This study was supported by grants from (1) National Key R&#38;D Program of China (2018YFC1704300 and 2020YFE0201600), (2) National Nature Science Foundation (81973877 and 82174408).

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&#160;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
<ol>
	<li>On the day of osteo...

<ol>
	<li>Bielack, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+in+high-grade+osteosarcoma+of+the+extremities+or+trunk:+an+analysis+of+1,702+patients+treated+on+neoadjuvant+cooperative+osteosarcoma+study+group+protocols.">Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols.</a> Journal of Clinical Oncology. 20 (3), 776-790 (2002).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+microenvironment+and+osteosarcoma+metastasis.">Bone microenvironment and osteosarcoma metastasis.</a> International Journal of Molecular Sciences. 21 (19), (2020).</li><li>Mirabello, L., Troisi, R. J., Savage, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma+incidence+and+survival+rates+from+1973+to+2004:+data+from+the+Surveillance,+Epidemiology,+and+End+Results+Program.">Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program.</a> Cancer. 115 (7), 1531-1543 (2009).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+and+safety+comparison+of+first-line+chemotherapeutic+agents+(high-dose+methotrexate,+doxorubicin,+cisplatin,+and+ifosfamide)+for+osteosarcoma:+a+network+meta-analysis.">The efficacy and safety comparison of first-line chemotherapeutic agents (high-dose methotrexate, doxorubicin, cisplatin, and ifosfamide) for osteosarcoma: a network meta-analysis.</a> Journal of Orthopaedic Surgery and Research. 15 (1), 51 (2020).</li><li>Tsukamoto, S., Errani, C., Angelini, A., Mavrogenis, A. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+and+heterotopic+murine+models+of+pancreatic+cancer+and+their+different+responses+to+FOLFIRINOX+chemotherapy.">Orthotopic and heterotopic murine models of pancreatic cancer and their different responses to FOLFIRINOX chemotherapy.</a> Disease Models &amp; Mechanisms. 11 (7), (2018).</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+for+osteosarcoma+in+the+mouse:+a+meta-analysis.">MicroRNAs for osteosarcoma in the mouse: a meta-analysis.</a> Oncotarget. 7 (51), 85650-85674 (2016).</li><li>Maloney, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratibial+injection+causes+direct+pulmonary+seeding+of+osteosarcoma+cells+and+is+not+a+spontaneous+model+of+metastasis:+A+mouse+osteosarcoma+model.">Intratibial injection causes direct pulmonary seeding of osteosarcoma cells and is not a spontaneous model of metastasis: A mouse osteosarcoma model.</a> Clinical Orthopaedics and Related Research. 476 (7), 1514-1522 (2018).</li><li>Yu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+reproducible+osteosarcoma+rat+model+using+orthotopic+implantation+technique.">Establishment of reproducible osteosarcoma rat model using orthotopic implantation technique.</a> Oncology Reports. 21 (5), 1175-1180 (2009).</li><li>Fidler, I. J., Naito, S., Pathak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+implantation+is+essential+for+the+selection,+growth+and+metastasis+of+human+renal+cell+cancer+in+nude+mice+[corrected].">Orthotopic implantation is essential for the selection, growth and metastasis of human renal cell cancer in nude mice [corrected].</a> Cancer Metastasis Reviews. 9 (2), 149-165 (1990).</li><li>Leacock, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+transgenic+model+of+Ewing's+sarcoma+reveals+conserved+mediators+of+EWS-FLI1+tumorigenesis.">A zebrafish transgenic model of Ewing's sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis.</a> Disease Models &amp; Mechanisms. 5 (1), 95-106 (2012).</li><li>Sharma, S., Boston, S. E., Riddle, D., Isakow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteosarcoma+of+the+proximal+tibia+in+a+dog+6+years+after+tibial+tuberosity+advancement.">Osteosarcoma of the proximal tibia in a dog 6 years after tibial tuberosity advancement.</a> The Canadian Veterinary Journal. 61 (9), 946-950 (2020).</li><li>Mohseny, A. B., Hogendoorn, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+human+osteosarcoma.">Zebrafish as a model for human osteosarcoma.</a> Advances in Experimental Medicine and Biology. 804, 221-236 (2014).</li><li>Hu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cantharidin+inhibits+osteosarcoma+proliferation+and+metastasis+by+directly+targeting+miR-214-3p/DKK3+axis+to+inactivate+&#946;-catenin+nuclear+translocation+and+LEF1+translation.">Cantharidin inhibits osteosarcoma proliferation and metastasis by directly targeting miR-214-3p/DKK3 axis to inactivate &#946;-catenin nuclear translocation and LEF1 translation.</a> International Journal of Biological Sciences. 17 (10), 2504-2522 (2021).</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphyllin+I+suppresses+human+osteosarcoma+growth+by+inactivation+of+Wnt/&#946;-catenin+pathway+in+vitro+and+in+vivo.">Polyphyllin I suppresses human osteosarcoma growth by inactivation of Wnt/&#946;-catenin pathway in vitro and in vivo.</a> Scientific Reports. 7 (1), 7605 (2017).</li><li>Lamar, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRC+tyrosine+kinase+activates+the+YAP/TAZ+axis+and+thereby+drives+tumor+growth+and+metastasis.">SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis.</a> The Journal of Biological Chemistry. 294 (7), 2302-2317 (2019).</li><li>Benton, G., Arnaoutova, I., George, J., Kleinman, H. K., Koblinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+from+discovery+and+ECM+mimicry+to+assays+and+models+for+cancer+research.">Matrigel: from discovery and ECM mimicry to assays and models for cancer research.</a> Advanced Drug Delivery Reviews. , 3-18 (2014).</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrine+inhibits+prostate+cancer+via+activation+of+the+unfolded+protein+response/endoplasmic+reticulum+stress+signaling+and+reversal+of+epithelial+to+mesenchymal+transition.">Matrine inhibits prostate cancer via activation of the unfolded protein response/endoplasmic reticulum stress signaling and reversal of epithelial to mesenchymal transition.</a> Molecular Medicine Reports. 18 (1), 945-957 (2018).</li><li>Fridman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+tumor+growth+of+both+primary+and+established+human+and+murine+tumor+cells+in+athymic+mice+after+coinjection+with+Matrigel.">Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.</a> Journal of the National Cancer Institute. 83 (11), 769-774 (1991).</li><li>Kocat&#252;rk, B., Versteeg, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+injection+of+breast+cancer+cells+into+the+mammary+fat+pad+of+mice+to+study+tumor+growth.">Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.</a> Journal of Visualized Experiments. (96), e51967 (2015).</li><li>Paschall, A. V., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+mouse+model+of+spontaneous+breast+cancer+metastasis.">An orthotopic mouse model of spontaneous breast cancer metastasis.</a> Journal of Visualized Experiments. (114), e54040 (2016).</li><li>Hildreth, B. E., Palmer, C., Allen, M. 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Orthotopic injection of osteosarcoma cells is an ideal model to study the function and mechanism of specific factors in osteosarcoma carcinogenesis and development to evaluate the therapeutic efficacy. To avoid differences in tumor growth, most active osteosarcoma cells at 80%-90% confluent with the same number are carefully injected into the tibia of each mouse, and the cell trypsinization time is strictly controlled without affecting the cell viability. As cell clumps affect cell counting leading to inaccurate cell num...

