In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Summary

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.

Abstract

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.

Introduction

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 — muscles and bones — 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.

Protocol

All procedures were approved by the Georgetown University Institutional Animal Care and Use Committee.

1. Cell Preparation for Orthotopic Injections

1. 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'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 — penicillin (100 units/ml), streptomycin (100 µg/ml) and fungizone (1 µg/ml).
2. Wash the ES cells with phosphate buffered saline (PBS) and trypsinize at 70% confluence with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) for 5 min.
3. 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.
4. 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.

2. Orthotopic Injection of ES cells into Gastrocnemius Muscle

1. Use 4-6 week old female SCID/beige mice.
2. 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.
3. Using a 28 G ½ 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). 
   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.
   1. Insert the needle into the gastrocnemius muscle anteriorly at approximately a 30 - 45 degree angle in the direction of the tibial crest/tuberosity.
   2. Slightly withdraw the needle once it touches the tibial crest/tuberosity. Slowly inject the cell suspension solution, gradually withdrawing the needle to release pressure.
4. Monitor the injected mice over the next 24 hr for signs of distress. 
   NOTE: Investigators with less experience in animal handling should consider anesthetizing mice for tumor cell injections. Some institutions may require anesthesia for safety reasons.

3. Monitoring Primary Tumor Growth

1. 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.
   1. Measure the calf size daily with digital calipers via its medial-lateral and anterior-posterior lengths.
   2. 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.

4. Femoral Artery Ligation (FAL) for Inducing Hypoxia in the Tumor-bearing Hindlimb

1. 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.
2. 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.
3. Place the mouse in an anesthesia induction chamber containing 3 - 5% isoflurane in 100% oxygen at a flow rate of 1 L/min.
4. 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.
   1. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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).
10. 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.
11. 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.

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.

1. 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.
2. 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.
3. 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
4. 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.
5. 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.
6. 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.
7. 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.
8. Upon surgery, collect tissue samples from primary tumors for RNA, DNA or protein isolation, snap freeze in liquid nitrogen and store at -80 ˚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.
9. 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. Remove the wound clips 10 days after amputation using a wound clip remover.

6. Monitoring Mice for the Presence of Metastases

1. Observe the mice daily and evaluate them for clinical signs of metastasis at least twice a week.
   1. 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.
   2. 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.
   3. Observe the animals for neurological symptoms, such as leg paralysis and ataxia suggesting metastases to the central nervous system.
   4. 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. 

7. Magnetic Resonance Imaging (MRI) for Detecting Metastases

1. 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.
2. Place the mouse into an anesthesia induction chamber containing 1 - 3% isoflurane in a gas mixture of 30% oxygen and 70% nitrous oxide.
3. Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber.
4. 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.
5. 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
6. 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.
7. Monitor the animals during the first 6 hr after imaging to make sure that there are no adverse effects of the anesthesia.

8. Euthanasia and Necropsy

1. 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.
2. 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.
3. 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 ½ needle with a 1 ml syringe and transfer to a blood collection tube containing 2 mg of EDTA.
4. 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
5. 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 ˚C for RNA, DNA or protein isolation.^25,26 For primary cell culture, collect tissue samples as described in section 9 below.

9. Primary Cell Culture

1. 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.
2. Prepare cell culture media appropriate for the cell line used for orthotopic injections, supplemented with penicillin (200 units/ml), streptomycin (200 µg/ml), fungizone (1 µg/ml), and 0.2% mycoplasma prophylactic antibiotic. Place 2.5 ml of the primary culture medium into a 6-cm cell culture plate.
3. 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
4. Transfer the isolated segments to a 6-cm cell culture plate containing primary culture medium described in step 9.2.
5. 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

Results

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...

Discussion

Our model involves the comparison of metastasis in two experimental groups — 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SK-ES1 Human Ewing sarcoma (ES) cells	ATCC
TC71 Human ES cells			Kindly provided from Dr. Toretsky
McCoy's 5A (modified) Medium	Gibco by Life Technologies	12330-031
RPMI-1640	ATCC	30-2001
PBS	Corning Cellgro	21-040-CV
FBS	Sigma-Aldrich	F2442-500mL
0.25% Trypsin-EDTA (1x)	Gibco by Life Technologies	25200-056
Penicillin-Streptomycin	Gibco by Life Technologies	15140-122
Fungizone® Antimycotic	Gibco by Life Technologies	15290-018
MycoZap™ Prophylactic	Lonza	VZA-2032
Collagen Type I Rat tail high concetration	BD Biosciences	354249
SCID/beige mice	Harlan or Charles River	250 (Charles River) or 18602F (Harlan)
1 ml Insulin syringes with permanently attached 28 G ½ needle	BD	329424
Saline (0.9% Sodium Chloride Injection, USP)	Hospira, INC	NDC 0409-7984-37
Digital calipers	World Precision Instruments, Inc	501601
Surgical Tools	Fine Science Tools
Rimadyl (Carprofen) Injectable	Zoetis
Hypoxyprobe-1 (Pimonidazole Hydrochloride solid)	HPI, Inc	HP-100mg
hypoxyprobe-2 (CCI-103F-250 mg)	HPI, Inc	CCI-103F-250mg
Povidone-iodine Swabstick	PDI	S41350
Sterile alcohol prep pad	Fisher HealthCare	22-363-750
LubriFresh P.M. (eye lubricant ointment)	Major Pharaceuticals	NDC 0904-5168-38
VWR Absorbent Underpads with Waterproof Moisture Barrier	VWR	56617-014
Oster Golden A5 Single Speed Vet Clipper with size 50 blade	Oster	078005-050-002 (clipper), 078919-006-005 (blade)
Nair Lotion with baby oil	Church & Dwight Co., Inc.
Silk 6-0	Surgical Specialties Corp	752B
Prolene (polypropylene) suture 6-0	Ethicon	8680G
Vicryl (Polyglactin 910) suture 4-0	Ethicon	J386H
Fisherbrand Applicators (Purified cotton)	Fisher Scientific	23-400-115
GelFoam Absorbable Dental Sponges - Size 4	Pfizer Pharmaceutical	9039605
Autoclip Wound Clip Applier	BD	427630
Stereo Microscope	Olympus	SZ61
Autoclip remover	BD	427637
Aound clip	BD	427631
MRI 7 Tesla	Bruker Corporation
Paravision 5.0 software	Bruker Corporation
CO2 Euthanasia system	VetEquip
25 G 5/8 Needle (for heart-puncture)	BD	305122
0.1 ml syringe (for heart-puncture)	Terumo	SS-01T
K3-EDTA Micro tube 1.3 ml	Sarstedt	41.1395.105
10% Neutral Buttered Formalin	Fisher Scientific	SF100-4