Name: in vivo model for testing effect of hypoxia on tumor metastasis
Uploaded: 2016-12-09T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis at JoVE.com

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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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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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>
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			<td>25G 5/8 Needle (for heart-puncture)</td>
			<td>BD</td>
			<td>305122</td>
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			<td>0.1 mL syringe (for heart-puncture)</td>
			<td>Terumo</td>
			<td>SS-01T</td>
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			<td>&#160;K3 EDTA Micro tube 1.3ml</td>
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			<td>41.1395.105</td>
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			<td>10% Neutral Buttered Formalin</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 ...

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