Research in Zhang lab was supported by X. H.-F. Z.'s NCI CA151293, CA183878, Breast Cancer Research Foundation, U.S. Department of Defense DAMD W81XWH-13-1-0195, a Pilot Award of CA149196-04, McNair Medical Institute and by H.W.'s U.S. Department of Defense DAMD W81XWH-13-1-0296.

All animal work was done in accordance to the animal care guidelines of the Baylor College of Medicine.
1. Cell Preparation
Note: Different cancer cell lines can be used for IIA injection depending on research purposes. We have used breast cancer cell lines MCF7, 4T1, 4T07, MDA-MB-361, MDA-MB-231, MDA-MB-436 and prostate cancer cell line C4-2 in our research. We typically use both GFP- and firefly luciferase-labeled cancer cells for our study and show some data here from the GFP-Luciferase-labeled MCF7 cell line.
<ol>
	<li>Maintain cells in DMEM containing 10% FBS and 0.1 mg/ml penicillin-streptomycin at 37 &#176;C in 5% CO2.</li>
	<li>Before IIA injection, trypsinize cells at 80 - 90% confluence with 0.25% Trypsin/EDTA solution. Use cold PBS to wash cells twice, and re-suspend cells in 10 ml PBS for cell counting. To remove PBS, centrifuge cells at 800 x g for 5 min.</li>
	<li>Re-suspend cells at a concentration of 5 x 106 cells/ml in ice-cold PBS.</li>
	<li>Use 100 &#181;l GFP- and firefly luciferase-labeled cancer cells for IIA injection of one mouse. 
	Note: Therefore, the number of cells received by each animal is 5 x 105. This number may need to be altered based upon the aggressiveness of cell lines.</li>
	<li>Keep cell suspensions on ice. 
	Note: Vibrations may be needed to prevent cell aggregation every few minutes until it is ready for injection. For some cell lines (such as MDA-MB-231), more stringent procedures (e.g., passing through 45 &#181;m cell strainer) may be needed to prevent aggregation. Please note that the clog of vessels may cause tissue necrosis, and therefore confounding the experiments.</li>
</ol>
2. Animal Preparation
<ol>
	<li>For animal pain management, give 5 mg/kg/day carprofen (or other analgesic) with dietary supplement to the mice no less than 24 hr before surgery. Administer a dose of 0.1 mg/ml buprenorphine subcutaneously 60 min prior to the surgery. Note: an optional procedure is to implant estradiol pallet into the back of animals to provide extra estradiol. This will accelerate the grow of ER+ cancer cells (e.g., MCF-7 cells)9.</li>
	<li>Anesthetize a 4 - 6 week old mouse (approximately 20 g) with ketamine (80 - 100 mg/kg) and xylazine (10 -12.5 mg/ml) by intraperitoneal injection.</li>
	<li>Confirm the appropriate level of sedation of a 20 g mouse by toe pinch and the observation of a 50% reduction in respiratory rate and pink mucous membranes (e.g., mouse ear skin and paw skin). Keep monitoring these vital signs every 15 min during the surgery. Note: No movement of the animal indicates that the animal is sufficiently anesthetized and ready for surgery.</li>
	<li>Use vet ointment on eyes to prevent dryness.</li>
	<li>Shave the mouse on the lower abdomen, especially the right groin area where the surgery and injection will take place. 
	Note: This is not necessary if athymic nude mice are used.</li>
	<li>Put the mouse on its back, spread the legs in their natural positions and stick the toes to mobile dissection cardboard with tape.</li>
	<li>Wipe the right groin area with 70% isopropyl ethanol soaked sterile cotton swabs, followed with betadine surgical scrub several times.</li>
</ol>
3. The Common Iliac Vein and Artery Location and Separation
<ol>
	<li>Make a skin incision of 1.0 - 1.2 cm long between the 4th and 5th nipples in the lower right abdomen using sterile, #10 carbon steel scalpel blades held in a No. 3 handle. Then use a sterile surgical drape to cover the animal body except the incision site.</li>
	<li>Move the animal to standard bench top dissection microscope. Use a magnification of 4X for injection.</li>
	<li>Under 4X magnification of the dissection microscope, insert blunt separation forceps between the fatty tissue and the peritoneum, and push the tissues outwards on both sides to expose the iliac vessels and nerves (see Figure 1). 
	Note: The artery between aorta from the midline and the lower part of artery branches is called common iliac artery. This is where the injection takes place.</li>
	<li>Use one pair of straight fine forceps to break through the connective tissue (like a membrane) between vessels and the nerve, closer to the vessels.</li>
	<li>Switch the straight fine forceps to the left hand. Grab a pair of angled fine forceps using the right hand. Insert the right hand forceps into same position where connective tissues have been broken. And then stick the right hand forceps through, beneath the vessels.</li>
	<li>At the same time, insert the left hand forceps into the connective tissues on the left side of the vessels to guide the right hand forceps going through.</li>