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&#39;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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automatic cell counter</td>
			<td>Shanghai Simo Biological Technology Co., Ltd</td>
			<td>IC1000</td>
			<td>Counting cells</td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>R500IP</td>
			<td>The Equipment of Anesthesia mice</td>
		</tr>
		<tr>
			<td>BALB/c athymic mice</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd.</td>
			<td>/</td>
			<td>animal</td>
		</tr>
		<tr>
			<td>Basement Membrane Matrix</td>
			<td>Shanghai Uning Bioscience Technology Co., Ltd</td>
			<td>356234, BD, Matrigel</td>
			<td>re-suspende cells</td>
		</tr>
		<tr>
			<td>Bioluminescence imaging system</td>
			<td>Shanghai Baitai Technology Co., Ltd</td>
			<td>Vieworks</td>
			<td>tracking the tumor growth and pulmonary metastasis, if the injection cell is labeled by luciferase</td>
		</tr>
		<tr>
			<td>Centrifuge tube (15 mL)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>&#160;430790, Corning</td>
			<td>Centrifuge the cells</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Shenzhen RWD Life Technology Co., Ltd</td>
			<td>VETEASY</td>
			<td>Anesthesia mice</td>
		</tr>
		<tr>
			<td>MEM media</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>LM-E1141</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Micro-volume syringe</td>
			<td>Shanghai high pigeon industry and trade Co., Ltd</td>
			<td>0-50 &#956;L</td>
			<td>Inject precise cells into the tibia</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Beyotime Biotechnology</td>
			<td>ST447</td>
			<td>wash the human osteosarcoma cells</td>
		</tr>
		<tr>
			<td>1ml syringes</td>
			<td>Shandong Weigao Group Medical Polymer Co., Ltd</td>
			<td>20200411</td>
			<td>drilling</td>
		</tr>
		<tr>
			<td>143B cell line</td>
			<td>ATCC</td>
			<td>CRL-8303</td>
			<td>osteosarcoma cell line</td>
		</tr>
		<tr>
			<td>Trypsin (0.25%)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>25200056, Gibco</td>
			<td>trypsin treatment of cells</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Beyotime Biotechnology</td>
			<td>ST798</td>
			<td>Staining cells to assess activity</td>
		</tr>
		<tr>
			<td>vector (pLV-luciferase)</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>VL3613</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Shanghai YueNian Biotechnology Co., Ltd</td>
			<td>11668027,Thermo fisher</td>
			<td>Plasmid transfection reagent</td>
		</tr>
		<tr>
			<td>X-ray imaging system</td>
			<td>Brook (Beijing) Technology Co., Ltd</td>
			<td>FX PRO</td>
			<td>X-ray images were obtained to detect tumor growth</td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models at JoVE.com

