This work was supported by National Institutes of Health (NIH) grants: UL1TR000101 (previously UL1RR031975) through the Clinical and Translational Science Awards Program, 1RO1CA123211, 1R03CA178809, R01CA197964 and 1R21CA198698 to JK. MRI was performed in the Georgetown-Lombardi Comprehensive Cancer Center's Preclinical Imaging Research Laboratory (PIRL) and tissue processing in the Georgetown-Lombardi Comprehensive Cancer Center's Histopathology &amp; Tissue Shared Resource, both supported by NIH/NCI grant P30-CA051008. The authors thank Dan Chalothorn and James E. Faber, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, for their assistance with postmortem x-ray angiography, and providing insight and expertise on collaterogenesis.

All procedures were approved by the Georgetown University Institutional Animal Care and Use Committee.
1. Cell Preparation for Orthotopic Injections
<ol>
	<li>Culture human ES cells under standard conditions. Use approximately one 15-cm cell culture plate not exceeding 70% of confluency for injection of 5 mice. 
	NOTE: For this study, SK-ES1 cells were cultured in McCoy&#39;s 5A medium with 15% fetal bovine serum (FBS) on collagen-coated plates and TC71 cells were cultured in RPMI with 10% FBS and 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Both media were supplemented with antibiotics &#8212; penicillin (100 units/ml), streptomycin (100 &#181;g/ml) and fungizone (1 &#181;g/ml).</li>
	<li>Wash the ES cells with phosphate buffered saline (PBS) and trypsinize at 70% confluence with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) for 5 min.</li>
	<li>Remove the ES cells from the plate with cell culture media, then centrifuge for 5 min at 200 x g at room temperature. Re-suspend the ES cells in 10 ml of cold PBS and then count the number of cells.</li>
	<li>Centrifuge ES cells for 5 min at 200 x g at room temperature, and then re-suspend at 2 x 107 (SK-ES1) or 107 (TC71) cells per ml in cold PBS. Keep the final cell suspension on ice while performing injections.</li>
</ol>
2. Orthotopic Injection of ES cells into Gastrocnemius Muscle
<ol>
	<li>Use 4-6 week old female SCID/beige mice.</li>
	<li>To inject ES cells, gently hold the mouse and stabilize its left leg between the fourth and fifth fingers, exposing the medial side of the lower hindlimb.</li>
	<li>Using a 28 G &#189; needle, inject 0.1 ml of the previously prepared cell suspension that contains either 2 x 106 (SK-ES1) or 106 (TC71) ES cells into the gastrocnemius muscle (Figure 2A).&#160; 
	NOTE: Although maximum volume for intramuscular injections is typically 0.05 ml, for this particular procedure the volume increase to 0.1 ml is necessary due to the high cell number injected. This procedure has been approved by the Georgetown University Institutional Animal Care and Use Committee.
	<ol>
		<li>Insert the needle into the gastrocnemius muscle anteriorly at approximately a 30 - 45 degree angle in the direction of the tibial crest/tuberosity.</li>
		<li>Slightly withdraw the needle once it touches the tibial crest/tuberosity. Slowly inject the cell suspension solution, gradually withdrawing the needle to release pressure.</li>
	</ol>
	</li>
	<li>Monitor the injected mice over the next 24 hr for signs of distress.&#160; 
	NOTE: Investigators with less experience in animal handling should consider anesthetizing mice for tumor cell injections. Some institutions may require anesthesia for safety reasons.</li>
</ol>
3. Monitoring Primary Tumor Growth
<ol>
	<li>Monitor the growth of primary tumors daily until the tumor size reaches the desired volume. 
	NOTE: In the current study, a calf volume of 250 mm3 was used as a starting point of the experiment (Figure 2B). Typically, it takes approximately 1 - 2 weeks for the tumors to reach this size.
	<ol>
		<li>Measure the calf size daily with digital calipers via its medial-lateral and anterior-posterior lengths.</li>
		<li>Determine the calf volume by the formula (D x d2/6) x 3.14, where D is the longer diameter and d is the shorter diameter of the tumor-bearing lower hindlimb. 
