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 ...

<ol>
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L., Ladanyi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+of+Ewing+sarcoma:+new+therapeutic+and+transcriptional+targets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+disseminated+multifocal+Ewing+sarcoma:+results+of+the+Euro-EWING+99+trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+impact+of+tumor+perfusion+in+MR-imaging+studies+in+Ewing+tumors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+modulates+EWS-FLI1+transcriptional+signature+and+enhances+the+malignant+properties+of+Ewing's+sarcoma+cells+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+hypoglycaemia+in+Ewing's+sarcoma+and+osteosarcoma:+regulation+and+phenotypic+effects+of+Hypoxia-Inducible+Factor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+shifts+activity+of+neuropeptide+Y+in+Ewing+sarcoma+from+growth-inhibitory+to+growth-promoting+effects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+high-resolution+animal+positron+emission+tomography+of+human+Ewing+tumours+and+their+metastases+in+a+murine+xenograft+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DKK2+mediates+osteolysis,+invasiveness,+and+metastatic+spread+in+Ewing+sarcoma.">DKK2 mediates osteolysis, invasiveness, and metastatic spread in Ewing sarcoma.</a> Cancer Research. 73, 967-977 (2012).</li><li>Manara, M. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+platelet-derived+growth+factor+beta+reduces+Ewing's+sarcoma+growth+and+metastasis+in+a+novel+orthotopic+human+xenograft+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronic+acid+as+a+new+adjuvant+therapeutic+strategy+for+Ewing's+sarcoma+patients.">Zoledronic acid as a new adjuvant therapeutic strategy for Ewing's sarcoma patients.</a> Cancer Research. 70, 7610-7619 (2010).</li><li>Merchant, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptide+Y+induces+ischemic+angiogenesis+and+restores+function+of+ischemic+skeletal+muscles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+neuropeptide+Y+is+critical+for+ischemic+revascularization+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+hypoxia+is+a+key+regulator+of+cancer+cell+and+endothelial+cell+interplay+in+tumours.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+enhances+tumor+stemness+by+increasing+the+invasive+and+tumorigenic+side+population+fraction.">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#174; Antimycotic</td>
			<td>Gibco by Life Technologies</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoZap&#8482; 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 28G&#189; needle&#160;</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&#160;</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-250mg)</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)&#160;</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&#160;</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 &#38; 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&#160;</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>CO2 Euthanasia system</td>
			<td>VetEquip</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25G 5/8 Needle (for heart-puncture)</td>
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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...

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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 ...

