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 osteosarcoma cell (143B-Luciferase) injection, wash 80%-90% confluent cells cultured in a 10 cm cell culture dish twice with PBS (pH 7.4) and trypsinize with 1.5 mL of 0.25% trypsin for 3 min. Then, add 6 mL of 10% serum-containing MEM media to quench the trypsin, and collect the cells in a 15 mL centrifuge tube. 
	NOTE: 143B-Luciferase cell line is obtained from 143B cell line transfect with pLV-luciferase vector23.</li>
	<li>Aspirate 20 &#181;L of cell suspension into the chamber of cell counting plate and calculate cell concentration using an automatic cell counter (see Table of Materials).</li>
	<li>Centrifuge the cells at 800 x g for 5 min at room temperature.</li>
	<li>Aspirate the supernatant with a pipette and resuspend the cell pellet in an 8.5 mg/mL basement membrane matrix (see Table of Materials) to a final concentration of 2 x 107 cells/mL.</li>
	<li>Keeping the cells on ice, bring them to the surgery room. The cells are to be used within 2 h. 
	NOTE: To avoid inaccurate injection doses (for example, due to the dead space in syringes), an extra cell suspension is prepared (usually two times the required volume of cell suspension). The basement membrane matrix is kept on ice all the time since it has coagulation property above room temperature24.</li>
</ol>
2. Surgery for orthotopic injection of the osteosarcoma cells
NOTE: The surgery tools are shown in Figure 1.
<ol>
	<li>Mice were raised in specific pathogen-free conditions. All procedures were done in an aseptic cabinet with sterile tools.</li>
	<li>Anesthetize the mice by exposing them to 2% isoflurane and 98% oxygen (oxygen flow rate, 2 L/min).</li>
	<li>Apply a small amount of ophthalmic ointment on the eyes to prevent dryness while under anesthesia. 
	NOTE: Perform the entire procedure in a well-ventilated area. Before osteosarcoma cell injection, ensure that each mouse is under deep anesthesia by a toe pinch; if the mouse still has responses, such as twitch or jerk, wait for a long time until the above responses disappear.</li>
	<li>Keep each mouse in a supine position. Hold the ankle of the mouse using the thumb and index finger and disinfect the injection site of the tibia with a 70% ethanol swab. 
	NOTE: To tightly hold the mouse ankle, both the thumb and index fingertips are of great importance for the subsequent procedures.</li>
	<li>Rotate the ankle joint of each mouse outward to move the tibia and fibula, and bend the knee joint to a suitable position until the proximal tibia plateau (the top of the tibia) is clearly visible through the skin (Figure 2A).</li>
	<li>Attach the needle to a 1 mL syringe and point the needle tip toward the injection site. Ensure that the syringe needle is parallel to the long axis of the tibia.
	<ol>
		<li>Percutaneously insert the needle through or adjacent to the patellar ligament as it goes through the skin/joint capsule; th0en, rotate the syringe (1/2 to 3/4-circle) to drill a hole through the tibia platform toward the distal end of the tibia (medullary cavity) for osteosarcoma cell injection with a micro-volume syringe (Figure 2B,C). 
		NOTE: Simultaneous rotation of the tibia can be felt while drilling if the needle tip is accurate. Ensure the needling moves forward with the syringe rotation rather than being directly pushed forward until about half of the needle is in the tibia.</li>
	</ol>
	</li>
	<li>Check whether the syringe needle made a prominent movement into the medullary canal to ensure successful drilling. 
	NOTE: Perform an X-ray examination (see Table of Materials) to confirm the proper position of the needle and collect the images.</li>
	<li>Load 143B osteosarcoma cell suspension (from step 1.5) into a micro-volume syringe and replace the 1 mL syringe in the tibia with the 143B cell-loaded micro-volume syringe (Figure 2D). Slowly inject ~10 &#181;L (ignore pre-existing solution in the needle) of 143B cell suspension into each athymic mouse&#39;s tibia (about 2 x 105 cells) without applying high pressure.</li>
	<li>Press the injection site with a cotton swab for 20-30 s when the micro-volume syringe is removed.</li>
	<li>Put each mouse back into a clean cage and closely monitor until the mouse is completely recovered from anesthesia (about 10 min).</li>
	<li>Monitor the tumor growth in vivo using an X-ray imaging system. Measure the longer diameter (a) and the short diameter (b) of the cancer mass every week with a caliper for tumor volume (V) calculation: V = 1/2 x a x b2. 
	&#8203;NOTE: Anesthetize the mice by exposing them to 2% isoflurane and 98% oxygen. The mice were anesthetized for x-ray imaging. Intratibia injection of luciferase or fluorescent protein labeled osteosarcoma cells enables tracking of primary and metastatic osteosarcoma lesions. 
	NOTE: Humane endpoints of the mice with osteosarcoma due to tumor growth of the knee and lung metastasis were based on the following criteria: (1) Body Condition Score, (2) weight loss threshold of 20%, (3) average maximum diameter of tumors of 2 cm, or (4) severely limited animal behavior.</li>
</ol>
3. Pathologic examination (collecting primary and pulmonary metastatic osteosarcoma specimen for analysis)
<ol>
	<li>Six weeks after osteosarcoma cell injection, sacrifice the mice by cervical dislocation after exposing them for CO2 inhalation.</li>
	<li>Keep the mouse in a supine position and stretch both the hind limbs.</li>
	<li>Separate the whole legs bearing osteosarcoma from the inguinal area. 
	NOTE: Ensure that all legs are separated from the same anatomical site.</li>
	<li>Prepare the histological specimen of legs bearing osteosarcoma by removing the skin, muscles, and feet, and then fix the specimen of each mouse in a 50 mL tube with 20 mL formalin solution (10%) for 24 h, followed by decalcification in 10% EDTA solution for 14 days with occasional buffer change.</li>
	<li>Embed the specimen in paraffin and prepare sections for histological examination following previously published work25.</li>
	<li>Gently separate the lungs and put them into a 50 mL tube filled with 20 mL formalin solution (10%). After 24 h, transfer the lungs of each mouse into a 15 mL tube with 70% ethanol. Embed the lungs in paraffin for Hematoxylin and Eosin (H&#38;E) staining and immunohistochemistry assay25.</li>
</ol>