		NOTE: The size of the normal adult mouse calf is of approximately 40 - 50 mm3. Its volume will increase due to tumor growth and at the later stages the calf will be mainly comprised of tumor tissue.</li>
	</ol>
	</li>
</ol>
4. Femoral Artery Ligation (FAL) for Inducing Hypoxia in the Tumor-bearing Hindlimb
<ol>
	<li>Prepare the surgical tools needed for this operation: curved or pointed fine forceps, pointed forceps, surgical scissors and a needle holder. Sterilize these tools prior to surgery using an autoclave or a hot-bead sterilizer. Additionally, have fine cotton swabs ready for this surgery. 
	NOTE: It is recommended that the tools be re-sterilized at the tips as needed during the procedure.</li>
	<li>Inject analgesic agent (Carprofen 5 mg/kg) subcutaneously (SQ). To detect and confirm hypoxia, inject hypoxyprobe-1 (pimonidazole, 60 mg/kg). 
	NOTE: This dose equates to 1.5 mg per mouse and is achieved by injecting 0.1 ml of 15-mg/ml hypoxyprobe solution in PBS intraperitoneally (IP). The hypoxyprobe is then detectable postmortem in animal tissues by immunohistochemistry.</li>
	<li>Place the mouse in an anesthesia induction chamber containing 3 - 5% isoflurane in 100% oxygen at a flow rate of 1 L/min.</li>
	<li>Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. Place the animal in the supine position on a sterile drape placed atop a warming pad on the operating surface. Use a nose cone to connect it to a continuous flow of 1 - 3% isoflurane in 100% oxygen at a flow rate of 0.8 L/min.
	<ol>
		<li>Apply sterile non-medicated ophthalmic ointment to each eye to prevent corneal drying. To thoroughly depilate the surgical area, apply hair removal cream, leaving it on the skin for no more than 10 sec. Then wipe off the hair removal cream using an ethanol prep pad.</li>
	</ol>
	</li>
	<li>Extend and secure the hindlimb with a piece of tape approximately 45 degrees from the midline of the mouse. Once the hindlimb is secure, wipe the exposed skin with 10% povidone/iodine swab/solution, followed by ethanol, repeating two more times each. For the remainder of the surgical procedure, use a stereo microscope to obtain an enlarged view of the hindlimb region.</li>
	<li>Using pointed forceps and surgical scissors, make an incision of the skin, approximately 1 cm long, from mid-thigh towards the inguinal region. Using saline-moistened fine cotton swabs, gently brush away subcutaneous fat tissue surrounding the thigh muscle.</li>
	<li>Carefully reveal the underlying femoral artery via blunt dissection through the subcutaneous fat tissue. Stabilize the wound and surgical field to expose the vasculature of the mid-upper adductor muscle.</li>
	<li>Using fine forceps, gently pierce through the membranous femoral sheath to expose the neurovascular bundle. Using a clean set of fine forceps, dissect and separate the femoral artery from the femoral vein and nerve at the proximal location near the groin, distal to the inguinal ligament. Use caution to avoid piercing the femoral vein wall.</li>
	<li>Following dissection, pass a strand of 6-0 silk suture underneath the femoral artery and distal to the branch of the lateral circumflex femoral artery (LCFA). Occlude the femoral artery using double knots (Figure 3).</li>
	<li>Close the incision using 6-0 polypropylene sutures. After closing the incision, inject SQ 0.5 ml of warm saline for fluid balance therapy. Place the animal on top of a draped warm pad in the recovery cage and monitor continuously until awake.</li>
	<li>Monitor the animals during first 6 hr after surgery and inject the analgesic agent (Carprofen 5 mg/kg, SQ) every day for 3 days. Remove sutures 10 days post-surgery using sterile scissors.</li>
</ol>
5. Primary Tumor Excision by Leg Amputation
NOTE: Amputate the tumor-bearing lower hindlimb when the calf size reaches 250 mm3 for the control group or 3 days after FAL for the hypoxic group.
<ol>
	<li>Shave hair from the tumor-bearing limb from the distal tibia to the pelvic region with hair clippers while gently holding the animal. Inject the analgesic agent (Carprofen 5 mg/kg, SQ) before the procedure.</li>
