CRL-1661</td><td></td></tr><tr><td>X-ray</td><td>Faxitron</td><td>UltraFocus</td><td></td></tr><tr><td>X-ray processor</td><td>Hope X-Ray Peoducts Inc</td><td>MicroMax X-ray Processor</td><td>Hope Processors are not available in USA anymore</td></tr><tr><td>wound clips</td><td>BECTON DICKINSON</td><td>427631</td><td></td></tr></tbody></table>

a syngeneic orthotopic osteosarcoma sprague dawley rat model with amputation to control metastasis rate

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.

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 (&#62;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.&#160;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.&#160;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62139/62139fig01.jpg" /> 
Figure 1: Tibia with tumor implantation needle inserted. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62139/62139fig02.jpg" /> 
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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62139/62139fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62139/62139fig04.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62139/62139fig05.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62139/62139fig06.jpg" /> 
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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62139/62139fig07.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62139/62139fig08.jpg" /> 
Figure 8: Immunohistochemistry of (A) CD68 immunostaining macrophages and (B) immunostaining T cells showing CD3 positive cells in OS in the tibia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62139/62139fig09.jpg" /> 
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 &#946;2-adrenergic receptors with actin as a loading control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62139/62139fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate at JoVE.com

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

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 ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

3.5K Views.  The Johns Hopkins University School of Medicine. Het implanteren van osteosarcoomcellen in de micro-omgeving van het scheenbeen bevordert longmetastase. Dit model kan ook nuttig zijn voor het bestuderen van de mechanismen van pulmonale metastase of nieuwe kankertherapieën die een intact immuunsysteem vereisen. Zodra de tumor is vastgesteld, is amputatie noodzakelijk om de snelheid van metastase in overlevingsstudies op lange termijn te beheersen.We gebruiken de jugulaire injecties om de visualisatie van intraveneuze medicijnafgifte ...

Video: A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Machine Learning Algorithms for Early Detection of Bone Metastases in an Experimental Rat Model

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy exceeding its constituents. This protocol describes the development of an ML algorithm to predict the growth of breast cancer bone macrometastases in a rat model before any abnormalities are observable with standard imaging methods. Such an algorithm can facilitate the detection of early metastatic disease (i.e., micrometastasis) that is regularly missed during staging examinations.
The applied metastasis model is site-specific, meaning that the rats develop metastases exclusively in their right hind leg. The model&#8217;s tumor-take rate is 60%&#8211;80%, with macrometastases becoming visible in magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a subset of animals 30 days after induction, whereas a second subset of animals exhibit no tumor growth.
Starting from image examinations acquired at an earlier time point, this protocol describes the extraction of features that indicate tissue vascularization detected by MRI, glucose metabolism by PET/CT, and the subsequent determination of the most relevant features for the prediction of macrometastatic disease. These features are then fed into a model-averaged neural network (avNNet) to classify the animals into one of two groups: one that will develop metastases and the other that will not develop any tumors. The protocol also describes the calculation of standard diagnostic parameters, such as overall accuracy, sensitivity, specificity, negative/positive predictive values, likelihood ratios, and the development of a receiver operating characteristic. An advantage of the proposed protocol is its flexibility, as it can be easily adapted to train a plethora of different ML algorithms with adjustable combinations of an unlimited number of features. Moreover, it can be used to analyze different problems in oncology, infection, and inflammation.

This protocol was designed to train a machine learning algorithm to use a combination of imaging parameters derived from magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a rat model of breast cancer bone metastases to detect early metastatic disease and predict subsequent progression to macrometastases.

Machine learning (ML) algorithms permit the integration of different features into a model to perform classification or regression tasks with an accuracy ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

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

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&#160;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.

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 ...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

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.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Medicine

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery.
Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies

In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant ...

Herb Decoction Formulation and Gavage Standardization in Osteosarcoma Mice

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine.&#160;Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.

Author Spotlight: Standardized Herbal Decoction Protocol for Enhanced Animal Studies

The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

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