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* 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 ....
All procedures were approved by the Georgetown University Institutional Animal Care and Use Committee.
1. Cell Preparation for Orthotopic Injections
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.......
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.......
The authors have nothing to disclose.
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....
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 |
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