	<li>Place the mouse into an anesthesia induction chamber containing 3 - 5% isoflurane in 100% oxygen at a flow rate of 1 L/min. Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber.</li>
	<li>Place the animal in the right lateral recumbent position on a sterile drape placed on a warming pad on the operating surface. Use a nose cone to connect it to a continuous flow of isoflurane 1 - 3% in 100% oxygen at a flow rate of 0.8 L/min. Apply sterile non-medicated ophthalmic ointment to each eye to prevent corneal drying</li>
	<li>Prepare the surgical site using 10% povidone/iodine swab/solution, followed by ethanol, repeating 3 times. Apply a sterile gauze (e.g., surgical drape) over the mouse to obtain a sterile surgical field.</li>
	<li>Make a middle femoral circumferential skin incision, followed by blunt dissection and retraction of the skin proximally. Expose the medial femoral neurovascular pedicle on the median side of the leg, and then ligate near the inguinal ligament using 4-0 coated (polyglactin 910) absorbable suture material.</li>
	<li>Perform a mid-femoral transection of muscle groups with scissors, followed by blunt dissection of soft tissue to the coxofemoral joint. Using a bone cutter, perform a mid-femoral osteotomy. Using a sterile fine cotton swab or an absorbable gelatin sponge, gently press the osteotomy site to minimize and prevent bleeding.</li>
	<li>Close the overlying skin using surgical wound clips and inject 0.5 ml of warm saline SQ for fluid balance therapy. Place the animal on top of a draped warm pad in the recovery cage and monitor continuously until awake.</li>
	<li>Upon surgery, collect tissue samples from primary tumors for RNA, DNA or protein isolation, snap freeze in liquid nitrogen and store at -80 &#730;C. For primary cell culture, collect tissue samples at this step, as described in section 9 below. Fix the remaining limb tissue in 10% neutral-buffered formalin for histology and immunochemistry, including hypoxyprobe-1 detection.</li>
	<li>Monitor the animals for locomotion, pain and food consumption during the first 6 hours after surgery, then every day for 3 days. Inject the analgesic agent (Carprofen 5 mg/kg, SQ) daily for 3 days.&#160;Remove the wound clips 10 days after amputation using a wound clip remover.</li>
</ol>
6. Monitoring Mice for the Presence of Metastases
<ol>
	<li>Observe the mice daily and evaluate them for clinical signs of metastasis at least twice a week.
	<ol>
		<li>Observe the animals for the presence of macrometastases presenting as masses that develop in various locations, typically shoulders, contralateral legs and jaws. To this end, carefully palpate head, neck and axillary regions, and contralateral hindlimb. Check for internal organ metastases via abdominal distension along with MRI scanning.</li>
		<li>Check for the presence of lung metastases by pressing the xyphoid process (the lower end of the sternum) with index finger.24 
		NOTE: This pressure diminishes the diaphragmatic respiration capacity. Mice with advanced lung metastases show signs of respiratory distress manifested by laborious breathing.</li>
		<li>Observe the animals for neurological symptoms, such as leg paralysis and ataxia suggesting metastases to the central nervous system.</li>
		<li>Monitor the mice at least once per week for weight loss, as an indication of potential disease progression. Body weight loss exceeding 15% of the pre-procedural weight is considered a humane endpoint.&#160;</li>
	</ol>
	</li>
</ol>
7. Magnetic Resonance Imaging (MRI) for Detecting Metastases
<ol>
	<li>Perform MRI to detect metastases at desired time points.18 
	NOTE: In the current study, a 7-Tesla horizontal spectrometer was used. MRI was performed at days 15 and 35 post-amputation for SK-ES1 cells and at day 15 for TC71 cells.</li>
	<li>Place the mouse into an anesthesia induction chamber containing 1 - 3% isoflurane in a gas mixture of 30% oxygen and 70% nitrous oxide.</li>