	<li>Once the right hand forceps gets beneath the vessels, try to separate the vessels from the surrounding tissue a little bit more, and then keep them on top of the right hand forceps.</li>
	<li>Use left hand forceps to deliver the tip of a 4-0 silk suture to the right hand forceps, and then pull the suture gently and slowly through beneath the vessels. Note: Now both common vein and artery vessels are lifted up by the suture (see Figure 2).</li>
</ol>
4. Injection and Post-injection Care
<ol>
	<li>Prepare the cells for injection in a 31 G insulin syringe. Use 100 &#181;l of cell suspension in PBS per injection. Make sure to pipette up and down several times to separate the cells and avoid aggregation so that the injected cells will not clog and block the blood flow.</li>
	<li>With the angled forceps in the left hand, put the forceps beneath the vessels along the guidance of the suture, and then open the two arms of the forceps, having the vessels held gently on the forceps. 
	Note: The interval between the two arms of the forceps will be the injection site.</li>
	<li>Hold the 31 G needle with 100 &#181;l cell suspensions in the right hand, with the bevel of the needle upward, ready for injection.</li>
	<li>Insert the needle into the artery lumen (blood will enter the needle tip). Then push cell suspension slowly in to inject the cells. The cell suspension will push away the red blood in the vessel. Try not to break the vessels or leak the cell suspension out.</li>
	<li>When the injection is finished, gently pull back the needle and keep holding the vessels up on the left hand forceps to stop bleeding. Then pull away the suture.</li>
	<li>Pull away the left hand forceps, and quickly use a cotton swab to press the artery incision area to stop bleeding. 
	Note: Usually 5 - 10 min continuous pressure is sufficient to stop bleeding.</li>
	<li>When bleeding stops, use skin glue to close the skin edges of the surgery area. Make an ear-tag, and check for bioluminescence signaling to examine the distribution of injected cells. 
	Note: A successful injection will result in an image similar to Figure 3.
	<ol>
		<li>Check bioluminescence signaling by injection of 100 &#181;l 15 mg/ml luciferin in PBS at 75 mg/kg mouse concentration via the intra-orbital sinus.
		<ol>
			<li>For intra-orbital injection, put fingers on both sides of the mouse eye, use pressure to make eye ball slightly pop out from the eye frame, insert 28 G insulin syringe needles between eye ball and nose, and then slowly push syringe to inject luciferin. 
			Note: At the correct position, the needle should be able to reach a depth of 2 - 3 mm before hitting hard tissue (bone).</li>
			<li>Place the mouse into imaging system for in vivo whole animal imaging for 10 - 60 sec within 5 min (see Figure 3). 
			Note: An optional procedure is to implant estradiol pellet into the back of animals to provide extra estradiol. This will accelerate the growth of ER+ cancer cells (e.g., MCF-7 cells)15.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Leave the mouse on a warm heating pad until it wakes up. Monitor the bleeding, swelling, signs of dehiscence and pain during the post-surgical period. Put the mice back to the cage with analgesic coverage (carprofin dietary supplement suggested) given for pain management until no signs of pain. Pay close attention and care to the animals during 7 days following surgery.</li>
</ol>
5. Monitoring Metastatic Growth
<ol>
	<li>Histomorphometry:
	<ol>
		<li>Harvest tumor bearing bone tissues, fix the bones in 4% paraformaldehyde for 24 hr, then decalcify them in pH 7.0 14% EDTA solution for 3 - 5 days, and subject them to paraffin embedding as previously described14.</li>
		<li>Section the paraffin-embed bone tissues at the thickness of 3 &#181;m and perform standard histological method of Hematoxylin/Eosin staining as previously described14.</li>
	</ol>
	</li>
	<li>Immunohistochemistry:
	<ol>
		<li>Subject paraffin-embedded tumor bearing bone slides to immunohistochemistry staining for various purposes as previously described14. Briefly, immunostain MCF7 cancer cells by anti-GFP antibodies, and immunostain osteogenic cells (pre-osteoblasts and osteoblasts) by anti-Osterix antibodies and anti-Alkaline phosphatase (ALP) antibodies, respectively.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Although only the iliac artery is the target of injection for cancer cells, we recommend the separation of both iliac vein and artery from surrounding tissues, and to lift them together as a bundle. This is because the vein and artery extensively contact with each other, and the venous vessel wall is thin and is easy to break. Therefore, for a successful injection, it saves time and effort to hold up the two vessels together, although cancer cells are injected only to the artery. A 4-0 silk suture is used to help this pr...

