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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 ....

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 .......

Representative 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.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

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 & 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 a....

Materials

NameCompanyCatalog NumberComments
SK-ES1 Human Ewing sarcoma (ES) cellsATCC
TC71 Human ES cellsKindly provided from Dr. Toretsky
McCoy's 5A (modified) MediumGibco by Life Technologies12330-031
RPMI-1640ATCC30-2001
PBSCorning Cellgro21-040-CV
FBSSigma-AldrichF2442-500mL
0.25% Trypsin-EDTA (1X)Gibco by Life Technologies25200-056
Penicillin-StreptomycinGibco by Life Technologies15140-122
Fungizone® AntimycoticGibco by Life Technologies15290-018
MycoZap™ ProphylacticLonzaVZA-2032
Collagen Type I Rat tail high concetrationBD Biosciences354249
SCID/beige miceHarlan or Charles River250 (Charles River) or 18602F (Harlan)
1 mL Insulin syringes with permanently attached 28G½ needle BD329424
Saline (0.9% Sodium Chloride Injection, USP)Hospira, INCNDC 0409-7984-37
Digital calipersWorld Precision Instruments, Inc501601
Surgical ToolsFine Science Tools
Rimadyl (Carprofen) Injectable Zoetis
Hypoxyprobe-1 (Pimonidazole Hydrochloride solid)HPI, IncHP-100mg
hypoxyprobe-2 (CCI-103F-250mg)HPI, IncCCI-103F-250mg
Povidone-iodine SwabstickPDIS41350
Sterile alcohol prep padFisher HealthCare22-363-750
LubriFresh P.M. (eye lubricant ointment) Major PharaceuticalsNDC 0904-5168-38
VWR Absorbent Underpads with Waterproof Moisture BarrierVWR56617-014 
Oster Golden A5 Single Speed Vet Clipper with size 50 bladeOster078005-050-002 (clipper), 078919-006-005 (blade)
Nair Lotion with baby oilChurch & Dwight Co., Inc.
Silk 6-0Surgical Specialties Corp752B
Prolene (polypropylene) suture 6-0Ethicon8680G
Vicryl (Polyglactin 910) suture 4-0 EthiconJ386H
Fisherbrand Applicators (Purified cotton)Fisher Scientific23-400-115
GelFoam Absorbable Dental Sponges - Size 4Pfizer Pharmaceutical9039605
Autoclip Wound Clip ApplierBD427630
Stereo MicroscopeOlympusSZ61
Autoclip removerBD427637
Aound clipBD427631
MRI 7 TeslaBruker Corporation
Paravision 5.0 softwareBruker Corporation
CO2 Euthanasia systemVetEquip
25G 5/8 Needle (for heart-puncture)BD305122
0.1 mL syringe (for heart-puncture)TerumoSS-01T
 K3 EDTA Micro tube 1.3mlSarstedt41.1395.105
10% Neutral Buttered FormalinFisher ScientificSF100-4

References

  1. Lessnick, S. L., Ladanyi, M. Molecular pathogenesis of Ewing sarcoma: new therapeutic and transcriptional targets. Annu Rev Pathol. 7, 145-159 (2012).
  2. Ladenstein, R. Primary disseminated....

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