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

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 ...

This protocol describes the technique to generate mouse models bearing primary osteosarcoma and the pulmonary metastasis lesions. By intratibial injection of osteosarcoma cells, this model best mimics the clinical development characteristics of tumor osteosarcoma. Accurate numbers of osteosarcoma cells are directly injected into the tibia using a microvolume syringe, allowing for identical tumor formation rates and tumor volumes.On the day of osteosarcoma cell injection, wash the cells twice with PBS. Trypsinize the cells with 1.5 milliliters of 0.25%trypsin for three minutes. Then add six milliliters of 10%serum-containing MEM media.Collect the cells in a 15-milliliter centrifuge tube. Aspirate 20 microliters of cell suspension into the chamber of the cell counting plate, and calculate cell concentration using an automatic cell counter. Centrifuge the cells at 800 times g for five minutes at room temperature.Aspirate the supernatant with a pipette, resuspend the cell pellet in an 8.5-milligram-per-milliliter basement membrane matrix, and place them on ice. Place the mouse in the supine position. Hold the ankle of the mouse using the thumb and index finger, and disinfect the injection site of the tibia with a 70%ethanol swab.Rotate the ankle joint of each mouse outward to move the tibia and fibula, and bend the knee joint to a suitable position until the proximal tibia plateau is visible through the skin. Attach the needle to a one-milliliter syringe, and point the needle tip toward the injection site, parallel to the axis of the tibia. Percutaneously insert the needle to drill a hole through the tibia platform toward the distal end of the tibia.Load the osteosarcoma cell suspension previously placed on ice into a microvolume syringe, and replace the one-milliliter syringe in the tibia with the osteosarcoma cell-loaded microvolume syringe. Slowly inject approximately 10 microliters of osteosarcoma cell suspension into each athymic mouse's tibia without applying much pressure. Then remove the microvolume syringe from the injection site, and press the site with the cotton swab for about 30 seconds.Place the mouse back into a clean cage, and observe its recovery for 10 minutes. This figure depicts the mouse model with orthotopic osteosarcoma developed from intratibial osteosarcoma cell injection and its isolated form. A gradual increase in the tumor volume is observed every week.The X-ray images obtained from the first through the sixth week after injection of luciferase-labeled osteosarcoma cells showed a gradual increase in volume. Furthermore, the image obtained after the injection into the mouse tibia showed orthotopic osteosarcoma growth in vivo. The pulmonary metastasis caused by intratibial injection of the osteosarcoma cells was successfully tracked in vivo using a bioluminescence live imaging system.Observation under a stereomicroscope revealed metastatic colonies in the isolated lung tissues. HE staining on paraffin-embedded lung tissues showed metastatic lesions. The needle should be percutaneously inserted into the joint capsule through the patellar ligament to drill a hole through the tibia platform toward the digital tibia by rotating the syringe.