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The authors declare that they have no competing financial interests.

Orthotopic injection of osteosarcoma cells is an ideal model to study the function and mechanism of specific factors in osteosarcoma carcinogenesis and development to evaluate the therapeutic efficacy. To avoid differences in tumor growth, most active osteosarcoma cells at 80%-90% confluent with the same number are carefully injected into the tibia of each mouse, and the cell trypsinization time is strictly controlled without affecting the cell viability. As cell clumps affect cell counting leading to inaccurate cell numbers being injected into the tibia of each mouse, the cell suspension needs to be appropriately mixed up and down with a pipette to avoid the formation of cell clumps.
Another critical aspect that must be taken into consideration is the resuspended solution for osteosarcoma cells. The injected cells are resuspended in a basement membrane matrix instead of in PBS or culture medium. Moreover, a high concentration of basement membrane matrix is challenging to be pipetted and affects the accurate volume; thus, an appropriate concentration of basement membrane matrix is required26. To drill a hole through the tibia platform for osteosarcoma cell injection, the needling moves forward with the syringe rotation rather than being directly pushed forward until about half of the needle is in the tibia. More particularly, immunodeficient mice are applied to establish an orthotopic osteosarcoma model using human osteosarcoma cells27. Meanwhile, the injection procedure is performed in biological safety cabinet using sterile surgical tools. Since mice may experience uneasiness after anesthesia and surgery, the mice must be closely monitored on the first week postsurgery.
Intratibia injection of osteosarcoma cells labeled with fluorescent protein or luciferase enables the tracking of primary and metastatic lesions using optical imaging28. Osteosarcoma is never allowed beyond the size limit as in the IACUC-approved protocol; meanwhile, ulcerations may occur in enormous size tumor mass, which may lead to failed immunohistochemical analyses. Although the primary bone tumors and bone metastasis have been recently reported to be achieved by implantation of solid tumor graft into bone, and the animals developed reproducible growth, as well as lung metastasis eventually29; however, the authors directly implanted fresh or cryopreserved tumor fragments into the proximal tibia, which showed the disadvantage of open surgery caused potential infection and failure of developing tumor engraftment. Moreover, the volume of implanted tumor fragments without strict control will result in a significant difference in produced tumor volume, which is difficult in following application, such as evaluating the therapeutic efficacy in vivo. Here, a simple and reproducible technique is reported to establish the intratibia primary osteosarcoma with later pulmonary metastasis mouse models via intratibia injection of osteosarcoma cells. This showed the advantages of best mimicking the clinical development characteristics of osteosarcoma in humans; accurate numbers of osteosarcoma cells being directly injected into tibia using micro-volume syringe allowing identical tumor formation rate (100%) and tumor volume. The method ensures avoiding the possibilities of infection or even death using open surgery techniques and allowing lively monitor and quantifying osteosarcoma growth and metastasis using the bioluminescence live imaging system after the injected osteosarcoma cells are labeled with bioluminescence. This prevents the injected osteosarcoma cells from directly reaching the bloodstream and colonizing in the lungs to form pulmonary embolism and/or false-positive pulmonary metastasis by resuspending the injected osteosarcoma cells in appropriate concentration of basement membrane matrix since the basement membrane matrix has the property of coagulation above room temperature. The immediate coagulation support and restrict osteosarcoma cells within the basement membrane matrix after being injected into the mouse tibia.
Another literature has reported the bone metastasis model establishment by intracardiac inoculation or intratibial inoculation of breast cancer cells30; however, cells used in this literature are breast cancer cells, which have different biological and clinical characteristics with osteosarcoma cells; moreover, both the intracardiac and the intratibial inoculation established cancer models in bone are formed by cancer cell colonizing directly or reaching through bloodstream rather than metastasis lesions formed by cancer cell dissemination from the primary cancer lesions.
There are several limitations of the current protocol. Mice used in this protocol are genetic immune system defect nude mice without thymus that prevents them from immunologically rejecting human cells and are widely used in preclinical trials, which are not applicable for immune functional research. Furthermore, we found that not all osteosarcoma cell lines are identically relevant in these models, and the tumorigenesis abilities of 143B, MNNG, MG-63, and U-2 OS cells are higher than the Saos-2 cells.
In conclusion, the present primary and pulmonary metastatic osteosarcoma models generated by orthotopic osteosarcoma cell injection are handy tools to study the tumor microenvironment, efficacy of therapeutics on osteosarcoma growth and/or metastasis. In addition, by intratibia injection of the genetically modified osteosarcoma cells specifically targeting a gene, the models are helpful to explore the key oncogenes and tumor suppressors in osteosarcoma growth and pulmonary metastasis.

