title: The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease.
abstract: BACKGROUND
  Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources.
  
  
  METHODS
  To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions.
  
  
  RESULTS
  Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p < 0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p = 0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p = 0.027). Evaluation of in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression.
  
  
  CONCLUSIONS
  Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.
authors.affiliation: Department of Pediatrics, Leiden University Medical Center, Albinusdreef 2, 2300, RC, Leiden, the Netherlands. a.lankester@lumc.nl.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Berghuis
authors.forename: Marco W
authors.initials: MW
authors.lastname: Schilham
authors.forename: Susy J
authors.initials: SJ
authors.lastname: Santos
authors.forename: Suvi
authors.initials: S
authors.lastname: Savola
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Knowles
authors.forename: Uta
authors.initials: U
authors.lastname: Dirksen
authors.forename: Karl-Ludwig
authors.initials: KL
authors.lastname: Schaefer
authors.forename: Jukka
authors.initials: J
authors.lastname: Vakkila
authors.forename: Pancras Cw
authors.initials: PC
authors.lastname: Hogendoorn
authors.forename: Arjan C
authors.initials: AC
authors.lastname: Lankester