The overall goal of this procedure is to directly test the effect of tumor hypoxia on metastasis using an animal model. This method can help to identify the mechanism underlying the prometastatic effects of hypoxia and potential novel therapeutic targets for preventing tumor dissemination. Two advantages of this technique are the ability to test the effects of tumor hypoxia in vivo, and to recapitulate the steps of metastasis and cell interactions that occur in cancer patients.In our model, tumor hypoxia is created by ligation of the femoral artery supplying blood to the tumor-bearing hilum of the mouse. Subsequent hind limb amputation prevents the mobility associated with repeat primary tumor growth, allowing for metastasis to form. Demonstrating the procedure will be Jason Tilan, Sung-Kyeok Hung, and Dr.Olga Rodriguez, co-director of the Preclinical Imaging Research Laboratory here at Georgetown.To inject the Ewing sarcoma cells, gently restrain a four to six week old female SCID Beige mouse, stabilizing the left leg between the fourth and fifth fingers to expose the medial side of the lower hind limb. Insert a 28 gauge 1/2"inch needle at an approximately 30-45 degree angle anteriorly into the gastrocnemius muscle toward the tibial crest. When the needle tip touches the crest, withdraw the needle slightly and slowly inject 0.1 milliliters of the Ewing sarcoma cells while continuing to withdraw the needle.Then use digital calipers to measure the calf size daily via the medial/lateral and anterior/posterior lengths until the tumor size reaches the desired volume. Before beginning the ligation, subcutaneously inject an analgesic agent followed by the administration of Hypoxyprobe-1 for hypoxia confirmation. Next, place the anesthetized mouse on a warming pad on the operating surface and apply ointment to the animal's eyes.Then, extend and secure the hind limb with a piece of tape approximately 45 degrees from the midline. Depilate the surgical area with hair-removal cream removing the cream with an ethanol prep pad after 10 seconds and disinfect the exposed skin with three consecutive 10%povidone-iodine and ethanol wipes. Transfer the mouse under a stereo microscope and make a 1 centimeter incision in the skin from the mid-thigh towards the inguinal region.Using saline-moistened fine cotton swabs, gently brush away the subcutaneous fat tissue surrounding the thigh muscle. Then carefully blunt dissect the subcutaneous fat tissue to reveal the underlying femoral artery. Stabilize the wound and surgical field to expose the vasculature of the mid-upper adductor muscle and use fine forceps to 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. Then, pass a 6-0 silk suture under the femoral artery distal to the brach of the lateral circumflex femoral artery, and occlude the femoral artery with double knots. Close the incision with 6-0 polypropylene sutures and subcutaneously inject 0.5 milliliters of warm saline for fluid balance therapy.Then place the animal on top of a draped warm pad in the recovery cage with monitoring until fully recovered. At the appropriate experimental time point, gently restrain the animal and use clippers to shave the hair from the tumor-bearing limb from the distal tibia to the pelvic region. Inject the analgesic agent and place the anesthetized animal in the right lateral recumbent position on a sterile drape placed on a warming pad.Apply ointment to the eyes and disinfect the surgical site as just demonstrated. Then, place a sterile drape over the animal and make a middle femoral circumferential skin incision followed by blunt dissection and proximal retraction of the skin. Expose the medial femoral neurovascular pedicle on the median side of the leg.Then use a 4-0 polyglactin 910 coated absorbable suture to ligate near the inguinal ligament. Using scissors, perform a mid-femoral transection of the muscle followed by blunt dissection of the soft tissue to the coxofemoral joint. Using a bone-cutter, perform a mid-femoral osteotomy followed by gentle pressure at the site of osteotomy with a absorbable gelatin sponge to minimize the bleeding.Then use surgical wound clips to close the overlying skin and subcutaneously inject 0.5 milliliters of warm saline for fluid balance therapy. At least twice a week, carefully palpate the head, neck, and axillary regions and contralateral hind limb of the tumor-injected animals. Check for internal organ metastases via abdominal distension and for the presence of lung metastases via gentle xiphoid process pressure with the index finger.To evaluate the internal organ metastasis by MRI at the appropriate experimental time points, turn on the pump connected to a warm water circulating system to maintain the mouse at a physiological body temperature. Then, place the respiratory sensor in contact with the upper abdomen of the animal and turn on the BIOPAC Systems respiratory monitoring equipment. Place the anesthetized mouse onto a custom-designed mouse stereotaxic holder and apply ophthalmic ointment to the animal's eyes.To reduce the motion artifacts and noise, select the gating option in the MRI protocol to synchronize the data collection with the respiratory cycle. Then, run a two dimensional T2-weighted anatomical imaging sequence to image the metastases. For primary tumor cell culture after amputation, incision, or necropsy, harvest two to three 2-3 millimeter segments of wet lustrous pink-red viable tumor tissue, from each tissue sample of interest and place the isolated segments in 6 centimeter cell culture plates containing fresh primary culture medium.Then, transfer the tissues to a cell culture incubator for the appropriate cellular outgrowth period. Following injection of the Ewing sarcoma cells into the gastrocnemius muscle, the primary tumors are allowed to grow to a calf size of 250 cubic millimeters. Control tumors at this volume exhibit a relatively low level of endogenous hypoxia solely in the cells distant from the vasculature as determined by Hypoxyprobe-1 staining.In contrast, femoral artery ligation induces profound hypoxia in the vast majority of the tumor cells, even those proximal to the blood vessels. The effectiveness of the femoral artery ligation is supported by the complete inhibition of the primary tumor growth over the three-day period between the femoral artery ligation and the amputation. Histopathological analysis reveals extensive areas of necrosis in femoral artery ligation treated primary tumors harvested three days post-surgery.Nevertheless, there are groups of viable tumor cells present within the tumor tissue located at the edges of the tumor close to the vasculature. These remaining viable tumor cells are highly invasive as evidenced by their massive intravasation. Many cells within the vessel lumen are Hypoxyprobe-1 positive, indicating that cells that are hypoxic post femoral artery ligation may have initiated angioinvasion.In these experiments, hypoxia significantly increased the frequency and multiplicity of the metastases, often observed in locations not affected in mice bearing control tumors. Once mastered, each surgical procedure, including preparation, anesthesia, and recovery can be completed in 30 minutes per animal. The length of the entire experiment depends on the cell line used and its rates of growth and metastasis.The MRI and euthanasia timelines vary according to the latency of the metastases, and must be established for each individual cell line in a pilot experiment. While attempting this procedure, it's important to remember to maintain the appropriate pain management and animal monitoring post-procedure. If the procedure is performed perfectly, no severe adverse effect or death related to the surgery should be observed.Following this procedure, tissues and cells from control and hypoxic primary tumors as well as from their corresponding metastasis can be subjected to further analysis, including global gene expression profiling for identifying the mechanisms of hypoxia-induced metastasis. This technique can be used for the researchers in the field of cancer metastasis for directly testing the effects of tumor hypoxia on disease dissemination. Although here we focused on Ewing sarcoma, a similar approach can be applied to other tumors.After watching this video, you should have a very good understanding of how to create an animal model of tumor hypoxia and how to monitor the resulting metastases both in vivo and ex vivo.

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12.2K visualizações.  Georgetown University Medical Center. The overall goal of this procedure is to directly test the effect of tumor hypoxia on metastasis using an animal model. This method can help to identify the mechanism underlying the prometastatic effects of hypoxia and potential novel therapeutic targets for preventing tumor dissemination. Two advantages of this technique are the ability to test the effects of tumor hypoxia in vivo, and to recapitulate the steps of metastasis and cell interactions that occur in cancer patients.In our m...