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JoVE Journal

Cancer Research

7.8K ビュー.  Shanghai University of Traditional Chinese Medicine. このプロトコルは、原発性骨肉腫および肺転移病変を有するマウスモデルを生成する技術を記述する。骨肉腫細胞の脛骨内注射によって、このモデルは腫瘍骨肉腫の臨床発達特性を最もよく模倣する。正確な数の骨肉腫細胞は、マイクロボリュームシリンジを使用して脛骨に直接注入され、同一の腫瘍形成速度および腫瘍体積を可能にする。骨肉腫細胞注射の日に?...

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

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

gavage strategy for decoction formula of traditional chinese medicine in osteosarcoma model mice

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...

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

Herb Decoction Formulation and Gavage Standardization in Osteosarcoma Mice

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

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...

practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

intracarotid cancer cell injection to produce mouse models of brain metastasis

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention...

establishment of patient-derived xenograft mouse model with human osteosarcoma tissues

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...

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

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brain metastasis mouse model: a method to generate brain metastasis mouse model by intracarotid cancer cell injection

This video demonstrates the establishment of a brain metastasis mouse model by intracarotid injection of fluorescently labeled cancer cells. The mouse model can be used to study the effects of novel therapeutics and the underlying physiological...

Brain Metastasis Mouse Model: A Method to Generate Brain Metastasis Mouse Model by Intracarotid Cancer Cell Injection

Then, place the mouse on a glass plate and secure with rubber bands. Clean the skin by applying 70% alcohol and povidone-iodine. Now, make an incision in the skin about 1 centimeter long with a surgical scalpel. Then, bluntly dissect the muscle with surgical forceps to expose the carotid artery...

establishment of pdx models by osteosarcoma tissue transplantation at the mouse flank region

Establishment of PDX Models by Osteosarcoma Tissue Transplantation at the Mouse Flank Region

a preclinical mouse model of osteosarcoma to define the extracellular vesicle-mediated communication between tumor and mesenchymal stem cells

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

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...

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

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

orthotopic homograft tumor transplantation: a technique to generate pancreatic tumor mouse models

The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more...

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

an orthotopic mouse model of spontaneous breast cancer metastasis

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

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...

primary tumor and mef cell isolation to study lung metastasis

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is...

establishment of cancer stem cell cultures from human conventional osteosarcoma

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent...

models of bone metastasis

Models of Bone Metastasis

Animal models are frequently utilized to study cancer metastasis to bone.  In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these...

patient-derived orthotopic xenograft models for human urothelial cell carcinoma and colorectal cancer tumor growth and spontaneous metastasis

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic...

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

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 ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...