Overall survival rates for pediatric high-grade sarcoma have improved greatly in the past few decades, but prevention and treatment of distant metastasis remain the most compelling problems facing these patients. Traditional preclinical mouse models have not proven adequate to study the biology and treatment of spontaneous distant sarcoma metastasis. To address this deficit, we developed an orthotopic implantation/amputation model in which patient-derived sarcoma xenografts are surgically implanted into mouse hindlimbs, allowed to grow, then subsequently amputated and the animals observed for development of metastases. NOD/SCID/IL-2Rγ-null mice were implanted with either histologically intact high grade sarcoma patient-derived xenografts or cell lines in the pretibial space and affected limbs were amputated after tumor growth. In contrast to subcutaneous flank tumors, we were able to consistently detect spontaneous distant spread of the tumors using our model. Metastases were seen in 27–90 % of animals, depending on the xenograft, and were repeatable and predictable. We also demonstrate the utility of this model for studying the biology of metastasis and present preliminary new insights suggesting the role of arginine metabolism and macrophage phenotype polarization in creating a tumor microenvironment that facilitates metastasis. Subcutaneous tumors express more arginase than inducible nitric oxide synthase and demonstrate significant macrophage infiltration, whereas orthotopic tumors express similar amounts of inducible nitric oxide synthase and arginase and have only a scant macrophage infiltrate. Thus, we present a model of spontaneous distant sarcoma metastasis that mimics the clinical situation and is amenable to studying the biology of the entire metastatic cascade.
Ewing sarcoma Osteosarcoma Rhabdomyosarcoma Arginase Metastasis Animal model
Bovine serum albumin
Inducible nitric oxide synthase
1×TBS and 0.05 % Tween 20
This is a preview of subscription content, log in to check access.
This work was supported by grants from the National Institutes of Health (1R01CA138212-01) and the Liddy Shriver Sarcoma Initiative (to DML), as well as from the Pablove Foundation (to MH). The authors also wish to acknowledge the support of the Giant Food Children’s Cancer Research Fund, the Heather Brooke Foundation, and the Love for Luca Foundation.
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no actual or potential conflicts of interest.
Research involving human and animal rights
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standard of the institution at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.
Wan L, Pantel K, Kang Y (2013) Tumor metastasis: moving new biological insights into the clinic. Nat Med 19:1450–1464CrossRefPubMedGoogle Scholar
Morton CL, Houghton PJ (2007) Establishment of human tumor xenografts in immunodeficient mice. Nat Protoc 2:247–250CrossRefPubMedGoogle Scholar
Johnson JI, Decker S, Zaharevitz D et al (2001) Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Br J Cancer 84:1424–1431PubMedCentralCrossRefPubMedGoogle Scholar
Kerbel RS (2003) Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved. Cancer Biol Ther 2:S134–s139PubMedGoogle Scholar
Norris RE (2012) Adamson PC Challenges and opportunities in childhood cancer drug development. Nat Rev Cancer 12:776–782CrossRefPubMedGoogle Scholar
Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving bioscience research reporting: The arrive guidelines for reporting animal research. PLoS Biol 8:e1000412PubMedCentralCrossRefPubMedGoogle Scholar
Hussain SP, Trivers GE, Hofseth LJ et al (2004) Nitric oxide, a mediator of inflammation, suppresses tumorigenesis. Cancer Res 64:6849–6853CrossRefPubMedGoogle Scholar
