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Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Published: December 9th, 2016



1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 2Department of Nursing, Georgetown University, School of Nursing and Health Studies, 3Department of Human Science, Georgetown University, School of Nursing and Health Studies, 4School of Medicine, Georgetown University Medical Center, 5Department of Pathology and Neuropathology, Medical University of Gdańsk, 6Department of Oncology, Georgetown University Medical Center, 7Department of Pathology, Georgetown University Medical Center
* These authors contributed equally

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 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.

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 ....

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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 .......

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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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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.......

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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 the....

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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 28G½ 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-250mg) 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
25G 5/8 Needle (for heart-puncture) BD 305122
0.1 mL syringe (for heart-puncture) Terumo SS-01T
 K3 EDTA Micro tube 1.3ml Sarstedt 41.1395.105
10% Neutral Buttered Formalin Fisher Scientific SF100-4

  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 multifocal Ewing sarcoma: results of the Euro-EWING 99 trial. J Clin Oncol. 28, 3284-3291 (2010).
  3. Dunst, J., Ahrens, S., Paulussen, M., Burdach, S., Jurgens, H. Prognostic impact of tumor perfusion in MR-imaging studies in Ewing tumors. Strahlenther Onkol. 177, 153-159 (2001).
  4. Aryee, D. N. Hypoxia modulates EWS-FLI1 transcriptional signature and enhances the malignant properties of Ewing's sarcoma cells in vitro. Cancer Research. 70, 4015-4023 (2010).
  5. Knowles, H. J., Schaefer, K. L., Dirksen, U., Athanasou, N. A. Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor. BMC cancer. 10, 372 (2010).
  6. Tilan, J. U. Hypoxia shifts activity of neuropeptide Y in Ewing sarcoma from growth-inhibitory to growth-promoting effects. Oncotarget. 4, 2487-2501 (2013).
  7. Franzius, C. Successful high-resolution animal positron emission tomography of human Ewing tumours and their metastases in a murine xenograft model. Eur J Nucl Med Mol Imaging. 33, 1432-1441 (2006).
  8. Hauer, K. DKK2 mediates osteolysis, invasiveness, and metastatic spread in Ewing sarcoma. Cancer Research. 73, 967-977 (2012).
  9. Manara, M. C. Preclinical in vivo study of new insulin-like growth factor-I receptor--specific inhibitor in Ewing's sarcoma. Clin Cancer Res. 13, 1322-1330 (2007).
  10. Scotlandi, K. Murine model for skeletal metastases of Ewing's sarcoma. J Orthop Res. 18, 959-966 (2000).
  11. Vormoor, J. Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice. Pediatr Res. 49, 332-341 (2001).
  12. Picarda, G. Preclinical evidence that use of TRAIL in Ewing's sarcoma and osteosarcoma therapy inhibits tumor growth, prevents osteolysis, and increases animal survival. Clin Cancer Res. 16, 2363-2374 (2010).
  13. Vormoor, B. Development of a preclinical orthotopic xenograft model of ewing sarcoma and other human malignant bone disease using advanced in vivo imaging. PLoS One. 9, e85128 (2014).
  14. Wang, Y. Platelet-derived growth factor receptor beta inhibition increases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity: imatinib and TRAIL dual therapy. Cancer. 116, 3892-3902 (2010).
  15. Wang, Y. X. Inhibiting platelet-derived growth factor beta reduces Ewing's sarcoma growth and metastasis in a novel orthotopic human xenograft model. In Vivo. 23, 903-909 (2009).
  16. Odri, G. A. Zoledronic acid as a new adjuvant therapeutic strategy for Ewing's sarcoma patients. Cancer Research. 70, 7610-7619 (2010).
  17. Merchant, M. S. Interferon gamma enhances the effectiveness of tumor necrosis factor-related apoptosis-inducing ligand receptor agonists in a xenograft model of Ewing's sarcoma. Cancer Research. 64, 8349-8356 (2004).
  18. Hong, S. H. High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases. Oncotarget. 6, 7151-7165 (2015).
  19. Lee, E. W. Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles. J Clin Invest. 111, 1853-1862 (2003).
  20. Tilan, J. U. Platelet neuropeptide Y is critical for ischemic revascularization in mice. FASEB J. , (2013).
  21. Toffoli, S., Michiels, C. Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours. The FEBS journal. 275, 2991-3002 (2008).
  22. Das, B. Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction. Stem cells (Dayton, Ohio). 26, 1818-1830 (2008).
  23. Arndt, C. A., Rose, P. S., Folpe, A. L., Laack, N. N. Common musculoskeletal tumors of childhood and adolescence. Mayo Clin Proc. 87, 475-487 (2012).
  24. Mendoza, A. A novel noninvasive method for evaluating experimental lung metastasis in mice. J Am Assoc Lab Anim Sci. 52, 584-589 (2013).
  25. Feldman, D. B., Seely, J. C. . Necropsy Guide: Rodents and the Rabbit. , (1988).
  26. Parkinson, C. M. Diagnostic necropsy and selected tissue and sample collection in rats and mice. J Vis Exp. , (2011).
  27. Raymond, A. K., Lazar, A. J., PP, L. i. n., S, P. a. t. e. l. . Bone Sarcoma. , (2013).
  28. Dietel, M., Arps, H., Gerding, D., Trapp, M., Niendorf, A. Establishment of primary cell cultures: experiences with 155 cell strains. Klin Wochenschr. 65, 507-512 (1987).
  29. Varghese, A. J., Gulyas, S., Mohindra, J. K. 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. Cancer Research. 36, 3761-3765 (1976).

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