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><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>&amp;nbsp;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 &amp;mu;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 once a week with a caliper and calculated as described in step 2.11 (Figure 3C). The orthotopic (primary) osteosarcoma growth in vivo was tracked by both the X-ray and the bioluminescence (when the injected cells were labeled with luciferase) live imaging system. The X-ray images were obtained from the first week to the sixth week after 143B osteosarcoma cell injection (Figure 3D). Furthermore, the image of orthotopic (primary) osteosarcoma growth in vivo was obtained after luciferase labeled 143B cells were injected into the mouse tibia (Figure 3E).
The pulmonary metastasis caused by the intratibial injection of luciferase labeled osteosarcoma cells was successfully tracked in vivo by a bioluminescence live imaging system&#160;(Figure 4A). The metastatic colonies in the isolated lung tissues were also visualized under the stereomicroscope (Figure 4B). The metastatic lesions were further confirmed by H&#38;E staining on paraffin-embedded lung tissues (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63072/63072fig01.jpg" /> 
Figure 1: Surgery Tools. (A) 1 mL scale syringe. (B) Micro-volume syringe. <a href="https://www.jove.com/files/ftp_upload/63072/63072fig01large.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/63072/63072fig02.jpg" /> 
Figure 2: Representation of the intratibial injection surgery. (A) The intratibial injection site of an athymic mouse. (B) A sterile 1 mL syringe with an accompanied needle was percutaneously inserted into the tibia toward the distal end via the proximal tibia plateau (the top of the tibia). (C) A lateral view of the drilling process. The syringe needle was parallel to the long tibia axis (solid line). (D) Intratibial injection with osteosarcoma cell loaded micro-volume syringe. <a href="https://www.jove.com/files/ftp_upload/63072/63072fig02large.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/63072/63072fig03.jpg" /> 
Figure 3: Visualization of the osteosarcoma growth in mice. (A) Successful mouse orthotopic osteosarcoma model. (B) Isolated orthotopic osteosarcoma. (C) Tumor volume was measured with a caliper and calculated using the following formula: tumor volume = 0.5 x longer diameter x short diameter x short diameter. Error bars stand for standard deviation (n = 8). (D) X-ray images were obtained from the same mouse at a different time (from 1-6 weeks). (E) Image obtained on the 28th day after luciferase labeled 143B cells were injected into the mouse tibia. The red arrows indicated the luminescence intensity of the orthotopic (primary) osteosarcoma. <a href="https://www.jove.com/files/ftp_upload/63072/63072fig03large.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/63072/63072fig04.jpg" /> 
Figure 4: Pulmonary metastasis of osteosarcoma. (A) Image obtained on the 28th day after luciferase labeled 143B cells were injected into the mouse tibia. The red arrows indicated the luminescence intensity of the pulmonary metastasis. (B) The isolated lungs with osteosarcoma metastases. The red arrows indicated the metastatic colonies (x20). (C) H&#38;E staining showed metastatic lesions in lung tissues (scale bar = 200 &#181;m). <a href="https://www.jove.com/files/ftp_upload/63072/63072fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

intratibial-osteosarcoma-cell-injection-to-generate-orthotopic

Universidad Científica del Sur

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Cancer Research

9.0K Views.  Shanghai University of Traditional Chinese Medicine. The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

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

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

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

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 ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

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 models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Medicine

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 ...

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.

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 ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

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, ...

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

Summary

Explore More Videos

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

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

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

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Models of Bone Metastasis

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

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

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

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