	<li>Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber.</li>
	<li>Transfer the anesthetized mouse onto a stereotaxic holder with respiration and temperature monitorization with continuous administration of 1.5% isoflurane and 30% nitrous oxide. Apply sterile non-medicated ophthalmic ointment to each eye to prevent corneal dryness. Image the animal either in a 40 or 23 mm Bruker mouse volume coil for whole body or brain imaging, respectively.</li>
	<li>Use a two-dimensional, T2-weighted RARE sequence: TR = 3,000 msec, TE = 24 msec, matrix = 256, FOV = 4.35 x 3.0 cm, slice thickness = 0.5 mm, RARE factor = 4 and averages = 4.18</li>
	<li>Place the animal in a warm recovery cage and monitor continuously until awake. 
	NOTE: The mice will recover rapidly since a shallow plane of anesthesia is used.</li>
	<li>Monitor the animals during the first 6 hr after imaging to make sure that there are no adverse effects of the anesthesia.</li>
</ol>
8. Euthanasia and Necropsy
<ol>
	<li>Euthanize the mice once the animals present with metastases detectable by MRI and/or clinical symptoms of disease progression. 
	NOTE: In the current study, the mice were sacrificed at days 50 and 25 post-amputation for animals bearing SK-ES1 and TC71 xenografts, respectively. In some cases, earlier euthanasia was necessary due to a high metastasis burden.</li>
	<li>Euthanize the mice by exposure to CO2 at 1.5 L/min (CO2 at 10 - 30% of the euthanasia chamber vol/min). To ensure animal death, perform cervical dislocation after CO2 exposure.</li>
	<li>Spray the entire mouse with 70% ethanol and place it into the laminar flow hood. Collect the blood by heart puncture using a 25 G &#189; needle with a 1 ml syringe and transfer to a blood collection tube containing 2 mg of EDTA.</li>
	<li>Collect the following tissues: spleen, adrenal glands, ovaries, kidneys, liver, lungs, brain, right leg, bone marrow from both humeri and spine, as well as macroscopic metastases present in other locations.25,26</li>
	<li>Fix half of each tissue in 10% neutral-buffered formalin for histology and immunochemistry, including hypoxyprobe-1 detection. Snap freeze the other half in liquid nitrogen, then store at -80 &#730;C for RNA, DNA or protein isolation.25,26 For primary cell culture, collect tissue samples as described in section 9 below.</li>
</ol>
9. Primary Cell Culture
<ol>
	<li>To perform primary cell culture, dissect tissues from the amputated limb (section 5 above) or during the necropsy (section 8 above) under sterile conditions in a laminar flow hood.</li>
	<li>Prepare cell culture media appropriate for the cell line used for orthotopic injections, supplemented with penicillin (200 units/ml), streptomycin (200 &#181;g/ml), fungizone (1 &#181;g/ml), and 0.2% mycoplasma prophylactic antibiotic. Place 2.5 ml of the primary culture medium into a 6-cm cell culture plate.</li>
	<li>Select viable tumor tissue areas from primary tumors or metastases, and then isolate two to three segments at 2-3 mm each using sterile scissors. 
	NOTE: Viable tumor tissue is usually found on the edges of the tumor and can be discriminated from necrosis by its pinkish or reddish color, significant luster and overall wet appearance. In contrast, necrotic tissue is commonly seen in the center of the tumor and presents as a whitish/cream color mass with a dull, cheesy appearance.27</li>
	<li>Transfer the isolated segments to a 6-cm cell culture plate containing primary culture medium described in step 9.2.</li>
	<li>Culture cells under standard conditions, as described in section 1 (NOTE) and 9.2.18,28 Check the culture for cellular outgrowth arising from the tissue pieces, which should be observed within a few days. Once cells reach confluence, trypsinize and propagate them according to standard cell culture techniques. 
	NOTE: The primary cell culture can be subsequently used to evaluate growth and metastatic properties of the cells derived from control and hypoxic tumors, as well as their molecular features, as previously described.18</li>
</ol>