Metastases account for over 90% of deaths caused by solid tumors. Bone is the most common organ affected by metastases of various cancer types, especially breast and prostate cancers. When diagnosed in the clinic, bone metastases usually have already entered advanced stages with either osteolytic or osteoblastic alterations in bone, often accompanied with neurological symptoms.
Previous studies predominantly focused on the overt osteolytic bone metastases1-3, however we currently have limited understanding of micrometastases in bones before the onset of the osteolytic process. This is at least partly due to lack of appropriate experimental models and approaches. Genetically engineered mouse models of breast cancer often metastasize to lungs, but much less efficiently to bones4. Likewise, the orthotopically transplanted tumors rarely develop spontaneous bone metastases, with some bone-tropical 4T1 mammary carcinoma sub-clones and MSP overexpressed PyMT transgenic mouse model as exceptions5-7. Intra-tibial drilling can deliver cancer cells to the bone8-10, but it also incurs damage and inflammation to local tissues. Currently intra-cardiac injection of breast cancer cell lines has been the major approach to investigate bone colonization11-13. However, after cancer cells are introduced into left ventricle only a limited proportion will finally reach bone and bone marrow, making it difficult to track microscopic metastases in a quantifiable fashion.
In this study, we establish a technique, namely intra-iliac artery (IIA) injection14, to selectively deliver cancer cells into hind limb tissues, thereby enriching cancer cells in bone and bone marrow without causing damage to local tissues. Because of the bone specificity, this approach also allows enough time for indolent cancer cells to eventually colonize before the animals succumb to primary tumors or metastases in other vital organs. When combined with a variety of other techniques, such as bioluminescence imaging, immunofluorescence staining and bone histomorphometry, IIA injection is potentially useful for a wide scope of research purposes related to bone metastases, especially to track the progression from single cancer cells to multi-cell micrometastases. In particular, we demonstrated that IIA injection enables us to visualize the interactions between cancer cells and various types of surrounding cells in the bone microenvironment.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>HyClone</td><td>SH30022.01</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000</td><td></td></tr><tr><td>Pen/Strep Amphatericin B</td><td>Lonza Biowhittaker</td><td>17-745E</td><td></td></tr><tr><td>PBS</td><td>Lonza Biowhittaker</td><td>17-516F</td><td></td></tr><tr><td>Trypsin/EDTA solution</td><td>HyClone</td><td>SH30042.01</td><td></td></tr><tr><td>45 &amp;mu;M cell strainer</td><td>VWR International Laboratory</td><td>195-2545</td><td></td></tr><tr><td>MediGel CPF with carprofen&amp;nbsp;</td><td>Controlled item from veterinary care in BCM</td><td></td><td>For pain management</td></tr><tr><td>Buprenorphine&amp;nbsp;</td><td>Controlled item from veterinary care in BCM</td><td></td><td>For pain management</td></tr><tr><td>Estradiol pellet</td><td>Innovative Research of America</td><td>SE-121</td><td></td></tr><tr><td>Ketamine and xylazine</td><td>Controlled item from veterinary care in BCM</td><td></td><td></td></tr><tr><td>Vet ointment</td><td>Controlled item from veterinary care in BCM</td><td></td><td>Avoid eye dryness</td></tr><tr><td>Shaver</td><td>Oster</td><td>78005-050</td><td>For furred mice</td></tr><tr><td>Isopropyl ethanol</td><td>ACROS</td><td>67-63-0</td><td></td></tr><tr><td>Betadine surgical scrub</td><td>Controlled item from veterinary care in BCM</td><td></td><td></td></tr><tr><td>#10 scalpel blades</td><td>Ted Pella, Inc</td><td>549-3CS-10</td><td>Multiple</td></tr><tr><td>No. 3 handle</td><td>Ted Pella, Inc</td><td>541-31</td><td>Need to be autoclaved</td></tr><tr><td>Sterile surgical drape</td><td>Sai Infusion Technology</td><td>PSS-SD1</td><td></td></tr><tr><td>Straight forceps&amp;nbsp;</td><td>Roboz Surgical Instrument</td><td>RS-5132</td><td>Need to be autoclaved</td></tr><tr><td>Straight fine forceps</td><td>Fine Science Tools</td><td>11253-20</td><td>Need to be autoclaved</td></tr><tr><td>Edged fine forceps</td><td>Fine Science Tools</td><td>11253-25</td><td>Need to be autoclaved</td></tr><tr><td>4-0 Vicryl silk suture</td><td>Johnson &amp;amp; Johnson Health Care</td><td>J214H</td><td></td></tr><tr><td>31 G insuline syringes</td><td>BD</td><td>328418</td><td>Multiple</td></tr><tr><td>Q-tips cotton swabs (Sterile)</td><td>VWR International Laboratory</td><td>89031-272</td><td></td></tr><tr><td>Skin glue</td><td>Henry Schein Animal Health</td><td>31477</td><td>For surgery site skin closure</td></tr><tr><td>Ear Tag Applicator</td><td>Fine Science Tools</td><td>24220-00</td><td></td></tr><tr><td>Ear tags</td><td>Fine Science Tools</td><td>24220-50</td><td></td></tr><tr><td>D-luciferin</td><td>Gold Biotechnology</td><td>LUCK</td><td>Avoid light and put on ice</td></tr><tr><td>28 G insulin syringes</td><td>BD</td><td>329410</td><td>For intra-orbital injection</td></tr><tr><td>Paraformadehyde</td><td>Alfa Aesar</td><td>30525-89-4</td><td>For tissue fixation</td></tr><tr><td>EDTA</td><td>OmniPur</td><td>4050</td><td>For bone tissue decalficication</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissection microscope</td><td>Leica</td><td>Leica S6E stereo</td><td></td></tr><tr><td>IVIS Lumina II imaging system</td><td>Advanced Molecular Vision</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-GFP antibodies (JL-8)</td><td>Clontech</td><td>632381</td><td></td></tr><tr><td>Anti-ALP antibodies</td><td>Abcam</td><td>ab108337</td><td></td></tr><tr><td>Anti-Osterix antibodies</td><td>Abcam</td><td>ab22552</td><td></td></tr></tbody></table>

