Advertisement

Clinical & Experimental Metastasis

, Volume 32, Issue 7, pp 703–715 | Cite as

An orthotopic xenograft model with survival hindlimb amputation allows investigation of the effect of tumor microenvironment on sarcoma metastasis

  • Seth D. Goldstein
  • Masanori Hayashi
  • Catherine M. Albert
  • Kyle W. Jackson
  • David M. Loeb
Research Paper

Abstract

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.

Keywords

Ewing sarcoma Osteosarcoma Rhabdomyosarcoma Arginase Metastasis Animal model 

Abbreviations

NSG

NOD/SCID/IL-2Rγ-null

PBS

Phosphate-buffered saline

BSA

Bovine serum albumin

iNOS

Inducible nitric oxide synthase

TBST

1×TBS and 0.05 % Tween 20

DAB

3,39-diaminobenzidine

NO

Nitric oxide

Notes

Acknowledgments

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.

References

  1. 1.
    Adamson PC, Blaney SM (2005) New approaches to drug development in pediatric oncology. Cancer J 11:324–330CrossRefPubMedGoogle Scholar
  2. 2.
    Khanna C, Fan TM, Gorlick R et al (2014) Toward a drug development path that targets metastatic progression in osteosarcoma. Clin Cancer Res 20:4200–4209PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Kern SE, Shibata D (2007) The fuzzy math of solid tumor stem cells: a perspective. Cancer Res 67:8985–8988CrossRefPubMedGoogle Scholar
  4. 4.
    Krishnan K, Khanna C, Helman LJ (2005) The biology of metastases in pediatric sarcomas. Cancer J 11:306–313CrossRefPubMedGoogle Scholar
  5. 5.
    Wan L, Pantel K, Kang Y (2013) Tumor metastasis: moving new biological insights into the clinic. Nat Med 19:1450–1464CrossRefPubMedGoogle Scholar
  6. 6.
    Morton CL, Houghton PJ (2007) Establishment of human tumor xenografts in immunodeficient mice. Nat Protoc 2:247–250CrossRefPubMedGoogle Scholar
  7. 7.
    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
  8. 8.
    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
  9. 9.
    Norris RE (2012) Adamson PC Challenges and opportunities in childhood cancer drug development. Nat Rev Cancer 12:776–782CrossRefPubMedGoogle Scholar
  10. 10.
    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
  11. 11.
    Hussain SP, Trivers GE, Hofseth LJ et al (2004) Nitric oxide, a mediator of inflammation, suppresses tumorigenesis. Cancer Res 64:6849–6853CrossRefPubMedGoogle Scholar
  12. 12.
    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
  13. 13.
    Yan X, Takahara M, Xie L et al (2011) Arginine metabolism in soft tissue sarcoma. J Dermatol Sci 61:211–215CrossRefPubMedGoogle Scholar
  14. 14.
    Williams EL (2005) Djamgoz MBA Nitric oxide and metastatic cell behaviour. Bioessays 27:1228–1238CrossRefPubMedGoogle Scholar
  15. 15.
    Quail DF, Joyce JA (2013) Microenvironmental regulation of tumor progression and metastasis. Nat Med 19:1423–1437PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    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
  17. 17.
    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
  18. 18.
    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
  19. 19.
    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
  20. 20.
    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
  21. 21.
    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
  22. 22.
    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
  23. 23.
    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
  24. 24.
    Chao T, Greager JA (1997) Experimental pulmonary sarcoma metastases in athymic nude mice. J Surg Oncol 65:123–126CrossRefPubMedGoogle Scholar
  25. 25.
    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
  26. 26.
    Talmadge JE, Singh RK, Fidler IJ, Raz A (2007) Murine models to evaluate novel and conventional therapeutic strategies for cancer. Am J Pathol 170:793–804PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    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
  28. 28.
    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
  29. 29.
    Hylander BL, Punt N, Tang H et al (2013) Origin of the vasculature supporting growth of primary patient tumor xenografts. J Transl Med. doi: 10.1186/1479-5876-11-110 PubMedCentralPubMedGoogle Scholar
  30. 30.
    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
  31. 31.
    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.2014.00533 Google Scholar
  32. 32.
    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
  33. 33.
    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
  34. 34.
    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
  35. 35.
    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
  36. 36.
    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
  37. 37.
    Harrell MI, Iritani BM, Ruddell A (2008) Lymph node mapping in the mouse. J Immunol Methods 332:170–174PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Seth D. Goldstein
    • 1
  • Masanori Hayashi
    • 1
  • Catherine M. Albert
    • 1
  • Kyle W. Jackson
    • 1
  • David M. Loeb
    • 1
  1. 1.Sidney Kimmel Comprehensive Cancer CenterJohns Hopkins UniversityBaltimoreUSA

Personalised recommendations