<ol><li>Lessnick, S. L., Ladanyi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+pathogenesis+of+Ewing+sarcoma%3A+new+therapeutic+and+transcriptional+targets%5BTitle%5D)+AND+%22Annu+Rev+Pathol%22%5BJournal%5D)">Molecular pathogenesis of Ewing sarcoma: new therapeutic and transcriptional targets.</a> Annu Rev Pathol. 7, 145-159 (2012).</li>
<li>Ladenstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+disseminated+multifocal+Ewing+sarcoma%3A+results+of+the+Euro-EWING+99+trial%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Primary disseminated multifocal Ewing sarcoma: results of the Euro-EWING 99 trial.</a> J Clin Oncol. 28, 3284-3291 (2010).</li>
<li>Dunst, J., Ahrens, S., Paulussen, M., Burdach, S., Jurgens, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prognostic+impact+of+tumor+perfusion+in+MR-imaging+studies+in+Ewing+tumors%5BTitle%5D)+AND+%22Strahlenther+Onkol%22%5BJournal%5D)">Prognostic impact of tumor perfusion in MR-imaging studies in Ewing tumors.</a> Strahlenther Onkol. 177, 153-159 (2001).</li>
<li>Aryee, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+modulates+EWS-FLI1+transcriptional+signature+and+enhances+the+malignant+properties+of+Ewing%27s+sarcoma+cells+in+vitro%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Hypoxia modulates EWS-FLI1 transcriptional signature and enhances the malignant properties of Ewing's sarcoma cells in vitro.</a> Cancer Research. 70, 4015-4023 (2010).</li>
<li>Knowles, H. J., Schaefer, K. L., Dirksen, U., Athanasou, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+and+hypoglycaemia+in+Ewing%27s+sarcoma+and+osteosarcoma%3A+regulation+and+phenotypic+effects+of+Hypoxia-Inducible+Factor%5BTitle%5D)+AND+%22BMC+cancer%22%5BJournal%5D)">Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.</a> BMC cancer. 10, 372(2010).</li>
<li>Tilan, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+shifts+activity+of+neuropeptide+Y+in+Ewing+sarcoma+from+growth-inhibitory+to+growth-promoting+effects%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects.</a> Oncotarget. 4, 2487-2501 (2013).</li>
<li>Franzius, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+high-resolution+animal+positron+emission+tomography+of+human+Ewing+tumours+and+their+metastases+in+a+murine+xenograft+model%5BTitle%5D)+AND+%22Eur+J+Nucl+Med+Mol+Imaging%22%5BJournal%5D)">Successful high-resolution animal positron emission tomography of human Ewing tumours and their metastases in a murine xenograft model.</a> Eur J Nucl Med Mol Imaging. 33, 1432-1441 (2006).</li>
<li>Hauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DKK2+mediates+osteolysis%2C+invasiveness%2C+and+metastatic+spread+in+Ewing+sarcoma%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">DKK2 mediates osteolysis, invasiveness, and metastatic spread in Ewing sarcoma.</a> Cancer Research. 73, 967-977 (2012).</li>
<li>Manara, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+in+vivo+study+of+new+insulin-like+growth+factor-I+receptor--specific+inhibitor+in+Ewing%27s+sarcoma%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Preclinical in vivo study of new insulin-like growth factor-I receptor--specific inhibitor in Ewing's sarcoma.</a> Clin Cancer Res. 13, 1322-1330 (2007).</li>
<li>Scotlandi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+model+for+skeletal+metastases+of+Ewing%27s+sarcoma%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Murine model for skeletal metastases of Ewing's sarcoma.</a> J Orthop Res. 18, 959-966 (2000).</li>
<li>Vormoor, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+an+in+vivo+model+for+pediatric+Ewing+tumors+by+transplantation+into+NOD%2Fscid+mice%5BTitle%5D)+AND+%22Pediatr+Res%22%5BJournal%5D)">Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice.</a> Pediatr Res. 49, 332-341 (2001).</li>
<li>Picarda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+evidence+that+use+of+TRAIL+in+Ewing%27s+sarcoma+and+osteosarcoma+therapy+inhibits+tumor+growth%2C+prevents+osteolysis%2C+and+increases+animal+survival%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Preclinical evidence that use of TRAIL in Ewing's sarcoma and osteosarcoma therapy inhibits tumor growth, prevents osteolysis, and increases animal survival.</a> Clin Cancer Res. 16, 2363-2374 (2010).</li>
<li>Vormoor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+preclinical+orthotopic+xenograft+model+of+ewing+sarcoma+and+other+human+malignant+bone+disease+using+advanced+in+vivo+imaging%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Development of a preclinical orthotopic xenograft model of ewing sarcoma and other human malignant bone disease using advanced in vivo imaging.</a> PLoS One. 9, e85128(2014).</li>
<li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet-derived+growth+factor+receptor+beta+inhibition+increases+tumor+necrosis+factor-related+apoptosis-inducing+ligand+%28TRAIL%29+sensitivity%3A+imatinib+and+TRAIL+dual+therapy%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">Platelet-derived growth factor receptor beta inhibition increases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity: imatinib and TRAIL dual therapy.</a> Cancer. 116, 3892-3902 (2010).</li>