Smith MA, Maris JM, Lock R et al (2011) Initial testing (stage 1) of the polyamine analog PG11047 by the pediatric preclinical testing program. Pediatr Blood Cancer 57:268–274PubMedCentralCrossRefPubMedGoogle Scholar
Tanaka S, Saito Y, Kunisawa J et al (2012) Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rγKO mice. J Immunol 188:6145–6155PubMedCentralCrossRefPubMedGoogle Scholar
Ito M, Hiramatsu H, Kobayashi K et al (2002) NOD/SCID/γc null mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100:3175–3182CrossRefPubMedGoogle Scholar
Berlin Ö, Samid D, Donthineni-Rao R, Akeson W, Amiel D, Woods VL Jr (1993) Development of a novel spontaneous metastasis model of human osteosarcoma transplanted orthotopically into bone of athymic mice. Cancer Res 53:4890–4895PubMedGoogle Scholar
Huang P, Allam A, Taghian A, Freeman J, Duffy M, Suit HD (1995) Growth and metastatic behavior of five human glioblastomas compared with nine other histological types of human tumor xenografts in SCID mice. J Neurosurg 83:308–315CrossRefPubMedGoogle Scholar
Khanna C, Prehn J, Yeung C, Caylor J, Tsokos M, Helman L (2000) An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential. Clin Exp Metastasis 18:261–271CrossRefPubMedGoogle Scholar
Luu HH, Kang Q, Jong KP et al (2005) An orthotopic model of human osteosarcoma growth and spontaneous pulmonary metastasis. Clin Exp Metastasis 22:319–329CrossRefPubMedGoogle Scholar
Yuan J, Ossendorf C, Szatkowski JP et al (2009) Osteoblastic and osteolytic human osteosarcomas can be studied with a new xenograft mouse model producing spontaneous metastases. Cancer Investig 27:435–442CrossRefGoogle Scholar
Duyverman AMMJ, Steller EJA, Fukumura D, Jain RK, Duda DG (2012) Studying primary tumor-associated fibroblast involvement in cancer metastasis in mice. Nat Protoc 7:756–762PubMedCentralCrossRefPubMedGoogle Scholar
Chao T, Greager JA (1997) Experimental pulmonary sarcoma metastases in athymic nude mice. J Surg Oncol 65:123–126CrossRefPubMedGoogle Scholar
Pocard M, Tsukui H, Salmon R, Dutrillaux B, Poupon MF (1996) Efficiency of orthotopic xenograft models for human colon cancers. In Vivo 10:463–469PubMedGoogle Scholar
Joo K, Kim J, Jin J et al (2013) Patient-specific orthotopic glioblastoma xenograft models recapitulate the histopathology and biology of human glioblastomas in situ. Cell Rep 3:260–273CrossRefPubMedGoogle Scholar
Kozlowski JM, Fidler IJ, Campbell D, Xu ZL, Kaighn ME, Hart IR (1984) Metastatic behavior of human tumor cell lines grown in the nude mouse. Cancer Res 44:3522–3529PubMedGoogle Scholar
Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29:13435–13444PubMedCentralCrossRefPubMedGoogle Scholar
Yang Z, Ming X (2014) Functions of arginase isoforms in macrophage inflammatory responses: Impact on cardiovascular diseases and metabolic disorders. Front Immunol. doi:10.3389/fimmu.2014.00533Google Scholar
Buddingh EP, Kuijjer ML, Duim RAJ et al (2011) Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clin Cancer Res 17:2110–2119CrossRefPubMedGoogle Scholar
Cheng RYS, Basudhar D, Ridnour LA et al (2014) Gene expression profiles of NO- and HNO-donor treated breast cancer cells: insights into tumor response and resistance pathways. Nitric Oxide Biol Chem 43:17–28CrossRefGoogle Scholar
Heinecke JL, Ridnour LA, Cheng RYS et al (2014) Tumor microenvironment-based feed-forward regulation of NOS2 in breast cancer progression. Proc Natl Acad Sci USA 111:6323–6328PubMedCentralCrossRefPubMedGoogle Scholar
Mayorek N, Naftali-Shani N, Grunewald M (2010) Diclofenac inhibits tumor growth in a murine model of pancreatic cancer by modulation of VEGF levels and arginase activity. PloS One 5(9):e12715PubMedCentralCrossRefPubMedGoogle Scholar
Kobayashi E, Masuda M, Nakayama R et al (2010) Reduced argininosuccinate synthetase is a predictive biomarker for the development of pulmonary metastasis in patients with osteosarcoma. Mol Cancer Ther 9:535–544CrossRefPubMedGoogle Scholar