In vivo-model for prøvetagning Virkning af Hypoxi på tumormetastase
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 sarkom (ES) er en aggressiv malignitet rammer børn og unge. 1 Tumorerne udvikle sig i blødt væv og knogler, almindeligvis i lemmerne. Mens tilstedeværelsen af metastaser er den mest kraftfulde negativ prognostisk faktor for ES patienter, mekanismerne bag deres udvikling er fortsat uklare. 2 Tumor hypoxia er en af de få faktorer er impliceret i ES progression. I ES-patienter, er tilstedeværelsen af ikke-perfunderede områder i tumorvævet forbundet med dårlig prognose. 3 In vitro hypoxi øger invasionsevne af ES-celler og udløser ekspression af pro-metastatiske gener. 4-6 på trods af disse linjer af beviser, eksisterer imidlertid ingen direkte beviser for hypoxi-induceret ES progression og spredning. Desuden er de mekanismer, hvormed hypoxi udøver sådanne virkninger er i øjeblikket, ukendte. Derfor har vi skabt en in vivo model for at udfylde hullet mellem de eksisterende in vitro data og klinisk obserbemærkninger. Denne model system muliggør direkte test af virkningerne af hypoxi på tumorer, der forekommer i deres naturlige miljø, ved hjælp af magnetisk resonans imaging (MRI) for at følge tumorprogression og metastase in vivo i kombination med ex vivo patologiske og molekylære analyser (figur 1).
Da ingen etableret transgen model af ES er i øjeblikket tilgængelig, in vivo undersøgelser af metastatiske egenskaber af disse tumorer er afhængige af injektioner af humane celler i immunkompromitterede mus. Mens anvendelsen af immunologisk svækkede dyr kan undervurdere virkningen af immunsystemet på sygdomsprogression, evnen til at anvende humane celler øger oversættelighed af sådanne undersøgelser. Blandt forskellige xenograftmodeller, systemiske injektioner i halen vene er de nemmeste at udføre, men de udelader de indledende trin i tumor celle intravasation og flygte fra det primære sted for vækst. 7-12 På den anden side orthoto pic xenografting, som involverer injektioner af tumorceller i knogler (femur, ribben) eller muskler, er mere teknisk udfordrende, men også mere biologisk relevant for human cancer. 13-16 Men i fortiden, den høje sygelighed forbundet med hurtig vækst af primære tumorer har ofte nødvendiggjort dyr eutanasi før metastase udvikling. I denne undersøgelse anvendte vi en tidligere etableret model af celle- injektioner i musculus gastrocnemius efterfulgt af udskæring af den resulterende primære tumor kombineret med langsgående overvågning af metastatisk progression af MRI. 17,18 Sådanne injektioner i musculus gastrocnemius i umiddelbar nærhed af tibia tillader tumorvækst i to naturlige ES miljøer – muskler og knogler – og resultere i fjerne metastaser til steder typisk ramt hos mennesker. 18 Derved denne model rekapitulerer nøjagtigt de metastatiske processer, der forekommer i ES-patienter under sygdomsprogression.
telt "> Lokaliseringen af primære tumorer i den nedre bagbenet letter også præcis styring af blodforsyningen til tumorvævet. femorale arterie ligering (FAL) er en veletableret teknik brugt i angiogenese forskning for at blokere blodstrømmen til distale regioner i benet og undersøge væv vaskularisering som reaktion på iskæmi. 19,20 Vigtigere er det indledende fald i blodstrømning er efterfulgt af sikkerhed beholderåbningen og væv reperfusion observeret ca. 3 dage efter FAL. 20 når således udføres i en tumor-bærende lemmer, denne model genskaber hypoxi / reperfusionshændelser, der forekommer naturligt i hurtigt voksende tumorer og muliggør udslip af metastatiske tumorceller på grund af restaurering af perfusion til den nedre bagbenet via nyligt åbnede kollaterale kar. 21 er vigtigt, denne fremgangsmåde skal udføres, når tumorstørrelsen er lille nok til at forhindre overdreven hypoxi i kontroltumorer (typisk ved tumorbærende kalv volume på 150 – 250 mm 3), sikrer signifikante forskelle i tumor hypoxi mellem kontrol og FAL-behandlede grupper.Foruden langsgående overvågning af effekten af hypoxi på ES latens og hyppigheden af metastaser, denne model giver også mulighed for indsamling af væv og udviklingen af nye cellelinjer fra både primære tumorer og metastaser. Vigtigere, tidligere arbejde fastslået, at metastaser-afledte cellelinier udvise forøget metastatisk potentiale ved genindførelse til dyr, hvilket indikerer at spredning tumor er forbundet med permanente ændringer i tumorcellen fænotype, og derved validere anvendelse af disse cellelinjer til at dechifrere de metastatiske processer. 18 Tilsammen kan disse modeller nu anvendes til de genetiske og molekylære analyser nødvendige for at identificere hypoxi-induceret metastatiske pathways.
Som hypoxi er en pro-metastatisk faktor øge malignitet af forskellige tumors, kan vores model bruges som en platform til at undersøge den rolle, hypoxi ved andre tumorer, der naturligt udvikler i lemmerne, såsom osteosarkom og rhabdomyosarcoma. 21-23 Desuden kan en lignende fremgangsmåde anvendes på maligniteter vokser i andre anatomiske steder med en veldefineret rute blodtilførsel. I sidste ende kan modellen ændres og dens anvendelighed yderligere udvidet, afhængigt af individuelle forskningsbehov.
Protocol
Representative Results
Discussion
Vores model involverer sammenligning af metastase i to forsøgsgrupper – en kontrolgruppe, hvor tumorerne får lov at udvikle sig i bagbenet efterfulgt af amputation efter at have nået en kalv volumen på 250 mm3, og en hypoxi-eksponerede gruppe, hvor tumor- bærende bagben udsættes for FAL ved samme volumen, efterfulgt af amputation 3 dage senere. Selvom i disse eksperimenter de FAL-behandlede tumorer er amputeret med en lille forsinkelse, sammenlignet med kontrol tumorer, er deres størrelse ikke stige i …
Declarações
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 assistance with postmortem x-ray angiography, and providing insight and expertise on collaterogenesis.
Materials
| 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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