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.

Figure 1 illustrates the anatomical location and relationship of common iliac artery (red) and vein (blue).
Figure 2 shows relative position of iliac vessels and nerves under dissection microscopy. As depicted in Figure 2A, the vessels and nerves are right beneath the peritoneal wall and can be revealed after the skin incision is made and the peritoneum is pushed away. The commo...

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

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 ...

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16.3K Views.  Baylor College of Medicine. 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. 

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

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Medicine

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 ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

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 ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

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

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

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

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Bioengineering

Intra-Cardiac Injection of Human Prostate Cancer Cells to Create a Bone Metastasis Xenograft Mouse Model

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is essential to understand the process and related mechanisms of prostate cancer bone metastasis for developing new therapeutics. For this, an animal model of bone metastasis is an essential tool. Here, we report detailed procedures to generate a bone metastasis mouse model via intra-cardiac injection of prostate cancer cells. A bioluminescence imaging system can determine whether prostate cancer cells have been accurately injected into the heart and monitor cancer cell metastasis since it has great advantages in monitoring metastatic lesion development. This model replicates the natural development of disseminated cancer cells to form micro-metastases in the bone and imitates the pathological process of prostate cancer bone metastasis. It provides an effective tool for further exploration of the molecular mechanisms and the in vivo therapeutic effects of this disease.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

As the most common male malignancy, prostate cancer (PC) ranks second in mortality, primarily due to a 65%-75% bone metastasis rate. Therefore, it is ...

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

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