<li>Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibiting+platelet-derived+growth+factor+beta+reduces+Ewing%27s+sarcoma+growth+and+metastasis+in+a+novel+orthotopic+human+xenograft+model%5BTitle%5D)+AND+%22In+Vivo%22%5BJournal%5D)">Inhibiting platelet-derived growth factor beta reduces Ewing's sarcoma growth and metastasis in a novel orthotopic human xenograft model.</a> In Vivo. 23, 903-909 (2009).</li>
<li>Odri, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zoledronic+acid+as+a+new+adjuvant+therapeutic+strategy+for+Ewing%27s+sarcoma+patients%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Zoledronic acid as a new adjuvant therapeutic strategy for Ewing's sarcoma patients.</a> Cancer Research. 70, 7610-7619 (2010).</li>
<li>Merchant, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interferon+gamma+enhances+the+effectiveness+of+tumor+necrosis+factor-related+apoptosis-inducing+ligand+receptor+agonists+in+a+xenograft+model+of+Ewing%27s+sarcoma%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Interferon gamma enhances the effectiveness of tumor necrosis factor-related apoptosis-inducing ligand receptor agonists in a xenograft model of Ewing's sarcoma.</a> Cancer Research. 64, 8349-8356 (2004).</li>
<li>Hong, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+neuropeptide+Y+release+associates+with+Ewing+sarcoma+bone+dissemination+-+in+vivo+model+of+site-specific+metastases%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases.</a> Oncotarget. 6, 7151-7165 (2015).</li>
<li>Lee, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropeptide+Y+induces+ischemic+angiogenesis+and+restores+function+of+ischemic+skeletal+muscles%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles.</a> J Clin Invest. 111, 1853-1862 (2003).</li>
<li>Tilan, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+neuropeptide+Y+is+critical+for+ischemic+revascularization+in+mice%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Platelet neuropeptide Y is critical for ischemic revascularization in mice.</a> FASEB J. , (2013).</li>
<li>Toffoli, S., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intermittent+hypoxia+is+a+key+regulator+of+cancer+cell+and+endothelial+cell+interplay+in+tumours%5BTitle%5D)+AND+%22The+FEBS+journal%22%5BJournal%5D)">Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours.</a> The FEBS journal. 275, 2991-3002 (2008).</li>
<li>Das, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+enhances+tumor+stemness+by+increasing+the+invasive+and+tumorigenic+side+population+fraction%5BTitle%5D)+AND+%22Stem+cells+%28Dayton%2C+Ohio%29%22%5BJournal%5D)">Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction.</a> Stem cells (Dayton, Ohio). 26, 1818-1830 (2008).</li>
<li>Arndt, C. A., Rose, P. S., Folpe, A. L., Laack, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Common+musculoskeletal+tumors+of+childhood+and+adolescence%5BTitle%5D)+AND+%22Mayo+Clin+Proc%22%5BJournal%5D)">Common musculoskeletal tumors of childhood and adolescence.</a> Mayo Clin Proc. 87, 475-487 (2012).</li>
<li>Mendoza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+noninvasive+method+for+evaluating+experimental+lung+metastasis+in+mice%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">A novel noninvasive method for evaluating experimental lung metastasis in mice.</a> J Am Assoc Lab Anim Sci. 52, 584-589 (2013).</li>
<li>Feldman, D. B., Seely, J. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFeldman%2C+DB+%22Necropsy+Guide%3A+Rodents+and+the+Rabbit%22">Necropsy Guide: Rodents and the Rabbit</a>. , CRC Press. (1988).</li>
<li>Parkinson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnostic+necropsy+and+selected+tissue+and+sample+collection+in+rats+and+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Diagnostic necropsy and selected tissue and sample collection in rats and mice.</a> J Vis Exp. , (2011).</li>
<li>Raymond, A. K., Lazar, A. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRaymond%2C+AK+%22Bone+Sarcoma%22">Bone Sarcoma</a>. PP, L. in, S, P. atel , Springer. (2013).</li>
<li>Dietel, M., Arps, H., Gerding, D., Trapp, M., Niendorf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+primary+cell+cultures%3A+experiences+with+155+cell+strains%5BTitle%5D)+AND+%22Klin+Wochenschr%22%5BJournal%5D)">Establishment of primary cell cultures: experiences with 155 cell strains.</a> Klin Wochenschr. 65, 507-512 (1987).</li>
<li>Varghese, A. J., Gulyas, S., Mohindra, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia-dependent+reduction+of+1-%282-nitro-1-imidazolyl%29-3-methoxy-2-propanol+by+Chinese+hamster+ovary+cells+and+KHT+tumor+cells+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Hypoxia-dependent reduction of 1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol by Chinese hamster ovary cells and KHT tumor cells in vitro and in vivo.</a> Cancer Research. 36, 3761-3765 (1976).</li>
<li>ATCC. , 
 <a target="_blank" href="http://www.atcc.org/products/all/HTB-86.aspx#characteristics">http://www.atcc.org/products/all/HTB-86.aspx#characteristics</a> (2014).</li>
</ol>

