Name: In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis (Video) | JoVE Journal
Uploaded: 2016-12-09T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Learn an in vivo model for testing tumor hypoxia effects on metastasis using femoral artery ligation and hind limb amputation in mice. This method helps identify prometastatic mechanisms and therapeutic targets.

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5 minutes

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>

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<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

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...

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.

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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Research

JoVE Journal

Cancer Research

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

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

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.

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

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

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.

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

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.

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

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).

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

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.

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

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.

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

Learn an in vivo model for testing tumor hypoxia effects on metastasis using femoral artery ligation and hind limb amputation in mice. This method helps identify prometastatic mechanisms and therapeutic targets.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

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