The authors have nothing to disclose.

Our model involves the comparison of metastasis in two experimental groups &#8212; a control group, where tumors are allowed to develop in the hindlimb followed by amputation upon reaching a calf volume of 250 mm3, and a hypoxia-exposed group, in which the tumor-bearing hindlimb is subjected to FAL at the same volume, followed by amputation 3 days later. Even though in these experiments the FAL-treated tumors are amputated with a slight delay, as compared to the control tumors, their size does not increase dur...

Ewing sarcoma (ES) is an aggressive malignancy affecting children and adolescents.1 The tumors develop in soft tissues and bones, commonly in limbs. While the presence of metastases is the single most powerful adverse prognostic factor for ES patients, the mechanisms underlying their development remain unclear.2 Tumor hypoxia is one of the few factors implicated in ES progression. In ES patients, the presence of non-perfused areas within the tumor tissue is associated with poor prognosis.3 In vitro, hypoxia increases invasiveness of ES cells and triggers expression of pro-metastatic genes.4-6 However, despite these lines of evidence, no direct proof for hypoxia-induced ES progression and spread exists. Moreover, the mechanisms by which hypoxia exerts such effects are, at present, unknown. Hence, we have created an in vivo model to fill the gap between existing in vitro data and clinical observations. This model system enables direct testing of the effects of hypoxia on tumors occurring in their natural environment, using magnetic resonance imaging (MRI) to follow tumor progression and metastasis in vivo in combination with ex vivo pathological and molecular analyses (Figure 1).
Since no established transgenic model of ES is currently available, the in vivo studies on metastatic properties of these tumors rely on injections of human cells into immunocompromised mice. While the use of immunologically impaired animals may underestimate the impact of the immune system on the disease progression, the ability to use human cells increases translatability of such studies. Among different xenograft models, systemic injections into tail vein are the easiest to perform, yet they omit the initial steps of tumor cell intravasation and escape from the primary site of growth.7-12 On the other hand, orthotopic xenografting, which involves injections of tumor cells into bones (femur, rib) or muscles, is more technically challenging, but also more biologically relevant to human cancer.13-16 However, in the past, the high morbidity associated with rapid growth of primary tumors has often necessitated animal euthanasia before metastasis development. In this study, we employed a previously established model of cell injections into the gastrocnemius muscle followed by excision of the resulting primary tumor combined with longitudinal monitoring of metastatic progression by MRI.17,18 Such injections into gastrocnemius muscle in close proximity to the tibia allow for tumor growth in two natural ES environments &#8212; muscles and bones &#8212; and result in distant metastases to locations typically affected in humans.18 Thereby, this model accurately recapitulates the metastatic processes occurring in ES patients during disease progression.
The localization of primary tumors in the lower hindlimb also facilitates the precise control of the blood supply to the tumor tissue. Femoral artery ligation (FAL) is a well-established technique utilized in angiogenesis research to block blood flow to distal regions of the leg and investigate tissue vascularization in response to ischemia.19,20 Importantly, the initial drop in blood flow is followed by collateral vessel opening and tissue reperfusion observed approximately 3 days after FAL.20 Thus, when performed in a tumor-bearing limb, this model recreates hypoxia/reperfusion events that occur naturally in rapidly growing tumors and enables the escape of metastatic tumor cells due to restoration of perfusion to the lower hindlimb via newly opened collateral vessels.21 Importantly, this procedure must be performed when the tumor size is small enough to prevent excessive hypoxia in control tumors (typically at the tumor-bearing calf volume of 150 - 250 mm3), ensuring significant differences in levels of tumor hypoxia between control and FAL-treated groups.
In addition to longitudinal monitoring of the effect of hypoxia on ES latency and the frequency of metastases, this model also allows for the collection of tissues and the development of new cell lines from both primary tumors and metastases. Importantly, previous work established that metastases-derived cell lines exhibit enhanced metastatic potential upon reintroduction to animals, indicating that tumor dissemination is associated with permanent changes in the tumor cell phenotype, and thereby validating the use of these cell lines to decipher the metastatic processes.18 Collectively, these models can now be used for the genetic and molecular analyses required for identifying hypoxia-induced metastatic pathways.
As hypoxia is a pro-metastatic factor enhancing the malignancy of various tumors, our model can be used as a platform to investigate the role of hypoxia in other tumor types that naturally develop in limbs, such as osteosarcoma and rhabdomyosarcoma.21-23 Moreover, a similar approach can be applied to malignancies growing in other anatomical locations with a well-defined route of blood supply. Ultimately, the model can be modified and its utility further extended, depending on individual research needs.

<table><tbody><tr><td>SK-ES1 Human Ewing sarcoma (ES) cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>TC71 Human ES cells</td><td></td><td></td><td>Kindly provided from Dr. Toretsky</td></tr><tr><td>McCoy&#039;s 5A (modified) Medium</td><td>Gibco by Life Technologies</td><td>12330-031</td><td></td></tr><tr><td>RPMI-1640</td><td>ATCC</td><td>30-2001</td><td></td></tr><tr><td>PBS</td><td>Corning Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F2442-500mL</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco by Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Fungizone&amp;reg; Antimycotic</td><td>Gibco by Life Technologies</td><td>15290-018</td><td></td></tr><tr><td>MycoZap&amp;trade; Prophylactic</td><td>Lonza</td><td>VZA-2032</td><td></td></tr><tr><td>Collagen Type I Rat tail high concetration</td><td>BD Biosciences</td><td>354249</td><td></td></tr><tr><td>SCID/beige mice</td><td>Harlan or Charles River</td><td>250 (Charles River) or 18602F (Harlan)</td><td></td></tr><tr><td>1 ml Insulin syringes with permanently attached 28&amp;nbsp;G &amp;frac12; needle</td><td>BD</td><td>329424</td><td></td></tr><tr><td>Saline (0.9% Sodium Chloride Injection, USP)</td><td>Hospira, INC</td><td>NDC 0409-7984-37</td><td></td></tr><tr><td>Digital calipers</td><td>World Precision Instruments, Inc</td><td>501601</td><td></td></tr><tr><td>Surgical Tools</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Rimadyl (Carprofen) Injectable&amp;nbsp;</td><td>Zoetis</td><td></td><td></td></tr><tr><td>Hypoxyprobe-1 (Pimonidazole Hydrochloride solid)</td><td>HPI, Inc</td><td>HP-100mg</td><td></td></tr><tr><td>hypoxyprobe-2 (CCI-103F-250 mg)</td><td>HPI, Inc</td><td>CCI-103F-250mg</td><td></td></tr><tr><td>Povidone-iodine Swabstick</td><td>PDI</td><td>S41350</td><td></td></tr><tr><td>Sterile alcohol prep pad</td><td>Fisher HealthCare</td><td>22-363-750</td><td></td></tr><tr><td>LubriFresh P.M. (eye lubricant ointment)&amp;nbsp;</td><td>Major Pharaceuticals</td><td>NDC 0904-5168-38</td><td></td></tr><tr><td>VWR Absorbent Underpads with Waterproof Moisture Barrier</td><td>VWR</td><td>56617-014&amp;nbsp;</td><td></td></tr><tr><td>Oster Golden A5 Single Speed Vet Clipper with size 50 blade</td><td>Oster</td><td>078005-050-002 (clipper), 078919-006-005 (blade)</td><td></td></tr><tr><td>Nair Lotion with baby oil</td><td>Church &amp;amp; Dwight Co., Inc.</td><td></td><td></td></tr><tr><td>Silk 6-0</td><td>Surgical Specialties Corp</td><td>752B</td><td></td></tr><tr><td>Prolene (polypropylene) suture 6-0</td><td>Ethicon</td><td>8680G</td><td></td></tr><tr><td>Vicryl (Polyglactin 910) suture 4-0&amp;nbsp;</td><td>Ethicon</td><td>J386H</td><td></td></tr><tr><td>Fisherbrand Applicators (Purified cotton)</td><td>Fisher Scientific</td><td>23-400-115</td><td></td></tr><tr><td>GelFoam Absorbable Dental Sponges - Size 4</td><td>Pfizer Pharmaceutical</td><td>9039605</td><td></td></tr><tr><td>Autoclip Wound Clip Applier</td><td>BD</td><td>427630</td><td></td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Autoclip remover</td><td>BD</td><td>427637</td><td></td></tr><tr><td>Aound clip</td><td>BD</td><td>427631</td><td></td></tr><tr><td>MRI 7 Tesla</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>Paravision 5.0 software</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Euthanasia system</td><td>VetEquip</td><td></td><td></td></tr><tr><td>25 G 5/8 Needle (for heart-puncture)</td><td>BD</td><td>305122</td><td></td></tr><tr><td>0.1 ml syringe (for heart-puncture)</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>K&lt;sub&gt;3&lt;/sub&gt;-EDTA Micro tube 1.3 ml</td><td>Sarstedt</td><td>41.1395.105</td><td></td></tr><tr><td>10% Neutral Buttered Formalin</td><td>Fisher Scientific</td><td>SF100-4</td><td></td></tr></tbody></table>

in vivo model for testing effect of hypoxia on tumor metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

Following injection of ES cells into gastrocnemius muscle, the primary tumors are allowed to grow to a calf size of 250 mm3 (Figure 1, 2). The time necessary for the tumors to reach this volume typically ranges from 10 - 15 days for TC71 to 20-25 days for SK-ES1 xenografts, respectively. Tumors at a calf volume of 250 mm3 exhibit a relatively low level of endogenous hypoxia (approximately 3% of tumor tissue), based on hypoxybrobe-1 (pimonidazole) sta...

Watch this Scientific Journal Video about In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis at JoVE.com

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

in-vivo-model-for-testing-effect-of-hypoxia-on-tumor-metastasis

Research

JoVE Journal

Cancer Research

12.4K Views.  Georgetown University Medical Center. This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Video: In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Medicine

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 ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

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 ...

Preclinical Imaging of Tumor Oxygen Levels Using EPRI and Ultrasound

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Samenvatting

Meer video's verkennen